Protein isoforms Research Papers - Academia.edu (original) (raw)

PH-20 is a glycoprotein located on the surface of the sperm plasma membrane and on the inner acrosomal membrane. The best understood function of sperm surface PH-20 is its hyaluronidase activity, which results in hydrolysis of the hyaluronic acid-rich cumulus matrix during sperm penetration of this extracellular oocyte investment. In this study, we investigated whether alterations in the secondary and tertiary structures of sperm surface PH-20 would affect its enzyme activity. Proteins were isolated from the sperm plasma membrane by treatment of living cells with phosphatidylinositol-specific phospholipase C (PI-PLC). PH-20 was purified from the PI-PLC released proteins by immunoaffinity chromatography. Two-dimensional electrophoresis of purified PH-20 revealed 6 isoforms with isoelectric points ranging from 5.1 to 6.0. Removal of the N-linked glycans from PH-20 with N-glycosidase F shifted the molecular weight from 64 kd to approximately 54 kd, its deduced molecular weight based on sequence analysis, suggesting that most if not all, of the potential N-glycosylation sites are linked to oligosaccharides. The lectins Con A and PSA recognized purified sperm surface PH-20 after Western blotting, suggesting that mannose is a major sugar within or at the terminal end of the linked glycan. The lectins UEA and LPA did not recognize PH-20 Western blot, suggesting that fucose and sialic acid are not terminal sugars of sperm surface PH-20. Deglycosylation of sperm surface PH-20 resulted in a complete loss of its hyaluronidase activity. The reduction of disulfide bonds with ␤-mercaptoethanol or dithiothreitol also resulted in loss of enzyme activity. We conclude that the hyaluronidase activity of sperm surface PH-20 is dependent on structural features established by sulfhydryl linkages, as well as glycosylation.

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- Andrology, Sequence Analysis, Protein structure, Glycosylation

Filamin, also called actin binding protein-280, is a dimeric protein that cross-links actin filaments in the cortical cytoplasm. In addition to this ubiquitously expressed isoform (FLN1), a second isoform (ABP-L/␥-filamin) was recently identified that is highly expressed in mammalian striated muscles. A monoclonal antibody was developed, that enabled us to identify filamin as a Z-disc protein in mammalian striated muscles by immunocytochemistry and immunoelectron microscopy. In addition, filamin was identified as a component of intercalated discs in mammalian cardiac muscle and of myotendinous junctions in skeletal muscle. Northern and Western blots showed that both, ABP-L/␥-filamin mRNA and protein, are absent from proliferating cultured human skeletal muscle cells. This muscle specific filamin isoform is, however, up-regulated immediately after the induction of differentiation. In cultured myotubes, ABP-L/␥-filamin localises in Z-discs already at the first stages of Z-disc formation, suggesting that ABP-L/␥-filamin might play a role in Z-disc assembly.

- by Peter F M van der Ven
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- Antibodies, Cell Differentiation, Mice, T cells

Fluorescent fusion proteins are an important tool for the study of vesicle trafficking and exocytosis, especially when combined with newer types of microscopy. We previously reported that the design of a vesicle-targeted fluorescent fusion construct strongly influences the kinetics of fluorescence change at exocytosis. In the present study we demonstrate that the cell in which a construct is expressed also affects the kinetics of fluorescence change at exocytosis. We fused enhanced green fluorescent protein to the carboxy terminus of the vesicular cargo protein rodent islet amyloid polypeptide. The two proteins were separated by a “linker” sequence of 18 amino acids. We then compared kinetics of fluorescence change at exocytosis for this fluorescent cargo protein expressed in three different types of peptidergic endocrine cell: pancreatic alpha cell, pancreatic beta cell, and adrenal chromaffin cell. In resting cells of all three types, fluorescent spots of similar size and membrane-proximal density appeared near the plasma membrane as expected if the probe is stored in large dense-core secretory vesicles. Upon stimulation, the fluorescent spots displayed sudden changes in fluorescence intensity that were consistent with exocytosis. In beta and alpha cells the fluorescent spots consistently brightened and persisted, whereas in chromaffin cells the fluorescent spots always dispersed rapidly. Thus, for fluorescent cargo proteins in peptidergic endocrine cells, cell type influences the kinetics of fluorescence change at exocytosis. Together with our previous findings, this observation strongly highlights the fact that the behavior of vesicle-targeted fluorescent cargo may be unrelated to that of native cargo, and it emphasizes the need for caution in interpreting fluorescence kinetics in terms of an exocytosis mechanism.

- by Joyce Rohan
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- Kinetics, Multidisciplinary, Pancreas, Peptides

The antimicrobial peptide human α-defensin 5 (HD5) is expressed in Paneth cells, secretory epithelial cells in the small intestine. Unlike other characterized defensins, HD5 is stored in secretory vesicles as a propeptide. The storage quantities of HD5 are ∼90-450 µg per cm 2 of mucosal surface area, which is sufficient to generate microbicidal concentrations in the intestinal lumen. HD5 peptides isolated from the intestinal lumen are proteolytically processed forms-HD5(56-94) and HD5(63-94)-that are cleaved at the Arg 55 -Ala 56 and Arg 62 -Thr 63 sites, respectively.We show here that a specific pattern of trypsin isozymes is expressed in Paneth cells, that trypsin colocalizes with HD5 and that this protease can efficiently cleave HD5 propeptide to forms identical to those isolated in vivo. By acting as a prodefensin convertase in human Paneth cells, trypsin is involved in the regulation of innate immunity in the small intestine.

- by satya to
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- Pharmacology, Biochemistry, Bioinformatics, Evolutionary Biology
- by Wenting Luo
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- Immunology, Transcription Regulation, Transcription Factors, Protein Stability

Mena, an actin regulatory protein, functions at the convergence of motility pathways that drive breast cancer cell invasion and migration in vivo. The tumor microenvironment spontaneously induces both increased expression of the Mena INV and decreased expression of Mena11a isoforms in invasive and migratory tumor cells. Tumor cells with this Mena expression pattern participate with macrophages in migration and intravasation in mouse mammary tumors in vivo. Consistent with these findings, anatomical sites containing tumor cells with high levels of Mena expression associated with perivascular macrophages were identified in human invasive ductal breast carcinomas and called TMEM. The number of TMEM sites positively correlated with the development of distant metastasis in humans. Here we demonstrate that mouse mammary tumors generated from EGFP-Mena INV expressing tumor cells are significantly less cohesive and have discontinuous cell-cell contacts compared to Mena11a xenografts. Using the mouse PyMT model we show that metastatic mammary tumors express 8.7 fold more total Mena and 7.5 fold more Mena INV mRNA than early non-metastatic ones. Furthermore, Mena INV expression in fine needle aspiration biopsy (FNA) samples of human invasive ductal carcinomas correlate with TMEM score while Mena11a does not. These results suggest that Mena INV is the isoform associated with breast cancer cell discohesion, invasion and intravasation in mice and in humans. They also imply that Mena INV expression and TMEM score measure related aspects of a common tumor cell dissemination mechanism and provide new insight into metastatic risk.

- by Frank Gertler
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- Cell Adhesion, Tumor microenvironment, Mice, Protein Function

We hypothesize that estrogen receptors (ERs) are differentially expressed in endometrial cancer. To test this hypothesis, we investigated the expression profile of ERalpha (ERalpha-A, ERalpha-B, ERalpha-C) and ERbeta genes and CpG methylation status in endometrial cancer cell lines and tissues using reverse transcription-PCR and methylation-specific PCR and direct DNA sequencing. The results demonstrated that ERalpha-A, ERalpha-B, and ERbeta were normally expressed whereas ERalpha-C gene was inactivated in all endometrial cancer cell lines. We further investigated the mechanisms of ERalpha-C gene inactivation through CpG methylation pathways. The treatment with demethylating agent (5'-aza-2'-deoxycytidine) restored ERalpha-C gene expression in all endometrial cancer cell lines. We further confirmed these findings with methylation-specific PCR and direct DNA sequencing and found that only ERalpha-C was methylated on all five different CpG sites in all cell lines. We further a...

- by Abhipsa Dharia
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- Cancer, Estrogen Receptor, Endometrial Cancer, Cell line

We describe a new method, Tag-seq, which employs ultra high-throughput sequencing of 21 base pair cDNA tags for sensitive and cost-effective gene expression profiling. We compared Tag-seq data to LongSAGE data and observed improved representation of several classes of rare transcripts, including transcription factors, antisense transcripts, and intronic sequences, the latter possibly representing novel exons or genes. We observed increases in the diversity, abundance, and dynamic range of such rare transcripts and took advantage of the greater dynamic range of expression to identify, in cancers and normal libraries, altered expression ratios of alternative transcript isoforms. The strand-specific information of Tag-seq reads further allowed us to detect altered expression ratios of sense and antisense (S-AS) transcripts between cancer and normal libraries. S-AS transcripts were enriched in known cancer genes, while transcript isoforms were enriched in miRNA targeting sites. We found that transcript abundance had a stronger GC-bias in LongSAGE than Tagseq, such that AT-rich tags were less abundant than GC-rich tags in LongSAGE. Tag-seq also performed better in gene discovery, identifying >98% of genes detected by LongSAGE and profiling a distinct subset of the transcriptome characterized by AT-rich genes, which was expressed at levels below those detectable by LongSAGE. Overall, Tag-seq is sensitive to rare transcripts, has less sequence composition bias relative to LongSAGE, and allows differential expression analysis for a greater range of transcripts, including transcripts encoding important regulatory molecules.

- by Helen McDonald
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- Algorithms, Biological Sciences, Genome, Next Generation

Cytochrome b561 family was characterized by the presence of ''b561 core domain'' that forms a transmembrane four helix bundle containing four totally conserved His residues, which might coordinate two heme b groups. We conducted BLAST and PSI-BLAST searches to obtain insights on structure and functions of this protein family. Analyses with CLUSTAL W on b561 sequences from various organisms showed that the members could be classified into 7 subfamilies based on characteristic motifs; groups A (animals/neuroendocrine), B (plants), C (insects), D (fungi), E (animals/TSF), F (plants + DoH), and G (SDR2). In group A, both motif 1, {FN(X)HP(X) 2 M(X) 2 G(X) 5 G(X)ALLVYR}, and motif 2, {YSLHSW(X)G}, were identified. These two motifs were also conserved in group B. There was no significant features characteristic to groups C and D. A modified version of motif 1, {LFSWHP(X) 2 M(X) 3 F(X) 3 M(X)EAIL(X)SP(X) 2 SS}, was found in group E with a high degree of conservation. Both motif 3, {DP(X)WFY(L)H(X) 3 Q}, and motif 4, {K(X)R(X)YWN(X)YHH(X) 2 G(R/Y)} ,were found in group F at different regions from those of motifs 1 and 2. The ''DoH'' domain common to the NH 2 -terminal region of dopamine h-hydroxylase was found to form fusion proteins with the b561 core domains in groups F and G. Based on these results, we proposed a hypothesis regarding structures and functions of the 7 subfamilies of cytochrome b561. D

- by Motonari Tsubaki
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- Biological Sciences, Phylogeny, Sequence alignment, Physical sciences

The Protein Ontology (PRO) provides a formal, logically-based classification of specific protein classes including structured representations of protein isoforms, variants and modified forms. Initially focused on proteins found in human, mouse and Escherichia coli, PRO now includes representations of protein complexes. The PRO Consortium works in concert with the developers of other biomedical ontologies and protein knowledge bases to provide the ability to formally organize and integrate representations of precise protein ...

- by Michael Caudy
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- Computer Science, Biology, Medicine, Biological Sciences

Bleb formation has been correlated with nonmuscle myosin II (NM-II) activity. Whether three isoforms of NM-II (NM-IIA, -IIB, and -IIC) have the same or differential roles in bleb formation is not well understood. Here, we report that ectopically expressed GFP-tagged NM-II isoforms exhibit different types of membrane protrusions such as multiple blebs, lamellipodia, combination of both or absence of any such protrusions in MCF-7 cells. Quantification suggests that 50% of NM-IIA-GFP, 29% of NM-IIB-GFP and 19% of NM-IIC1-GFP expressing MCF-7 cells show multiple blebs formation compared with 36% of GFP alone expressing cells. Interestingly, NM-IIB has almost 50% lesser rate of dissociation from actin filament compared with NM-IIA and -IIC1 as determined by FRET analysis both at cell and bleb cortices. When we induce bleb formation by disruption of the cortex, we find that all three NM-II-GFP isoforms can re-appear and form filaments to a different degree in the growing bleb. NM-IIB-GFP ...

- by Kankan Bhattacharyya
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- Cytoskeleton, Biological Sciences, Actin Cytoskeleton, Protein isoforms

Objectives In experimental pharmacology, drug effect studies currently establish and analyse cumulative concentration-response curves (CCRC) under repeated measurements designs. Usually the CCRC parameters are estimated using the Hill's function in a nonlinear regression for independent data. The two-way analysis of variance is generally used to identify a statistical difference between the responses for two treatments but that analysis does not take into account the nonlinearity of the model and the heteroscedasticity (uneven distribution) of the data. We presently tested the possibility of finding a statistical solution for the nonlinear response in repeated measurements data using the nonlinear mixed effects (nlme) models. Methods Experimental data sets, originating from studies on b-adrenoceptor-induced relaxation in rat thoracic aorta ring, were analysed using the nlme methods. Key findings Comparison with classical methods showed the superiority of the nlme models approach. For each pharmacological parameter (E m , n, pD 2), a point estimate, a standard error and a confidence interval are returned by the nlme procedures respecting the assumption of independency and normality of the residuals. Conclusions Using the method presently described, it is now possible to detect significant differences for each pharmacological parameter estimated in different situations, even for designs with small samples size (i.e. at least six complete curves).

Current evidence supports the notion that the amyloid β-peptide (Aβ) plays a major role in the neurotoxicity observed in the brain in Alzheimer's disease. However, the signal transduction mechanisms involved still remain unknown. In the present work, we analyzed the effect of protein kinase C (PKC) on some members of the Wnt signaling pathway and its implications for Aβ neurotoxicity. Activation of PKC by phorbol 12-myristate 13-acetate protected rat hippocampal neurons from Aβ toxicity. This effect was accomplished by inhibition of glycogen synthase kinase-3β (GSK-3β) activity, which led to the accumulation of cytoplasmic β-catenin and transcriptional activation via β-catenin/T-cell factor/lymphoid enhancer factor-1 (TCF/LEF-1) of Wnt target genes, which in the present study were engrailed-1 (en-1) and cyclin D1 (cycD1). In contrast, inhibition of Ca 2+ -dependent PKC isoforms activated GSK-3β and offered no protection from Aβ neurotoxicity. Wnt-3a and lithium salts, classical activators of the Wnt pathway, mimicked PKC activation. Our results suggest that regulation of members of the Wnt signaling pathway by Ca 2+ -dependent PKC isoforms may be important in controlling the neurotoxic process induced by Aβ.

- by Alejandra Alvarez
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- Physiology, Transcription Factors, Signal Transduction, Hippocampus

Elevated glucose concentration increases oxidation and Advanced Glycation End product (AGE) formation. The binding of circulatory AGEs or AGEs included in erythrocyte membrane to the receptor for AGEs (RAGE) generates in endothelial cells an oxidative stress and enhances the expression of inflammatory molecules. Engagement of RAGE by AGEs and subsequent signaling plays an important role in the development of diabetic complications. Soluble RAGE isoforms (sRAGE) neutralize the ligand-mediated damage by acting as a decoy. If the expression of RAGE is upregulated during the pathogenesis of inflammatory diseases, sRAGE mostly found decreased when complications ensue. By modulating RAGE isoform expression, it could be possible to reduce the incidence of complications. This review focused on the capability of Angiotensin Receptor Blockers (ARBs), which are used to treat patients with hypertension and/or diabetes, to modulate RAGE isoform expression because some data reported the interfere...

The dynamic actin cytoskeleton, consisting of six actin isoforms in mammals and a variety of actin binding proteins is essential for all developmental processes and for the viability of the adult organism. Actin isoform specific functions have been proposed for muscle contraction, cell migration, endo-and exocytosis and maintaining cell shape. However, these specific functions for each of the actin isoforms during development are not well understood. Based on transgenic mouse models, we will discuss the expression patterns of the six conventional actin isoforms in mammals during development and adult life. Ablation of actin genes usually leads to lethality and affects expression of other actin isoforms at the cell or tissue level. A good knowledge of their expression and functions will contribute to fully understand severe phenotypes or diseases caused by mutations in actin isoforms.

- by Christophe Ampe
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- Smooth muscle, Mice, Deafness, Clinical Sciences

We earlier identified a developmental series of seven isoforms/polymorphs of microparticulate inulin by comparing non-covalent bonding strengths. Their pharmaceutical utility lies in modulation of cellular immunity, exploited as vaccine adjuvants Advax™ especially for delta inulin (DI). As such particles cannot be sterilized by filtration we explore the effect of 60Co gamma radiation (GR) on inulin isoforms, particularly DI. Its adjuvant activity and overt physical properties were unaffected by normal GR sterilizing doses (up to 25 kGy). Heating irradiated isoform suspensions near their critical dissolution temperature revealed increased solubility deduced to reflect a single lethal event in one component of a multi-component structure. Local oxidative effects of GR on DI were not found. The observed DI loss was almost halved by re-annealing at the critical temperature: surviving inulin chains apparently reassemble into smaller amounts of the original type of structure. Colorimetric tetrazolium assay revealed increases in reducing activity after GR of raw inulin powder, which yielded DI with normal physical properties but only 25% normal recovery yet 4x normal reducing ability, implying final retention of some GR-changed inulin chains. These findings suggest minimal inulin chain cleavage and confirm that GR may be a viable strategy for terminal sterilization of microparticulate inulin adjuvants. Highlights 1. Advax™ adjuvant (delta inulin-based) has uniform particles ~1-2 µm diameter 2. Micro particles this size cannot be terminally sterile-filtered 3. 60 Co sterilization (25 kGy) does not overtly affect adjuvant action or structure 4. Isoform and reducing-end analyses show single-hit cleavage of some inulin chains 5. Gamma irradiation appears a viable strategy for Advax™ terminal sterilisation *Highlights (for review)

- by Nikolai Petrovsky and +1
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- Gamma Rays, Biological Sciences, Mice, Vaccine

Filamentous tau deposits are a defining feature of a number of human neurodegenerative diseases. Apes and monkeys have been reported to be differentially susceptible to developing tau pathology. Despite this, only little is known about the organisation and sequence of Tau from nonhuman primates. Here we have sequenced Tau exons 1-13, including flanking intronic regions, and the region in intron 9 that contains Saitohin in chimpanzees, gorillas, and gibbons. Partial sequences were obtained for cynomolgus macaque and green monkey. Chimpanzee brain tau was 100% identical to human tau. Identities were 99.5% for gorilla tau and 99.0% for gibbon tau. Chimpanzee DNA was polymorphic for a repeat in intron 9, which was present in human and gorilla tau, and for the nucleotide at position +29 of the intron that follows exon 10. As was the case of the other nonhuman primates examined, chimpanzee DNA was homozygous for nucleotides used to define the H2 haplotype in human Tau . These differences between human and chimpanzee Tau may contribute to the apparent resistance of chimpanzee brain to developing tau pathology. Sequencing of Saitohin revealed an intact open reading frame in chimpanzee and gorilla, but not in gibbon or macaque. D

- by Thomas Arendt
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- Genetics, RNA, Polymorphism, Western blotting

Certain drug classes alleviate the symptoms of Willis-Ekbom's disease, whereas others aggravate them. The pharmacological profiles of these drugs suggest that drugs that alleviate Willis-Ekbom's disease inhibit thyroid hormone activity, whereas drugs that aggravate Willis-Ekbom's disease increase thyroid hormone activity. These different effects may be secondary to the opposing actions that drugs have on the CYP4503A4 enzyme isoform. Drugs that worsen the symptoms of the…

- by Rosana Alves
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- Treatment Outcome, Adolescent, Child, Young Adult

Variable environmental availability of metal ions represents a constant challenge for most organisms, so that during evolution, they have optimised physiological and molecular mechanisms to cope with this particular requirement. Metallothioneins (MTs) are proteins that play a major role in metal homeostasis and as a reservoir. The MT gene/protein systems of terrestrial helicid snails are an invaluable model for the study of metal-binding features and MT isoform-specific functionality of these proteins. In the present study, we characterised three paralogous MT isogenes and their expressed products in the escargot (Cantareus aspersus). The metal-dependent transcriptional activation of the three isogenes was assessed using quantitative Real Time PCR. The metal-binding capacities of the three isoforms were studied by characterising the purified native complexes. All the data were analysed in relation to the trace element status of the animals after metal feeding. Two of the three C. aspersus MT (CaMT) isoforms appeared to be metal-specific, (CaCdMT and CaCuMT, for cadmium and copper respectively). A third isoform (CaCd/CuMT) was non-specific, since it was natively recovered as a mixed Cd/Cu complex. A specific role in Cd detoxification for CaCdMT was revealed, with a 80–90% contribution to the Cd balance in snails exposed to this metal. Conclusive data were also obtained for the CaCuMT isoform, which is involved in Cu homeostasis, sharing about 30–50% of the Cu balance of C. aspersus. No apparent metal-related physiological function was found for the third isoform (CaCd/CuMT), so its contribution to the metal balance of the escargot may be, if at all, of only marginal significance, but may enclose a major interest in evolutionary studies.

- by Silvia De Palacios
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- Mass Spectrometry, Molecular Mechanics, Sequence alignment, Copper

Human interleukin 6 (IL-6) is a potent cytokine with immunomodulatory properties. As the influence of N-glycosylation on the in vivo activities of IL-6 could not be elucidated so far, a semisynthesis of homogeneous glycoforms of IL-6 was established by sequential native chemical ligation. The four cysteines of IL-6 are convenient for ligations and require only the short synthetic glycopeptide 43-48. The Cys-peptide 49-183 could be obtained recombinantly by cleavage of a SUMO tag. The fragment 1-42 was accessible by the simultaneous cleavage of two inteins, leading to the 1-42 thioester with the native Nterminus. Ligation and refolding studies showed that the inherently labile AspÀPro bond 139-140 was detrimental for the sequential C-to N-terminal ligation. A reversed ligation sequence using glycopeptide hydrazides gave full-length IL-6 glycoproteins, which showed full bioactivity after efficient refolding and purification.

- by Daniela Gradinaru
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- Cognitive Science, Aging, Drug metabolism, Cerebral Cortex

Imatinib currently represents the standard treatment in the early chronic phase of chronic myelogenous leukemia (CML), thanks to the high percentage of cytogenetic complete remission achieved, but it is yet unclear to what extent it can eradicate leukemia. Therefore, different vaccination strategies have been suggested, mainly based on the exploitment of the junctional peptides spanning the fusion region of the Bcr/Abl proteins. To identify new potential immunologic targets, 63 Philadelphia chromosome-positive patients and 6 BCR/ABL-positive cell lines were tested in nested reverse transcriptase PCR to detect the presence of BCR/ABL transcripts arising from the alternative splicing of the main BCR/ ABL transcripts. We could detect BCR/ABL transcripts with junctions between BCR exon 1, 13, or 14 and ABL exon 4 in f80% of patients and 84% of cell lines, beside the main fusion transcripts. Translation products of these transcripts were characterized at their COOH terminus by a large amino acid portion derived from the out of frame (OOF) reading of ABL gene. These proteins were detected in BCR/ABL-positive cell lines by immunoprecipitation and immunohistochemistry. Finally, we determined whether OOF-specific CD8 + T cells could be found in the peripheral blood of CML patients and whether they could acquire effector function following in vitro sensitization with OOF-derived peptides predicted to bind to human leucocyte antigen (HLA)-A2 and HLA-A3 molecules. We detected the presence of OOF-specific CD8 + T cells in four of four patients studied, and in one case, these T cells exhibited specific cytotoxic activity against both peptide-pulsed targets and autologous primary CML cells.

- by Giancarlo Tonon and +1
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- Cancer, Immunohistochemistry, Cell line, Immunotherapy

Estrogen (17b-estradiol, E2) plays pivotal roles in the function and maintenance of the skeleton, including the bone-forming osteoblasts (OBs). The functions of E2 are largely mediated through two distinct estrogen receptor isoforms, ERa and ERb, both of which are expressed in OBs. The level of each isoform dominates at early or late stages of OB differentiation. To date, only a limited comparison between the transcriptional targets of ERa and ERb on endogenous gene expression has been reported. We have developed new stable cell lines, which contain doxycycline (Dox)-inducible ERa and ERb, in the U2OS human osteosarcoma to determine the global transcriptional profile of ERa-and ERb-regulation of endogenous gene expression. The U2OS-ERa and U2OS-ERb cell lines were treated with Dox and either vehicle control or E2 for 24 h. Gene expression analysis was performed using a microarray containing $6,800 full-length genes. We detected 63 genes that were regulated solely by ERa and 59 genes that were only regulated solely by ERb. Of the ERaregulated genes, 81% were upregulated and 19% were inhibited. Similarly 76% of the ERb-regulated genes were upregulated whereas 24% were inhibited by E2. Surprisingly, only 17 genes were induced by both ERa and ERb. Real-time PCR was employed to confirm the expression of a selected number of genes. The regulation of a number of known E2responsive genes in human OBs, as well as many interesting novel genes, is shown. The distinct patterns of E2-dependent gene regulation in the U2OS cells by ERa and ERb shown here suggest that during OB differentiation, when either isoform dominates, a unique pattern of gene responses will occur, partially due to the differentiation state and the ER isoform present.

- by Thomas Spelsberg
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- Gene expression, Cell Division, Estrogen Receptor, Cell line
- by Sylvain Bourgoin
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- Immunology, Macrophages, Immunology of the Gut, Confocal Microscopy

Inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4) is a 120 kDa acute-phase glycoprotein produced primarily in the liver, secreted into the blood, and identified in serum. ITIH4 is involved in liver development and stabilization of the extracellular matrix (ECM), and its expression is altered in liver disease. In this study, we aimed to characterize glycosylation of recombinant and serum-derived ITIH4 using analytical mass spectrometry. Recombinant ITIH4 was analyzed to optimize glycopeptide analyses, followed by serum-derived ITIH4. First, we confirmed that the four ITIH4 N-X-S/T sequons (N81, N207, N517, and N577) were glycosylated by treating ITIH4 tryptic/ GluC glycopeptides with PNGaseF in the presence of 18 O water. Next, we performed glycosidase-assisted LC−MS/MS analysis of ITIH4 trypsin-GluC glycopeptides enriched via hydrophilic interaction liquid chromatography to characterize ITIH4 N-glycoforms. While microheterogeneity of N-glycoforms differed between ITIH4 protein expressed in HEK293 cells and protein isolated from serum, occupancy of N-glycosylation sites did not differ. A fifth N-glycosylation site was discovered at N274 with the rare nonconsensus NVV motif. Site N274 contained high-mannose N-linked glycans in both serum and recombinant ITIH4. We also identified isoform-specific ITIH4 O-glycoforms and documented that utilization of O-glycosylation sites on ITIH4 differed between the cell line and serum.

- by Zuzana Brnakova
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- Biological Sciences, Glycoproteins, Glycosylation, CHEMICAL SCIENCES
- by Paul Rosch
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- Immunology, Protein Folding, Molecular Mechanics, Food Allergy

The adaptor protein Tks5/FISH (tyrosine kinase substrate 5/five SH3 domains, hereafter termed Tks5) is a crucial component of a protein network that controls the invasiveness of cancer cells and progression of Alzheimer's disease. Tks5 consists of an amino-terminal PX domain that is followed by five SH3 domains (SH3A-E), and two different splice variants are expressed. We identified son of sevenless-1 (Sos1) as a novel binding partner of Tks5 and found colocalization of Tks5 with Sos1 in human epithelial lung carcinoma (A549) cells and in podosomes of Src-transformed NIH 3T3 cells. We observe synergistic binding of SH3A and SH3B to Sos1 when peptide arrays are used, indicating that the tandem SH3A and SH3B domains of Tks5 can potentially bind in a superSH3 binding mode, as was described for the homologous protein p47phox. These results are further corroborated by pull-down assays and isothermal titration calorimetry showing that both intact SH3 domains are required for efficient binding to the entire proline-rich domain of Sos1. The presence of a basic insertion between the SH3A and SH3B domains in the long splice variant of Tks5 decreases the affinity to Sos1 isoforms about 10-fold as determined by analytical ultracentrifugation. Furthermore, it leads to an alteration in the recognition of binding motifs for the interaction with Sos1: While the insertion abrogates the interaction with the majority of peptides derived from the proline-rich domains of Sos1 and dynamin that are recognized by the short splice isoform, it enables binding to a different set of peptides including a sequence comprising the splice insertion in the long isoform of Sos1 (Sos1_2). In the absence of the basic insertion, Tks5 was found to bind a range of Sos1 and dynamin peptides including conventional proline-rich motifs and atypical recognition sequences. Hereby, the tandem SH3 domains in Tks5 employ two distinct types of binding modes: One class of peptides is recognized by single SH3 domains, whereas a second class of peptides requires the presence of both domains to bind synergistically. We conclude that the tandem SH3A and SH3B domains of Tks5 constitute a versatile module for the implementation of isoform-specific protein-protein interactions.

- by Sandra Beer-hammer
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- Molecular Biology, Signal Transduction, Molecular, Protein-Protein Interaction

A role for the small G protein rho and rho-kinase has been shown in smooth muscle contraction regarding Ca ++ sensitivity. However, there are no data in the literature assessing how this system operates in human umbilical arteries (HUA). Therefore, we evaluated the effects of HA-1077 and Y-27632, two rho-kinase inhibitors, on agonist-(5-hydroxytryptamine [5-HT]) and depolarizationinduced (KCl) contractions of HUA. HA-1077 and Y-27632 inhibited 5-HT-induced contractile responses at 10 -4 M concentration but not at 10 -5 M. HA-1077 at 10 -4 M also significantly attenuated contractions induced by 20 mM KCl. In addition, HUA precontracted with 5-HT relaxed concentration dependently in response to HA-1077 and Y-27632. When precontracted with KCl, HUA also relaxed dose-dependently in response to HA-1077, but the maximal relaxation was significantly smaller than the response obtained when precontracted with 5-HT. To determine possible involvement of rho-kinase on agonist-induced intracellular calcium-mediated contractions, tissues were precontracted with 5-HT in Ca ++ -free Krebs solution before cumulative addition of HA-1077 or Y-27632 (10 -7 to 10 -4 M). Both rho-kinase inhibitors relaxed HUA completely. Maximum relaxations of HUA to HA-1077 and Y-27632 were significantly larger than the responses seen in normal Krebs solution and were obtained with lower concentrations of the drugs considered to be more specific for rho-kinase inhibition. However, preincubation of HUA with HA-1077 or Y-27632 (10 -5 M for both) did not affect the 5-HT-induced contractions in this medium. Finally, immunoblot experiments revealed the expression of rho-kinase isoform rockII protein in HUA. These results indicate that rhoA/rho-kinase pathway can contribute to agonist-induced contractions of HUA. However, this effect appears to be limited to intracellular calcium-induced contractions and may be more important in sustaining contractions rather than the initial phase of force development.

- by Mustafa Ark
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- Biophysics, Calcium, Serotonin, Smooth muscle

Previous studies in rats have indicated that a diet enriched with Bisphenol A adversely effects metabolism and reproductive success. In rats exposed to BPA by maternal gavage, alteration in the developmental programming, higher obesity rates and reproductive anomalies were induced. Starting with this evidence, the aim of this study was to provide important insights on the effects induced by a BPA enriched diet, on the reproductive physiology and metabolism of juvenile fish, simulating the scenario occurring when wild fish fed on prey contaminated with environmental BPA. Seabream was chosen as model, as it is one of the primary commercial species valued by consumers and these results could provide important findings on adverse effects that could be passed on to humans by eating contaminated fish. A novel method for measuring BPA in the food and water by affinity chromatography was developed. Analysis of signals involved in reproduction uncovered altered levels of vtg and Zp, clearly indicating the estrogenic effect of BPA. Similarly, BPA up-regulated catd and era gene expression. A noteworthy outcome from this study was the full length cloning of two vtg encoding proteins, namely vtgA and vtgB, which are differently modulated by BPA. Cyp1a1 and EROD activity were significantly downregulated, confirming the ability of estrogenic compounds to inhibit the detoxification process. GST activity was unaffected by BPA contamination, while CAT activity was down regulated. These results collectively confirm the estrogenic effect of BPA and provide additional characterization of novel vtg genes in Sparus aurata.

- by Damiano Mita
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- Fish Diseases, Aquaculture, Biological Sciences, Environmental Sciences

Two isoforms of ADPglucose pyrophosphatase/phosphodiesterase (AGPPase) have been characterized using barley leaves (Hordeum vulgare L.). Whilst one of the isoforms, designated as soluble AGPPase1 (SAGPPase1), is soluble in low ionic strength buffers, the other, SAGPPase2, is extractable using cell wall hydrolytic enzymes or high salt concentration solutions, thus indicating that it is adventitiously bound to the cell wall. Both AGPPase isoforms are highly resistant to SDS, this characteristic being utilized to purify them to homogeneity after zymographic detection of AGPPase activity in SDS-containing gels. N-terminal and internal amino acid sequencing analyses revealed that both SAGPPase1 and SAGPPase2 are distinct oligomers of the previously designated HvGLP1, which is a member of the ubiquitously distributed group of proteins of unknown function designated as germin-like proteins (GLPs).

- by Jose MUÑOZ LOPEZ
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- Kinetics, Glucose, Oxidoreductases, Hordeum vulgare

N-methyl-d-aspartate receptors (NMDARs) are present at many excitatory glutamate synapses in the central nervous system and display unique properties that depend on their subunit composition. Biophysical, pharmacological and molecular methods have been used to determine the key features conferred by the various NMDAR subunits, and have helped to establish which NMDAR subtypes are present at particular synapses. Recent studies are

- by Stuart Cull-candy
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- Pharmacology, Neuroscience, Cognitive Science, Schizophrenia

Memory impairment is a process associated with alterations in neuronal plasticity, synapses formation, and stabilization. As the neural cell adhesion molecule (NCAM) plays a key role in synaptic bond stabilization, we analyzed the usefulness of soluble NCAM isoforms in the diagnosis of patients with dementia of the Alzheimer type (DAT).NCAM was measured in the sera of 70 control subjects and

- by JosÉ Lastiri
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- Neurobiology Of Disease, Neurobiology, Brain, Neuronal Plasticity

Testicular descent corresponds to migration of the testis from the abdominal cavity to the scrotum and is essential for proper functioning of the testis. Recent advances in the characterization of estrogen receptor (ESR) subtypes and isoforms in various tissues prompted us to study ESRs within the gubernaculum testis, a structure involved in testicular descent. In the rat gubernaculum, we searched for ESR alpha (Esr1) and beta (Esr2) and for the androgen receptor (Ar), androgens being known to regulate testicular descent. Reverse transcription-polymerase chain reaction (RT-PCR) revealed that Esr1, Esr2, and Ar mRNAs were all expressed in the gubernaculum. Using PEETA (Primer extension, Electrophoresis, Elution, Tailing, and Amplification), we established that all Esr1 leader exons, previously identified in other organs, such as the uterus and pituitary, were transcribed in the gubernaculum, with the major form being O/B. The RNA protection assays, RT-PCR, and Western blot experiments revealed that isoform-specific mRNA transcripts generated by alternative splicing of the C-leader sequence on coding exons 1 and 2 of the Esr1 gene gave the 46-and 66-kDa ESR1 proteins. The ESR1 and AR proteins were found to colocalize in the parenchymal cells of the gubernaculum early in development, whereas AR also was strongly expressed in the muscular cells, both during fetal and postnatal life. The ESR2 protein was weakly expressed, principally in the muscular cells, but only once testicular descent had occurred. The levels of the 46-kDa ESR1 variant (ER46) exceeded those of the 66-kDa ESR1 form (ER66) at periods when the gubernaculum developed. Conversely, the 66-kDa form appears to predominate clearly when the gubernaculum growth was low or completed. The possible role of estrogens on the modulation of the androgen-dependent growth of the gubernaculum and, more widely, on testicular descent is discussed.

- by Philippa Saunders
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- Estrogen Receptor, Biological Sciences, Alternative splicing, tESTIS

Claudin-18 isoform 2 (CLDN18.2) is one of the few members of the human claudin family of tight junction molecules with strict restriction to one cell lineage. The objective of the current study was to compare molecular structure and tissue distribution of this gastrocyte specific molecule in mammals. We show here that the CLDN18.2 protein sequence is highly conserved, in particular with regard to functionally relevant domains in mouse, rat, rabbit, dog, monkey and human and also in lizards. Moreover, promoter regions of orthologs are highly homologous, including the binding site of the transcription factor cyclic AMP-responsive element binding protein (CREB), which is known to regulate activation of human CLDN18.2. Employing RT-PCR and immunohistochemistry, we found that, analogous to the human gene, all orthologous CLDN18.2 transcripts and proteins are exclusively expressed in differentiated gastric cells. Gene structure, promoter elements and RNA expression pattern of the lung-tissue specific Claudin-18 isoform 1 (CLDN18.1) as well, are homologous across species. These findings exemplify phylogenetic conservation of lineage-specific members of a multigene family. Given that CLDN18.2 is a novel drug target candidate, our data is also relevant for drug development as it reveals all six investigated mammalian species as suitable models for testing safety of CLDN18.2 targeting regimen.

- by Ugur Sercan SAHIN
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- Genetics, Molecular Evolution, Membrane Proteins, Immunohistochemistry

HUVEC or mouse 3T3 cells infected with SV-40 generate within 3 to 5 days post-infection an ENOX2 species corresponding to the exon-4 minus splice variant of a tumor-associated NADH oxidase (ENOX2 or tNOX) expressed at the cancer cell surface. This study was to seek evidence for splicing factors that might direct formation of the exon 4 minus ENOX2 splice variant. To determine if silencing of ENOX2 exon 4 occurs because of motifs located in exon 4, transfections were performed on MCF-10A (mammary non-cancer), BT-20 (mammary cancer), and HeLa (cervical cancer) cells using a GFP minigene construct containing either a constitutively spliced exon (albumin exon 2) or the alternatively spliced ENOX2 exon 4 between the two GFP halves. Removal of exon 4 from the processed RNA of the GFP minigene construct occurred with HeLa and to a lesser extent with BT-20 but not in non-cancer MCF-10A cells. The Splicing Rainbow Program was used to identify all of the possible hnRNPs binding sites of exon 4 of ENOX2. There are 8 Exonic Splicing Silencers (ESSs) for hnRNP binding in the exon 4 sequences. Each of these sites were mutated by site-directed mutagenesis to test if any were responsible for the splicing skip. Results showed MutG75 ESS mutation changed the GFP expression which is a sign of splicing silence, while other mutations did not. As MutG75 changed the ESS binding site for hnRNP F, this result suggests that hnRNP F directs formation of the exon 4 minus variant of ENOX2.

- by Xiaoyu Tang
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- Membrane Proteins, Gene expression, Cell line, Molecular and cellular biology
- by N. Howlett and +1
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- Science, DNA damage, Multidisciplinary, Gene Silencing

Glycoproteins of the scavenger receptor cysteine-rich (SRCR) superfamily contain one or more protein modules homologous to the membrane-distal domain of macrophage scavenger receptor I. These domains can be found in the extracellular regions of membrane proteins and in secreted glycoproteins, from the most primitive species to vertebrates. A systematic, bioinformatics-based search for putative human proteins related to the forty-seven known human group B SRCR domains identified a new family member that we have called Soluble Scavenger with 5 Domains (SSc5D). SSc5D is a new soluble protein whose expression is restricted to monocytes/macrophages and T-lymphocytes, and is particularly enriched in the placenta. The gene encoding SSc5D spans 30 kb of genomic DNA, and contains fourteen exons producing a 4.8 kb-long mRNA. The mature polypeptide is predicted to consist of 1573 amino acids comprising, towards the N-terminus, five very similar SRCR domains that are highly conserved among non-marsupial mammals, and a large (>250 nm), very heavily glycosylated, mucin-like sequence towards the C-terminus. Each of the SRCR domains is encoded by a single exon, and contains eight cysteine residues, as observed for all other group B SRCR domains. A shorter isoform encoded by a weakly expressed, alternatively spliced transcript, which lacks the mucin-like C-terminal region, was also identified. It seems likely that SSc5D has a role at the interface between adaptive and innate immunity, or in placental function.

- by Marta Oliveira
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- Immunology, Macrophages, Innate immunity, Molecular

Kinases constitute an important class of therapeutic targets being explored by both academia and the pharmaceutical industry. The major focus of this effort has been directed towards the identification of ATP-competitive inhibitors. Although it has long been recognized that the intracellular concentration of ATP is very different from the concentrations utilized in biochemical enzyme assays, little thought has been devoted to incorporating this discrepancy into our understanding of translation from enzyme inhibition to cellular function. Significant work has been dedicated to the discovery of JAK kinase inhibitors; however, a disconnect between enzyme and cellular function is prominently displayed in the literature for this class of inhibitors. Herein we demonstrate utilizing the four JAK family members that the difference in the ATP K M of each individual kinase has a significant impact on the enzyme to cell inhibition translation. We evaluated a large number of JAK inhibitors in enzymatic assays utilizing either 1 mM ATP or K M ATP for the four isoforms as well as in primary cell assays. This dataset provided the opportunity to examine individual kinase contribution to the heterodimeric kinase complexes mediating cellular signaling. In contrast to a recent study, we demonstrate that for IL-15 cytokine signaling it is sufficient to inhibit either JAK1 or JAK3 to fully inhibit downstream STAT5 phosphorylation. This additional data thus provides a critical piece of information explaining why JAK1 has incorrectly been thought as having a dominant role over JAK3. Beyond enabling a deeper understanding of JAK signaling, conducting similar analyses for other kinases by taking into account potency at high ATP rather than K M ATP may provide crucial insights into a compound's activity and selectivity in cellular contexts.

- by Atli Thorarensen
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- Signal Transduction, Biological Sciences, CHEMICAL SCIENCES, Protein isoforms

Aims: Glial fibrillary acidic protein (GFAP)-d i san o v e l isoform that differs in its C-terminal sequence from other GFAP isoforms. Previous studies suggest restriction of expression to the subpial layer, subventricular zone and the subgranular zone astrocytes, with an absence in pathological conditions causing reactive gliosis. GFAP-d is speculated to have roles in regulation of astrocyte size and motility and a subpopulation of GFAP-d-positive glia may be multipotent stem cells. The aim of this study was to investigate its expression in common causes of lesionrelated refractory epilepsy. Methods: Hippocampal sclerosis (HS), focal cortical dysplasia (FCD) type IIB, cortical tuberous sclerosis (TSC) lesions, gangliogliomas, grey matter heterotopias and hemimegalencephaly from a wide age range of patients using both surgical and post mortem tissue specimens were studied. Results: GFAP-d expression was observed in CA4 and CA1 astrocytes in HS with less frequent labelling in the granule cell layer, even where granule cell dispersion was present. No significant labelling was noted in the subiculum in HS cases or in any subfields in non-HS epilepsy cases. Balloon cells in FCDIIB and hemimegalencephaly, giant cells in TSC and the astrocytic component of gangliogliomas showed immunoreactivity, colocalizing with conventional GFAP. No neuronal expression for GFAP-d was seen in any of the pathologies. Quantitative analysis in 10 FCDIIB and five TSC cases revealed greater numbers of GFAP-d-positive balloon cells than conventional GFAP. There was no GFAP-d expression within nodular heterotopia. Conclusions: GFAP-d expression patterns in HS overall appears to mirror regional reactive gliosis. It is a useful marker for the demonstration of balloon cells in FCD and TSC, which may be relevant to their abnormal size and localization. The lack of GFAP-d within heterotopia supports their composition from cells destined for deeper cortical layers.

- by waney squier
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- Cognitive Science, Malformations of Cortical Development, Epilepsy, Adolescent

The NMR structure of the horse (Equus caballus) cellular prion protein at 25°C exhibits the typical PrP C [cellular form of prion protein (PrP)] global architecture, but in contrast to most other mammalian PrP C s, it contains a well-structured loop connecting the β2 strand with the α2 helix. Comparison with designed variants of the mouse prion protein resulted in the identification of a single amino acid exchange within the loop, D167S, which correlates with the high structural order of this loop in the solution structure at 25°C and is unique to the PrP sequences of equine species. The β2-α2 loop and the α3 helix form a protein surface epitope that has been proposed to be the recognition area for a hypothetical chaperone, "protein X," which would promote conversion of PrP C into the disease-related scrapie form and thus mediate intermolecular interactions related to the transmission barrier for transmissible spongiform encephalopathies (TSEs) between different species. The present results are evaluated in light of recent indications from in vivo experiments that the local β2-α2 loop structure affects the susceptibility of transgenic mice to TSEs and the fact that there are no reports on TSE in horses.

- by daniel perez
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- Molecular Biology, Transgenic Mice, Prion Diseases, Sequence alignment

Aquaporins are water channel proteins belonging to the major intrinsic protein (MIP) superfamily of membrane proteins. More than 150 MIPs have been identified in organisms ranging from bacteria to animals and plants. In plants, aquaporins are present in the plasma membrane and in the vacuolar membrane where they are abundant constituents. Functional studies of aquaporins have hitherto mainly been performed by heterologous expression in Xenopus oocytes. A main issue is now to understand their role in the plant, where they are likely to be important both at the cellular and at the whole plant level. Plants contain a large number of aquaporin isoforms with distinct cell type-and tissue-specific expression patterns. Some of these are constitutively expressed, whereas the expression of others is regulated in response to environmental factors, such as drought and salinity. At the protein level, regulation of water transport activity by phosphorylation has been reported for some aquaporins. ß Major intrinsic protein; Plasma membrane intrinsic protein; Tonoplast intrinsic protein 0005-2736 / 00 / $^see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 5 -2 7 3 6 ( 0 0 ) 0 0 1 4 7 -4 * Corresponding

Several inborn errors of metabolism with abnormal polyol concentrations in body fluids are known to date. Most of these defects can be diagnosed by the assessment of urinary concentrations of polyols. We present two methods using tandem mass spectrometry for screening for inborn errors affecting polyol metabolism. Urine samples supplemented with internal standards ([ 13 C 4 ]erythritol, [ 13 C 2 ]arabitol and [ 2 H 3 ]sorbitol) were desalted by a mixed-bed ion-exchange resin. Separation was achieved by two different columns. Sugar isomers could not be separated using a Prevail Carbohydrate ES 54 column (method 1), whereas with the other column (Aminex HPX-87C) separation of the isomers was achieved (method 2). Multiple reaction monitoring polyol detection was achieved by tandem mass spectrometry with an electron ion-spray source operating in the negative mode. Age-related reference ranges of polyols (erythritol, treitol, arabitol, ribitol, xylitol, galactitol, mannitol, sorbitol, sedoheptitol and perseitol) in urine were established. The applicability of the method was demonstrated by the abnormal polyol concentrations observed in patients with transaldolase deficiency, ribose-5-phosphate isomerase deficiency and classical galactosaemia. This paper describes two methods for the analysis of urinary polyols by liquid chromatography-tandem mass spectrometry. Method 1 is a fast screening method with the quantification of total isomers and method 2 is a more selective method with the separate quantification of the polyols. Both methods can be used for diagnosing inborn errors of metabolism affecting polyol metabolism. Polyols, or polyhydric alcohols, can be formed by the reduction of sugars and are classified on the basis of the numbers of carbon atoms: erythritol and threitol are C 4-polyols (tetritols);

- by M. Wamelink
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- Genetics, Nutrition, Mass Spectrometry, Polymers

The morphological appearance of the vastus lateralis (VL) muscle from high-level power-lifters on long-term anabolic steroid supplementation (PAS) and power-lifters never taking anabolic steroids (P) was compared. The effects of long-and short-term supplementation were compared. Enzyme-immunohistochemical investigations were performed to assess muscle fiber type composition, fiber area, number of myonuclei per fiber, internal myonuclei, myonuclear domains and proportion of satellite cells. The PAS group had larger type I, IIA, IIAB and IIC fiber areas (p<0.05). The number of myonuclei/fiber and the proportion of central nuclei were significantly higher in the PAS group (p<0.05). Similar results were seen in the trapezius muscle (T) but additionally, in T the proportion of fibers expressing developmental myosin isoforms was higher in the PAS group compared to the P group. Further, in VL, the PAS group had significantly larger nuclear domains in fibers containing ‡5 myonuclei. The results of AS on VL morphology in this study were similar to previously reported short-term effects of AS on VL. The initial effects from AS appear to be maintained for several years.

- by Christer Malm
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- Skeletal muscle biology, Weight Lifting, Enzyme, Cell nucleus

Automated eukaryotic gene structure annotation

EVidenceModeler (EVM) is an automated annotation tool that predicts protein-coding regions, alternatively spliced transcripts and untranslated regions of eukaryotic genes.

Abstract... more

Automated eukaryotic gene structure annotation

EVidenceModeler (EVM) is an automated annotation tool that predicts protein-coding regions, alternatively spliced transcripts and untranslated regions of eukaryotic genes.

Abstract EVidenceModeler (EVM) is presented as an automated eukaryotic gene structure annotation tool that reports eukaryotic gene structures as a weighted consensus of all available evidence. EVM, when combined with the Program to Assemble Spliced Alignments (PASA), yields a comprehensive, configurable annotation system that predicts protein-coding genes and alternatively spliced isoforms. Our experiments on both rice and human genome sequences demonstrate that EVM produces automated gene structure annotation approaching the quality of manual curation.

- by Jennifer Wortman
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- Computational Biology, Biological Sciences, Software, Plant Genome Project

Disrupting inositol 1,4,5-trisphosphate (IP 3 ) receptor (IP 3 R)/B-cell lymphoma 2 (Bcl-2) complexes using a cell-permeable peptide (stabilized TAT-fused IP 3 R-derived peptide (TAT-IDP S )) that selectively targets the BH4 domain of Bcl-2 but not that of B-cell lymphoma 2-extra large (Bcl-Xl) potentiated pro-apoptotic Ca 2 þ signaling in chronic lymphocytic leukemia cells. However, the molecular mechanisms rendering cancer cells but not normal cells particularly sensitive to disrupting IP 3 R/Bcl-2 complexes are poorly understood. Therefore, we studied the effect of TAT-IDP S in a more heterogeneous Bcl-2-dependent cancer model using a set of 'primed to death' diffuse large B-cell lymphoma (DL-BCL) cell lines containing elevated Bcl-2 levels. We discovered a large heterogeneity in the apoptotic responses of these cells to TAT-IDP S with SU-DHL-4 being most sensitive and OCI-LY-1 being most resistant. This sensitivity strongly correlated with the ability of TAT-IDP S to promote IP 3 R-mediated Ca 2 þ release. Although total IP 3 R-expression levels were very similar among SU-DHL-4 and OCI-LY-1, we discovered that the IP 3 R2-protein level was the highest for SU-DHL-4 and the lowest for OCI-LY-1. Strikingly, TAT-IDP S -induced Ca 2 þ rise and apoptosis in the different DL-BCL cell lines strongly correlated with their IP 3 R2-protein level, but not with IP 3 R1-, IP 3 R3-or total IP 3 R-expression levels. Inhibiting or knocking down IP 3 R2 activity in SU-DHL-4-reduced TAT-IDP S -induced apoptosis, which is compatible with its ability to dissociate Bcl-2 from IP 3 R2 and to promote IP 3 -induced pro-apoptotic Ca 2 þ signaling. Thus, certain chronically activated B-cell lymphoma cells are addicted to high Bcl-2 levels for their survival not only to neutralize pro-apoptotic Bcl-2-family members but also to suppress IP 3 R hyperactivity. In particular, cancer cells expressing high levels of IP 3 R2 are addicted to IP 3 R/Bcl-2 complex formation and disruption of these complexes using peptide tools results in pro-apoptotic Ca 2 þ signaling and cell death.

- by Giovanni Monaco and +3
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- Calcium, Apoptosis, RNA interference, Peptides

CapZ is a heterodimeric Ca 2؉ -independent actin binding protein which plays an important role in organizing the actin filament lattice of cross-striated muscle cells. It caps the barbed end of actin filaments and promotes nucleation of actin polymerization, thereby regulating actin filament length. Here we report the expression of the two muscle-specific isoforms ␣2 and ␤1, from chicken in Escherichia coli as individual subunits using the pQE60 expression vector and the subsequent renaturation of the functional CapZ heterodimer from inclusion bodies. Optimal renaturation conditions were obtained both by simultaneous refolding of urea-solubilized subunits and by rapid dilution into a buffer containing 20% glycerol, 5 mM EGTA, 2 mM DTT, 1 mM PMSF, and 100 mM Tris, pH 7.4. The refolding mixture was incubated for 24 h at 15°C and the protein was concentrated by ultrafiltration. Biochemical characterization of the recombinant heterodimer revealed actin binding activities indistinguishable from those of native CapZ as purified from chicken skeletal muscle. Using the same protocol, we were able to refold the ␤1, but not the ␣2 isoform as a single polypeptide, indicating a role for ␤1 as a molecular template for the folding of ␣2. The reported recombinant approach leads to high yields of active heterodimer and allows the renaturation and characterization of the ␤ subunit.

- by Kirsten Remmert
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- Protein Folding, Gene expression, Escherichia coli, Solubility

Starting from the very simple molecule sulfamic acid, O-substituted-, N-substituted-, or di-/tri-substituted sulfamates may be obtained, which show specific biological activities which were or started to be exploited for the design of many types of therapeutic agents. Among them, sulfamate inhibitors of aminoacyl-tRNA synthetases (aaRSs) were recently reported, constituting completely new classes of antibiotics, useful in the fight of drug-resistant infections. Anti-viral agents incorporating sulfamate moieties have also been obtained, with at least two types of such derivatives investigated: the nucleoside/nucleotide human immunodeficiency virus (HIV) reverse transcriptase inhibitors, and the HIV protease inhibitors (PIs). In the increasing armamentarium of anti-cancer drugs, the sulfamates occupy a special position, with at least two important targets evidenced so far: the steroid sulfatases (STSs) and the carbonic anhydrases (CAs). An impressing number of inhibitors of STSs of the sulfamate type have been reported in the last years, with several compounds, such as 667COUMATE among others, progressing to clinical trials for the treatment of hormone-dependent tumors (breast and prostate cancers). This field is rapidly evolving, with many types of new inhibitors being constantly reported and designed in such a way as to increase their anti-tumor properties, and decrease undesired features (for example, estrogenicity, a problem encountered with the first generation such inhibitors, such as EMATE). Among the many isozymes of CAs, at least two, CA IX and CA XII, are highly overexpressed in tumors, being generally absent in the normal tissues. Inhibition of tumor-associated CAs was hypothesized to lead to novel therapeutic approaches for the treatment of cancer. Many sulfamates act as very potent (low nanomolar) CA inhibitors. The X-ray crystal structure of the best-studied isozyme, CA II, with three sulfamates (sulfamic acid, topiramate, and EMATE) has recently been reported, which Contract grant sponsor: 6th Framework Programme of EU (EUROXY Project).

- by Andrea Scozzafava
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- Chemistry, Medicine, Escherichia coli, Fructose

The protein encoded by the Nce103 gene of Saccharomyces cerevisiae, a b-carbonic anhydrase (CA, EC 4.2.1.1) designated as scCA, was investigated for its activation with amines and amino acids. scCA was poorly activated by amino acids such as L-/D-His, Phe, DOPA, Trp (K A s of 82-90 lM) and more effectively activated by amines such as histamine, dopamine, serotonin, pyridyl-alkylamines, aminoethyl-piperazine/morpholine (K A s of 10.2-21.3 lM). The best activator was L-adrenaline, with an activation constant of 0.95 lM. This study may help to better understand the catalytic/activation mechanisms of the b-CAs and eventually to design modulators of CA activity for similar enzymes present in pathogenic fungi, such as Candida albicans and Cryptococcus neoformans.

- by MELTEM AYDIN
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- Organic Chemistry, Catalysis, Kinetics, Saccharomyces cerevisiae

Cut-off values of serum growth hormone (GH) in pharmacological stimulation tests (PhT) evaluated in short-statured children using a chemiluminescent immunometric assay (ICMA) calibrated with the International Recombinant Human GH Standard... more

- by Alicia Belgorosky and +1
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- Cognitive Science, Adolescent, Linear models, Child