Cricetinae Research Papers - Academia.edu (original) (raw)

This work reports the validation of a simple CZE method to be used in quality control of recombinant human glycosylated interleukin-7 (rhIL-7) batches produced in Chinese Hamster Ovary (CHO) cells. The separation buffer was a 25mM sodium borate at pH 10 containing 12mM diaminobutane (DAB) used as a dynamic coating agent of the capillary. This method allowed the separation of seven peaks ranging from low to high sialylated glycoforms. An extensive study on conditioning methods of the capillary has been conducted to yield repeatable results. Excellent RSD of EOF mobility (less than 0.6%) was obtained when conditioning included capillary equilibration under virtual analyses and storage in 0.1M NaOH overnight. Method specificity has been demonstrated to be able to discriminate different rhIL-7 glycoforms produced in CHO from formulation matrix. Linearity was demonstrated between 0.5 and 4mg/mL. LOQ was 0.5mg/mL. Repeatability (RSD<1.4 and 3.3% for t(m) and A%, respectively), intermed...

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- Chemistry, Analytical Chemistry, Capillary electrophoresis, Comparative Study
- by Pheravut Wongsawad
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- Microbiology, Polymorphism, Experimental parasitology, Identification

To characterize the muscarinic receptor present in guinea-pig uterus smooth muscle the affinities of a series of 27 muscarinic receptor antagonists for M 1 (rat cortex), M 2 (rat heart), M 3 (rat submandibular gland), m4 (transfected in CHO cells) and muscarinic binding sites in guinea-pig uterus smooth muscle were determined in radioligand binding studies. In addition, functional experiments were performed to assess pK B values of the antagonist for muscarinic receptors in guinea-pig atrium and uterus. The results obtained are consistent with the presence of M 2 receptors in the uterus through which the functional contractile response is mediated. Correlation coefficients of 0.98, 0.91 and 0.91 were calculated for the following linear regressions: pK i uterus vs. pK i M2, pK B uterus vs. pK i M 2 and pK B uterus vs. pK B atrium. This study also revealed that the compounds dicyclomine, DAU 5884, DAU 6202 as well as AQ-RA 721 could distinguish m4 from M E sites and are therefore important tools to characterize muscarinic receptor subtypes. In addition, DAU 5884 and DAU 6202 have been identified as highly potent M 1 selective antagonists.

- by Hendrikus W. G. M. Boddeke
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- Smooth muscle, Cerebral Cortex, Rats, Myocardium

Prostacyclin (PGI 2 ) possesses various physiological functions, including modulation of nociception, inflammation and cardiovascular activity. Elucidation of these functions has been hampered by the absence of selective IP receptor... more

- by Dinesh Srinivasan
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- British, cyclic AMP, Rats, Imidazoles

Tuberculosis (TB) is one of the leading infectious causes of death due to single infectious agent after HIV/AIDS. Rifampicin (RIF), Isoniazid (INH), Ethambutol (EMB), Pyrazinamide (PZA) and/or their combinations are extensively prescribed to treat TB. Despite several therapeutic implications, these drugs also produce several toxic effects at cellular level. MTT assay and Ames test were adopted in this study for the determination of cytotoxic and mutagenic potential of these anti-TB drugs. Among all tested drugs, cytotoxic potential of RIF was strongest with highly significant decline (p < 0.001) in cell numbers at the concentration of 250 g/ml with LC 50 at 325 g/ml, while significant decline (p < 0.01) in cell count was observed in INH treated group at the concentration 500 g/ml with LC 50 at 1000 g/ml. Moreover, combination RIPE demonstrated significant reduction (p < 0.01) in cell number at the concentration of 25-500-500-500 g/ml with LC 50 at 60-1200-1200-1200 g/ml. It is apparent from the data that almost all drugs represented identical mutagenic pattern i.e., more significant results were achieved in TA100 with metabolic activation (+S9). RIF proved to be highly mutagenic of all tested drugs with significant mutagenicity (p < 0.01) at 0.0525 g/plate against TA98 strain with S9. The combination RIPE exhibited highly significant mutagenic activity (p < 0.01) at concentration 0.125-3-3-3 g/plate without S9, while addition of S9 resulted in similar activity at lower doses, i.e., 0.0525-1-1-1 g/plate. It was concluded from the data that all anti-TB drugs possess significant cytotoxic and mutagenic potential, especially in combination, making TB patient more vulnerable to cytotoxic and mutagenic effects of anti-TB drugs, which could produce further health complications in TB patients.

- by Zainab Batool
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- Drug interactions, Risk assessment, Cell line, Mutation

h i g h l i g h t s Combination of Sm14 and Sm29 antigens was tested in S. mansoni infection model. Significant reduction of S. mansoni adult worms and tissue egg burden was attained. Reduced liver histopathology and granuloma size was... more

- by Shereen Mossallam
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- Microbiology, Vaccines, Biology, Medicine

A series of biochemical, staining and electron microscopy techniques were utilized to investigate the mechanisms of C-and G-banding. These led to the following conclusions. I. The treatment of fixed chromosomes with 0.07 N NaOH for 30 to 180 set removes from 16 to 81% of the DNA from the chromosomes. 2. On average, the complete C-band technique removes 60% of the DNA. 3. This DNA is preferentially extracted from the non-C-band regions. 4. In marked contrast to this, all G-band techniques (except 1) removed less than 9 o,, of the chromosomal DNA. 5. Most of the G-band techniques, including those using trypsin, remove very little protein from the chromosomes. 6. Feulgen staining indicated that neither C-nor G-banding can be explained on the basis of different amounts of DNA along the length of the chromatid. 7. Treatment of chromosomes witkalkali or prolonged treatment with trypsin tends to destroy G-bands, while C-bands remain. 8. The combined use of acridine orange and Giemsa staining indicate that, (a) repetitious DNA in situ renatures in seconds while non-repetitious DNA renatures in minutes; (6) Neither C-nor G-banding depends upon the differential renaturation of DNA for its effect. 9. G-banding is more delicate and relatively mild conditions allow staining of both C-and G-bands. To obtain only C-bands the chromosome must be treated more harshly to disrupt or destroy the G-bands. 10. DNA-non histone protein interactions probably play an important role in the production of both C-and G-banding.

- by David Comings
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- RNA, Transmission Electron Microscopy, DNA, Mice

Recently c-Cbl has been reported to be phosphorylated upon CSF-1 stimulation. The product of the c-cbl protooncogene (c-Cbl) is a 120 kDa protein harboring several docking sites for Src homology 2 (SH2) domain containing proteins and proline-rich regions that have been shown to allow its constitutive association with the SH3 domains of Grb2. We demonstrate here that CSF-1 exposure of stable transfectant CHO cells expressing the CSF-1 receptor induced the sustained tyrosine phosphorylation of c-Cbl and its subsequent association with Crk-II and the p85 kDa subunit of the PI 3-kinase, while it constitutively associates with Grb2. We demonstrate by in vitro experiments that these associations require the SH2 domain of Crk-II and both the C-and N-terminal SH2 domains of the p85 subunit of the PI 3-kinase. c-Cbl is the major PI 3-kinase-containing protein in c-Fms expressing CHO cells upon CSF-1 stimulation. Thus c-Cbl behaves as a core protein, allowing the formation of a quaternary complex including, Crk-II, PI 3-kinase and Grb2. We provide evidence that this multiprotein complex can interact with the tyrosine phosphorylated CSF-1 receptor through the unoccupied SH2 domain of Grb2.

- by Heidy Antomarchi
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- Oncogene, Phosphorylation, Proteins, Clinical Sciences

ErbB tyrosine kinase receptors mediate mitogenic signal cascade by binding a variety of ligands and recruiting the different cassettes of adaptor proteins. In the present study, we examined heregulin (HRG)-induced signal transduction of ErbB4 receptor and found that the phosphatidylinositol 3′-kinase (PI3K)-Akt pathway negatively regulated the extracellular signal-regulated kinase (ERK) cascade by phosphorylating Raf-1 on Ser259. As the time-course kinetics of Akt and ERK activities seemed to be transient and complex, we constructed a mathematical simulation model for HRG-induced ErbB4 receptor signalling to explain the dynamics of the regulation mechanism in this signal transduction cascade. The model reflected well the experimental results observed in HRG-induced ErbB4 cells and in other modes of growth hormone-induced cell signalling that involve Raf-Akt cross-talk. The model suggested that HRG signalling is regulated by protein phosphatase 2A as well as Raf-Akt cross-talk, and p...

- by Takashi Naka
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- Kinetics, Enzyme Inhibitors, Signal Transduction, Biological Sciences

Serum albumin is the most abundant plasma protein and has a long half-life due to neonatal Fc receptor (FcRn) mediated transcytosis by many cell types including proximal tubule cells of the kidney. Albumin also interacts with, and is modified by, many small and large molecules. Therefore, the focus of this study was to address the impact of specific known biologic albumin modifications on albumin-FcRn binding and cellular handling. Binding at pH 6.0 and 7.4 was performed since FcRn binds albumin strongly at acidic pH and releases it following transcytosis at physiological pH. Equilibrium dissociation constants (KDs) were measured using microscale thermophoresis. Since studies have shown that glycated albumin is excreted in the urine at a higher rate than unmodified albumin we studied glucose and methylgloxal modified albumins (21day). All had reduced affinity to FcRn at pH 6.0 suggesting these albumins would not be returned to the circulation via the transcytotic pathway. To address...

- by Sudhanshu Kumar
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- Physiology, Small Angle X Ray Scattering, Medical Physiology, Rats

For long-term growth of mammalian cells in perfused bioreactors, it is essential to monitor the concentration of dissolved oxygen (DO) present in the culture medium to ascertain the health of the cells. An optical oxygen sensor based on dynamic fluorescent quenching was developed for long-term continuous measurement of DO for NASA-designed rotating perfused bioreactors. Tris(4,7diphenyl-1,10-phenanthroline) ruthenium(II) chloride is employed as the fluorescent dye indicator. A pulsed, blue LED was chosen as the excitation light source. The sensor can be sterilized using an autoclave. The sensors were tested in a perfused rotating bioreactor supporting a BHK-21 (baby hamster kidney) cell culture over one 28-day, one 43-day, and one 180-day cell runs. The sensors were initially calibrated in sterile phosphate-buffered saline (PBS) against a blood-gas analyzer (BGA), and then used continuously during the entire cell culture without recalibration. In the 180-day cell run, two oxygen sensors were employed; one interfaced at the outlet of the bioreactor and the other at the inlet of the bioreactor. The DO concentrations determined by both sensors were compared with those sampled and measured regularly with the BGA reference. The sensor outputs were found to correlate well with the BGA data throughout the experiment using a single calibration, where the DO of the culture medium varied between 25 and 60 mm Hg at the bioreactor outlet and 80-116 mm Hg at the bioreactor inlet. During all 180 days of culture, the precision and the bias were F 5.1 mm Hg and À 3.8 mm Hg at the bioreactor outlet, and F 19 mm Hg and À 18 mm Hg at inlet. The sensor dynamic range is between 0 and 200 mm Hg and the response time is less than 1 minute. The resolution of the sensor is 0.1 mm Hg at 50 mm Hg, and 0.25 mm Hg at 130 mm Hg.

- by antony jeevarajan
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- Bioengineering, Biotechnology, Multidisciplinary, Cell Culture

Four new polysulfur aromatic alkaloids, lissoclibadins 4 (1), 5 (2), 6 (3), and 7 (4), were isolated from the ascidian Lissoclinum cf. badium collected in Indonesia, together with seven known alkaloids, lissoclibadins 1 (5), 2 (6), and 3 (7), lissoclinotoxins E (8) and F (9), 3,4-dimethoxy-6-(2′-N,N-dimethylaminoethyl)-5-(methylthio)benzotrithiane (10), and N,N-dimethyl-5-(methylthio)varacin (11). Compounds 1-11 were isolated from the ascidian collected in March (wet season), while 5-11 have been obtained previously from the organism collected in September (dry season) at the same site. The structures of the new compounds were assigned on the basis of their spectroscopic data. Lissoclibadins 4-7 (1-4) inhibited the colony formation of Chinese hamster V79 cells with EC 50 values of 0.71, 0.06, 0.06, and 0.17 µM, respectively. Compounds 1-4 showed also weak antimicrobial activity against Staphylococcus aureus, Escherichia coli, and Saccharomyces cereVisiae.

- by Remy Mangindaan
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- Natural Products, Indonesia, Pharmacognosy, Biological Sciences
- by Clay Scott
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- Kinetics, Calcium, Molecular Pharmacology, Mode of action

Specific regions in the rodent larynx exhibit cellular changes in response to inhaled xenobiotics. These regions include the base of the epiglottis, ventral pouch, and medial surfaces of the vocal processes of the arytenoid cartilages. There are interspecies differences among laboratory rodents in the microscopic anatomy of these sensitive areas of the laryngeal mucosa. In CRL:CD strain Sprague-Dawley rats, the

- by Roger A Renne
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- Clinical Sciences, Rats, Epithelium, Xenobiotics

Polyamines are ubiquitous molecules, which, like iron, are essential for cell growth. All eukaryotic cells are equipped with a specific polyamine transport system (PTS). Polyamines have primary and secondary amino groups which chelate bivalent metal cations such as Fe and Cu. In the present study, we investigated the potential contribution of naturally occurring polyamines and their active transport system to iron uptake. In presence of subtoxic Fe(III) (10 mM), treatment of CHO cells with spermine, and to a lesser extent with spermidine (10-100 mM), resulted in a marked cytotoxic effect. This cytotoxicity was prevented by the addition of an iron-chelator, deferioxamine, and was not observed in CHO-MG cells, a mutant cell line devoid of polyamine transport activity. Experiments using 14 C-polyamines and 55 Fe(III) revealed that these toxic effects were related to polyamine-modulation of iron uptake, and were dependent on the presence of the active PTS. These results demonstrated active uptake of polyamine-iron complexes via the PTS. The number of amino groups affected the efficacy of the studied natural polyamines to transport iron via the PTS. Spermine, a tetramine, was more efficient than the triamine spermidine. Co-transport of iron by the diamine putrescine was not observed. These results demonstrate that the cell polyamine transport system is a potential cell entry pathway for iron. The studied polyamines, spermine and spermidine, may be components of the pool of transferrin-independent iron-chelating vectors, which have recently attracted the attention of many investigators.

- by jean-guy Delcros
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- Chemistry, Medicine, Polyamines, Cell line
- by Christian Reichel
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- Mass Spectrometry, Bioanalysis, Erythropoietin, Anemia

Intratracheal (i.t.) or intravenous (i.v.) administration of FR90145 1, a potent inhibitor of human leukocyte elastase (HLE) prevented HLE-induced lung hemorrhage in hamsters with ED50 values of 10.5 /ig/site and 8. 1 mg/kg, respectively. al-Antitrypsin (al-AT) also showed inhibitory effect in this model. However, the ED50 value by i.t. injection of FR901451was 20-fold lower than that of al-AT. Moreover, FR901451 i.t. significantly modulated porcine pancreas elastase (PPE)-induced changes of the respiratory mechanics in hamsters. The ED50 values were 529 /zg/site and 244 //g/site, which were expressed by static lung compliance (Cst) and vital capacity (VC) of the lungs, respectively. These results suggest that FR90145 1 could be clinically useful agent for the treatment of the destructive lung disease such as pulmonary emphysema.

We describe a method for the generation of high-affinity monoclonal antibodies, which combines the power of natural immune responses with in vitro panning, B cell culture, RT-PCR and expression of the recombinant product. B cells from immunised rabbits were incubated at approximately 1000-10,000 cells per well with solid phase antigen coated on the surface of 96-well ELISA plates. Extensive washing removed non-binding cells as well as those B cells, which bound with low affinity. Retained B cells were cultured for 7 days in the presence of activated rabbit splenocyte supernatant and irradiated EL-4-B5 mouse thymoma cells, to induce proliferation and secretion of immunoglobulin. Supernatants were screened to confirm the presence of specific antibody, before the cells were harvested en masse from individual positive wells. Single heavy-and lightchain variable region genes were recovered from individual wells by RT-PCR, critically without the need for isolation of single B cells. Paired VH and VL genes were subsequently expressed as recombinant antibodies and shown to retain the original activity and specificity of the B cell culture supernatants. The method has also been successfully applied to the generation of high-affinity antibodies to antigen expressed on the surface of target cells.

- by Alistair Henry
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- Immunology, Immune response, RNA, Phage display

We describe a rapid method for monitoring the cell growth and decline phases in suspension cultures of animal cells. During the cell growth phase, ultraviolet (UV)-absorbing components in the medium are consumed, but at later times as cells begin to die, UV-absorbing molecules such as proteins are released into the medium. Measuring the absorbance at 280 nm (A 280) with a NanoDrop spectrophotometer, an inverse correlation between the onset of the cell decline phase and A 280 was observed. This simple method can be applied to quickly determine the beginning of the decline phase of cultures of mammalian and insect cells in suspension.

- by F. Wurm
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- Analytical Chemistry, Nanotechnology, Cell Culture, Cell Viability

A human galactoside-binding protein with an M, of 31,000 was cloned from the human III-1(180 fibrosarcoma complementary DNA library. A partial complementary DNA clone containing the complete coding region was characterized and the deduced sequence encodes a polypeptide of 242 amino acids with the characteristics of a carbohydrate-binding pro tein. The gene coding for the human galactoside-binding protein was mapped to the chromosomal band Ipl3. The deduced amino acid sequence of the human galactoside-binding protein revealed 95 residues at the amino terminus, homologous to the predicted amino acid sequence of the second exon of the human l.-myc gene.

- by Rebecca Keough
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- Cancer, Comparative Study, DNA, Alternative splicing

This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier's archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright

- by Angelina Fong
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- Aging, Biology, Medicine, Longevity

This paper describes a proactive product stewardship program for glass fibers. That effort included epidemiological studies of workers, establishment of stringent workplace exposure limits, liaison with customers on safe use of products and, most importantly, a research program to evaluate the safety of existing glass fiber products and guide development of new even safer products. Chronic inhalation exposure bioassays were conducted with rodents and hamsters. Amosite and crocidolite asbestos produced respiratory tract cancers as did exposure to ''biopersistent'' synthetic vitreous fibers. ''less biopersistent'' glass fibers did not cause respiratory tract cancers. Corollary studies demonstrated the role of slow fiber dissolution rates and biopersistence in cancer induction. These results guided development of safer glass fiber products and have been used in Europe to regulate fibers and by IARC and NTP in classifying fibers. IARC concluded special purpose fibers and refractory ceramic fibers are ''possibly carcinogenic to humans'' and insulation glass wool, continuous glass filament, rock wool and slag wool are ''not classifiable as to their carcinogenicity to human.'' The NTP's 12th report on carcinogens lists ''Certain Glass Wool Fibers (Inhalable)'' as ''reasonably anticipated to be a human carcinogen.'' ''Certain'' in the descriptor refers to ''biopersistent'' glass fibers and excludes ''less biopersistent'' glass fibers.

- by Roger McClellan
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- Glass, Risk assessment, Industry, Carcinogens

To determine whether the addition of a pre-S/S human vaccine increases the antiviral activity of lamivudine, four woodchucks were treated with a daily dose of 100 mg/kg lamivudine and four 50 g doses of CHO-derived pre-S/S human vaccine. WHV DNA titres decreased up to two logarithms in three woodchucks. At week 4, in three of the animals, the sequence analysis showed a predominant strain containing a nucleotide change from A to T at position 1696 of domain B of the WHV DNA polymerase. Vaccination did not further suppress WHV DNA, despite anti-HBs production in three animals. The woodchuck remains a useful model for characterising the biology and kinetics of the emergence of drug-resistant variants and could be used for pre-clinical studies of combinations of new antiviral drugs.

- by Giampiero Palmieri
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- Kinetics, Inflammation, Biological Sciences, Hepatitis B

Markov chain Monte Carlo (MCMC) method was applied to model kinetics of a fed-batch Chinese hamster ovary cell culture process in 5,000-L bioreactors. The kinetic model consists of six differential equations, which describe dynamics of viable cell density and concentrations of glucose, glutamine, ammonia, lactate, and the antibody fusion protein B1 (B1). The kinetic model has 18 parameters, six of which were calculated from the cell culture data, whereas the other 12 were estimated from a training data set that comprised of seven cell culture runs using a MCMC method. The model was confirmed in two validation data sets that represented a perturbation of the cell culture condition. The agreement between the predicted and measured values of both validation data sets may indicate high reliability of the model estimates. The kinetic model uniquely incorporated the ammonia removal and the exponential function of B1 protein concentration. The model indicated that ammonia and lactate play critical roles in cell growth and that low concentrations of glucose (0.17 mM) and glutamine (0.09 mM) in the cell culture medium may help reduce ammonia and lactate production. The model demonstrated that 83% of the glucose consumed was used for cell maintenance during the late phase of the cell cultures, whereas the maintenance coefficient for glutamine was negligible. Finally, the kinetic model suggests that it is critical for B1 production to sustain a high number of viable cells. The MCMC methodology may be a useful tool for modeling kinetics of a fed-batch mammalian cell culture process. V

- by Zizhuo Xing
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- Engineering, Technology, Kinetics, Cell Culture

Sialylated glycans serve as key elements of receptors for many viruses, bacteria, and bacterial toxins. The microbial recognition and their binding specificity can be affected by the linkage of the terminal sugar residue, types of underlying sugar chains, and the nature of the entire glycoconjugate. Owing to the pathobiological significance of sialylated glycans, we have engineered Chinese hamster ovary (CHO) cells to secrete mucin-type immunoglobulin-fused proteins carrying terminal Į2,3-or Į2,6-linked sialic acid on defined O-glycan core saccharide chains. Besides stably expressing P-selectin glycoprotein ligand-1/mouse immunoglobulin G2b cDNA (PSGL-1/mIgG2b), CHO cells were stably transfected with plasmids encoding glycosyltransferases to synthesize core 2 (GCNT1), core 3 (B3GNT6), core 4 (GCNT1 and B3GNT6), or extended core 1 (B3GNT3) chains with or without the type 1 chain-encoding enzyme B3GALT5 and ST6GAL1. Western blot and liquid chromatography-mass spectrometry analysis confirmed the presence of core 1, 2, 3, 4, and extended core 1 chains carrying either type 1 (Galβ3GlcNAc) or type 2 (Galβ4GlcNAc) outer chains with or without Į2,6-linked sialic acids. This panel of recombinant mucins carrying a repertoire of sialylated O-glycans

- by Jan Holgersson
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- Biology, Polysaccharides, Medicine, Mice

Efficient expression of innate immunity is critically dependent upon the capacity of the neutrophil to be activated rapidly in the face of an acute threat and to involute once that threat has been eliminated. Here we report a novel mechanism regulating neutrophil survival dynamically through the tyrosine phosphorylation or dephosphorylation of caspase-8. Caspase-8 is tyrosine-phosphorylated in freshly isolated neutrophils but spontaneously dephosphorylates in culture, in association with the progression of constitutive apoptosis. Phosphorylation of caspase-8 on Tyr-310 facilitates its interaction with the Src-homology domain 2 containing tyrosine phosphatase-1 (SHP-1) and enables SHP-1 to dephosphorylate caspase-8, permitting apoptosis to proceed. The non-receptor tyrosine kinase, Lyn, can phosphorylate caspase-8 on Tyr-397 and Tyr-465, rendering it resistant to activational cleavage and inhibiting apoptosis. Exposure to lipopolysaccharide reduces SHP-1 activity and binding to caspase-8, caspase-8 activity, and rates of spontaneous apoptosis. SHP-1 activity is reduced and Lyn increased in neutrophils from patients with sepsis, in association with profoundly delayed apoptosis; inhibition of Lyn can partially reverse this delay. Thus the phosphorylation and dephosphorylation of caspase-8, mediated by Lyn and SHP-1, respectively, represents a novel, dynamic post-translational mechanism for the regulation of neutrophil apoptosis whose dysregulation contributes to persistent neutrophil survival in sepsis.

- by Andras Kapus
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- Biology, Biological Chemistry, Innate immunity, Apoptosis

To exclude that aromatization plays a role in the estrogenic activity of tibolone, we studied the effect tibolone and metabolites on the aromatization of androstenedione and the aromatization of tibolone and its metabolites to 7␣-methyl-17␣-ethynylestradiol (7␣-MEE) by human recombinant aromatase. Testosterone (T), 17␣-methyltestosterone (MT), 19-nortestosterone (Nan), 7␣-methyl-19-nortestosterone (MENT) and norethisterone (NET) were used as reference compounds. Sensitive in vitro bioassays with steroid receptors were used to monitor the generation of product and the reduction of substrate. LC-MSMS without derivatization was used for structural confirmation. A 10 times excess of tibolone and its metabolites did not inhibit the conversion of androstenedione to estrone by human recombinant aromatase as determined by estradiol receptor assay whereas T, MT, Nan, and MENT inhibited the conversion for 75, 53, 85 and 67%, respectively. Tibolone, 3␣-and 3␤-hydroxytibolone were not converted by human aromatase whereas the estrogenic activity formed with the 4-isomer suggests a conversion rate of 0.2% after 120 min incubation. In contrast T, MT, Nan, and MENT were completely converted to their A-ring aromates within 15 min while NET could not be aromatized. Aromatization of T, MT, Nan and MENT was confirmed with LC-MSMS. Structure/function analysis indicated that the 17␣-ethynyl-group prevents aromatization of (19-nor)steroids while 7␣-methyl substitution had no effect. Our results with the sensitive estradiol receptor assays show that in contrast to reference compounds tibolone and its metabolites are not aromatized.

- by Lenus Kloosterboer
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- Mass Spectrometry, STEROIDS, Medicine, Cell line

Microvascular hematocrit and its possible relation to oxygen supply were systematically examined. We studied the red cell volume fraction (hematocrit) in arterial blood and in capillaries under a variety of circumstances. Control capillary hematocrit averaged 10.4 +/- 2.0% (SE) and arteriolar (14.2 micrometer ID) hematocrit averaged 13.9 +/- 1.2% in cremaster muscles of pentobarbital-anesthetized hamsters. Carotid artery hematocrit was 53.2 +/- 0.6%. The low microvessel hematocrit could not be entirely explained by a high red cell flux through arteriovenous channels other than capillaries (shunting). Hematocrit was not only low at rest, but varied with physiological stimuli. A 1-Hz muscle contraction increased capillary hematocrit to 18.5 +/- 2.4%, and maximal vasodilation induced a rise to 39.3 +/- 9.5%. The quantitative relations between capillary red cell flux, arterial hematocrit, and total blood flow could be explained by a two-element model of microvascular blood flow that inc...

- by Bruce Klitzman
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- Physiology, Mathematics, Capillaries, Muscles

Fli-1 is a proto-oncogene which is rearranged in tumors induced by three dierent retroviruses, Cas-Br-E, F-MuLV, and 10A1. This gene is a member of the Ets gene family, a class of transcription factors that recognize and bind to a DNA motif known as the Ets binding site (EBS). Our laboratory has previously cloned and characterized the promoter region of both human and mouse Fli-1 genes. We had then identi®ed several regulatory elements conserved between the two species. Two of them, an exon 1 GATA/EBS dual element and an EBS element located in the 5' end of intron 1, were analysed in the present study. EMSA analysis performed with nuclear extracts from dierent cell lines showed that the EBS element in intron 1 (EBSi) was bound by one potential Ets-related ubiquitous factor. The GATA/ EBS element was bound by several factors that seemed Ets-related, one of which was found to be speci®cally expressed in hematopoietic cells. the GATA/EBS dual element was thus chosen for further analysis. A human Fli-1-derived genomic fragment containing the GATA/ EBS led to enhanced transcription when positioned upstream of the SV40 promoter in the erythroleukemic HEL cell line. In addition, an increasing number of GATA/EBS oligonucleotides upstream of this same promoter resulted in a copy number-dependent increase in luciferase activity which was greatly reduced when the EBS consensus sequence was mutated. One of the factors binding to the GATA/EBS region was identi®ed to be Spi-1 by supershift analysis and was also shown to bind to the EBS element of the human Ets-2 gene. Supershift analysis also demonstrated the binding of the GATA-1 factor to the GATA/EBS dual element. Our results suggest that Spi-1 and GATA-1 might play a key role in the regulation of Fli-1.

- by E. Rassart
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- Regulation, Leukemia, Gene expression, Cercopithecus aethiops

An excess of intracellular free Cholesterol (Chol) is cytotoxic, and its homeostasis is crucial for cell viability. Apolipoprotein A-I (apoA-I) is a highly efficient Chol acceptor as it activates complex cellular pathways that tend to mobilize and export Chol from cellular depots. Here we hypothesize that membrane composition and/or organization is strongly involved in Chol homeostasis. To test this hypothesis, we constructed a cell line over expressing Stearoyl CoA desaturase (SCD-cells), which modifies plasma membrane (PM) composition by the enrichment of monounsaturated fatty,acids and determined this effect on membrane properties, cell viability and cholesterol homeostasis. PM in SCD-cells has a higher phospholipids/sphingomyelin ratio and is slightly enriched in Chol. These cells showed an increase in the cholesteryl esters/free Chol ratio, they were more resistant to Chol toxicity and in addition, they exported more caveolin than Control cells. The data suggest that cell functionality is preserved by regulating membrane fluidity and Chol exportation and storage.

- by María Soledad Jaureguiberry
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- Biochemistry, Biophysics, Medical Microbiology, Membrane Biology

Two different classes of hamster cell mutants (25RA cells and M1 cells) express opposite phenotypes in sterol dependent regulation. In 25RA cells, sterols added in growth medium fail to cause down-regulation of sterol synthesis rate and low density lipoprotein (LDL) receptor activity, while in M1 cells, removal of lipids from growth medium fail to cause up-regulation of sterol synthesis rate

- by Mazahir T Hasan
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- Genetics, Mutation, Cholesterol, Hybrid Solar Cells

Scope: It has been shown that calcium supplementation favorably modifies plasma lipoprotein profile in postmenopausal women. The present study investigated the interaction of dietary calcium with genes of transporters, receptors and enzymes involved in cholesterol metabolism. Methods and results: Forty-eight ovariectomized hamsters were fed one of the four diets containing 0, 2, 6 and 8 g calcium per kg. Plasma total cholesterol (TC), triacylglycerols (TG), and non-high density lipoprotein cholesterol were dose-dependently decreased, whereas high-density lipoprotein cholesterol (HDL-C) was dose-dependently increased with the increasing dietary calcium levels. Dietary calcium had no effect on protein mass of hepatic sterol regulatory element binding protein-2 (SREBP), liver X receptor-alpha (LXR), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), LDL receptor (LDLR) and cholesterol-7a-hydroxylase (CYP7A1). However, dietary calcium up-regulated the mRNA levels of hepatic CYP7A1 and intestinal ATP binding cassette transporters (ABCG5/8) whereas it down-regulated the intestinal Niemann-Pick C1 like 1 (NPC1L1) and microsomal triacylglycerol transport protein (MTP). In addition, dietary calcium increased the activity of intestinal acyl coenzyme A: cholesterol acyltransferase 2, while it decreased plasma cholesteryl ester transport protein (CETP). Conclusion: Beneficial modification of lipoprotein profile by dietary calcium was mediated by sequestering bile acid absorption and enhancing excretion of fecal cholesterol, via up-regulation of mRNA CYP7A1 and intestinal ABCG 5/8 with down-regulation of mRNA NPC1L1 and MTP.

- by Katerina Malshina
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- Nutrition and Dietetics, Food and Nutrition, Liver, Cholesterol

- by Jean-Guy Delcros
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- Polyamines, Cell line, Iron, Spermine

During entrance into torpor heart and respiration rates are greatly reduced in parallel with the reduction of metabolic rate, suggesting an involvement of parasympathetic control. We compared the effect of parasympathetic inhibition with the effect of sympathetic inhibition on spontaneous torpor behaviour in the Djungarian hamster. Hamsters were acclimated to short photoperiod and displayed their standard torpor pattern as observed from T b records. Parasympathetic inhibition was achieved by a subcutaneous implant of 21-day release pellets with Atropine and the sympathetic noradrenergic pathway was inhibited with a single injection of 6-Hydroxydopamine. Atropine treatment did not affect the occurrence and quality of spontaneous daily torpor at all. However, the reversible sympathetic inhibition by 6-Hydroxydopamine injection resulted in a complete disappearance of torpor for about 6 days. These results conclude that the onset of daily torpor requires an intact noradrenergic signalling of the sympathetic nervous system. We further observed that parasympathetic as well as sympathetic blockade resulted in an immediate abolishment of ultradian rhythms of body temperature. This suggests that the expression of ultradian oscillations in body temperature require a continued interaction of sympathetic and parasympathetic activity.

- by Gerhard Heldmaier
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- Cryobiology, Physiology, Signal Transduction, Hibernation

To test the activity of novel hydroxyvitamin D(3) analogs (20(OH)D(3), 20,23(OH)(2)D and 1,20(OH)(2)D(3)) on normal and malignant melanocytes in comparison to 1,25(OH)(2)D(3). Human epidermal melanocytes and human and hamster melanoma cells were used to measure effects on proliferation and colony formation in monolayer and soft agar. Cell morphology and melanogenesis were also analyzed. QPCR was used to measure gene expression. Novel secosteroids inhibited proliferation and colony formation by melanoma cells in a similar fashion to 1,25(OH)(2)D(3), having no effect on melanogenesis. These effects were accompanied by ligand-induced translocation of VDR to the nucleus. In normal melanocytes 1α-hydroxyderivatives (1,25(OH)(2)D(3) and 1,20(OH)(2)D(3)) had stronger anti-proliferative effects than 20(OH)D(3) and 20,23(OH)(2)D(3), and inhibited dendrite formation. The cells tested expressed genes encoding VDR and enzymes that activate or inactivate vitamin D(3). Novel secosteroids show pot...

- by Robert Tuckey
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- Vitamins, Melanoma, Calcitriol, Anticancer

Acini of human parotid gland consist of pyramidal secretory cells and of flattened, basally placed, myoepithelial cells. Since secretory cells show a cytological organization intermediate between that of exocrine pancreas and that of mucous glands, human parotid is considered to be a seromucous gland. Short intercellular canaliculi extend between apical portions of secretory cells; more basally intercellular spaces, constantly separated from the lumen, are seen, Variations observed in cytology of secretory cells are believed to be more likely related to the presence of stages of a maturative cycle rather than to the existence of different types of cells. Secretory granules often show a bipartite structure consisting of a main portion and of a dense body (or core) contained in it. On the basis of the appearance of the main portion, dark and pale granules are recognizable. Dark granules are quite dense and homogeneous while pale granules, which tend to coalesce, are formed by a clear flocculent material. Together with granules endowed with intermediate characters, dark and pale granules are often present even in the same cell. Discharge of secretion into the lumen seems to take place following the fusion of the membrane of the secretory granule with the plasmalemma. In fact, bulging secretory granules and subluminal clefts are frequently seen. The shape of nuclei and the pattern of their chromatin content vary apparently with the cell cycle. Inside the nuclei, a distinct fibrous lamina, nucleoli and nuclear bodies are present. In addition, some nuclei exhibit a laminated inclusion arranged concentrically. Myoepithelial cells have an elongated body and thin processes extending among secretory cells. They are characterized by a filamentous cytoplasm poor in organelles.

- by Alessandro Lodovico Riva
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- Electron Microscopy, Cats, Biological Sciences, Endoplasmic Reticulum

The dynamics of G protein heterotrimer complex formation and disassembly in response to nucleotide binding and receptor activation govern the rate of responses to external stimuli. We use a novel flow cytometry approach to study the effects of lipid modification, isoform specificity, lipid environment, and receptor stimulation on the affinity and kinetics of G protein subunit binding. Fluoresceinlabeled myristoylated GR i1 (F-R i1 ) was used as the ligand bound to G γ in competition binding studies with differently modified GR subunit isoforms. In detergent solutions, the binding affinity of GR i to γ was 2 orders of magnitude higher than for GR o and GR s (IC 50 of 0.2 nM vs 17 and 27 nM, respectively), while in reconstituted bovine brain lipid vesicles, binding was slightly weaker. The effects of receptor on the G protein complex were assessed in R 2A AR receptor expressing CHO cell membranes into which purified γ subunits and F-R i1 were reconstituted. These cell membrane studies led to the following observations: (1) binding of R subunit to the γ was not enhanced by receptor in the presence or absence of agonist, indicating that γ contributed essentially all of the binding energy for R i1 interaction with the membrane; (2) activation of the receptor facilitated GTPγS-stimulated detachment of F-R i1 from γ and the membrane. Thus flow cytometry permits quantiatitive and real-time assessments of protein-protein interactions in complex membrane environments.

- by William Lim
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- Biochemistry, Kinetics, Flow Cytometry, Phospholipids

OBJECTIVE: Chagas' disease has spread throughout Latin America because of the high rate of migration among these countries. Approximately 30% of Chagas' patients will develop cardiomyopathy, and 10% of these will develop severe cardiac damage leading to heart failure. Beta-blockade improves symptoms and survival in heart failure patients; however, its efficacy has not been well established in Chagas' disease. We evaluated the role of carvedilol in cardiac remodeling and mortality in a Chagas' cardiomyopathy animal model. METHODS: We studied Trypanosoma cruzi infection in 55 Syrian hamsters that were divided into three groups: control (15), infected (20), and infected + carvedilol (20). Animals underwent echocardiography, electrocardiography, and morphometry for collagen evaluation in ventricles stained with picrosirius red. RESULTS: The left ventricular diastolic diameter did not change between groups, although it was slightly larger in infected groups, as was left ventricular systolic diameter. Fractional shortening also did not change between groups, although it was slightly lower in infected groups. Collagen accumulation in the interstitial myocardial space was significantly higher in infected groups and was not attenuated by carvedilol. The same response was observed in the perivascular space. The survival curve showed significantly better survival in the control group compared with the infected groups; but no benefit of carvedilol was observed during the study. However, in the acute phase (up to 100 days of infection), carvedilol did reduce mortality. CONCLUSION: Carvedilol did not attenuate cardiac remodeling or mortality in this model of Chagas' cardiomyopathy. The treatment did improve survival in the acute phase of the disease.

- by Eudes Oliveira Cunha
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- Treatment Outcome, Echocardiography, Collagen, Heart rate

We have investigated the displacement into the sol phase of inhaled particles deposited in the intrapulmonary conducting airways. Hamsters inhaled an aerosol of monodisperse polystyrene particles of 6 Fm diameter. Their lungs were fixed by intravascular perfusion, and light and electron microscopy was used to study the epithelial coating. The surfactant film at the wall-air interface was investigated by measuring its surface tension. The number of particles retained was determined stereologically. In addition we investigated the displacement of spherical particles in vitro on a DPPC monolayer in a Langmuir-Wilhelmy surface balance and determined the surface tension in vivo in the horse trachea by video bronchoscopy, applying the droplet spreading method. We found that particles deposited onto a surfactant film were pulled into the aqueous subphase, and we concluded that surface forces due to the airway surfactant likely displace deposited particles into the periciliary fluid (sol phase). Comparing lungs fixed immediately after inhalation with lungs fixed 24 hr after inhalation revealed that 86% of the particles retained in the intrapulmonary conducting airways immediately after inhalation had been cleared within 24 hr. One-third of the particles of the lungs fixed immediately after inhalation was phagocytized. The combination of structural and stereological analyses with in vitro and in vivo measurements has led to new insights into the role of airway surfactant with respect to the fate of inhaled particles, which may have important consequences regarding the effects of hazardous particles. These studies may also help to evaluate the deposition pattern and clearance of therapeutic particles, with important implications for their therapeutic use.

- by Peter Gehr
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- Materials Engineering, Biophysics, Microscopy, Horses

Many bioactive peptides, such as hormones and neuropeptides, require amidation at the C terminus for their full biological activity. Peptidylglycine α-hydroxylating monooxygenase (PHM) performs the first step of the amidation reaction-the hydroxylation of peptidylglycine substrates at the Cα position of the terminal glycine. The hydroxylation reaction is copper-and O 2dependent and requires 2 equiv of exogenous reductant. The proposed mechanism suggests that O 2 is reduced by two electrons, each provided by one of two nonequivalent copper sites in PHM (Cu H and Cu M). The characteristics of the reduced oxygen species in the PHM reaction and the identity of the reactive intermediate remain uncertain. To further investigate the nature of the key

- by Dario Estrin
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- Chemistry, Inorganic Chemistry, Quantum Theory, Medicine

Differential display-ribosomal protein SIB-NADH-ubiquinone oxidoreductase-translational initiation factor 61p27 BBP-Kritl Chemical reverse transformation of CHO-KI and other cells is a well-established phenomenon, in which oncogenically transformed cells re-acquire fibroblastoid morphology, contact inhibition and anchorage-dependent growth, in response to cyclic AMP and other agents. A limited number of changes in gene transcription and enzyme activity have been demonstrated to coincide with these morphological and physiological changes. We have used a partial differential display to identify four genes that are transcriptionally modulated in reverse transformation. One of these, encoding ribosomal protein SIS, is transcriptionally suppressed, probably as a result of the detransforming process. Three others are transcriptionally activated. One has homology to NADH-ubiquinone oxidoreductase chain 4 protein, and is also probably changed as a result of the detransforming process. Another is homologous to a human sequence which encodes a 27 kDa protein, p27BBP/eIF6, that is involved in the biogenesis of 60S ribosomal subunit, and in cell lines of epithelial origin binds to fl integrin. This has not previously been described as transformation-related, and could have a causative role in reverse transformation. The third has homology, with transcriptional or processing variations, to a human genomic sequence, a positional candidate for a tumour suppressor gene, encoding the Kritl protein which interacts with the Ras-family GTPase Krev-I.

- by István Raskó
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- RNA, Biology, Medicine, Expressed Sequence Tags

The synthesis of a series of conjugated spermine derivatives with benzoxadiazole, phenylxanthene or bodipy fluorophores is described. These fluorescent probes were used to identify the activity of the polyamine transport system (PTS). N 1-Methylspermine NBD conjugate 5 proved to have the optimal fluorescence characteristics and was used to show a selectivity for PTS-proficient CHO versus PTS-deficient CHO-MG cells. It can therefore be used as a tool for the selection of cells sensitive to cytotoxic compounds vectored through the PTS.

- by Jean-Marc Barret
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- Organic Chemistry, Pharmaceutical Chemistry, Polyamines, Drug Design

A comprehensive study of cholesterol, bile acid, and lipoprotein metabolism was undertaken in two strains of hamster that differed markedly in their response to a sucrose-rich/low fat diet. Under basal conditions, hamsters from the LPN strain differed from Janvier hamsters by a lower cholesterolemia, a higher postprandial insulinemia, a more active cholesterogenesis in both liver [3- to 4-fold higher 3-hydroxy 3-methylglutaryl coenzyme A reductase (HMG-CoAR) activity and mRNA] and small intestine, and a lower hepatic acyl-coenzyme A:cholesterol acyltransferase activity. Cholesterol saturation indices in the gallbladder bile were similar for both strains, but the lipid concentration was 2-fold higher in LPN than in Janvier hamsters. LPN hamsters had a lower capacity to transform cholesterol into bile acids, shown by the smaller fraction of endogenous cholesterol converted into bile acids prior to fecal excretion (0.34 vs. 0.77). In LPN hamsters, the activities of cholesterol 7alpha-h...

- by Maâmar Souidi
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- Kinetics, Lipids, Derivatives, Cholesterol

Bupropion is an atypical monocyclic antidepressant (Wellbutrin Ò) and smoking cessation adjuvant (Zyban Ò), belonging to the aminoketone class, structurally related to amphetamines. It is a selective inhibitor of dopamine, norepinephrine, and serotonin reuptake [1]. It also possesses anticholinergic activity. Concerning cardiovas-cular side effects, bupropion has always been perceived as safer than tricyclic antidepressants. Indeed, sinus tachycardia is the most common cardiovascular side effect associated with bupropion, mostly during overdosage [1-17]. Nonetheless, a number of bupropion-related QTc interval prolongation have been reported [1,5,6,8-13,18]. Again, most of these occurred during overdosage, and there has been no report of

- by Sylvie Pilote
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- Gap Junctions, Patch-clamp and imaging techniques, Electrocardiography, Cell line

Human artificial chromosomes (HACs), which carry a fully functional centromere and are maintained as a single-copy episome, are not associated with random mutagenesis and offer greater control over expression of ectopic genes on the HAC. Recently, we generated a HAC with a conditional centromere, which includes the tetracycline operator (tet-O) sequence embedded in the alphoid DNA array. This conditional centromere can be inactivated, loss of the alphoid tet-O (tet-O HAC) by expression of tet-repressor fusion proteins. In this report, we describe adaptation of the tet-O HAC vector for gene delivery and gene expression in human cells. A loxP cassette was inserted into the tet-O HAC by homologous recombination in chicken DT40 cells following a microcell-mediated chromosome transfer (MMCT). The tet-O HAC with the loxP cassette was then transferred into Chinese hamster ovary cells, and EGFP transgene was efficiently and accurately incorporated into the tet-O HAC vector. The EGFP transgene was stably expressed in human cells after transfer via MMCT. Because the transgenes inserted on the tet-O HAC can be eliminated from cells by HAC loss due to centromere inactivation, this HAC vector system provides important novel features and has potential applications for gene expression studies and gene therapy.

- by V. Larionov
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- Genetics, Fluorescence, Gene expression, In Situ Hybridization

The scavenger receptor-BI (SR-BI) delivers sterols from circulating lipoproteins to tissues, but the relative potency of individual lipoproteins and the transported cholesterol has not been studied in detail. In this study, we used Chinese hamster ovary cells that express recombinant mouse SR-BI but have no functional low density lipoprotein (LDL) receptors (ldlA7-SRBI cells) to compare the fate of lipids transferred from high or low density lipoproteins to cells by SR-BI. HDL and LDL were equally effective in mediating the transfer of [ 3 H]cholesterol to cells. Only 5% of the free cholesterol transferred to cells was esterified, in direct contrast to the findings in the cells that express LDL receptors in which 50% of the transported cholesterol was esterified. Almost all the free cholesterol transferred from lipoproteins to cells was rapidly excreted when the ldlA7-SRBI cells were switched to media containing unlabeled lipoproteins. SR-BI expression was associated with an increase in selective cholesteryl ester uptake from both lipoproteins, but HDL was a more effective donor. HDL and LDL were equally effective in delivering cholesterol to the intracellular regulatory pool via SR-BI. These data indicate that SR-BI is able to exchange cholesterol rapidly between lipoproteins and cell membranes and can mediate the uptake of cholesteryl esters from both classes of lipoproteins.

- by H. Stangl
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- Membrane Proteins, Biological Chemistry, Transcription Factors, Biological Sciences
- by Anna Lysakowski
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- Cognitive Science, Electron Microscopy, Biology, Transmission Electron Microscopy

Cell volume affects diverse functions including cytoskeletal organization, but the underlying signaling pathways remained undefined. We have shown previously that shrinkage induces Fyn-dependent tyrosine phosphorylation of the cortical actin-binding protein, cortactin. Because FER kinase was implicated in the direct phosphorylation of cortactin, we investigated the osmotic responsiveness of FER and its relationship to Fyn and cortactin. Shrinkage increased FER activity and tyrosine phosphorylation. These effects were abolished by the Src family inhibitor PP2 and strongly mitigated in Fyn-deficient but not in Src-deficient cells. FER overexpression caused cortactin phosphorylation that was further enhanced by hypertonicity. Exchange of tyrosine residues 421, 466, and 482 for phenylalanine prevented cortactin phosphorylation by hypertonicity and strongly decreased it upon FER overexpression, suggesting that FER targets primarily the same osmo-sensitive tyrosines. Because constituents of the cell-cell contacts are substrates of Fyn and FER, we investigated the effect of shrinkage on the adherens junctions. Hypertonicity provoked Fyn-dependent tyrosine phosphorylation in ␤-catenin, ␣-catenin, and p120 Cas and caused the dissociation of ␤-catenin from the contacts. This process was delayed in Fyn-deficient or PP2-treated cells. Thus, FER is a volume-sensitive kinase downstream from Fyn, and the Fyn/FER pathway may contribute to the cell size-dependent reorganization of the cytoskeleton and the cell-cell contacts.

- by Andras Kapus
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- Chemistry, Biological Chemistry, Medicine, Cytoskeleton