Juan Carlos Huamani Contreras - Academia.edu (original) (raw)
Papers by Juan Carlos Huamani Contreras
Journal of Virology, 1999
An adenovirus vector encoding the human Bcl-2 gene (hBcl-2) was derived. In vivo expression of hB... more An adenovirus vector encoding the human Bcl-2 gene (hBcl-2) was derived. In vivo expression of hBcl-2 in murine livers enhanced and prolonged adenovirus-mediated gene expression. Furthermore, in the hBcl-2-treated group a significant reduction in the apoptosis induced by the adenovirus vector was observed. Thus, the cytoprotection of the vector-infected cells with antiapoptotic genes appears promising for successful in vivo gene therapy.
Biochemical Journal, 1996
Hormone-sensitive lipase (HSL) plays a key role in lipid metabolism and overall energy homoeostas... more Hormone-sensitive lipase (HSL) plays a key role in lipid metabolism and overall energy homoeostasis, by controlling the release of fatty acids from stored triglycerides in adipose tissue. Lipases and esterases form a protein superfamily with a common structural fold, called the α/β-hydrolase fold, and a catalytic triad of serine, aspartic or glutamic acid and histidine. Previous alignments between HSL and lipase 2 of Moraxella TA144 have been extended to cover a much larger part of the HSL sequence. From these extended alignments, possible sites for the catalytic triad and α/β-hydrolase fold are suggested. Furthermore, it is proposed that HSL contains a structural domain with catalytic capacity and a regulatory module attached, as well as a structural N-terminal domain unique to this enzyme. In order to test the proposed domain structure, rat HSL was overexpressed and purified to homogeneity using a baculovirus/insect-cell expression system. The purification, resulting in > 99% p...
Nature Structural Biology, 1999
Brefeldin A esterase (BFAE), a detoxifying enzyme isolated from Bacillus subtilis, hydrolyzes and... more Brefeldin A esterase (BFAE), a detoxifying enzyme isolated from Bacillus subtilis, hydrolyzes and inactivates BFA, a potent fungal inhibitor of intracellular vesicle-dependent secretory transport and poliovirus RNA replication. We have solved the crystal structure of BFAE and we discovered that the previously reported amino acid sequence was in serious error due to frame shifts in the cDNA sequence. The correct
Methods in Enzymology, 1997
Publisher Summary This chapter discusses the production and purification of recombinant hormone-s... more Publisher Summary This chapter discusses the production and purification of recombinant hormone-sensitive lipase (HSL) on a large scale using a baculovirus-insect cell expression system, and also provides an update on the available activity assays for HSL, including the use of monomolecular films. A three-dimensional model of the catalytic domain of HSL was generated on the basis of secondary structure predictions and subsequent sequence alignments to members of the cholinesterase family. The model implies that exons 5, 6, and 9 encode the core of the α/β-hydrolase fold, found in all known esterases and lipases, and constituting the catalytic domain of HSL. It has been proposed that parts of exons 7 and 8 encode a regulatory module, containing the phosphorylation sites of HSL, probably inserted into this lipase during the course of evolution. The available HSL cDNAs have been used to establish transient expression systems in COS cells for the purpose of mapping functional domains of the HSL protein using site-directed mutagenesis. The cDNAs have also been used to establish large-scale expression systems for both rat and human HSL utilizing baculovirus-insect cell technology.
Transplantation, 2002
Background. Pancreatic islets are susceptible to myriad insults that occur during islet isolation... more Background. Pancreatic islets are susceptible to myriad insults that occur during islet isolation and transplantation. Studies demonstrated the role of Akt in regulating pancreatic -cell growth and survival. Activation of Akt maintains Bad phosphorylation and prevents its binding to mitochondrial targets, decreases caspase-9 activity, and prevents the translocation of forkhead transcription factors (FKHR). Simvastatin activates Akt in mammalian cells; therefore, we investigated the role of simvastatin on human pancreatic islets (HPI) survival. Methods. HPI were treated with simvastatin, with and without LY294002, an inhibitor of phosphoinositide 3-kinase. PI viability was examined with ethidium bromide-acridine orange, and apoptosis was examined using a quantitative assay. Akt, Bad, FKHR phosphorylation, and mitochondrial cytochrome c release were analyzed by Western blots. Caspase-9 activity was assessed by a fluorometric assay. A limited number of HPI were transplanted after simvastatin treatment in diabetic NOD-SCID mice. Results. Low levels of Akt phosphorylation (activation) were demonstrated early after islet isolation. Akt activation; increase in islet viability; and decrease in Bad phosphorylation, cytochrome c release, caspase-9 activation, and translocation of FKHR were observed after simvastatin treatment, effects reversed by LY294002. Among recipients of islets without simvastatin, none demonstrated reversal of diabetes after the transplant. In contrast, 58% of the recipients given islets treated with simvastatin remained euglycemic 30 days after the transplant. Conclusions. Targeting the survival pathway with simvastatin exerts a cytoprotective effect on isolated PI. Activation of the Akt pathway is a potential new therapeutic approach to reduce loss of functional islet mass to bolster success in clinical islet transplantation.
Transplantation, 2002
INTRODUCTION Proinflammatory cytokines (PIC) (interleukin-1beta, interferon-gamma, and tumor necr... more INTRODUCTION Proinflammatory cytokines (PIC) (interleukin-1beta, interferon-gamma, and tumor necrosis factor alpha) are released after intraportal islet transplantation lead to functional suppression and islet apoptosis. Estradiol has been shown to promote survival of cells undergoing PIC-induced apoptosis. In this study, we evaluated the effects of estradiol on isolated human pancreatic islet (IHPI) survival after exposure to PIC and analyzed potential mechanisms of action. METHODS Hand-picked, freshly isolated IHPI were incubated with PIC and estradiol. Viability was analyzed from single islet cells stained with ethidium bromide and acridine orange, apoptosis using a quantitative kit, NF-kappaB nuclear translocation using a promoter-Luciferase NF-kappaB responsive construct, mitochondrial permeability transition using the ApoAlert Mitochondrial kit, and caspase 9 by a fluorometric assay. In vitro functionality was examined by static incubation, and a limited number of islets were transplanted in nonobese diabetic, severe combined immunodeficient mice. RESULTS 17beta-Estradiol induced a dose-dependent increase in islet viability, an effect partially reversed by the estrogen receptor antagonist ICI 182,780. In vitro, islets treated with estradiol presented higher stimulation index. Euglycemia was achieved in 6 of 12 animals that received estradiol-treated islets compared with 1 of 12 control animals. Lower NF-kappaB nuclear translocation, cytochrome release, and caspase 9 activation occurred in islets treated with estradiol. CONCLUSIONS Estradiol promoted IHPI survival and improved functionality after PIC exposure in vitro and in vivo after transplantation. The molecular mechanisms involved included a decrease in NF-kappaB nuclear translocation, decrease in mitochondrial cytochrome release, and caspase 9 activation. The use of estradiol might be beneficial in clinical islet transplantation.
Virology Journal, 2007
Background Human adenovirus serotype 5 (Ad5) has been widely explored as a gene delivery vector f... more Background Human adenovirus serotype 5 (Ad5) has been widely explored as a gene delivery vector for a variety of diseases. Many target cells, however, express low levels of Ad5 native receptor, the Coxsackie-Adenovirus Receptor (CAR), and thus are resistant to Ad5 infection. The Protein Transduction Domain of the HIV Tat protein, namely PTDtat, has been shown to mediate protein transduction in a wide range of cells. We hypothesize that re-targeting Ad5 vector via the PTDtat motif would improve the efficacy of Ad5-mediated gene delivery. Results In this study, we genetically incorporated the PTDtat motif into the knob domain of Ad5 fiber, and rescued the resultant viral vector, Ad5.PTDtat. Our data showed the modification did not interfere with Ad5 binding to its native receptor CAR, suggesting Ad5 infection via the CAR pathway is retained. In addition, we found that Ad5.PTDtat exhibited enhanced gene transfer efficacy in all of the cell lines that we have tested, which included both...
Transplant Immunology, 2003
The immune decision toward allograft tolerance in non-human primates requires early inhibition of... more The immune decision toward allograft tolerance in non-human primates requires early inhibition of innate immunity and induction of immune regulation
Journal of Cell Biology, 2003
Akey step in lipolytic activation of adipocytes is the translocation of hormone-sensitive lipase ... more Akey step in lipolytic activation of adipocytes is the translocation of hormone-sensitive lipase (HSL) from the cytosol to the surface of the lipid storage droplet. Adipocytes from perilipin-null animals have an elevated basal rate of lipolysis compared with adipocytes from wild-type mice, but fail to respond maximally to lipolytic stimuli. This defect is downstream of the β-adrenergic receptor–adenylyl cyclase complex. Now, we show that HSL is basally associated with lipid droplet surfaces at a low level in perilipin nulls, but that stimulated translocation from the cytosol to lipid droplets is absent in adipocytes derived from embryonic fibroblasts of perilipin-null mice. We have also reconstructed the HSL translocation reaction in the nonadipocyte Chinese hamster ovary cell line by introduction of GFP-tagged HSL with and without perilipin A. On activation of protein kinase A, HSL-GFP translocates to lipid droplets only in cells that express fully phosphorylatable perilipin A, con...
Protein Expression and Purification, 1998
Plasmid, 2010
Integrative and replicative plasmids for the expression driven by the P(43) promoter and secretio... more Integrative and replicative plasmids for the expression driven by the P(43) promoter and secretion of recombinant proteins in Bacillus subtilis were constructed. The plasmids named pInt and pRep respectively were tested for the production of recombinant human interferon gamma (rhIFN-γ). A synthetic hIFN-γ gene employing the optimized B. subtilis codon usage was fused with the Bacillus licheniformis α-amylase signal peptide (sp-amyL) encoding sequence. The integrative construct produced 2.5±0.2mgl(-1) and the replicative system produced 20.3±0.8mgl(-1) of total recombinant rhIFN-γ. The results showed that secretion of hIFN-γ was the bottleneck for the overexpression of mature rhIFN-γ by B. subtilis.
Molecular Biotechnology, 2002
The delineation of the molecular basis of cancer allows for the possibility of specific intervent... more The delineation of the molecular basis of cancer allows for the possibility of specific intervention at the molecular level for therapeutic purposes. To a large extent, the genetic lesions associated with malignant transformation and progression are being identified. Thus, not only in the context of inherited genetic diseases, but also for many acquired disorders, characteristic aberrancies of patterns of gene expression may be precisely defined. It is therefore clear that elucidation of the genetic basis of inherited and acquired diseases has rendered gene therapy both a novel and rational approach for these disorders. To this end, three main strategies have been developed: mutation compensation, molecular chemotherapy, and genetic immunopotentiation. Mutation compensation relies on strategies to ablate activated oncogenes at the level of DNA (triplex), messenger RNA (antisense or ribozyme), or protein (intracellular single-chain antibodies), and augment tumor suppressor gene expression. This article will review in detail practical procedures to generate a single-chain intracellular antibody (scFv). We will emphasize in this article the different steps in our protocol that we have employed to develop scFvs to a variety of target proteins. Index Entries: Intracellular single-chain antibody (scFv); gene therapy; colony lift assay; light chain (VL); heavy chain (VH).
Journal of Biological Chemistry, 2002
The identity of the enzymes responsible for lipase and cholesterol esterase activities in the sma... more The identity of the enzymes responsible for lipase and cholesterol esterase activities in the small intestinal mucosa is not known. Because hormone-sensitive lipase (HSL) catalyzes the hydrolysis of acylglycerols and cholesteryl esters, we sought to determine whether HSL could be involved. HSL mRNA and protein were detected in all segments of the small intestine by Northern and Western blot analyses, respectively. Immunocytochemistry experiments revealed that HSL was expressed in the differentiated enterocytes of the villi and was absent in the undifferentiated cells of the crypt. Diacylglycerol lipase and cholesterol esterase activities were found in the different segments. Analysis of gut from HSL-null mice showed that diacylglycerol lipase activity was unchanged in the duodenum and reduced in jejunum. Neutral cholesterol esterase activity was totally abolished in duodenum, jejunum, and ileum of HSL-null mice. Analysis of HSL mRNA structure showed two types of transcripts expressed in equal amounts with alternative 5-ends transcribed from two exons. This work demonstrates that HSL is expressed in the mucosa of the small intestine. The results also reveal that the enzyme participates in acylglycerol hydrolysis in jejunal enterocytes and cholesteryl ester hydrolysis throughout the small intestine.
Journal of Biological Chemistry, 1997
Monoglyceride lipase catalyzes the last step in the hydrolysis of stored triglycerides in the adi... more Monoglyceride lipase catalyzes the last step in the hydrolysis of stored triglycerides in the adipocyte and presumably also complements the action of lipoprotein lipase in degrading triglycerides from chylomicrons and very low density lipoproteins. Monoglyceride lipase was cloned from a mouse adipocyte cDNA library. The predicted amino acid sequence consisted of 302 amino acids, corresponding to a molecular weight of 33,218. The sequence showed no extensive homology to other known mammalian proteins, but a number of microbial proteins, including two bacterial lysophospholipases and a family of haloperoxidases, were found to be distantly related to this enzyme. By means of multiple sequence alignment and secondary structure prediction, the structural elements in monoglyceride lipase, as well as the putative catalytic triad, were identified. The residues of the proposed triad, Ser-122, in a GXSXG motif, Asp-239, and His-269, were confirmed by site-directed mutagenesis experiments. Northern blot analysis revealed that monoglyceride lipase is ubiquitously expressed among tissues, with a transcript size of about 4 kilobases.
Journal of Biological Chemistry, 2003
Lipolysis in adipocytes governs the release of fatty acids for the supply of energy to various ti... more Lipolysis in adipocytes governs the release of fatty acids for the supply of energy to various tissues of the body. This reaction is mediated by hormone-sensitive lipase (HSL), a cytosolic enzyme, and perilipin, which coats the lipid droplet surface in adipocytes. Both HSL and perilipin are substrates for polyphosphorylation by protein kinase A (PKA), and phosphorylation of perilipin is required to induce HSL to translocate from the cytosol to the surface of the lipid droplet, a critical step in the lipolytic reaction (Sztalryd C.,
Journal of Biological Chemistry, 2003
Lipid metabolism plays an important role in glucose homeostasis under normal and pathological con... more Lipid metabolism plays an important role in glucose homeostasis under normal and pathological conditions. In adipocytes, skeletal muscle, and pancreatic -cells, lipids are mobilized from acylglycerides by the hormone-sensitive lipase (HSL). Here, the consequences of a targeted disruption of the HSL gene for glucose homeostasis were examined. HSL null mice were slightly hyperglycemic in the fasted, but not fed state, which was accompanied by moderate hyperinsulinemia. During glucose challenges, however, disposal of the sugar was not affected in HSL null mice, presumably because of release of increased amounts of insulin. Impaired insulin sensitivity was further indicated by retarded glucose disposal during an insulin tolerance test. A euglycemic hyperinsulinemic clamp revealed that hepatic glucose production was insufficiently blocked by insulin in HSL null mice. In vitro, insulin-stimulated glucose uptake into soleus muscle, and lipogenesis in adipocytes were moderately reduced, suggesting additional sites of insulin resistance. Morphometric analysis of pancreatic islets revealed a doubling of -cell mass in HSL null mice, which is consistent with an adaptation to insulin resistance. Insulin secretion in vitro, examined by perifusion of isolated islets, was not impacted by HSL deficiency. Thus, HSL deficiency results in a moderate impairment of insulin sensitivity in multiple target tissues of the hormone but is compensated by hyperinsulinemia.
Expert Opinion on Therapeutic Patents, 1997
Delineation of the molecular basis of cancer affords the possibility of specific intervention at ... more Delineation of the molecular basis of cancer affords the possibility of specific intervention at the molecular level for therapeutic purposes. To this end, viral and non-viral vectors have been designed for delivery and expression of genes into target malignant and non-malignant cells. Gene transfer by available vectors, applied in both the ex vivo and in vivo contexts, has resulted frequently in the desired cellular phenotypical changes. In this regard, recombinant adenoviruses have been particularly efficient for in vivo gene transfer. Importantly, numerous human clinical protocols using adenoviruses have rapidly entered into Phase I clinical trials. However, major vector-related problems remain to be solved before the transfer of therapeutic genes by adenoviruses can become an effective and common place strategy for cancer treatment. An overriding obstacle is the basic ability to deliver therapeutic genes quantitatively, and specifically, into tumour cells. In addition, transgene expression in transduced target cells has not been prolonged enough for certain applications. The short-term expression is due both to the adenoviral non-integrative life cycle and to potent inflammatory and immunological responses against the vector and transgene. Here we review a number of diverse advances in the design of adenoviral vectors for overcoming these obstacles. As vector technology fulfils these requirements for obtaining the 'targetable-injectable' vector, it is anticipated that promising results already observed in preclinical studies will translate quickly into the clinic.
Expert Opinion on Therapeutic Patents, 1999
It is well established that most cancers result from a series of accumulated, acquired genetic le... more It is well established that most cancers result from a series of accumulated, acquired genetic lesions in somatic cells that are faithfully reproduced until a malignant clone is created, which is ultimately able to destroy the host. To a larger and larger extent, the genetic lesions associated with malignant transformation and progression in a wide variety of human cancers are being identified. Armed with this knowledge of the molecular anatomy of the cancer cell, gene therapy has emerged as a new method of therapeutic and possibly preventive intervention against cancer targeted at the level of cellular gene expression. This review highlights current strategies and significant developments being employed in gene therapy for neoplastic diseases. Three main approaches currently being investigated are mutation compensation, molecular chemotherapy, and genetic immunotherapy. Mutation compensation relies on strategies to ablate activated oncogenes at the level of DNA (triplex), messenger RNA (antisense or ribozyme) or protein (intracellular single chain antibodies), and augment tumour suppresser gene expression. Molecular chemotherapy uses the delivery of a toxin gene to tumour cells for eradication. This can be accomplished by either transductional targeting, whereby the toxin is specifically delivered to the tumour, or by transcriptional targeting, whereby tumour specific transcriptional activators are employed to selectively 'turn on' the toxin gene exclusively within the tumour. Genetic immunotherapy refers to the treatment based upon the induction of a specific immune response against tumour associated antigens (TAAs). The main objective of this therapy is to reinforce and bolster the immune system of the cancer-bearing host resulting in rejection of the tumour. In this context, for each of these conceptual approaches, human clinical protocols have entered testing in Phase I, II and III to assess dose escalation, safety, and toxicity issues, and more recently to evaluate efficacy, respectively.
Diabetes, 2003
Although ∼1 million islets exist in the adult human pancreas, current pancreas preservation and i... more Although ∼1 million islets exist in the adult human pancreas, current pancreas preservation and islet isolation techniques recover <50%. Presently, cadaveric donors remain the sole source of pancreatic tissue for transplantation. Brain death is characterized by activation of proinflammatory cytokines and organ injury during preservation and reperfusion. In this study, we assessed the effects of brain death on islet isolation yields and functionality. Brain death was induced in male 250- to 350-g Lewis rats by inflation of a Fogarty catheter placed intracranially. The rats were mechanically ventilated for 2, 4, and 6 h before removal of the pancreas (n = 6). In controls, the catheter was not inflated (n = 6). Shortly after brain death induction, a significant increase in serum tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 was demonstrated in a time-dependent manner. Upregulation of TNF-α, IL-1β, and IL-6 mRNA was noted in the pancreas. Brain death donors presented...
Cytogenetic and Genome Research, 1967
The chromosomes of the field mouse Akodon azarae have been investigated. In males the diploid chr... more The chromosomes of the field mouse Akodon azarae have been investigated. In males the diploid chromosome number is 38, with XY sex chromosomes. The four types of chromosome constitution in females are as follows: (a) 38 chromosomes with XX sex chromosomes; (b) 38 chromosomes Xx, the x being a grossly deleted X chromosome; (c) 38/37 Xx/XO sex chromosome mosaic; and (d) 37 chromosomes with an XO sex chromosome complement. This variability in females is believed to result from a dosage compensation mechanism ranging from X chromosome inactivation to total elimination of the X chromosome.
Journal of Virology, 1999
An adenovirus vector encoding the human Bcl-2 gene (hBcl-2) was derived. In vivo expression of hB... more An adenovirus vector encoding the human Bcl-2 gene (hBcl-2) was derived. In vivo expression of hBcl-2 in murine livers enhanced and prolonged adenovirus-mediated gene expression. Furthermore, in the hBcl-2-treated group a significant reduction in the apoptosis induced by the adenovirus vector was observed. Thus, the cytoprotection of the vector-infected cells with antiapoptotic genes appears promising for successful in vivo gene therapy.
Biochemical Journal, 1996
Hormone-sensitive lipase (HSL) plays a key role in lipid metabolism and overall energy homoeostas... more Hormone-sensitive lipase (HSL) plays a key role in lipid metabolism and overall energy homoeostasis, by controlling the release of fatty acids from stored triglycerides in adipose tissue. Lipases and esterases form a protein superfamily with a common structural fold, called the α/β-hydrolase fold, and a catalytic triad of serine, aspartic or glutamic acid and histidine. Previous alignments between HSL and lipase 2 of Moraxella TA144 have been extended to cover a much larger part of the HSL sequence. From these extended alignments, possible sites for the catalytic triad and α/β-hydrolase fold are suggested. Furthermore, it is proposed that HSL contains a structural domain with catalytic capacity and a regulatory module attached, as well as a structural N-terminal domain unique to this enzyme. In order to test the proposed domain structure, rat HSL was overexpressed and purified to homogeneity using a baculovirus/insect-cell expression system. The purification, resulting in > 99% p...
Nature Structural Biology, 1999
Brefeldin A esterase (BFAE), a detoxifying enzyme isolated from Bacillus subtilis, hydrolyzes and... more Brefeldin A esterase (BFAE), a detoxifying enzyme isolated from Bacillus subtilis, hydrolyzes and inactivates BFA, a potent fungal inhibitor of intracellular vesicle-dependent secretory transport and poliovirus RNA replication. We have solved the crystal structure of BFAE and we discovered that the previously reported amino acid sequence was in serious error due to frame shifts in the cDNA sequence. The correct
Methods in Enzymology, 1997
Publisher Summary This chapter discusses the production and purification of recombinant hormone-s... more Publisher Summary This chapter discusses the production and purification of recombinant hormone-sensitive lipase (HSL) on a large scale using a baculovirus-insect cell expression system, and also provides an update on the available activity assays for HSL, including the use of monomolecular films. A three-dimensional model of the catalytic domain of HSL was generated on the basis of secondary structure predictions and subsequent sequence alignments to members of the cholinesterase family. The model implies that exons 5, 6, and 9 encode the core of the α/β-hydrolase fold, found in all known esterases and lipases, and constituting the catalytic domain of HSL. It has been proposed that parts of exons 7 and 8 encode a regulatory module, containing the phosphorylation sites of HSL, probably inserted into this lipase during the course of evolution. The available HSL cDNAs have been used to establish transient expression systems in COS cells for the purpose of mapping functional domains of the HSL protein using site-directed mutagenesis. The cDNAs have also been used to establish large-scale expression systems for both rat and human HSL utilizing baculovirus-insect cell technology.
Transplantation, 2002
Background. Pancreatic islets are susceptible to myriad insults that occur during islet isolation... more Background. Pancreatic islets are susceptible to myriad insults that occur during islet isolation and transplantation. Studies demonstrated the role of Akt in regulating pancreatic -cell growth and survival. Activation of Akt maintains Bad phosphorylation and prevents its binding to mitochondrial targets, decreases caspase-9 activity, and prevents the translocation of forkhead transcription factors (FKHR). Simvastatin activates Akt in mammalian cells; therefore, we investigated the role of simvastatin on human pancreatic islets (HPI) survival. Methods. HPI were treated with simvastatin, with and without LY294002, an inhibitor of phosphoinositide 3-kinase. PI viability was examined with ethidium bromide-acridine orange, and apoptosis was examined using a quantitative assay. Akt, Bad, FKHR phosphorylation, and mitochondrial cytochrome c release were analyzed by Western blots. Caspase-9 activity was assessed by a fluorometric assay. A limited number of HPI were transplanted after simvastatin treatment in diabetic NOD-SCID mice. Results. Low levels of Akt phosphorylation (activation) were demonstrated early after islet isolation. Akt activation; increase in islet viability; and decrease in Bad phosphorylation, cytochrome c release, caspase-9 activation, and translocation of FKHR were observed after simvastatin treatment, effects reversed by LY294002. Among recipients of islets without simvastatin, none demonstrated reversal of diabetes after the transplant. In contrast, 58% of the recipients given islets treated with simvastatin remained euglycemic 30 days after the transplant. Conclusions. Targeting the survival pathway with simvastatin exerts a cytoprotective effect on isolated PI. Activation of the Akt pathway is a potential new therapeutic approach to reduce loss of functional islet mass to bolster success in clinical islet transplantation.
Transplantation, 2002
INTRODUCTION Proinflammatory cytokines (PIC) (interleukin-1beta, interferon-gamma, and tumor necr... more INTRODUCTION Proinflammatory cytokines (PIC) (interleukin-1beta, interferon-gamma, and tumor necrosis factor alpha) are released after intraportal islet transplantation lead to functional suppression and islet apoptosis. Estradiol has been shown to promote survival of cells undergoing PIC-induced apoptosis. In this study, we evaluated the effects of estradiol on isolated human pancreatic islet (IHPI) survival after exposure to PIC and analyzed potential mechanisms of action. METHODS Hand-picked, freshly isolated IHPI were incubated with PIC and estradiol. Viability was analyzed from single islet cells stained with ethidium bromide and acridine orange, apoptosis using a quantitative kit, NF-kappaB nuclear translocation using a promoter-Luciferase NF-kappaB responsive construct, mitochondrial permeability transition using the ApoAlert Mitochondrial kit, and caspase 9 by a fluorometric assay. In vitro functionality was examined by static incubation, and a limited number of islets were transplanted in nonobese diabetic, severe combined immunodeficient mice. RESULTS 17beta-Estradiol induced a dose-dependent increase in islet viability, an effect partially reversed by the estrogen receptor antagonist ICI 182,780. In vitro, islets treated with estradiol presented higher stimulation index. Euglycemia was achieved in 6 of 12 animals that received estradiol-treated islets compared with 1 of 12 control animals. Lower NF-kappaB nuclear translocation, cytochrome release, and caspase 9 activation occurred in islets treated with estradiol. CONCLUSIONS Estradiol promoted IHPI survival and improved functionality after PIC exposure in vitro and in vivo after transplantation. The molecular mechanisms involved included a decrease in NF-kappaB nuclear translocation, decrease in mitochondrial cytochrome release, and caspase 9 activation. The use of estradiol might be beneficial in clinical islet transplantation.
Virology Journal, 2007
Background Human adenovirus serotype 5 (Ad5) has been widely explored as a gene delivery vector f... more Background Human adenovirus serotype 5 (Ad5) has been widely explored as a gene delivery vector for a variety of diseases. Many target cells, however, express low levels of Ad5 native receptor, the Coxsackie-Adenovirus Receptor (CAR), and thus are resistant to Ad5 infection. The Protein Transduction Domain of the HIV Tat protein, namely PTDtat, has been shown to mediate protein transduction in a wide range of cells. We hypothesize that re-targeting Ad5 vector via the PTDtat motif would improve the efficacy of Ad5-mediated gene delivery. Results In this study, we genetically incorporated the PTDtat motif into the knob domain of Ad5 fiber, and rescued the resultant viral vector, Ad5.PTDtat. Our data showed the modification did not interfere with Ad5 binding to its native receptor CAR, suggesting Ad5 infection via the CAR pathway is retained. In addition, we found that Ad5.PTDtat exhibited enhanced gene transfer efficacy in all of the cell lines that we have tested, which included both...
Transplant Immunology, 2003
The immune decision toward allograft tolerance in non-human primates requires early inhibition of... more The immune decision toward allograft tolerance in non-human primates requires early inhibition of innate immunity and induction of immune regulation
Journal of Cell Biology, 2003
Akey step in lipolytic activation of adipocytes is the translocation of hormone-sensitive lipase ... more Akey step in lipolytic activation of adipocytes is the translocation of hormone-sensitive lipase (HSL) from the cytosol to the surface of the lipid storage droplet. Adipocytes from perilipin-null animals have an elevated basal rate of lipolysis compared with adipocytes from wild-type mice, but fail to respond maximally to lipolytic stimuli. This defect is downstream of the β-adrenergic receptor–adenylyl cyclase complex. Now, we show that HSL is basally associated with lipid droplet surfaces at a low level in perilipin nulls, but that stimulated translocation from the cytosol to lipid droplets is absent in adipocytes derived from embryonic fibroblasts of perilipin-null mice. We have also reconstructed the HSL translocation reaction in the nonadipocyte Chinese hamster ovary cell line by introduction of GFP-tagged HSL with and without perilipin A. On activation of protein kinase A, HSL-GFP translocates to lipid droplets only in cells that express fully phosphorylatable perilipin A, con...
Protein Expression and Purification, 1998
Plasmid, 2010
Integrative and replicative plasmids for the expression driven by the P(43) promoter and secretio... more Integrative and replicative plasmids for the expression driven by the P(43) promoter and secretion of recombinant proteins in Bacillus subtilis were constructed. The plasmids named pInt and pRep respectively were tested for the production of recombinant human interferon gamma (rhIFN-γ). A synthetic hIFN-γ gene employing the optimized B. subtilis codon usage was fused with the Bacillus licheniformis α-amylase signal peptide (sp-amyL) encoding sequence. The integrative construct produced 2.5±0.2mgl(-1) and the replicative system produced 20.3±0.8mgl(-1) of total recombinant rhIFN-γ. The results showed that secretion of hIFN-γ was the bottleneck for the overexpression of mature rhIFN-γ by B. subtilis.
Molecular Biotechnology, 2002
The delineation of the molecular basis of cancer allows for the possibility of specific intervent... more The delineation of the molecular basis of cancer allows for the possibility of specific intervention at the molecular level for therapeutic purposes. To a large extent, the genetic lesions associated with malignant transformation and progression are being identified. Thus, not only in the context of inherited genetic diseases, but also for many acquired disorders, characteristic aberrancies of patterns of gene expression may be precisely defined. It is therefore clear that elucidation of the genetic basis of inherited and acquired diseases has rendered gene therapy both a novel and rational approach for these disorders. To this end, three main strategies have been developed: mutation compensation, molecular chemotherapy, and genetic immunopotentiation. Mutation compensation relies on strategies to ablate activated oncogenes at the level of DNA (triplex), messenger RNA (antisense or ribozyme), or protein (intracellular single-chain antibodies), and augment tumor suppressor gene expression. This article will review in detail practical procedures to generate a single-chain intracellular antibody (scFv). We will emphasize in this article the different steps in our protocol that we have employed to develop scFvs to a variety of target proteins. Index Entries: Intracellular single-chain antibody (scFv); gene therapy; colony lift assay; light chain (VL); heavy chain (VH).
Journal of Biological Chemistry, 2002
The identity of the enzymes responsible for lipase and cholesterol esterase activities in the sma... more The identity of the enzymes responsible for lipase and cholesterol esterase activities in the small intestinal mucosa is not known. Because hormone-sensitive lipase (HSL) catalyzes the hydrolysis of acylglycerols and cholesteryl esters, we sought to determine whether HSL could be involved. HSL mRNA and protein were detected in all segments of the small intestine by Northern and Western blot analyses, respectively. Immunocytochemistry experiments revealed that HSL was expressed in the differentiated enterocytes of the villi and was absent in the undifferentiated cells of the crypt. Diacylglycerol lipase and cholesterol esterase activities were found in the different segments. Analysis of gut from HSL-null mice showed that diacylglycerol lipase activity was unchanged in the duodenum and reduced in jejunum. Neutral cholesterol esterase activity was totally abolished in duodenum, jejunum, and ileum of HSL-null mice. Analysis of HSL mRNA structure showed two types of transcripts expressed in equal amounts with alternative 5-ends transcribed from two exons. This work demonstrates that HSL is expressed in the mucosa of the small intestine. The results also reveal that the enzyme participates in acylglycerol hydrolysis in jejunal enterocytes and cholesteryl ester hydrolysis throughout the small intestine.
Journal of Biological Chemistry, 1997
Monoglyceride lipase catalyzes the last step in the hydrolysis of stored triglycerides in the adi... more Monoglyceride lipase catalyzes the last step in the hydrolysis of stored triglycerides in the adipocyte and presumably also complements the action of lipoprotein lipase in degrading triglycerides from chylomicrons and very low density lipoproteins. Monoglyceride lipase was cloned from a mouse adipocyte cDNA library. The predicted amino acid sequence consisted of 302 amino acids, corresponding to a molecular weight of 33,218. The sequence showed no extensive homology to other known mammalian proteins, but a number of microbial proteins, including two bacterial lysophospholipases and a family of haloperoxidases, were found to be distantly related to this enzyme. By means of multiple sequence alignment and secondary structure prediction, the structural elements in monoglyceride lipase, as well as the putative catalytic triad, were identified. The residues of the proposed triad, Ser-122, in a GXSXG motif, Asp-239, and His-269, were confirmed by site-directed mutagenesis experiments. Northern blot analysis revealed that monoglyceride lipase is ubiquitously expressed among tissues, with a transcript size of about 4 kilobases.
Journal of Biological Chemistry, 2003
Lipolysis in adipocytes governs the release of fatty acids for the supply of energy to various ti... more Lipolysis in adipocytes governs the release of fatty acids for the supply of energy to various tissues of the body. This reaction is mediated by hormone-sensitive lipase (HSL), a cytosolic enzyme, and perilipin, which coats the lipid droplet surface in adipocytes. Both HSL and perilipin are substrates for polyphosphorylation by protein kinase A (PKA), and phosphorylation of perilipin is required to induce HSL to translocate from the cytosol to the surface of the lipid droplet, a critical step in the lipolytic reaction (Sztalryd C.,
Journal of Biological Chemistry, 2003
Lipid metabolism plays an important role in glucose homeostasis under normal and pathological con... more Lipid metabolism plays an important role in glucose homeostasis under normal and pathological conditions. In adipocytes, skeletal muscle, and pancreatic -cells, lipids are mobilized from acylglycerides by the hormone-sensitive lipase (HSL). Here, the consequences of a targeted disruption of the HSL gene for glucose homeostasis were examined. HSL null mice were slightly hyperglycemic in the fasted, but not fed state, which was accompanied by moderate hyperinsulinemia. During glucose challenges, however, disposal of the sugar was not affected in HSL null mice, presumably because of release of increased amounts of insulin. Impaired insulin sensitivity was further indicated by retarded glucose disposal during an insulin tolerance test. A euglycemic hyperinsulinemic clamp revealed that hepatic glucose production was insufficiently blocked by insulin in HSL null mice. In vitro, insulin-stimulated glucose uptake into soleus muscle, and lipogenesis in adipocytes were moderately reduced, suggesting additional sites of insulin resistance. Morphometric analysis of pancreatic islets revealed a doubling of -cell mass in HSL null mice, which is consistent with an adaptation to insulin resistance. Insulin secretion in vitro, examined by perifusion of isolated islets, was not impacted by HSL deficiency. Thus, HSL deficiency results in a moderate impairment of insulin sensitivity in multiple target tissues of the hormone but is compensated by hyperinsulinemia.
Expert Opinion on Therapeutic Patents, 1997
Delineation of the molecular basis of cancer affords the possibility of specific intervention at ... more Delineation of the molecular basis of cancer affords the possibility of specific intervention at the molecular level for therapeutic purposes. To this end, viral and non-viral vectors have been designed for delivery and expression of genes into target malignant and non-malignant cells. Gene transfer by available vectors, applied in both the ex vivo and in vivo contexts, has resulted frequently in the desired cellular phenotypical changes. In this regard, recombinant adenoviruses have been particularly efficient for in vivo gene transfer. Importantly, numerous human clinical protocols using adenoviruses have rapidly entered into Phase I clinical trials. However, major vector-related problems remain to be solved before the transfer of therapeutic genes by adenoviruses can become an effective and common place strategy for cancer treatment. An overriding obstacle is the basic ability to deliver therapeutic genes quantitatively, and specifically, into tumour cells. In addition, transgene expression in transduced target cells has not been prolonged enough for certain applications. The short-term expression is due both to the adenoviral non-integrative life cycle and to potent inflammatory and immunological responses against the vector and transgene. Here we review a number of diverse advances in the design of adenoviral vectors for overcoming these obstacles. As vector technology fulfils these requirements for obtaining the 'targetable-injectable' vector, it is anticipated that promising results already observed in preclinical studies will translate quickly into the clinic.
Expert Opinion on Therapeutic Patents, 1999
It is well established that most cancers result from a series of accumulated, acquired genetic le... more It is well established that most cancers result from a series of accumulated, acquired genetic lesions in somatic cells that are faithfully reproduced until a malignant clone is created, which is ultimately able to destroy the host. To a larger and larger extent, the genetic lesions associated with malignant transformation and progression in a wide variety of human cancers are being identified. Armed with this knowledge of the molecular anatomy of the cancer cell, gene therapy has emerged as a new method of therapeutic and possibly preventive intervention against cancer targeted at the level of cellular gene expression. This review highlights current strategies and significant developments being employed in gene therapy for neoplastic diseases. Three main approaches currently being investigated are mutation compensation, molecular chemotherapy, and genetic immunotherapy. Mutation compensation relies on strategies to ablate activated oncogenes at the level of DNA (triplex), messenger RNA (antisense or ribozyme) or protein (intracellular single chain antibodies), and augment tumour suppresser gene expression. Molecular chemotherapy uses the delivery of a toxin gene to tumour cells for eradication. This can be accomplished by either transductional targeting, whereby the toxin is specifically delivered to the tumour, or by transcriptional targeting, whereby tumour specific transcriptional activators are employed to selectively 'turn on' the toxin gene exclusively within the tumour. Genetic immunotherapy refers to the treatment based upon the induction of a specific immune response against tumour associated antigens (TAAs). The main objective of this therapy is to reinforce and bolster the immune system of the cancer-bearing host resulting in rejection of the tumour. In this context, for each of these conceptual approaches, human clinical protocols have entered testing in Phase I, II and III to assess dose escalation, safety, and toxicity issues, and more recently to evaluate efficacy, respectively.
Diabetes, 2003
Although ∼1 million islets exist in the adult human pancreas, current pancreas preservation and i... more Although ∼1 million islets exist in the adult human pancreas, current pancreas preservation and islet isolation techniques recover <50%. Presently, cadaveric donors remain the sole source of pancreatic tissue for transplantation. Brain death is characterized by activation of proinflammatory cytokines and organ injury during preservation and reperfusion. In this study, we assessed the effects of brain death on islet isolation yields and functionality. Brain death was induced in male 250- to 350-g Lewis rats by inflation of a Fogarty catheter placed intracranially. The rats were mechanically ventilated for 2, 4, and 6 h before removal of the pancreas (n = 6). In controls, the catheter was not inflated (n = 6). Shortly after brain death induction, a significant increase in serum tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 was demonstrated in a time-dependent manner. Upregulation of TNF-α, IL-1β, and IL-6 mRNA was noted in the pancreas. Brain death donors presented...
Cytogenetic and Genome Research, 1967
The chromosomes of the field mouse Akodon azarae have been investigated. In males the diploid chr... more The chromosomes of the field mouse Akodon azarae have been investigated. In males the diploid chromosome number is 38, with XY sex chromosomes. The four types of chromosome constitution in females are as follows: (a) 38 chromosomes with XX sex chromosomes; (b) 38 chromosomes Xx, the x being a grossly deleted X chromosome; (c) 38/37 Xx/XO sex chromosome mosaic; and (d) 37 chromosomes with an XO sex chromosome complement. This variability in females is believed to result from a dosage compensation mechanism ranging from X chromosome inactivation to total elimination of the X chromosome.