Cell Proteins Interacting with the Human Immunodeficiency Virus in Immunoblotting can be Detected by R5- or X4- Tropic Human Immunodeficiency Virus Particles.

INTRODUCTION: The present study reported a new immunoblot assay, with revelation by R5- or X4-whole free human immunodeficiency virus (HIV) particles or recombinant gp160. MATERIALS AND METHODS: The assay was optimized to identify cell proteins interacting with HIV. Whole cell lysates were prepared from peripheral blood lymphocytes (PBLs), dendritic cells (DC), monocyte-derived macrophage (MDM), and Henrietta Lacks (Hela, wild-type or transfected with DC-specific intracellular adhesion molecule-3-Grabbing Non-Integrin, HeLa) and Human endometrial cells (HEC-1A) lines; HIV particles used were the R5-tropic HIV-1JRCSF and the X4-tropic HIV-1NDK. RESULTS: Experiments with PBL lysates and both viruses demonstrated different bands, including a unique band at 105-117 kDa in addition to nonspecific bands. The 105-117 kDa band migrated at the same level of that observed in controls using total PBL lysate and anti-CD4 mAb for detection and thus likely corresponds to the cluster difference (CD) 4 complex. Blots using lysates of DCs, MDM, HeLa cell line, and HEC-1A cell line allowed identifying several bands that positions were similar to that seen by recombinant gp160 or whole R5- or X4-HIV particles. CONCLUSION: Blot of whole lysates of various HIV target cells is recognized by free HIV particles and allows identifying a wide range of HIV-interacting cell proteins. Such optimized assay could be useful to recognize new cellular HIV attachment proteins.

Functional activity of murine intestinal mucosal cells is regulated by the glucagon-like peptide-1 receptor.

To determine whether the glucagon-like peptide-1 receptor (GLP-1r) plays a role in the regulation of intestinal functional activity, we analyzed the distribution of the GLP-1r in mouse tissues and tested if tissues expressing the receptor respond to exendin-4 and exendin (9-39) amide, a GLP-1r agonist and antagonist respectively. In ileum, Glp1r mRNA level was two fold higher in extracts from epithelial cells than non-epithelial tissues. By immunohistochemistry, the receptor was localized to the mucosal cell layer of villi of ileum and colon, to the myenteric and submucosal plexus and to Paneth cells. Intravenous administration of exendin-4 to CD-1 mice induced expression of the immediate early gene c-fos in mucosal cells but not in cells of the enteric plexuses or in L cells of ileum. The induction of c-fos was inhibited by the voltage-gated sodium channel blocker tetrodotoxin. Exendin-4 also increased c-fos expression in ileal segments in vitro, suggesting that this action of the analog was independent of an extrinsic input. The induction of c-fos expression by exendin-4 was inhibited by exendin (9-39) amide, indicating that the action of exendin-4 was mediated by activation of the receptor. Our findings indicate that the GLP-1r is involved in ileal enterocyte and Paneth cell function, that the GLP-1 analog activates c-fos expression in the absence of an extrinsic input and that some of the actions of the receptor is/are mediated by voltage-gated Na channels.

Phenotype of entero-endocrine L cells becomes restricted during development.

BACKGROUND: Glucagon-like peptide (GLP)-1 and glucose-dependent insulinotropic polypeptide (GIP) are hormones secreted by L and K cells, respectively, and by LK cells. To characterize L and K cells during development, we examined ileum from embryonic (e)- 12 to e-17. RESULTS: GLP-1 cells were first seen at e-15 and their number increased at e-17. At e-17, most GLP-1 cells co-expressed GIP. The transcription factors Pax6 and Pdx-1 are required for GIP expression, while Pax6 activates the expression of GLP-1. At e-17, the mucosa has GIP+ Pax6+, GIP+ Pdx-1+, GLP-1+ Pax6+, and GLP-1+ Pdx-1+ cells. Unlike ileal L cells of postnatal and adult mice, a subset of ileal L cells of e-17 embryos co-expressed GLP-1 and glucagon (Glu). Glu-positive cells contain proprotein-convertase 2 (PC2) and PC3/1, the enzymes responsible for Glu and GLP-1 synthesis, respectively. CONCLUSIONS: Our findings indicate that most GLP-1+ cells of ileum of e-17 embryos co-express GIP and, therefore, are LK cells. In addition, a subset of GLP-1+ cells of embryos but not of neonates co-express glucagon, indicating that the expression of Glu in GLP-1+ cells disappears after birth.

Regulation of mouse intestinal L cell progenitors proliferation by the glucagon family of peptides.

Glucagon like peptide-1 (GLP-1) and GLP-2 are hormones secreted by intestinal L cells that stimulate glucose-dependent insulin secretion and regulate intestinal growth, respectively. Mice with deletion of the glucagon receptor (Gcgr) have high levels of circulating GLP-1 and GLP-2. We sought to determine whether the increased level of the glucagon-like peptides is due to L cell hyperplasia. We found, first, that high levels of the glucagon-like peptides increase L cell number but does not affect the number of other intestinal epithelial cell types. Second, a large proportion of ileal L cells of Gcgr(-/-) mice coexpressed glucose-dependent insulinotropic peptide (GIP). Cells coexpressing GIP and GLP-1 are termed LK cells. Third, the augmentation in L cell number was due to a higher rate of proliferation of L cell progenitors rather than to the entrance of mature L cells into the cell cycle. Fourth, a high concentration of the glucagon-like peptides in the circulation augmented the mRNA levels of transcription factors expressed by late but not early enteroendocrine progenitors. Fifth, the administration of exendin 9-39, a GLP-1 receptor antagonist, resulted in a decrease in the rate of L cell precursor proliferation. Finally, we determined that L cells do not express the GLP-1 receptor, suggesting that the effect of GLP-1 is mediated by paracrine and/or neuronal signals. Our results suggest that GLP-1 plays an important role in the regulation of L cell number.

Inhibition of connective tissue growth factor by small interfering ribonucleic acid prevents increase in extracellular matrix molecules in a rodent model of diabetic retinopathy.

PURPOSE: Connective tissue growth factor (CTGF) is a profibrotic factor that induces extracellular matrix (ECM) production and angiogenesis, two processes involved in diabetic retinopathy (DR). In this study, we examined whether insulin therapy or a CTGF-specific small interfering RNA (siRNA) administered to diabetic rats decreased the levels of CTGF and of selected putative downstream genes in the retina. METHODS: Rats with streptozotocin-induced diabetes were used. Animals received either no treatment for 12 weeks or were administered constant insulin therapy. MRNA and protein levels of CTGF and select ECM genes were determined using real-time PCR and western blotting of the retina. Localization of CTGF in the retina was visualized using immunohistochemistry. A group of diabetic rats received intravitreal injection of CTGF siRNA, and the retinas were examined three days later. RESULTS: CTGF mRNA and protein significantly increased in the retinas of diabetic rats. Immunohistochemistry indicated that retinal Müller cells of diabetic rats expressed CTGF. Hyperglycemia upregulated mRNA levels of fibronectin, laminin ß1, collagen IVα3, and vascular endothelial growth factor (VEGF), and this increase was prevented by insulin therapy. Treatment of diabetic rats with CTGF siRNA decreased laminin ß1, collagen IVα3 mRNA, and CTGF mRNA and protein but did not affect fibronectin or vascular endothelial growth factor mRNA levels. CONCLUSIONS: These results indicate that CTGF and ECM genes can be regulated using insulin. Importantly, these results also suggest that CTGF regulates changes in ECM molecules in DR.

Involvement of ERK1/2 kinase in insulin-and thrombin-stimulated vascular smooth muscle cell proliferation.

It is well recognized that the proliferation of vascular smooth muscle cells (VSMCs) is a key event in the pathogenesis of various vascular diseases, including atherosclerosis and hypertension. We have previously shown that among extracellular signal-regulated protein kinases (ERKs), the 42- and 44-kDa isoforms (ERK1/2) participate in the cellular mitogenic machinery triggered by several VSMCs activators, including insulin (INS) and thrombin (Thr). However, understanding of the intracellular signal transduction pathways involved is incomplete. This review considers the recent findings in INS and Thr signaling mechanisms that modulate the proliferation of VSMCs with particular emphasis on the ERK1/2 signaling pathway, an important mediator of VSMCs hypertrophy and vascular disease. Moreover, because the ERK1/2 pathway have been acknowledged as an important mediator of VSMCs hypertrophy, ERK1/2 is identified as a key target for novel therapeutic interventions to minimize irreversible tissue damage associated with hypertension and atherosclerosis.

Differential expression of glucagon and glucagon-like peptide 1 receptors in mouse pancreatic alpha and beta cells in two models of alpha cell hyperplasia.

Glucose homeostasis is determined by a balance between insulin and glucagon, produced by beta and alpha cells of the pancreas respectively. The levels of circulating hormones is partly determined by the mass of these two endocrine cell types. However, in contrast to beta cells, the identity of the signals regulating alpha cell number is not known. Mice with a global deletion of the glucagon receptor (Gcgr-/-) and mice with ablation of prohormone convertase 2 (PC2), the enzyme involved in the conversion of proglucagon into mature glucagon, develop alpha cell hyperplasia. These observations and the fact that Gcgr-/- mice exhibit high levels of circulating glucagon-like peptide-1 (GLP-1) suggested that members of the glucagon family of peptides could be directly involved in the regulation of alpha cell number. In this study we sought to determine whether alpha cells express receptors for Glucagon (Gcgr) and/or the glucagon-like peptide-1 (GLP1r). We examined the expression of these receptors in islets of Gcgr-/-, PC2-/- mice and control littermates, in an alpha (alphaTC1/9) and in a beta (betaTC3) cell line. Gcgr was expressed exclusively by islet beta cells, but not by alpha cells, of the two lines of mice lacking glucagon signaling. Similarly, betaTC but not alphaTC cells, expressed Gcgr. The expression of GLP1r by alpha cells was determined by the genotype and age of the mice. In embryos, GLU+ cells of Gcgr+/+ mice cells express GLP1r during early development, but not in adults. In contrast, alpha cells of Gcgr-/- mice were GLP1r+ throughout life, reflecting the immature state of GLU+ cells when Gcgr is deleted. Unlike alpha cells, beta cells of all mice lines examined initiate GLP1r expression after birth. These results suggest that GLP-1 may affect the maturation of postnatal but not prenatal beta cells. In addition, they also suggest that the incretin could mediate alpha cell proliferation, inducing the development of alpha cell hyperplasia in Gcgr-/- mice.

Role of PI3K/AKT, cPLA2 and ERK1/2 signaling pathways in insulin regulation of vascular smooth muscle cells proliferation.

Vascular smooth muscle cells (VSMCs) respond to arterial wall injury by intimal proliferation and play a key role in atherogenesis by proliferating and migrating excessively in response to repeated injury, such as hypertension and atherosclerosis. In contrast, fully differentiated, quiescent VSMCs allow arterial vasodilatation and vasoconstriction. Exaggerated and uncontrolled VSMCs proliferation appears therefore to be a common feature of both atherosclerosis and hypertension. Phosphorylation/dephosphorylation reactions of enzymes belonging to the family of mitogen-activated protein kinases (MAPKs), phosphatidylinositol 3-kinase (PI3K) and protein kinase B (Akt) play an important role in the transduction of mitogenic signal. We have previously shown that among extracellular signal-regulated protein kinases (ERKs), the 42 and 44 kDa isoforms (ERK1/2) as well as Akt and cytosolic phospholipase 2 (cPLA2) participate in the cellular mitogenic machinery triggered by several VSMCs activators, including insulin (INS). The ability of INS to significantly increase VSMCs proliferation has been demonstrated in several systems, but understanding of the intracellular signal transduction pathways involved is incomplete. Signal transduction pathways involved in regulation of the VSMCs proliferation by INS remains poorly understood. Thus, this review examines recent findings in signaling mechanisms employed by INS in modulating the regulation of proliferation of VSMCs with particular emphasis on PI3K/Akt, cPLA2 and ERK1/2 signaling pathways that have been identified as important mediators of VSMCs hypertrophy and vascular diseases. These findings are critical for understanding the role of INS in vascular biology and hyperinsulinemia.

Adenoviral-mediated inhibition of connective tissue growth factor in Rat-2 cells.

Connective tissue growth factor (CTGF) is a profibrotic factor shown to induce extracellular matrix production and angiogenesis, two processes involved in the development of diabetic retinopathy (DR). In this study we tested the effect of a recombinant adenovirus encoding for a CTGF antisense oligonucleotide (rAdASO) on the levels of transforming growth factor-beta (TGF-beta) induced expression of CTGF in Rat-2 fibroblasts. Using semi-quantitative RT-PCR, there was a 2-fold increase in CTGF message induced by TGF-beta. Western blot and immunocytochemical analyses revealed a significant increase in CTGF protein level. This upregulation of CTGF by TGF-beta was inhibited by infection with rAdASO. These findings indicate that infection of the Rat-2 cells with rAdASO was effective in decreasing TGF-beta-induced CTGF expression. These results indicate that this viral vector might have therapeutic potential to control elevated CTGF levels that occur in DR.

Nestin expression in pancreatic endocrine and exocrine cells of mice lacking glucagon signaling.

Nestin, a marker of neural stem cells, is also expressed by cells located in the epithelium of the pancreatic primordium and by a subpopulation of exocrine cells but not by endocrine cells. These findings raised the possibility that the pancreatic epithelium is heterogeneous and comprised of subpopulations of exocrine/nestin-positive and endocrine/nestin-negative precursor cells. We examined this issue in two mutant mouse models characterized by protracted expression of several embryonal properties in islet cells. One mutant line comprises mice lacking mature glucagon due to abrogation of proprotein convertase-2 (PC2(-/-)), responsible for the conversion of proglucagon into glucagon, while the second line consists of mice with a global deletion of the glucagon receptor (Gcgr(-/-)). We demonstrate that nestin is transiently expressed by acinar cells and by insulin and glucagon cells of islets of both lines of mice. In addition, the lack of glucagon signaling increased nestin mRNA levels in pancreas of mutant embryos and adult mice. We conclude that nestin+ cells located in the pancreatic primordium generate the cells of the endocrine and exocrine lineages. Furthermore, our results suggest that nestin expression is regulated by glucagon signaling.

Ablation of the glucagon receptor gene increases fetal lethality and produces alterations in islet development and maturation.

Although glucagon (GLU) plays a pivotal role in glucose homeostasis, its role in the regulation of fetal growth and maturation is poorly understood. These issues were examined in a line of mice with a global deletion of the GLU receptor (Gcgr-/-), which are characterized by lower blood glucose levels and by alpha- and delta-cell hyperplasia in adults. Ablation of Gcgr was deleterious to fetal survival; it delayed beta-cell differentiation and perturbed the proportion of beta- to alpha-cells in embryonic islets. In adults, the mutation inhibited the progression of alpha-cells to maturity, affected the expression of several beta-cell-specific genes, and resulted in an augmentation of the alpha-, beta-, and delta-cell mass. This increase was due to an augmentation in both islet number and in the rate of proliferation of cells expressing GLU or insulin. These findings suggest that GLU participates in a feedback loop that regulates the proportion of the different endocrine cell types in islets, the number of islets per pancreas, and development of the mature alpha-cell phenotype.

Expression of a recombinant protein of the platelet F11 receptor (F11R) (JAM-1/JAM-A) in insect cells: F11R is naturally phosphorylated in the extracellular domain.

The F11 receptor (F11R/JAM) is a member of the immunoglobulin superfamily localized on the membrane surface of human platelets and a component of tight junctions of endothelial and epithelial cells. F11R was demonstrated to participate in the adhesion of human platelets to cytokine-inflamed endothelial cells (EC), indicating an important role for F11R in inflammatory thrombosis and atherosclerosis. Domains responsible for the formation of tight junctions, the adhesion of platelets to EC, activation of platelets resulting in granule release, the activation of IIb/3 integrin and platelet aggregation, were identified in the external portion of F11R. To further examine critical sites of F11R, we utilized the baculovirus system to generate the F11R recombinant protein with the sequence of the extracellular domain, in two types of insect cells, Sf9 and H5. The F11R recombinant protein was detected in the cytoplasm of both infected Sf9 and H5 insect cells, but only infected H5 cells secreted a soluble F11R protein. The purified recombinant F11R proteins, obtained from both types of insect cells, were recognizeable by a conformation-dependent monoclonal antibody, M.Ab.F11, directed against domains within the N-terminus and the first Ig-like fold of F11R. Assessment of the phosphorylation state in the recombinant F11R protein revealed phosphorylation of serine, threonine and tyrosine amino acid residues within the external domain. Real-time biomolecular interaction analysis, performed to assess kinetic constants associated with the binding of active molecules to the purified recombinant F11R protein revealed high affinity binding of the phosphorylated recombinant protein by M.Ab.F11 with K(a) of 5.47 x 10(6) and K(d) of 1.83 x 10(-7), comparable to values measured with intact human platelets. The findings reported here provide new information on specific domains of F11R that can lead to the generation of therapeutic agents expected to be useful in the treatment of cardiovascular diseases.

The role of pregnancy hormones in the regulation of Pdx-1 expression.

During pregnancy, pancreatic beta cells undergo changes that are probably due to an increase in the lactogenic hormones prolactin (PRL) and placental lactogen (PL). Since the transcription factor PDX-1 is involved in the regulation of the beta cell function and phenotype, we tested the possibility that the effect of PRL on beta cells was mediated by PDX-1. Exposure of islet cells to PRL in vitro resulted in increased levels of PDX-1 protein and mRNA and a stimulation of pdx-1 transcription. However, PDX-1 levels in islets exposed in vivo to high concentration of prolactin was similar to controls. In vitro studies suggested that the up-regulation of PDX-1 by PRL was opposed by glucocorticoids (GC) at concentrations similar to those present in pregnant and control female mice. We conclude that, although pdx-1 is a key regulator of beta cell specific genes, it does not appear to play a central role in the up-regulation of islet cell function during pregnancy.

Angiotensin II regulation of the Na+ pump involves the phosphatidylinositol-3 kinase and p42/44 mitogen-activated protein kinase signaling pathways in vascular smooth muscle cells.

This investigation used primary cultured rat vascular smooth muscle cells to examine angiotensin II (Ang II) regulation of Na(+), K(+)-ATPase (Na(+) pump) activity, and Na(+) pump alpha(1)- and beta(1)-subunit gene transcription. This regulation was mediated through both phosphatidylinositol-3 kinase (PI3K) and p42/44 mitogen-activated protein kinase (p42/44(MAPK)) signaling pathways. Both acute (10 min) and prolonged (24 h) treatment with Ang II stimulated Na(+) pump activity. Also, prolonged exposure to Ang II (24 h) increased promoter transcription of the Na(+) pump alpha(1)- and beta(1)-subunits. Furthermore, PI3K activities because well because p42/44(MAPK) phosphorylation were increased within 10 min after Ang II treatment. To determine whether these stimulatory activities of Ang II are acting through Ang II receptors 1 and/or 2 (AT(1), AT(2)), cells were pretreated with either AT(1) receptor blocker losartan or the AT(2) receptor blocker PD 123,319. Indeed, these treatments prevented the stimulatory effect of Ang II on Na(+) pump activity at both acute and 24-h time points. Furthermore, the Ang II-stimulated alpha(1)-subunit promoter transcription was inhibited by losartan but not by the AT(2) receptor blocker. These results indicate that Ang II acts through both the AT(1) and AT(2) receptor to up-regulate Na(+) pump activity; however, Ang II regulates alpha(1)-gene transcription through AT(1) but not AT(2) receptors. It was also observed that the Ang II-stimulated beta(1)-subunit gene transcription is not mediated through either AT(1) or AT(2) receptors. To examine whether the Na(+)/H(+) exchanger is involved in Ang II-stimulated Na(+) pump activity, cells were pretreated with amiloride, a specific inhibitor of the Na(+)/H(+) exchanger. This pretreatment prevented 24 h, but not acute, Ang II-stimulated Na(+) pump activity. The 24-h Ang II-stimulated alpha(1)-subunit promoter transcription was also inhibited by amiloride. This suggests that the prolonged effect of Ang II on Na(+) pump activity is dependent on increased Na(+)/H(+) exchange. Because Ang II treatment for 10 min increased PI3K activity because well because p42/44(MAPK) phosphorylation, studies were performed to determine the involvement of PI3K and p42/44(MAPK) signaling pathways in both Ang II-stimulated Na(+) pump activity and alpha(1)- and beta(1)-gene transcription. Cells were pretreated with either the PI3K inhibitor wortmannin or the p42/44(MAPK) inhibitor PD 98059. Ang II-stimulated PI3K or p42/44(MAPK) activity was inhibited by these pretreatments. Furthermore, pretreatment of cells with the PI3K inhibitors wortmannin and LY29404 or the MAPK inhibitors U0126 and PD 98059 were all observed to inhibit Ang II-stimulated Na(+) pump activity. To more specifically determine the role of PI3K in Ang II-regulation of alpha(1)-and beta(1)-gene transcription, cells were cotransfected with a dominant-negative p85 construct. Cotransfection with dominant-negative p85 reduced Ang II-stimulated alpha(1)-but not beta(1)-gene transcription in vascular smooth muscle cells. These results indicate that Ang II acts through PI3K/p42/44(MAPK) signaling pathways to up-regulate Na(+) pump activity and alpha(1)-gene transcription and that Ang II-regulated beta(1)-gene transcription is not mediated through either PI3K or p42/44 (MAPK) signaling pathways.

F11-receptor (F11R/JAM) mediates platelet adhesion to endothelial cells: role in inflammatory thrombosis.

The F11 receptor (F11R) is a cell adhesion molecule (CAM), member of the immunoglobulin superfamily found on the surface of human platelets, and determined to play a role in platelet aggregation, secretion, adhesion and spreading. The same molecule is present also at tight junctions of endothelial cells (EC) where it is known as JAM and acts as a CAM through homophilic interactions. The role of F11R/JAM in the interaction of platelets with endothelial cells was investigated in the current studies. We report here that washed human platelets adhere specifically to a matrix made of immobilized, recombinant sF11R. Furthermore, platelets adhere to cytokine- (TNF-alpha, INF-gamma) stimulated human umbilical vein endothelial cells (HUVEC), and approximately 40-60% of the adhesive force is exerted by homophilic interactions between the F11R of platelets and EC. This is evidenced by the inhibition of platelet adhesion to endothelial cells by recombinant soluble form of the F11R, and by two F11R peptides with amino acid sequences of the N-terminal region, and in the 1(st) Ig fold of the F11R, respectively. This study suggests a role for F11R in the adhesion of platelets to cytokine-inflamed endothelial cells and thus in thrombosis and atherosclerosis induced in non-denuded blood vessels by inflammatory processes. Agents that block the F11R-mediated adhesion of platelets to EC may be of therapeutic value in controlling thrombosis and preventing heart attacks and stroke.

Plasmodium falciparum: glycosylation status of Plasmodium falciparum circumsporozoite protein expressed in the baculovirus system.

We expressed the main surface antigen of Plasmodium falciparum sporozoites, the circumsporozoite protein (CSP), in High Five (Trichoplusia ni) insect cells using the baculovirus system. Significant amounts of the recombinant protein could be obtained, as judged by SDS-PAGE, Western blot, and immunofluorescence analysis. The cellular localization for recombinant CSP was determined by immunofluorescence. The high fluorescence signal of the permeabilized cells, relative to that of fixed nonpermeabilized cells, revealed a clear intracellular localization of this surface antigen. Analysis of possible posttranslational modifications of CSP showed that this recombinant protein is only N-glycosylated in the baculovirus system. Although DNA-sequence analysis revealed a GPI-cleavage/attachment site, no GPI anchor could be demonstrated. These analyses show that the glycosylation status of this recombinant protein may not reflect its native form in P. falciparum. The impact of these findings on vaccine development will be discussed.

Differential, tissue-specific, transcriptional regulation of apolipoprotein B secretion by transforming growth factor beta.

Apolipoprotein B (apoB) is required for the assembly and secretion of triglyceride-rich lipoproteins. ApoB synthesis is constitutive, and post-translational mechanisms modulate its secretion. Transforming growth factor beta (TGF-beta) increased apoB secretion in both differentiated and nondifferentiated Caco-2 cells and decreased secretion in HepG2 cells without affecting apolipoprotein A-I secretion. TGF-beta altered apoB secretion by changing steady-state mRNA levels and protein synthesis. Expression of SMAD3 and SMAD4 differentially regulated apoB secretion in these cells. Thus, SMADs mediate dissimilar secretion of apoB in both the cell lines by affecting gene transcription. We identified a 485-bp element, 55 kb upstream of the apob gene that contains a SMAD binding motif. This motif increased the expression of chloramphenicol acetyltransferase in Caco-2 cells treated with TGF-beta or transfected with SMADs. Hence, TGF-beta activates SMADs that bind to the 485-bp intestinal enhancer element in the apob gene and increase its transcription and secretion in Caco-2 cells. This is the first example showing differential transcriptional regulation of the apob gene by cytokines and dissimilar regulation of one gene in two different cell lines by TGF-beta. In this regulation, the presence of cytokine-responsive motif in the tissue-specific enhancer element confers cell-specific response.

Two regions of the human platelet F11-receptor (F11R) are critical for platelet aggregation, potentiation and adhesion.

The F11 receptor (F11R) was first identified on the surface of human platelets as a target for a stimulatory monoclonal antibody (M.Ab.F11) that induces secretion, followed by exposure of fibrinogen receptors and aggregation. Cloning of the gene of F11R has revealed that this protein is a cell adhesion molecule (CAM), a member of the Ig superfamily and an ortholog of the murine protein called junctional adhesion molecule (JAM). The present study has identified two domains through which M.Ab.F11 triggers a platelet response culminating with aggregation. M.Ab.F11-mediated platelet adhesion, and the potentiation of collagen and ADP-induced platelet aggregation by M.Ab.F11, were found to involve the same two domains. A F11R recombinant protein (sF11R) completely inhibited platelet aggregation, adhesion and potentiation induced by M.Ab.F11, indicative that the active conformation of the external domain of F11R is present in the soluble, secreted recombinant protein. Furthermore, a specific peptide containing the sequence of the N-terminal amino acids S-1 to C-23 of F11R, and a peptide with the sequence of K-70 to C-82 in the 1st immunoglobulin-like (Ig) fold of F11R, both inhibited M.Ab.F11-induced aggregation, adhesion and potentiation of the aggregation of human platelets. Modeling of the 3D structure of the extracellular domain of the human platelet F11R suggests that these two regions form an active site within the conformation of this CAM. The sequence of these functional domains of F11R (in the N-terminus and 1st Ig-fold) provide the basis for new drug development in the treatment of certain types of thrombocytopenia and inflammatory thrombosis.

The GPI1 homologue from Plasmodium falciparum complements a Saccharomyces cerevisiae GPI1 anchoring mutant.

Glycosylphosphatidylinositol (GPI) represents an important anchoring molecule for cell surface proteins. The first step in its synthesis is the transfer of N-acetylglucosamine (GlcNAc) from UDP-N-acetylglucosamine to phosphatidylinositol (PI). This chemically simple step is genetically complex because three or four genes are required in both yeast (GPI1, GPI2 and GPI3) and mammals (GPI1, PIG A, PIG H and PIG C), respectively. Here, we report cloning of a Plasmodium falciparum (P. falciparum) homologue of GPI1 (PfGPI1). Analysis showed that P. falciparum Gpi1p is somewhat more similar to the yeast proteins than human Gpi1p, showing 26 and 20% amino acid sequence identity with the Saccharomyces cerevisiae and Homo sapiens proteins, respectively. Multiple sequence alignment demonstrates also that the C-terminal half GPI1 proteins is much better conserved than the N-terminal half. The P. falciparum Gpi1p has a calculated molecular weight of 65 kDa and a predicted potential tyrosine phosphorylation site. The potential tyrosine phosphorylation site seems to occur in all other known Gpi1 proteins. Like the other GPI1 proteins, the predictive software revealed the absence of targeting signals such as organelle transit peptides, DNA binding sites, or N-terminal secretory signals. Hydrophobicity plots revealed multiple hydrophobic regions that could function as transmembrane segments. The cloned P. falciparum GPI1 gene complemented a gpi1 yeast mutant.