OBJECTIVE

Sedentary lifestyle and a western diet promote subacute-chronic inflammation, obesity, and subsequently dysglycemia. The aim of the current study was to evaluate the efficacy of the anti-inflammatory drug salsalate to improve glycemia by reducing systemic inflammation in obese adults at risk for the development of type 2 diabetes.

METHODS

In a double-masked, placebo controlled trial, we evaluated 20 obese nondiabetic adults at baseline and after 1 month of salsalate or placebo.

RESULTS

Compared with placebo, salsalate reduced fasting glucose 13% (P < 0.002), glycemic response after an oral glucose challenge 20% (P < 0.004), and glycated albumin 17% (P < 0.0003). Although insulin levels were unchanged, fasting and oral glucose tolerance test C-peptide levels decreased in the salsalate-treated subjects compared with placebo (P < 0.03), consistent with improved insulin sensitivity and a known effect of salicylates to inhibit insulin clearance. Adiponectin increased 57% after salsalate compared with placebo (P < 0.003). Additionally, within the group of salsalate-treated subjects, circulating levels of C-reactive protein were reduced by 34% (P < 0.05).

CONCLUSIONS

This proof-of-principle study demonstrates that salsalate reduces glycemia and may improve inflammatory cardiovascular risk indexes in overweight individuals. These data support the hypothesis that subacute-chronic inflammation contributes to the pathogenesis of obesity-related dysglycemia and that targeting inflammation may provide a therapeutic route for diabetes prevention.

In tumour cells, elevated levels of mitochondria-bound isoforms of hexokinase (HK-I and HK-II) result in the evasion of apoptosis, thereby allowing the cells to continue proliferating. The molecular mechanisms by which bound HK promotes cell survival are not yet fully understood. Our studies relying on the purified mitochondrial outer membrane protein VDAC (voltage-dependent anion channel), isolated mitochondria or cells in culture suggested that the anti-apoptotic activity of HK-I occurs via modulation of the mitochondrial phase of apoptosis. In the present paper, a direct interaction of HK-I with bilayer-reconstituted purified VDAC, inducing channel closure, is demonstrated for the first time. Moreover, HK-I prevented the Ca(2+)-dependent opening of the mitochondrial PTP (permeability transition pore) and release of the pro-apoptotic protein cytochrome c. The effects of HK-I on VDAC activity and PTP opening were prevented by the HK reaction product glucose 6-phosphate, a metabolic intermediate in most biosynthetic pathways. Furthermore, glucose 6-phosphate re-opened both the VDAC and the PTP closed by HK-I. The HK-I-mediated effects on VDAC and PTP were not observed using either yeast HK or HK-I lacking the N-terminal hydrophobic peptide responsible for binding to mitochondria, or in the presence of an antibody specific for the N-terminus of HK-I. Finally, HK-I overexpression in leukaemia-derived U-937 or vascular smooth muscle cells protected against staurosporine-induced apoptosis, with a decrease of up to 70% in cell death. These results offer insight into the mechanisms by which bound HK promotes tumour cell survival, and suggests that its overexpression not only ensures supplies of energy and phosphometabolites, but also reflects an anti-apoptotic defence mechanism.

Liraglutide is a glucagon-like peptide-1 (GLP-1) analog developed for type 2 diabetes. Long-term liraglutide exposure in rodents was associated with thyroid C-cell hyperplasia and tumors. Here, we report data supporting a GLP-1 receptor-mediated mechanism for these changes in rodents. The GLP-1 receptor was localized to rodent C-cells. GLP-1 receptor agonists stimulated calcitonin release, up-regulation of calcitonin gene expression, and subsequently C-cell hyperplasia in rats and, to a lesser extent, in mice. In contrast, humans and/or cynomolgus monkeys had low GLP-1 receptor expression in thyroid C-cells, and GLP-1 receptor agonists did not activate adenylate cyclase or generate calcitonin release in primates. Moreover, 20 months of liraglutide treatment (at >60 times human exposure levels) did not lead to C-cell hyperplasia in monkeys. Mean calcitonin levels in patients exposed to liraglutide for 2 yr remained at the lower end of the normal range, and there was no difference in the proportion of patients with calcitonin levels increasing above the clinically relevant cutoff level of 20 pg/ml. Our findings delineate important species-specific differences in GLP-1 receptor expression and action in the thyroid. Nevertheless, the long-term consequences of sustained GLP-1 receptor activation in the human thyroid remain unknown and merit further investigation.

The attachment of pathogenic microorganisms to host cells and tissues is often mediated through the expression of surface receptors recognizing components of the extracellular matrix (ECM). Here, we investigate the ability of Borrelia spirochaetes to bind the ECM constituent, fibronectin. Borrelia lysates were separated by SDS-PAGE, transferred to nitrocellulose and probed with alkaline phosphatase-labelled fibronectin (fibronectin-AP). Five of six Borrelia species and four of eight B. burgdorferi sensu lato isolates expressed one or more fibronectin-binding proteins. Borrelia burgdorferi isolate B31 expressed a 47 kDa (P47) fibronectin-binding protein that was localized to the outer envelope based on susceptibility to proteinase K. The interaction of P47 with fibronectin was specific, and the region of fibronectin bound by P47 mapped to the gelatin/collagen binding domain. P47 was purified by affinity chromatography, digested with endoproteinase Lys-C, and the peptide fragments analysed by liquid chromatography/tandem mass spectroscopy. A search of protein databases disclosed that the P47 peptide mass profile matched that predicted for the bbk32 gene product of B. burgdorferi isolate B31. The bbk32 gene was cloned into Escherichia coli, and the ability of recombinant BBK32 to bind fibronectin and inhibit the attachment of B. burgdorferi was demonstrated. The identification of BBK32 as a receptor for fibronectin binding may enhance our understanding of the pathogenesis and chronic nature of Lyme disease.

Reports that interleukin-8 (IL-8) induces the infiltration of neutrophils followed by T-cells into injection sites led us to postulate that by stimulation of neutrophil degranulation IL-8 may cause the release of factors with chemoattractant activity for T-lymphocytes. Extracts of human neutrophil granules were chromatographed to isolate and purify T-lymphocyte chemoattractant factors. Two major peaks of T-cell chemotactic activity were purified by C18 reversed phase high pressure liquid chromatography (HPLC). The first peak was resolved further by C4 reversed phase HPLC and yielded an active fraction shown by NH2-terminal amino acid sequence analysis to contain defensins HNP-1, HNP-2, and HNP-3. Purified defensins HNP-1 and HNP-2 (kindly provided by Dr. R. I. Lehrer, UCLA) were also potent chemoattractants for human T-cells, while HNP-3 was inactive. The second peak of T-cell chemoattractant activity was also further purified to homogeneity by C4 reversed phase HPLC and identified by NH2-terminal sequence analysis as CAP37/azurocidin, a protein with sequence homology to serine proteases. 0.1 100 ng of defensins and 1.0 100 ng/ml CAP37 were able to stimulate in vitro T-cell chemotaxis. Neutrophil activating factors, i.e. IL-8, phorbol 12-myristate 13-acetate/ionomycin, and formylmethionylleucylphenylalanine each induced the release of CAP37 and defensins from neutrophil granules. Subcutaneous administration of defensins or CAP37/azurocidin into BALB/c mice resulted in a moderate neutrophil and mononuclear cell infiltrate by 4 h, which was greater by 24 h at the site of injection. Additionally, subcutaneous injection of defensins into chimeric huPBL-SCID mice resulted in significant infiltration by human CD3+ cells within 4 h. These results identify the antimicrobial proteins, CAP37/azurocidin and defensins HNP-1 and HNP-2, as potent neutrophil-derived chemoattractants for T-cells. These proteins represent primordial antimicrobial peptides which may have evolved into acute inflammatory cell-derived signals that mobilize immunocompetent T-cells and other inflammatory cells.

All streptococcal genomes encode the alternative sigma factor SigX and 21 SigX-dependent proteins required for genetic transformation, yet no pyogenic streptococci are known to develop competence. Resolving this paradox may depend on understanding the regulation of sigX. We report the identification of a regulatory circuit linked to the sigX genes of mutans, pyogenic, and bovis streptococci that uses a novel small, double-tryptophan-containing sigX-inducing peptide (XIP) pheromone. In all three groups, the XIP gene (comS), and sigX have identical, non-canonical promoters consisting of 9 bp inverted repeats separated from a -10 hexamer by 19 bp. comS is adjacent to a gene encoding a putative transcription factor of the Rgg family and is regulated by its product, which we designate ComR. Deletion of comR or comS in Streptococcus mutans abolished transformability, as did deletion of the oligopeptide permease subunit oppD, suggesting that XIP is imported. Providing S. mutans with synthetic fragments of ComS revealed that seven C-terminal residues, including the WW motif, cause robust induction of both sigX and the competent state. We propose that this circuit is the proximal regulator of sigX in S. mutans, and we infer that it controls competence in a parallel way in all pyogenic and bovis streptococci.

Inflammation is a key mechanism in the initiation, progression, and clinical sequelae of cardiovascular diseases (CVDs), including atherosclerosis, nephropathy, and cardiomyopathy. Angiotensin II, the major effector peptide of the renin-angiotensin-aldosterone system (RAAS), plays a significant role in the advent and perpetuation of these inflammatory diseases, most notably in atherogenesis. Consequently, suppression of the influence of angiotensin II by angiotensin-converting enzyme inhibitors and angiotensin II receptor blockers may reduce or potentially reverse atherosclerosis and other inflammation-associated CVDs. Angiotensin II receptor blockers and angiotensin-converting enzyme inhibitors exert anti-inflammatory actions and prevent or reduce the development of atherosclerosis in animal models. Clinically, RAAS suppression reduces common carotid and femoral artery intima-media thickness, thus indicating moderation of the vascular disease process. These clinical benefits likely involve restraint of the deleterious effects of angiotensin II in addition to, or independent of, lowering blood pressure. Increasing evidence that the detection and monitoring of vascular inflammation are important tools in the management of atherosclerosis also implicates the RAAS in this pathogenic process. Inflammatory molecules such as intercellular adhesion molecule-1, vascular cell adhesion molecule-1, monocyte chemoattractant protein-1, tumor necrosis factor-alpha, and C-reactive protein have potential diagnostic and prognostic values in CVD and are modified by angiotensin-converting enzyme inhibitors and angiotensin II receptor blockers. Monitoring these markers may be crucial for determining which agents, or combinations of agents, will result in the most clinically beneficial outcomes for patients. Large-scale trials are still required to determine the effects of the long-term suppression of inflammation on CVDs through the use of RAAS modulating agents, as well as to determine how closely markers of inflammatory activity may correlate with CVD outcomes.

The incidence of Alzheimer disease (AD) and vascular dementia is greatly increased following cerebral ischemia and stroke in which hypoxic conditions occur in affected brain areas. beta-Amyloid peptide (Abeta), which is derived from the beta-amyloid precursor protein (APP) by sequential proteolytic cleavages from beta-secretase (BACE1) and presenilin-1 (PS1)/gamma-secretase, is widely believed to trigger a cascade of pathological events culminating in AD and vascular dementia. However, a direct molecular link between hypoxic insults and APP processing has yet to be established. Here, we demonstrate that acute hypoxia increases the expression and the enzymatic activity of BACE1 by up-regulating the level of BACE1 mRNA, resulting in increases in the APP C-terminal fragment-beta (betaCTF) and Abeta. Hypoxia has no effect on the level of PS1, APP, and tumor necrosis factor-alpha-converting enzyme (TACE, an enzyme known to cleave APP at the alpha-secretase cleavage site). Sequence analysis, mutagenesis, and gel shift studies revealed binding of HIF-1 to the BACE1 promoter. Overexpression of HIF-1alpha increases BACE1 mRNA and protein level, whereas down-regulation of HIF-1alpha reduced the level of BACE1. Hypoxic treatment fails to further potentiate the stimulatory effect of HIF-1alpha overexpression on BACE1 expression, suggesting that hypoxic induction of BACE1 expression is primarily mediated by HIF-1alpha. Finally, we observed significant reduction in BACE1 protein levels in the hippocampus and the cortex of HIF-1alpha conditional knock-out mice. Our results demonstrate an important role for hypoxia/HIF-1alpha in modulating the amyloidogenic processing of APP and provide a molecular mechanism for increased incidence of AD following cerebral ischemic and stroke injuries.

BACKGROUND

In 2007, the effects of the autologous nonmyeloablative hematopoietic stem cell transplantation (HSCT) in 15 patients with type 1 diabetes mellitus (DM) were reported. Most patients became insulin free with normal levels of glycated hemoglobin A(1c) (HbA(1c)) during a mean 18.8-month follow-up. To investigate if this effect was due to preservation of beta-cell mass, continued monitoring was performed of C-peptide levels after stem cell transplantation in the 15 original and 8 additional patients.

OBJECTIVE

To determine C-peptide levels after autologous nonmyeloablative HSCT in patients with newly diagnosed type 1 DM during a longer follow-up.

METHODS

A prospective phase 1/2 study of 23 patients with type 1 DM (aged 13-31 years) diagnosed in the previous 6 weeks by clinical findings with hyperglycemia and confirmed by measurement of serum levels of anti-glutamic acid decarboxylase antibodies. Enrollment was November 2003-April 2008, with follow-up until December 2008 at the Bone Marrow Transplantation Unit of the School of Medicine of Ribeirão Preto, Ribeirão Preto, Brazil. Hematopoietic stem cells were mobilized via the 2007 protocol.

METHODS

C-peptide levels measured during the mixed-meal tolerance test, before, and at different times following HSCT. Secondary end points included morbidity and mortality from transplantation, temporal changes in exogenous insulin requirements, and serum levels of HbA(1c).

RESULTS

During a 7- to 58-month follow-up (mean, 29.8 months; median, 30 months), 20 patients without previous ketoacidosis and not receiving corticosteroids during the preparative regimen became insulin free. Twelve patients maintained this status for a mean 31 months (range, 14-52 months) and 8 patients relapsed and resumed insulin use at low dose (0.1-0.3 IU/kg). In the continuous insulin-independent group, HbA(1c) levels were less than 7.0% and mean (SE) area under the curve (AUC) of C-peptide levels increased significantly from 225.0 (75.2) ng/mL per 2 hours pretransplantation to 785.4 (90.3) ng/mL per 2 hours at 24 months posttransplantation (P < .001) and to 728.1 (144.4) ng/mL per 2 hours at 36 months (P = .001). In the transient insulin-independent group, mean (SE) AUC of C-peptide levels also increased from 148.9 (75.2) ng/mL per 2 hours pretransplantation to 546.8 (96.9) ng/mL per 2 hours at 36 months (P = .001), which was sustained at 48 months. In this group, 2 patients regained insulin independence after treatment with sitagliptin, which was associated with increase in C-peptide levels. Two patients developed bilateral nosocomial pneumonia, 3 patients developed late endocrine dysfunction, and 9 patients developed oligospermia. There was no mortality.

CONCLUSIONS

After a mean follow-up of 29.8 months following autologous nonmyeloablative HSCT in patients with newly diagnosed type 1 DM, C-peptide levels increased significantly and the majority of patients achieved insulin independence with good glycemic control.

BACKGROUND

clinicaltrials.gov Identifier: NCT00315133.

Actin depolymerizing factor (ADF) occurs naturally in two forms, one of which contains a phosphorylated Ser and does not bind G-actin or depolymerize F-actin. Removal of this phosphate in vitro by alkaline phosphatase restores full F-actin depolymerizing activity. To identify the phosphorylation site, [32P]pADF was purified and digested with endoproteinase Lys-C. The digest contained only one 32P-labeled peptide. Further digestion with endoproteinase Asp-N and mass spectrometric analysis showed that this peptide came from the N terminus of ADF. Alkaline phosphatase treatment of one Asp-N peptide (mass 753) converted it to a peptide of mass 673, demonstrating that this peptide contains the phosphate group. Tandem mass spectrometric sequence analysis of this peptide identified the phosphorylated Ser as the encoded Ser3 (Ser2 in the processed protein). HeLa cells, transfected with either chick wild-type ADF cDNA or a cDNA mutated to code for Ala in place of Ser24 or Thr25, express and phosphorylate the exogenous ADF. Cells also expressed high levels of mutant ADF when Ser3 was deleted or converted to either Ala or Glu. However, none of these mutants was phosphorylated, confirming that Ser3 in the encoded ADF is the single in vivo regulatory site.

Antibodies to citrullinated proteins (anti-cyclic-citrullinated peptide [anti-CCP] antibodies) are highly specific for rheumatoid arthritis (RA) and precede the onset of disease symptoms, indicating a pathogenetic role for these antibodies in RA. We recently showed that distinct genetic risk factors are associated with either anti-CCP-positive disease or anti-CCP-negative disease. These data are important as they indicate that distinct pathogenic mechanisms are underlying anti-CCP-positive disease or anti-CCP-negative disease. Likewise, these observations raise the question of whether anti-CCP-positive RA and anti-CCP-negative RA are clinically different disease entities. We therefore investigated whether RA patients with anti-CCP antibodies have a different clinical presentation and disease course compared with patients without these autoantibodies. In a cohort of 454 incident patients with RA, 228 patients were anti-CCP-positive and 226 patients were anti-CCP-negative. The early symptoms, tender and swollen joint count, and C-reactive protein level at inclusion, as well as the swollen joint count and radiological destruction during 4 years of follow-up, were compared for the two groups. There were no differences in morning stiffness, type, location and distribution of early symptoms, patients' rated disease activity and C-reactive protein at inclusion between RA patients with and without anti-CCP antibodies. The mean tender and swollen joint count for the different joints at inclusion was similar. At follow-up, patients with anti-CCP antibodies had more swollen joints and more severe radiological destruction. Nevertheless, the distribution of affected joints, for swelling, bone erosions and joint space narrowing, was similar. In conclusion, the phenotype of RA patients with or without anti-CCP antibodies is similar with respect to clinical presentation but differs with respect to disease course.

Concentrations of insulin, proinsulin, and C-peptide were measured in portal and peripheral venous blood in six nondiabetic, nonobese subjects. Portal vein samples were obtained by umbilical vein catheterization. Three subjects were studied with intravenous infusion of 25 g glucose, and three with 30 g arginine. Insulin and proinsulin were determined in the insulin immunoassay after separation by gel filtration, and C-peptide was measured by direct immunoassay. With both glucose and arginine stimulation, portal vein levels of all three peptides peaked at 90-120 s after the onset of the stimulus. Relative increases in insulin concentration were greater than those of proinsulin or C-peptide. In peripheral venous blood, maximal levels of the three peptides were observed later (2-5 min), and the increase in insulin relative toproinsulin and C-peptide was not as great. At the time of peak secretion, portal vein insulin and C-peptide approached equimolar concentrations, and proinsulin, as measured against an insulin standard, comprised approximately 2.5% of the total immunoreactive insulin. After stimulation by glucose or arginine, portal insulin, proinsulin and C-peptide levels were not correlated with the concentrations measured in simultaneously drawn peripheral samples. At all sampling times, however, significant correlation was found between insulin and C-peptide in both peripheral and portal blood. The results indicate that under the conditions studied, insulin and C-peptide are secreted in equimolar concentrations in man, and that proinsulin is secreted in the same proportion to insulin as found in the pancreas. Consideration of the relative secretory and metabolic rates of the three beta cell peptides explains their peripheral concentrations. The data further support the use of plasma C-peptide as an indicator of beta cell secretory function.

In mammalian tissues the C-terminal amide structure has been found to occur only in neuroactive or hormonally-active peptides. About half known neuropeptide and peptide hormones have this unique chemical feature. Using a chemical detection method, a search for previously unknown peptides that possess the C-terminal amide structure in extracts of brain and intestine was carried out and a number of novel neuropeptides and hormonal peptides, designated neuropeptide Y, PHI, peptide YY, galanin and neuropeptide K were isolated. We recently performed a similar search in porcine pancreas and found a high concentration of a peptide having a glycine amide at its C-terminus. Here we report the isolation, primary structure and biological activity of this novel peptide. The 49-residue peptide strongly inhibits glucose-induced insulin release from the isolated perfused pancreas and was therefore named pancreastatin. It may be important in the regulation of insulin secretion and in the pathogenesis and treatment of diabetes mellitus.

Epidemiological, clinical and experimental evidence suggests a link between type 2 diabetes and Alzheimer's disease (AD). Insulin modulates metabolism of beta-amyloid precursor protein (APP) in neurons, decreasing the intracellular accumulation of beta-amyloid (Abeta) peptides, which are pivotal in AD pathogenesis. The present study investigates whether the widely prescribed insulin-sensitizing drug, metformin (Glucophage(R)), affects APP metabolism and Abeta generation in various cell models. We demonstrate that metformin, at doses that lead to activation of the AMP-activated protein kinase (AMPK), significantly increases the generation of both intracellular and extracellular Abeta species. Furthermore, the effect of metformin on Abeta generation is mediated by transcriptional up-regulation of beta-secretase (BACE1), which results in an elevated protein level and increased enzymatic activity. Unlike insulin, metformin exerts no effect on Abeta degradation. In addition, we found that glucose deprivation and various tyrphostins, known inhibitors of insulin-like growth factors/insulin receptor tyrosine kinases, do not modulate the effect of metformin on Abeta. Finally, inhibition of AMP-activated protein kinase (AMPK) by the pharmacological inhibitor Compound C largely suppresses metformin's effect on Abeta generation and BACE1 transcription, suggesting an AMPK-dependent mechanism. Although insulin and metformin display opposing effects on Abeta generation, in combined use, metformin enhances insulin's effect in reducing Abeta levels. Our findings suggest a potentially harmful consequence of this widely prescribed antidiabetic drug when used as a monotherapy in elderly diabetic patients.

The Arabidopsis genes FT and TERMINAL FLOWER1 (TFL1) encode related proteins with similarity to human Raf kinase inhibitor protein. FT, and likely also TFL1, is recruited to the promoters of floral genes through interaction with FD, a bZIP transcription factor. FT, however, induces flowering, while TFL1 represses flowering. Residues responsible for the opposite activities of FT and TFL1 were mapped by examining plants that overexpress chimeric proteins. A region important in vivo localizes to a 14-amino-acid segment that evolves very rapidly in TFL1 orthologs, but is almost invariant in FT orthologs. Crystal structures show that this segment forms an external loop of variable conformation. The only residue unambiguously distinguishing the FT and TFL1 loops makes a hydrogen bond with a residue near the entrance of a potential ligand-binding pocket in TFL1, but not in FT. This pocket is contacted by a C-terminal peptide, which also contributes to the opposite FT and TFL1 activities. In combination, these results identify a molecular surface likely to be recognized by FT- and/or TFL1-specific interactors.

Clinical islet transplantation is gaining acceptance as a potential therapy, particularly for subjects who have labile diabetes or problems with hypoglycemic awareness. The risks of the procedure and long-term outcomes are still not fully known. We have performed 54 islet transplantation procedures on 30 subjects and have detailed follow-up in 17 consecutive Edmonton protocol-treated subjects who attained insulin independence after transplantation of adequate numbers of islets. Subjects were assessed pretransplant and followed prospectively posttransplant for immediate and long-term complications related to the procedure or immunosuppressive therapy. The 17 patients all became insulin independent after a minimum of 9,000 islets/kg were transplanted. Of 15 consecutive patients with at least 1 year of follow-up after the initial transplant, 12 (80%) were insulin independent at 1 year. In 14 subjects who have maintained demonstrable C-peptide secretion, glucose control has been stable and glycemic lability and problems with hypoglycemic reactions have been corrected. After 2 of the 54 procedures, some thrombosis was detected in the portal vein circulation. Five subjects had bleeding related to the percutaneous portal vein access procedures: three required transfusion alone, and in one subject, who had a partial thrombosis of the portal vein, an expanding intrahepatic and subscapular hemorrhage occurred while on anticoagulation, requiring transfusion and surgery. Elevated liver function test results were found in 46% of subjects but resolved in all. Complications related to the therapy have been hypercholesterolemia requiring statin therapy in 65%; a rise in creatinine in two patients, both of whom had preexisting renal disease; a rise in protein in four, all of whom had preexisting proteinuria; and antihypertensive therapy increased or started in 53%. Three of the 17 patients have required retinal laser photocoagulation. There have been no cases of posttransplant lymphoproliferative disorder or cytomegalovirus infection, and no deaths. The acute insulin response to arginine correlated better with transplanted islet mass than acute insulin response to glucose (AIR(g)) and area under the curve for insulin (AUC(i)), but the AIR(g) and AUC(i) were more closely related to glycemic control. The AUC(i) directly posttransplant was lower in those who eventually became C-peptide deficient. Our results, with a maximum follow-up of 34 months, indicate that prolonged insulin independence can be achieved after islet transplantation. There are some risks associated acutely with the procedure, and hypercholesterolemia and hypertension are treatable concerns on longer-term follow-up. All patients with persisting C-peptide secretion have had a resolution of both glycemic lability and problems with hypoglycemic reactions. Apart from the rise in serum creatinine in two subjects, no serious consequences of immunosuppressive therapy have been encountered. Islet transplantation is a reasonable option in those with severe problems with glycemic lability or hypoglycemia.

Regulation of mitochondrial outer membrane (MOM) permeability has dual importance: in normal metabolite and energy exchange between mitochondria and cytoplasm and thus in control of respiration, and in apoptosis by release of apoptogenic factors into the cytosol. However, the mechanism of this regulation, dependent on the voltage-dependent anion channel (VDAC), the major channel of MOM, remains controversial. A long-standing puzzle is that in permeabilized cells, adenine nucleotide translocase (ANT) is less accessible to cytosolic ADP than in isolated mitochondria. We solve this puzzle by finding a missing player in the regulation of MOM permeability: the cytoskeletal protein tubulin. We show that nanomolar concentrations of dimeric tubulin induce voltage-sensitive reversible closure of VDAC reconstituted into planar phospholipid membranes. Tubulin strikingly increases VDAC voltage sensitivity and at physiological salt conditions could induce VDAC closure at <10 mV transmembrane potentials. Experiments with isolated mitochondria confirm these findings. Tubulin added to isolated mitochondria decreases ADP availability to ANT, partially restoring the low MOM permeability (high apparent K(m) for ADP) found in permeabilized cells. Our findings suggest a previously unknown mechanism of regulation of mitochondrial energetics, governed by VDAC and tubulin at the mitochondria-cytosol interface. This tubulin-VDAC interaction requires tubulin anionic C-terminal tail (CTT) peptides. The significance of this interaction may be reflected in the evolutionary conservation of length and anionic charge in CTT throughout eukaryotes, despite wide changes in the exact sequence. Additionally, tubulins that have lost significant length or anionic character are only found in cells that do not have mitochondria.

Efficient expansion of hematopoietic progenitor cells requires, at least, the simultaneous stimulation of the receptors c-kit and gp130. While c-kit is activated by SCF; gp130, in cells which do not express sufficient amounts of IL-6R, can be activated by the complex of soluble IL-6R (sIL-6R) and IL-6. The therapeutic use of IL-6/sIL-6R, however, has been hampered by the high concentrations of the sIL-6R protein required. We have designed a fusion protein of sIL-6R and IL-6, linked by a flexible peptide chain, that was expressed to high levels. On gp130 expressing cells the fusion protein turned out to be fully active at 100 to 1,000-fold lower concentration than the combination of unlinked IL-6 and IL-6R. The fusion protein was used to effectively expand human hematopoietic progenitor cells ex vivo in a dose dependent fashion.

Although the Ca2+/phospholipid-dependent protein kinase, protein kinase C, has a broad substrate specificity in vitro, the enzyme appears considerably less promiscuous in vivo. To date only a handful of proteins have been identified as physiological substrates for this protein kinase. In order to determine the basis for this selectivity for substrates in intact cells, we have probed the substrate primary sequence requirements of protein kinase C using synthetic peptides corresponding to sites of phosphorylation from four of the known physiological substrates. We have also identified the acetylated N-terminal serine of chick muscle lactate dehydrogenase as an in vitro site of phosphorylation for this protein kinase. These comparative studies have demonstrated that, in vivo, the enzyme exhibits a preference for one basic residue C-terminal to the phosphorylatable residue, as in the sequence: Ser/Thr-Xaa-Lys/Arg, where Xaa is usually an uncharged residue. Additional basic residues, both N and C-terminal to the target amino acid, enhance the Vmax and Km parameters of phosphorylation. None of the peptides based on physiological phosphorylation sites of protein kinase C was an efficient substrate of cAMP-dependent protein kinase, emphasizing the distinct site-recognition selectivities of these two pleiotropic protein kinases. The favorable kinetic parameters of several of the synthetic peptides, coupled with their selectivity for phosphorylation by protein kinase C, will facilitate the assay of this enzyme in the presence of other protein kinases in tissue and cell extracts.

The tyrosine kinase activity of c-Src is stimulated during mitosis by dephosphorylation of its regulatory tyrosine residue. This is associated with increased accessibility of its Src homology-2 (SH2) domain for binding a phosphotyrosine-containing peptide. But physiological targets of activated c-Src in mitosis have not yet been identified. Here we report that a 68K protein (p68) becomes tyrosine-phosphorylated and physically associates with Src during mitosis in mouse fibroblasts. p68 independently binds the Src SH2 and SH3 domains in vitro and both domains are required for p68 phosphorylation and binding in vivo. p68 is closely related to the p62 protein that is associated with the Ras GTPase-activating protein (GAP) and selectively binds, directly or indirectly, polyribonucleotides. Because the Src SH3 domain also binds heterogeneous nuclear ribonucleoprotein K, these results raise the intriguing possibility that c-Src may regulate the processing, trafficking or translation of RNA in a cell-cycle-dependent manner.

Phospholipase C-gamma (PLC-gamma) is a substrate of the fibroblast growth factor receptor (FGFR; encoded by the flg gene) and other receptors with tyrosine kinase activity. It has been demonstrated that the src homology region 2 (SH2 domain) of PLC-gamma and of other signalling molecules such as GTPase-activating protein and phosphatidylinositol 3-kinase-associated p85 direct their binding toward tyrosine-autophosphorylated regions of the epidermal growth factor or platelet-derived growth factor receptor. In this report, we describe the identification of Tyr-766 as an autophosphorylation site of flg-encoded FGFR by direct sequencing of a tyrosine-phosphorylated tryptic peptide isolated from the cytoplasmic domain of FGFR expressed in Escherichia coli. The same phosphopeptide was found in wild-type FGFR phosphorylated either in vitro or in living cells. Like other growth factor receptors, tyrosine-phosphorylated wild-type FGFR or its cytoplasmic domain becomes associated with intact PLC-gamma or with a fusion protein containing the SH2 domain of PLC-gamma. To delineate the site of association, we have examined the capacity of a 28-amino-acid tryptic peptide containing phosphorylated Tyr-766 to bind to various constructs containing SH2 and other domains of PLC-gamma. It is demonstrated that the tyrosine-phosphorylated peptide binds specifically to the SH2 domain but not to the SH3 domain or other regions of PLC-gamma. Hence, Tyr-766 and its flanking sequences represent a major binding site in FGFR for PLC-gamma. Alignment of the amino acid sequences surrounding Tyr-766 with corresponding regions of other FGFRs revealed conserved tyrosine residues in all known members of the FGFR family. We propose that homologous tyrosine-phosphorylated regions in other FGFRs also function as binding sites for PLC-gamma and therefore are involved in coupling to phosphatidylinositol breakdown.

BACKGROUND

The genome of Mycobacterium tuberculosis H37Rv has five copies of a cluster of genes known as the ESAT-6 loci. These clusters contain members of the CFP-10 (lhp) and ESAT-6 (esat-6) gene families (encoding secreted T-cell antigens that lack detectable secretion signals) as well as genes encoding secreted, cell-wall-associated subtilisin-like serine proteases, putative ABC transporters, ATP-binding proteins and other membrane-associated proteins. These membrane-associated and energy-providing proteins may function to secrete members of the ESAT-6 and CFP-10 protein families, and the proteases may be involved in processing the secreted peptide.

RESULTS

Finished and unfinished genome sequencing data of 98 publicly available microbial genomes has been analyzed for the presence of orthologs of the ESAT-6 loci. The multiple duplicates of the ESAT-6 gene cluster found in the genome of M. tuberculosis H37Rv are also conserved in the genomes of other mycobacteria, for example M. tuberculosis CDC1551, M. tuberculosis 210, M. bovis, M. leprae, M. avium, and the avirulent strain M. smegmatis. Phylogenetic analyses of the resulting sequences have established the duplication order of the gene clusters and demonstrated that the gene cluster known as region 4 (Rv3444c-3450c) is ancestral. Region 4 is also the only region for which an ortholog could be found in the genomes of Corynebacterium diphtheriae and Streptomyces coelicolor.

CONCLUSIONS

Comparative genomic analysis revealed that the presence of the ESAT-6 gene cluster is a feature of some high-G+C Gram-positive bacteria. Multiple duplications of this cluster have occurred and are maintained only within the genomes of members of the genus Mycobacterium.

AKT was originally identified as a proto-oncogene with a pleckstrin homology and Ser/Thr protein kinase domains. Recent studies revealed that AKT regulates a variety of cellular functions including cell survival, cell growth, cell differentiation, cell cycle progression, transcription, translation, and cellular metabolism. To clarify the substrate specificity of AKT, we have used an oriented peptide library approach to determine optimal amino acids at positions N-terminal and C-terminal to the site of phosphorylation. The predicted optimal peptide substrate (Arg-Lys-Arg-Xaa-Arg-Thr-Tyr-Ser*-Phe-Gly where Ser* is the phosphorylation site) has similarities to but is distinct from optimal substrates that we previously defined for related basophilic protein kinases such as protein kinase A, Ser/Arg-rich kinases, and protein kinase C family members. The positions most important for high V(max)/K(m) ratio were Arg-3>Arg-5>Arg-7. The substrate specificity of AKT was further investigated by screening a lambdaGEX phage HeLa cell cDNA expression library. All of the substrates identified by this procedure contained Arg-Xaa-Arg-Xaa-Xaa-(Ser/Thr) motifs and were in close agreement with the motif identified by peptide library screening. The results of this study should help in prediction of likely AKT substrates from primary sequences.

The A4 or beta-peptide (39 to 43 amino acid residues) is the principal proteinaceous component of amyloid deposits in Alzheimer's disease. Using circular dichroism (c.d.), we have studied the secondary structures and aggregational properties in solution of 4 synthetic amyloid beta-peptides: beta-(1-28), beta-(1-39), beta-(1-42) and beta-(29-42). The natural components of cerebrovascular deposits and extracellular amyloid plaques are beta-(1-39) and beta-(1-42), while beta-(1-28) and beta-(29-42) are unnatural fragments. The beta-(1-28), beta-(1-39) and beta-(1-42) peptides adopt mixtures of beta-sheet, alpha-helix and random coil structures, with the relative proportions of each secondary structure being strongly dependent upon the solution conditions. In aqueous solution, beta-sheet structure is favored for the beta-(1-39) and beta-(1-42) peptides, while in aqueous solution containing trifluoroethanol (TFE) or hexafluoroisopropanol (HFIP), alpha-helical structure is favored for all 3 peptides. The alpha-helical structure unfolds with increasing temperature and is favored at pH 1 to 4 and pH 7 to 10; the beta-sheet conformation is temperature insensitive and is favored at pH 4 to 7. Peptide concentration studies showed that the beta-sheet conformation is oligomeric (intermolecular), whereas the alpha-helical conformation is monomeric (intramolecular). The rate of aggregation to the oligomeric beta-sheet structure (alpha-helix----random coil----beta-sheet) is also dependent upon the solution conditions such as the pH and peptide concentration; maximum beta-sheet formation occurs at pH 5.4. These results suggest that beta-peptide is not an intrinsically insoluble peptide. Thus, solution abnormalities, together with localized high peptide concentrations, which may occur in Alzheimer's disease, may contribute to the formation of amyloid plaques. The hydrophobic beta-(29-42) peptide adopts exclusively an intermolecular beta-sheet conformation in aqueous solution despite changes in temperature or pH. Therefore, this segment may be the first region of the beta-peptide to aggregate and may direct the folding of the complete beta-peptide to produce the beta-pleated sheet structure found in amyloid deposits. Differences between the solution conformations of the beta-(1-39) and beta-(1-42) peptides suggests that the last 3 C-terminal amino acids are crucial to amyloid deposition.