Discordant effects of a chronic physiological increase in plasma FFA on insulin signaling in healthy subjects with or without a family history of type 2 diabetes

Muscle insulin resistance develops when plasma free fatty acids (FFAs) are acutely increased to supraphysiological levels (approximately 1,500-4,000 micromol/l). However, plasma FFA levels >1,000 micromol/l are rarely observed in humans under usual living conditions, and it is unknown whether insulin action may be impaired during a sustained but physiological FFA increase to levels seen in obesity and type 2 diabetes mellitus (T2DM) (approximately 600-800 micromol/l). It is also unclear whether normal glucose-tolerant subjects with a strong family history of T2DM (FH+) would respond to a low-dose lipid infusion as individuals without any family history of T2DM (CON). To examine these questions, we studied 7 FH+ and 10 CON subjects in whom we infused saline (SAL) or low-dose Liposyn (LIP) for 4 days. On day 4, a euglycemic insulin clamp with [3-3H]glucose and indirect calorimetry was performed to assess glucose turnover, combined with vastus lateralis muscle biopsies to examine insulin signaling. LIP increased plasma FFA approximately 1.5-fold, to levels seen in T2DM. Compared with CON, FH+ were markedly insulin resistant and had severely impaired insulin signaling in response to insulin stimulation. LIP in CON reduced insulin-stimulated glucose disposal (Rd) by 25%, insulin-stimulated insulin receptor tyrosine phosphorylation by 17%, phosphatidylinositol 3-kinase activity associated with insulin receptor substrate-1 by 20%, and insulin-stimulated glycogen synthase fractional velocity over baseline (44 vs. 15%; all P < 0.05). In contrast to CON, a physiological elevation in plasma FFA in FH+ led to no further deterioration in Rd or to any additional impairment of insulin signaling. In conclusion, a 4-day physiological increase in plasma FFA to levels seen in obesity and T2DM impairs insulin action/insulin signaling in CON but does not worsen insulin resistance in FH+. Whether this lack of additional deterioration in insulin signaling in FH+ is due to already well-established lipotoxicity, or to other molecular mechanisms related to insulin resistance that are nearly maximally expressed early in life, remains to be determined.     

Sorption of heavy metals from electroplating effluent using immobilized biomass Trichoderma viride in a continuous packed-bed column

The sorption of heavy metals ions by immobilized Trichoderma viride biomass in a packed-bed column was studied. Fungal biomass T. viride was immobilized to Ca-alginate used for removal of Cr(VI), Ni(II) and Zn(II) ions from synthetic solutions and electroplating effluent. The experiments were conducted to study the effect of important design parameters such as bed height, flow rate and initial concentration of metal ions. The maximum sorption capacity was observed at flow rate 5 ml/min, bed height 20 cm and metal ions concentration 50 mg/L with immobilized biomass. Whereas, breakthrough time and saturation time decreased with increase flow rate and metal ions concentration and an inverse condition was found in bed height. The bed depth service time (BDST) Adams-Bohart model was used to analyze the experimental data. The regeneration efficiency was observed 40.1%, 75% and 53% for Cr(VI), Ni(II) and Zn(II) without any significant alteration in sorption capacity after 5th sorption-desorption cycles.     

Reversal of 5-fluorodeoxyuridine-caused growth inhibition in intact and excised etiolated hypocotyls of Sinapis alba L. by thymidine

Summary The elongation growth in etiolated, intact seedlings and excised hypocotyl segments of Sinapis alba is inhibited by FdUrd in the same fashion, and in either case there is a direct correlation between FdUrd concentration and inhibition of elongation growth. Removal of the roots reduced elongation; however, the percentage inhibition by FdUrd remained the same. Therefore, the growth inhibition by FdUrd is not a consequence of root growth inhibition.The growth inhibition in excised hypocotyls cultured on synthetic media is inversely proportional to the size of the segments, and of the seedlings from which they are taken. Elongation of the smaller segments is more sensitive to FdUrd than that of the larger ones. Anatomical observations showed that the inhibition of growth elongation by FdUrd in the hypocotyl segments is due to inhibition of cell elongation, and not of cell division. Root formation is inhibited in all isolated segments.The growth inhibition by FdUrd could be reversed by dThd but not by uridine, and this reversibility depended upon the FdUrd concentration. When FdUrd inhibition is partial (up to 10^-7M) relatively high dThd concentration (up to 100 fold) are required for complete reversal; when inhibition is maximal relatively lower dThd concentrations effect a complete or near-complete reversal. Irreversible, unspecific effects of FdUrd were not found.These experiments confirm that DNA synthesis is involved in cell elongation of the hypocotyls even when the apical meristem and roots are removed, and that the growth inhibition by FdUrd is not a nonspecific, toxic effect.Abbreviations FdUrd 5-fluorodeoxyuridine - dThd thymidine     

Salmonella typhimurium secreted invasion determinants are homologous to Shigella lpa proteins

Salmonella typhimurium secreted proteins (Ssp) were previously implicated in epithelial cell invasion. Here we describe four genes ( sspB , sspC , sspD , and sspA ), located between spaT and prgH , which encode proteins of 63, 42, 36, and 87 kDa, respectively. These Ssp are homologous to Shigella flexneri secreted proteins lpaB, lpaC, lpaD and lpaA. A non-invasive mutant with a transposon insertion in sspC lacks Ssp of 87,42 and 36 kDa. Complementation analyses show that sspC and sspD encode the 42 and the 36 kDa Ssp, while the 87 kDa Ssp is encoded by sspA . sspC and sspD , but not sspA are required for invasion. Amino-terminal sequencing shows that SspC and SspA are secreted without amino-terminal processing. We further demonstrate that Ssp secretion requires proteins encoded by prgHIJK , homologous to the Shigella lpa secretion system, since SspA is abundantly secreted by wild-type bacteria but is completely retained within the cellular fraction of a prgHIJK mutant. A precipitate containing abundant SspC and three other major Ssp of 63,59 and 22 kDa was isolated from culture supernatants of wild-type bacteria. These data indicate that major secreted invasion determinants of S. typhimurium are structurally and functionally homolgous to S. flexneri lpa proteins.     

Free fatty acid-induced peripheral insulin resistance augments splanchnic glucose uptake in healthy humans

To investigate the effect of elevated plasma free fatty acid (FFA) concentrations on splanchnic glucose uptake (SGU), we measured SGU in nine healthy subjects (age, 44 +/- 4 yr; body mass index, 27.4 +/- 1.2 kg/m(2); fasting plasma glucose, 5.2 +/- 0.1 mmol/l) during an Intralipid-heparin (LIP) infusion and during a saline (Sal) infusion. SGU was estimated by the oral glucose load (OGL)-insulin clamp method: subjects received a 7-h euglycemic insulin (100 mU x m(-2) x min(-1)) clamp, and a 75-g OGL was ingested 3 h after the insulin clamp was started. After glucose ingestion, the steady-state glucose infusion rate (GIR) during the insulin clamp was decreased to maintain euglycemia. SGU was calculated by subtracting the integrated decrease in GIR during the period after glucose ingestion from the ingested glucose load. [3-(3)H]glucose was infused during the initial 3 h of the insulin clamp to determine rates of endogenous glucose production (EGP) and glucose disappearance (R(d)). During the 3-h euglycemic insulin clamp before glucose ingestion, R(d) was decreased (8.8 +/- 0.5 vs. 7.6 +/- 0.5 mg x kg(-1) x min(-1), P < 0.01), and suppression of EGP was impaired (0.2 +/- 0.04 vs. 0.07 +/- 0.03 mg x kg(-1) x min(-1), P < 0.01). During the 4-h period after glucose ingestion, SGU was significantly increased during the LIP vs. Sal infusion study (30 +/- 2 vs. 20 +/- 2%, P < 0.005). In conclusion, an elevation in plasma FFA concentration impairs whole body glucose R(d) and insulin-mediated suppression of EGP in healthy subjects but augments SGU.     

C-5 substituted heteroaryl-3-pyridinecarbonitriles as PKCθ inhibitors: Part II

We previously reported that a 3-pyridinecarbonitrile analog with a furan substituent at C-5 and a 4-methylindol-5-ylamino substituent at C-4, 1, was a potent inhibitor of PKCθ (IC₅₀ = 4.5 nM). Replacement of the C-5 furan ring of 1 with bicyclic heteroaryl rings, led to compounds with significantly improved potency against PKCθ. Analog 6b with a 4-methylindol-5-ylamino group at C-4 and a 5-[(4-methylpiperazin-1-yl)methyl]-1-benzofuran-2-yl group at C-5 had an IC₅₀ value of 0.28 nM for the inhibition of PKCθ.     

An antibody against SSEA-5 glycan on human pluripotent stem cells enables removal of teratoma-forming cells

An important risk in the clinical application of human pluripotent stem cells (hPSCs), including human embryonic and induced pluripotent stem cells (hESCs and hiPSCs), is teratoma formation by residual undifferentiated cells. We raised a monoclonal antibody against hESCs, designated anti-stage-specific embryonic antigen (SSEA)-5, which binds a previously unidentified antigen highly and specifically expressed on hPSCs--the H type-1 glycan. Separation based on SSEA-5 expression through fluorescence-activated cell sorting (FACS) greatly reduced teratoma-formation potential of heterogeneously differentiated cultures. To ensure complete removal of teratoma-forming cells, we identified additional pluripotency surface markers (PSMs) exhibiting a large dynamic expression range during differentiation: CD9, CD30, CD50, CD90 and CD200. Immunohistochemistry studies of human fetal tissues and bioinformatics analysis of a microarray database revealed that concurrent expression of these markers is both common and specific to hPSCs. Immunodepletion with antibodies against SSEA-5 and two additional PSMs completely removed teratoma-formation potential from incompletely differentiated hESC cultures.     

Protein model discrimination using mutational sensitivity derived from deep sequencing

1. Download : Download high-res image (318KB)
2. Download : Download full-size image

Highlights? Individual mutant phenotypes in a saturation library derived via deep sequencing ? Residue depth and active-site residue derivation from mutant phenotypes ? Mutant-phenotype-derived parameters used for protein model discrimination ? Derivation of sequence-structure-function relationships without protein isolation     

Full engagement of liganded maltose‐binding protein stabilizes a semi‐open ATP‐binding cassette dimer in the maltose transporter

MalFGK₂ is an ATP‐binding cassette (ABC) transporter that mediates the uptake of maltose/maltodextrins into Escherichia coli. A periplasmic maltose‐binding protein (MBP) delivers maltose to the transmembrane subunits (MalFG) and stimulates the ATPase activity of the cytoplasmic nucleotide‐binding subunits (MalK dimer). This MBP‐stimulated ATPase activity is independent of maltose for purified transporter in detergent micelles. However, when the transporter is reconstituted in membrane bilayers, only the liganded form of MBP efficiently stimulates its activity. To investigate the mechanism of maltose stimulation, electron paramagnetic resonance spectroscopy was used to study the interactions between the transporter and MBP in nanodiscs and in detergent. We found that full engagement of both lobes of maltose‐bound MBP unto MalFGK₂ is facilitated by nucleotides and stabilizes a semi‐open MalK dimer. Maltose‐bound MBP promotes the transition to the semi‐open state of MalK when the transporter is in the membrane, whereas such regulation does not require maltose in detergent. We suggest that stabilization of the semi‐open MalK₂ conformation by maltose‐bound MBP is key to the coupling of maltose transport to ATP hydrolysis in vivo, because it facilitates the progression of the MalK dimer from the open to the semi‐open conformation, from which it can proceed to hydrolyze ATP.     

Conformational behavior of polypeptides derived through simultaneous global conservative site-directed mutagenesis of chymotrypsin inhibitor 2

The natural occurrence of conservative residue substitutions in proteins suggests that side-chain packing schemes in protein interiors can accommodate mutational replacements of residues by others of similar nature. To explore the extent to which such substitutions are tolerated, especially when introduced simultaneously and globally over the entire length of a polypeptide chain, we examined the conformational behavior of a model 65 residues-long protein, wild-type chymotrypsin inhibitor 2 (WTCI2), and two globally-mutated (GM) variants named GMCI2-1 and GMCI2-2, each incorporating 55 conservative residue substitutions. GMCI2-1, was soluble over a wide range of pH, and folded into a compact, spherical, monomer marked by (i) complete absence of surface hydrophobicity, (ii) a WTCI2-like betaII-type CD spectrum, (iii) high WTCI2-like thermal stability, and (d) 1D and 2D NMR spectra characteristic of folded protein structure. GMCI2-2 was insoluble over a wide range of pH, and could be solubilized only at pH 4.0, showing non-WTCI2-like far-UV CD spectra characterized by high helical content. These results tentatively indicate that polypeptides incorporating residues of identical nature at equivalent chain locations can show the potential to fold with similar characteristics. However, further detailed investigations would be required to determine whether indeed the structural fold of GMCI2-1 resembles that of WTCI2, and to evaluate the extent to which it does so.