Effects of pioglitazone and metformin on beta-cell function in nondiabetic subjects at high risk for type 2 diabetes

Thiazolidinediones (TZDs) and metformin decreased the incidence of diabetes in subjects at risk for developing diabetes and improved peripheral or hepatic insulin sensitivity, respectively. Whether they also directly improved beta-cell function is not clear. In vitro studies showed improved beta-cell function in response to TZDs and metformin; however, the effects of TZDs or metformin on beta-cell function in humans are still uncertain. We hypothesized that both TZDs and metformin directly affect beta-cell function. We evaluated beta-cell function and insulin sensitivity (S(I)) in subjects with impaired glucose tolerance or a history of gestational diabetes using oral and intravenous glucose tolerance tests in addition to the glucose-potentiated arginine stimulation test. In contrast to metformin, pioglitazone improved S(I), glucose tolerance, and insulin-independent glucose disposal [glucose effectiveness (S(G))]. Neither pioglitazone nor metformin significantly improved beta-cell compensation for insulin resistance [disposition index (DI)], but the change in DI significantly correlated with baseline S(I). Insulin secretion in response to arginine at maximally potentiating glucose levels (AIR(max)) tended to increase after metformin and to decrease after pioglitazone; however, when adjusted for S(I), the changes were not significant. Our results demonstrate that, in nondiabetic subjects at risk for diabetes, pioglitazone, but not metformin, significantly improved glucose tolerance by improving S(I) and S(G). We did not find any evidence that either pioglitazone or metformin improved beta-cell function. Improved beta-cell compensation was observed primarily in the subgroup of subjects that had the lowest S(I) at baseline.     

Cloning, expression and characterization of the pig liver GDP-mannose pyrophosphorylase. Evidence that GDP-mannose and GDP-Glc pyrophosphorylases are different proteins.

GDP-Man, the mannosyl donor for most Man-containing polymers is formed by the transfer of Man-1-P to GTP to form GDP-Man and PPi. This reaction is catalyzed by the widespread and essential enzyme, GDP-Man pyrophosphorylase (GMPP). The pig liver GMPP consists of an alpha subunit (43 kDa) and a beta subunit (37 kDa). Purified pig GMPP catalyzes the synthesis of GDP-Glc (from Glc-1-P and GTP) and GDP-Man (from Man-1-P and GTP), but has higher activity for the formation of GDP-Glc than for synthesis of GDP-Man. In the present study, we report the cloning of the cDNA for the beta subunit of GMPP, and its expression in a bacterial system resulting in the formation of active enzyme. The full length cDNA encoding the beta subunit was isolated from a porcine cDNA library, and its predicted gene product showed high amino-acid sequence homology to GMPPs from other species. The gene was expressed in Escherichia coli cells, and a 37-kDa protein was over-produced in these cells. This gene product reacted strongly with antibody reactive to the native beta subunit of pig GMPP. Most interestingly, this recombinant protein had high activity for synthesizing GDP-Man (from Man-1-P and GTP), but very low activity for the formation of GDP-Glc (from Glc-1-P and GTP). Other properties of the recombinant protein were also analyzed. This study suggests that the beta subunit is the GMPP, whereas the alpha subunit, or a combination of both subunits, may have the GDP-Glc pyrophosphorylase activity.     

Effects of sex chromosome dosage on corpus callosum morphology in supernumerary sex chromosome aneuploidies

Background

Supernumerary sex chromosome aneuploidies (sSCA) are characterized by the presence of one or more additional sex chromosomes in an individual’s karyotype; they affect around 1 in 400 individuals. Although there is high variability, each sSCA subtype has a characteristic set of cognitive and physical phenotypes. Here, we investigated the differences in the morphometry of the human corpus callosum (CC) between sex-matched controls 46,XY (N =99), 46,XX (N =93), and six unique sSCA karyotypes: 47,XYY (N =29), 47,XXY (N =58), 48,XXYY (N =20), 47,XXX (N =30), 48,XXXY (N =5), and 49,XXXXY (N =6).

Methods

We investigated CC morphometry using local and global area, local curvature of the CC boundary, and between-landmark distance analysis (BLDA). We hypothesized that CC morphometry would vary differentially along a proposed spectrum of Y:X chromosome ratio with supernumerary Y karyotypes having the largest CC areas and supernumerary X karyotypes having significantly smaller CC areas. To investigate this, we defined an sSCA spectrum based on a descending Y:X karyotype ratio: 47,XYY, 46,XY, 48,XXYY, 47,XXY, 48,XXXY, 49,XXXXY, 46,XX, 47,XXX. We similarly explored the effects of both X and Y chromosome numbers within sex. Results of shape-based metrics were analyzed using permutation tests consisting of 5,000 iterations.

Results

Several subregional areas, local curvature, and BLDs differed between groups.Moderate associations were found between area and curvature in relation to the spectrum and X and Y chromosome counts. BLD was strongly associated with X chromosome count in both male and female groups.

Conclusions

Our results suggest that X- and Y-linked genes have differential effects on CC morphometry. To our knowledge, this is the first study to compare CC morphometry across these extremely rare groups.

     

The effect of tsushimycin on the synthesis of lipid-linked saccharides in aorta.

The antibiotic, tsushimycin, inhibits the formation of dolichyl phosphate mannose, dolichyl phosphate glucose and dolichyl pyrophosphate N-acetylglucosamine in the particulate enzyme preparation from pig aorta. Although this antibiotic also inhibits the incorporation of mannose and glucose into lipid-linked oligosaccharides, these reactions are less sensitive to antibiotic than those involved in the synthesis of lipid-linked monosaccharides. In the presence of tsushimycin, most of the mannose incorporated into lipid-linked oligosaccharides is into one oligosaccharide that has the properties of the heptasaccharide Man5GlcNAc2, whereas in the absence of antibiotic most of the mannose is in larger-sized oligosaccharides. On the other hand, the glucose-labelled lipid-linked oligosaccharides appear to be similar in size in the presence or absence of antibiotic. Tsushimycin also inhibits the formation of lipid-linked monosaccharides by the solubilized enzyme preparation of aorta. Various concentrations of dolichyl phosphate or the detergent, Nonidet P40, had no effect on antibiotic inhibition. Some evidence indicates that tsushimycin binds to the particulate enzyme.     

Purification, crystallization, and properties of D-ribose isomerase from Mycobacterium smegmatis.

D-Ribose isomerase, which catalyzes the conversion of D-ribose to D-ribulose, was purified from extracts of Mycobacterium smegmatis grown on D-ribose. The purified enzyme crystalized as hexagonal plates from a 44% solution of ammonium sulfate. The enzyme was homogenous by disc gel electrophoresis and ultracentrifugal analysis. The molecular weight of the enzyme was between 145,000 and 174,000 by sedimentation equilibrium analysis. Its sedimentation constant of 8.7 S indicates it is globular. On the basis of sodium dodecyl sulfate gel electrophoresis in the presence of Mn2+, the enzyme is probably composed of 4 identical subunits of molecular weight about 42,000 to 44,000. The enzyme was specific for sugars having the same configuration as D-ribose at carbon atoms 1 to 3. Thus, the enzyme could also utilize L-lyxose, D-allose, and L-rhamnose as substrates. The Km for D-ribose was 4 mM and for L-lyxose it was 5.3 mM. The enzyme required a divalent cation for activity with optimum activity being shown with Mn2+. the Km for the various cations was as follows: Mn2+, 1 times 10(-7) M, Co2+, 4 times 10(-7) M, and Mg2+, 1.8 times 10(-5) M. The pH optimum for the enzyme was 7.5 to 8.5. Polyols did not inhibit the enzyme to any great extent. The product of the reaction was identified as D-ribulose by thin layer chromatography and by preparation of the O-nitrophenylhydrazone derivative.     

Trehalose-phosphate synthase of Mycobacterium tuberculosis. Cloning, expression and properties of the recombinant enzyme.

The trehalose-phosphate synthase (TPS) of Mycobacterium smegmatis was previously purified to apparent homogeneity and several peptides from the 58 kDa protein were sequenced. Based on that sequence information, the gene for TPS was identified in the Mycobacterium tuberculosis genome, and the gene was cloned and expressed in Escherichia coli with a (His)6 tag at the amino terminus. The TPS was expressed in good yield and as active enzyme, and was purified on a metal ion column to give a single band of approximately 58 kDa on SDS/PAGE. Approximately 1.3 mg of purified TPS were obtained from a 1-L culture of E. coli ( approximately 2.3 g cell paste). The purified recombinant enzyme showed a single band of approximately 58 kDa on SDS/PAGE, but a molecular mass of approximately 220 kDa by gel filtration, indicating that the active TPS is probably a tetrameric protein. Like the enzyme originally purified from M. smegmatis, the recombinant enzyme is an unusual glycosyltransferase as it can utilize any of the nucleoside diphosphate glucose derivatives as glucosyl donors, i.e. ADP-glucose, CDP-glucose, GDP-glucose, TDP-glucose and UDP-glucose, with ADP-glucose, GDP-glucose and UDP-glucose being the preferred substrates. These studies prove conclusively that the mycobacterial TPS is indeed responsible for catalyzing the synthesis of trehalose-P from any of the nucleoside diphosphate glucose derivatives. Although the original enzyme from M. smegmatis was greatly stimulated in its utilization of UDP-glucose by polyanions such as heparin, the recombinant enzyme was stimulated only modestly by heparin. The Km for UDP-glucose as the glucosyl donor was approximately 18 mm, and that for GDP-glucose was approximately 16 mm. The enzyme was specific for glucose-6-P as the glucosyl acceptor, and the Km for this substrate was approximately 7 mm when UDP-glucose was the glucosyl donor and approximately 4 mm with GDP-glucose. TPS did not show an absolute requirement for divalent cations, but activity was increased about twofold by 10 mm Mn2+. This recombinant system will be useful for obtaining sufficient amounts of protein for structural studies. TPS should be a valuable target site for chemotherapeutic intervention in tuberculosis.     

Osteoclast cell-surface changes during the egg-laying cycle in Japanese quail

The medullary bone serves as a source of labile calcium mobilized during calcification of the egg shell in birds. Quantitative histological methods demonstrate that the numbers of medullary bone osteoclasts and nuclei per osteoclast remain unchanged during the egg cycle in the Japanese quail (Coturnix). Therefore, cyclic changes in bone resorption cannot be explained by modulations of osteoclasts from and into other bone cells, a mechanism previously suggested for certain species of birds. Rather, dramatic changes in osteoclast cell-surface features occur during the egg cycle, which might account for cyclic variations in resorptive activity. During egg shell calcification, osteoclasts with ruffled borders are closely apposed to bone surfaces; the cytoplasm is rich in vacuoles that contain mineral crystals and seem to derive from the ruffled border. At the completion of egg shell calcification, the ruffled borders and vacuoles move away from the bone surface, although the osteoclast remains attached to the bone along the filamentous or "clear" zone. Associated with the disappearance of the ruffled borders is the appearance of extensive interdigitated cell processes along the peripheral surface of the osteoclast away from the bone. These unusual structures, which may serve as a reservoir of membrane, largely disappear when ruffled borders and associated structures reappear. Therefore, in these hens, the osteoclasts modulate their cell surface rather than their population during the egg cycle.     

Exercise therapy for prevention of falls in people with Parkinson's disease: A protocol for a randomised controlled trial and economic evaluation

Background

People with Parkinson's disease are twice as likely to be recurrent fallers compared to other older people. As these falls have devastating consequences, there is an urgent need to identify and test innovative interventions with the potential to reduce falls in people with Parkinson's disease. The main objective of this randomised controlled trial is to determine whether fall rates can be reduced in people with Parkinson's disease using exercise targeting three potentially remediable risk factors for falls (reduced balance, reduced leg muscle strength and freezing of gait). In addition we will establish the cost effectiveness of the exercise program from the health provider's perspective.

Methods/Design

230 community-dwelling participants with idiopathic Parkinson's disease will be recruited. Eligible participants will also have a history of falls or be identified as being at risk of falls on assessment. Participants will be randomly allocated to a usual-care control group or an intervention group which will undertake weight-bearing balance and strengthening exercises and use cueing strategies to address freezing of gait. The intervention group will choose between the home-based or support group-based mode of the program. Participants in both groups will receive standardized falls prevention advice. The primary outcome measure will be fall rates. Participants will record falls and medical interventions in a diary for the duration of the 6-month intervention period. Secondary measures include the Parkinson's Disease Falls Risk Score, maximal leg muscle strength, standing balance, the Short Physical Performance Battery, freezing of gait, health and well being, habitual physical activity and positive and negative affect schedule.

Discussion

No adequately powered studies have investigated exercise interventions aimed at reducing falls in people with Parkinson's disease. This trial will determine the effectiveness of the exercise intervention in reducing falls and its cost effectiveness. This pragmatic program, if found to be effective, has the potential to be implemented within existing community services.

Trial registration

The protocol for this study is registered with the Australian New Zealand Clinical Trials Registry (ACTRN12608000303347).     

Effect of castanospermine on the structure and secretion of glycoprotein enzymes in Aspergillus fumigatus.

Aspergillus fumigatus secretes a number of glycosidases into the culture medium when the cells are grown in a mineral salts medium containing guar flour (a galactomannan) as the carbon source. At least some of these glycosidases have been reported to be glycoproteins having N-linked oligosaccharides. In this study, we examined the effect of the glycoprotein processing inhibitor, castanospermine, on the structures of the N-linked oligosaccharides and on the secretion of various glycosidases. Cells were grown in the presence of various amounts of castanospermine; at different times of growth, samples of the media were removed for the measurement of enzymatic activity. Of the three glycosidases assayed, beta-hexosaminidase was most sensitive to castanospermine; and its activity was depressed 30 to 40% at 100 micrograms of alkaloid per ml and even more at higher alkaloid concentrations. On the other hand, beta-galactosidase activity was hardly diminished at castanospermine levels of up to 1 mg/ml, but significant inhibition was observed at 2 mg/ml. beta-Galactosidase was intermediate in sensitivity. Cells were grown in the presence or absence of castanospermine and labeled with [2-3H]mannose, [6-3H]glucosamine, or [1-3H]galactose to label the sugar portion of the glycoproteins. The secreted glycoproteins were digested with pronase to obtain glycopeptides, and these were identified on Bio-Gel P-4 (Bio-Rad Laboratories). The glycopeptides were then digested with endoglucosaminidase H to release the peptide portion of susceptible structures, and the released oligosaccharides were reisolated and identified on Bio-Gel P-4. The oligosaccharides from control and castanospermine-grown cells were identified by a combination of enzymatic and chemical studies. In control cells, the oligosaccharide appeared to be mostly Man8GlcNAc and Man9GlcNAc, whereas in the presence of alkaloid, the major structures were Glc3Man7GlcNAc and Glc3Man8GlcNAc. These data fit previous observations that castanospermine inhibits glucosidase I.     

Castanospermine inhibits glucosidase I and glycoprotein secretion in human hepatoma cells.

We studied the effect of the plant alkaloid castanospermine on the biosynthesis and secretion of human hepatoma glycoproteins. The HepG-2 cells, grown in the presence or absence of the alkaloid, were labelled with [2-3H]mannose and then the labelled glycopeptides were prepared by Pronase digestion. This material was analysed by gel filtration on Bio-Gel P-4 before and after treatment with endo-beta-N-acetylglucosaminidase H. Castanospermine caused an accumulation of high-mannose oligosaccharides, by 70-75% over control. The major accumulated product, which could also be labelled with [3H]galactose and was only partially susceptible to alpha-mannosidase digestion, was identified by h.p.l.c. as a Glc3Man9GlcNAc. Thus the alkaloid inhibits glucosidase I in the human hepatoma cells. Analysis of total glycoproteins secreted by the cells into the medium revealed the presence of only complex oligosaccharides in both control and treated cultures, and the amount of the oligosaccharides labelled with radioactive mannose, galactose or N-acetylmannosamine, secreted by treated cells, was decreased by about 60%. The rate of secretion of total protein labelled with [35S]methionine and precipitated from the medium with trichloroacetic acid was inhibited by up to 40% in the presence of castanospermine. Pulse-chase studies utilizing [35S]methionine labelling were performed to study the effect of the alkaloid on secretion of individual plasma proteins. Immunoprecipitation at different chase times with monospecific antisera showed that castanospermine markedly decreased the secretion rates of alpha 1-antitrypsin, caeruloplasmin and, to a lesser extent, that of antithrombin-III. Secretions of apolipoprotein E, a glycoprotein containing only O-linked oligosaccharide(s), and albumin, a non-glycosylated protein, were not affected by the drug. It is suggested that castanospermine inhibits secretion of at least some glycoproteins containing N-linked oligosaccharides, owing to the inhibition of oligosaccharide processing.