Is Serum Zinc Associated with Pancreatic Beta Cell Function and Insulin Sensitivity in Pre-Diabetic and Normal Individuals? Findings from the Hunter Community Study

Aim

To determine if there is a difference in serum zinc concentration between normoglycaemic, pre-diabetic and type-2 diabetic groups and if this is associated with pancreatic beta cell function and insulin sensitivity in the former 2 groups.

Method

Cross sectional study of a random sample of older community-dwelling men and women in Newcastle, New South Wales, Australia. Beta cell function, insulin sensitivity and insulin resistance were calculated for normoglycaemic and prediabetes participants using the Homeostasis Model Assessment (HOMA-2) calculator.

Result

A total of 452 participants were recruited for this study. Approximately 33% (N = 149) had diabetes, 33% (N = 151) had prediabetes and 34% (N = 152) were normoglycaemic. Homeostasis Model Assessment (HOMA) parameters were found to be significantly different between normoglycaemic and prediabetes groups (p<0.001). In adjusted linear regression, higher serum zinc concentration was associated with increased insulin sensitivity (p = 0.01) in the prediabetic group. There was also a significant association between smoking and worse insulin sensitivity.

Conclusion

Higher serum zinc concentration is associated with increased insulin sensitivity. Longitudinal studies are required to determine if low serum zinc concentration plays a role in progression from pre-diabetes to diabetes.     

Multilocus Variable-Number Tandem-Repeat Analysis and Plasmid Profiling To Study the Occurrence of blaCMY-2 within a Pulsed-Field Gel Electrophoresis-Defined Clade of Salmonella enterica Serovar Typhimurium

Salmonella enterica serovar Typhimurium circulating in food animal populations and carrying resistance to antimicrobial agents represents a human health risk. Recently, a new clade of S. Typhimurium, WA-TYP035/187, was reported in cattle and humans in the Pacific Northwest, United States of America. The objective of this study was to describe a possible mechanism of acquisition of expanded-spectrum cephalosporin resistance in this clade. Ceftazidime resistance increased steadily among WA-TYP035/187 isolates, from 0% (0/2) in 1999 to 77.8% (28/36) in 2006 (χ² for linear trend, P value of <0.001). Among 112 bovine-source and 18 human-source isolates, 49 (43.8%) and 12 (66.7%) were resistant to ceftazidime, respectively. Multiple-locus variable-number tandem-repeat analysis (MLVA) and plasmid profiling suggested that resistance was acquired by multiple independent genetic events within the WA-TYP035/187 clade. Given the lack of an obvious reservoir in species other than cattle and a parallel rise in ceftiofur resistance in the bovine-specific serovar Salmonella enterica serovar Dublin in the same time frame and region, selection pressure due to the use of the expanded-spectrum cephalosporin drug ceftiofur in cattle is a likely factor driving the increasing cephalosporin resistance of WA-TYP035/187.Salmonella enterica serovar Typhimurium is one of the leading causes of food-borne bacterial disease in the United States (5). The proportion of human clinical isolates with resistance to ceftiofur, an expanded-spectrum cephalosporin, increased from 0% in 1996 to 4.5% in 2004 (5). Expanded-spectrum cephalosporin resistance in Salmonella is of particular concern because these drugs are commonly used in treatment of pediatric salmonellosis. In addition to limiting the effectiveness of antibiotics in treating multidrug-resistant (MDR) Salmonella infections, these infections may be more likely to cause prolonged or severe illnesses than are infections with antimicrobial-susceptible strains (31). Cattle are known to be a reservoir for pathogenic nontyphoidal Salmonella and most likely contribute to human cases of MDR salmonellosis (1, 24, 26). Furthermore, recent studies of Salmonella isolates from veterinary diagnostic laboratory submissions suggest that antimicrobial resistance, particularly resistance to expanded-spectrum cephalosporins, has increased among northwestern U.S. cattle isolates (11, 12).We have observed that humans and cattle frequently have infections with the same strains as defined by PFGE and that many of these shared strains have multidrug resistance, including resistance to the expanded-spectrum cephalosporin ceftazidime. One of these shared strains (designated TYP035 at the Washington State Department of Health Public Health Laboratories) emerged among cattle in the late 1990s and was subsequently detected in human infections. In 2004 a Salmonella Typhimurium pulsed-field gel electrophoresis (PFGE) type designated TYP187 was detected in cattle (and subsequently in humans); it was distinguishable from TYP035 by only one band and shared a similar resistance phenotype, and its SpeI-PFGE profile was similar to or indistinguishable from that of TYP035 (11). This cattle-associated MDR clade is referred to hereafter as WA-TYP035/187.These observations indicate that cattle contribute to MDR Salmonella infections in humans and that an understanding of the mechanisms of emergence, dissemination, or acquisition of MDR would be of public health importance. This study investigates the increasing incidence of expanded-spectrum cephalosporin resistance in WA-TYP035/187. Variable-number tandem-repeat (VNTR) loci provide polymorphic markers that are the basis of a powerful molecular technique to discriminate isolates within clonal complexes (17, 18, 29, 32). Therefore, we used a combination of multilocus VNTR analysis (MLVA) and bla_CMY-2 plasmid profiling to investigate antimicrobial resistance within the WA-TYP035/187 clade.     

Definition of the structural elements in plasminogen required for high-affinity binding to apolipoprotein(a): a study utilizing surface plasmon resonance

Lipoprotein(a) [Lp(a)] is suggested to link atherosclerosis and thrombosis owing to the similarity between the apolipoprotein(a) [apo(a)] moiety of Lp(a) and plasminogen. Lp(a) may interfere with tPA-mediated plasminogen activation in fibrinolysis, thereby generating a hypercoaguable state in vivo. The present study employed surface plasmon resonance (SPR) to examine the binding interaction between plasminogen and a physiologically relevant, 17-kringle recombinant apo(a) species [17K r-apo(a)] in real time. Native, intact Glu(1)-plasminogen bound to apo(a) with substantially higher affinity (K(D) approximately 0.3 microM) compared to a series of plasminogen fragments (K1-5, K1-3, K4, K5P, and tail domain) that interacted weakly with apo(a) (K(D) > 50 microM). Treatment of Glu(1)-plasminogen with citraconic anhydride (a lysine modification reagent) completely abolished binding to wild-type 17K r-apo(a), whereas citraconylated 17K r-apo(a) decreased binding to wild-type Glu(1)-plasminogen by approximately 50%; inhibition of binding was also observed using the lysine analogue epsilon-aminocaproic acid. Whereas native Glu(1)-plasminogen exhibited monophasic binding to 17K r-apo(a), truncated Lys(78)-plasminogen exhibited biphasic binding. Altering Glu(1)-plasminogen from its native, closed conformation (in chloride buffer) to an open conformation (in acetate buffer) also yielded biphasic isotherms. These SPR data are consistent with a two-state kinetic model in which a conformational change in the plasminogen-apo(a) complex may occur following the initial binding event. Differential binding kinetics between Glu(1)-/Lys(78)-plasminogen and apo(a) may explain why Lp(a) is a stronger inhibitor of tPA-mediated Glu(1)-plasminogen activation compared to Lys(78)-plasminogen activation.     

Hormone-regulated expression and distribution of versican in mouse uterine tissues

Background  

Remodeling of the extracellular matrix is one of the most striking features observed in the uterus during the estrous cycle and after hormone replacement. Versican (VER) is a hyaluronan-binding proteoglycan that undergoes RNA alternative splicing, generating four distinct isoforms. This study analyzed the synthesis and distribution of VER in mouse uterine tissues during the estrous cycle, in ovariectomized (OVX) animals and after 17beta-estradiol (E2) and medroxyprogesterone (MPA) treatments, either alone or in combination.     

Monitoring of glycoprotein products in cell culture lysates using lectin affinity chromatography and capillary HPLC coupled to electrospray linear ion trap-Fourier transform mass spectrometry (LTQ/FTMS)

In this paper, we describe the combination of lectin chromatography with capillary LC coupled to a linear ion trap-Fourier transform mass spectrometer (LTQ/FTMS) to enrich and characterize overexpressed glycoproteins from a cell culture lysate. A well-characterized glycoprotein, recombinant tissue plasminogen activator (rt-PA), was used as a standard, and we demonstrated that the three N-linked glycopeptides (including glycan structures) present in a tryptic digest of the rt-PA standard could be characterized in the new hybrid MS platform. A feature of this approach is that a significant amount of information can be obtained about the carbohydrate structures by direct analysis of the tryptic digest without the need for additional time-consuming sample preparation protocols. A combination of lectins was then studied for improved recovery of captured glycopeptides and was related to the selectivity of different lectins for specific glycosylation motifs. This approach was then extended to the lysate of a cell line routinely used in biotechnology manufacture (Chinese hamster ovary, CHO). This study showed that the combinations of lectins could enrich glycoproteins significantly from a CHO cell lysate. We also demonstrated that with this level of enrichment and with the new hybrid mass spectrometer, we could study the structures of N-linked glycopeptides of rt-PA present in a crude CHO cell lysate, at a ratio of 1:200 (rtPA:total cell lysate protein, w/w) by accurate mass measurement in the FTMS and tandem MSn in the linear ion trap. The generic and high throughput nature of the lectin approach combined with the ability to directly analyze the glycan structures in the tryptic digest suggest that this platform has the potential to routinely monitor glycoprotein products at early stage manufacturing in the biotech industry.     

Longitudinal Study of Fecal Shedding of Escherichia coli O157:H7 in Feedlot Cattle: Predominance and Persistence of Specific Clonal Types despite Massive Cattle Population Turnover

Identification of the sources and methods of transmission of Escherichia coli O157:H7 in feedlot cattle may facilitate the development of on-farm control measures for this important food-borne pathogen. The prevalence of E. coli O157:H7 in fecal samples of commercial feedlot cattle in 20 feedlot pens between April and September 2000 was determined throughout the finishing feeding period prior to slaughter. Using immunomagnetic separation, E. coli O157:H7 was isolated from 636 of 4,790 (13%) fecal samples in this study, with highest prevalence earliest in the feeding period. No differences were observed in the fecal or water trough sediment prevalence values of E. coli O157:H7 in 10 pens supplied with chlorinated drinking water supplies compared with nonchlorinated water pens. Pulsed-field gel electrophoresis of XbaI-digested bacterial DNA of the 230 isolates obtained from eight of the pens revealed 56 unique restriction endonuclease digestion patterns (REDPs), although nearly 60% of the isolates belonged to a group of four closely related genetic subtypes that were present in each of the pens and throughout the sampling period. The other REDPs were typically transiently detected, often in single pens and on single sample dates, and in many cases were also closely related to the four predominant REDPs. The persistence and predominance of a few REDPs observed over the entire feeding period on this livestock operation highlight the importance of the farm environment, and not necessarily the incoming cattle, as a potential source or reservoir of E. coli O157:H7 on farms.     

Interphase chromosomal deoxyribonucleoprotein isolated as a discrete structure from cultured cells

A new procedure is described for the preparation of interphase chromatin from cultured mouse cells (line P815). The primary objective of this procedure was to eliminate exchanges of histones between deoxynucleoprotein molecules; this objective is shown experimentally to have been attained. The chromatin is released from cells by the non-ionic detergent Nonidet P40 in medium of low ionic strength (0.1 mM-KNa₂PO₄), and may then be sedimented as a structure which conserves the general form and ultrastructural characteristics of chromatin within the cell. The nuclear envelope cannot be detected in these structures by electron microscopy, and their content of choline-containing phospholipids is less than 10% of that of nuclei. The maintenance of form in this structure must thus depend on properties of the chromatin itself, and possibly on the more compact peripheral chromatin.Soluble DNP² prepared by shearing these structures has the same relative contents of DNA, histones, non-histone proteins and RNA as DNP prepared by standard methods. Analyses by electrophoresis on polyacrylamide gels of the non-histone proteins reveals certain differences from the pattern of these proteins in DNP prepared by a salt precipitation method. The template activity for RNA synthesis, in the presence of Escherichia coli RNA polymerase of sheared, soluble DNP prepared by this procedure, is comparable to that of DNP prepared by other methods. However, in the absence of exogenous RNA polymerase the rate of RNA synthesis by structured (unsheared) chromatin is about ten times higher than the rate using sheared DNP.The rapid removal of the nuclear envelope in this lysis procedure allowed experimental examination of the origin of the histones and non-histone proteins of DNP. When DNP was prepared from a mixture of two populations of cells, one containing DNA distinguishable by a density label and the other containing radioactively labelled proteins, radioactive proteins were found exclusively in DNP of normal density, and not in dense DNP and vice versa. It is concluded that the proteins of DNP prepared in this way are not acquired during the preparation procedure but were already associated with DNA in vivo, and that other proteins are not bound non-specifically to DNA during the preparation of DNP. When a mixture of DNP molecules prepared, in this way is precipitated in 150 mm-NaCl and redissolved, some radioactively labelled histones migrate onto dense DNA molecules.This procedure is suitable for routine, quantitative isolation of chromosomal DNP from small numbers of cells; it is also applicable to cells of other cultured lines.     

Cutting edge: cationic antimicrobial peptides block the binding of lipopolysaccharide (LPS) to LPS binding protein

We investigated the mechanism by which cationic antimicrobial peptides block the activation of macrophages by LPS. The initial step in LPS signaling is the transfer of LPS to CD14 by LPS binding protein (LBP). Because many cationic antimicrobial peptides bind LPS, we asked whether these peptides block the binding of LPS to LBP. Using an assay that measures the binding of LPS to immobilized LBP, we show for the first time that a variety of structurally diverse cationic antimicrobial peptides block the interaction of LPS with LBP. The relative ability of different cationic peptides to block the binding of LPS to LBP correlated with their ability to block LPS-induced TNF-alpha production by the RAW 264.7 macrophage cell line.     

Impaired proteolytic processing of lysosomal N-acetyl-beta-hexosaminidase in cultured fibroblasts from patients with infantile generalized N-acetylneuraminic acid storage disease

Cultured skin fibroblasts from patients suffering with infantile generalized N-acetylneuraminic acid (NeuAc) storage disease accumulate free NeuAc in a population of lysosomes less dense than those observed in normal fibroblasts (1.035 vs. greater than 1.07 mean density), as assessed by the distribution of lysosomal enzyme activities and NeuAc on Percoll gradients after subcellular fractionation. In the present study, normal and affected fibroblasts were labeled with [35S]methionine, and cell homogenates or subcellular fractions from Percoll gradients were immunoprecipitated with polyclonal antibodies to lysosomal N-acetyl-beta-hexosaminidase (Hex); immunoprecipitated polypeptides were analyzed by SDS-polyacrylamide gel electrophoresis. The synthesis and initial processing of Hex polypeptides were comparable in normal and affected fibroblasts, but mature polypeptides were quantitatively localized in "buoyant" lysosomes of affected cells, along with Hex activity; moreover, mature alpha-chain of Hex was approximately 2 kDa larger than that observed in normal cells. The molecular weight difference was apparently due to impaired proteolytic processing of alpha-chain in affected fibroblasts, since treatment of immunoprecipitated alpha-chain from normal and affected cells with neuraminidase and endo-beta-N-acetylglucosaminidase H failed to resolve the molecular weight difference. The impaired processing was observed to be persistent (after a chase of up to 200 h), but had no apparent effect on the turnover or activity of Hex in affected fibroblasts. The observed proteolytic processing defect may be primary or secondary in infantile NeuAc storage disease.