Chronic Endotoxemia in Subjects with Type-1 Diabetes Is Seen Much before the Onset of Microvascular Complications

Background

Lipopolysaccharide (LPS)/Endotoxin is hypothesized to play an important role in chronic inflammation associated with Type-1 diabetes (T1DM) and its complications. Endotoxin core antibodies (EndoCAb), LPS binding protein (LBP) and soluble CD14 (sCD14) act as modulators of LPS induced activation of innate immune system in vivo. For the present study we estimated the levels of LPS and its translocation markers in T1DM subjects with and without microvascular complications (MVC) and correlate them with clinical parameters of T1DM and serum inflammatory cytokine levels (TNF-α, IL-6, IL-1β and GM-CSF).

Methods

A total of 197 subjects (64 normal glucose tolerance (NGT) subjects, 97 T1DM subjects without MVC and 36 with MVC) were included in this study and the levels of serum LPS, its translocation markers and cytokines measured by immunoassays.

Results

Compared to NGT, T1DM subjects (both with and without MVC) had significantly higher levels of LPS, reduced levels of LBP and EndoCAb along with significant increase in the levels of IL-1β, IL-6, TNF-α and GM-CSF (p<0.05). No significant change was seen in the levels of these biomarkers between T1DM subjects with and without MVC.

Conclusions

Decreased levels of EndoCAb and LBP suggest sustained endotoxin activity in T1DM subjects even before the onset of microvascular complications.     

Antioxidant and anti-stress compounds improve regrowth of cryopreserved <Emphasis Type="Italic">Rubus</Emphasis> shoot tips

Regrowth of plants after cryopreservation varies, and resulting regrowth ranges from poor to excellent. Oxidative stress is a potential cause of damage in plant tissues. Antioxidants and anti-stress compounds may improve regrowth by preventing or repairing the damage. Lipoic acid (LA), glutathione (GSH), glycine betaine (GB), and polyvinylpyrrolidone (PVP) were tested during cryopreservation of shoot tips using the plant vitrification solution 2 (PVS2) protocol. Two in vitro-grown blackberry cultivars were cold acclimated and then cryopreserved in liquid nitrogen (LN). The antioxidant and anti-stress compounds were added at four critical steps of the protocol: pretreatment, loading, rinsing, and regrowth. Three out of the four compounds significantly improved regrowth of cryopreserved shoot tips. Regrowth ranged from 40% to 50% for controls to >80% for treated shoot tips. LA (4-8 mM) produced high regrowth at pretreatment, loading, and rinsing for ‘Chehalem’ and at all steps for ‘Hull Thornless’. Recovery improved at all steps with GSH (0.16 mM) and GB (10 mM). PVP had a neutral or negative impact on regrowth. Overall addition of LA, GSH, and GB improved regrowth by ∼25% over the shoot tips cryopreserved using the regular PVS2 protocol (control). This study shows that adding non-vitamin antioxidants and anti-stress compounds during the PVS2-vitrification protocol improves regrowth of shoot cultures following cryopreservation. We recommend inclusion of antioxidants as part of standard cryopreservation protocols.     

Targeted deletion of fatty acid transport protein-4 results in early embryonic lethality

Fatty acid transport protein-4 (FATP4) is the major FATP in the small intestine. We previously demonstrated, using in vitro antisense experiments, that FATP4 is required for fatty acid uptake into intestinal epithelial cells. To further examine the physiological role of FATP4, mice carrying a targeted deletion of FATP4 were generated. Deletion of one allele of FATP4 resulted in 48% reduction of FATP4 protein levels and a 40% reduction of fatty acid uptake by isolated enterocytes. However, loss of one FATP4 allele did not cause any detectable effects on fat absorption on either a normal or a high fat diet. Deletion of both FATP4 alleles resulted in embryonic lethality as crosses between heterozygous FATP4 parents resulted in no homozygous offspring; furthermore, no homozygous embryos were detected as early as day 9.5 of gestation. Early embryonic lethality has been observed with deletion of other genes involved in lipid absorption in the small intestine, namely microsomal triglyceride transfer protein and apolipoprotein B, and has been attributed to a requirement for fat absorption early in embryonic development across the visceral endoderm. In mice, the extraembryonic endoderm supplies nutrients to the embryo prior to development of a chorioallantoic placenta. In wild-type mice we found that FATP4 protein is highly expressed by the epithelial cells of the visceral endoderm and localized to the brush-border membrane of extraembryonic endodermal cells. This localization is consistent with a role for FATP4 in fat absorption in early embryogenesis and suggests a novel requirement for FATP4 function during development.     

Cu-mediated N-arylation of 1,2,3-triazin-4-ones: synthesis of fused triazinone derivatives as potential inhibitors of chorismate mutase

A rapid and direct access to N-aryl substituted fused triazinone derivatives has been accomplished via N-arylation of 1,2,3-triazin-4-one ring involving a Cu-mediated coupling between triazinone derivatives and aryl boronic acids. A combination of Cu(OAc)(2)-Et(3)N in 1,2-dichloroethane was found to be effective and various fused triazinone derivatives have been prepared by using this methodology. Molecular structure of a representative compound was confirmed by single crystal X-ray diffraction study. The scope and limitations of this reaction is discussed. Some of the compounds synthesized were tested for chorismate mutase inhibitory properties in vitro. The in vitro dose response study of an active compound is presented.     

Azelnidipine prevents cardiac dysfunction in streptozotocin-diabetic rats by reducing intracellular calcium accumulation, oxidative stress and apoptosis

Background

In the general population, peripheral metabolic complications (MC) increase the risk for left ventricular dysfunction. Human immunodeficiency virus infection (HIV) and combination anti-retroviral therapy (cART) are associated with MC, left ventricular dysfunction, and a higher incidence of cardiovascular events than the general population. We examined whether myocardial nutrient metabolism and left ventricular dysfunction are related to one another and worse in HIV infected men treated with cART vs. HIV-negative men with or without MC.

Methods

Prospective, cross-sectional study of myocardial glucose and fatty acid metabolism and left ventricular function in HIV+ and HIV-negative men with and without MC. Myocardial glucose utilization (GLUT), and fatty acid oxidation and utilization rates were quantified using ¹¹C-glucose and ¹¹C-palmitate and myocardial positron emission tomography (PET) imaging in four groups of men: 23 HIV+ men with MC+ (HIV+/MC+, 42 ± 6 yrs), 15 HIV+ men without MC (HIV+/MC-, 41 ± 6 yrs), 9 HIV-negative men with MC (HIV-/MC+, 33 ± 5 yrs), and 22 HIV-negative men without MC (HIV-/MC-, 25 ± 6 yrs). Left ventricular function parameters were quantified using echocardiography.

Results

Myocardial glucose utilization was similar among groups, however when normalized to fasting plasma insulin concentration (GLUT/INS) was lower (p < 0.01) in men with metabolic complications (HIV+: 9.2 ± 6.2 vs. HIV-: 10.4 ± 8.1 nmol/g/min/μU/mL) than men without metabolic complications (HIV+: 45.0 ± 33.3 vs. HIV-: 60.3 ± 53.0 nmol/g/min/μU/mL). Lower GLUT/INS was associated with lower myocardial relaxation velocity during early diastole (r = 0.39, p < 0.001).

Conclusion

Men with metabolic complications, irrespective of HIV infection, had lower basal myocardial glucose utilization rates per unit insulin that were related to left ventricular diastolic impairments, indicating that well-controlled HIV infection is not an independent risk factor for blunted myocardial glucose utilization per unit of insulin.

Trial Registration

NIH Clinical Trials NCT00656851     

A sensitive assay for ABCA1-mediated cholesterol efflux using BODIPY-cholesterol

Studies have shown a negative association between cellular cholesterol efflux and coronary artery disease (CAD). Standard protocol for quantitating cholesterol efflux involves labeling cells with [(3)H]cholesterol and measuring release of the labeled sterol. Using [(3)H]cholesterol is not ideal for the development of a high-throughput assay to screen large numbers of serum as would be required in studying the link between efflux and CAD. We compared efflux using a fluorescent sterol (boron dipyrromethene difluoride linked to sterol carbon-24, BODIPY-cholesterol) with that of [(3)H]cholesterol in J774 macrophages. Fractional efflux of BODIPY-cholesterol was significantly higher than that of [(3)H]cholesterol when apo A-I, HDL(3), or 2% apoB-depleted human serum were used as acceptors. BODIPY-cholesterol efflux correlated significantly with [(3)H]cholesterol efflux (p < 0.0001) when apoB-depleted sera were used. The BODIPY-cholesterol efflux correlated significantly with preβ-1 (r(2) = 0.6) but not with total HDL-cholesterol. Reproducibility of the BODIPY-cholesterol efflux assay was excellent between weeks (r(2) = 0.98, inter-assay CV = 3.31%). These studies demonstrate that BODIPY-cholesterol provides an efficient measurement of efflux compared with [(3)H]cholesterol and is a sensitive probe for ABCA1-mediated efflux. The increased sensitivity of BODIPY-cholesterol assay coupled with the simplicity of measuring fluorescence results in a sensitive, high-throughput assay that can screen large numbers of sera, and thus establish the relationship between cholesterol efflux and atherosclerosis.     

Purification and characterization of a motility initiating protein from caprine epididymal plasma

Numerous reports have appeared on the occurrence of undefined protein factors in male reproductive fluids that promote motility of mature sperm and initiate forward motility in the immature (immotile) caput‐epididymal sperm. This study reports for the first time purification to apparent homogeneity of a motility initiating protein (MIP) from epididymal plasma and its characterization using the caprine sperm model. It is a 125 kDa (approximately) dimeric protein made up of two subunits: 70 and 54 kDa. MIP is an acidic protein with an isoelectric point of 4.75. The motility protein at 30 µg/ml (240 nM) level showed nearly maximal motility‐promoting activity. MIP is heat stable and it is maximally active at pH 8. It is a glycoprotein that binds with high affinity to concanavalin A and it contains mannose, galactose, and N‐acetyl glucosamine approximately in the ratios of 6:1:6. It is sensitive to the actions of α‐mannosidase and β‐N‐acetylglucoseaminidase thereby demonstrating that the sugar side chain of the glycoprotein is essential for its biological activity. Epididymal plasma is its richest source. It is also capable of enhancing forward motility of mature cauda‐sperm. Its antibody markedly inhibits sperm motility. MIP antibody is highly immunospecific and it recognizes both the subunits. MIP causes significant increase of the intrasperm level of cyclic AMP. MIP: the physiological motility‐activating protein has potential for use as a contraceptive vaccine and for solving some of the problems of human infertility and animal breeding. J. Cell. Physiol. 222:254–263, 2010. © 2009 Wiley‐Liss, Inc.