Ultrastructure of islet microcirculation, pericytes and the islet exocrine interface in the HIP rat model of diabetes

CONTEXT: The transgenic human islet amyloid polypeptide (HIP) rat model of type 2 diabetes mellitus (T2DM) parallels the functional and structural changes in human islets with T2DM. OBJECTIVE: The transmission electron microscope (TEM) was utilized to observe the ultrastructural changes in islet microcirculation. METHODS: Pancreatic tissue from male Sprague Dawley rats (2, 4, 8, 14 months) were used as controls (SDC) and compared to the 2-, 4-, 8- and 14-month-old HIP rat models. RESULTS: The 2-month-old HIP model demonstrated no islet or microcirculation remodeling changes when compared to the SDC models. The 4-month-old HIP model demonstrated significant pericapillary amyloid deposition and diminution of pericyte foot processes as compared to the SDC models. The 8-month-old model demonstrated extensive islet amyloid deposition associated with pericyte and beta-cell apoptosis when compared with SDC. The 14-month-old HIP model demonstrated a marked reduction of beta-cells and intra-islet capillaries with near complete replacement of islets by amyloidoses. Increased cellularity in the region of the islet exocrine interface was noted in the 4- to 14-month-old HIP models as compared to SDC. In contrast to intra-islet capillary rarefaction there was noticeable angiogenesis in the islet exocrine interface. Pericytes seemed to be closely associated with collagenosis, intra-islet adipogenesis and angiogenesis in the islet exocrine interface. CONCLUSION: The above novel findings regarding the microcirculation and pericytes could assist researchers and clinicians in a better morphological understanding of T2DM and lead to new strategies for prevention and treatment of T2DM.     

Modulation of expression of LDH isoenzymes in endothelial cells by laminin: implications for angiogenesis

Endothelial cell (EC) matrix interaction is critical in angiogenesis. Although matrix components can regulate the process of angiogenesis by acting as a reservoir of various cytokines, it is not clear if extracellular matrix (ECM) can modulate the production and activity of angiogenic cytokines. Investigations were therefore carried out to study the influence of the basement membrane (BM) protein, laminin (Ln) on the activity of vascular endothelial growth factor (VEGF), the major angiogenic cytokine, using isolated human umbilical vein ECs (HUVECs) in culture. Analysis of the biochemical markers of angiogenesis confirmed proangiogenic effect of Ln. The levels of VEGF protein and mRNA were not different in cells maintained on Ln, collagen I or polylysine substrata. Chorioallantoic membrane assay using VEGF isolated from cell extracts however revealed that Ln increased its angiogenic potency. Immunoblotting and HPLC analysis showed considerable reduction in poly adenosyl ribosylation of VEGF associated with a significant decrease in the levels of NAD+, in cells maintained on Ln substrata. Further, a shift in the isoenzymic pattern of LDH towards the B rich forms and an upregulation of LDH B gene were observed in cells maintained on Ln. Ln modulates expression of LDH gene through alpha(6)beta(4) integrin mediated downstream signaling involving p38 mitogen activated protein kinases (MAPK) pathway. It thus appears that Ln can affect aerobic metabolism of ECs by modulating the expression of LDH isoenzymes resulting in a decrease in the level of NAD+ that can cause a reduction in the poly adenosyl ribosylation of VEGF altering its angiogenic potency.     

Modulation of endothelial nitric oxide synthase by fibronectin

Nitric oxide (NO) produced by the action of endothelial nitric oxide synthase (eNOS) plays an important role in the regulation of vascular tone, cell survival, and angiogenesis. Interaction of endothelial cells (ECs) with a fibronectin (FN) rich matrix is important in the regulation of EC function and survival during angiogenesis. The present study was carried out to examine if FN can regulate eNOS and thereby NO levels in ECs. The activity and the levels of mRNA and protein of eNOS were significantly low in HUVECs maintained in culture on FN. Inhibition of p38 MAPK and blocking the interaction of FN with α₅β₁ integrin using antibody caused the reversal of the FN effect. Immunoblot analysis of Ser/Thr phosphorylation of purified eNOS suggested that FN downregulates post-translational phosphorylation of eNOS at Ser residues. These results suggest that FN negatively modulates eNOS in an α₅β₁ integrin-p38 MAPK-dependent pathway.     

3-Keto-22-epi-28-nor-cathasterone, a brassinosteroid-related metabolite from Cystoseira myrica

Bioassay-guided purification of an ethanolic extract of Cystoseira myrica against HEPG-2 (liver) and HCT116 (colon) human cancer cell lines led to the isolation of 3-keto-22-epi-28-nor-cathasterone, 1 and cholest-4-ene-3,6-dione, 2. This finding allowed us to report for the first time that a brassinosteroid-related metabolite occurs in seaweed. These compounds showed activity in the range of 12.38–1.16 μM with selective activity of compound 2 to liver cancer cell lines.     

Characterization of the smallest dimeric bile salt hydrolase from a thermophile Brevibacillus sp.

A thermophilic microorganism producing bile salt hydrolase was isolated from hot water springs, Pali, Maharashtra, India. This microorganism was identified as Brevibacillus sp. by 16S rDNA sequencing. Bile salt hydrolase (BSH) was purified to homogeneity from this thermophilic source using Q-sepharose chromatography and its enzymatic properties were characterized. The subunit molecular mass of the purified enzyme was estimated to be 28 kDa by SDS-PAGE and, 28.2 kDa by MALDI-TOF analysis. The native molecular mass was estimated to be 56 kDa by gel filtration chromatography, indicating the protein to be a homodimer. The pH and temperature optimum for the enzyme catalysis were 9.0 and 60°C, respectively. Even though BSH from Brevibacillus sp. hydrolyzed all of the six major human bile salts, the enzyme preferred glycine conjugated substrates with apparent K _M and k _cat values of 3.08 μM and 6.32 × 10² s⁻¹, respectively, for glycodeoxycholic acid. The NH₂-terminal sequence of the purified enzyme was determined and it did not show any homology with other bacterial bile salt hydrolases. To our knowledge, this is the first report describing the purification of BSH to homogeneity from a thermophilic source. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Synthesis of Highly Immunogenic Multiple Antigenic Peptides for Epitopes of Viral Antigen to Use in ELISA

Synthesis of multiple antigenic peptides (MAPs) for predicted antigenic determinants of a viral antigen is described. The method includes prediction of linear epitopes using predictive computer algorithms, synthesis of peptides for the predicted regions, testing of peptides to find the most reactive sites, synthesis of MAPs and their testing. The procedure involves manual synthesis of MAPs by solid phase peptide synthesis with Wang resin as solid support. The MAPs were prepared in eight copies and used for immunization of rabbits to generate anti-peptide antibodies. Further, the reactivity of MAPs in detecting the native cognate antigen in the whole virus was confirmed by ELISA. The MAP and anti-peptide antibodies could serve as diagnostic tools for viral diseases. MAPs have efficiently been used to confirm the presence of linear antigenic and immunogenic epitopes on viral proteins, for possible use in diagnostic and vaccine. It was suggested that this method could help in epitope mapping of dreadful human or animal pathogens as it involves production of safe, chemically defined and non-infectious materials for use as antigen as well as immunogen.     

Modulation of glucose transporter (GLUT4) by vanadate and Trigonella in alloxan-diabetic rats

Oral administration of vanadate is an effective treatment for diabetes in animal models. However, vanadate exerts these effects at high doses and several toxic effects are produced. Low doses of vanadate are relatively safe but are unable to elicit any antidiabetic effect. The present study explored the prospect of using low doses of vanadate in combination with Trigonella seed powder (TSP) to evaluate their antidiabetic effect in alloxan-diabetic rats. Alloxan-diabetic rats were treated with insulin, vanadate, TSP and vanadate and TSP in combination for 3 weeks. The effect of these antidiabetic compounds was examined on general physiological parameters and distribution of glucose transporter (GLUT4) in skeletal muscle by immunoblotting and immunohistochemistry. Treatment of alloxan-diabetic rats with insulin, vanadate, TSP and vanadate in combination with TSP revived normoglycemia and restored the disturbances in the distribution of GLUT4 in skeletal muscle. TSP treatment was only partially effective in the restoration of diabetic alterations. The treatment of diabetic rats with combined doses of vanadate and TSP was most effective in the normalization of plasma glucose levels and correction of altered GLUT4 distribution.     

Hx, a novel fluorescent, minor groove and sequence specific recognition element: design, synthesis, and DNA binding properties of p-anisylbenzimidazole-imidazole/pyrrole-containing polyamides

With the aim of incorporating a recognition element that acts as a fluorescent probe upon binding to DNA, three novel pyrrole (P) and imidazole (I)-containing polyamides were synthesized. The compounds contain a p-anisylbenzimidazolecarboxamido (Hx) moiety attached to a PP, IP, or PI unit, giving compounds HxPP (2), HxIP (3), and HxPI (4), respectively. These fluorescent hybrids were tested against their complementary nonfluorescent, non-formamido tetraamide counterparts, namely, PPPP (5), PPIP (6), and PPPI (7) (cognate sequences 5'-AAATTT-3', 5'-ATCGAT-3', and 5'-ACATGT-3', respectively). The binding affinities for both series of polyamides for their cognate and noncognate sequences were ascertained by surface plasmon resonance (SPR) studies, which revealed that the Hx-containing polyamides gave binding constants in the 10(6) M(-1) range while little binding was observed for the noncognates. The binding data were further compared to the corresponding and previously reported formamido-triamides f-PPP (8), f-PIP (9), and f-PPI (10). DNase I footprinting studies provided additional evidence that the Hx moiety behaved similarly to two consecutive pyrroles (PP found in 5-7), which also behaved like a formamido-pyrrole (f-P) unit found in distamycin and many formamido-triamides, including 8-10. The biophysical characterization of polyamides 2-7 on their binding to the abovementioned DNA sequences was determined using thermal melts (ΔT(M)), circular dichroism (CD), and isothermal titration calorimetry (ITC) studies. Density functional calculations (B3LYP) provided a theoretical framework that explains the similarity between PP and Hx on the basis of molecular electrostatic surfaces and dipole moments. Furthermore, emission studies on polyamides 2 and 3 showed that upon excitation at 322 nm binding to their respective cognate sequences resulted in an increase in fluorescence at 370 nm. These low molecular weight polyamides show promise for use as probes for monitoring DNA recognition processes in cells.