Regulation of endogenous glucose production after a mixed meal in type 2 diabetes

The extent and time course of suppression of endogenous glucose production (EGP) in type 2 diabetes after a mixed meal have been determined using a new tracer methodology. Groups of age-, sex-, and weight-matched normal controls (n = 8) and diet-controlled type 2 diabetic subjects (n = 8) were studied after ingesting a standard mixed meal (550 kcal; 67% carbohydrate, 19% fat, 14% protein). There was an early insulin increment in both groups such that, by 20 min, plasma insulin levels were 266 +/- 54 and 190 +/- 53 pmol/l, respectively. EGP was similar basally [2.55 +/- 0.12 mg x kg(-1) x min(-1) in control subjects vs. 2.92 +/- 0.16 mg x kg(-1) x min(-1) in the patients (P = 0.09)]. After glucose ingestion, EGP declined rapidly in both groups to approximately 50% of basal within 30 min of the meal. Despite the initial rapid decrease, the EGP was significantly greater in the diabetic group at 60 min (1.75 +/- 0.12 vs. 1.05 +/- 0.14 mg x kg(-1) x min(-1); P < 0.01) and did not reach nadir until 210 min (0.96 +/- 0.17 mg x kg(-1) x min(-1)). Between 60 and 240 min, EGP was 47% higher in the diabetic group (0.89 +/- 0.09 vs. 1.31 +/- 0.13 mg x kg(-1) x min(-1), P < 0.02). These data quantitate the initial rapid suppression of EGP after a mixed meal in type 2 diabetes and the contribution of continuing excess glucose production to subsequent hyperglycemia.     

Molecular genetic testing of beta-thalassemia patients of Indian origin and a novel 8-bp deletion mutation at codons 36/37/38/39

Hemoglobinopathies are the most commonly inherited genetic disorders in India. Certain communities in India have a high predisposition to beta-thalassemia. To offer prenatal diagnosis and to prevent the birth of an affected child, mutation testing in clinically diagnosed beta-thalassemia patients/carriers is a prerequisite. Over a period of 4 years, we have conducted DNA analysis in 385 carriers for 15 beta-thalassemia mutations, HbD, HbE, and HbS. Using reverse dot blot (RDB) and amplification refractory mutation system (ARMS), we have been able to identify mutations in 381 of 385 thalassemia chromosomes. The study included the analysis of five common mutations found in Asian Indians, namely IVS1-5 (G-C), 619-bp deletion, IVS1-1 (G-T), and the frameshifts at CD8/9(+G) and CD41/42(-CTTT). The occurrence of these five mutations was seen in 299 (91.2%) carriers referred to us, the percentage of mutations varying between 4.0 and 68.9%. We also found Cd16 (-C) in 2.1%, CD30 (G-C) in 1.5%, and CD 15(G-A) in 0.6%; these are considered common mutations in the Indian population, as well. The beta-thalassemia anomaly in 4 (0.6%) carriers remained uncharacterized by RDB and ARMS analysis. During delineation of the mutations in uncharacterized carriers by single-stranded conformational polymorphism (SSCP) and sequencing analysis, we have also been able to identify two unusual mutations, one involving an initiation codon and the second involving a novel 8-bp deletion, in Indian families of Uttar Pradesh origin.     

Angiotensin II-induced mesangial cell apoptosis: role of oxidative stress

BACKGROUND: Angiotensin II (ANG II) has been shown to play a role in the induction of glomerular injury. In the present study, we evaluated the effects of ANG II on mesangial cell apoptosis and the involved molecular mechanism. MATERIALS AND METHODS: The effect of ANG II on apoptosis of mouse mesangial cells (MC) was evaluated by morphologic, DNA fragmentation and TUNEL assays. To evaluate the role of oxidative stress and involved mechanisms, we studied the effect of antioxidants, anti-TGF-beta antibody, inhibitors of nitric oxide synthase and modulators of cytosolic calcium/heme oxygenase (HO) activity. In addition, we studied the effect of ANG II on the generation of reactive oxygen species (ROS) by MCs. RESULTS: ANG II promoted apoptosis of MCs in a dose dependent manner. This effect of ANG II was not only associated with ROS production, but also inhibited by antioxidants. Both Anti-TGF-beta antibody and propranolol inhibited ANG II-induced ROS generation and apoptosis. BAPTA inhibited both ANG II- and TGF-beta-induced apoptosis. On the other hand, thapsigargin stimulated MC apoptosis under basal as well as ANG II/TGF-beta stimulated states. ANG II receptor types 1 and 2 antagonists attenuated the proapoptotic effect of ANG II. Hemin inhibited but zinc protoporphyrin enhanced the proapoptotic effect of ANG II. Propranolol increased HO activity; whereas pre-treatment with propranolol prevented ANG II-induced apoptosis. CONCLUSIONS: ANG II promotes MC apoptosis. This effect of ANG II is mediated through downstream signaling involving TGF-beta, phospholipase D, and Ca(2+), contributing to the activation of NADPH oxidase and generation of ROS. HO activity plays a modulatory role in ANG II- induced MC apoptosis.     

HIV-1 gp120-induced tubular epithelial cell apoptosis is mediated through p38-MAPK phosphorylation

BACKGROUND: HIV-associated nephropathy is accompanied by significant tubular alterations in the form of tubular cell proliferation, apoptosis, and microcystic dilatation. In the present study we evaluated the role of CD4 receptors in HIV-1-induced tubular cell injury. METHODS: To confirm the presence of CD4 receptors in tubular cells, immunocytochemical, Western and Northern blot studies were carried out. To determine the downstream effect of CD4 and gp120 interaction, we evaluated the effect of gp120 on tubular cell p38 mitogen-activated protein kinase (MAPK) activity and phosphorylation. To establish causal relationships between gp120, CD4, and p38 MAPK pathways, we studied the effect of anti-CD4 antibody and SB 202190 (an inhibitor of p38 MAPK) on gp120-induced tubular cell apoptosis. RESULTS: Proximal tubular cells in culture as well as in intact tissue showed expression of CD4 (immunocytochemical and Western blot studies). Cultured tubular cells also showed mRNA expression for CD4 (Northern blot studies). Gp120, at concentrations of 10-100 ng/ ml, triggered tubular cell apoptosis; however, this effect of gp120 was inhibited by anti-CD4 antibody. SB 202190 also inhibited gp120-induced tubular cell apoptosis. In addition, gp120 promoted tubular cell p38 MAPK phosphorylation in a time- and dose- dependent manner. CONCLUSION: Gp120 through interaction with CD4 triggers tubular cell apoptosis. This effect of gp120 on tubular cells is mediated through phosphorylation of p38 MAPK.     

The role of PKCalpha and RLIP76 in transport-mediated doxorubicin-resistance in lung cancer

In deletion mutant analyses of potential phosphorylation sites in RLIP76, we identified T297 and S509 as targets for phosphorylation by PKCalpha. Phosphorylation at T297 increased doxorubicin (DOX)-transport activity approximately 2-fold for RLIP76 purified from recombinant source, or from three small (H69, H1417, H1618) and three non-small cell, one each derived from H226 (squamous), H358 (bronchio alveolar), and H1395 (adenocarcinoma) lung cancer cell lines. T297 phosphorylation conferred sensitivity to tryptic digestion at R293. The specific activity for DOX-transport by RLIP76 purified from non-small cell, which was primarily in the phosphorylated form, was approximately twice that in small cell lung cancer cell lines. These finding offer a novel explanation for the observed intrinsic differences in sensitivity to DOX between non-small cell and small cell lung cancer cell lines.     

Chemically modified papain for applications in detergent formulations

Papain was modified using succinic anhydride, and the modified papain so obtained was compared with the native papain for its activity and stability in detergents. This study was done using commercial enzyme detergents as references. It was found that modified papain retained activity comparable to the commercial enzyme detergents. Chemically modified papain may prove to be an inexpensive alternative to alkaline proteases that are used in detergents.     

DNA annealing and DNA-protein interactions by capillary electrophoresis

This work deals with annealing of single-stranded DNA and the binding of a serum respond factor to a DNA probe containing specific binding site. Capillary electrophoresis (CE) method is explored and compared with the mobility-shift gel electrophoresis (GE) procedure. The results indicate the CE method offers direct and rapid annealing of the DNA strands. It requires no prior incubation with additives (polynucleotides, proteins) to reduce nonspecific DNA-protein interactions. Unwanted nonspecific interactions are not observed in the CE method. The presence of a fluorescein tag to the DNA probe yields identical results to those with the radioactive label. A fluorescein tag in the CE work can be used without any adverse effects. The dissociation constant (Kd) of this protein-DNA complex by the CE method was similar to those determined by the GE method (approximately 10(-6) M). The proposed method is extremely powerful, highly sensitive, quantitative, and fast. It can determine even very small conformational differences of the DNA probe.     

Activity of allelic variants of Pi class human glutathione S-transferase toward chlorambucil

Clinical efficacy of alkylating anticancer drugs, such as chlorambucil, is often limited by the emergence of drug resistant tumor cells. Increased glutathione (GSH) conjugation (inactivation) of alkylating anticancer drugs or their activated metabolites due to overexpression of the Pi class GSH S-transferase (hGSTP1-1) is believed to be an important mechanism in tumor cell resistance to alkylating agents. Interestingly, the hGSTP1 locus is polymorphic in human populations and involves amino acid residues in positions 104 (isoleucine or valine) and/or 113 (alanine or valine). Here, we report that the allelic variants of hGSTP1-1 significantly differ in their efficiency in catalyzing the GSH conjugation of chlorambucil. Catalytic efficiency of the hGSTP1-1(I104,A113) isoform toward chlorambucil was approximately 2.5-, 7.5- and 15-fold higher compared with I104,V113, V104,A113 and V104,V113 variants of hGSTP1-1, respectively. The results of the present study suggest that hGSTP1-1 polymorphism may be an important factor in GST-mediated tumor cell resistance to some alkylating agents.     

Spectral Light Quality Affects Protein Profile of Synechococcus sp. PCC 7942: A Comparative 2-Dimensional Gel Electrophoresis (2-DGE) Analysis

We report here a comparative analysis of the effect of blue (450 nm), red (660 nm), and white light (400–700 nm) on the protein profile of cyanobacteria Synechococcus sp. PCC 7942. In vivo labeling of cells with [³⁵S] methionine and their subsequent analysis by two-dimensional gel electrophoresis (2-DGE) showed that eight polypeptides were unique to dark adapted cells, ten were blue light specific, and four were specifically induced in red light. The results show that Synechococcus sp. respond to various light treatments rapidly and synthesize new polypeptides in dark and blue/red light. Received: 12 October 1999 / Accepted: 16 November 1999