Differential gene expression of NADPH oxidase (p22phox) and hemoxygenase-1 in patients with Type 2 diabetes and microangiopathy.

AIMS: While the downstream effects of increased reactive oxygen species (ROS) in the pathogenesis of diabetes were well studied, only a few studies have explored the cellular sources of ROS. We examined whether protection against oxidative stress is altered in patients with diabetes and microangiopathy by examining changes in NADPH oxidase (p22(phox)) and hemoxygenase-1 (HO-1) levels. METHODS: NADPH oxidase (p22(phox)) and HO-1 gene expression were probed by RT-PCR using leucocytes from patients with Type 2 diabetes without (n = 19) and with microangiopathy (n = 20) and non-diabetic subjects (n = 17). Levels of lipid peroxidation as measured by thiobarbituric reactive substances (TBARS) and protein carbonyl content (PCO) were determined by fluorimetric and spectrophotometric methods, respectively. RESULTS: p22(phox) gene expression (mean +/- SE) was significantly (P < 0.05) higher in diabetic patients with (0.99 +/- 0.04) and without microangiopathy (0.86 +/- 0.05) compared with control subjects (0.66 +/- 0.05). Consistent with the mRNA data, the p22(phox) protein expression and NADPH oxidase activity was also increased in cells from diabetic patients compared with control subjects. However, HO-1 gene expression was significantly (P < 0.05) lower in patients with (0.73 +/- 0.03) and without microangiopathy (0.85 +/- 0.02) compared with control subjects (1.06 +/- 0.03). The mean (+/- SE) levels of TBARS were significantly (P < 0.05) higher in diabetic patients with (14.36 +/- 1.3 nM/ml) and without microangiopathy (12.20 +/- 1.3 nM/ml) compared with control subjects (8.58 +/- 0.7 nM/ml). The protein carbonyl content was also significantly (P < 0.05) higher in diabetic patients with (1.02 +/- 0.04 nmol/mg protein) and without microangiopathy (0.84 +/- 0.06 nmol/mg protein) compared with control subjects (0.48 +/- 0.02 nmol/mg protein). In diabetic subjects, increased p22(phox) gene expression was negatively correlated with HO-1 and positively correlated with TBARS, PCO, HbA(1c) and diabetes duration. In contrast, HO-1 gene expression was correlated negatively with p22phox, TBARS, PCO, HbA(1c) and diabetes duration. CONCLUSION: Our results indicate that increased oxidative damage is seen in Asian Indians with Type 2 diabetes and microangiopathy and is associated with increased NADPH oxidase (p22(phox)) and decreased HO-1 gene expression.     

Abnormal guanine nucleotide regulatory protein in MVP dysautonomia: evidence from reconstitution of Gs

We and others have used the term MVP dysautonomia for a particular subset of hyperadrenergic dysautonomia patients. The role of the stimulatory guanine nucleotide regulatory protein (Gs) in this dysautonomia was studied by cholate extraction of Gs from erythrocytes from 11 normal subjects and 14 symptomatic dysautonomic patients and reconstitution into cyc-S49 lymphoma membranes, which have normal receptor and adenylyl cyclase but lack Gs. Isoproterenol-stimulated adenylyl cyclase activity in the dysautonomia group was increased compared to that in controls [3.66 +/- 0.20 (mean +/- SE; n = 14) vs. 2.87 +/- 0.14 (n = 11) U cyc- reconstituted activity/mg erythrocyte protein; P less than 0.05]. beta-Adrenergic receptor high affinity state formation was greatest in the severely symptomatic group [KL/KH: severe symptoms, 130 +/- 48 (n = 6); mild symptoms, 33 +/- 7 (n = 7); control, 27 +/- 6 (n = 11); severe dysautonomia distinct, P less than 0.017]. Sodium dodecyl sulfate-polyacrylamide gels of cholera toxin-dependent ADP-ribosylated G-proteins yielded no gross distinction between severely symptomatic and control groups. This subset of hyperadrenergic dysautonomia patients, thus, has supercoupled beta 2-adrenergic receptors (increase in both agonist binding and cyclase activation) conferred by an abnormal Gs, whose effects on agonist binding reflect the severity of illness.     

α,β-Unsaturated carbonyl system of chalcone-based derivatives is responsible for broad inhibition of proteasomal activity and preferential killing of human papilloma virus (HPV) positive cervical cancer cells

Proteasome inhibitors have potential for the treatment of cervical cancer. We describe the synthesis and biological characterization of a new series of 1,3-diphenylpropen-1-one (chalcone) based derivatives lacking the boronic acid moieties of the previously reported chalcone-based proteasome inhibitor 3,5-bis(4-boronic acid benzylidene)-1-methylpiperidin-4-one and bearing a variety of amino acid substitutions on the amino group of the 4-piperidone. Our lead compound 2 (RA-1) inhibits proteasomal activity and has improved dose-dependent antiproliferative and proapoptotic properties in cervical cancer cells containing human papillomavirus. Further, it induces synergistic killing of cervical cancer cell lines when tested in combination with an FDA approved proteasome inhibitor. Exploration of the potential mechanism of proteasomal inhibition by our lead compound using in silico docking studies suggests that the carbonyl group of its oxopiperidine moiety is susceptible to nucleophilic attack by the γ-hydroxythreonine side chain within the catalytic sites of the proteasome.     

In vivo assessment of genotoxic effects of Annona squamosa seed extract in rats

Widespread use of pesticides represents a potential risk to human and environmental health. Hence, biopesticides from plants are some of the future strategies for plant protection. In this regard, a seed extract of Annona squamosa was prepared and found to be a promising pesticide. In order to establish the inherent toxicity and non-target safety required for registration and marketing of pesticides, toxicological studies are conducted. The genotoxicity potential was evaluated in rats with 75, 150 and 300 mg/kg Annona squamosa by the comet assay in leucocytes, micronucleus and chromosomal aberration tests in bone marrow. We also studied the effects of 300 mg/kg of extract on lipid peroxidation, reduced glutathione level and glutathione S transferase activity in liver, lungs, brain, kidneys, heart and spleen of treated rats. The comet assay showed a statistically significant dose related increase in DNA migration. The micronucleus and chromosomal aberration tests revealed a significant induction in frequency of micronuclei and chromosomal aberrations at 150 and 300 mg/kg. Annona squamosa treatment significantly enhanced lipid peroxidation, decreased glutathione and glutathione S transferase levels revealing the oxidative stress condition. Our results warrant careful use of Annona squamosa seed extract as a biopesticide till more tests are carried out.     

Comparison of MMP-2 and MMP-9 secretion from T helper 0, 1 and 2 lymphocytes alone and in coculture with macrophages

Metalloproteinases (MMPs) participate in extracellular matrix remodelling and regulatory signalling during chronic inflammatory states such as atherosclerosis formation. However, the sources and mediators of MMP upregulation need clarification. We investigated whether proinflammatory mouse T helper type 1 (Th1) lymphocytes are more active in MMP secretion than naïve Th0 or anti-inflammatory Th2 phenotypes, in the absence of specific antigenic stimulation, under baseline conditions and after contact with irradiated macrophages. We also compared the effect of Th0, Th1 or Th2 lymphocyte-conditioned medium and irradiated lymphocytes on MMP production from macrophages. Finally, we investigated whether CD40–CD40 ligand (CD40L) interactions were involved in T-cell-stimulated MMP secretion from macrophages. Under baseline conditions, MMP-2 messenger RNA (mRNA) and protein levels were greater in Th1 than Th0 or Th2 lymphocytes; MMP-9 mRNA, but not protein, was also upregulated. In the presence of irradiated macrophages MMP-2 and MMP-9 production from Th1 and Th2 was greater than from Th0 lymphocytes. Conditioned media from Th1 but not Th0 or Th2 cells increased MMP-9 secretion from macrophages. Irradiated Th1 lymphocytes stimulated both MMP-2 and MMP-9 secretion from macrophages more than irradiated Th2 or Th0 cells; this activation was independent of CD40–CD40L interaction. These findings demonstrate for the first time greater MMP secretion by Th1 than Th2 or Th0 lymphocytes and their greater ability to upregulate macrophage MMP secretion in the absence of specific antigenic stimulation. These mechanisms could promote matrix turnover in inflammatory states and, for example, promote atherosclerotic plaque rupture.