Acetoxy drug: protein transacetylase of buffalo liver-characterization and mass spectrometry of the acetylated protein product

The purification and characterization of the buffalo liver microsomal transacetylase (TAase) catalyzing the transfer of acetyl groups from a model acetoxy drug: 7,8-diacetoxy-4-methylcoumarin (DAMC) to GST3-3 has been described here. The enzyme was routinely assayed using DAMC and cytosolic GST as the substrates and was partially purified from microsomes of the buffalo liver. The enzyme was found to have approximate molecular of weight 65 kDa. The action of TAase and DAMC on liver cytosolic GST resulted in the formation of monoacetoxymonohydroxy-4-methylcoumarin (MAMHC) and 7,8-dihydroxy-4-methylcoumarin (DHMC), although the former was the major metabolite. The buffalo liver microsomal TAase exhibited hyperbolic kinetics and yielded K(m) (1667 microM) and V(max) (192 units) when the concentration of DAMC was varied keeping the concentration of GST constant. After having characterized the nature of the substrates and a product of the TAase-catalyzed reaction, we set out to identify the acetylated protein which is another product of the reaction. GST3-3 was used as a model protein substrate for the action of TAase using DAMC as the acetyl donor. The subunit of control and modified GST3-3 were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and digested with trypsin. The tryptic peptides were extracted from the gel pieces and analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOFMS). The data search for calibrated and labeled mass peaks of peptides was performed on the Matrix Science Server using the search engine Mascot. The peptide maps so obtained covered 97% of the GST3-3 sequence. On comparison of MALDI peptide maps of modified and control GST, seven new peaks were recognized corresponding to the potentially acetylated peptides in peptide map. The mass value of each of them was 42 Da higher than the theoretical mass of a non-modified GST3-3 tryptic peptide, strongly suggesting acetylation. By examining the fragmentation patterns and by comparing experimental and predicted values for MS/MS daughter ions, the identity of the seven acetylated GST tryptic peptides could be confirmed by the application of LC/MS/MS. In the modified GST, N-terminal proline and six lysines (Lys(51), Lys(82), Lys(123), Lsy(181), Lys(191) and Lys(210)) were found to be acetylated. The structure of acetylated GST revealed that the lysines that underwent acetylation were peripheral in positions.     

Evidences showing ultraviolet-B radiation-induced damage of DNA in cyanobacteria and its detection by PCR assay

Impact of ultraviolet-B radiation in causing the damages to the DNA of the cyanobacterium, Anabaena strain BT2 has been investigated. Exposure of genomic DNA (in vitro) to UV-B radiation for 1 h did not cause any shift in the absorption peak (lambda(max)) but more than 30% increase in absorbance was noticed in comparison to untreated control DNA (no exposure to UV-B). This increase in absorbance in a way may be comparable to typical hypochromic effect but there was no decrease in absorbance following transfer of UV-B-treated DNA to fluorescent light or in the dark. That the damaging effect of UV-B radiation on native structure of DNA is indeed real was also evident from the PCR-based assay such as RAPD, rDNA amplification, and ARDRA. Template activity of UV-B-treated genomic DNA was drastically inhibited, there was no amplification in RAPD assay after prior exposure of DNA to UV-B for 60 min. Only one band of approximately 400 bp was observed even after 60 min of exposure which suggests that certain segment of DNA strand is resistant to UV-B effects. Similar to the effects on RAPD profile, amplification of rDNA was significantly inhibited following exposure of genomic DNA to UV-B. Our findings clearly demonstrate that UV-B does affect the DNA of cyanobacteria and the killings of these microbes might be due to the irreversible damages caused to DNA by this high energy radiation. It is felt that PCR assay may be conveniently used for screening the damages caused to DNA by UV-B radiation in cyanobacteria and other microorganisms.     

Homocysteine causes cerebrovascular leakage in mice

Elevated plasma homocysteine (Hcy) is associated with cerebrovascular disease and activates matrix metalloproteinases (MMPs), which lead to vascular remodeling that could disrupt the blood-brain barrier. To determine whether Hcy administration can increase brain microvascular leakage secondary to activation of MMPs, we examined pial venules by intravital video microscopy through a craniotomy in anesthetized mice. Bovine serum albumin labeled with fluorescein isothiocyanate (BSA-FITC) was injected into a carotid artery to measure extravenular leakage. Hcy (30 microM/total blood volume) was injected 10 min after FITC-BSA injection. Four groups of mice were examined: 1) wild type (WT) given vehicle; 2) WT given Hcy (WT + Hcy); 3) MMP-9 gene knockout given Hcy (MMP-9-/- + Hcy); and 4) MMP-9-/- with topical application of histamine (10(-4) M) (MMP-9-/- + histamine). In the WT + Hcy mice, leakage of FITC-BSA from pial venules was significantly (P < 0.05) greater than in the other groups. There was no significant leakage of pial microvessels in MMP-9-/- + Hcy mice. Increased cerebrovascular leakage in the MMP-9-/- + histamine group showed that microvascular permeability could still increase by a mechanism independent of MMP-9. Treatment of cultured mouse microvascular endothelial cells with 30 microM Hcy resulted in significantly greater F-actin formation than in control cells without Hcy. Treatment with a broad-range MMP inhibitor (GM-6001; 1 microM) ameliorated Hcy-induced F-actin formation. These data suggest that Hcy increases microvascular permeability, in part, through MMP-9 activation.     

Pioglitazone prevents cardiac remodeling in high-fat, high-calorie-induced Type 2 diabetes mellitus

The agonists of peroxisome proliferator-activated receptor-gamma (PPARgamma) ameliorate cardiovascular complications associated with diabetes mellitus. We tested the hypothesis that recovery from ailing to failing myocardium in diabetes by PPARgamma agonist is in part due to decreased matrix metalloproteinase-9 (MMP-9) activation and left ventricular (LV) tissue levels of homocysteine (Hcy). C57BL/6J mice were made diabetic (D) by feeding them a high-fat calorie diet. PPARgamma was activated by adding pioglitazone (Pi) to the diet. After 6 wk, mice were grouped into: normal calorie diet (N), D, N + Pi and D + Pi (n = 6 in each group). LV variables were measured by echocardiography, endothelial-myocyte (E-M) coupling was measured in cardiac rings, and MMP-9 activation was measured by zymography. Blood glucose levels were twofold higher in D mice compared with N mice. Pi decreased the levels of glucose in D mice to the levels in N mice. LV Hcy levels were 3.5 +/- 0.5 microM in N groups compared with 12.4 +/- 0.6 microM in D groups. Treatment with Pi normalized the LV levels of Hcy but had no effect on plasma levels of Hcy. In the D group, LV contraction was reduced compared with that of the N group and was ameliorated by treatment with Pi. LV wall thickness was reduced to 0.25 +/- 0.02 mm in the D group compared with 0.42 +/- 0.01 mm in the N group. LV diastolic diameter was 3.05 +/- 0.01 mm in the D group compared with 2.20 +/- 0.02 mm in the N group. LV systolic diameter was 1.19 +/- 0.02 mm in the D group and 0.59 +/- 0.01 mm in the N group. Pi normalized the LV variables in D mice. The responses to ACh and nitroprusside were attenuated in diabetic hearts, suggesting that there was E-M uncoupling in the D group compared with the N group, which was ameliorated by Pi. Plasma and LV levels of MMP-2 and -9 activities were higher in the D group than in the N group but normalized after Pi treatment. These results suggest that E-M uncoupling in the myocardium, in part, is due to increased MMP activities secondary to suppressing PPARgamma activity in high-fat, calorie-induced Type 2 diabetes mellitus.     

Mitochondrial mechanism of microvascular endothelial cells apoptosis in hyperhomocysteinemia

An elevated level of homocysteine (Hcy) limits the growth and induces apoptosis. However, the mechanism of Hcy-induced programmed cell death in endothelial cells is largely unknown. We hypothesize that Hcy induces intracellular reactive oxygen species (ROS) production that leads to the loss of transmembrane mitochondrial potential (Deltapsi(m)) accompanied by the release of cytochrome-c from mitochondria. Cytochrome-c release contributes to caspase activation, such as caspase-9, caspase-6, and caspase-3, which results in the degradation of numerous nuclear proteins including poly (ADP-ribose) polymerase (PARP), which subsequently leads to the internucleosomal cleavage of DNA, resulting cell death. In this study, rat heart microvascular endothelial cells (MVEC) were treated with different doses of Hcy at different time intervals. Apoptosis was measured by DNA laddering and transferase-mediated dUTP nick-end labeling (TUNEL) assay. ROS production and MP were determined using fluorescent probes (2,7-dichlorofluorescein (DCFH-DA) and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzamidazolocarbocyanin iodide (JC-1), respectively, by confocal microscopy. Differential gene expression for apoptosis was analyzed by cDNA array. The results showed that Hcy-mediated ROS production preceded the loss of MP, the release of cytochrome-c, and the activation of caspase-9 and -3. Moreover the Hcy treatment resulted in a decrease in Bcl(2)/Bax ratio, evaluated by mRNA levels. Caspase-9 and -3 were activated, causing cleavage of PARP, a hallmark of apoptosis and internucleosomal DNA fragmentation. The cytotoxic effect of Hcy was blocked by using small interfering RNA (siRNA)-mediated suppression of caspase-9 in MVEC. Suppressing the activation of caspase-9 inhibited the activation of caspase -3 and enhanced the cell viability and MP. Our data suggested that Hcy-mediated ROS production promotes endothelial cell death in part by disturbing MP, which results in subsequent release of cytochrome-c and activation of caspase-9 and 3, leading to cell death.     

Role of nitric oxide in ovarian follicular development and egg production in Japanese quail (Coturnix coturnix japonica)

Role of nitric oxide (NO) in regulating the reproductive functions at hypothalamo-hypophysealovarian axis in Japanese quail was studied. In first experiment, metabolites of NO, i.e. nitrite and nitrate (NO2 and NO3) were estimated together in hypothalamus, serum and ovarian follicles of good and poor layers. In the second experiment, different NO modulators such as L-arginine (L-Arg), sodium nitroprusside (SNP) and N(G)-nitro-L-arginine methyl ester, HCl (L-NAME) were administered to the birds. In the first experiment, significantly higher (P < 0.01) NO2 and NO3 levels in serum, hypothalamus and largest (F1) ovarian follicles were observed in good layers as compared to poor layers. Higher (P < 0.05) NO2 and NO3 concentration was observed in F1 follicles than smaller follicles (F2) only in good layers. The NO2 and NO3 concentration was significantly reduced (P < 0.05) in post ovulatory follicles (POFs) in comparison to F1 and F2 follicles. In the second experiment, the serum NO2 and NO3 concentrations were higher (P < 0.05) in the SNP, lower (P < 0.05) in the L-Name group and unchanged in the L-Arg treated group in comparison to control group. compared to control, L-Arg and SNP increased (P < 0.05) the hypothalamic NO2 and NO3 concentration where as L-NAME reduced (P < 0.05) these levels. The NO2 and NO3 concentration was increased (P < 0.05) as the follicle size increased and it was significantly reduced (P < 0.05) in POFs. The higher (P < 0.05) follicular NO2 and NO3 concentration was observed in L-Arg group in comparison to control group. Egg production was also found to be higher (P < 0.05) in L-Arg group whereas it was not different (P > 0.05) in SNP and L-NAME treated groups. The yolk weight and yolk to albumin ratio was reduced (P < 0.05) in L-NAME group in comparison to control group. It may be concluded from the present study that NO plays a key role in regulating follicular development, ovulatory mechanisms and egg production in Japanese quail.     

GABA receptors ameliorate Hcy-mediated integrin shedding and constrictive collagen remodeling in microvascular endothelial cells

Mammalian endothelial cells are deficient in cystathionine β synthetase (CBS) activity, which is responsible for homocysteine (Hcy) clearance. This deficiency makes the endothelium theprime target for Hcy toxicity. Hcy induces integrin shedding in microvascular endothelial cells (MVEC) by increasing matrix metalloproteinase (MMP). Hcy competes with inhibitory neurotransmitter γ aminobutyric acid (GABA)-A receptor. We hypothesized that Hcy transduces MVEC remodeling by increasing metalloproteinase activity and shedding β-1 integrin by inactivating the GABA-A/B receptors, thus behaving as an excitatory neurotransmitter. MVEC were isolated from mouse brain. The presence of GABA-A receptor was determined by immunolabeling. It was induced by muscimol, an agonist of GABA-A receptors as measured by Western blot analysis. Hcy induced MMP-2 activity in a dose- and time-dependent maner, measured by zymography. GABA-A/B receptors ameliorated the Hcy-mediated MMP-2 activation. Hcy selectively increased the levels of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-3 but decreased the levels of TIMP-4. Treatment with muscimol decreased the levels of TIMP-1 and TIMP-3 and increased the levels of TIMP-4 to control. Hcy caused a robust increae in the levels of a disintegrin and metalloproteinase (ADAM)-12. In the medium of MVEC reated with Hcy, the levels of β-1 integrin were significantly increased. Treatment with muscimol or baclofen (GABA-B receptor agonist) ameliorated the levels significantly increased. Treatment with muscimol or baclofen (GABA-B receptor agonist) ameliorated the levels of β-1 integrin in the medium. These results suggested that Hcy induced DAM-12. Significantly, Hcy facilitated the β-1 integrin shedding. Treatment of MVEC with muscimole or baclofen during Hcy administration ameliorated the expression of metalloproteinase, integrin-shedding, and constrictive collagen remodeling, suggesting a role of Hcy in GABA receptor-mediated cerebrovascular remodeling.