State of the antioxidant protection system of rat liver in ischemia and reperfusion

Purpose: determination of the state of the antioxidant protection system of the cytosolic fraction and suspension of rat liver mitochondria after experimental ischemia and reperfusion. Materials and methods: the study was conducted using white mature rats, divided into 3 groups: the control group (n = 15); The 2nd group of animals (n = 15), from which the liver was taken after 15 minutes of liver ischemia; the 3rd group of rats (n = 15), from which the liver was taken after a 15-minute reperfusion period, followed by a 15-minute ischemic period. Mitochondrial suspension and cytosolic fraction were isolated from liver tissue. Results: the obtained research results showed the presence of certain pathobiochemical changes in the suspension of mitochondria and the cytosolic fraction after ischemia or reperfusion. In the mitochondrial suspension during the reperfusion period it was found an adaptive increase in the activity of glutathione peroxidase by 39% and glutathione reductase by 61%. In the cytosolic fraction, it was the most remarkable increase of the total antioxidant capacity by 38% already during ischemia and a progressive decrease in the level of reduced glutathione form by 26% in ischemic and 55% in reperfusion period. The change in the state of the antioxidant system occurred against the background of an increase in the number of products of oxidative modifications of biomolecules by 40% during ischemia and 2.2 times after reperfusion. Conclusion: The results indicate the need to develop not only a mitochondria-oriented correction of oxidative disorders, but also active support for the components of the cytosol, which provide the main accumulation of free radical damage products and their subsequent removal from the cell, which is essential for survival.

Evidence for a Novel Key Regulatory Step in the Triglyceride Synthetic Pathway of Human Adipose Tissue

The triglyceride (TG) synthetic pathway is believed to be regulated at the level of the rate-limiting enzyme acyl-CoA:1,2-diacylglycerol O-acyltransferase (DGAT). Recent reports using rat hepatic tissue suggest a kinase-dependent mechanism for the regulation of DGAT activity. To examine this process further, the present study investigates the regulatory mechanisms involved in the modulation of DGAT in human adipocytes. Adipocytes were fractionated into a microsomal fraction containing DGAT and a cytosolic fraction containing a putative regulatory kinase. DGAT activity was determined bymeasuring the incorporation of 14C-oleoyl-CoA into TG with exogenously supplied 1,2-dioleoyl-sn-glycerol. Kinase activity was assayed by addition of the cytosolic fraction in the presence of Mg2+ and ATP. The results indicate a significant inhibition of human adipose tissue DGAT activity by as much as 43% (avg: 17.5% ± 10.4%, p < 0.01) via a mechanism consistent with a phosphorylation event. Partial purification of the putative cytosolic kinase was achieved by multidimensional chromatography. This study thus provides evidence for a novel and key regulatory step in the human TG biosynthetic pathway. Further research is necessary to determine whether the model outlined here is a physiologic conduit through which extracellular hormones exert a regulatory influence on TG synthesis.

Purification of a Src family tyrosine protein kinase from bovine retinas

Since tyrosine phosphorylation appears to play important functions in photoreceptor cells, we searched here for retinal nonreceptor tyrosine kinases of the Src family. We demonstrated that Src family tyrosine kinases were present in the cytosolic fraction of extracted bovine retinas. A Src family tyrosine kinase with an apparent molecular mass of about 62 kDa was purified to homogeneity from the soluble fraction of dark-adapted bovine retinas after three consecutive purification steps: ω-aminooctyl-agarose hydrophobic chromatography, Cibacron blue 3GA-agarose pseudo-affinity chromatography, and α-casein-agarose affinity chromatography. The purified protein was subjected to N-terminal amino acid sequencing and the sequence Gly-Ile-Ile-Lys-Ser-Glu-Glu was obtained, which displayed homology with the first seven residues of the Src family tyrosine kinase c-Yes from Bos taurus (Gly-Cys-Ile-Lys-Ser-Lys-Glu). Although the cytosolic fraction from dark-adapted retinas contained tyrosine kinases of the Src family capable of phosphorylating the α-subunit of transducin, which is the heterotrimeric G protein involved in phototransduction, the purified tyrosine kinase was not capable of using transducin as a substrate. The cellular role of this retinal Src family member remains to be found.

Effect of laser irradiation on xanthine oxidase activity and superoxide radical generation in rat liver cytosol fraction

The effect of tumor growth in the body and laser irradiation on the enzymatic activity of xanthine oxidase, in particular its D- and O-forms, and also the rate of generation of the superoxide radical (O2–) and the level of protein sulfhydryl groups in the liver rat cytosolic fraction has been investigated. It has been found that in the cytosolic fraction of rats with transplanted Guerin’s carcinoma decreases the enzymatic activity of the D-form of xanthine oxidase with a simultaneous increase in its O-form during the period of intensive (14 days, which corresponds to the logarithmic phase of on cogenesis) and the period of final tumor growth (21 days, which corresponds to the stationary phase of oncogenesis). The increase in the enzymatic activity of the O-form of xanthine oxidase was accompanied by an increase the rate of superoxide radical generation and a decrease in the level of protein SH-groups in the liver cytosolic fraction of tumor-bearing rats. Daily directed action of laser irradiation on the area of growth of Guerin’s carcinoma leads to less destructive changes in the liver. Thus, there is an increase in the enzymatic activity of the D-form of xanthine oxidase, a decrease the rate of superoxide radical formation and an increase the content of protein SH-groups in the cytosolic fraction of the liver of experimental animals compared with non-irradiated tumor-bearing rats.

Correction: Keratinocyte differentiation promotes ER stress-dependent lysosome biogenesis

Following publication of this article, the authors realized there was an error in Figure 2b that needed correction. The TFEB panel of Figure 2b (total lysate) appears to be the same as the TFEB panel of Figure 2e (cytosolic fraction); the TFE3 panels of Figure 2b (total lysate) appear to be the same as the TFE3 panels of Figure 2e (cytosolic fraction) which happened during image assembly. This error did not impact the scientific conclusions of the article.

Serine Proteinases in Leishmania (Viannia) braziliensis Promastigotes Have Distinct Subcellular Distributions and Expression

Serine proteinases in Leishmania (Viannia) braziliensis promastigotes were assessed in this work. This study included the investigation of the enzymatic activity of subcellular fractions obtained from benzamidine affinity chromatography, reverse transcription polymerase chain reactions, and in silico assays of subcellular localization of subtilisin. Promastigote serine proteinases showed gelatinolytic activity with molecular masses of 43 kDa to 170 kDa in the cytosolic fraction and 67 kDa to 170 kDa in the membranous fraction. Serine proteinase activities were detected using N-benzyloxycarbonyl-l-phenylalanyl-l-arginine 7-amino-4-methylcoumarin (Z-FR-AMC) and N-succinyl-l-alanine-l-phenylalanine-l-lysine 7-amino-4-methylcoumarin (Suc-AFK-AMC) as substrates in the cytosolic fraction (Z-FR-AMC = 392 ± 30 µmol.min−1 mg of protein−1 and Suc-AFK-AMC = 252 ± 20 µmol.min−1 mg of protein−1) and in the membranous fraction (Z-FR-AMC = 53 ± 5 µmol.min−1 mg of protein−1 and Suc-AFK-AMC = 63.6 ± 6.5 µmol.min−1 mg of protein−1). Enzyme specificity was shown by inhibition with aprotinin (19% to 80% inhibition) and phenylmethanesulfonyl fluoride (3% to 69%), depending on the subcellular fraction and substrate. The expression of subtilisin (LbrM.13.0860 and LbrM.28.2570) and tryparedoxin peroxidase (LbrM.15.1080) genes was observed by the detection of RNA transcripts 200 bp, 162 bp, and 166 bp long, respectively. Subsequent in silico assays showed LbrM.13.0860 can be located in the cytosol and LbrM.28.2570 in the membrane of the parasite. Data obtained here show the subcellular distribution and expression of serine proteinases, including the subtilisin-like serine proteinases in L. (V.) braziliensis promastigotes.

BLOOD

All forms of mercury are global pollutants having no environmental limits.Human exposure to mercury occurs basically through food chain due to accumulation oforganic forms of mercury in fish. Objectives: The purpose of the present study was to analyzethe effect of phenyl mercuric acetate on plasma and cytosolic fraction GSH. Study Design:Experimental Study. Setting: Department of Pharmaceutical Chemistry, Faculty of Pharmacy,Gomal University, Dera Ismail Khan. Period: 29 January 2011 to 11 march 2012 .StatisticalAnalysis: One-way ANOVA followed by Dunnet’s HSD test. Results: For the estimation ofthiols Ellman’s method was used and was found statistically significant (p < 0.001) decreasein plasma and cytosolic fraction GSH which was dose and time dependent. The plasma GSHcontents drop in 0 to 120 minutes by various concentrations of phenyl mercuric acetate were64.45%, 59.33%, 50.89%, 41.56%, 33.63% and 32.99% while drop in cytosolic fraction GSHlevel from 0 to 120 minutes by different concentrations of phenyl mercuric acetate (PMA) was53.86%, 48.60%, 45.41%, 36.11%, 29.38% and 27.06%.Conclusions: It is clear that duringorganic mercury toxicity the blood components are also affected which is proved from ourresults. With the increase of time ,the mercury toxicity would be more harmful so detoxificationof organic mercury should be done on emergency bases at the earliest with the help of suitablechelating agents along with antioxidant therapy.