The use of biochemical parameters for a qualitative and quantitative assessment of ischemic damage to the small-intestinal mucosa

Lactate dehydrogenase has been measured in the small-intestinal mucosa in order to assess its value as a marker for the effects of ischemia and of reperfusion. The decrease in specific activity of the enzyme illustrates the deleterious effect of reperfusion on the quality of the remaining epithelial cells. However, this parameter fails to detect the loss of epithelial cells, which is the major event during ischemia as well as during reperfusion. In contrast, the expression of enzyme activity per g protein of the underlying intestinal muscle allowed us, in addition, to assess quantitatively the loss of epithelial cells, in good agreement with the histological data.     

In vitro dissociation-recombination of malate dehydrogenase subunits in Corydalis solida

Summary Two allelic forms of NAD specific malate dehydrogenase were found in samples of a wild population of Corydalis solida. The dimeric nature and the origin of the heterodimeric form has been demonstrated by in vitro dissociation and recombination of the subunits detected by subsequent electrophoresis. The method is applicable for polyacrylamide gel electrophoresis of crude leaf extracts of individual MDH isozyme forms.     

Five Cyanophora (Cyanophorales,Glaucophyta) species delineated based on morphological and molecular data

Cyanophora is an important glaucophyte genus of unicellular biflagellates that may have retained ancestral features of photosynthetic eukaryotes. The nuclear genome of Cyanophora was recently sequenced, but taxonomic studies of more than two strains are lacking for this genus. Furthermore, no study has used molecular methods to taxonomically delineate Cyanophora species. Here, we delimited the species of Cyanophora using light and electron microscopy, combined with molecular data from several globally distributed strains, including one newly established. Using a light microscope, we identified two distinct morphological groups: one with ovoid to ellipsoidal vegetative cells and another with dorsoventrally flattened or broad, bean‐shaped vegetative cells containing duplicated plastids. Our light and scanning electron microscopy clearly distinguished three species with ovoid to ellipsoidal cells (C. paradoxa Korshikov, C. cuspidata Tos.Takah. & Nozaki sp. nov., and C. kugrensii Tos.Takah. & Nozaki sp. nov.) and two species with broad, bean‐shaped cells (C. biloba Kugrens, B.L.Clay, C.J.Mey. & R.E.Lee and C. sudae Tos.Takah. & Nozaki sp. nov.) based on differences in cell shape and surface ornamentations of the vegetative cells under the field‐emission scanning electron microscope. Molecular phylogenetic analyses of P700 chl a apoprotein A2 (psaB) genes and internal transcribed spacer (ITS) regions of nuclear ribosomal DNA (rDNA), as well as a comparison of secondary structures of nuclear rDNA ITS‐2 and genetic distances of psaB genes, supported the delineation of five morphological species of Cyanophora.     

Discovery of quinazolin-4-amines bearing benzimidazole fragments as dual inhibitors of c-Met and VEGFR-2

Both c-Met and VEGFR-2 are important targets for the treatment of cancers. In this study, a series of N-(2-phenyl-1H-benzo[d]imidazol-5-yl)quinazolin-4-amine derivatives were designed and identified as dual c-Met and VEGFR-2 inhibitors. Among these compounds bearing quinazoline and benzimidazole fragments, compound 7j exhibited the most potent inhibitory activity against c-Met and VEGFR-2 with IC₅₀ of 0.05 μM and 0.02 μM, respectively. It also showed the highest anticancer activity against the tested cancer cell lines with IC₅₀ of 1.5 μM against MCF-7 and 8.7 μM against Hep-G2. Docking simulation supported the initial pharmacophoric hypothesis and suggested a common mode of interaction at the ATP-binding site of c-Met and VEGFR-2, which demonstrates that compound 7j is a potential agent for cancer therapy deserving further researching.     

High Expression of SOX2 and OCT4 Indicates Radiation Resistance and an Independent Negative Prognosis in Cervical Squamous Cell Carcinoma

Radiotherapy (RT) as a preoperative or postoperative adjuvant or primary treatment is the most common management modality for locally advanced cervical cancer. Radioresistance of tumor cells remains a major therapeutic problem. Consequently, we aimed to explore if the stem cell biomarkers SOX2 and OCT4 protein could be used to predict radioresistance in patients with locally advanced cervical squamous cell carcinoma (LACSCC). These 132 patients were divided into two groups (radiation-resistant and radiation-sensitive groups) according to progress-free survival (PFS). Using pretreatment paraffin-embedded tissues, we evaluated SOX2 and OCT4 expression using immunohistochemical staining. The percentage of overexpression of SOX2 and OCT4 in the radiation-resistant group was much higher than that in the radiation-sensitive group (p<0.001 and p <0.001, respectively). The patients with high expression of SOX2 and OCT4 showed a shorter PFS than those with low expression. Our study suggests that the expression of SOX2 and OCT4 in tumor cells indicates resistance to radiotherapy and that these two factors were important predictors of poor survival in patients with LACSCC (hazard ratio [95% CI], 2.294 [1.013, 5.195] and 2.300 [1.050, 5.037], respectively; p=0.046 and p=0.037, respectively).     

HS‐SPME‐GC‐FID method for detection and quantification of Bacillus cereus ATCC 10702 mediated 2‐acetyl‐1‐pyrroline

A rapid micro‐scale solid‐phase micro‐extraction (SPME) procedure coupled with gas‐chromatography with flame ionized detector (GC‐FID) was used to extract parts per billion levels of a principle basmati aroma compound “2‐acetyl‐1‐pyrroline” (2‐AP) from bacterial samples. In present investigation, optimization parameters of bacterial incubation period, sample weight, pre‐incubation time, adsorption time, and temperature, precursors and their concentrations has been studied. In the optimized conditions, detection of 2‐AP produced by Bacillus cereus ATCC10702 using only 0.5 g of sample volume was 85 μg/kg. Along with 2‐AP, 15 other compounds produced by B. cereus were also reported out of which 14 were reported for the first time consisting mainly of (E)?2‐hexenal, pentadecanal, 4‐hydroxy‐2‐butanone, n‐hexanal, 2–6‐nonadienal, 3‐methoxy‐2(5H) furanone and 2‐acetyl‐1‐pyridine and octanal. High recovery of 2‐AP (87 %) from very less amount of B. cereus samples was observed. The method is reproducible fast and can be used for detection of 2‐AP production by B. cereus. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1356–1363, 2014     

Optimization of a phenol extraction‐based protein preparation method amenable to downstream 2DE and MALDI‐MS based analysis of bacterial proteomes

2DE is one of the most efficient and widely used methods for resolving complex protein mixtures. For efficient analysis of complex samples, high‐resolution separation of proteins on 2D gel is essential, and for that purpose good sample preparation is crucial. In this study, we have improvized a method for preparing bacterial total cellular proteome, from a strategy applied earlier to recalcitrant plant tissues, which gave high‐quality resolution on 2DE. The method involving phenol extraction followed by methanol/ammonium acetate precipitation was first optimized for the chemolithotrophic proteobacteria Tetrathiobacter kashmirensis WT001 and Pseudaminobacter salicylatoxidans KCT001 that did not yield quality protein preps in conventional trichloroacetic acid/acetone precipitation method. Subsequently, to validate its general applicability, the method was evaluated against the trichloroacetic acid/acetone precipitation method for two other model bacteria, i.e. Escherichia coli DH5α and Mycobacterium smegmatis mc²6. Identification of at least four proteins each from the outer membrane, periplasm, and cytoplasm of T. kashmirensis by MALDI‐MS not only proved the efficiency of the method in extracting proteins from the different cellular compartments but also the amenability of the obtained protein spots toward MALDI‐MS based identification.     

Fibroblasts derived from long‐lived insulin receptor substrate 1 null mice are not resistant to multiple forms of stress

Reduced signalling through the insulin/insulin-like growth factor-1 signalling (IIS) pathway is a highly conserved lifespan determinant in model organisms. The precise mechanism underlying the effects of the IIS on lifespan and health is currently unclear, although cellular stress resistance may be important. We have previously demonstrated that mice globally lacking insulin receptor substrate 1 (Irs1^−/−) are long-lived and enjoy a greater period of their life free from age-related pathology compared with wild-type (WT) controls. In this study, we show that primary dermal fibroblasts and primary myoblasts derived from Irs1^−/− mice are no more resistant to a range of oxidant and nonoxidant chemical stressors than cells derived from WT mice.     

Genetic Background Influences P/Q-type Ca2+ Channel α1A Subunit mRNA Expression in Olfactory Bulb and Reproductive Ability of N-type Ca2+ Channel α1B Subunit-Deficient Mice

The Ca²⁺ channel α_1B subunit is a pore-forming component capable of generating N-type Ca²⁺ channel activity. Although the N-type Ca²⁺ channel plays a role in a variety of neuronal functions, α_1B-deficient mice did not show apparent behavioral abnormality. In a previous study, we observed a compensatory increase of mRNA expression of the P/Q-type Ca²⁺ channel α_1A subunit gene in olfactory bulb of α_1B-deficient mice with a CBA × C57BL/6 background; these mice showed a normal reproductive ability. In this study, we found that the mRNA expression level of the α_1A subunit was the same in olfactory bulb of wild, heterozygous, and homozygous α_1B-deficient mice with a CBA/JN background, and the homozygous male mice produced no offspring. These results suggest that the genetic background influences α_1A subunit mRNA expression and reproductive ability in α_1B-deficient mice.