The Detection of Hamster Connexins: A Comparison of Expression Profiles with Wild-Type Mouse and the Cancer-Prone Min Mouse

The open reading frames of 17 connexins from Syrian hamster (using tissues) and 16 connexins from the Chinese hamster cell line V79, were fully (Cx30, Cx31, Cx37, Cx43 and Cx45) or partially sequenced. We have also detected, and partially sequenced, seven rat connexins that previously were unavailable. The expression of connexin genes was examined in some hamster organs and cultured hamster cells, and compared with wild-type mouse and the cancer-prone Min mouse. Although the expression patterns were similar for most organs and connexins in hamster and mouse, there were also some prominent differences (Cx29 and 30.3 in testis; Cx31.1 and 32 in eye; Cx46 in brain, kidney and testis; Cx47 in kidney). This suggests that some connexins have species-specific expression profiles. In contrast, there were minimal differences in expression profiles between wild type and Min mice. Species-specific expression profiles should be considered in attempts to make animal models of human connexin-associated diseases.     

Investigating the effect of cell substrate on cancer cell stiffness by optical tweezers

The mechanical properties of cells are influenced by their microenvironment. Here we report cell stiffness alteration by changing the cell substrate stiffness for isolated cells and cells in contact with other cells. Polydimethylsiloxane (PDMS) is used to prepare soft substrates with three different stiffness values (173, 88 and 17 kPa respectively). Breast cancer cells lines, namely HBL-100, MCF-7 and MDA-MB-231 with different level of aggressiveness are cultured on these substrates and their local elasticity is investigated by vertical indentation of the cell membrane. Our preliminary results show an unforeseen behavior of the MDA-MB-231 cells. When cultured on glass substrate as isolated cells, they are less stiff than the other two types of cells, in agreement with the general statement that more aggressive and metastatic cells are softer. However, when connected to other cells the stiffness of MDA-MB-231 cells becomes similar to the other two cell lines. Moreover, the stiffness of MDA-MB-231 cells cultured on soft PDMS substrates is significantly higher than the stiffness of the other cell types, demonstrating thus the strong influence of the environmental conditions on the mechanical properties of the cells.     

Cryptic species identification: a simple diagnostic tool for discriminating between two problematic bumblebee species

Distinguishing between cryptic species is a perennial problem for biologists. Bombus ruderatus and Bombus hortorum are two species of bumblebee, which can be indistinguishable from their morphology. The former species is in decline, whereas the latter is ubiquitous. In the UK, isolated records of B. ruderatus occur amongst many for B. hortorum. For ecological studies of B. ruderatus to be feasible, the two species need to be reliably distinguishable. We present a diagnostic tool for quick and reliable identification of problematic individuals based on a restriction enzyme digest of the cytochrome b region of mitochondrial DNA.     

Cardiolipin biosynthesis and remodeling enzymes are altered during development of heart failure

Cardiolipin (CL) is responsible for modulation of activities of various enzymes involved in oxidative phosphorylation. Although energy production decreases in heart failure (HF), regulation of cardiolipin during HF development is unknown. Enzymes involved in cardiac cardiolipin synthesis and remodeling were studied in spontaneously hypertensive HF (SHHF) rats, explanted hearts from human HF patients, and nonfailing Sprague Dawley (SD) rats. The biosynthetic enzymes cytidinediphosphatediacylglycerol synthetase (CDS), phosphatidylglycerolphosphate synthase (PGPS) and cardiolipin synthase (CLS) were investigated. Mitochondrial CDS activity and CDS-1 mRNA increased in HF whereas CDS-2 mRNA in SHHF and humans, not in SD rats, decreased. PGPS activity, but not mRNA, increased in SHHF. CLS activity and mRNA decreased in SHHF, but mRNA was not significantly altered in humans. Cardiolipin remodeling enzymes, monolysocardiolipin acyltransferase (MLCL AT) and tafazzin, showed variable changes during HF. MLCL AT activity increased in SHHF. Tafazzin mRNA decreased in SHHF and human HF, but not in SD rats. The gene expression of acyl-CoA: lysocardiolipin acyltransferase-1, an endoplasmic reticulum MLCL AT, remained unaltered in SHHF rats. The results provide mechanisms whereby both cardiolipin biosynthesis and remodeling are altered during HF. Increases in CDS-1, PGPS, and MLCL AT suggest compensatory mechanisms during the development of HF. Human and SD data imply that similar trends may occur in human HF, but not during nonpathological aging, consistent with previous cardiolipin studies.     

Studies on the structure of yeast phosphofructokinase

In this paper, we describe an efficient procedure for the purification of yeast phosphofructokinase. This procedure eliminates any time delay and enables to obtain an enzyme with minimum proteolytic alterations. The molecular weights of the oligomeric enzyme and of its constitutive subunits were both evaluated by means of several independent methods. However, the accuracy of each measurement was not sufficient to discriminate between an hexameric and an octameric structure of the enzyme oligomer. On the other hand, crosslinking experiments demonstrated the octameric structure of yeast phosphofructokinase. Obviously, some methods of molecular weight determination have led to erroneous results. In particular, our experiments show that the reliability of molecular weight determinations performed by gel filtration of native proteins must be considered with caution.     

Variation of Pleurotus ostreatus (Jacq. Ex Fr.) P. Kumm. (1871) performance subjected to differentdoses and timings of nano-urea

Supplementation of the growing substrate by nitrogenous additives has been known to improve the production of oyster mushroom (Pleurotus ostreatus (Jacq. ex Fr.) P. Kumm. (1871)). However, the application of nano-additives has not been reported in such cultivation yet. The study investigated the effect of nano-urea added in two different doses (3 g and 5 g per kg substrate), once (at spawning or after first flush) or twice (at spawning and after first flush) to the growing substrate consisting of wheat straw and spent oyster substrate (1:1, w/w). Results showed that the application of nano-urea once has induced the highest number of mushroom flushes (four flushes) despite the dose applied. Contrarily to early findings, where high doses of nitrogen have caused inhibition of mushroom growth and production, nano-urea application has had better effects when applied twice. With 5 g/kg, it induced the shortest period between the first and the third flush (15 days). With 3 g/kg, it resulted in the highest biological and economic yields at the third flush (332.7 g/bag and 283.1 g/bag respectively), in total (973.4 g/bag and 854.0 g/bag respectively), the highest biological efficiency (109.6%), and pileus diameter/stipe length ratio (2.8). Experimental findings of the current study may be potentially applied at commercial scale.     

Cloning,over expression and functional attributes of serine proteases from Oceanobacillus iheyensis O.M.A18 and Haloalkaliphilic bacterium O.M.E12

Cloning, over-expression, characterization and structural and functional analysis of two alkaline proteases from the newly isolated haloalkaliphilic bacteria: Oceanobacillus iheyensis O.M.A₁8 and Haloalkaliphilic bacterium O.M.E₁2 were carried out. The cloned protease genes were over-expressed in Escherichia coli within 6 h of the IPTG induction. The protease genes were sequenced and the sequence submitted to the GenBank with the accession numbers, HM219179 and HM219182. The recombinant proteases were active in the range of pH 8–11 and temperature 30–50 °C. The amino acid sequences of the alkaline proteases displayed hydrophobic character and stable configurations. The amino acids Asp 141, His 171 and Ser 324 formed the catalytic triad, while Ile, Leu and Ser were other amino acid moieties present in the active site. The characteristics of the recombinant proteases were compared and found to be similar to their native counterparts. On the basis of the in-silico analysis and inhibitor studies, the enzymes were confirmed as serine proteases. The study hold significance as only limited enzymes from the haloalkaliphilic bacteria have been cloned, sequenced and analyzed for the structure and function analysis.     

Oryzalexin S,a Novel Stemarane-type Diterpene Rice Phytoalexin

TTUR 2-2, an alkalophilic Bacillus strain isolated from soil, grew well in media containing cholic acid (CA) at 5% or higher and efficiently converted 7α- and 12α-hydroxyl groups of CA to keto groups, with the conversion rate for both hydroxyl groups reaching 100% by 72 hours of cultivation. The strain also converted a 3α-hydroxyl group to a keto group, but the conversion rate was about 5% at 72 hours. The strain neither affected any other part of the CA molecule, nor oxidized 7β- or 12 β -hydroxyl groups.

By NTG mutagenesis, the following mutants were acquired; (1) converting only the 7α- and 12α-hydroxyl groups, (2) converting only the 12α-hydroxyl group, and (3) converting only the 7α-hydroxyl group. These mutants selectively produce 12-ketochenodeoxycholic acid (12KCDCA), 7-ketodeoxycholic acid (7KDOCA), and 7,12-diketolithocholic acid (7,12DKLCA), from CA; and 7-ketolithocholic acid (7KLCA) from cheno-deoxycholic acid (CDCA), respectively, at high yields, close to 100%.     

Proteomic Analysis of Specific Proteins in the Root of Salt-Tolerant Barley

Comparative two-dimensional electrophoresis showed six proteins, which were significantly produced in the root of salt-tolerant barley. These proteins were identified as stress/defense-related proteins that do not scavenge reactive oxygen species directly, suggesting that salt-tolerant barley develops not only an antioxidative system, but also physical and biochemical changes to cope with salt stress.