Measurement of extracellular pH,K(+), and lactate in ischemic heart

Simultaneous and continuous measurements of extracellular pH, potassium (K(+)), and lactate (L(-)) in ischemic rabbit papillary muscle are presented for the first time. Potentiometric pH and K(+) sensors and an amperometric lactate biosensor were used. These miniature electrodes were previously developed and individually tested for this purpose. The pH sensor was based on an iridium oxide layer electrodeposited on a planar platinum electrode fabricated on a flexible substrate. The potentiometric K(+) sensor was based on a polymeric membrane and valinomycin ionophore. The L(-) biosensor was based on lactate oxidase and an organic conducting salt polarized at 0.15V vs Ag/AgCl reference electrode. The utility of this novel analytical system to cardiovascular research was demonstrated by using the system to study the interrelationship of cellular K(+) and lactate loss in ischemic myocardium, and the role of extracellular pH and buffer capacity on this relationship. The results indicated: (i) sequential brief episodes of ischemia produced reproducible trends of L(-), pH, and K(+) changes during the first three episodes, (ii) extracellular L(-) increased with increasing buffer capacity of extracellular compartment, (iii) the patterns of extracellular L(-) and K(+) changes were not related directly, and (iv) L(-) transport and lactic acid diffusion were not the primary cause of extracellular acidosis during ischemia.     

Synthesis and anti-HBV activity of thiouracils linked via S and N-1 to the 5-position of methyl beta-D-ribofuranoside

Reverse nucleoside derivatives of 2-(methylsulfanyl)uracils 6a-d were prepared by treating of the sodium salt of 2-(methylsulfanyl)uracils (5a-d) with methyl 2,3-O-isopropylidene-5-O-p-toluenesulfonyl-beta-D-ribofuranoside (2). The alkylation of 2-thiouracils 4a-d with methyl 5-deoxy-5-iodo-2,3-O-isopropylidene-D-ribofuranoside (3) afforded the corresponding S-ribofuranoside derivatives 8a-d. Deisopropylidenation of 6a-d and 8a-d afforded the corresponding deprotected derivatives 7a-d and 9a-d, respectively. The Anti-HBV activity of selected compounds was studied.     

Membrane redistribution of the Escherichia coli MinD protein induced by MinE

Escherichia coli cells contain potential division sites at midcell and adjacent to the cell poles. Selection of the correct division site at midcell is controlled by three proteins: MinC, MinD, and MinE. It has previously been shown (D. Raskin and P. de Boer, Cell 91:685-694, 1997) that MinE-Gfp localizes to the midcell site in an MinD-dependent manner. We use here Gfp-MinD to show that MinD associates with the membrane around the entire periphery of the cell in the absence of the other Min proteins and that MinE is capable of altering the membrane distribution pattern of Gfp-MinD. Studies with the isolated N-terminal and C-terminal MinE domains indicated different roles for the two MinE domains in the redistribution of membrane-associated MinD.     

Effect of Al, Co, and Pb ions on growth ofFrankia spp. in a mineral medium

Growth of three Frankia strains associated with roots of Casuarina tree, treated with different concentrations of metal ions, was measured as total protein content. One strain was highly resistant to different aluminum ion concentrations up to 10 mmol/L. The other two strains were more sensitive to the higher aluminum concentrations (1.25-10 mmol/L). Growth inhibition by cobalt and lead concentrations varied, depending on the tested strain. Stimulation occurred only at cobalt concentrations of 0.33 and 0.65 mmol/L for one strain.     

Protein-protein interactions in receptor activation and intracellular signalling

We review here signalling complexes that we have defined using X-ray analysis in our laboratory. They include growth factors and their receptors: nerve growth factor (NGF) and its hetero-hexameric 7S NGF storage complex, hepatocyte growth factor/scatter factor (HGF/SF) NK1 dimers and fibroblast growth factor (FGF1) in complex with its receptor (FGFR2) ectodomain and heparin. We also review our recent structural studies on intracellular signalling complexes, focusing on phosducin transducin GPry, CK2 protein kinase and its complexes, and the cyclin D-dependent kinase, Cdk6, bound to the cell cycle inhibitor p19INK4d. Comparing the structures of these complexes with others we show that the surface area buried in signalling interactions does not always give a good indication of the strength of the interactions. We show that conformational changes are often important in complexes with intermediate buried surface areas of 1500 to 2000 A2, such as Cdk6INK4 interactions. Some interactions involve recognition of continuous epitopes, where there is no necessity for a tertiary structure and very often the binding conformation is induced during the process of interaction, for example phosducin binding to the betagamma subunits (Gtbetagamma) of the heterotrimeric G protein transducin.     

The hydrophobic lock-and-key intersubunit motif of glutathione transferase A1-1: implications for catalysis, ligandin function and stability

A hydrophobic lock-and-key intersubunit motif involving a phenylalanine is a major structural feature conserved at the dimer interface of classes alpha, mu and pi glutathione transferases. In order to determine the contribution of this subunit interaction towards the function and stability of human class alpha GSTA1-1, the interaction was truncated by replacing the phenylalanine 'key' Phe-51 with serine. The F51S mutant protein is dimeric with a native-like core structure indicating that Phe-51 is not essential for dimerization. The mutation impacts on catalytic and ligandin function suggesting that tertiary structural changes have occurred at/near the active and non-substrate ligand-binding sites. The active site appears to be disrupted mainly at the glutathione-binding region that is adjacent to the lock-and-key intersubunit motif. The F51S mutant displays enhanced exposure of hydrophobic surface and ligandin function. The lock-and-key motif stabilizes the quaternary structure of hGSTA1-1 at the dimer interface and the protein concentration dependence of stability indicates that the dissociation and unfolding processes of the mutant protein remain closely coupled.     

Potential protective effects of melatonin on bone marrow of rats exposed to cytotoxic drugs

Myelosuppression is the most serious, dose limiting, toxicity of cytotoxic drugs. Efforts to protect the bone marrow have been only variably successful, and no agreement exists on how to approach this problem. Melatonin, the major hormonal product of the pineal gland, is supposed to have both chemoprotective and myelostimulatory effects. This experimental study was carried out to test these two effects on the bone marrow of rats, daily intraperitoneally injected with 100 microg melatonin. Injection of 10 mg aracytin for 10 days produced a significant (P < 0.01) decrease in red blood cells count (RBCs), total leucocytic count, as well as platelets count. When melatonin was injected along with aracytin, it would significantly increase (P < 0.05) RBC count and (P < 0.01) blood platelet count. Injection of melatonin after aracytin treatment would significantly increase (P < 0.01) RBC, total leucocytic and platelet counts in comparison with rats treated with aracytin only. The effects of melatonin were more clear in rats treated with it after aracytin injection than those treated with melatonin and aracytin at the same time. Furthermore, it was found that aracytin produced a significant (P < 0.01) decrease in serum total proteins, albumin, and significantly increased the (P < 0.01) albumin/globulin ratio. Melatonin injection would significantly increase (P < 0.01) total protein, globulin, and significantly decrease (P < 0.01) the albumin/glubulin ratio when injected either with aracytin or after aracytin treatment. These results indicate that melatonin protects bone marrow, lymphoid tissues from damaging effect of cytotoxic drugs, as well as stimulating the suppressed bone marrow.     

Purification of the two major proteins from whey concentrate using a cation‐exchange selective adsorption process

The packed‐bed adsorption and elution of aqueous solutions of whey concentrate powders were investigated at pH 3.7 using a 5‐mL SP Sepharose FF column to separate and isolate two major proteins namely, α‐lactalbumin (ALA) and β‐lactoglobulin (BLG) from these solutions. ALA displaced and eluted BLG from the column in a pure form. Pure ALA could then be eluted with good recovery. A novel consecutive two‐stage separation process was developed to separate ALA and BLG from whey concentrate mixtures. Almost all of the BLG in the feed was recovered, with 78% being recovered at 95% purity and a further 20% at 86% purity. In addition, 67% of ALA was recovered, 48% at 54% purity and 19% at 60% purity. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Efficient isolation of high quality nucleic acids from different tissues of <Emphasis Type="Italic">Taxus baccata</Emphasis> L.

Improved and efficient methods were developed for isolating high quality DNA and RNA from different sources of Iranian Yew (Taxus baccata L.). The methods were based on CTAB extraction buffer added with high levels of polyvinylpyrrolidone (PVP) and β-mercaptoethanol to properly remove polysaccharides and prevent oxidation of phenolics. The pellets obtained by ethanol precipitation were washed only with Chloroform: isoamyl alcohol (24:1). So, we could successfully eliminate the dangerous phenol/chloroform extraction steps from the isolation procedure. Both spectrophotometric (A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀ ratios) and agarose electrophoresis analysis of isolated nucleic acids (DNA and RNA) indicated good results. DNA with the average yield of 100–300 μg/g leaf and stem tissue and total RNA with an average yield of 20–30 μg/g cell culture and 80–100 μg/g leaf and stem tissue of Iranian yew could be obtained. Successful amplification of pam and pds by PCR and RT-PCR, showed the integrity of isolated DNA and RNA, respectively.     

Sperm status and DNA dose play key roles in sperm/ICSI‐mediated gene transfer in caprine

In relation to the growing recent interest in the establishment of sperm‐mediated gene transfer (SMGT) technology as a convenient and effective method for the simple production of transgenic animals, in this study the possibility of using SMGT to produce transgenic caprine embryos was investigated for the first time. Buck sperm were directly incubated with different concentrations (0–500 ng) of pcDNA/his/Lac‐Z plasmid and used for IVF or ICSI. Sperm used for ICSI were categorized into motile or live‐immotile group before being injected into oocytes. In a separate experiment, dead sperm prepared by repeated freezing/thawing were used for DNA‐incubation before ICSI. Sham injection was carried out by intracytoplasmic injection of approximately the same volume of media containing different doses of DNA using an ICSI needle. Transgene expression and transmission were detected by X‐Gal staining and PCR analysis of developed embryos, respectively. A reasonable blastocyst rate was observed in all the groups. Only embryos in the sham group were negative for transgene transmission. Transgene expression was completely dependent on the delivery technique and status of sperm, and was only observed in the live‐immotile and dead ICSI groups. The results of this study showed that the technique (IVF vs. ICSI vs. sham injection), sperm status (motile vs. live‐immotile vs. dead) and to some extent DNA concentration affect embryo development, transgene transmission and expression. Mol. Reprod. Dev. 77:868–875, 2010. © 2010 Wiley‐Liss, Inc.