Evaluation of GABA Production and Probiotic Activities of Enterococcus faecium BS5

Probiotics and Antimicrobial Proteins - Gamma-aminobutyric acid (GABA) is a principal inhibitory neurotransmitter in the central nervous system and is produced by irreversible decarboxylation of...     

Trypanosoma brucei brucei: biochemical characterization of ecto-nucleoside triphosphate diphosphohydrolase activities

In this work we describe the ability of living cells of Trypanosoma brucei brucei to hydrolyze extracellular ATP. In these intact parasites there was a low level of ATP hydrolysis in the absence of any divalent metal (4.72+/-0.51 nmol Pi x 10(-7) cells x h(-1)). The ATP hydrolysis was stimulated by MgCl(2) and the Mg-dependent ecto-ATPase activity was 27.15+/-2.91 nmol Pi x 10(-7) cells x h(-1). This stimulatory activity was also observed when MgCl(2) was replaced by MnCl(2). CaCl(2) and ZnCl(2) were also able to stimulate the ATPase activity, although less than MgCl(2). The apparent K(m) for ATP was 0.61 mM. This ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities. To confirm that this Mg-dependent ATPase activity is an ecto-ATPase activity, we used an impermeable inhibitor, DIDS (4, 4'-diisothiocyanostylbene 2'-2'-disulfonic acid), as well as suramin, an antagonist of P(2) purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg(2+)-dependent ATPase activity in a dose-dependent manner. Living cells sequentially hydrolyzed the ATP molecule generating ADP, AMP and adenosine, and supplementation of the culture medium with ATP was able to sustain the proliferation of T. brucei brucei as well as adenosine supplementation. Furthermore, the E-NTPDase activity of T. brucei brucei is modulated by the availability of purines in the medium. These results indicate that this surface enzyme may play a role in the salvage of purines from the extracellular medium in T. brucei brucei.     

Monolayer formation of human osteoblastic cells on vertically aligned multiwalled carbon nanotube scaffolds

Monolayer formation of SaOS‐2 (human osteoblast‐like cells) was observed on VACNT (vertically aligned multiwalled carbon nanotubes) scaffolds without purification or functionalization. The VACNT were produced by a microwave plasma chemical vapour deposition on titanium surfaces with nickel or iron as catalyst. Cell viability and morphology studies were evaluated by LDH (lactate dehydrogenase) release assay and SEM (scanning electron microscopy), respectively. The non‐toxicity and the flat spreading with monolayer formation of the SaOs‐2 on VACNT scaffolds surface indicate that they can be used for biomedical applications.     

Serine racemase modulates intracellular D-serine levels through an alpha,beta-elimination activity

Mammalian brain contains high levels of d-serine, an endogenous co-agonist of N-methyl D-aspartate type of glutamate receptors. D-Serine is synthesized by serine racemase, a brain enriched enzyme converting L- to D-serine. Degradation of D-serine is achieved by D-amino acid oxidase, but this enzyme is not present in forebrain areas that are highly enriched in D-serine. We now report that serine racemase catalyzes the degradation of cellular D-serine itself, through the alpha,beta-elimination of water. The enzyme also catalyzes water alpha,beta-elimination with L-serine and L-threonine. alpha,beta-Elimination with these substrates is observed both in vitro and in vivo. To investigate further the role of alpha,beta-elimination in regulating cellular D-serine, we generated a serine racemase mutant displaying selective impairment of alpha,beta-elimination activity (Q155D). Levels of D-serine synthesized by the Q155D mutant are several-fold higher than the wild-type both in vitro and in vivo. This suggests that the alpha,beta-elimination reaction limits the achievable D-serine concentration in vivo. Additional mutants in vicinal residues (H152S, P153S, and N154F) similarly altered the partition between the alpha,beta-elimination and racemization reactions. alpha,beta-Elimination also competes with the reverse serine racemase reaction in vivo. Although the formation of L- from D-serine is readily detected in Q155D mutant-expressing cells incubated with physiological D-serine concentrations, reversal with wild-type serine racemase-expressing cells required much higher D-serine concentration. We propose that alpha,beta-elimination provides a novel mechanism for regulating intracellular D-serine levels, especially in brain areas that do not possess D-amino acid oxidase activity. Extracellular D-serine is more stable toward alpha,beta-elimination, likely due to physical separation from serine racemase and its elimination activity.     

Immunophenotyping of human HT29 colon cancer cell primary tumours and their metastases in severe combined immunodeficient mice

The immunophenotype of HT29 human colon cancer cells implanted into severe combined immunodeficient mice was assessed in primary tumours and their metastases in the lungs using an indirect immunohistochemical method. After primary tumours were surgically removed, the metastases were given time to develop, thus paralleling the clinical situation. While vimentin was negative in both primary and secondary tumours, E-cadherin was present as membrane-bound labelling in the primary tumours only. Whereas the markers p53, MIB1, PCNA and CEA were consistently positive in both primary and metastatic tumours, CD44 variant 6 and CA125 were negative in metastases but positive in the primary tumours. There was a significant increase in the percentage of cells labelled for p53 in the primary tumours compared with the metastases. For the proliferation markers, there was no significant difference in labelling between primary tumours and metastases for MIB1. Of the cytokeratins examined, CK 20 gave the strongest and most consistent reaction in both primary and secondary tumours. The results indicate that, for certain immunohistochemical markers, results are the same in both primary tumours and metastases. Hence, in these cases, antigens that are expressed on the primary tumour as well as on the metastases can serve as target molecules for immunologically based forms of treatment of metastases. This revised version was published online in November 2006 with corrections to the Cover Date.     

Comparisons of the molecular evolutionary process at rbcL and ndhF in the grass family (Poaceae)

We examine rate heterogeneity among evolutionary lineages of the grass family at two plasmid loci, ndhF and rbcL, and we introduce a method to determine whether patterns of rate heterogeneity are correlated between loci. We show both that rates of synonymous evolution are heterogeneous among grass lineages and that are heterogeneity is correlated between loci at synonymous sites. At nonsynonymous sites, the pattern of rate heterogeneity is not correlated between loci, primarily due to an aberrant pattern of rate heterogeneity at nonsynonymous sites of rbcL. We compare patterns of synonymous rate heterogeneity to predictors based on the generation time effect and the speciation rate hypotheses. Although there is some evidence for generation time effects, neither generation time effects nor speciation rates appear to be sufficient to explain patterns of rate heterogeneity in the grass plastid sequences.      

Neurobiology through the looking-glass: D-serine as a new glial-derived transmitter

D-Amino acids have been known to be present in bacteria for more than 50 years, but only recently they were identified in mammals. The occurrence of D-amino acids in mammals challenge classic concepts in biology in which only L-amino acids would be present or thought to play important roles. Recent discoveries uncovered a role of endogenous D-serine as a putative glial-derived transmitter that regulates glutamatergic neurotransmission in mammalian brain. Free D-serine levels in the brain are about one third of L-serine values and its extracellular concentration is higher than many common L-amino acids. D-Serine occurs in protoplasmic astrocytes, a class of glial cells that ensheath the synapses and modulate neuronal activity. Biochemical and electrophysiological studies suggest that endogenous D-serine is a physiological modulator at the co-agonist site of NMDA-type of glutamate receptors. We previously showed that D-serine is synthesized by a glial serine racemase, a novel enzyme converting L- to D-serine in mammalian brain. The enzyme requires pyridoxal 5'-phosphate and it was the first racemase to be cloned from eucaryotes. Inhibitors of serine racemase have therapeutic implications for pathological processes in which over-stimulation of NMDA receptors takes place, such as stroke and neurodegenerative diseases. Here, we review the role of endogenous D-serine in modulating NMDA neurotransmission, its biosynthetic apparatus and the potential usefulness of serine racemase inhibitors as a novel neuroprotective strategy to decrease glutamate/NMDA excitotoxicity.