Development of Genus/Species-Specific PCR Analysis for Identification of <Emphasis Type="Italic">Carnobacterium</Emphasis> Strains

Heterofermentative lactic acid bacteria belonging to the genus Carnobacterium are currently divided into seven different species, C. piscicola, C. mobile, C. gallinarum, C. inhibens, C. divergens, C. funditum, and C. alterfunditum. 16S rDNA-targeted PCR assay was carried out for the identification of the genus Carnobacterium. In addition, type strains of all Carnobacterium species were analyzed by 16S–23S rDNA intergenic spacer analysis in comparison with type strains of phylogenetically related lactic acid bacteria. These methods enabled the identification and the discrimination among Carnobacterium species and the other phylogenetically related lactic acid bacteria. Likewise, analogous results were obtained by restriction analysis of amplified 16S rDNA performed with HaeIII and HinfI as restriction enzymes. Received: 25 July 2001 / Accepted: 19 October 2001     

Molecular dynamics study of a hyperthermophilic and a mesophilic rubredoxin

In recent years, increased interest in the origin of protein thermal stability has gained attention both for its possible role in understanding the forces governing the folding of a protein and for the design of new highly stable engineered biocatalysts. To study the origin of thermostability, we have performed molecular dynamics simulations of two rubredoxins, from the mesophile Clostridium pasteurianum and from the hyperthermophile Pyrococcus furiosus. The simulations were carried out at two temperatures, 300 and 373 K, for each molecule. The length of the simulations was within the range of 6-7.2 ns. The rubredoxin from the hyperthermophilic organism was more flexible than its mesophilic counterpart at both temperatures; however, the overall flexibility of both molecules at their optimal growth temperature was the same, despite 59% sequence homology. The conformational space sampled by both molecules was larger at 300 K than at 373 K. The essential dynamics analysis showed that the principal overall motions of the two molecules are significantly different. On the contrary, each molecule showed similar directions of motion at both temperatures.     

Ionic network at the C-terminus of the beta-glycosidase from the hyperthermophilic archaeon Sulfolobus solfataricus: Functional role in the quaternary structure thermal stabilization

Biochemical, crystallographic, and computational data support the hypothesis that electrostatic interactions are among the dominant forces in stabilizing hyperthermophilic proteins. The thermostable beta-glycosidase from the hyperthermophile Sulfolobus solfataricus (Ssbeta-gly) is an interesting model system for the study of protein adaptation to high temperatures. The largest ion-pair network of Ssbeta-gly is located at the tetrameric interface of the molecule; in this paper, key residues in this region were modified by site-directed mutagenesis and the stability of the mutants was analyzed by kinetics of thermal denaturation. All mutations produced faster enzyme inactivation, suggesting that the C-terminal ionic network prevents the dissociation into monomers, which is the limiting step in the mechanism of Ssbeta-gly inactivation. Moreover, the calculated reaction order showed that the mechanism of inactivation depends on the mutation introduced, suggesting that intermediates maintaining enzymatic activity are produced during the inactivation transition of some, but not all, mutants. Molecular models of each mutant allow us to rationalize the experimental evidence and give support to the current theories on the mechanism of ion pair stabilization in proteins from hyperthermophiles.     

The nucleotide-independent Fe(III)-binding site is located on beta subunit of the mitochondrial F(1)-ATPase

The need for methods to identify disease biomarkers is underscored by the survival-rate of patients diagnosed at early stages of cancer progression. Surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) is a novel approach to biomarker discovery that combines two powerful techniques: chromatography and mass spectrometry. One of the key features of SELDI-TOF MS is its ability to provide a rapid protein expression profile from a variety of biological and clinical samples. It has been used for biomarker identification as well as the study of protein-protein, and protein-DNA interaction. The versatility of SELDI-TOF MS has allowed its use in projects ranging from the identification of potential diagnostic markers for prostate, bladder, breast, and ovarian cancers and Alzheimer's disease, to the study of biomolecular interactions and the characterization of posttranslational modifications. In this minireview we discuss the application of SELDI-TOF MS to protein biomarker discovery and profiling.     

NMDA receptor stimulation induces temporary alpha-tubulin degradation signaled by nitric oxide-mediated tyrosine nitration in the nervous system of Sepia officinalis

Biochemical and immunohistochemical evidence is reported, showing basal protein nitration in specific regions of the optic lobes of Sepia officinalis, mainly in the fiber layers of the plexiform zone. SDS-PAGE analysis of optic lobe extracts revealed an intense 3-nitrotyrosine immunoreactive band identified as alpha-tubulin by immunoprecipitation and partial purification. Stimulation of NMDA receptors resulted in a selective decrease in alpha-tubulin levels within 30 min with partial recovery after 4 h. The effect was suppressed by the NO synthase (NOS) inhibitor L-nitroarginine. Incubation of optic lobes with free 3-nitrotyrosine resulted likewise in a selective loss of alpha-tubulin, due apparently to incorporation of the amino acid into the C-terminus of detyrosinated alpha-tubulin to give the nitrated protein purportedly more susceptible to degradation. Overall, these results point to a novel potential physiologic role of NO and free 3-nitrotyrosine in the control of the alpha-tubulin tyrosination/detyrosination cycle and turnover in Sepia nervous tissue.     

Comparative cytotoxic and cytoprotective effects of taurohyodeoxycholic acid (THDCA) and tauroursodeoxycholic acid (TUDCA) in HepG2 cell line

This study was performed to compare the effects of two hydrophilic bile acids, taurohyodeoxycholic acid (THDCA) and tauroursodeoxycholic acid (TUDCA), on HepG2 cells. Cytotoxicity was evaluated at different times of exposure by incubating cells with increasing concentrations (50-800 micromol/l) of either bile acid, while their cytoprotective effect was tested in comparison with deoxycholic acid (DCA) (350 micromol/l and 750 micromol/l)-induced cytotoxicity. Culture media, harvested at the end of each incubation period, were analyzed to evaluate aspartate transaminase (AST), alanine transaminase and gamma-glutamyltranspeptidase release. In addition, the hemolytic effect of THDCA and TUDCA on human red blood cells was also determined. At 24 h of incubation neither THDCA nor TUDCA was cytotoxic at concentrations up to 200 and 400 micromol/l. At 800 micromol/l both THDCA and TUDCA induced a slight increase in AST release. At this concentration and with time of exposure prolonged up to 72 h, THDCA and TUDCA induced a progressive increase of AST release significantly (P<0.05) higher than that of controls being AST values for THDCA (2.97+/-0.88 time control value (tcv) at 48 h and 4.50+/-1.13 tcv at 72 h) significantly greater than those of TUDCA (1.50+/-0.20 tcv at 48 h and 1.80+/-0.43 tcv at 72 h) (P<0.01). In cytoprotection experiments, the addition of 50 micromol/l THDCA decreased only slightly (-5%) AST release induced by 350 micromol/l DCA, while the addition of 50 micromol/l TUDCA was significantly effective (-23%; P<0.05). Higher doses of THDCA or TUDCA did not reduce toxicity induced by 350 micromol/l DCA, but were much less toxic than an equimolar dose of DCA alone. At the concentration used in this experimental model neither THDCA nor TUDCA was hemolytic; however at a very high concentration (6 mmol/l) both bile acids induced 5-8% hemolysis. We conclude that bile acid molecules with a similar degree of hydrophilicity may show different cytotoxic and cytoprotective properties.     

A calcium-permeable channel activated by muscarinic acetylcholine receptors and InsP3 in developing chick ciliary ganglion neurons

The electrical responses elicited by the muscarinic cholinergic pathway have been studied in cultured embryonic chick ciliary ganglion (CG) neurons. Neurons obtained from E7-E8 ganglia were maintained in serum-free medium for 1 to 3 days. Stimulation with 50 microM muscarine induced depolarizing responses in about 30% of the cells tested. In voltage clamp experiments at a holding potential of -50 mV, an inward current could be recorded in the same percentage of cells in response to muscarinic stimulation. In single channel experiments, with standard physiological solution in the pipette, muscarine transiently activated an inward conducting channel. Cell-attached recordings with 100 mM CaCl(2) in the pipette provided evidence that muscarinic agonists can activate a cationic calcium-permeable channel. Two main conductance levels could be detected, of 2.3+/-0.6 and 5.6+/-0.6 pS, respectively. In excised patches, addition of 5-20 microM inositol 1,4,5-trisphosphate (InsP(3)) to the bath reactivated a channel that could be blocked by heparin and whose characteristics were very similar to those of the channel seen in response to muscarinic stimulation. A channel with similar properties has been previously shown to be activated by basic fibroblast growth factor (bFGF) and InsP(3) in the same preparation.     

Glutathione transferase isoenzymes from frog (Xenopus laevis) liver and embryo

The expression of glutathione transferase isoenzymes has been investigated in embryo and adult liver of the frog Xenopus laevis. By analysing the GST isoenzymes recovered from GSH-affinity chromatography in terms of electrophoretic mobility, HPLC elution profile, immunological reactivity, N-terminal amino acid sequence and mass spectrometry molecular mass no significant difference in the GST subunit composition between embryos and liver was found. In both tissues the same three subunits, showing similarity to mu, alpha and sigma class GSTs, are present. These results, together with those previously reported for toad (Bufo bufo), strongly support the notion that the transition from an aquatic environment to a terrestrial atmosphere containing high oxygen concentration has accompanied specific GST gene expression.     

Sequence and functional similarities between pro-apoptotic Bid and plant lipid transfer proteins

Pro-apoptotic proteins of the Bcl-2 family are known to act on mitochondria and facilitate the release of cytochrome c, but the biochemical mechanism of this action is unknown. Association with mitochondrial membranes is likely to be important in determining the capacity of releasing cytochrome c. The present work provides new evidence suggesting that some pro-apoptotic proteins like Bid have an intrinsic capacity of binding and exchanging membrane lipids. Detailed analysis indicates a significant sequence similarity between a subset of Bcl-2 family proteins including Bid and Nix and plant lipid transfer proteins. The similar structural signatures could be related to common interactions with membrane lipids. Indeed, isolated Bid shows a lipid transfer activity that is even higher than that of plant lipid transfer proteins. To investigate the possible relevance of these structure-function correlations to the apoptotic action of Bid, cell free assays were established with isolated mitochondria, recombinant Bid and a variety of exogenous lipids. Micromolar concentrations of lysolipids such as lysophosphatidylcholine were found to change the association of Bid with mitochondria and also stimulate the release of cytochrome c promoted by Bid. The changes in mitochondrial association and cytochrome c release were enhanced by the presence of liposomes of lipid composition similar to that of mitochondrial membranes. Thus, a mixture of liposomes, mitochondria and key lysolipids could reproduce the conditions enabling Bid to transfer lipids between donor and acceptor membranes, and also change its reversible association with mitochondria. Bid was also found to enhance the incorporation of a fluorescent lysolipid, but not of a related fatty acid, into mitochondria. On the basis of the results presented here, it is hypothesised that Bid action may depend upon its capacity of exchanging lipids and lysolipids with mitochondrial membranes. The hypothesis is discussed in relation to current models for the integrated action of pro-apoptotic proteins of the Bcl-2 family.