Halophilic archaea isolated from man-made young (200 years) salt lakes in Slănic, Prahova, Romania

We isolated a number of extremely halophilic archaea from four salt lakes (Red Bath, Green Bath, Shepherd Bath and Bride Cave) located in Slanic Prahova, Romania. The characterization of 12 representative isolates by polyphasic approach revealed that 11 strains were members of the genus Haloferax and only one was a member of the genus Haloarcula. The 11 Haloferax isolates possessed sulfated diglycosylarchaeol-1 as the major membrane glycolipid, and G+C contents of total DNA were 63.4–65.8 mol%. The predominant isolation of Haloferax species from the lakes may suggest that the underground salt deposit possesses Haloferax species as the major biota of ancient origin. To the best of the authors’ knowledge, this is the first paper on the survey of halophilic archaea of man-made young salt lakes.     

定向进化人α型干扰素基因家族的研究

应用DNA改组(DNAshuffling)技术重组了12种人α型干扰素基因,并结合噬菌体表面呈现(phagedisplay)技术构建了噬菌体干扰素改组文库,采用简便的、功能性的定向竞争筛选方法,获得了比活达1 0×109IU/mg的新型α型基因工程复合干扰素,是目前国际上已投产的α2型干扰素的5倍,具有生产应用前景,上述策略也为其它细胞因子或酶的改造提供了借鉴方法。     

Mechanism of 4-(beta-D-ribofuranosyl)aminobenzene 5'-phosphate synthase, a key enzyme in the methanopterin biosynthetic pathway

The first committed step in methanopterin biosynthesis is catalyzed by 4-(beta-D-ribofuranosyl)aminobenzene 5'-phosphate (RFA-P) synthase. Unlike all known phosphoribosyltransferases, beta-RFA-P synthase catalyzes the unique formation of a C-riboside instead of an N-riboside in the condensation of p-aminobenzoic acid (pABA) and 5-phospho-alpha-D-ribosyl-1-pyrophosphate (PRPP) to produce 4-(beta-D-ribofuranosyl)aminobenzene 5'-phosphate (beta-RFA-P), CO(2), and inorganic pyrophosphate (PP(i)). Here we report the successful cloning, active overexpression in Escherichia coli, and purification of this homodimeric enzyme containing two 36.2-kDa subunits from the methanogen Methanococcus jannaschii. Steady-state initial velocity and product inhibition kinetic studies indicate an ordered Bi-Ter mechanism involving binding of PRPP, then pABA, followed by release of the products CO(2), then beta-RFA-P, and finally PP. The Michaelis parameters are as follows: K(m)pABA, 0.15 mm; K(m)PRPP, 1.50 mm; V(max), 375 nmol/min/mg; k(cat), 0.23 s(-1). CO(2) showed uncompetitive inhibition, K(i) = 0.990 mm, under varied PRPP and saturated pABA, and a mixed type of inhibition, K(1) = 1.40 mm and K = 3.800 mm, under varied pABA and saturated PRPP. RFA-P showed uncompetitive inhibition, K(i) = 0.210 mm, under varied PRPP and saturated pABA, and again uncompetitive, K(i) = 0.300 mm, under saturated PRPP and varied pABA. PP(i) exhibits competitive inhibition, K(i) = 0.320 mm, under varied PRPP and saturated pABA, and a mixed type of inhibition, K(1) = 0.60 mm and K(2) = 1.900 mm, under saturated PRPP and varied pABA. Synthase lacks any chromogenic cofactor, and the presence of pyridoxal phosphate and the mechanistically related pyruvoyl cofactors has been strictly excluded.     

Gene expression alterations in connexin null mice extend beyond the gap junction

Connexin43 (Cx43) is the principal gap junction protein between astrocytes in the neonatal brain and also interconnects neural precursor cells during CNS development. In an attempt to understand global effects of expression of the Cx43 gap junction gene on development and function of the nervous system, we have compared gene expression patterns in cultured astrocytes and brains from wildtype mice with those in which Cx43 is deleted as well as in spinal cords of experimental autoimmune encepahlomyelitis (EAE) mice. One surprising result obtained from high densitity mouse cDNA studies was the large number of genes that were statistically altered in mice with decreased expression of Cx43. These altered genes encode proteins with a wide range of functions within cells, and thus deletion of normal gap junction expression appears to result in globally altered glial functions in addition to disruption of intercellular communication. Here we discuss those results in the context of the strategies and data analysis paradigms that we are using in such studies.     

3-D anatomically based dynamic modeling of the human knee to include tibio-femoral and patello-femoral joints

An anatomical dynamic model consisting of three body segments, femur, tibia and patella, has been developed in order to determine the three-dimensional dynamic response of the human knee. Deformable contact was allowed at all articular surfaces, which were mathematically represented using Coons' bicubic surface patches. Nonlinear elastic springs were used to model all ligamentous structures. Two joint coordinate systems were employed to describe the six-degrees-of-freedom tibio-femoral (TF) and patello-femoral (PF) joint motions using twelve kinematic parameters. Two versions of the model were developed to account for wrapping and nonwrapping of the quadriceps tendon around the femur. Model equations consist of twelve nonlinear second-order ordinary differential equations coupled with nonlinear algebraic constraint equations resulting in a Differential-Algebraic Equations (DAE) system that was solved using the Differential/Algebraic System Solver (DASSL) developed at Lawrence Livermore National Laboratory. Model calculations were performed to simulate the knee extension exercise by applying non-linear forcing functions to the quadriceps tendon. Under the conditions tested, both "screw home mechanism" and patellar flexion lagging were predicted. Throughout the entire range of motion, the medial component of the TF contact force was found to be larger than the lateral one while the lateral component of the PF contact force was found to be larger than the medial one. The anterior and posterior fibers of both anterior and posterior cruciate ligaments, ACL and PCL, respectively, had opposite force patterns: the posterior fibers were most taut at full extension while the anterior fibers were most taut near 90 degrees of flexion. The ACL was found to carry a larger total force than the PCL at full extension, while the PCL carried a larger total force than the ACL in the range of 75 degrees to 90 degrees of flexion.     

High phosphate (up to 600 mM) induces pseudohyphal development in five wild type Candida albicans

A method is described for the formation of nearly 100% pseudohyphae populations of wild-type Candida albicans A72. The method employs fungal growth at 37 degrees C (ca. 5x10(6) cells/ml) in a glucose-proline-N-acetyl-glucosamine medium supplemented with up to 600 mM phosphate (KH(2)PO(4)/K(2)HPO(4) 1:1) at pH 6.5. Four other strains of C. albicans (MEN, 10261, SG5314 and CAI-4) also formed pseudohyphae under these conditions, although the phosphate response profiles differed in the concentration required for each strain to form pseudohyphae.     

Stability and instability properties of aggregation of single chain amphiphiles into binary mixtures

In a lysophospholipid binary mixture, there are three ways of association between the mixture components of single-chain amphiphiles: (a) between two identical molecules each of the first and second component (self-association process) and (b) between two different molecules (cross-association process). Association probabilities for three binary mixtures were analysed as functions depending on the electric dipole moments of the polar head groups. A 3-D view representation is most suitable for this analysis. The most important finding is that for certain values of the electric dipole moments there are molecular couples which have a maximum stability to the changes in the external electrolytic medium. This fact confirms the formation of clusters and their stability, which is equivalent to the existence of micro-heterogeneities within the lipid bilayers. On the other hand, there are unstable molecular associations, and this fact influences the appearance of some phase transitions. Generally, the increase of the electric dipole moment or the increase of the acyl-chain length of one component from a binary lipid mixture decreases the self-association probability between its own molecules, but it increases the self-association probability of the other mixture components. Furthermore, the cross-association probability has high values for any binary lipid mixture of single-chain amphiphiles.     

Bacterial and Fungal Diversity of Quaternary Cave Sediment Deposits

The bacterial and fungal assemblages of clastic sediments collected from two caves located in north-western Romania were investigated by assessing ITS and 16S rRNA gene diversity. Bacterial members belonging to Chloroflexi, Nitrospirae, Proteobacteria, Firmicutes, Acidobacteria, Gemmatimonadetes, and fungal members of Ascomycota were identified. Except for Bacillus sp., all bacteria were related to uncultured or unknown species and the majority (86%) of the bacterial sequences from one of the caves had no close GenBank relatives. The bacterial sequences obtained clustered with species found in extreme environments. Half of the bacterial operational taxonomic units were clustered with clones isolated from deep subsurface sediments of a radioactively contaminated site in the USA. The present study represents the first attempt to identify microorganisms in Quaternary cave sediments.

Supplemental materials are available for this article. Go to the publisher's online edition of Geomicrobiology Journal to view the supplemental file.     

2SNP: scalable phasing based on 2-SNP haplotypes

2SNP software package implements a new very fast scalable algorithm for haplotype inference based on genotype statistics collected only for pairs of SNPs. This software can be used for comparatively accurate phasing of large number of long genome sequences, e.g. obtained from DNA arrays. As an input 2SNP takes genotype matrix and outputs the corresponding haplotype matrix. On datasets across 79 regions from HapMap 2SNP is several orders of magnitude faster than GERBIL and PHASE while matching them in quality measured by the number of correctly phased genotypes, single-site and switching errors. For example, 2SNP requires 41 s on Pentium 4 2 Ghz processor to phase 30 genotypes with 1381 SNPs (ENm010.7p15:2 data from HapMap) versus GERBIL and PHASE requiring more than a week and admitting no less errors than 2SNP.