THE HOST RANGE OF THE CHLORELLAVOROUS BACTERIUM ("VAMPIROVIBRIO CHLORELLVORUS")

The chlorellavorous bacterium . "Vampirovibrio chlorellavorus" Gromov et Mamkaeva, grows only by killing and consuming the cell contents of species of the green alga Chlorella Beijerinck. Of the 76 algal strains examined in this study, the bacterium attacks all 31 strains of the species C. vulgaris. C. sorokiniana, and C. kessleri, but attacks only two of 39 strains of nine other Chlorella species. Neither of two Prototheca strains was susceptible to attack. This narrow host specificity may be related to cell surface properties .     

Ovarian and haemolymph titres of ecdysteroid during the gonadotrophic cycle in Diploptera punctata

The free (non-conjugated) ecdysteroid in the ovaries during the first gonadotrophic cycle of Diploptera punctata was identified as 20-hydroxyecdysone. The hormone, quantified by radioimmunoassay and by ultraviolet absorbance, was detectable in the ovary toward the end of vitellogenesis; the quantity increased rapidly during chorion formation. Ovaries with chorionated eggs contained 67 μg of 20-hydroxyecdysone per g fresh weight. The haemolymph free-ecdysteroid, not identified physicochemically, was quantified by radioimmunoassays. The highest concentration was observed at adult emergence; the titre declined between days 1–3 and then remained at a relatively constant level through oviposition (which occurs between day 7 and 8); titres in pregnant females were higher. Ovariectomized females exhibited the same pattern of ecdysteroid titres in the haemolymph as the sham operated controls throughout the period corresponding to the first gonadotrophic cycle. Thus the ovary may not be the only source of haemolymph ecdysteroid related to reproduction in adult females.     

A novel osteoclast precursor cell line, 4B12, recapitulates the features of primary osteoclast differentiation and function: Enhanced transfection efficiency before and after differentiation

Osteoclasts are bone‐resorbing multinucleated cells differentiated from monocyte/macrophage lineage precursors. A novel osteoclast precursor cell line, 4B12 was established from Mac‐1⁺c‐Fms⁺RANK⁺ cells from calvaria of 14‐day‐old mouse embryos using immunofluorescence and cell‐sorting methods. Like M‐CSF‐dependent bone marrow macrophages (M‐BMMs), M‐CSF is required for 4B12 cells to differentiate into TRAP‐positive multinucleated cells [TRAP(+) MNCs] in the presence of RANKL. Bone‐resorbing osteoclasts differentiated from 4B12 cells on dentine slices possess both a clear zone and ruffled borders and express osteoclast‐specific genes. Bone‐resorbing activity, but not TRAP, was enhanced in the presence of IL‐1α. The number of TRAP(+) MNCs and the number of pits formed from 4B12 cells on dentine slices was fourfold higher than that from M‐BMMs. 4B12 cells were identified as macrophages with Mac‐1 and F4/80, yet lost these markers upon differentiation into osteoclasts as determined by confocal laser scanning microscopy. The 4B12 cells do not have the potential to differentiate into dendritic cells indicating commitment to the osteoclast lineage. 4B12 cells are readily transfectable with siRNA transfection before and after differentiation. These data show that 4B12 cells faithfully replicate the properties of primary cells and are a useful and powerful model for analyzing the molecular and cellular regulatory mechanisms of osteoclastogenesis and osteoclast function. J. Cell. Physiol. 221: 40–53, 2009. © 2009 Wiley‐Liss, Inc     

Effect of salinity stress on the life history variables of Branchipus schaefferi Fisher, 1834 (Crustacea: Anostraca)

Background

Freshwater anostracans inhabit ephemeral water bodies in which as the water level decreases due to evaporation the salt concentration increases. Thus, for most anostracans salinity becomes the major stress factor.

Results

We tested five concentrations of NaCl (0 to 8 g/l) on the life table demography of Branchipus schaefferi fed Chlorella (alga). Age-specific survivorship curves of male and female B. schaefferi showed nearly a similar pattern in that increased salt concentration resulted in decreased survivorship. The age-specific reproduction (m_x) of females showed several peaks of cyst production at 0 and 1 g/l salinity while in treatments containing salt at 4 or 8 g/l, there were fewer peaks. Average lifespan, life expectancy at birth, gross and net reproductive rates, generation time and the rate of population increase were all significantly influenced by the salt concentration in the medium. The highest value of net reproductive rate (970 cysts/female) was in treatments containing 0 g/l of salt, while the lowest was 13 cysts/female at 8 g/l. The rate of population increase (r) varied from 0.52 to 0.32 per day depending on the salt concentration in the medium.

Conclusion

The low survival and offspring production of B. schaefferi at higher salinity levels suggests that this species is unlikely to colonize inland saline water bodies. Therefore, the temporary ponds in which it is found, proper conservative measures must be taken to protect this species.     

Gaussian and linear deconvolution of LC-MS/MS chromatograms of the eight aminobutyric acid isomers

Isomeric molecules present a challenge for analytical resolution and quantification, even with MS-based detection. The eight aminobutyric acid (ABA) isomers are of interest for their various biological activities, particularly γ-aminobutyric acid (GABA) and the d- and l-isomers of β-aminoisobutyric acid (β-AIBA; BAIBA). This study aimed to investigate LC-MS/MS-based resolution of these ABA isomers as their Marfey's (Mar) reagent derivatives. HPLC was able to separate three Mar-ABA isomers l-β-ABA (l-BABA), and l- and d-α-ABA (AABA) completely, with three isomers (GABA, and d/l-BAIBA) in one chromatographic cluster, and two isomers (α-AIBA (AAIBA) and d-BABA) in a second cluster. Partially separated cluster components were deconvoluted using Gaussian peak fitting except for GABA and d-BAIBA. MS/MS detection of Marfey's derivatized ABA isomers provided six MS/MS fragments, with substantially different intensity profiles between structural isomers. This allowed linear deconvolution of ABA isomer peaks. Combining HPLC separation with linear and Gaussian deconvolution allowed resolution of all eight ABA isomers. Application to human serum found a substantial level of l-AABA (13 μM), an intermediate level of l-BAIBA (0.8 μM), and low but detectable levels (<0.2 μM) of GABA, l-BABA, AAIBA, d-BAIBA, and d-AABA. This approach should be useful for LC-MS/MS deconvolution of other challenging groups of isomeric molecules.     

Penicillin-binding proteins of clostridia

The penicillin-binding protein (PBP) profiles of 33Clostridium perfringens and sixClostridium species isolated from clinically significant infections were analyzed. Three new PBPs—PBPs 2B, 4B, and 5B (84, 70, and 49 kDa respectively)—and a high-molecular-weight PBP 6 (45 kDa) were demonstrated in theC. perfringens isolates. In addition to PBPs 1 and 2, PBPs 2B and 4B were seen to show low binding affinities for penicillin, although further studies are required to determine their possible roles in the development of penicillin resistance. The PBP profiles of theC. perfringens isolates were complex. Variations in apparent molecular weights (M _r s) of all PBPs, with the exception of PBP 5 and the presence or absence of PBPs 2, 3, and 4B, gave rise to nine different PBP patterns. The high-M _rPBPs 5 and 6, which exhibited high-penicillin-binding affinities, were with only one exception consistent within theC. perfringens isolates. These PBPs 5 and 6 of theC. perfringens isolates and independent PBPs found in the otherClostridium species studied indicate that PBP analysis may assist in the differentiation ofClostridium spacies.     

Linkage mapping of powdery mildew and greenbug resistance genes on recombinant 1RS from 'Amigo' and 'Kavkaz' wheat-rye translocations of chromosome 1RS.1AL.

Cultivated rye (Secale cereale L., 2n = 2x = 14, RR) is an important source of genes for insect and disease resistance in wheat (Triticum aestivum L., 2n = 6x = 42). Rye chromosome arm 1RS of S. cereale 'Kavkaz' originally found as a 1BL.1RS translocation, carries genes for disease resistance (e.g., Lr26, Sr31, Yr9, and Pm8), while 1RS of the S. cereale 'Amigo' translocation (1RSA) carries a single resistance gene for greenbug (Schizaphis graminum Rondani) biotypes B and C and also carries additional disease-resistance genes. The purpose of this research was to identify individual plants that were recombinant in the homologous region of.1AL.1RSV and 1AL.1RSA using both molecular and phenotypic markers. Secale cereale 'Nekota' (1AL.1RSA) and S. cereale 'Pavon 76' (1AL.1RSV) were mated and the F1 was backcrossed to 'Nekota' (1AL.1AS) to generate eighty BC1F2:3 families (i.e., ('Nekota' 1AL.1RSA x 'Pavon 76' 1AL.1RSV) x 'Nekota' 1AL.1AS). These families were genotyped using the secalin-gliadin grain storage protein banding pattern generated with polyacrylamide gel electrophoresis to discriminate 1AL.1AS/1AL.1RS heterozygotes from the 1AL.1RSA+V and 1AL.1AS homozygotes. Segregation of the secalin locus and PCR markers based on the R173 family of rye specific repeated DNA sequences demonstrated the presence of recombinant 1AL.1RSA+V families. Powdery mildew (Blumeria graminis) and greenbug resistance genes on the recombinant 1RSA+V arm were mapped in relation to the Sec-1 locus, 2 additional protein bands, 3 SSRs, and 13 RFLP markers. The resultant linkage map of 1RS spanned 82.4 cM with marker order and spacing showing reasonable agreement with previous maps of 1RS. Fifteen markers lie within a region of 29.7 cM next to the centromere, yet corresponded to just 36% of the overall map length. The map position of the RFLP marker probe mwg68 was 10.9 cM distal to the Sec-1 locus and 7.8 cM proximal to the powdery mildew resistance locus. The greenbug resistance gene was located 2.7 cM proximal to the Sec-1 locus.     

The University of Minnesota Biocatalysis/Biodegradation Database: post-genomic data mining

The University of Minnesota Biocatalysis/Biodegradation Database (UM-BBD, http://umbbd.ahc.umn.edu/) provides curated information on microbial catabolism and related biotransformations, primarily for environmental pollutants. Currently, it contains information on over 130 metabolic pathways, 800 reactions, 750 compounds and 500 enzymes. In the past two years, it has increased its breath to include more examples of microbial metabolism of metals and metalloids; and expanded the types of information it includes to contain microbial biotransformations of, and binding interactions with many chemical elements. It has also increased the ways in which this data can be accessed (mined). Structure-based searching was added, for exact matches, similarity, or substructures. Analysis of UM-BBD reactions has lead to a prototype, guided, pathway prediction system. Guided prediction means that the user is shown all possible biotransformations at each step and guides the process to its conclusion. Mining the UM-BBD's data provides a unique view into how the microbial world recycles organic functional groups. UM-BBD users are encouraged to comment on all aspects of the database, including the information it contains and the tools by which it can be mined. The database and prediction system develop under the direction of the scientific community.     

Structural requirements for the inhibitory action of the CD9 large extracellular domain in sperm/oocyte binding and fusion

CD9 has been shown to be essential for sperm/oocyte fusion in mice, the only non-redundant role found for a member of the tetraspanin family. CD9 can act in cis, reconstituting sperm/oocyte fusion when ectopically expressed in oocytes from CD9 null mice, or in trans, inhibiting sperm fusion when the large extracellular domain (LED) is added to CD9-positive oocytes as a soluble protein. In contrast to cis inhibition, the structural requirements of the trans inhibition by soluble CD9 LED are unknown. Here we show that human CD9 LED is as potent an inhibitor as mouse CD9 LED in mouse sperm/oocyte fusion assays and that CD9 LED can also inhibit sperm/oocyte binding. The two disulphide bridges that define membership of the tetraspanin family are critical for structure and function of human CD9 LED and mutation of a pentapeptide sequence in the hypervariable region further defines the critical region for trans inhibition.