Differences in regulation of pH(i) in large (>/=10 nuclei) and small (</=5 nuclei) osteoclasts

Osteoclasts aremultinucleated cells that resorb bone by extrusion of protons andproteolytic enzymes. They display marked heterogeneity in cell size,shape, and resorptive activity. Because high resorptive activity invivo is associated with an increase in the average size of osteoclastsin areas of greater resorption and because of the importance of protonextrusion in resorption, we investigated whether the activity of thebafilomycin A₁-sensitive vacuolar-typeH⁺-ATPase (V-ATPase) and amiloride-sensitiveNa⁺/H⁺ exchanger differed between large andsmall osteoclasts. Osteoclasts were obtained from newborn rabbit bones,cultured on glass coverslips, and loaded with the pH-sensitiveindicator2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF).Intracellular pH (pH_i) was recorded in single osteoclasts by monitoring fluorescence. Large (10 nuclei) and small (5 nuclei) osteoclasts differed in that large osteoclasts had a higher basal pH_i, their pH_i was decreased by bafilomycinA₁ addition or removal of extracellular Na⁺,and the realkalinization upon readdition of Na⁺ wasbafilomycin A₁ sensitive. After acid loading, asubpopulation of large osteoclasts (40%) recovered by V-ATPaseactivity alone, whereas all small osteoclasts recovered byNa⁺/H⁺ exchanger activity. Interestingly, in60% of the large osteoclasts, pH_i recovery was mediated byboth the Na⁺/H⁺ exchanger and V-ATPaseactivity. Our results show a striking difference betweenpH_i regulatory mechanisms of large and small osteoclaststhat we hypothesize may be associated with differences in the potentialresorptive activity of these cells.

     

The diversity of small eukaryotic phytoplankton (< or =3 microm) in marine ecosystems

Small cells dominate photosynthetic biomass and primary production in many marine ecosystems. Traditionally, picoplankton refers to cells ≤2 μm. Here we extend the size range of the organisms considered to 3 μm, a threshold often used operationally in field studies. While the prokaryotic component of picophytoplankton is dominated by two genera, Prochlorococcus and Synechococcus , the eukaryotic fraction is much more diverse. Since the discovery of the ubiquitous Micromonas pusilla in the early 1950s, just over 70 species that can be <3 μm have been described. In fact, most algal classes contain such species. Less than a decade ago, culture-independent approaches (in particular, cloning and sequencing, denaturing gradient gel electrophoresis, FISH) have demonstrated that the diversity of eukaryotic picoplankton is much more extensive than could be assumed from described taxa alone. These approaches revealed the importance of certain classes such as the Prasinophyceae but also unearthed novel divisions such as the recently described picobiliphytes. In the last couple of years, the first genomes of photosynthetic picoplankton have become available, providing key information on their physiological capabilities. In this paper, we discuss the range of methods that can be used to assess small phytoplankton diversity, present the species described to date, review the existing molecular data obtained on field populations, and end up by looking at the promises offered by genomics.     

A mitochondrial carrier gene, <Emphasis Type="Italic">CG32103</Emphasis>, is highly expressed in the corpora allata in the fruit fly <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> (Diptera: Drosophilidae)

Here we describe a novel gene that is highly expressed in the corpora allata, an endocrine organ responsible for synthesizing juvenile hormones (JHs), in the fruit fly, Drosophila melanogaster Meigen. We isolated an enhancer-trap line in which the transgene was inserted at the locus CG32103, which encodes a mitochondrial carrier family protein with calcium-binding motifs. RNA in situ hybridization revealed that CG32103 is predominantly expressed in the corpora allata in D. melanogaster larvae. Putative orthologs of CG32103 are conserved in many insect species. Mitochondrial carriers are responsible for transporting metabolites across the inner mitochondrial membrane. Given that both mitochondrial membrane transport and cytoplasmic calcium signaling are important for JH biosynthesis regulation, we speculated that CG32103 represents a new member of the family of JH biosynthesis regulators in insects.     

The rabies virion-associated 100-kDa polypeptide (VAP100) is a host-derived minor component of the viral envelope

We investigated a minor polypeptide component of 100-kDa detected in the rabies virion (referred to as VAP100) by using a monoclonal antibody (mAb), #16743, which was shown to recognize the SDS-denatured VAP100 antigen by immunoblot analyses. Although the VAP100 antigen was hardly detectable in the cell by usual immunoblot methods with this mAb, we could detect the antigen by a luminescent immunoblot method as well as by immunoprecipitation from the metabolically radiolabeled cell lysates and virions. Fluorescent antibody (FA) staining with mAb #16743 detected the uniformly distributed antigen on the formalin-fixed normal BHK-21 cells, while slight accumulation of the antigen was also seen in the Golgi area when the cells were permeabilized by treatment with Triton X-100 after fixation. Rabies virus infection induced alteration of the behavior of VAP100 to show a spotted distribution pattern in virus-infected cells. Double FA staining with mAb #16743 and rabbit antibody against the rabies virus envelope antigen demonstrated colocalized distribution of the viral envelope antigens and VAP100 in the cell. From these results, we think that VAP100 is a membrane-associated component of the cell, and its colocalized distribution with the viral envelope antigens in the cell implicates an intimate association of the VAP100 with viral envelope protein(s) and a reflection of possible involvement in the efficient incorporation of VAP100 into the virion.     

<Emphasis Type="Italic">PVAS3</Emphasis>, a class-II ubiquitous asparagine synthetase from the common bean (<Emphasis Type="Italic">Phaseolus vulgaris)</Emphasis>

A gene encoding a putative asparagine synthetase (AS; EC 6.3.5.4) has been isolated from common bean (Phaseolus vulgaris). A 2.4 kb cDNA clone of this gene (PVAS3) encodes a protein of 570 amino acids with a predicted molecular mass of 64,678 Da, an isoelectric point of 6.45, and a net charge of −5.9 at pH 7.0. The PVAS3 protein sequence conserves all the amino acid residues that are essential for glutamine-dependent AS, and PVAS3 complemented an E. coli asparagine auxotroph, that demonstrates that it encodes a glutamine-dependent AS. PVAS3 displayed significant similarity to other AS. It showed the highest similarity to soybean SAS3 (92.9% identity), rice AS (73.7% identity), Arabidopsis ASN2 (73.2%) and sunflower HAS2 (72.9%). A phylogenetic analysis revealed that PVAS3 belongs to class-II asparagine synthetases. Expression analysis by real-time RT-PCR revealed that PVAS3 is expressed ubiquitously and is not repressed by light.     

The isolation and analysis of a soybean <Emphasis Type="Italic">CO</Emphasis> Homologue <Emphasis Type="Italic">GmCOL10</Emphasis>

The CONSTANS (CO) gene is a key regulator of the response to photoperiod in the model plant Arabidopsis thaliana, and its homologues are present in many plant species. We describe here the isolation of the CO homologue for zinc finger protein gene GmCOL10 (Glycine max CONSTANS-Like 10) from the soybean cultivar Kennong18. Sequence comparisons showed that the closest A. thaliana gene to GmCOL10 is COL5. The expression of GmCOL10 was regulated in a circadian manner, especially under short-day conditions. The expression of GmCOL10 was concentrated in vegetative organs, and in particular in the unifoliolates and cotyledons. An analysis of subcellular localization found GmCOL10 in the nucleus. Our data suggested that GmCOL10 was not related to the photoperiodic pathway of floral transition as Arabidopsis CO does.     

The efficacy of small cell foundation as a varroa mite (<Emphasis Type="Italic">Varroa destructor</Emphasis>) control

Due to a continuing shift toward reducing/minimizing the use of chemicals in honey bee colonies, we explored the possibility of using small cell foundation as a varroa control. Based on the number of anecdotal reports supporting small cell as an efficacious varroa control tool, we hypothesized that bee colonies housed on combs constructed on small cell foundation would have lower varroa populations and higher adult bee populations and more cm² brood. To summarize our results, we found that the use of small cell foundation did not significantly affect cm² total brood, total mites per colony, mites per brood cell, or mites per adult bee, but did affect adult bee population for two sampling months. Varroa levels were similar in all colonies throughout the study. We found no evidence that small cell foundation was beneficial with regard to varroa control under the tested conditions in Florida.     

Synthesis of orthogonally protected (<Emphasis Type="Italic">3R</Emphasis>,<Emphasis Type="Italic">4S</Emphasis>)- and (<Emphasis Type="Italic">3S</Emphasis>,<Emphasis Type="Italic">4S</Emphasis>)-4,5-diamino-3-hydroxypentanoic acids

Summary.  The paper describes two methods of the synthesis of ethyl (3R,4S)- and (3S,4S)-4-[(benzyloxycarbonyl)amino]-5-[(tert-butyloxycarbonyl)amino]-3-hydroxypentanoates, useful for the syntheses of edeine analogs. Differently N-protected (S)-2,3-diaminopropanoic acid was used as a substrate in both procedures. The absolute configuration of newly generated asymmetric carbon atoms C-3 in β-hydroxy-γ,δ-diamino products was assigned by means of ¹H NMR spectroscopy after their transformation into corresponding piperidin-2-ones. Received May 24, 2002 Accepted October 10, 2002 Published online December 18, 2002 Acknowledgment The authors are indebted to the Faculty of Chemistry, Technical University of Gdańsk for financial support. Authors' address: Zbigniew Czajgucki, M. Sc., Department of Pharmaceutical Technology and Biochemistry, Faculty of Chemistry, Technical University of Gdańsk, 11/12 Narutowicza St., 80-952 Gdańsk, Poland, Fax +48 58 347 11 44, E-mail: zmczaj@wp.pl     

Molecular nature of allelic polymorphism of highly variable microsatellite locus <Emphasis Type="Italic">Du161(arm)</Emphasis> in unisexual lizard <Emphasis Type="Italic">Darevskia armeniaca</Emphasis> (lacertidae)

In the genome of unisexual (parthenogenetic) lizard Darevskia armeniaca, highly variable locus Du161 (arm) was discovered. Analysis of allelic polymorphism was carried out using locus-specific PCR of the lizard DNA specimens from 13 isolated Armenian populations (N = 138). In the sample examined, a total of 12 Du161(arm) alleles were identified, and their differences at the level of primary DNA structure were determined. Sequence analysis of the Du161(arm) alleles showed that their microsatellite clusters contained repeats of one type (GATA repeats). Allelic Du161(arm) variants differed in the number of GATA monomers in microsatellite, point mutations of transition and transversion types, located at fixed distances from micro-satellite cluster, and by single nucleotide insertions, as well as by longer insertions located within and outside of the microsatellite cluster. Moreover, point mutations formed different combinations (haplotypes), typical of certain alleles. These combinations can be used for the analysis of the origin and inheritance of these alleles in D. armeniaca, as well as for investigation of their interspecific variation in the representatives of the genus Darevskia.     

The neglected hypogeous fungus <Emphasis Type="Italic">Hydnotrya bailii</Emphasis> Soehner (1959) is a widespread sister taxon of <Emphasis Type="Italic">Hydnotrya tulasnei</Emphasis> (Berk.) Berk. &; Broome (1846)

The neglected false truffle species Hydnotrya bailii Soehner (Ascomycetes, Discinaceae) is re-described and separated from its sister taxon Hydnotrya tulasnei by morphological and phylogenetic analyses based on internal transcribed spacer rDNA sequences. The most distinct morphological and ecological characters are small globose, rather than kidney-like, ascomata as known from the sister taxon H. tulasnei, strictly monoseriate ascospores and montane habitats. Phylogenetic analyses resulted in two clearly separated clusters that revealed the ectomycorrhizal specificity of H. bailii to Picea abies and that H. tulasnei is preferably associated to Fagus sylvatica. We also show that H. bailii was already present in mycorrhizal samples but until now could not be correctly assigned. Our analyses also indicate cryptic diversity within H. cerebriformis and other, morphologically not yet characterized, Hydnotrya groups. An emended determination key for all Hydnotrya species known from Central Europe is provided.     

Identification of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a binding protein for a 68-kDa <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> parasporal protein cytotoxic against leukaemic cells

Background  

Bacillus thuringiensis (Bt), an ubiquitous gram-positive spore-forming bacterium forms parasporal proteins during the stationary phase of its growth. Recent findings of selective human cancer cell-killing activity in non-insecticidal Bt isolates resulted in a new category of Bt parasporal protein called parasporin. However, little is known about the receptor molecules that bind parasporins and the mechanism of anti-cancer activity. A Malaysian Bt isolate, designated Bt18 produces parasporal protein that exhibit preferential cytotoxic activity for human leukaemic T cells (CEM-SS) but is non-cytotoxic to normal T cells or other cancer cell lines such as human cervical cancer (HeLa), human breast cancer (MCF-7) and colon cancer (HT-29) suggesting properties similar to parasporin. In this study we aim to identify the binding protein for Bt18 in human leukaemic T cells.     

<Emphasis Type="Italic">Rhynchospora galeana</Emphasis>, a new name for <Emphasis Type="Italic">Rhynchospora breviseta</Emphasis> (Gale) Channell (Cyperaceae)

We propose the new name Rhynchospora galeana to replace the illegitimate later homonym R. breviseta (Gale) Channell. Rhynchospora galeana is a member of section Plumosae. It grows in savannas and pinelands in the southeastern U.S.A. and the Greater Antilles.     

DNAzyme-mediated recovery of small recombinant RNAs from a 5S rRNA-derived chimera expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Manufacturing large quantities of recombinant RNAs by overexpression in a bacterial host is hampered by their instability in intracellular environment. To overcome this problem, an RNA of interest can be fused into a stable bacterial RNA for the resulting chimeric construct to accumulate in the cytoplasm to a sufficiently high level. Being supplemented with cost-effective procedures for isolation of the chimera from cells and recovery of the recombinant RNA from stabilizing scaffold, this strategy might become a viable alternative to the existing methods of chemical or enzymatic RNA synthesis.     

Rapid linkage disequilibrium decay in the <Emphasis Type="Italic">Lr10</Emphasis> gene in wild emmer wheat (<Emphasis Type="Italic">Triticum dicoccoides</Emphasis>) populations

Introduction  

Recombination is a key evolutionary factor enhancing diversity. However, the effect of recombination on diversity in inbreeding species is expected to be low. To estimate this effect, recombination and diversity patterns of Lr10 gene were studied in natural populations of the inbreeder species, wild emmer wheat (Triticum dicoccoides). Wild emmer wheat is the progenitor of most cultivated wheats and it harbors rich genetic resources for disease resistance. Lr10 is a leaf rust resistance gene encoding three domains: a coiled-coil, nucleotide-binding site, and leucine-rich repeat (CC-NBS-LRR).     

Secretion of biologically active interferon-gamma inducible protein-10 (IP-10) by <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

Background  

Chemokines are a large group of chemotactic cytokines that regulate and direct migration of leukocytes, activate inflammatory responses, and are involved in many other functions including regulation of tumor development. Interferon-gamma inducible-protein-10 (IP-10) is a member of the C-X-C subfamily of the chemokine family of cytokines. IP-10 specifically chemoattracts activated T lymphocytes, monocytes, and NK cells. IP-10 has been described also as a modulator of other antitumor cytokines. These properties make IP-10 a novel therapeutic molecule for the treatment of chronic and infectious diseases. Currently there are no suitable live biological systems to produce and secrete IP-10. Lactococcus lactis has been well-characterized over the years as a safe microorganism to produce heterologous proteins and to be used as a safe, live vaccine to deliver antigens and cytokines of interest. Here we report a recombinant strain of L. lactis genetically modified to produce and secrete biologically active IP-10.