Taxonomic description of Methanococcoides euhalobius and its transfer to the Methanohalophilus genus

A sequence analysis of the 16S-rRNA of Methanococcoides euhalobius revealed that this organism was highly related to members of the genus Methanohalophilus. On the basis of sequence data, an oligonucleotide probe specific to Methanohalophilus species was designed. Hybridization studies with this probe confirmed close relationship of Methanococcoides euhalobius to Methanohalophilus species. Therefore, we propose that Methanococcoides euhalobius should be transferred to the genus Methanohalophilus as Methanohalophilus euhalobius.     

Dna insertional mutagenesis for the elucidation of a Photosystem II repair process in the green alga Chlamydomonas reinhardtii

The work outlines the isolation of transformant Chlamydomonas reinhardtii cells that appear to be unable to repair Photosystem II from photoinhibitory damage. A physiological and biochemical characterization of three mutants is presented. The results show differential stability for the D1 reaction center protein in the three mutants compared to the wild type and suggest lesions that affect different aspects of the Photosystem II repair mechanism. In the ag16.2 mutant, significantly greater amounts of D1 accumulate in the thylakoid membrane than in the wild type under steady-state growth conditions, and D1 loss is significantly retarded in the presence of the protein biosynthesis inhibitor chloramphenicol. Moreover, aberrant electrophoretic mobility of D1 in the ag16.2 suggests that this protein is modified to an as yet unknown configuration. These results indicate that the biosynthesis and/or degradation of D1 is altered in this strain. A different type of mutation occurred in the kn66.7 and kn27.4 mutants of C. reinhardtii. The stability of D1 declined much faster as a function of light intensity in these mutants than in the wild type. Thereby, the threshold of photoinhibition in these mutants was significantly lower than that in the wild type. It appears that kn66.7 and kn27.4 are similar conditional mutants, with the only difference between them being the amplitude of the chloroplast response to the mutation and the differential sensitivity they display to the level of irradiance.     

Characterization of the cyclophilin gene family of Arabidopsis thaliana and phylogenetic analysis of known cyclophilin proteins

We have isolated four members of the Arabidopsis cyclophilin (CyP) gene family, designated ROC1 to ROC4 (rotamase CyP). Deduced peptides of ROC1, 2 and 3 are 75% to 91% identical to Brassica napus cytosolic CyP, contain no leader peptides and include a conserved seven amino-acid insertion relative to mammalian cytosolic CyPs. Two other Arabidopsis CyPs, ROC5 (43H1; ATCYP1) and ROC6 (ATCYP2), share these features. ROC1, ROC2, ROC3 and ROC5 are expressed in all tested organs of light-grown plants. ROC2 and ROC5 show elevated expression in flowers. Expression of ROC1, ROC2, and ROC3 decreases in darkness and these genes also exhibit small elevations in expression upon wounding. The five Arabidopsis genes encoding putative cytosolic CyPs (ROC1, 2, 3, 5 and 6) contain no introns. In contrast, ROC4, which encodes a chloroplast stromal CyP, is interrupted by six introns. ROC4 is not expressed in roots, and is strongly induced by light. Phylogenetic trees of all known CyPs and CyP-related proteins provide evidence of possible horizontal transfer of CyP genes between prokaryotes and eukaryotes and of a possible polyphyletic origin of these proteins within eukaryotes. These trees also show significant grouping of eukaryotic CyPs on the basis of subcellular localization and structure. Mitochondrial CyPs are closely related to cytosolic CyPs of the source organism, but endoplasmic reticulum CyPs form separate clades. Known plant CyPs fall into three clades, one including the majority of higher-plant cytosolic CyPs, one including only ROC2 and a related rice CyP, and one including only chloroplast CyPs.     

Leishmanial antigens in the diagnosis of active lesions and ancient scars of American tegumentary leishmaniasis patients.

Cutaneous biopsies (n = 94) obtained from 88 patients with American tegumentary leishmaniasis were studied by conventional and immunohistochemical techniques. Specimens were distributed as active lesions of cutaneous leishmaniasis (n = 53) (Group I), cicatricial lesions of cutaneous leishmaniasis (n = 35) (Group II) and suggestive scars of healed mucosal leishmaniasis patients (n = 6) (Group III). In addition, active cutaneous lesions of other etiology (n = 24) (Group C1) and cutaneous scars not related to leishmaniasis (n = 10) (Group C2) were also included in the protocol. Amastigotes in Group I biopsies were detected by routine histopathological exam (30.2%), imprint (28.2%), culture (43.4%), immunofluorescence (41.4%) and immunoperoxidase (58.5%) techniques; and by the five methods together (79.3%). In Group II, 5.7% of cultures were positive. Leishmanial antigen was also seen in the cytoplasm of macrophages and giant cells (cellular pattern), vessel walls (vascular pattern) and dermal nerves (neural pattern). Positive reaction was detected in 49 (92.5%), 20 (57%) and 4 (67%) biopsies of Groups I, II and III, respectively. Antigen persistency in cicatricial tissue may be related to immunoprotection or, on the contrary, to the development of late lesions. We suggest that the cellular, vascular and neural patterns could be applied in the immunodiagnosis of active and cicatricial lesions in which leishmaniasis is suspected.     

Evidence for the presence of ferritin in plant mitochondria.

In this work, evidence for the presence of ferritins in plant mitochondria is supplied. Mitochondria were isolated from etiolated pea stems and Arabidopsis thaliana cell cultures. The proteins were separated by SDS/PAGE. A protein, with an apparent molecular mass of approximately 25-26 kDa (corresponding to that of ferritin), was cross-reacted with an antibody raised against pea seed ferritin. The mitochondrial ferritin from pea stems was also purified by immunoprecipitation. The purified protein was analyzed by MALDI-TOF mass spectrometry and the results of both mass finger print and peptide fragmentation by post source decay assign the polypeptide sequence to the pea ferritin (P < 0.05). The mitochondrial localization of ferritin was also confirmed by immunocytochemistry experiments on isolated mitochondria and cross-sections of pea stem cells. The possible role of ferritin in oxidative stress of plant mitochondria is discussed.     

Covariation among Alaskan chrysophyte stomatocyst assemblages and environmental gradients: A comparison with diatom assemblages

Northern lakes, for which we have little long-term data, are predicted to experience profound impacts with greenhouse warming. In order to assess the usefulness of chrysophytes as paleoenvironmental indicators, stomatocysts were enumerated from the surface sediments of 51 Alaskan lakes distributed along a strong climatic gradient. A total of 142 cyst morphotypes were described using light microscopy, of which 13 are believed to be new forms.
Principal components analysis of the stomatocyst assemblages showed a limited amount of variation (Λ₁= 0.11, Λ₂= 0.09). However, redundancy analysis identified total phosphorus (TP), sodium (Na), and altitude (ALT) as significant variables in terms of their ability to describe the distribution of stomatocysts. Major ion and nutrient concentrations were also found to be strong predictors of diatom variation from the same lake set. In comparison to the diatom assemblages from the same lakes, the cysts were less abundant and showed a lower amount of species variation. Reasons for the lack of variation may be due to the predominance of unornamented and collective category cysts (i. e. cysts which cannot be differentiated under the light microscope).     

Radioiodination of proteins using prosthetic group: a convenient way to produce labelled proteins with in vivo stability.

Radiolabelled peptides can provide new approaches for radiopharmaceutical development. Several prosthetic groups have been developed for radioiodination of proteins in order to minimize in vivo dehalogenation. In this work, the prosthetic group N-succinimidyl 4-[131I]iodobenzoate ([131I]SIB) was obtained by an alternative procedure that employs Cu(I) assisted radioiododebromination to produce p-[131I]iodobenzoic acid with a radiochemical yield of 92.73 +/- 1.51% (N = 6), followed by the reaction with TSTU (O-(N-succinimidyl)-N,N,N'N'-tetramethyluronium) in alkaline medium. The HPLC profile of the final product, revealed that [131I]SIB was obtained with a radiochemical purity of 98.19 +/- 1.14% (N = 6 Swiss mices (normal group) and animals with inflammation focus developed on the right thigh by tupertine injection) were injected with human immunoglobulin (IgG) radioiodinated with [131I]SIB and by direct method (Iodogen). The comparison of results showed a fast blood clearance, better target organ/background relation and low uptake in thyroid and stomach (p < 0.01) for the protein labelled with [131I]SIB, what suggests a greater in vivo stability.     

Hystological analysis of ovarian development of the characiform Oligosarcus hepsetus (Cuvier, 1829) in a Brazilian reservoir.

A histological analysis was performed of the ovarian development of Oligosarcus hepsetus (Cuvier, 1829), a medium-sized carnivorous Characiform species in the Lajes reservoir, Brazil. A total of 125 fishes, collected monthly between April 2001 and June 2002 were examined by routine macroscopic and histological techniques. Eight phases of the oocyte development were described, and 4 stages and 4 substages of gonadal development were proposed. Spawning in reservoirs, similarly to that in rivers, is in batches, which favors juvenile survival and suggests that the impoundment of this area did not influence the reproductive strategy of this very successful species in a dammed environment.     

Molecular characterization and mapping of ALMT1, the aluminium-tolerance gene of bread wheat (Triticum aestivum L.).

The major aluminum (Al) tolerance gene in wheat ALMT1 confers. An Al-activated efflux of malate from root apices. We determined the genomic structure of the ALMT1 gene and found it consists of 6 exons interrupted by 5 introns. Sequencing a range of wheat genotypes identified 3 alleles for ALMT1, 1 of which was identical to the ALMT1 gene from an Aegilops tauschii accession. The ALMT1 gene was mapped to chromosome 4DL using 'Chinese Spring' deletion lines, and loss of ALMT1 coincided with the loss of both Al tolerance and Al-activated malate efflux. Aluminium tolerance in each of 5 different doubled-haploid populations was found to be conditioned by a single major gene. When ALMT1 was polymorphic between the parental lines, QTL and linkage analyses indicated that ALMT1 mapped to chromosome 4DL and cosegregated with Al tolerance. In 2 populations examined, Al tolerance also segregated with a greater capacity for Al-activated malate efflux. Aluminium tolerance was not associated with a particular coding allele for ALMT1, but was significantly correlated with the relative level of ALMT1 expression. These findings suggest that the Al tolerance in a diverse range of wheat genotypes is primarily conditioned by ALMT1.     

Different proteins bind to the butyrolactone receptor protein ARE sequence located upstream of the regulatory ccaR gene of Streptomyces clavuligerus

Cell-free extracts from Streptomyces clavuligerus, purified by elution from heparin-agarose with an ARE-containing DNA fragment or by salt elution chromatography, bind to a 26 nt ARE sequence, for butyrolactone receptor proteins (ARE_ccaR). This sequence is located upstream of the ccaR gene, encoding the activator protein CcaR required for clavulanic acid and cephamycin C biosynthesis. The binding is specific for the ARE sequence as shown by competition with a 34 nt unlabelled probe identical to the ARE sequence. A brp gene, encoding a butyrolactone receptor protein, was cloned from S. clavuligerus. Sixty-one nucleotides upstream of brp another ARE sequence (ARE_brp) was found, suggesting that Brp autoregulates its expression. Pure recombinant rBrp protein binds specifically to the ARE sequences present upstream of ccaR and brp. A brp-deleted mutant, S. clavuligerus Δbrp::neo1, produced 150–300% clavulanic acid and 120–220% cephamycin C as compared with the parental strain, suggesting that Brp exerts a repressor role in antibiotic biosynthesis. EMSA assays using affinity chromatography extracts from the deletion mutant S. clavuligerus Δbrp::neo1 lacked a high-mobility band-shift due to Brp but still showed a slow-mobility band-shift observed in the wild-type strain. These results indicate that two different proteins bind specifically to the ARE sequence and modulate clavulanic acid and cephamycin C biosynthesis by its action on ccaR gene expression.