Effect of protective agents, rehydration media and initial cell concentration on viability of Pantoea agglomerans strain CPA-2 subjected to freeze-drying

The effect of initial cell density, protective agents and rehydration media on the viability of biocontrol agent Pantoea agglomerans CPA-2 when subjected to freeze-drying was studied. Several additives were tested as protective agents against freeze-drying injury. Maximum viability of the bacterial cells was obtained with disaccharides (survival levels > 60%). Freeze-dried samples were rehydrated with several media; the highest percentage viability was obtained with 10% non-fat skim milk (100%+). The effect of initial bacterial load on the final recovery was dependent on protectant but not on rehydration media. Sucrose was an effective protectant when a high initial concentration (10(10) cfu ml(-1) was used; the opposite occurred with non-fat skim milk. The use of 10(10) cfu ml(-1) as an initial concentration, sucrose as a protectant and non-fat skim milk as a rehydration medium enabled 100% of P. agglomerans viability to be conserved after freeze-drying. Results suggest the possibility of achieving a good formulation system for the studied biocontrol agent with a high number of viable cells to be used toward pathogens, which is desirable for the industrial development of the product.     

The ionic basis of cardiac activity in the bivalve mollusc Perna perna

The ionic basis of cardiac activity and aspects of excitation-contraction (E-C) coupling were investigated in the isolated heart of the bivalve mollusc Perna perna, using the sucrose-gap technique. The role of the principal ions was established employing artificial seawater, in which specific ion concentrations were modified, and ion channel blockers. The mean membrane resting potential (MP) and the action potential (AP) were -33+/-0.7 mV (n=89) and 13+/-0.3 mV (n=71), respectively. The MP potential was primarily dependent on K(+) ions. Three types of cardiac APs were identified: fast, slow and spike-plateau potentials. Cardiac activity was maintained in Na(+)- or Ca(2+)-free salines but ceased when either Cd(2+) or EDTA was added to these salines. Other Ca(2+) channel blockers reduced the amplitude and increased duration of the cardiac APs. Tetrodotoxin (TTX) and procaine did not alter the AP. The data showed that the depolarizing phase of the AP was dependent on Ca(2+) influx while the plateau phase, when present, resulted from Na(+) influx that was modulated by Ca(2+). The mechanical responses were more sensitive to changes in extracellular Ca(2+) concentration than were the electrical responses.     

Surfactant components modulate fibroblast apoptosis and type I collagen and collagenase-1 expression

During lung injury, fibroblasts migrate into the alveolar spaces where they can be exposed to pulmonary surfactant. We examined the effects of Survanta and surfactant protein A (SP-A) on fibroblast growth and apoptosis and on type I collagen, collagenase-1, and tissue inhibitor of metalloproteinase (TIMP)-1 expression. Lung fibroblasts were treated with 100, 500, and 1,000 microg/ml of Survanta; 10, 50, and 100 microg/ml of SP-A; and 500 microg/ml of Survanta plus 50 microg/ml of SP-A. Growth rate was evaluated by a formazan-based chromogenic assay, apoptosis was evaluated by DNA end labeling and ELISA, and collagen, collagenase-1, and TIMP-1 were evaluated by Northern blotting. Survanta provoked fibroblast apoptosis, induced collagenase-1 expression, and decreased type I collagen affecting mRNA stability approximately 10-fold as assessed with the use of actinomycin D. Collagen synthesis and collagenase activity paralleled the gene expression results. SP-A increased collagen expression approximately 2-fold and had no effect on collagenase-1, TIMP-1, or growth rate. When fibroblasts were exposed to a combination of Survanta plus SP-A, the effects of Survanta were partially reversed. These findings suggest that surfactant lipids may protect against intraluminal fibrogenesis by inducing fibroblast apoptosis and decreasing collagen accumulation.     

Defined nuclear changes accompany the reprogramming of the microspore to embryogenesis

The switch of the gametophytic developmental program toward pollen embryogenesis to form a haploid plant represents an important alternative for plant breeding. In the present study, the switch of the gametophytic developmental program toward a sporophytic pathway, "embryogenesis," has been studied in three different plant species, Brassica, tobacco, and pepper. The switch has been induced by stress (heat shock) at the very responsive stage of the microspore, which is the vacuolate period. As a result, the cell nucleus undergoes striking structural changes with regard to late gametophytic development, including alterations of biosynthetic activities and proliferative activity. An enrichment in HSP70 heat-shock protein and in the presence of Ntf6-MAP kinase was observed after inductive treatment in the nuclei during early embryogenesis. This apparently reflected the possible roles of these proteins, specifically the protective role of HSP70 for the nuclear machinery, and signal transduction of Ntf6-MAPK for the entry of cells into proliferation. Importantly, the observed nuclear changes were similar in the three species investigated and represented convenient markers for early monitoring of embryogenesis and selection purposes for obtaining double-haploid plants in plant breeding.     

Compounds Extracted from Phyllantus and Jatropha elliptica Inhibit the Binding of [3H]Glutamate and [3H]GMP-PNP in Rat Cerebral Cortex Membrane

Glutamate is to be considered a nociceptive neurotransmitter and glutamatergic antagonists present antinoceptive activity. In this study we investigated the effects of the naturally occurring antinociceptive compounds rutin, geraniin and quercetine extracted from Phyllanthus, as well as the diterpene jatrophone, extracted from Jatropha elliptica on the binding of [³H]glutamate and [³H]GMP-PNP [a GTP analogue which binds to extracellular site(s), modulating the glutamatergic transmission] in rat brain membrane. Jatrophone inhibited [³H]glutamate binding and geraniin inhibited [³H]GMP-PNP binding. Quercetine inhibited the binding of both ligands. These results may indicate a neurochemical parameter possibly related to the antinoceptive activity of these natural compounds.     

Some oncogene and tumour suppressor gene protein products expression in B-cell chronic lymphocytic leukaemia

The expression of Bcl-2, P53 proteins and known markers of proliferation, namely proliferating cell nuclear antigen (PCNA) and Ki67, in 29 patients with B-cell chronic lymphocytic leukaemia (B-CLL) was investigated. All leukaemic patients were classified, and immunophenotyped by the two-colour immunofluorescence method with the use of fluorocytometry. B-CLL was heterogeneous in the range of biological parameters of tumour cells. B-CLL patients manifested 34% positive Ki67 and 61% PCNA expression, whereas Bcl-2 and P53 positivity was 81% and 42%, respectively. The level of intracellular expression of Bcl-2 and P53 proteins did not depend on the stage of disease estimated by routine methods. Ki67 and PCNA expression was significantly higher in B-CLL patients with more advanced stages of the disease. A statistically significant correlation was established between their mutual expression.     

Nucleolar Organizing Regions, 18S and 5S rDNA in Astyanax Scabripinnis (Pisces, Characidae): Populations Distribution and Functional Diversity

Astyanax scabripinnis specimens from four distinct populations in Brazil were studied with respect to their karyotype macrostructure, nucleolar organizer regions, and 18S and 5S rRNA genes. The four populations showed a 2n = 50 chromosomes (3 M + 11 SM + 5 ST + 6 A pairs) and 1–2 B chromosomes. No chromosomal differentiations were observed between sexes. Although a karyotypic diversity has been characterized in this fish group, the populations now analyzed presented the same macrokaryotypic pattern. Chromosome mapping of 5S rDNA showed a total of eight sites located in four distinct chromosomal pairs, with no apparent differences among populations. A comparative study on 18S rDNA locations and Ag-NORs showed some secondary NOR sites that are not usually expressed in karyotypes and a probable differential NOR activity among populations. Correlations between these data, environmental conditions and B chromosomes are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Heme synthesis in yeast does not require oxygen as an obligatory electron acceptor

In a previous paper (Krawiec, Z., Biliński, T., Schüller, C. & Ruis, H., 2000, Acta Biochim. Polon. 47, 201-207) we have shown that catalase T holoenzyme is synthesized in the absence of oxygen after treatment of anaerobic yeast cultures with 0.3 M. NaCl, or during heat shock. This finding suggests that heme moiety of the enzyme can either be formed de novo in the absence of oxygen, or derives from the preexisting heme pool present in cells used as inoculum. The strain bearing hem1 mutation, resulting in inability to form delta-aminolevulinate (ALA), the first committed precursor of heme, was used in order to form heme-depleted cells used as inocula. The cultures were supplemented with ALA at the end of anaerobic growth prior the stress treatment. The appearance of active catalase T in the stressed cells strongly suggests that heme moiety of catalase T is formed in the absence of oxygen. This finding suggests the necessity to reconsider current opinions concerning mechanisms of heme synthesis and the role of heme as an oxygen sensor.     

Dirigent-mediated podophyllotoxin biosynthesis in Linum flavum and Podophyllum peltatum

Given the importance of the antitumor/antiviral lignans, podophyllotoxin and 5-methoxypodophyllotoxin, as biotechnological targets, their biosynthetic pathways were investigated in Podophyllum peltatum and Linum flavum. Entry into their pathways was established to occur via dirigent mediated coupling of E-coniferyl alcohol to afford (+)-pinoresinol; the encoding gene was cloned and the recombinant protein subsequently obtained. Radiolabeled substrate studies using partially purified enzyme preparations next revealed (+)-pinoresinol was enantiospecifically converted sequentially into (+)-lariciresinol and (-)-secoisolariciresinol via the action of an NADPH-dependent bifunctional pinoresinol/lariciresinol reductase. The resulting (-)-secoisolariciresinol was enantiospecifically dehydrogenated into (-)-matairesinol, as evidenced through the conversion of both radio- and stable isotopically labeled secoisolariciresinol into matairesinol, this being catalyzed by the NAD-dependent secoisolariciresinol dehydrogenase. (-)-Matairesinol was further hydroxylated to afford 7'-hydroxymatairesinol, this being efficiently metabolized into 5-methoxypodophyllotoxin. Thus much of the overall biosynthetic pathway to podophyllotoxin has been established, that is, from the dirigent mediated coupling of E-coniferyl alcohol to the subsequent conversions leading to 7'-hydroxymatairesinol.