Cloning,functional expression and expression studies of the nitrate transporter gene from Chlorella sorokiniana (strain 211-8k)

The nitrate transporter from Chlorella sorokiniana (accession number AY026523) has been cloned by screening a cDNA library based on mRNA isolated after 30 min treatment of Chlorella with 5 mM nitrate and with a RT-PCR product (730 bp) as a probe. The Chlorella sequence has similarity to known nitrate transporters of the NRT2 family (high-affinity nitrate transporters). The cDNA clone was used for functional expression in Xenopus oocytes and a nitrate-dependent current was measured at pH 5.5 but not at pH 7.4. A second algal gene or a second gene product was not needed for functional expression in Xenopus. Inhibitor studies in Chlorella indicated that protein phosphorylation/dephosphorylation is involved in nitrate induction of ChNRT2.1. In addition to nitrate, ChNRT2.1 expression is induced by nitroprusside, a NO donor, and is affected by glucose.     

Annual, daily and intradiurnal variation of Celtis pollen in the city of La Plata, Argentina

The behaviour of Celtis airborne pollenwas studied for a period of three years(1998–2000) in the city of La Plata. The pollengrains were captured with a Lanzoni trap andthe maximum pollination period was observed tooccur during the end of winter and spring inthe three years. The annual values of pollenconcentration varied, and a significantdecrease was observed during 2000. The greatestairborne pollen record was in October with anaverage of 82.3% in relation to its totalconcentration during 1998–2000. Based on theintradiurnal behaviour analysis, it wasobserved that the maximum pollination peakoccurs at 2H when the temperaturereaches its maximum values. Considering thethree sampling years, the meteorologicalvariables that most influenced the processes of pollen emission,dispersion and transportation in the atmospherewere: maximum and minimum temperature andrainfall.     

Oxidative damage of the gastric mucosa in Helicobacter pylori positive chronic atrophic and nonatrophic gastritis, before and after eradication

Background. Helicobacter pylori is the main cause of gastritis and a primary carcinogen. The aim of this study was to assess oxidative damage in mucosal compartments of gastric mucosa in H. pylori positive and negative atrophic and nonatrophic gastritis. Materials and methods. Five groups of 10 patients each were identified according to H. pylori positive or negative chronic atrophic (Hp‐CAG and CAG, respectively) and nonatrophic gastritis (Hp‐CG and CG, respectively), and H. pylori negative normal mucosa (controls). Oxidative damage was evaluated by nitrotyrosine immunohistochemistry in the whole mucosa and in each compartment at baseline and at 2 and 12 months after eradication. Types of intestinal metaplasia were classified by histochemistry. Results. Total nitrotyrosine levels appeared significantly higher in H. pylori positive than in negative patients, and in Hp‐CAG than in Hp‐CG (p < .001); no differences were found between H. pylori negative gastritis and normal mucosa. Nitrotyrosine were found in foveolae and intestinal metaplasia only in Hp‐CAG. At 12 months after H. pylori eradication, total nitrotyrosine levels showed a trend toward a decrease in Hp‐CG and decreased significantly in Hp‐CAG (p = .002), disappearing from the foveolae (p = .002), but remaining unchanged in intestinal metaplasia. Type I and II of intestinal metaplasia were present with the same prevalence in Hp‐CAG and CAG, and did not change after H. pylori eradication. Conclusions. Oxidative damage of the gastric mucosa increases from Hp‐CG to Hp‐CAG, involving the foveolae and intestinal metaplasia. H. pylori eradication induces a complete healing of foveolae but not of intestinal metaplasia, reducing the overall oxidative damage in the mucosa.     

Expression of 60 kDa heat shock protein (Hsp60) on plasma membrane of Daudi cells

In Daudi cells, a fraction of the 60 kDa heat shock protein (Hsp60), which is typically a mitochondrial protein, is located on cell membrane. This was demonstrated by the recovery of biotinylated Hsp60 in the anti-Hsp60 immunoprecipitate obtained from cells in which surface-exposed proteins were selectively labeled with biotin. In further experiments, isolated membrane proteins (obtained by two different biochemical methods) were probed in Western blot with two antibodies (N-20 and K-19) directed against different epitopes located, respectively, at the amino- and at the carboxyl-terminus of the Hsp60. Both these antibodies recognized, among the isolated membrane proteins, a unique band with an electrophoretic mobility identical to that of the cytoplasmic Hsp60, thus demonstrating that Hsp60 is present on cell surface as an intact, full-length, protein. FACS analysis, performed with the same two highly specific anti-Hsp60 antibodies, confirmed that both the N-terminus and the C-terminus of the Hsp60 are exposed outside the cell and are accessible for recognition by the corresponding antibody. Moreover, quantitative analysis of the data showed that constitutive cell surface expression of the Hsp60 is limited to a small fraction (about 10%) of the whole cell population.     

Mutagenic effects of short term action of N-methyl-N’-nitro-N-nitrosoguanidine onChlamydomonas geitleri

A possible method of finding the best concentration of a chemical mutagen or a shortterm action is demonstrated by the results obtained withChlamydomonas geitleri. Mostly two cases occur. We must either use the shortest time of the mutagen action with a sufficiently high effect or utilize the whole limited time for this action using a suitable concentration with a similar effect. It is possible to choose such a structure of the doae of the chemical mutagen which in the mutation spectrum ensures either the optimal frequencies of all components or of only some of them chosen beforehand.     

Nuclear-cytoplasmic interactions affect in utero developmental capacity, phenotype, and cellular metabolism of bovine nuclear transfer fetuses

We generated a clone of bovine somatic cell nuclear transfer embryos using oocyte pools from defined maternal sources to study nuclear-cytoplasmic interactions. Nucleocytoplasmic hybrids were reconstructed with Bos taurus (Brown Swiss) granulosa cells and oocytes that contained B. taurus A (Simmental), B. taurus B (Simmental), or Bos indicus (Dwarf Zebu) cytoplasm. Another set of embryos was reconstructed with randomly selected Brown Swiss (B. taurus R) oocytes. Embryo transfer resulted in nine (12.5%), nine (13.8%), three (50%), and 11 (16.7%) Day 80 fetuses, of which eight (11.1%), three (4.6%), three (50%), and 10 (15.2%) were viable, respectively. The proportion of viable fetuses was affected by cytoplasm (likelihood ratio test, P < 0.02) and was higher for embryos with B. indicus cytoplasm than for the B. taurus A (P < 0.05) and B (P < 0.01) groups. Furthermore, the proportion of surviving Day 80 fetuses was reduced for B. taurus B as compared with B. taurus A and B. taurus R cytoplasm (P < 0.05 and P < 0.02). Body weight of nucleocytoplasmic hybrid fetuses was not significantly different from Brown Swiss control fetuses produced by artificial insemination (AI), but fetuses reconstructed with random cytoplasts of the same breed as the nuclear donor exhibited overgrowth (P < 0.01) and a higher coefficient of variation in weight. Furthermore, body weight, crown rump length, thorax circumference (P < 0.05), and femur length (P < 0.01) of fetuses with B. taurus A cytoplasm differed from fetuses with B. taurus R cytoplasms. Fetal skin, heart, and liver cells with B. indicus cytoplasm showed a greater increase in number per time period (P < 0.001) and oxygen consumption rate per cell (skin and liver, P < 0.001; heart, P < 0.08) in comparison with their counterparts with B. taurus A cytoplasm. These data point to complex oocyte cytoplasm-dependent epigenetic modifications and/or nuclear DNA-mitochondrial DNA interactions with relevance to nuclear transfer and other reproductive technologies such as ooplasmic transfer in human assisted reproduction.     

Synthesis and evaluation of phenylcarbamate derivatives as ligands for nicotinic acetylcholine receptors

Phenylcarbamate derivatives were synthesized and evaluated in radioligand binding assays for different nicotinic acetylcholine receptor (nAChR) subtypes. Carbamate derivatives bearing a pyrrolidine or piperidine moiety 8-20 exhibited much lower affinity for alpha7* nAChR than the analogues in the quinuclidine series 21-25, although the same structural elements are present. Furthermore, in contrast to the quinuclidine analogues 21-25, all (S)-pyrrolidine derivatives 8-12 and the piperidine analogues 15 and 16 exhibited higher affinities for alpha4beta2* nAChR.