Importance of some factors on the dimorphism of Candida albicans

The passage between the yeast and mycelial forms of Candida albicans B 311-10 was studied by using the minimal syntehtic medium of Shepherd et al. [19] modified without biotin and with low glucose concentrations. It was observed that biotin, aminoacids and particularly pH are not important factors in the dimorphism of C. albicans. The only factor of notable importance in the passage of yeast form to mycelial form in C. albicans was glucose concentration.     

Ribonucleic acid transfer from nucleus to cytoplasm during interphase and mitosis in mouse somatic cells cultured in vitro

Summary Kidney cells from primary cultures of 15-day old mouse embryos were incubated for 2, 5 or 10 min with H³-uridine, then either fixed immediately or incubated again for various periods in a chase medium containing an excess of unlabeled uridine and cytidine. The number of grains over the non-nucleolar part of the nucleus (chromatin), the nucleolus and the cytoplasm were counted on the autoradiograms.The grain count showed that both chromatin and nucleolus incorporate very rapidly H³-uridine from the medium, whereas a time lag elapses before any H³-radioactivity above background is detected in the cytoplasm. Incorporation of H³-uridine into the RNA of the nucleus and the nucleolus is not immediately blocked after chase, suggesting that the labeled precursor pool is not completely washed out from the living cell, or diluted by the excess of unlabeled uridine present in the medium. The grain count over the nucleus and the nucleolus rises for a certain time after chase and then gradually declines; H³-radioactivity appears in the cytoplasm 10 min after chase and keeps rising through a 110-min interval. The experiment, then — even though it suggests that the bulk of cellular RNA is synthesized in the chromatin and the nucleolus and then continuously released into the cytoplasm — does not rule out the possibility that some RNA fraction, characterized by a low turnover rate, is synthesized independently in the cytoplasm.Synthesis of RNA is a continuous process throughout the cell cycle, except during metaphase and anaphase. It ceases at prometaphase after the disappearance of the nucleolus and disintegration of the nuclear membrane, and resumes in early telophase. Part of the chromosomal RNA does not remain associated with the chromosomes through division, but is suddenly released into the cytoplasm when the cell enters metaphase.     

Dopaminergic Inhibition of Prolactin Release and Calcium Influx Induced by Neurotensin in Anterior Pituitary Is Independent of Cyclic AMP System

The present study demonstrates that 3,4-dihydroxyphenylethylamine (DA, dopamine) prevents neurotensin (NT) stimulation of both prolactin (PRL) release and calcium influx by interacting with specific receptors that are functionally linked to calcium channels. As shown by the studies with dispersed cells from rat anterior pituitary, the pharmacology of the control of PRL release and calcium influx, both induced by NT, was found to be typical of a DAergic process. This was demonstrated by the order of potency of agonists in inhibiting PRL release and calcium influx (DA greater than epinephrine greater than norepinephrine much greater than isoproterenol); by the high affinity of antagonists such as haloperidol and fluphenazine for this process; and by the high degree of stereoselectivity of sulpiride. Specific D2 receptor agonists, such as bromocriptine and lisuride, and the specific D2 receptor antagonist (-)-sulpiride were found to be highly potent on the DA receptors negatively coupled with calcium channels and PRL release. DA was found to lack the capacity to change the influx of calcium induced by either the sodium channel activator veratridine or high extracellular potassium levels, thus indicating a specific action of this amine on calcium channels sensitive to NT. In a range of concentrations that are effective in inhibiting either the calcium influx or the PRL release, both induced by NT, DA did not alter the cyclic AMP generating system. DA (from 1.0 nM to 50 nM) did not affect adenylate cyclase activity in rat pituitary gland homogenates and did not modify intracellular cyclic AMP levels in pituitary cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular Characterization of Microbial Population Dynamics during Sildenafil Citrate Degradation

Little is known about pharmaceutical and personal care products pollutants (PPCPs), but there is a growing interest in how they might impact the environment and microbial communities. The widespread use of Viagra (sildenafil citrate) has attracted great attention because of the high usage rate, the unpredictable disposal and the unknown potential effects on wildlife and the environment. Until now information regarding the impact of Viagra on microbial community in water environment has not been reported. In this research, for the first time, the genetic profile of the microbial community, developing in a Viagra polluted water environment, was evaluated by means of the 16S and 18S rRNA genes, for bacteria and fungi, respectively, amplified by polymerase chain reaction (PCR) and separated using the denaturing gradient gel electrophoresis (DGGE) technique. The DGGE results revealed a complex microbial community structure with most of the population persisting throughout the experimental period. DNA sequences from bands observed in the different denaturing gradient gel electrophoresis profiles exhibited the highest degree of identity to uncultured bacteria and fungi found previously mainly in polluted environmental and treating bioreactors. Biotransformation ability of sildenafil citrate by the microbial pool was studied and the capability of these microorganisms to detoxify a polluted water ecosystem was assessed. The bacterial and fungal population was able to degrade sildenafil citrate entirely. Additionally, assays conducted on Daphnia magna, algal growth inhibition assay and cell viability determination on HepG2 human cells showed that biotransformation products obtained from the bacterial growth was not toxic. The higher removal efficiency for sildenafil citrate and the lack of toxicity by the biotransformation products obtained showed that the microbial community identified here represented a composite population that might have biotechnological relevance to retrieve sildenafil citrate contaminated sites.     

The Dendritic Cell Major Histocompatibility Complex II (MHC II) Peptidome Derives from a Variety of Processing Pathways and Includes Peptides with a Broad Spectrum of HLA-DM Sensitivity

The repertoire of peptides displayed in vivo by MHC II molecules derives from a wide spectrum of proteins produced by different cell types. Although intracellular endosomal processing in dendritic cells and B cells has been characterized for a few antigens, the overall range of processing pathways responsible for generating the MHC II peptidome are currently unclear. To determine the contribution of non-endosomal processing pathways, we eluted and sequenced over 3000 HLA-DR1-bound peptides presented in vivo by dendritic cells. The processing enzymes were identified by reference to a database of experimentally determined cleavage sites and experimentally validated for four epitopes derived from complement 3, collagen II, thymosin β4, and gelsolin. We determined that self-antigens processed by tissue-specific proteases, including complement, matrix metalloproteases, caspases, and granzymes, and carried by lymph, contribute significantly to the MHC II self-peptidome presented by conventional dendritic cells in vivo. Additionally, the presented peptides exhibited a wide spectrum of binding affinity and HLA-DM susceptibility. The results indicate that the HLA-DR1-restricted self-peptidome presented under physiological conditions derives from a variety of processing pathways. Non-endosomal processing enzymes add to the number of epitopes cleaved by cathepsins, altogether generating a wider peptide repertoire. Taken together with HLA-DM-dependent and-independent loading pathways, this ensures that a broad self-peptidome is presented by dendritic cells. This work brings attention to the role of “self-recognition” as a dynamic interaction between dendritic cells and the metabolic/catabolic activities ongoing in every parenchymal organ as part of tissue growth, remodeling, and physiological apoptosis.     

Transposon Tc1 of the nematode Caenorhabditis elegans jumps in human cells.

The transposon Tc1 of the nematode Caenorhabditis elegans is a member of the widespread family of Tc1/mariner transposons. The distribution pattern of virtually identical transposons among insect species that diverged 200 million years ago suggested horizontal transfer of the elements between species. Thishypothesis gained experimental support when it was shown that Tc1 and later also mariner transposons could be made to jump in vitro , with their transposase as the only protein required. Later it was shown that mariner transposons from one fruit fly species can jump in other fruit fly species and in a protozoan and, recently, that a Tc1-like transposon from the nematode jumps in fish cells and that a fish Tc1-like transposon jumps in human cells. Here we show that the Tc1 element from the nematode jumps in human cells. This provides further support for the horizontal spread hypothesis. Furthermore, it suggests that Tc1 can be used as vehicle for DNA integration in human gene therapy.     

Preparation of O-phosphotyrosine-containing peptides by Fmoc solid-phase synthesis: Evaluation of several Fmoc-Tyr(PO3R2)-OH derivatives

Summary The synthesis of two model Tyr(P)-containing peptides using Fmoc-Tyr(PO₃ tBu₂)-OH, Fmoc-Tyr(PO₃Bzl₂)-OH and Fmoc-Tyr(PO₃H₂)-OH established that the t-butylphosphate-protected derivative was the preferred derivative for use in Fmoc solid-phase peptide synthesis, since it afforded phosphopeptides in high purity and with the lowest amount of Tyr-peptide contamination. In addition, this study confirmed that commercially available Fmoc-Tyr(PO₃H₂)-OH is also suitable for use in Fmoc solid-phase synthesis but gives less pure phosphopeptides, along with the generation of 1–4% of the tyrosine-containing peptide for the model sequences studied. In view of the good performance of Fmoc-Tyr(PO₃ tBu₂)-OH, a large-scale three-step synthetic procedure was developed which involved phenacyl protection of the carboxyl group, phosphite-triester phosphorylation of the tyrosyl hydroxyl using di-t-butyl N,N-diethylphosphoramidite, and final removal of the phenacyl group by zinc reduction in acetic acid.Abbreviations BOP benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate - tBu t-butyl - Bzl benzyl - DBU 1,8-diazabicyclo[5,4,0]undec-7-ene - DMF N,N-dimethylformamide - EDT ethanedithiol - Fmoc 9-fluorenylmethoxycarbonyl - HOBt N-hydroxybenzotriazole - HPLC high performance liquid chromatography - NMM N-methylmorpholine - Pac phenacyl - TFA trifluoroacetic acid - THF tetrahydrofuran - Tyr(P) O-phosphotyrosine