Phosphorylation of fibrinogen by casein kinase 2.

Casein kinase 2 from rat liver cytosol phosphorylated human fibrinogen in a reaction that was not stimulated by Ca2+ or cyclic AMP, but was markedly inhibited by heparin, and proceeded at a similar rate when either ATP or GTP was used as phosphate donor. Analysis of casein kinase 2 by glycerol-density-gradient centrifugation showed that the activities towards fibrinogen, casein, phosvitin, high-mobility-group protein 14 and glycogen synthase coincided. Maximal incorporation into fibrinogen by casein kinase 2 averaged 1 mol of phosphate/mol of protein substrate, most of it in the alpha-chain, although some phosphorylation of the beta-chain was also detected. Analysis of phosphorylated alpha-chain revealed that most of the phosphate was incorporated on serine. Phosphorylation of human fibrinogen was also performed by casein kinase 2 from human polymorphonuclear leucocytes, lymphocytes and platelets.     

Antibodies Specific for GABAA Receptor α Subunits Reveal that Chronic Alcohol Treatment Down-Regulates α-Subunit Expression in Rat Brain Regions

Abstract— Chronic administration of ethanol results in the development of tolerance and dependence. The molecular mechanism underlying these behavioral actions of ethanol is poorly understood. Several lines of evidence have suggested that some of the pharmacological actions of ethanol are mediated via a potentiation of GABAergic transmission. Chronic ethanol administration results in a reduction in the GABA_A receptor-mediated ³⁶Cl^? uptake in cortical synaptoneurosomes and primary cultured neurons. We and others have shown that it also results in a 40-50% reduction in GABA_A receptor α-subunit mRNA levels in the rat cerebral cortex. In the present study, we investigated the expression of α₁, α₂, and α₃ subunits of the GABA_A receptor in the cerebral cortex and the α₁ subunit in the cerebellum by immunoblotting using polyclonal antibodies raised against α₁-, α₂-, and α₃-subunit polypeptides following chronic ethanol treatment. These results reveal that chronic ethanol administration to rats results in a 61 ± 4% reduction in level of the GABA_A receptor α₁subunit (51 kDa), 47 ± 8% reduction in level of the α₂subunit (53 kDa), and 30 ± 7% reduction in level of the α₃subunit (59 kDa) in the cerebral cortex and a 56 ± 5% reduction in content of the α₁ subunit in the cerebellum. In summary, this ethanol-induced reduction in content of the GABA_A receptor α subunits may underlie alterations in the GABA_A receptor function and could be related to cellular adaptation to the functional disturbance caused by ethanol.     

Changes in marriage seasonality among some european rural populations

The preservation or the change of the population structure is often strictly related to the acquisition or loss of behavioural models: the choice of the wedding period is among them. Analysing the period in which people marry, it is possible to point out modifications in the economy, in the work calendar and it also can be helpful to determine the importance of religious traditions. In this work, the authors study the marriage seasonality of nine rural European populations in France, Spain and Italy and its evolution during the last two centuries. The research has allowed to point out three different patterns of marriage seasonality: the Autumn marriage, the Winter marriage and populations with no remarkable seasonal variations. The first distribution results from the combination of work calendar, harsh climate in winter and religious traditions (the month of May consacrated to Maria, the Lent and Advent period), while the second one is due mostly to summer migrations. The importance of these factors tend to reduce during the XX century, making the distributions more homogeneous.     

Mitochondrial D-loop "signatures" produced by low-stringency single specific primer PCR constitute a simple comparative human identity test.

We have developed a technique called "LSSP-PCR" (low-stringency single specific primer PCR) that detects single or multiple mutations in DNA. A purified DNA fragment is submitted to PCR by using a single primer specific for one of the extremities of the fragment, under conditions of very low stringency. The primer hybridizes specifically to its complementary extremity and nonspecifically to multiple sites within the fragment, in a sequence-dependent manner. A complex set of reaction products is thus created that, when separated by electrophoresis, constitutes a unique "gene signature." We here report the application of LSSP-PCR to the detection of sequence variation in the control (D-loop) region of human mtDNA, which is known to differ significantly between unrelated individuals. We prepared human DNA samples from blood and amplified a 1024-bp portion of the mtDNA control region, using primers L15996 and H408. The amplified mtDNA fragments were then reamplified under LSSP-PCR conditions by using L15996 or H408 as drivers to produce complex signatures that always differed between unrelated individuals and yet were highly reproducible. In contrast, all mother-child pairs tested were identical, as expected from the matrilineal inheritance of mtDNA. Thus, the use of LSSP-PCR to produce D-loop signatures constitutes a powerful new technique for mtDNA-based comparative identity testing.     

Changes in Dehydrodiferulic Acids and Peroxidase Activity against Ferulic Acid Associated with Cell Walls during Growth of Pinus pinaster Hypocotyl

Hydroxycinnamic acids associated with hypocotyl cell walls of dark-grown seedlings of Pinus pinaster Aiton were extracted with 1 N NaOH and identified by gas chromatography-mass spectrometry. The main hydroxycinnamic acid found was ferulic acid. Diferulic acid dehydrodimers were also found, with the 8,8-coupled isomer (compound 11) being the dehydrodiferulate present in the highest amount. However, the 5,5-coupled isomer, commonly referred to referred to as diferulic acid, was not detected. Two truxillic acids, 4-4[prime]-dihydroxy-3-3[prime]-dimethoxy-[alpha]-truxillic acids I and II, were tentatively identified. The 8,8-coupled dehydrodiferulic acid (compound 11) was the phenolic acid that showed the most conspicuous changes with hypocotyl age as well as along the hypocotyl axis. Peroxidase activity against ferulic acid was found in the apoplastic fluid as well as being ionically and covalently bound to the cell walls. The peroxidase activity increased with hypocotyl age as well as from the subapical toward the basal region of the hypocotyls. A key role in the cell-wall stiffening of 8,8 but not 5,5 dimerization of ferulic acid catalyzed by cell-wall peroxidases is proposed.     

Theiler’s Virus Infection of Perforin-Deficient Mice

Theiler’s virus, a murine picornavirus, infects the central nervous systems of C57BL/6 mice and is cleared after approximately 10 days by a process which requires CD8⁺ cytotoxic T cells. We used perforin-deficient C57BL/6 mice to test the role of this protein in viral clearance. Perforin-deficient mice died from viral encephalomyelitis between days 12 and 18 postinoculation. They had high levels of viral RNA in their central nervous systems until the time of death. In contrast, viral RNA had disappeared by day 11 postinoculation in wild-type C57BL/6 mice. Cytotoxic T cells can kill infected cells by two main mechanisms: the secretion of the pore-forming protein perforin or the interaction of the Fas ligand with the apoptosis-inducing Fas molecule on the target cell. Our results demonstrate that clearance of Theiler’s virus from the central nervous system in C57BL/6 mice is perforin dependent.     

Influence of gender and the endocrine environment on the distribution of androgen receptors in the lacrimal gland

Androgens are known to regulate both the structure and function of lacrimal tissue in a variety of species. To explore the endocrine basis for this hormone action, the following study was designed to: (1) determine the cellular distribution of androgen receptors in the lacrimal gland; and (2) examine the influence of gender and the endocrine environment on the glandular content of these binding sites. Lacrimal glands were obtained from intact, castrated, hypophysectomized, diabetic or sham-operated male or female adult rats, mice or hamsters, as well as from orchiectomized rats exposed to placebo compounds or physiological levels of testosterone. The cellular of androgen receptors was evaluated by utilizing an immunoperoxidase protocol, in which a purified rabbit polyclonal antibody to the rat androgen receptor was used as the first antibody. Our findings with lacrimal glands showed that: (1) androgen receptors are located almost exclusively in nuclei of epithelial cells; (2) the cellular distribution or intranuclear density of these binding sites is far more extensive in glands of males, as compared to females; (3) orchiectomy or hypophysectomy, but not sham-surgery or diabetes, lead to a dramatic reduction in the immunocytochemical expression of androgen receptors; and (4) testosterone administration to orchiectomized rats induces a marked increase in androgen receptor content, relative to that in placebo-exposed glands. Our results also reveal that a 10 kb androgen receptor mRNA exists in the rat lacrimal gland. Overall, these findings demonstrate that gender and the endocrine system may significantly influence the distribution of androgen binding sites in rat lacrimal tissue. Moreover, our results show that androgens up-regulate their own lacrimal gland receptors.     

Cell culture and animal infection with distinct Trypanosoma cruzi strains expressing red and green fluorescent proteins

Different strains of Trypanosoma cruzi were transfected with an expression vector that allows the integration of green fluorescent protein (GFP) and red fluorescent protein (RFP) genes into the beta-tubulin locus by homologous recombination. The sites of integration of the GFP and RFP markers were determined by pulse-field gel electrophoresis and Southern blot analyses. Cloned cell lines selected from transfected epimastigote populations maintained high levels of fluorescent protein expression even after 6 months of in vitro culture of epimastigotes in the absence of drug selection. Fluorescent trypomastigotes and amastigotes were observed within Vero cells in culture as well as in hearts and diaphragms of infected mice. The infectivity of the GFP- and RFP-expressing parasites in tissue culture cells was comparable to wild type populations. Furthermore, GFP- and RFP-expressing parasites were able to produce similar levels of parasitemia in mice compared with wild type parasites. Cell cultures infected simultaneously with two cloned cell lines from the same parasite strain, each one expressing a distinct fluorescent marker, showed that at least two different parasites are able to infect the same cell. Double-infected cells were also detected when GFP- and RFP-expressing parasites were derived from strains belonging to two distinct T. cruzi lineages. These results show the usefulness of parasites expressing GFP and RFP for the study of various aspects of T. cruzi infection including the mechanisms of cell invasion, genetic exchange among parasites and the differential tissue distribution in animal models of Chagas disease.     

Mismatch repair in Trypanosoma brucei: heterologous expression of MSH2 from Trypanosoma cruzi provides new insights into the response to oxidative damage

Trypanosomes are unicellular eukaryotes that cause disease in humans and other mammals. Trypanosoma cruzi and Trypanosoma brucei are the causative agents, respectively, of Chagas disease in the Americas and sleeping sickness in sub-Saharan Africa. To better comprehend the interaction of these parasites with their hosts, understanding the mechanisms involved in the generation of genetic variability is critical. One such mechanism is mismatch repair (MMR), which has a crucial, evolutionarily conserved role in maintaining the fidelity of DNA replication, as well as acting in other cellular processes, such as DNA recombination. Here we have attempted to complement T. brucei MMR through the expression of MSH2 from T. cruzi. Our results show that T. brucei MSH2-null mutants are more sensitive to hydrogen peroxide (H2O2) than wild type cells, suggesting the involvement of MSH2 in the response to oxidative stress in this parasite. This phenotype is reverted by the expression of either the T. cruzi or the T. brucei MSH2 protein in the MSH2-null mutants. In contrast, MMR complementation, as assessed by resistance to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and microsatellite instability, was not achieved by the heterologous expression of T. cruzi MSH2. This finding, associated to the demonstration that mutation of MLH1, another component of the MMR system, did not affect sensitivity of T. brucei cells to H2O2, suggests an additional role of MSH2 in dealing with oxidative damage in these parasites, which may occur independently of MMR.     

Influence of antibiotics on bacterial load and sperm parameters during short-term preservation of collared peccary semen

Studies on semen and sperm cells are critical to develop assisted reproductive technologies for the conservation of the collared peccary. The objective of the study was to compare the effect of different antibiotics on the bacterial load and sperm quality during short-term storage of peccary semen. Fresh semen samples from 10 males were extended in Tris-egg yolk or Tris-Aloe vera supplemented with streptomycin-penicillin (SP; 1 mg/mL - 1000 IU/mL or 2 mg/mL - 2000 IU/mL) or gentamicin (30 µg/mL or 70 µg/mL) before storage at 5°C. Bacterial load and sperm motility, membrane integrity and function, mitochondrial activity, and morphology, were evaluated at different time points for 36 h. The SP and gentamicin treatments concentration inhibited (p < 0.05) bacterial growth for 36 h regardless of the extender. Compared to the other treatments, Tris-egg yolk plus 70 µg/mL gentamicin maintained the sperm parameters for longer, including total motility (41.9 ± 6.1%) at 24 h, and membrane integrity (58.3 ± 2.1%) at 36 h. In contrast, the highest SP concentration in both extenders impaired sperm membrane integrity at 36 h (p < 0.05). For the liquid storage of collared peccary semen, it therefore is recommended to use Tris extender supplemented with egg yolk and gentamicin (70 µg/mL).