Modulation of trophoblast stem cell and giant cell phenotypes: analyses using the Rcho-1 cell model

Trophoblast giant cells are located at the maternal-embryonic interface and have fundamental roles in the invasive and endocrine phenotypes of the rodent placenta. In this report, we describe the experimental modulation of trophoblast stem cell and trophoblast giant cell phenotypes using the Rcho-1 trophoblast cell model. Rcho-1 trophoblast cells can be manipulated to proliferate or differentiate into trophoblast giant cells. Differentiated Rcho-1 trophoblast cells are invasive and possess an endocrine phenotype, including the production of members of the prolactin (PRL) family. Dimethyl sulfoxide (DMSO), a known differentiation-inducing agent, was found to possess profound effects on the in vitro development of trophoblast cells. Exposure to DMSO, at non-toxic concentrations, inhibited trophoblast giant cell differentiation in a dose-dependent manner. These concentrations of DMSO did not significantly affect trophoblast cell proliferation or survival. Trophoblast cells exposed to DMSO exhibited an altered morphology; they were clustered in tightly packed colonies. Trophoblast giant cell formation was disrupted, as was the expression of members of the PRL gene family. The effects of DMSO were reversible. Removal of DMSO resulted in the formation of trophoblast giant cells and expression of the PRL gene family. The phenotype of the DMSO-treated cells was further determined by examining the expression of a battery of genes characteristic of trophoblast stem cells and differentiated trophoblast cell lineages. DMSO treatment had a striking stimulatory effect on eomesodermin expression and a reciprocal inhibitory effect on Hand1 expression. In summary, DMSO reversibly inhibits trophoblast differentiation and induces a quiescent state, which mimics some but not all aspects of the trophoblast stem cell phenotype.     

Kinin and opioid receptors in the paratrigeminal nucleus modulate the somatosensory reflex to rat sciatic nerve stimulation

The influence of kinin and opioid receptor blockade in the paratrigeminal nucleus (Pa5) on the somatosensory reflex (SSR) to sciatic nerve stimulation (SNS) was assessed in anaesthetized-paralyzed rats. SNS (square 1 ms pulses at 0.6 mA and 20 Hz for 10s) increased mean arterial pressure from 87+/-3 to 106+/-3 mmHg. Pressor responses to SNS were reduced 40-60% by HOE-140 and LF 16-0687 (B2 receptor antagonists; 20 and 100 pmol respectively), CTOP or nor-binaltorphimine (mu and kappa opioid receptor antagonists, respectively; 1 microg) but potentiated by naltrindole (delta opioid receptor antagonist) receptor antagonist microinjections into the contralateral (but not ipsilateral) Pa5. The SSR to sciatic nerve stimulation was not changed by B1 kinin receptor or NK1, NK2 and NK3 tachykinin receptor antagonists administered to the Pa5. Capsaicin pretreatment (40 mg/kg/day, 3 days) abolished the effects of the opioid receptor antagonists, but did not change the effect of kinin B2 receptor blockade on the SSR. Thus, the activity of B2 and opioid receptor-operated mechanisms in the Pa5 contribute to the SSR in the rat, suggesting a role for these endogenous peptides in the cardiovascular responses to SNS.     

Effects of prepartum lipid supplementation on FSH superstimulation and transferable embryo recovery in multiparous beef cows

The objective of this experiment was to determine the effect of prepartum lipid supplementation on the number and quality of embryos recovered following ovarian super-ovulation in postpartum suckled beef cows. Mature cows (n = 40) were assigned to one of two treatments (lipid versus. no lipid) and supplemented for approximately 40 days prior to calving. Supplements provided to cows were isocaloric and isonitrogenous. The treatment group was fed 1.6 kg hd(-1) per day of whole soybeans (WSB; 19.8% ether extract, and 41.8% crude protein) and the control group received a supplement consisting of 1.8 kg hd(-1) day of a soybean meal and soy-hull combination (SBS; 2.15% EE and 36.81% CP). Cows were synchronized using a GnRH [Cystorelin((R)) 100 microg im]-GnRH-PGF(2alpha) [Lutalyse 25 mg im] protocol. Cows were administered two injections of GnRH seven days apart and PG seven days after the second GnRH injection. Twenty-eight cows (WSB, n = 15; SBS, n = 13) responded to estrus synchronization and were superstimulated. Super-ovulation was initiated on day 8-10 of the synchronized cycle by twice-daily injections of pFSH (Pluset) over four days in decreasing doses using a total of 608.4 IU per cow. Prostaglandin F(2alpha) was administered 96 and 108 h after super-stimulation was initiated with FSH. Days postpartum (WSB = 59 days; SBS = 57 days) at initiation of FSH treatments were similar (P > 0.10) for both treatments. Cows were monitored for estrus activity by the HeatWatch Estrus Detection System. Twenty-seven cows (WSB, n = 15; SBS, n = 12) exhibited estrus after FSH and inseminated at 0, 12, and 24 h after the onset of estrus with 1, 2, and 1 units of semen, respectively. Embryos were recovered and evaluated 7-8 days later. Only cows that responded to FSH and that were inseminated were used for statistical analysis. Data were analyzed using the General Linear Models Procedure of SAS. Body condition scores did not differ (P > 0.10) between treatments when cows were evaluated at the initiation of the experiment, two weeks prior to calving, and at initiation of superovulation with FSH. Estrous cyclicity prior to the initiation of estrus synchronization did not differ (P > 0.10) between treatments. There was no difference (P > 0.10) between treatments in recovery of total embryos (WSB, 14.7 +/- 3.5; SBS, 17.5 +/- 3.0), transferable embryos (WSB, 10.3 +/- 2.5; SBS, 13.6 +/- 2.6), degenerate embryos (WSB, 3.3 +/- 1.1; SBS, 1.6 +/- 1.7) or unfertilized ova (WSB, 1.1 +/- 0.5; SBS, 2.3 +/- 1.2). Cows that were supplemented with whole soybeans prior to parturition failed to produce an increased total number of ova or transferable embryos following super-ovulation.     

Polyvalent DNA prime and envelope protein boost HIV-1 vaccine elicits humoral and cellular responses and controls plasma viremia in rhesus macaques following rectal challenge with an R5 SHIV isolate

Immunization of macaques with multivalent DNA encoding gp120 genes from HIV-1 subtypes A, B, C and E and a gag gene followed by boosting with homologous gp120 proteins elicited strong anti-gp120 antibodies capable of neutralizing homologous and to a lesser degree heterologous HIV-1 isolates. Both Env- and Gag-specific cell mediated immune (CMI) responses were detected in the immunized animals. Following rectal challenge with an SHIV isolate encoding HIV-1(Ba-L)env, plasma viremia in the infected immunized animals was significantly lower than that observed in the na?ve animals. Further, one of six immunized animals was completely protected whereas all six na?ve animals were infected. These results demonstrate that a vaccine based on priming with a polyvalent DNA vaccine from multiple HIV-1 subtypes followed by boosting with homologous Env proteins elicits anti-HIV-1 immune responses capable of controlling rectal transmission of SHIV(Ba-L).     

The effect of genetic variation on the lipid response to dietary change: recent findings

PURPOSE OF REVIEW: Dyslipidaemia is an important risk factor for cardiovascular disease, and can be modified by diet. However, the lipid response to dietary change may be influenced by genetic variation. This review examines recent research (published since August 2003) on the effect of genetic variation on the lipid response to dietary change. RECENT FINDINGS: In 10 reports describing intervention studies and seven reports describing observational studies, the lipid response to diet was modified by polymorphisms within the genes for apoE, apoB, apoCIII, lipoprotein lipase, hepatic lipase, endothelial lipase, the liver fatty acid-binding protein, the beta3-adrenergic receptor, adipsin and the peroxisome proliferator-activated receptor gamma. The studies varied widely in terms of the number and type of study participants, the composition and duration of the dietary interventions, the nutrients studied and dietary assessment methods used in the observational studies, and the polymorphisms analysed--some of which had not been studied before with regard to the lipid response to diet. SUMMARY: The lipid response to dietary change is highly complex. Future studies will have to be large in order to assess the effects of multiple polymorphisms, and will have to control for many factors other than diet. At present, it is premature to recommend the use of genotyping in the design of therapeutic diets. However, such studies may be useful in identifying the mechanisms by which dietary components influence lipid levels.     

Histone H2A ubiquitination does not preclude histone H1 binding, but it facilitates its association with the nucleosome

Histone H2A ubiquitination is a bulky posttranslational modification that occurs at the vicinity of the binding site for linker histones in the nucleosome. Therefore, we took several experimental approaches to investigate the role of ubiquitinated H2A (uH2A) in the binding of linker histones. Our results showed that uH2A was present in situ in histone H1-containing nucleosomes. Notably in vitro experiments using nucleosomes reconstituted onto 167-bp random sequence and 208-bp (5 S rRNA gene) DNA fragments showed that ubiquitination of H2A did not prevent binding of histone H1 but it rather enhanced the binding of this histone to the nucleosome. We also showed that ubiquitination of H2A did not affect the positioning of the histone octamer in the nucleosome in either the absence or the presence of linker histones.     

Bradykinin microinjection in the paratrigeminal nucleus triggers neuronal discharge in the rat rostroventrolateral reticular nucleus

A small collection of neurons in the dorsal lateral medulla, the paratrigeminal nucleus (Pa5), projects directly to the rostroventrolateral reticular nucleus (RVL). Bradykinin (BK) microinjections in the Pa5 produce marked pressor responses. Also, the Pa5 is believed to be a component of the neuronal substrates of the somatosensory response and the baroreflex arc. Considering the developing interest in the functional physiology of the Pa5, the present study was designed to characterize RVL neuronal activity in response to BK microinjections in the Pa5 as well as to phenylephrine-induced blood pressure increases in freely behaving rats. Of the 46 discriminated RVL neurons, 82% responded with a 180% mean increase in firing rate after BK application to the paratrigeminal nucleus, before the onset of the blood pressure increase. Thirty (79%) of the RVL BK-excited neurons were baroreceptor-inhibited units that responded with a 30% decrease in firing rate in response to a phenylephrine-produced increase of blood pressure. Twenty-seven (71%) units of the latter population displayed cardiac-cycle-locked rhythmic activity. The findings demonstrate a BK-stimulated functional connection between the Pa5 and RVL that may represent the neural pathway in the BK-mediated pressor response. This pathway may be relevant to baroreflex mechanisms since it relates to cardiovascular pressure-sensitive neurons.