Einfluß des Phasenstatus der Vorkulturzellen auf die Ascosporenbildung von Saccharomyces cerevisiae

Summary Cells from three growth phases were examined for their ability to sporulate: cells from a) phase II (first phase of exponential growth with glucose as carbon source), b) phase III (second lag-phase during adaptation to oxidative metabolism), and c) phase IV (second phase of almost exponential growth with ethanol as carbon source). 1. Cells from phase III showed the best sporulation ability because they reached the highest percentage of asci and also of 4-spored asci. 2. Cells of phase II exhibited the highest and those of phase IV the lowest rate of sporulation (Fig. 3). 3. The longer the cells remained in the presporulation medium the more abbreviated was the time in the sporulation culture before the first asci appeared, and this abbreviation was just equal to the time of elongation in the preculture. This clearly demonstrates the different degree of respiratory adaptation. — After transfer to the sporulation medium O₂-consumption arose to a steep maximum within the first 10 hours followed by medium values which dropped again rapidly at the onset of ascospore formation (Fig. 4). Only during the time of high and medium O₂-consumption there was an increase in dry weight reflecting the assimilation of acetate. In cells of phase II compared with those of phase IV this assimilation of acetate showed the same delay as the onset of sporulation, whereas full capacity of respiration was reached much sooner.     

THE CENTRIOLE CYCLE IN SYNCHRONIZED HELA CELLS

Progression of the HeLa cell through its life cycle is accompanied by centriolar replication and pericentriolar changes that are in synchrony with DNA synthesis and mitosis. The first signs of preparation for replication occur during G₁ at which time the two orthogonal centrioles separate. Replication by budding begins at/or near the initiation of DNA synthesis and is completed by G₂. Pericentriolar changes which probably are causally related to spindle tubule formation occur at this time and include the appearance of vesicles, electron-opaque bodies, and an amorphous pericentriolar halo. These phenomena begin to disappear by late prophase, and the remainder of mitosis manifests decreasing centriolar and pericentriolar activity.     

Currents through Hv1 channels deplete protons in their vicinity

Proton channels have evolved to provide a pH regulatory mechanism, affording the extrusion of protons from the cytoplasm at all membrane potentials. Previous evidence has suggested that channel-mediated acid extrusion could significantly change the local concentration of protons in the vicinity of the channel. In this work, we directly measure the proton depletion caused by activation of Hv1 proton channels using patch-clamp fluorometry recordings from channels labeled with the Venus fluorescent protein at intracellular domains. The fluorescence of the Venus protein is very sensitive to pH, thus behaving as a genetically encoded sensor of local pH. Eliciting outward proton currents increases the fluorescence intensity of Venus. This dequenching is related to the magnitude of the current and not to channel gating and is dependent on the pH gradient. Our results provide direct evidence of local proton depletion caused by flux through the proton-selective channel.     

Role of Estradiol in the Regulation of Prolactin Secretion During Late Pregnancy

Estrogen action is necessary for evidencing the stimulatory action of mifepristone and naloxone on prolactin (PRL) secretion during late pregnancy. Our aim is to determine the mechanism mediating this facilitator action of estrogens. To investigate the hypothalamic mechanisms involved in estrogen actions in PRL secretion at the end of pregnancy, we measured the effect of pretreatment with the estrogen antagonist tamoxifen on the expression of tyrosine hydroxylase (TH), hormone receptors (ERα and β, PRs, PRLR_(long)), and μ- and κ- opioid receptors (ORs) at mRNA (by semiquantitative RT-PCR) and protein (by western blot for TH, PRLR_(long), ERα, PRs, μ- and ORs) levels in extracts of medial basal hypothalamus (MBH) and serum PRL, E₂ and P₄ levels (by RIA) in mifepristone- and naloxone-treated rats. Tamoxifen administration partially prevented PRL release induced by the combined treatment. TH expression diminished and ERα expression increased in mifepristone-treated rats at mRNA and protein levels and tamoxifen partially prevented these changes with no effect on PRs expression. Mifepristone increased PRLR_(long) mRNA levels; this increase was blocked by tamoxifen. Combined tamoxifen and mifepristone treatment decreased μ- and k-ORs mRNA but not protein levels. In conclusion, E₂ induces neuroadaptive mechanisms necessary to facilitate PRL release preceding delivery. Acting through ERα, E₂ modulates hypothalamic dopaminergic neurons activity, regulating TH, μ- and κ-ORs and PRLR_(long) expression, and is necessary for evidencing the effects of P₄ withdrawal. Its presence on days 14 and 15 of pregnancy is crucial to facilitate the opioid system modulation of PRL secretion at the end of pregnancy in the rat.     

Development and validation of an immunoreceptor assay for simulect based on surface plasmon resonance.

Simulect is a chimeric human/mouse antibody directed against interleukin-2 (IL-2) receptor. A combined immuno- and receptor assay has been developed and validated to characterize the production of Simulect batches. This assay is based on surface plasmon resonance (SPR) technology. In each experiment two successive interactions were monitored: the direct binding of Simulect to an anti-human IgG antibody, followed by the direct binding of IL-2-soluble receptor to the preformed anti-human IgG antibody/Simulect complex. Based on the first interaction a direct immunoassay for Simulect was optimized and validated. Based on the second interaction a direct receptor assay for Simulect biological activity was optimized and validated. The assays were validated by performing three independent assays on 3 different days. The intra- and interday variations of the immunoassay (expressed as % CV) were, respectively, 1.7 and 1.6%. The overall accuracy for the immunoassay was 98.5% +/- 1. The intra- and interday variations of the receptor assay (% CV) were, respectively, 1.6 and 3.7%. The overall accuracy of the receptor assay was 100% +/- 2. Four batches of Simulect were compared to a reference batch. The results did not show significant differences for the immunoreactivity. However, the results of the receptor assay showed accuracies which were apparently higher than 100%. This was explained by a slight degradation of the reference batch after few years of storage. These results demonstrate the advantage of this method combining evaluation of the immunological and biological integrity of the drug and a high reproducibility in accuracy and precision of the biosensor-based technology.     

Synthesis and Characterization of Binding of 5-[76Br] Bromo-3-[[2(S)-Azetidinyl]methoxy]pyridine, a Novel Nicotinic Acetylcholine Receptor Ligand, in Rat Brain

5-[76Br]Bromo-3-[[2(S)-azetidinyl]methoxy]pyridine ([76Br]BAP), a novel nicotinic acetylcholine receptor ligand, was synthesized using [76Br]bromide in an oxidative bromodestannylation of the corresponding trimethylstannyl compound. The radiochemical yield was 25%, and the specific radioactivity was on the order of 1 Ci/micromol. The binding properties of [76Br]BAP were characterized in vitro and in vivo in rat brain, and positron emission tomography (PET) experiments were performed in two rhesus monkeys. In association experiments on membranes of the cortex and thalamus, >90% of maximal specific [76Br]BAP binding was obtained after 60 min. The dissociation half-life of [76Br]BAP was 51 +/- 6 min in cortical membranes and 56 +/- 3 min in thalamic membranes. Saturation experiments with [76Br]BAP revealed one population of binding sites with dissociation constant (K(D)) values of 36 +/- 9 and 30 +/- 9 pM in membranes of cortex and thalamus, respectively. The maximal binding site density (Bmax) values were 90 +/- 17 and 207 +/- 33 fmol/mg in membranes of cortex and thalamus, respectively. Scatchard plots were nonlinear, and the Hill coefficients were <1, suggesting the presence of a lower-affinity binding site. In vitro autoradiography studies showed that binding of [76Br]BAP was high in the thalamus and presubiculum, moderate in the cortex and striatum, and low in the cerebellum and hippocampus. A similar pattern of [76Br]BAP accumulation was observed by ex vivo autoradiography. In vivo, binding of [76Br]BAP in whole rat brain was blocked by preinjection of (S)(-)-nicotine (0.3 mg/kg) by 27, 52, 68, and 91% at survival times of 10, 25, 40, 120, and 300 min, respectively. In a preliminary PET study in rhesus monkeys, the highest [76Br]BAP uptake was found in the thalamus, and radioactivity was displaceable by approximately 60% with cytisine and by 50% with (S)(-)-nicotine. The data of this study indicate that [76Br]BAP is a promising radioligand for the characterization of nicotinic acetylcholine receptors in vivo.     

Molting phenology of the Pacific harbor seal (Phoca vitulina richardii) on two islands off the Baja California Peninsula,Mexico

We document and compare the annual molt of the Pacific harbor seal (Phoca vitulina richardii) on two islands off the west coast of the Baja California Peninsula that are the northern and southern extremes of its distribution in Mexico. During 2014, observations were made from March to July on Todos Santos Island (northern extreme) and from January to June on San Roque Island (southern extreme). On Todos Santos, the premolt lasted 15 wk (March–June) and the molt 12 wk (April–July). On San Roque, the premolt lasted 22 wk (January–June) and the molt 17 wk (February–June). The proportion of seals undergoing molt peaked on 26 May on Todos Santos and on 7 June on San Roque. Shedding of old hair most commonly initiated on the torso and progressed to the head and flippers (reverse molting pattern). The period when the highest number of harbor seals haul out in Mexico is in late April on the more southerly islands and in early May on the more northerly islands, when a large proportion of seals are in premolt.     

Physiological roles of the mitochondrial permeability transition pore

Neurons experience high metabolic demand during such processes as synaptic vesicle recycling, membrane potential maintenance and Ca²⁺ exchange/extrusion. The energy needs of these events are met in large part by mitochondrial production of ATP through the process of oxidative phosphorylation. The job of ATP production by the mitochondria is performed by the F₁F_O ATP synthase, a multi-protein enzyme that contains a membrane-inserted portion, an extra-membranous enzymatic portion and an extensive regulatory complex. Although required for ATP production by mitochondria, recent findings have confirmed that the membrane-confined portion of the c-subunit of the ATP synthase also houses a large conductance uncoupling channel, the mitochondrial permeability transition pore (mPTP), the persistent opening of which produces osmotic dysregulation of the inner mitochondrial membrane, uncoupling of oxidative phosphorylation and cell death. Recent advances in understanding the molecular components of mPTP and its regulatory mechanisms have determined that decreased uncoupling occurs in states of enhanced mitochondrial efficiency; relative closure of mPTP therefore contributes to cellular functions as diverse as cardiac development and synaptic efficacy.