Studies of sperm from mutant mice suggesting that two neurotransmitter receptors are important to the zona pellucida-initiated acrosome reaction

Two sperm neurotransmitter receptor/channels, the glycine receptor (GlyR) and a nicotinic acetylcholine receptor containing an alpha7 subunit (alpha7nAChR) were previously shown to be important to the mouse acrosome reaction (AR) initiated by solubilized egg zona pellucida (ZP). Here, we investigated whether sperm from homozygous mutant mice with a single amino acid mutation in the alpha subunit of their GlyR and sperm from homozygous mutant mice with an engineered disruption of the gene for the nicotinic acetylcholine receptor alpha7 subunit could undergo the AR on ZP-intact eggs. Wild-type and mutant sperm were treated with 3-quinuclidinyl benzilate (QNB), known to be an inhibitor of the ZP-initiated AR (but shown in the present work not to inhibit the acetylcholine-initiated AR). The ZP-initiated AR on ZP-intact eggs should occur only in sperm not treated with QNB. The absence of such an increase in the untreated mutant sperm would demonstrate that such sperm were unable to respond to the intact ZP. The results demonstrated for the first time that GlyR mutant sperm do not undergo the AR on ZP-intact mouse eggs, and that their ability to fertilize is inhibited by 63% in vitro. Moreover, we found that GlyR mutant sperm exhibited normal capacitation and confirmed that they not undergo the AR initiated by solubilized mouse ZP. Our studies demonstrated for the first time that sperm from mutant alpha7nAChR mice exhibit normal capacitation, do not undergo the AR in response to acetylcholine, solubilized ZP or on ZP-intact eggs, and display a 25% reduction in fertilization in vitro. This is the first genetic evidence for the importance of the alpha7nAChR in the ZP-initiated AR. While defects in either the GlyR or the alpha7nAChR completely inhibit the ZP-initiated AR, fertilization by these mutant sperm can still occur in vitro, probably due to sperm that complete spontaneous AR on the ZP.     

Regulation of cuticular and postpharyngeal hydrocarbons in the slave-making ant Polyergus rufescens: effect of Formica rufibarbis slaves

The purpose of this study was to compare cuticular hydrocarbon profiles of slave-making Polyergus rufescens ants reared alone or with their Formica rufibarbis slaves. Chemical analyses showed that due to the close contacts occurring when the Formica were tending the Polyergus, the synthesis of the cuticular hydrocarbons carried by the slaves was enhanced in the slave-makers. The postpharyngeal hydrocarbon levels increased during the first 15 days of life, whether or not the Polyergus were exposed to Formica. Our findings suggest that Polyergus is able to secrete all components of their cuticular hydrocarbon blend and that none are acquired through contact with Formica. In addition to presenting our experimental evidence, several hypotheses are proposed to explain the synthesis and regulation of hydrocarbon blends borne when these two species cohabitate within a single colony.     

Differential modulation of evoked and spontaneous glycine release from rat spinal cord glycinergic terminals by the cyclic AMP/protein kinase A transduction cascade

The mechanisms underlying cyclic AMP modulation of action potential-dependent and -independent (spontaneous) release of glycine from terminals synapsing onto sacral dorsal commissural nucleus neurons of lamina X were studied in spinal cord slices using conventional patch-clamp recordings. 3-Isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor, and forskolin increased the amplitude of evoked inhibitory postsynaptic currents (eIPSCs) in a sensitive manner to protein kinase A (PKA) inhibition (with KT-5720). Direct activation (with adenosine 3',5'-cyclic-monophosphothioate, Sp-isomer) and inhibition (with adenosine 3',5'-cyclic-monophosphothioate, Rp-isomer) of PKA increased and decreased the eIPSC amplitude, respectively. Paired pulse experiments and direct injection of PKA inhibitor fragment 6-22 amide (PKI(6-22)) into the recording neuron revealed that these effects on eIPSC amplitude occurred presynaptically, indicating that evoked glycine release is regulated by presynaptic cAMP via changes in PKA activity. Increasing cAMP also increased spontaneous release of glycine, causing an increased frequency of miniature IPSCs (mIPSCs). In contrast to the effects on evoked release, this response was not solely mediated via PKA, as it was not occluded by PKA inhibition, and both direct inhibition and direct activation of PKA actually enhanced mIPSC frequency. Direct inhibition of cAMP (with SQ 22536) did, however, reduce mIPSC frequency. These results suggest cAMP modulation of evoked and spontaneous release involves different presynaptic mechanisms and proteins.     

Glutamate and Schizophrenia: Beyond the Dopamine Hypothesis

1. After 50 years of antipsychotic drug development focused on the dopamine D2 receptor, schizophrenia remains a chronic, disabling disorder for most affected individuals.2. Studies over the last decade demonstrate that administration of low doses of NMDA receptor antagonists can cause in normal subjects the negative symptoms, cognitive impairments and physiologic disturbances observed in schizophrenia.3. Furthermore, a number of recently identified risk genes for schizophrenia affect NMDA receptor function or glutamatergic neurotransmission.4. Placebo-controlled trials with agents that directly or indirectly activate the glycine modulatory site on the NMDA receptor have shown reduction in negative symptoms, improvement in cognition and in some cases reduction in positive symptoms in schizophrenic patients receiving concurrent antipsychotic medications.5. Thus, hypofunction of the NMDA receptor, possibly on critical GABAergic inter-neurons, may contribute to the pathophysiology of schizophrenia.     

Respiratory pattern of captive Yangtze finless porpoises (Neophocaena phocaenoides asiaeorientalis)

Cetacean respiration usually happen in bouts. The most widely applied quantitative method used to analyze the structure of these bouts is the log_e-survivorship analysis, based on the assumption that the respiratory intervals are distributed as negative exponentials. However, for the data collected from three captive Yangtze finless porpoises (Neophocaena phocaenoides asiaeorientalis), we failed to obtain a convergent result with the application of log_e-survivorship analysis. However, the two-Gaussian model, which was recently proposed to analyze the feeding behavior of cows, was successfully fitted to the data. According to the fitting results, the overall respiratory pattern of the captive Yangtze finless porpoises can be described as a dive with a mean duration of around 30–40 s, followed by two or three ventilations with a mean interval of approximately 9 s. The average intra-bout intervals during both active and inactive periods are constant at 7.7–9.9 s for all individuals. However, when shifting from active to inactive states, the adult male and female decrease their mean numbers of respirations per bout and average length of inter-bout respiratory intervals, while the estimates of both parameters increase for the juvenile female. It was pointed out that the two-Gaussian model might be more adequate for cetacean respiratory-bout structure analyses than the log_e-survivorship technique.     

A quantitative analysis on latent heat of an aqueous binary mixture

The latent heat during phase change of water-NaCl binary mixture was measured using a differential scanning calorimeter, and the magnitude for two distinct phase change events, water/ice and eutectic phase change, were analyzed considering the phase change characteristics of a binary mixture. During the analysis, the latent heat associated with each event was calculated by normalizing the amount of each endothermic peak with only the amount of sample participating in each event estimated from the lever rule for the phase diagram. The resulting latent heat of each phase change measured is 303.7 +/- 2.5 J/g for water/ice phase change, and 233.0 +/- 1.6 J/g for eutectic phase change, respectively regardless of the initial concentration of mixture. Although the latent heats of water/ice phase change in water-NaCl mixtures are closely correlated, further study is warranted to investigate the reason for smaller latent heat of water/ice phase change than that in pure water (335 J/g). The analysis using the lever rule was extended to estimate the latent heat of dihydrate as 115 J/g with the measured eutectic and water/ice latent heat values. This new analysis based on the lever rule will be useful to estimate the latent heat of water-NaCl mixtures at various concentrations, and may become a framework for more general analysis of latent heat of various biological solutions.     

Differential in-gel electrophoresis (DIGE) analysis of CHO cells under hyperosmotic pressure: osmoprotective effect of glycine betaine addition

The use of glycine betaine combined with hyperosmolality is known to be an efficient means for achieving high protein production in recombinant Chinese hamster ovary (rCHO) cells. In order to understand the intracellular events and identify the key factors in rCHO cells cultivated with glycine betaine under hyperosmotic conditions, two-dimensional differential in-gel electrophoresis (2D-DIGE) followed by mass spectrometric analysis was applied. Differentially expressed 19 protein spots were selected and 16 different kinds of proteins were successfully identified. The identified proteins were associated with cellular metabolism (PEPCK, GAPDH, and PK), cellular architecture (β-tubulin and β-actin), protein folding (GRP78 and OSP94), mRNA processing (Rbm34, ACF, and IPMK), and protein secretion (γ-COP). 2D-Western blot analysis of β-tubulin, GAPDH, Peroxidoxin-1, and GRP78 confirmed the proteomic findings. The proteins identified from this study, which are related to cell growth and antibody production, can be applied to cell engineering for maximizing the efficacy of the use of glycine betaine combined with hyperosmolality in rCHO cells.     

Glycine decarboxylase controls photosynthesis and plant growth

Photorespiration makes oxygenic photosynthesis possible by scavenging 2-phosphoglycolate. Hence, compromising photorespiration impairs photosynthesis. We examined whether facilitating photorespiratory carbon flow in turn accelerates photosynthesis and found that overexpression of the H-protein of glycine decarboxylase indeed considerably enhanced net-photosynthesis and growth of Arabidopsis thaliana. At the molecular level, lower glycine levels confirmed elevated GDC activity in vivo, and lower levels of the CO₂ acceptor ribulose 1,5-bisphosphate indicated higher drain from CO₂ fixation. Thus, the photorespiratory enzyme glycine decarboxylase appears as an important feed-back signaller that contributes to the control of the Calvin-Benson cycle and hence carbon flow through both photosynthesis and photorespiration.