Potassium-stimulated release of GABA,glycine, and taurine from the chick retina

The effect of depolarizing potassium concentration on the release of [¹⁴C]glycine, [³H]GABA, and [³⁵S]taurine was investigated in the whole chick retina and in a synaptosomal fraction prepared from the chick retina. In the whole retina, increasing potassium concentration above 40 mM resulted in an increased release of the three amino acids. The release of glycine was the most stimulated and that of taurine, the least. The potassium-evoked release of glycine and GABA was calcium dependent. In the synaptosomal fraction, 68.5 mM potassium significantly stimulated the efflux of GABA and glycine by a calcium-dependent mechanism. The release of taurine from this fraction was unaffected by high potassium.     

Chlorine transfer between glycine, taurine, and histamine: reaction rates and impact on cellular reactivity

Hypochlorous acid formed by activated neutrophils reacts with amines to produce chloramines. Chloramines vary in stability, reactivity, and cell permeability. We have examined whether chloramine exchange occurs between physiologically important amines or amino acids and if this affects interactions of chloramines with cells. We have demonstrated transchlorination reactions between histamine, glycine, and taurine chloramines by measuring chloramine decay rates with mixtures as well as by mass spectrometry. Kinetic analysis suggested the formation of an intermediate complex with a high Km. Apparent second-order rate constants, determined for concentrations 相似文献   

Chlorine transfer between glycine, taurine, and histamine: reaction rates and impact on cellular reactivity

Hypochlorous acid formed by activated neutrophils reacts with amines to produce chloramines. Chloramines vary in stability, reactivity, and cell permeability. We have examined whether chloramine exchange occurs between physiologically important amines or amino acids and if this affects interactions of chloramines with cells. We have demonstrated transchlorination reactions between histamine, glycine, and taurine chloramines by measuring chloramine decay rates with mixtures as well as by mass spectrometry. Kinetic analysis suggested the formation of an intermediate complex with a high K(m). Apparent second-order rate constants, determined for concentrations 相似文献   

p53 checkpoint-defective cells are sensitive to X rays, but not hypoxia

X-ray-induced damage leads to cell-cycle "checkpoint" arrest by p53-dependent induction of the cyclin-dependent kinase inhibitor p21 (Waf1/Cip1/Sdi1). Human tumor cells that lack this response fail to arrest after exposure to DNA-damaging agents, undergo multiple rounds of endoreduplicative DNA synthesis, and eventually commit to an apoptotic cell death. Since low oxygen tension can also induce p53 protein accumulation, and can lead to cell-cycle arrest or apoptosis, we examined the expression of p21 in tumor cells under normoxic and hypoxic conditions. In a survey of cells, mRNA for the p21 gene was induced two- to threefold in response to hypoxia in a seemingly p53-independent manner. We therefore examined genetically matched cells that differ in their p21 and p53 status for response to ionizing radiation and hypoxia. We found that both p21-deficient and p53-deficient cells exhibit an increase in chromosome instability, an increased level of apoptosis, and a failure to arrest after exposure to ionizing radiation. However, cells that lack either p21 or p53 exhibit no increase in chromosome instability or elevated apoptosis and still arrest in response to hypoxia. Thus, the mechanism responsible for the differential response to either hypoxia or X rays presumably lies in the control of cell-cycle progression in response to stress and its dependence on p21. Since the loss of a DNA-damage-dependent checkpoint does not sensitize cells to killing by stresses that elicit a DNA-damage-independent checkpoint, targeting the function of p21 pharmacologically will not kill tumor cells in situ in the absence of a DNA damage signal.     

Heat shock protein levels are not elevated in heat-resistant B16 melanoma cells

Heat-resistant variants have been selected from B16 melanoma cells and from surface mutants previously derived from them. The aim of the present study was to explore the possible role of heat shock proteins in the manifestation of this heat resistance. The major heat shock proteins evident after heating have subunit molecular weights of 68, 70, 89, and 110K on sodium dodecyl sulfate-polyacrylamide gels. The 68-kDa protein is not evident in any of the unheated B16 cell lines while the levels of the other heat shock proteins are elevated after heating. The constitutive levels of the 70, 89, and 110-kDa heat shock proteins were assessed after gel electrophoretic separation of proteins in several of the heat-resistant variants. No major differences were found in the levels of these proteins between the heat-sensitive parent lines and the heat-resistant variants. We therefore conclude that heat shock proteins are not a determining factor in the heat-resistant phenotype of B16 melanoma cells.     

Methylgroups of ribosomal protein L11 are not related to the synthesis of ppGpp

Summary Ribosomes carrying either a normally methylated or an undermethylated L11, respectively, were tested with respect to the stringency reaction in the presence of crude stringent factor. Systems with either kind of ribosomes synthesize ppGpp with the same efficiency. The ppGpp synthesis in presence of ribosomes with undermethylated L11 depends also on stringent factor, mRNA and deacylated tRNA whereas aminoacyl-tRNA and peptidyl-analogous tRNA show no effect. Thus, the absence of methylation in L11 does not influence the stringency reaction.     

In the Archaea Haloferax volcanii, membrane protein biogenesis and protein synthesis rates are affected by decreased ribosomal binding to the translocon

In the haloarchaea Haloferax volcanii, ribosomes are found in the cytoplasm and membrane-bound at similar levels. Transformation of H. volcanii to express chimeras of the translocon components SecY and SecE fused to a cellulose-binding domain substantially decreased ribosomal membrane binding, relative to non-transformed cells, likely due to steric hindrance by the cellulose-binding domain. Treatment of cells with the polypeptide synthesis terminator puromycin, with or without low salt washes previously shown to prevent in vitro ribosomal membrane binding in halophilic archaea, did not lead to release of translocon-bound ribosomes, indicating that ribosome release is not directly related to the translation status of a given ribosome. Release was, however, achieved during cell starvation or stationary growth, pointing at a regulated manner of ribosomal release in H. volcanii. Decreased ribosomal binding selectively affected membrane protein levels, suggesting that membrane insertion occurs co-translationally in Archaea. In the presence of chimera-incorporating sterically hindered translocons, the reduced ability of ribosomes to bind in the transformed cells modulated protein synthesis rates over time, suggesting that these cells manage to compensate for the reduction in ribosome binding. Possible strategies for this compensation, such as a shift to a post-translational mode of membrane protein insertion or maintained ribosomal membrane-binding, are discussed.     

Effects of the ionophores X537A and A23187 on GABA,glycine, and taurine release from chick retinal subcellular fractions

The ionophore X537A at concentrations of 5–20 M stimulated the release of [³H]GABA and [³⁵S]taurine, from retinal subcellular crude nuclear (P₁) and crude synaptosomal (P₂) fractions. The release of [³H]GABA increased 114% and 136% over control values in P₁ and P₂ fractions, respectively. The efflux of [³⁵S]taurine from P₁ was increased by 45% and that from P₂ by 21%. X537A increased⁴⁵Ca²⁺ uptake in the P₂ fraction but not in the P₁ fraction. The effect of X537A on the amino acid release was not dependent on the presence of exogenous calcium. X537A did not affect [³H]GABA or [³⁵S]taurine uptake by the retinal fractions. A23187 enhanced [³H]GABA release from P₁ and P₂ by 52% and 105%, respectively. The ionophore also increased [¹⁴C]glycine liberation in both P₁ (35%) and P₂ (50%) but failed to stimulate [³⁵S]taurine release. A23187 produced a transient increase of⁴⁵Ca²⁺ uptake of 38% in P₁ and 30% in P₂. The effects of A23187 on the release of amino acids were calcium dependent. The amino acid uptake was not affected by the ionophore. These results are consisent with the suggested neurotransmitter role for GABA at the outer synaptic layer and for GABA and glycine at the inner synaptic layer of the retina. A neurotransmitter role for taurine is not supported by the present results.     

Nonessential amino acids are not necessary to stimulate net muscle protein synthesis in healthy volunteers

The present study was performed to test the hypothesis that orally administered essential amino acids, in combination with carbohydrate, will stimulate net muscle protein synthesis in resting human muscle in vivo. Four volunteers ingested 500 mL of a solution containing 13.4 g of essential amino acids and 35 g sucrose (EAA). Blood samples were taken from femoral arterial and venous catheters over a 2-hour period following the ingestion of EAA to measure arteriovenous concentrations of amino acids across the muscle. Two muscle biopsies were taken during the study, one before administration of the drink and one approximately 2 hours after consumption of EAA. Serum insulin increased from normal physiologic levels at baseline (9.2 +/- 0.8 microU/mL) and peaked (48 +/- 7.1 microU/mL) 30 minutes after EAA ingestion. Arterial essential amino acid concentrations increased approximately 100 to 400% above basal levels between 10 and 30 minutes following drink ingestion. Net nitrogen (N) balance changed from negative (-495 +/- 128 nmol/mL) prior to consumption of EAA to a peak positive value (416 +/- 140 nmol/mL) within 10 minutes of ingestion of the drink. EAA resulted in an estimated positive net N uptake of 307.3 mg N above basal levels over the 2-hour period. Muscle amino acid concentrations were similar prior to and 2 hours following ingestion of EAA. We conclude that ingestion of a solution composed of carbohydrates to stimulate insulin release and a small amount of essential amino acids to increase amino acid availability for protein synthesis is an effective stimulator of muscle protein anabolism.     

Synthesis and characteristics of the specific monosulfates of chenodeoxycholate,deoxycholate and their taurine or glycine conjugates

The isomeric monosulfates of chenodeoxycholate, deoxycholate, and their taurine or glycine conjugates, were synthesized and characterized. Reaction with chlorosulfonic acid in pyridine for 2 minutes mainly afforded the 3-monosulfates. To prepare the 7- or the 12-monosulfates, the 3-hydroxyl group was protected by carbethoxylation prior to sulfation of the 7- or 12-hydroxyl group for 24 h to 5 days; after sulfation, the protecting 3-carbethoxy function was removed by mild alkaline hydrolysis. The crude bile salt monosulfates were purified by chromatography on silica gel and on Sephadex LH-20 and were crystallized from methanolethanol-ethyl acetate. The results of elemental analysis demonstrated that the compounds were disodium dihydroxy bile salt monosulfates. Thin layer chromatography of the sulfates, and gas-liquid chromatography after oxidation and solvolysis, showed that the substances were pure and that the sulfate group was at the expected position.     

K+ currents through SV-type vacuolar channels are sensitive to elevated luminal sodium levels

Non-selective slow vacuolar (SV) channels mediate uptake of K+ and Na+ into vacuolar compartment. Under salt stress plant cells accumulate Na+ in the vacuole and release vacuolar K+ into the cytoplasm. It is, however, unclear how plants mediate transport of K+ from the vacuole without concomitant efflux of toxic Na+. Here we show by patch-clamp studies on isolated Arabidopsis thaliana cell culture vacuoles that SV channels do not mediate Na+ release from the vacuole as luminal Na+ blocks this channel. Gating of the SV channel is dependent on the K+ gradient across the vacuolar membrane. Under symmetrical K+ concentrations on both sides of the vacuolar membrane, SV channels mediate potassium uptake. When cytoplasmic K+ decreases, SV channels allow K+ release from the vacuole. In contrast to potassium, Na+ can be taken up by SV channels, but not released even in the presence of a 150-fold gradient (lumen to cytoplasm). Accumulation of Na+ in the vacuole shifts the activation potential of SV channels to more positive voltages and prevents gradient-driven efflux of K+. Similar to sodium, under physiological conditions, vacuolar Ca2+ is not released from vacuoles via SV channels. We suggest that a major Arabidopsis SV channel is equipped with a positively charged intrinsic gate located at the luminal side, which prevents release of Na+ and Ca2+, but permits efflux of K+. This property of the SV channel guarantees that K+ can shuttle across the vacuolar membrane while maintaining Na+ and Ca2+ stored in this organelle.     

N-Propynyl analogs of beta-phenylethylidenehydrazines: synthesis and evaluation of effects on glycine, GABA, and monoamine oxidase

A group of beta-phenylethylidenehydrazines possessing a variety of substituents (Me, OMe, Cl, F, and CF(3)) at the ortho-, meta-, or para-positions of the phenyl ring, in conjunction with either a N-bis-(2-propynyl) or a N-mono-(2-propynyl) moiety, were synthesized and compared to the novel neuroprotective drug beta-phenylethylidenehydrazine (PEH) with regard to their ability to inhibit the enzymes GABA-transaminase (GABA-T) and monoamine oxidase (MAO)-A and -B in vitro in brain tissue. Two of the analogs synthesized (mono- and bis-N-propynylPEH) were also studied exvivo in rats to compare their effects to those of PEH with regard to ability to inhibit GABA-T and MAO and to change brain levels of several important amino acids. Unlike PEH, none of the new drugs inhibited GABA-T in vitro at 10 or 100 microM, and all of the drugs (including PEH) were poor inhibitors (at 10 microM) of MAO-A and -B invitro. The two analogs studied exvivo inhibited GABA-T to a lesser extent than PEH, unlike PEH that did not elevate brain levels of GABA, and inhibited MAO-A and -B more potently than PEH. Interestingly, unlike PEH, the two analogs caused marked increases in brain levels of glycine; because of the current interest in drugs that increase glycine availability in the brain as potential antipsychotic drugs, these two analogs now warrant further investigation.     

Rheb-TOR signaling promotes protein synthesis, but not glucose or amino acid import, in Drosophila

Background  

The Ras-related GTPase, Rheb, regulates the growth of animal cells. Genetic and biochemical tests place Rheb upstream of the target of rapamycin (TOR) protein kinase, and downstream of the tuberous sclerosis complex (TSC1/TSC2) and the insulin-signaling pathway. TOR activity is regulated by nutritional cues, suggesting that Rheb might either control, or respond to, nutrient availability.     

Development of sensitivity to glycine, GABA and beta-alanine in cultured spinal neurons

Whole cell patch-clamp recording of cultured chick spinal neurons, presumed to be motoneurons, revealed currents elicited in these cells by GABA, glycine and beta-alanine, corresponding to the opening of chloride channels. During maturation, sensitivity to all three transmitters were first detected on the day 6 of culture, and appeared in most neurons by day 8. However, at all stages of development, a fraction of the cells were sensitive only to GABA; this observation supports the notion that the GABA and the glycine receptors are distinct. On the other hand, separate activation by glycine and beta-alanine was never observed, in agreement with the postulate that these amino acids bind to the same receptor.     

Pinaceae show elevated rates of gene turnover that are robust to incomplete gene annotation

Gene duplications and gene losses are major determinants of genome evolution and phenotypic diversity. The frequency of gene turnover (gene gains and gene losses combined) is known to vary between organisms. Comparative genomic analyses of gene families can highlight such variation; however, estimates of gene turnover may be biased when using highly fragmented genome assemblies resulting in poor gene annotations. Here, we address potential biases introduced by gene annotation errors in estimates of gene turnover frequencies in a dataset including both well‐annotated angiosperm genomes and the incomplete gene sets of four Pinaceae, including two pine species, Norway spruce and Douglas‐fir. We show that Pinaceae experienced higher gene turnover rates than angiosperm lineages lacking recent whole‐genome duplications. This finding is robust to both known major issues in Pinaceae gene sets: missing gene models and erroneous annotation of pseudogenes. A separate analysis limited to the four Pinaceae gene sets pointed to an accelerated gene turnover rate in pines compared with Norway spruce and Douglas‐fir. Our results indicate that gene turnover significantly contributes to genome variation and possibly to speciation in Pinaceae, particularly in pines. Moreover, these findings indicate that reliable estimates of gene turnover frequencies can be discerned in incomplete and potentially inaccurate gene sets. Because gymnosperms are known to exhibit low overall substitution rates compared with angiosperms, our results suggest that the rate of single‐base pair mutations is uncoupled from the rate of large DNA duplications and deletions associated with gene turnover in Pinaceae.     

Believe it or not, QTLs are accurate!

It is generally believed that mapping quantitative trait loci (QTLs) does not accurately position genes underlying polygenic traits on the genome, which limits the application of QTL analysis in marker-assisted selection and gene discovery. However, now that a few plant QTLs have been cloned or accurately tagged, it appears that they might be accurate to within 2cM or less. This means that there will be circumstances when map-based cloning using only original mapping data would be a realistic option that avoids time-consuming and expensive fine mapping. Acceptance of this view would enhance the value of past and future mapping experiments, particularly those revealing small and environmentally sensitive QTLs that are often considered intractable at the molecular level.     

Injected mRNA does not increase protein synthesis in unfertilized, fertilized, or ammonia-activated sea urchin eggs

We have investigated whether the rate of protein synthesis in unfertilized and fertilization-activated sea urchin eggs is limited by the availability of mRNA by injecting eggs, zygotes, and ammonia-activated eggs with globin mRNA. Message-injected and buffer-injected cells were labeled with radioactive amino acids and the proteins separated on a polyacrylamide gel. The relative amounts of newly synthesized globin and endogenous proteins were obtained by scanning the gel fluorograph. Globin mRNA is translated poorly in Strongylocentrotus droebachiensis eggs and does not significantly increase or decrease endogenous protein synthesis. In zygotes and ammonia-activated eggs, however, globin mRNA is translated well and appears to compete with endogenous mRNAs for the limiting component of the translational machinery as it is released. Our results are consistent with the hypothesis that either ribosomes or recruitment factors are gradually activated after fertilization or ammonia treatment, that such components are the rate-limiting factor, and that they impart the typical sigmoidal increase in protein synthesis rate observed in fertilized eggs before the first cleavage.