Deficits in the Activation and Phosphorylation of Hippocampal Tyrosine Hydroxylase in the Aged Fischer 344 Rat Following Intraventricular Administration of 6-Hydroxydopamine

Abstract: Tyrosine hydroxylase activity was measured under optimal and suboptimal assay conditions in hippocampal extracts from young (2 month), mature (12 month), and old (24 month) Fischer 344 male rats 72 h after the infusion of 200 µg of the neurotoxin 6-hydroxydopamine or vehicle into the lateral ventricle. The lesion resulted in a 45–55% decrease of tyrosine hydroxylase activity measured under optimal conditions (pH 6.1, 3.0 m M 6-methyl-5,6,7,8-tetrahydropterin) and an ∼35% decrease in the relative concentration of immunoreactive tyrosine hydroxylase. When measured under suboptimal conditions (pH 6.6, 0.7 m M 6-methyl-5,6,7,8-tetrahydropterin), tyrosine hydroxylase activity in 2- and 12-month-old lesioned animals was twice that measured in vehicle-treated animals. However, in the old lesioned animals, tyrosine hydroxylase activity measured under suboptimal conditions was not different from that measured in age-matched vehicle-treated animals. Isoforms of tyrosine hydroxylase were identified on immunoblots after two-dimensional gel electrophoresis using enhanced chemiluminescence. The relative proportion of lower pl isoforms of tyrosine hydroxylase in the 2-month-old lesioned animals was greater than that observed in vehicle-treated controls. In contrast, no difference was seen in the relative proportion of tyrosine hydroxylase isoforms in the 24-month-old lesioned versus control animals. These data indicate that the ability of locus ceruleus neurons to rapidly respond to and compensate for insult is attenuated in 24-month-old Fischer 344 rats due to a deficit in stimulus-evoked enzyme phosphorylation.     

马内菲青霉蛋白酶的体外诱导及其活性测定

目的 建立马内菲青霉蛋白酶的体外诱导方法并进行活性测定。方法 制作酶诱导培养基, 在不同pH 条件下培养马内菲青霉, 在280 nm紫外光下分光光度法检测不同诱导时间的蛋白酶活性。结果 成功诱导并定量测定了马内菲青霉在不同pH条件下、不同诱导时间的体外蛋白酶产出量。pH 4. 0 时, 菌株在第6 天出现分泌高峰; pH 5. 6 时, 菌株在第3 天出现分泌高峰; 而pH 7. 2 时, 菌株几乎无蛋白酶产出。结论 建立了马内菲青霉蛋白酶产出的体外诱导和检测方法, 为进一步研究马内菲青霉体外蛋白酶的理化特性提供了途径。     

Fehlermöglichkeiten der Gelöstsauerstoffmessung beim Betrieb von Bioreaktoren

A review of errors, which can influence the measured data of electrochemical oxygen sensors (OS) in fermentation technique is presented. The specifities of various sensor constructions are pointed out. References are given for selection of sensors to application in fermenters. Influences on the measured values can take place by fermentation conditions, arrangement of sensors in the fermenter and composition of the fermentation broth. The oxygen measurement in the multi-phase system of fermentation fluids can cause a remarkable deviation of the measured values to the real ones. Practical hints are given for use, calibration and sterilization of OS. Restricting conditions for measurements with this sensors are enumerated.     

Unidirectional sodium and potassium fluxes through the sodium channel of squid giant axons.

Unidirectional 22Na-traced sodium influx or 42K-traced potassium efflux across the membranes of voltage-clamped squid giant axons was measured at various membrane potentials under bi-ionic conditions. Tetrodotoxin almost entirely eliminated the extra K+ efflux induced by short repetitive depolarizations in the presence of tetraethylammonium or 3,4-diaminopyridine. A method of determining the voltage dependence of the unidirectional flux through voltage-gated channels is described. This technique was used to obtain the unidirectional flux-voltage relation for the sodium channel in bi-ionic and single-ion conditions. It allows the determination of the unidirectional flux at the zero-current potential which, for influx, was found to be approximately 20% of the value measured 80 mV negative to the zero-current potential. The unidirectional flux ratio under bi-ionic conditions was also measured and the flux ratio exponent found to average 1.15 with an external sodium and an internal potassium solution. A three-barrier, two-site, multi-occupancy model previously obtained for other conditions was found to predict a similar non-unity average for the flux ratio exponent. It is also shown that some single-occupancy models can predict non-unity values for the flux ratio exponent in bi-ionic conditions.     

The lipF promoter of Mycobacterium tuberculosis is upregulated specifically by acidic pH but not by other stress conditions

The lipF gene of Mycobacterium tuberculosis has been implicated in pathogenesis and its promoter has been shown to be upregulated by acidic stress. To further define the acidic pH that upregulates the lipF promoter from M. tuberculosis and to establish that it is specifically upregulated by acid stress and not by other environmental stresses, promoter expression levels were measured under a variety of conditions. The conditions measured were pH, temperature, oxidative stress, and hypoxic stress.     

ADP increases the affinity for cytochrome c by interaction with the matrix side of bovine heart cytochrome c oxidase

The effect of intraliposomal ADP and ATP on the kinetics of cytochrome c oxidation in reconstituted bovine heart cytochrome c oxidase was measured by the photometric and polarographic method: 1. Intraliposomal ADP decreases and intraliposomal ATP increases the Km for cytochrome c when measured by the photometric assay under uncoupled conditions. 2. The above described effects are not obtained when the kinetics are measured with the polarographic assay. 3. Extraliposomal ATP increases the Km for cytochrome c similar to intraliposomal ATP, but this effect is measured with both methods of assay. 4. Under coupled conditions only a small decrease of the Km for cytochrome c by intraliposomal ADP is found.     

Improved frequency response of pneumotachometers by digital compensation

To measure impedance one measures or estimates flow, which is commonly done by measuring the pressure drop across a pneumotachometer. The frequency response characteristics of standard pneumotachometer/pressure transducers (PPT) limit their use to relatively low frequencies. Also, the frequency response of PPTs has been reported to be "load" dependent. Thus, the frequency response characteristics measured under "no-load" conditions, which theoretically could be used to compensate subsequent measurements, may not be appropriate for measurements made under loaded conditions. Another method of measuring impedance exists which depends on a reference impedance element other than a pneumotachometer. In this method, an oscillatory flow signal with known amplitude is generated and used to force the system being tested. Unlike PPTs, this oscillatory flow generator (OFG) is a closed system that allows measurements to be made only during breath holding. Our objective was to determine whether the frequency response of a PPT could be compensated using measurements made under no-load conditions, such that it accurately measured an impedance load. The frequency response of the PPT under no-load conditions was measured by the OFG and used to compensate the output of the PPT in subsequent impedance measurements. The compensated PPT was used to measure the impedance of a mechanical structure and the impedances of four human subjects. The impedances of the mechanical structure and the subjects were also measured using the OFG.(ABSTRACT TRUNCATED AT 250 WORDS)     

Unimpaired formation of hormone-stimulated inositol trisphosphate in human mesangial cells under hyperglycemic conditions.

The relationship between bulk cellular myo-inositol content and phosphatidylinositol metabolism was evaluated in a human mesangial cell line under euglycemic and hyperglycemic conditions. Mesangial cells maintained in high glucose medium displayed a concentration-dependent fall in myo-inositol as measured by gas-liquid chromatography. Measurements of phosphatidylinositol, phosphatidylinositol 4-monophosphate and phosphatidylinositol 4,5-bisphosphate mass revealed slight but statistically insignificant increases in cells exposed to high glucose containing medium. CDP-diacylglycerol: myo-inositol 3-phosphatidylinositol transferase activity, measured in plasma membranes from mesangial cells grown under control and hyperglycemic conditions, was kinetically similar with Michaelis constants (Km values) for myo-inositol of 2.9 and 2.1 mM, respectively. Finally, hormone-stimulated intracellular calcium mobilization and myo-inositol 1,4,5-trisphosphate mass was measured from mesangial cells grown under normal and hyperglycemic conditions. Both intracellular calcium and inositol trisphosphate formation were unchanged in cells previously exposed to high glucose conditions (400 mg/dl) compared to cells grown under normal glucose concentration (100 mg/dl). These data indicate that bulk changes in myo-inositol induced by hyperglycemia are neither associated with alterations in basal levels of inositol containing glycerolipids nor with changes in hormone-stimulated calcium mobilization and inositol trisphosphate formation under conditions of short term changes in extracellular glucose.     

Behavioral indexes of poor welfare in laboratory rats

A study compared the behavior of rats kept under standard conditions with the behavior of those from enriched, semi-enriched, and single-housed conditions to determine whether standard conditions have detrimental effects on nonhuman animal welfare. The subjects were 35 female Hooded Norway rats. Using an emergence box, open field, and Hebb-Williams maze, the study measured the subjects' behaviors. Housing conditions significantly affected the behavior of the rats as measured by all 3 procedures. The results of the rats from standard conditions were intermediate between those of the single-housed and the more enriched groups. Thus, enrichng the standard environment would ameliorate behavioral deficits in rats kept under standard conditions.     

QTLs mapping of morphological traits related to salt tolerance in <Emphasis Type="Italic">Medicago truncatula</Emphasis>

To understand the complex inheritance of tolerance to salt stress in Medicago truncatula, quantitative trait loci (QTLs) analysis was performed using a set of recombinant inbred lines (RILs) derived from a cross between the tolerant line Jemalong A17 and susceptible line F83005.5. The RILs and parental lines were grown in individual pots filled with sterilized sand in a greenhouse under 0 and 50 mM NaCl. Plants were harvested after a period of 60 days. Fourteen quantitative traits related to aerial and root growths were measured. Broad-sense heritability of measured traits ranged from 0.21 to 0.83 and from 0.05 to 0.62 in control and in salt-stressed conditions, respectively. Established correlations between measured traits are dependent on treatment effect. We identified and mapped 10 QTLs in control conditions and 19 in salt stress. No major QTL was identified indicating that tolerance to salt stress is governed by several genes with low effects. The QTLs detected under control and under salt-stressed conditions almost did not share the same map locations suggesting that the loci that are not stable across treatments reflect adaptation to this constraint. The maximum of QTLs was observed on the chromosome 8. The usefulness of these QTLs, identified in greenhouse conditions, for marker-assisted selection should therefore be evaluated under field conditions, and validated in other genetic backgrounds.     

Isotope analysis as a natural reaction probe to determine mechanisms of biodegradation of 1,2-dichloroethane

1,2-Dichloroethane (1,2-DCA), a chlorinated aliphatic hydrocarbon, is a well-known groundwater contaminant. In this study, fractionation of stable carbon isotope values of 1,2-DCA during biodegradation was used as a novel reaction probe to provide information about the mechanism of 1,2-DCA biodegradation under both aerobic (O2-reducing) and anaerobic (NO3-reducing) conditions. Under O2-reducing conditions, an isotopic enrichment value (epsilon) of -25.8 +/- 1.1 per thousand (+/-95% confidence intervals) was measured for the enrichment culture. Under NO3-reducing conditions, an epsilon-value of -25.8 +/- 3.5 per thousand was measured. The microbial culture produced isotopic enrichment values (epsilon) that are not only large and reproducible, but also are the same whether O2 or NO3 was used as an electron acceptor. Combining data measured under both O2- and NO3-reducing conditions, an isotopic enrichment value (epsilon) of -25.8 +/- 1.6 per thousand is measured for the microbial culture during 1,2-DCA degradation. The epsilon-value can be converted into a kinetic isotope effect (KIE) value to relate the observed isotopic fractionation to the mechanism of degradation. This KIE value (1.05) is consistent with degradation via hydrolytic dehalogenation under both electron-accepting conditions. This study demonstrates the added value of compound-specific isotope analysis not only as a technique to verify the occurrence and extent of biodegradation in the field, but also as a natural reaction probe to provide insight into the enzymatic mechanism of contaminant degradation.     

The metabolism of halothane by hepatocytes: a comparison between free radical spin trapping and lipid peroxidation in relation to cell damage

The technique of free radical spin trapping has been applied to demonstrate the formation of free radicals produced during the metabolism of halothane by rat liver hepatocytes under hypoxic conditions. The results obtained support previous findings that reported sex differences in the metabolic activation of halothane by rats in vivo. Cell viability under hypoxic conditions, as judged by trypan blue staining and lactate dehydrogenase release, shows a correlation with the extent of metabolism of halothane as measured by electron spin resonance spectroscopy. The extent of lipid peroxidation was measured by diene conjugation, malondialdehyde production and chemiluminescence. The latter technique allowed the demonstration of lipid peroxidation during incubations of hepatocytes under aerobic conditions. The magnitude of the aerobic chemiluminescence showed a similar sex dependency to the extent of free radical formation under hypoxic conditions. Cell viability measurements show that halothane metabolism in both hypoxic and aerobic conditions can lead to cell death. Consequently, oxidative lipid damage could be a cause of cell damage, as judged by cell viability, additional to covalent binding.     

Behavioral Indexes of Poor Welfare in Laboratory Rats

A study compared the behavior of rats kept under standard conditions with the behavior of those from enriched, semi-enriched, and single-housed conditions to determine whether standard conditions have detrimental effects on nonhuman animal welfare. The subjects were 35 female Hooded Norway rats. Using an emergence box, open field, and Hebb-Williams maze, the study measured the subjects' behaviors. Housing conditions significantly affected the behavior of the rats as measured by all 3 procedures. The results of the rats from standard conditions were intermediate between those of the single-housed and the more enriched groups. Thus, enrichng the standard environment would ameliorate behavioral deficits in rats kept under standard conditions.     

The pressure fluctuation spectrum as a measure of mixing and emulsification in a biochemical reactor

Summary Pressure fluctuation spectra were measured inside a biochemical reactor at various mixing speeds and aeration rates. These spectra change considerably depending on the mixing conditions and appear to provide a detailed picture of the turbulent conditions within the reactor. Droplet size distributions were measured for 4% (v/v) hydrocarbon in the reactor. It was found that these droplet size distributions could be qualitatively predicted by analysis of the Rayleigh-Taylor instabilities at the oil-water interface in conjunction with the observed pressure fluctuation spectra.     

Effect of calcitonin on the prolactin surge of proestrus

The effect of calcitonin (CT) on the prolactin (PRL) surge of proestrus in rats was investigated under normal and perturbed lighting conditions. Salmon calcitonin (SCT) was injected i.p. on diestrus 2 or on proestrus, plasma PRL levels were measured by radioimmunoassay. SCT had no effect on the PRL surge under normal lighting conditions but it induced a small drop in PRL level measured on proestrus morning, 3 hours after CT injection. Animals submitted to perturbed light conditions had higher PRL levels than those kept under normal lighting. These data would indicate that for the female rats on proestrus the sensitivity to stress due to injection and blood sampling may be modulated by changing the photoperiod. SCT injection under these conditions may facilitate this destabilization in PRL level.     

Dispersal of motile bacteria from a plane layer.

The dispersal of an initially well-defined concentration of the motile bacterium Escherichia coli was measured under nonchemotactic conditions. The distribution of bacteria along a glass observation cell was measured by recording the intensity of light scattered by the organisms. For comparison, the diffusion of fluorescein was also measured by determining the distribution of fluorescence throughout the observation cell. The dispersal of bacteria from a plane layer, under nonchemotactic conditions, can be adequately described by the Gaussian solution of the diffusion equation.     

Testing biochemistry revisited: how in vivo metabolism can be understood from in vitro enzyme kinetics

A decade ago, a team of biochemists including two of us, modeled yeast glycolysis and showed that one of the most studied biochemical pathways could not be quite understood in terms of the kinetic properties of the constituent enzymes as measured in cell extract. Moreover, when the same model was later applied to different experimental steady-state conditions, it often exhibited unrestrained metabolite accumulation.Here we resolve this issue by showing that the results of such ab initio modeling are improved substantially by (i) including appropriate allosteric regulation and (ii) measuring the enzyme kinetic parameters under conditions that resemble the intracellular environment. The following modifications proved crucial: (i) implementation of allosteric regulation of hexokinase and pyruvate kinase, (ii) implementation of V(max) values measured under conditions that resembled the yeast cytosol, and (iii) redetermination of the kinetic parameters of glyceraldehyde-3-phosphate dehydrogenase under physiological conditions.Model predictions and experiments were compared under five different conditions of yeast growth and starvation. When either the original model was used (which lacked important allosteric regulation), or the enzyme parameters were measured under conditions that were, as usual, optimal for high enzyme activity, fructose 1,6-bisphosphate and some other glycolytic intermediates tended to accumulate to unrealistically high concentrations. Combining all adjustments yielded an accurate correspondence between model and experiments for all five steady-state and dynamic conditions. This enhances our understanding of in vivo metabolism in terms of in vitro biochemistry.     

Ploidy evolution in the yeast Saccharomyces cerevisiae: a test of the nutrient limitation hypothesis

The nutrient limitation hypothesis provides a nongenetic explanation for the evolution of life cycles that retain both haploid and diploid phases: differences in nutrient requirements and uptake allow haploids to override the potential genetic advantages provided by diploidy under certain nutrient limiting conditions. The relative fitness of an isogenic series of haploid, diploid and tetraploid yeast cells (Saccharomyces cerevisiae), which were also equivalent at the mating type locus, was measured. Fitness was measured both by growth rate against a common competitor and by intrinsic growth rate in isolated cultures, under four environmental conditions: (1) rich medium (YPD) at the preferred growth temperature (30 °C); (2) nutrient poor medium (MM) at 30 °C; (3) YPD at a nonpreferred temperature (37 °C); and (4) MM at 37 °C. In contrast to the predictions of the nutrient limitation hypothesis, haploids grew significantly faster than diploids under nutrient rich conditions, but there were no apparent differences between them when fitness was determined by relative competitive ability. In addition, temperature affected the relative growth of haploids and diploids, with haploids growing proportionately faster at higher temperatures. Tetraploids performed very poorly under all conditions compared. Cell geometric parameters were not consistent predictors of fitness under the conditions measured.     

Microbially — related redox changes in a subtropical lake 2. Simulation of metalinmetic conditions in a chemostat

The individual influence of sulfate reducing and brown phototrophic sulfur bacteria from Lake Kinneret on the metalimnetic redox conditions was investigated by simulating the hydrochemical and microbiological conditions in a specially designed chemostat. The results show a strong correlation between measured redox values and the prevailing hydrogen sulfide concentration leading to a linear relationship. Changes in this relationship allowed differentiation between the on-going microbial processes, sulfate reduction and sulfide oxidation. The comparison ofin vitro andin situ redox values shows that the results of the simulation experiments agree with the data previously measured in the metalimnion of Lake Kinneret.     

Population of Aerobic Heterotrophic Nitrogen-Fixing Bacteria Associated with Wetland and Dryland Rice

Nitrogen-fixing activity and populations of nitrogen-fixing bacteria associated with two varieties of rice grown in dryland and wetland conditions were measured at various growth stages during the dry season. Acetylene reduction activities were measured both in the field and for the hydroponically grown rice, which was transferred from the field to water culture 1 day before assay. The activities measured by both methods were higher in wetland than in dryland rice. The population of nitrogen-fixing heterotrophic bacteria associated with rhizosphere soil, root, and basal shoots was determined by the most probable number method with semisolid glucose-yeast extract and semisolid malate-yeast extract media. The number of nitrogen-fixing bacteria was higher in wetland conditions than in dryland conditions. The difference between two conditions was most pronounced in the population associated with the basal shoot. The glucose medium gave higher counts than did the malate medium. Colonies were picked from tryptic soy agar plates, and their nitrogen-fixing activity was tested on a semisolid glucose-yeast extract medium. The incidence of nitrogen-fixing bacteria among aerobic heterotrophic bacteria in association with rhizosphere soil, root, and basal shoots was much lower in dryland rice than in wetland rice.