Pyruvate Metabolism in the Brain of Young Rats Intoxicated with Organic and Inorganic Lead

Abstract: The activities of lipoyl dehydrogenase, aspartate transaminase, and alanine transaminase, and levels of lactate were estimated in cerebral cortex, cerebellum, and brainstem of rats intoxicated acutely with tetraethyl lead and chronically with lead acetate. A significant inhibition of lipoyl dehydrogenase was observed in both groups of animals, whereas transaminase activities were increased in inorganic lead toxicity. Oxidative decarboxylation and anaplerosis of pyruvate was assessed in brain slices using [l-¹⁴C]pyruvate. Pyruvate dehydrogenase activity was decreased in both organic and inorganic lead toxicity, whereas labelling of aspartate and alanine was increased in inorganic lead toxicity. In studies in vitro , lead acetate showed a more significant effect than tetraethyl lead. The higher anaerobic metabolism in inorganic lead toxicity, as evidenced by increased anaerobic lactate production by brain slices, could either be an adaptive mechanism or be due to the delayed maturation of brain in the developing rat. Such a mechanism does not occur in acute organic lead toxicity, as the compound brings about massive and rapid degenerative changes in brain, resulting in convulsive seizures and death of the animals.     

Changes in heated and autoclaved forest soils of S.E. Australia. II. Phosphorus and phosphatase activity

The effect of soil heat and autoclaving on labile inorganic P (Bray I), microbial P (P-flush) and on phosphatase activity was studied by heating five forest soils in the laboratory, which simulated the effects of heat during bushfires. Top soil was heated to 60 °C, 120 °C and 250 °C or autoclaved for 30 minutes. Soils were analysed immediately after heating and during seven months of incubation to assess immediate and longer-term effects of heating.Labile inorganic P increased immediately after heating and autoclaving soils, with the highest amount recorded for the 250 °C treatment. Phosphorus associated with microbial biomass decreased with heat, and none or small amounts were detected in soils heated to 250 °C and autoclaved, because high temperatures killed the microbial population. Most of the P released from microbes acted as a source of labile inorganic P in soils low in inorganic P, and some of the released P was fixed by the soil. In one soil high in inorganic labile P and with undetectable amounts of microbial-P, the increase in Bray P on heating could only be assigned to solubilisation of other sources of total P Because high temperatures denature enzymatic proteins, phosphatase activity diminished with the increase in temperature, and no activity was detected in 250 °C and autoclaved soils.Phosphorus released by heating decreased during incubation in three of the five soils studied, approaching values observed in unheated soils. Simultaneously, an increase in microbial P was observed in these heated soils, indicating that the partial recovery of microbial biomass acted as a sink for the decrease in Bray-P measured. Phosphatase activity recovered only partially during incubation of heated soils.     

Distribution and characterization of mineral-binding phosphoprotein particles in Bivalvia

Representative species of four bivalve subclasses were examined for the presence of mineral-binding phosphoprotein particles in the physiological fluids. The particles were identified in Heterodont bivalves only, and particles from nine different Heterodont species were isolated and characterized. All phosphoprotein particles are internally cross-linked via histidinoalanine residues. In all species over 80% of the amino acid residues in the particles are aspartic acid, phosphoserine (and/or phosphothreonine), and histidine. These amino acids are probably the only residues directly related to mineral ion binding, since all phosphoprotein particles bind mineral irrespective of the minor amino acid content, which is species dependent. In their native state the phosphoprotein particles contain large amounts of calcium, magnesium, and inorganic phosphate ions (up to 45 metal ions and 8 phosphate ions per 100 amino acid residues) and trace amounts of transition elements. Evidence for the presence of calcium phosphate complexes in the native phosphoprotein particles was obtained by observing a concomitant increase in the inorganic phosphate and calcium ion content of the particles with pH in vivo.     

固氮红细菌(Rhodobacter azotoformans) YLK20去除无机氮的影响因素

【背景】不产氧光合细菌(Anoxygenicphototrophicbacteria,APB)作为一类重要的微生物资源,在水产养殖水体氮污染的修复方面已有广泛研究与应用。养殖水体环境复杂,含多种有机物,尤其是有机氮显著影响菌体除氮功效。【目的】在高浓度无机三态氮(氨氮、硝氮和亚硝氮)共存体系中,阐明小分子有机碳、有机氮和盐度对固氮红细菌(Rhodobacter azotoformans) YLK20去除无机三态氮的影响规律及机制,挖掘针对性强和适应性广的高效除氮菌株。【方法】采用RAST和KEGG方法分析YLK20基因组碳氮代谢途径及耐盐机制;采用次溴酸钠氧化法、紫外和N-(1-萘基)-乙二胺分光光度法分别测定氨氮、硝氮和亚硝氮含量。【结果】基因组显示,YLK20拥有EMP、HMP、TCA、固氮、氨化、氨同化和反硝化碳氮代谢途径,含有soh B、nha C、bet B和gbs A等多种耐盐基因。丙酮酸钠、乙酸钠、柠檬酸钠、乙醇和甘露醇是YLK20生长和去除无机三态氮的良好有机碳,葡萄糖和果糖的存在降低了无机三态氮去除能力,蔗糖体系中硝氮和亚硝氮能被良好去除,但氨氮去除能力较低。在高浓度蛋白胨(3.21 g/L)和尿素(1.43 g/L)体系中,YLK20仍能高效去除无机三态氮。YLK20能在3%盐度内生长良好,低盐度时该菌株能良好去除无机三态氮,高盐度时亚硝氮去除能力受到严重抑制。YLK20对海水和淡水实际养殖水体中的无机三态氮有良好去除效果。【结论】YLK20主要通过氨同化和反硝化途径去除无机三态氮,尤其在高浓度有机氮环境中也能高效去除;该菌株适应盐度范围广,兼可适用于淡水和海水养殖水体;该菌株生长和无机三态氮去除影响因素、规律及除氮机制的阐明,可为APB微生物制剂的合理应用提供指导。     

Effects of lead on luteal function in rhesus monkeys

Exposure to lead in the workplace or home environment has been implicated as a cause of decreased fertility in women. In a previous study, as part of our effort to determine effects of lead in primates, female rhesus monkeys were exposed to lead acetate in drinking water (n = 10) or provided water with no added lead (n = 7) for 33 mo. Lead was administered at levels between 2 and 8 mg/kg/day, with doses adjusted to keep blood lead values near a target of 70 micrograms/dl (observed mean +/- SEM = 68.9 +/- 6.54 micrograms/dl). Blood lead concentrations in control animals were less than 10 micrograms/dl. No significant differences were detected between control and experimental animals in body weight, hematocrit, or general health. Female monkeys receiving lead exhibited longer and more variable menstrual cycles and shorter menstrual flow. In the present study, circulating amounts of progesterone (P4) were determined to evaluate luteal function during the final 7 mo of treatment with lead. Several characteristics were altered as a result of lead treatment: circulating amounts of P4 were reduced as indicated by relative units of area under the concentration-time curve, maximal amounts of P4 were reduced, and P4 levels were greater than 1 ng/ml on fewer days. There were no significant differences between groups in mean percent of anovulatory cycles. Therefore, although chronic treatment with the levels of lead used in this study did not prevent ovulation, luteal function was suppressed. These results extend previous observations of adverse effects of lead on ovarian activity and fertility in monkeys.     

Automated determination of organic sulfur compounds. I. Spectrophotometric measurement of inorganic sulfate by low-pressure exchange chromatography

A method for determination of inorganic sulfate is described which represents the terminal step of an automated assay of organic sulfur compounds in biological fluids. Nanomole quantities of inorganic sulfate were applied to a barium chloranilate column. Corresponding amounts of chloranilate ion, released as a result of an exchange reaction, were then measured by uv absorption at 313 nm. The reproducibility and sensitivity of the method and a calibration curve are reported.     

Loss of cell components during rehydration of dried Rhodotorula glutinis and its implications for lead uptake

Microbial cells are routinely dried and ground before they are used in metal biosorption studies. In this work, a metal biosorbent was prepared by drying biomass of the yeast Rhodotorula glutinis in an oven at 70°C for 24 h followed by grinding. Two forms of the prepared biosorbent particles, washed and unwashed, were examined for their ability to remove lead from solution. It was found that the unwashed biosorbent exhibited higher lead uptake than the washed biosorbent. Analysis of the supernatant of washed cells incubated in water and that of unwashed cells incubated in lead solution revealed the presence of protein, carbohydrates, organic acids and inorganic phosphate. Overall, the washed and unwashed cells leached, respectively, 14.5 and 13.4% of their initial dry weight (100 mg). Acid‐base titration data revealed that the leached components contained several potential binding sites for metal cations with carboxyl and phosphoryl groups being particularly important. The higher level of lead uptake exhibited by the unwashed biomass was attributed to the fact that it leached smaller amounts of cell constituents with proton binding sites relative to the washed cells.     

Inhibition of cytokinesis by lead (Pb2+) in a centric diatom.

Effect of inorganic and organo lead has been studied on the mitosis of a centric diatom Cyclotella meneghiniana f. unipunctata. Binucleate cells were formed in the presence of different concentrations of Pb2+ (1.0, 2.0, 3.0 and 5.0 mM) due to inhibition of cell plate formation. Lead at 5.0 mM concentration was more inhibitory than the other concentrations. Organo lead was a powerful depressant of cytokinesis than inorganic lead. Failure of cytokinesis might be due to disruption of microtubules. Formation of distinct nuclei delayed post incubation cell divisions suggest partial damage of mitotic spindles.     

A novel staining method for detecting phytase activity

The level of microbial resistance to heavy metals is an important issue for the microbial ecology of heavy metal-contaminated habitats. However, assays based upon growth in nutrient media will overestimate the resistance level due to metal ion interactions with inorganic and organic components. The analysis of Pb-resistant bacteria isolated from soils containing up to 38 mmol total Pb x kg(-1) indicated that PYT80B medium which did not contain inorganic salts, contained low amounts of organic matter, and was buffered with a molecule that did not interact with metal ions (2-N-morpholinoethanesulfonic acid (MES)) provided the lowest estimates of lead resistance. However, better results were obtained by assaying metabolic activity (aerobic respiration) of resting cells suspended in 10 mM MES. By this criterion, 50% inhibition of Arthrobacter JS7 was found at 37 microM Pb(NO3)2. The effects of Pb+2 concentrations upon respiration of resting cells and growth rate in PYT80B medium were similar. The activity assay also showed that metal resistance was induced to higher levels when Arthrobacter JS7 was grown in the presence of Pb.     

Glutamate metabolism in the brain of young rats exposed to organic and inorganic lead

The synthesis of glutamate and its conversion to glutamine and GABA were studied using labelled glucose in cerebral cortex, cerebellum and brainstem of rats intoxicated acutely with tetraethyl lead and chronically with lead acetate. To assess the interconversion and the synaptosomal accumulation of these amino acids, the labelling of glutamate, glutamine and GABA were measured in whole tissue and synaptosomes after giving labelled glutamate. The radioactive carbon dioxide production from labelled glutamate by brain slices was measured to evaluate the oxidation of glutamate. The tissue levels of glutamate, glutamine and GABA and the activity of glutamate decarboxylase were also measured in both conditions.In inorganic lead toxicity, even though the glutamate pool size was reduced, the glutamate-glutamine cycling between synaptosomes and astrocytes was increased. The oxidation of glutamate and the glutamate-GABA cycling were reduced. These findings suggest that brain tries to maintain the endogenous glutamate levels by decreasing the oxidation of glutamate and increasing the uptake systems and the cycling through glutamine in inorganic lead toxicity. In organic lead toxicity, the glutamate pool as well as glutamate turnover was reduced markedly resulting in complete distortion of glutamate metabolism.     

Lead accumulation in the roots of grass pea (Lathyrus sativus L.): a novel plant for phytoremediation systems?

Eleven day-old grass pea plants (Lathyrus sativus L.) were grown hydroponically for 96 h in the presence of 0.5 mM lead nitrate (Pb(NO(3))(2)). The survival rate was 100%. The mean lead content (measured by ICP-OES) in root tissues was 153 mg Pb g(-1) dry matter. Over three quarters of the lead was not labile. Compared with control plants, lead-exposed plants showed a six-fold, two-fold and three and a half-fold reduction in their root calcium, zinc and copper contents, respectively. Together, these results suggested that Lathyrus sativus L. was tolerant to a deficiency in essential nutrients and able to store large amounts of lead in its root tissues. Therefore, it could be used for the development of new rhizofiltration systems.     

The role of apoptosis in mineralizing murine versus avian micromass culture systems

Chondrocyte apoptosis is thought to be an important step in the calcification of cartilage in vivo; however, there are conflicting reports as to whether or not this apoptosis is a necessary precursor to mineralization. The goal of this study was to determine whether or not apoptosis is necessary for mineralization in an in vitro murine micromass model of endochondral ossification. C3H10T1/2 murine mesenchymal stem cells were plated in micromass culture in the presence of 4 mM inorganic phosphate with the addition of the apoptogens, camptothecin, or staurosporine, to induce apoptosis. The rate and total accumulation of mineralization was measured with ⁴⁵Ca uptake. In these studies, both apoptogens increased the rate of mineralization, with staurosporine increasing ⁴⁵Ca accumulation by about 2.5 times that of controls and camptothecin increasing total amounts of mineralization about 1.5 times that of controls. Inhibiting cell apoptosis with the caspase inhibitor, ZVAD‐fmk, to prevent apoptosis, caused slower rates of ⁴⁵Ca uptake; however, total amounts of ⁴⁵Ca accumulation reached the same values by day 30 of culture. FTIR data showed mineralization in all samples treated with 4 mM inorganic phosphate, with the highest mineral to matrix ratios in the camptothecin treated samples. J. Cell. Biochem. 111: 653–658, 2010. © 2010 Wiley‐Liss, Inc.     

Effects of Organic and Inorganic Lead on Synaptosomal Uptake, Release, and Receptor Binding of Glutamate in Young Rats

Chemical changes in central glutamate neurotransmission were assessed by measuring synaptic membrane receptor binding, the uptake and release by synaptosomes of glutamate in rats treated acutely with tetraethyl lead and chronically with lead acetate. The activity of Na+, K+-ATPase in synaptosomes was measured to correlate with the changes in uptake/release studies. The affinity of receptor binding and uptake systems was significantly reduced although the number of receptor sites and the capacity of uptake systems were increased. The activity of Na+, K+-ATPase was also found to be increased in synaptosomes. The changes were more marked in inorganic lead toxicity, and all three regions studied--cerebral cortex, cerebellum, and brainstem--showed significant alterations.     

Regulatory effects of adenosine diphosphate on the activity of the plasma membrane ATPase of corn roots

Plasma membrane enriched microsomal fraction was isolated from corn root cells by sucrose density centrifugation. The ATPase activity as measured by the release rate of inorganic phosphate, was decreased by the presence of modifiers which included diethylstilbestrol, vanadate, N,N'-dicyclohexylcarbodiimide, and miconazole. The presence of ADP also decreased the rate of ATP hydrolysis. Furthermore, a preincubation of the membrane with ADP significantly reduced the inhibitory effects of these membrane ATPase modifiers. Since the modes of interaction of these modifiers with the enzyme are different, the results suggest that the binding of ADP may stabilize the plasma membrane ATPase in a modifier insensitive state.     

Association of inorganic pyrophosphatase activity with normal calcification of rat costal cartilage in vivo

1. Dialysed extracts of rat costal cartilage were shown to possess an enzyme that hydrolyses inorganic pyrophosphate. 2. Inorganic pyrophosphatase activity assayed in the presence of 2mm substrate was maximal at pH6.8. 3. Mg(2+) was essential for activity, which was greatest with 10mm or higher concentrations of Mg(2+). 4. Extracts prepared from cartilage taken from suckling rats (<20g.) showed little or no hydrolytic activity, but as rat weight increased inorganic pyrophosphatase activity was detected, increased to a maximum in tissue from animals weighing about 40g., and then rapidly declined. 5. The increase in inorganic pyrophosphatase activity was associated with an increase in the uptake of (45)Ca by the cartilage in vivo. 6. Accumulation of calcium, inorganic phosphate and magnesium occurred when inorganic pyrophosphatase activity was at its maximum. 7. Alkaline phosphatase activity, measured in the same extracts used to determine pyrophosphatase activity, was highest in the tissues of the animals weighing <20g., and decreased as inorganic pyrophosphatase activity increased to its maximum. 8. There was no direct relationship between alkaline phosphatase activity and the onset of calcification.     

Photosynthesis and respiration of a diatom biofilm cultured in a new gradient growth chamber

Abstract A diatom biofilm was grown in a chamber developed for culture of biofilms in chemical gradients. The diatoms grew on a polycarbonate membrane filter which separated a sterile reservoir, with added phosphate, from a reservoir without phosphate. Within 3 weeks of inoculation, a thick biofilm developed on the surface of the filter. The biofilms were homogeneous and therefore suitable for calculations of O₂ diffusion fluxes from concentration profiles of O₂. Profiles of O₂, pH, and gross photosynthesis at different light intensities and liquid medium concentrations of dissolved inorganic carbon and O₂ were measured with microelectrodes. Respiratory activity in a layer of the biofilm was determined as the difference between gross photosynthesis and outflux of O₂ from that layer. The photosynthetic activity in a well-developed biofilm grown at 360 μEinst m⁻² s⁻¹ and 2.4 mM HCO₃⁻ was limited by the supply of inorganic carbon. Exposure to light above 360 μEinst m⁻² s⁻¹ stimulated gross photosynthesis as well as respiratory processes without affecting net outflux of O₂. Higher concentrations of inorganic carbon, on the other hand, enhanced gross photosynthesis without concurrent increase in respiratory rate, resulting in an increased outflux of O₂. High concentrations of O₂ in the liquid medium decreased the net outflux of O₂ with little effect on the gross photosynthesis. The effects of inorganic carbon and O₂ on the metabolic activities of the biofilm were consistent with the presence of photorespiratory activity.     

Phosphonoacetic acid utilization by fungal isolates: occurrence and properties of a phosphonoacetate hydrolase in some penicillia

Among a collection of 18 fungal strains representing eight genera, only two strains (Penicillium oxalicum and P. minioluteum) were capable of growth on phosphonoacetic acid as sole phosphorous source. Enrichment liquid cultures in minimal medium with the compound as the only P-source selected four isolates, that were also identified as Penicillium spp. Phosphonoacetate metabolism did not lead to extracellular release of inorganic phosphate. In all cases phosphonoacetate hydrolase activity was detected in partially purified extracts, and a protein of the expected molecular mass reacted with polyclonal antibodies raised against the enzyme from P. oxalicum. There was no relation between phosphonoacetate hydrolase specific activity and growth rate or yield. Phosphonoacetic acid was the inducer of the hydrolase, independently of the concurrent availability of inorganic phosphate. Notwithstanding this, the utilization of the phosphonate was significantly inhibited in the presence of phosphate, suggesting an interference of the latter with phosphonoacetic acid uptake.     

A comparison of the distribution of enzymatically and non-enzymatically produced lead phosphate in insect flight muscle

Lead phosphate precipitates were produced in indirect flight muscles of Phormia regina by sequential incubation in solutions containing lead and inorganic phosphate and their distribution was compared with those produced by ATP hydrolysis in the presence of lead. Enzymatically produced precipitates were associated almost exclusively with thick filaments. Non-enzymatically produced precipitates were associated with thick filaments but were also found associated with thin filaments in significant numbers.