Effect of dietary seaweed (Ascophyllum nodosum) supplementation on milk mineral concentrations, transfer efficiency, and hematological parameters in lactating Holstein cows.

This study investigated the effect of feeding seaweed (Ascophyllum nodosum) to dairy cows on milk mineral concentrations, feed-to-milk mineral transfer efficiencies, and hematological parameters. Lactating Holstein cows (n = 46) were allocated to 1 of 2 diets (n = 23 each): (1) control (CON; without seaweed) and (2) seaweed (SWD; replacing 330 g/d of dried corn meal in CON with 330 g/d dried A. nodosum). All cows were fed the CON diet for 4 wk before the experiment (adaptation period), and animals were then fed the experimental diets for 9 wk. Samples included sequential 3-wk composite feed samples, a composite milk sample on the last day of each week, and a blood sample at the end of the study. Data were statistically analyzed using a linear mixed effects model with diet, week, and their interaction as fixed factors; cow (nested within diet) as a random factor; and data collected on the last day of the adaptation period as covariates. Feeding SWD increased milk concentrations of Mg (+6.6 mg/kg), P (+56 mg/kg), and I (+1,720 µg/kg). It also reduced transfer efficiency of Ca, Mg, P, K, Mn, and Zn, and increased transfer efficiency of Mo. Feeding SWD marginally reduced milk protein concentrations, whereas there was no effect of SWD feeding on cows' hematological parameters. Feeding A. nodosum increased milk I concentrations, which can be beneficial when feed I concentration is limited or in demographics or populations with increased risk of I deficiency (e.g., female adolescents, pregnant women, nursing mothers). However, care should also be taken when feeding SWD to dairy cows because, in the present study, milk I concentrations were particularly high and could result in I intakes that pose a health risk for children consuming milk.

A new approach to establish safe levels of available metals in soil with respect to potential health hazard of human.

Safe levels of extractable pollutant elements in soil have not been universally established. Prediction of metal solubility in polluted soils and the subsequent transfer of these metals from soil pore water to the human food supply via crops are required for effective risk assessment from polluted soils. Thus an attempt has been made to develop a novel approach to protect human health from exposure to toxic metals through assessing risk from metal polluted soils utilised for agriculture. In this study, we assess the relative efficacy of various forms of 'free ion activity model' (FIAM) for predicting the concentration of cadmium (Cd), lead (Pb), nickel (Ni), zinc (Zn) and copper (Cu) in spinach and wheat as example crops, thereby providing an assessment of risk to human health from consumption of these crops. Free metal ion activity in soil solution was estimated using the Windermere Humic Aqueous Model VII (WHAM-VII) and the Baker soil test. Approximately 91, 81, 75, 94 and 70% of the variability in Cd, Pb, Ni, Zn and Cu content, respectively, of spinach could be described by a FIAM using an estimate of the free ion activity of the metals provided by WHAM-VII. Owing to the different concentration of ethylenediamine tetraacetic acid (EDTA) and diethylenetriamine pentaacetic acid (DTPA) used in the present experiment, higher prediction coefficients were obtained using EDTA (0.05 M), rather than DTPA (0.005 M), as the metal extractant in an integrated solubility-FIAM model. Out of three formulations, the FIAM, based on free ion activity of metals in soil pore water, determined from solution extracted with Rhizon samplers, was distinctly superior to the other formulations in predicting metal uptake by spinach and wheat. A safe level of extractable metal in soil was prescribed using a hazard quotient derived from predicted plant metal content and estimated dietary intake of wheat and spinach by a human population. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12665-021-09988-7.

Phosphorus feeding practices, barriers to and motivators for minimising phosphorus feeding to dairy cows in diverse dairy farming systems.

Minimising phosphorus (P) feeding to dairy cows can reduce feed costs and minimise water pollution without impairing animal performance. This study aimed to determine current P feeding practices and identify the barriers to and motivators for minimising P feeding on dairy farms, using Great Britain (GB) dairy farming as an example of diverse systems. Farmers (n = 139) and feed advisers (n = 31) were involved simultaneously in independent questionnaire surveys on P feeding in dairy farms. Data on the herd size, milk yield and concentrate fed were analysed using ANOVA to investigate the effect of farm classification, region, and feed professional advice. Chi-square tests were used to investigate associations between farm characteristics and implemented P feeding and management practices. Most farmers (72%) did not know the P concentration in their lactating cow's diet and did not commonly adopt precision P feeding practices, indicating that cows might have been offered dietary P in excess of recommended P requirement. Farmers' tendency to feed P in excess of recommendations increased with herd size, but so did their awareness of P pollution issues and likeliness of testing manure P. However, 68% of farmers did not analyse manure P, indicating that mineral P fertiliser application rates were not adjusted accordingly, highlighting the risk of P being applied beyond crops' requirement. Almost all farmers (96%) were willing to lower dietary P concentration but the uncertainty of P availability in feed ingredients (30%) and concerns over reduced cow fertility (22%) were primary barriers. The willingness to reduce dietary P concentrations was driven by the prospect of reducing environmental damage (28%) and feed costs (27%) and advice from their feed professionals (25%). Most farmers (70%) relied on a feed professional, and these farmers had a higher tendency to analyse their forage P. However, farmers of pasture-based systems relied less on feed professionals. Both farmers (73%) and feed advisers (68%) were unsatisfied with the amount of training on P management available. Therefore, the training on P management needs to be more available and the influence that feed professionals have over P feeding should be better utilised. Study findings demonstrate the importance of considering type of dairy farming systems when developing precision P feeding strategies and highlight the increasing importance of feed professionals in minimising P feeding.

Loss of Gut Microbiota Alters Immune System Composition and Cripples Postinfarction Cardiac Repair.

BACKGROUND: The impact of gut microbiota on the regulation of host physiology has recently garnered considerable attention, particularly in key areas such as the immune system and metabolism. These areas are also crucial for the pathophysiology of and repair after myocardial infarction (MI). However, the role of the gut microbiota in the context of MI remains to be fully elucidated. METHODS: To investigate the effects of gut microbiota on cardiac repair after MI, C57BL/6J mice were treated with antibiotics 7 days before MI to deplete mouse gut microbiota. Flow cytometry was applied to examine the changes in immune cell composition in the heart. 16S rDNA sequencing was conducted as a readout for changes in gut microbial composition. Short-chain fatty acid (SCFA) species altered after antibiotic treatment were identified by high-performance liquid chromatography. Fecal reconstitution, transplantation of monocytes, or dietary SCFA or Lactobacillus probiotic supplementation was conducted to evaluate the cardioprotective effects of microbiota on the mice after MI. RESULTS: Antibiotic-treated mice displayed drastic, dose-dependent mortality after MI. We observed an association between the gut microbiota depletion and significant reductions in the proportion of myeloid cells and SCFAs, more specifically acetate, butyrate, and propionate. Infiltration of CX3CR1+ monocytes to the peri-infarct zone after MI was also reduced, suggesting impairment of repair after MI. Accordingly, the physiological status and survival of mice were significantly improved after fecal reconstitution, transplantation of monocytes, or dietary SCFA supplementation. MI was associated with a reorganization of the gut microbial community such as a reduction in Lactobacillus. Supplementing antibiotic-treated mice with a Lactobacillus probiotic before MI restored myeloid cell proportions, yielded cardioprotective effects, and shifted the balance of SCFAs toward propionate. CONCLUSIONS: Gut microbiota-derived SCFAs play an important role in maintaining host immune composition and repair capacity after MI. This suggests that manipulation of these elements may provide opportunities to modulate pathological outcome after MI and indeed human health and disease as a whole.

Short communication: Effect of abomasal inorganic phosphorus infusion on phosphorus absorption in large intestine, milk production, and phosphorus excretion of dairy cattle.

The objective of the study was to evaluate the effect of inorganic phosphorus (Pi) infusion on P absorption in large intestine, milk production, and phosphorus excretion. Four ruminally and ileally cannulated crossbred cows were used in a 4 × 4 Latin square with 21-d periods. Cows were fed a total mixed ration containing 0.21% P, providing 50% of the cows' P requirement. Cobalt-EDTA was used as marker to measure large intestine digesta flow. On d 13 to 21 of each period, each cow was infused daily with 0, 20.1, 40.2, or 60.3 g of Pi into the abomasum and total collection was conducted on d 18 to 21. Ileal samples were collected every 9 h on d 18 to 21. Feed, digesta, and fecal samples were analyzed for total P and Pi using the molybdovanadate yellow method and blue method, respectively. All data were analyzed using PROC GLIMMIX in SAS 9.3 (SAS Institute Inc., Cary, NC) using contrasts to evaluate linear, quadratic, and cubic effects of Pi infusion dose. Dry matter intake, apparent dry matter digestibility, milk yield, and milk total P were unaffected by Pi infusion. Ileal flow and fecal excretion of total P and Pi increased linearly with increasing infused Pi. In the large intestine, net absorption of TP and Pi was increased linearly with increasing infused Pi. The magnitude of absorption from the large intestine was greater than reflected in current models, raising questions that could be evaluated with longer infusion periods or dietary alteration.

Neurophysiological study on the effect of various short durations of deep breathing: A randomized controlled trial.

The study aims to study the effects of short duration deep breathing on the EEG power with topography based on parallel group randomized controlled trial design which was lacking in prior reports. 50 participants were split into 4 groups: control (CONT), deep breathing (DB) for 5 (DB5), 7 (DB7), and 9 (DB9) minutes. EEG recordings were obtained during baseline, deep breathing session, after deep breathing, and a follow-up session after 7 days of consecutive practice. Frontal theta power of DB5 and DB9 was significantly larger than that of CONT after the deep breathing session (p = 0.027 and p = 0.006, respectively) and the profound finding showed that the theta topography obtained a central-focused distribution for DB7 and DB9. The result obtained was consistent with previous literature, albeit for certain deep breathing durations only, indicating a possible linkage between the deep breathing duration and the neurophysiology of the brain.

Fixed-dose combinations of antimicrobials: A need for special attention.

OBJECTIVE: To highlight the issue of freely available fixed-dose combinations (FDCs) of antimicrobials. METHODS: A critique of two such antimicrobial FDCs was undertaken wherein the following aspects were assessed - rational and regulatory issues and justification for clinical use. Available in vitro, in vivo (animals and humans) evidence from published literature was analysed. CONCLUSIONS: There are several inadequately addressed aspects of the considered FDCs which are available in Indian market. In view of the growing problem of antimicrobial resistance, this issue must get the required attention.

The effects of forage particle length and exogenous phytase inclusion on phosphorus digestion and absorption in lactating cows.

Accurate estimates of phosphorus (P) availability from feed are needed to allow P requirements to be met with reduced P intake, thus reducing P excretion by livestock. Exogenous phytase supplementation in poultry and swine diets improves bioavailability of P, and limited research suggests that this strategy may have some application in dairy cattle rations. The effects of exogenous phytase and forage particle length on site and extent of P digestion were evaluated with 5 ruminally and ileally cannulated lactating cows (188 ± 35 d in milk). Cows were assigned in a 2 × 2 factorial arrangement of treatments in 2 incomplete Latin squares with four 21-d periods. Diets contained P slightly in excess of National Research Council requirements with all P from feed sources. During the last 4d of each period, total mixed ration, refusals, omasal, ileal, and fecal samples were collected and analyzed for total P, inorganic P (Pi), and phytate (Pp). Total P intake was not influenced by dietary treatments but Pp intake decreased and Pi intake increased with supplemental phytase, suggesting rapid action of the enzyme in the total mixed ration after mixing. Omasal flow of Pi decreased with phytase supplementation, but we observed no effect of diet in ileal flow or small intestinal digestibility of any P fraction. Fecal excretion of total P was slightly higher and Pp excretion was lower for cows receiving diets supplemented with phytase. Milk yield and composition were unaffected by diets. When phytase was added to the mixed ration, dietary Pp was rapidly degraded before intake and total-tract Pp digestion was increased. The lack of effect of phytase supplementation on dietary P utilization was probably because these late-lactation cows had a low P requirement and were fed P-adequate diets.

Effect of dietary phytate on phosphorus digestibility in dairy cows.

The objective was to evaluate the effect of dietary phytate P (Pp) supply on ruminal and postruminal Pp digestion and net disappearance of P from the lower digestive tract of lactating cows. Six ruminally and ileally cannulated crossbred lactating cows were used in 2 incomplete Latin squares with four 21-d periods (17d of diet adaptation, 4d of total collection). Dietary treatments were low Pp, medium Pp, and high Pp, and a high inorganic P (Pi) diet with the same total P content as the highest Pp diet but with P mostly from inorganic sources. The diets contained 0.10, 0.18, 0.29, and 0.11% Pp and 0.43, 0.48, 0.54, and 0.53% total P on a dry matter basis, with cottonseed meal used to increase Pp content. Ytterbium-labeled corn silage and Co-EDTA were used as particulate and liquid phase markers to measure omasal and ileal digesta flow. Omasal and ileal digesta were collected every 6h on d 20 and 21 and rumen contents were collected on d 21. Samples were analyzed for total P (molybdovanadate yellow method), Pi (blue method), and Pp (high performance ion chromatography). Phytate P and total P intake increased linearly with increasing dietary Pp. Ruminal Pp disappearance also increased linearly with dietary Pp but the magnitude of change was small. Small intestinal net disappearance of Pi was not affected by dietary Pp. Phytate P was hydrolyzed in the large intestine but its hydrolysis was not influenced by dietary Pp. Net disappearance of Pi form the large intestine did not vary with dietary Pp. Dry matter digestibility decreased linearly with increasing dietary Pp, as did apparent digestion of P, and fecal P increased linearly. Dry matter digestibility was higher for high Pi than for high Pp, likely due to the effect of cottonseed meal in the latter diet. Replacing a portion of Pp with Pi resulted in decreased P excretion but this effect was confounded with increased fecal dry matter for the high-Pp (high-cottonseed meal) diet. In lactating cows Pp digestibility was not negatively influenced by dietary Pp and fecal P excretion was regulated by dietary total P rather than by form of dietary P.

Disappearance of infused phytate from the large intestine of dairy heifers.

The objective of this study was to investigate the disappearance of phytate from the large intestine of dairy heifers. Uncertainty about the availability of phosphorus (P) in different feeds may limit implementation of dietary strategies to reduce fecal P excretion by dairy cows. Increased understanding of the dynamics of phytate degradation and disappearance of P in the large intestine may improve prediction of intestinal P digestion and absorption. Eight ruminally- and ileally-cannulated crossbred dairy heifers were used in two 4×4 Latin square designs with 9-d periods, including 3d of washout. All heifers were fed a high-forage diet containing 0.14% P throughout the study. Ytterbium-labeled corn silage and Co-EDTA were dosed to the rumen 4 times daily as particulate and liquid phase markers, respectively, to measure ileal digesta flow. Ond 4 to 7 of each period, each heifer was infused ileally with 0, 5, 15, or 25 g/d of phytate (phytic acid) in solution and total fecal collection was conducted. When infusion ceased (d 8 and 9) ileal digesta was sampled to measure P flow to the ileum from the basal diet. Feed, digesta, and feces were dried, ground, and analyzed for phytate P, inorganic P, and total P using high performance ion chromatography, inductively coupled plasma atomic emission spectroscopy, and the molybdovanadate yellow method, respectively. Phytate degradation in the large intestine was observed but was not complete, and the amount of infused phytate did not influence the degradability of phytate. Fecal excretion of total P increased with increasing total P infused. The slope coefficient for ileal P flow (dietary only) to feces was 0.56 ± 0.26 (mean ± SE), whereas the slope coefficient for infused P was 0.75 ± 0.13. These indicate net absorption of P from the large intestine and greater disappearance of P from dietary P flowing to the ileum than from the infused pure phytate (44 vs. 25%). This data will support mechanistic modeling efforts to improve prediction of P digestion, allowing more accurate estimation of P bioavailability in feeds.

Analgesic activity of Amorphophallus campanulatus tuber.

The methanol extract of Amorphophallus campanulatus tuber, given orally at the doses of 250 and 500 mg/kg, showed significant analgesic activity in mice.

Does drug abuse influence the microglial response in AIDS and HIV encephalitis?

OBJECTIVES: To investigate the pathological evidence for a possible interaction between drugs of abuse and HIV infection in terms of microglial responses in early and late HIV/AIDS, and to discuss the possible long-term consequences of microglial activation in chronic HIV infection. DESIGN: This brain pathology study compared age and sex-matched control patients with HIV-negative intravenous drug users, and with HIV-positive drug users both in the presymptomatic stage and with AIDS. A further group of non-drug-using AIDS patients was included. All the AIDS patients had HIV encephalitis (HIVE) but no other significant HIV-associated brain pathology. METHODS: Microglia/macrophages were identified in the grey and white matter of the frontal and temporal lobes and the thalamus, using antibodies to CD68 and MHCII. Objective quantitation was used to compare subjects in the different groups. RESULTS: AIDS patients showed a significant increase in activated microglia/macrophages in both the grey and white matter of all areas compared with non-AIDS patients. Drug users with HIVE tended to have more activated microglia than non-drug-using comparison groups, but this difference was not found in all brain areas studied. CONCLUSION: Drug misuse appears to enhance the microglial activation resulting from HIV infection in some individuals. Other factors such as the severity of HIVE, or systemic immune factors, are also likely to affect the degree of microglial activation. The implications for drug-using patients who survive long term with HIV/AIDS are discussed, particularly in relation to premature neurodegeneration.

Electrical treatment of tibial non-union: a prospective, randomised, double-blind trial.

Electrical stimulation in the treatment of bony non-union has been used in different forms for many years. However, there is still a lot of uncertainty about its efficacy. We, therefore, undertook a prospective, randomised, double-blind trial to try and determine its effect. Over a period of 5 years, 34 consecutive patients with a tibial non-union met our "criteria for inclusion". Each patient had an oblique fibular osteotomy, followed by a unilateral external fixator. They were then randomly allocated one of two machines. Group 1, the active group, received electrical stimulation from an active machine. Group 2, the dummy group, had an identical machine but without any current passing through the active coils. They were then followed up for 6 months and evaluated clinically and radiologically for bony union. Unfortunately, there was by chance, an imbalance in smoking habit between the two groups. The union rate in the subgroup that smoked was 75% (6/8) in the active group as compared to 46% (6/13) in the dummy group. The active group of non-smokers had 100% (10/10) union rate, compared to 67% (2/3) in the dummy group. Overall 24 out of the 34 patients progressed to union. Out of 18, 16 (89%) in the active group showed bony union as compared to 8/16 (50%) in the dummy. There was, thus, a statistically significant positive association between tibial union and electrical stimulation (odds ratio 8, 95% CI: 1.5-41, P=0.02).

Change in distribution & antifungal susceptibility of Candida species isolated from candidaemia cases in a tertiary care centre during 1996-2000.

BACKGROUND & OBJECTIVES: As a marked increase in the number of patients with candidaemia was reported in the first half (1991-1995) of the last decade at the Postgraduate Institute of Medical Education & Research, Chandigarh, India, the present study was aimed at determining further change if any, in the incidence and distribution of Candida species and their antifungal resistance pattern during the second half (1996-2000) of the same decade. METHODS: The patients with candidaemia were studied to determine the frequency of candidaemia and Candida species isolated during 1996-2000. One hundred Candida strains other than Pichia anomala (C. pelliculosa) were randomly selected from those isolates to evaluate antifungal susceptibility pattern against amphotericin B, 5-fluorocytosine, ketoconazole, fluconazole and itraconazole. The results were compared with our previous study. RESULTS: An increase in the number of patients with candidaemia was observed during 1996 (538) and 1997 (421) compared to 1998-2000 due to P. anomala outbreak. With the control of the outbreak, a substantial decrease in the incidence of candidaemia was observed from 1998 (251 in 1998, 122 in 1999 and 165 in 2000). A higher isolation of non-C. albicans Candida species (89.8%) was observed, with C. tropicalis being the most common (541, 36.1%) agent. No major change in the isolation rate of other non-C. albicans Candida species (C. guilliermondii, C. krusei, C. glabrata and C. parapsilosis) was observed. An emergence of resistance to amphotericin B in 15.4 per cent C. albicans, 8.1 per cent C. tropicalis and 33.3 per cent C. krusei strains was observed. An increase in resistance to ketoconazole (from 0% to 13%) and 5-fluorocytosine (from 1% to 8%) and a decrease to fluconazole (from 13% to 6%) were observed. Resistance to itraconazole was observed in 17 per cent of Candida strains by broth macro-dilution method. INTERPRETATION & CONCLUSION: A change in the isolation of Candida species was observed i.e. in the incidence and isolation of non-C. albicans Candida species. Emergence of resistance to amphotericin B and increase of resistance to most other antifungals are cause for concern.

Cardioprotection of red wine: role of polyphenolic antioxidants.

Epidemiological studies suggest that the consumption of wine, particularly of red wine, reduces the incidence of mortality and morbidity from coronary heart disease. This has given rise to what is now popularly termed the "French paradox". The cardioprotective effect has been attributed to antioxidants present in the polyphenol fraction of red wine. Grapes contain a variety of antioxidants, including resveratrol, catechin, epicatechin and proanthocyanidins. Of these, resveratrol is present mainly in grape skin while proanthocyanidin is present in the seeds. In this report, we provide evidence that red wine extract as well as resveratrol and proanthocyanidins are equally effective in reducing myocardial ischemic reperfusion injury, which suggests that these red wine polyphenolic antioxidants play a crucial role in cardioprotection.

Purification of 1,3-beta-D-glucan synthase activity from pea tissue. Two polypeptides of 55 kDa and 70 kDa copurify with enzyme activity.

From pea plasma membranes isolated by aqueous polymer two-phase partitioning we have purified 1,3-beta-D-glucan synthase [glucan synthase-II (GS-II) or callose synthase], an enzyme that several reports have suggested consists of between six and nine different subunits. The procedure involves (a) preliminary removal of peripheral proteins by 0.1% digitonin; (b) solubilization of GS-II with 0.5% digitonin; (c) precipitation of activity-irrelevant proteins from the digitonin extract by Ca2+, spermine and cellobiose, which are GS-II effectors needed in step (d); (d) product entrapment by formation of 1,3-beta-D-glucan from UDP-Glc by GS-II in the presence of the mentioned effectors, followed by centrifugal sedimentation of product micelles and elution of proteins therefrom with buffer; (e) preparative isoelectric focusing (IEF) of product-entrapped proteins; and (f) glycerol gradient centrifugation of the fractions of peak GS-II activity from IEF. The procedure yields 300-fold enrichment of GS-II specific activity over that in isolated plasma membranes, and 5500-fold over that in the original homogenate. Out of approximately six principal polypeptides that occur after the product entrapment step, the glycerol gradient GS-II activity peak contains only two major polypeptides, one of 55 kDa and another of 70 kDa, plus minor amounts of one or two others whose distribution and occurrence indicate are not responsible for GS-II activity. Antisera against either the 55-kDa or the 70-kDa polypeptide adsorb more than 60% of the GS-II activity from a product-entrapped preparation. After native gel electrophoresis, GS-II activity is associated with a single protein band of very large molecular mass, whose principal components are the 55-kDa and 70-kDa polypeptides, accompanied by minor amounts of a few other polypeptides most of which do not occur in enzyme preparations purified by the previously described procedure. The 55-kDa but not the 70-kDa component can be labeled by ultraviolet irradiation of the plasma membranes in the presence of [alpha-32P]UDP-Glc under GS-II assay conditions. It seems likely, therefore, that the 55-kDa and 70-kDa polypeptides form a large catalytic complex of which the 55-kDa component is the UDP-Glc-binding subunit.

Inhibition of bioenergetics alters intracellular calcium, membrane composition, and fluidity in a neuronal cell line.

The effect of inhibited bioenergetics and ATP depletion on membrane composition and fluidity was examined in cultured neuroblastoma-glioma hybrid NG108-15 cells. Sodium cyanide (CN) and 2-deoxyglucose (2-DG) were used to block, oxidative phosphorylation and anaerobic glycolysis, respectively. Endoplasmic reticulum (ER) Ca(2+)-pump activity measured by 45Ca2+ uptake was > 92% inhibited in intact cells incubated with CN (1 mM) and 2-DG (20 mM) for 30 min. In addition, exposure of cells to CN and 2-DG caused a 134% increased release of isotopically labeled arachidonic acid (3H-AA) or arachidonate-derived metabolites from membranes. Removal of Ca2+ from the incubation medium ablated the CN/2-DG induced release of 3H-AA or its metabolites. Membrane fluidity of intact cells was measured by electron spin resonance spectroscopy using the spin label 12-doxyl stearic acid. The mean rotational correlation time (tau c) of the spin label increased 49% in CN/2-DG exposed cells compared to controls, indicating a decrease in membrane fluidity. These results show that depletion of cellular ATP results in inhibition of the ER Ca(2+)-pump, loss of AA from membranes, and decreased membrane fluidity. We propose that impaired bioenergetics can increase intracellular Ca2+ as a result of Ca(2+)-pump inhibition and thereby activate Ca(2+)-dependent phospholipases causing membrane effects. Since neurons derive energy predominantly from oxidative metabolism, ATP depletion during brain hypoxia may initiate a similar cytotoxic mechanism.

Diazepam effects on the P3 event-related potential.

Cerebral event-related potentials arise from synchronous neural activity associated with cognitive processing. The P3 is a late positive component that is related to task complexity and is directly proportional to stimulus evaluation time. P3 latency and amplitude were examined after oral administration of diazepam, a commonly prescribed medication with known cognitive side effects. Latency of the P3 was significantly increased after a single dose of both 5 and 10 mg of diazepam but not after placebo. P3 amplitude decreased slightly, but not significantly after 10 mg of diazepam. Changes in P3 occurred in the absence of significant changes in the preceding evoked waves (N1, P2). These findings should be considered when the P3 is obtained for clinical or research purposes from medicated patients.

Cloning, sequence analysis, and overexpression of Escherichia coli folK, the gene coding for 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase.

The gene coding for the Escherichia coli enzyme 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase has been cloned and sequenced. This gene, designated folK, codes for a protein of 159 amino acids, including an amino-terminal methionine. The protein was overexpressed in E. coli MC4100 by cloning the gene behind the lacUV5 promoter in a high-copy-number plasmid. The enzyme was purified to homogeneity. Amino-terminal analysis of the purified protein showed that the amino-terminal methionine had been removed. The compositional molecular mass (17,945 Da) was identical to the molecular mass determined by mass spectrometry. The enzyme was observed to have a large number of proline residues and migrated anomalously in sodium dodecyl sulfate-polyacrylamide gels, with an apparent molecular mass of 23,000 Da.