Pharmacokinetics of Glutamate–Oxaloacetate Transaminase and Glutamate–Pyruvate Transaminase and Their Blood Glutamate-Lowering Activity in Na?ve Rats

Traumatic brain injury (TBI) and stroke lead to elevated levels of glutamate in the brain that negatively affect the neurological outcomes in both animals and humans. Intravenous administration of glutamate-oxaloacetate transaminase (GOT) and glutamate-pyruvate transaminase (GPT) enzymes can be used to lower the blood glutamate levels and to improve the neurological outcome following TBI and stroke. The objective of this study was to analyze the pharmacokinetics and to determine the glutamate-lowering effects of GOT and GPT enzymes in na?ve rats. We determined the time course of serum GOT, GPT, and glutamate levels following a single intravenous administration of two different doses of each one of the studied enzymes. Forty-six male rats were randomly assigned into one of 5 treatment groups: saline (control), human GOT at dose 0.03 and 0.06?mg/kg and porcine GPT at dose 0.6 and 1.2?mg/kg. Blood samples were collected at baseline, 5?min, and 2, 4, 8, 12, and 24?h after the drug injection and GOT, GPT and glutamate levels were determined. The pharmacokinetics of both GOT and GPT followed one-compartment model, and both enzymes exhibited substantial glutamate-lowering effects following intravenous administration. Analysis of the pharmacokinetic data indicated that both enzymes were distributed predominantly in the blood (central circulation) and did not permeate to the peripheral organs and tissues. Several-hour delay was present between the time course of the enzyme levels and the glutamate-lowering effects (leading to clock-wise hysteresis on concentration-effect curves), apparently due to the time that is required to affect the pool of serum glutamate. We conclude that the interaction between the systemically-administered enzymes (GOT and GPT) and the glutamate takes place in the central circulation. Thus, glutamate-lowering effects of GOT and GPT apparently lead to redistribution of the excess glutamate from the brain's extracellular fluid into the blood and can reduce secondary brain injury due to glutamate neurotoxicity. The outcomes of this study regarding the pharmacokinetic and pharmacodynamic properties of the GOT and GPT enzymes will be subsequently verified in clinical studies that can lead to design of effective neuroprotective treatment strategies in patients with traumatic brain diseases and stroke.     

Independent and Coordinate Regulation of β1- and β2-Adrenergic Receptors in Rat C6 Glioma Cells

Abstract

Rat C6 glioma cells have both β₁- and β₂-adrenergic receptors in ～ 7:3 ratio. When the cells were exposed to the β-adrenergic agonist isoproterenol, there was a rapid sequestration of up to 50% of the surface receptor population over a 30-min period as measured by the loss of binding of the hydrophilic ligand [³H] CGP-12177 to intact cells. Using the β₂-selective antagonist CGP 20712A to quantify the proportion of the two subtypes, it was found that although both β₁ and β₂ receptors were sequestered, the latter were sequestered initially twice as fast as the former. More prolonged agonist exposure led to a down-regulation of ～ 90% of the total receptor population by 6 h as measured by the loss of binding of the more hydrophobic ligand [¹²⁵I] iodocyanopindolol to cell lysates. The two subtypes, however, underwent down-regulation with similar kinetics. Treatment of the cells with agents that raise cyclic AMP levels such as cholera toxin and forskolin resulted in a slower, but still coordinated down-regulation of both subtypes. Thus, there appears to be both independent and coordinate regulation of endogenous β₁-and β₂-adrenergic receptors in the same cell line.     

Methylglyoxal Causes Swelling and Activation of a Volume-Sensitive Anion Conductance in Rat Pancreatic β-Cells

Membrane potential and whole-cell current were studied in rat pancreatic β-cells using the `perforated patch' technique and cell volume measured by a video-imaging method. Exposure of β-cells to the α-ketoaldehyde methylglyoxal (1 mm) resulted in depolarization and electrical activity. In cells voltage-clamped at −70 mV, this effect was accompanied by the development of inward current noise. In voltage-pulse experiments, methylglyoxal activated an outwardly rectifying conductance which was virtually identical to the volume-sensitive anion conductance previously described in these cells. Two inhibitors of this conductance, 4,4′-dithiocyanatostilbene-2,2′-disulfonic acid (DIDS) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), also inhibited the depolarization and inward current evoked by methylglyoxal. Methylglyoxal increased β-cell volume to a relative value of 1.33 after 10 min with a gradual return towards basal levels following withdrawal of the α-ketoaldehyde. None of the effects of methylglyoxal was observed in response to t-butylglyoxal which, unlike methylglyoxal, is a poor substrate for the glyoxalase pathway. Methylglyoxal had no apparent effect on β-cell K⁺ channel activity. It is suggested that the metabolism of methylglyoxal to d-lactate causes β-cell swelling and activation of the volume-sensitive anion channel, leading to depolarization. These findings could be relevant to the stimulatory action of d-glucose, the metabolism of which generates significant quantities of l-lactate. Received: 15 May 1998/Revised: 25 September 1998     

Use of Naïve Bayes Classifier to Assess the Effects of Antipsychotic Agents on Brain Electrical Activity Parameters in Rats

Journal of Evolutionary Biochemistry and Physiology - Research and development of novel methods to determine the effects of antipsychotic agents is an important challenge for experimental...     

Role of Alveolar β2-Adrenergic Receptors on Lung Fluid Clearance and Exercise Ventilation in Healthy Humans

Background

In experimental conditions alveolar fluid clearance is controlled by alveolar β₂-adrenergic receptors. We hypothesized that if this occurs in humans, then non-selective β-blockers should reduce the membrane diffusing capacity (D_M), an index of lung interstitial fluid homeostasis. Moreover, we wondered whether this effect is potentiated by saline solution infusion, an intervention expected to cause interstitial lung edema. Since fluid retention within the lungs might trigger excessive ventilation during exercise, we also hypothesized that after the β₂-blockade ventilation increased in excess to CO₂ output and this was further enhanced by interstitial edema.

Methods and Results

22 healthy males took part in the study. On day 1, spirometry, lung diffusion for carbon monoxide (DLCO) including its subcomponents D_M and capillary volume (V_Cap), and cardiopulmonary exercise test were performed. On day 2, these tests were repeated after rapid 25 ml/kg saline infusion. Then, in random order 11 subjects were assigned to oral treatment with Carvedilol (CARV) and 11 to Bisoprolol (BISOPR). When heart rate fell at least by 10 beats·min⁻¹, the tests were repeated before (day 3) and after saline infusion (day 4). CARV but not BISOPR, decreased D_M (−13±7%, p = 0.001) and increased V_Cap (+20±22%, p = 0.016) and VE/VCO₂ slope (+12±8%, p<0.01). These changes further increased after saline: −18±13% for D_M (p<0.01), +44±28% for V_Cap (p<0.001), and +20±10% for VE/VCO₂ slope (p<0.001).

Conclusions

These findings support the hypothesis that in humans in vivo the β₂-alveolar receptors contribute to control alveolar fluid clearance and that interstitial lung fluid may trigger exercise hyperventilation.     

The Arginine of the DRY Motif in Transmembrane Segment III Functions as a Balancing Micro-switch in the Activation of the β2-Adrenergic Receptor

Recent high resolution x-ray structures of the β2-adrenergic receptor confirmed a close salt-bridge interaction between the suspected micro-switch residue ArgIII:26 (Arg3.50) and the neighboring AspIII:25 (Asp3.49). However, neither the expected "ionic lock" interactions between ArgIII:26 and GluVI:-06 (Glu6.30) in the inactive conformation nor the interaction with TyrV:24 (Tyr5.58) in the active conformation were observed in the x-ray structures. Here we find through molecular dynamics simulations, after removal of the stabilizing T4 lysozyme, that the expected salt bridge between ArgIII:26 and GluVI:-06 does form relatively easily in the inactive receptor conformation. Moreover, mutational analysis of GluVI:-06 in TM-VI and the neighboring AspIII:25 in TM-III demonstrated that these two residues do function as locks for the inactive receptor conformation as we observed increased G(s) signaling, arrestin mobilization, and internalization upon alanine substitutions. Conversely, TyrV:24 appears to play a role in stabilizing the active receptor conformation as loss of function of G(s) signaling, arrestin mobilization, and receptor internalization was observed upon alanine substitution of TyrV:24. The loss of function of the TyrV:24 mutant could partly be rescued by alanine substitution of either AspIII:25 or GluVI:-06 in the double mutants. Surprisingly, removal of the side chain of the ArgIII:26 micro-switch itself had no effect on G(s) signaling and internalization and only reduced arrestin mobilization slightly. It is suggested that ArgIII:26 is equally important for stabilizing the inactive and the active conformation through interaction with key residues in TM-III, -V, and -VI, but that the ArgIII:26 micro-switch residue itself apparently is not essential for the actual G protein activation.     

The Role of Interchain Heterodisulfide Formation in Activation of the Human Common β and Mouse βIL-3 Receptors

The cytokines, interleukin-3 (IL-3), interleukin-5 (IL-5), and granulocyte-macrophage colony-stimulating factor (GM-CSF), exhibit overlapping activities in the regulation of hematopoietic cells. In humans, the common β (βc) receptor is shared by the three cytokines and functions together with cytokine-specific α subunits in signaling. A widely accepted hypothesis is that receptor activation requires heterodisulfide formation between the domain 1 D-E loop disulfide in human βc (hβc) and unidentified cysteine residues in the N-terminal domains of the α receptors. Since the development of this hypothesis, new data have been obtained showing that domain 1 of hβc is part of the cytokine binding epitope of this receptor and that an IL-3Rα isoform lacking the N-terminal Ig-like domain (the “SP2” isoform) is competent for signaling. We therefore investigated whether distortion of the domain 1-domain 4 ligand-binding epitope in hβc and the related mouse receptor, β_IL-3, could account for the loss of receptor signaling when the domain 1 D-E loop disulfide is disrupted. Indeed, mutation of the disulfide in hβc led to both a complete loss of high affinity binding with the human IL-3Rα SP2 isoform and of downstream signaling. Mutation of the orthologous residues in the mouse IL-3-specific receptor, β_IL-3, not only precluded direct binding of mouse IL-3 but also resulted in complete loss of high affinity binding and signaling with the mouse IL-3Rα SP2 isoform. Our data are most consistent with a role for the domain 1 D-E loop disulfide of hβc and β_IL-3 in maintaining the precise positions of ligand-binding residues necessary for normal high affinity binding and signaling.     

LPS Exposure Increases Maternal Corticosterone Levels,Causes Placental Injury and Increases IL-1Β Levels in Adult Rat Offspring: Relevance to Autism

Maternal immune activation can induce neuropsychiatric disorders, such as autism and schizophrenia. Previous investigations by our group have shown that prenatal treatment of rats on gestation day 9.5 with lipopolysaccharide (LPS; 100 μg/kg, intraperitoneally), which mimics infections by gram-negative bacteria, induced autism-like behavior in male rats, including impaired communication and socialization and induced repetitive/restricted behavior. However, the behavior of female rats was unchanged. Little is known about how LPS-induced changes in the pregnant dam subsequently affect the developing fetus and the fetal immune system. The present study evaluated the hypothalamic-pituitary-adrenal (HPA) axis activity, the placental tissue and the reproductive performance of pregnant Wistar rats exposed to LPS. In the adult offspring, we evaluated the HPA axis and pro-inflammatory cytokine levels with or without a LPS challenge. LPS exposure increased maternal serum corticosterone levels, injured placental tissue and led to higher post-implantation loss, resulting in fewer live fetuses. The HPA axis was not affected in adult offspring. However, prenatal LPS exposure increased IL-1β serum levels, revealing that prenatal LPS exposure modified the immune response to a LPS challenge in adulthood. Increased IL-1β levels have been reported in several autistic patients. Together with our previous studies, our model induced autistic-like behavioral and immune disturbances in childhood and adulthood, indicating that it is a robust rat model of autism.     

The Relationship of Children’s Intelligence Quotient and Blood Lead and Zinc Levels: a Meta-analysis and System Review

This study aimed to evaluate the concentrations of copper, iron, and selenium in elderly people with Alzheimer disease (AD), comparing the same parameters in a paired group of healthy people, in order to verify if the amount of these metals may influence the cognitive impairment progression. Patients’ cognitive impairment was evaluated by Clinical Dementia Rating (CDR). The elementary quantification of erythrocytes was performed by inductively coupled plasma mass spectrometry technique. The statistical analyses were carried out by SPSS software 20.0 version, employing Shapiro-Wilk, Wilcoxon, Kruskall-Wallis, and Spearman correlation tests, considering significant results of p < 0.05. The sample was composed of 34% (n = 11) of women and 66% (n = 21) of men in each group. The AD group was characterized by a higher concentration of copper (p < 0.0001) and iron (p < 0.0001); however, there is no significant difference in selenium level. The analyses of the metal levels in different stages of AD were not significant in CDR-1, however in CDR-2 and CDR-3, elevated levels of copper and iron were observed; in CDR-3 patients, the level of selenium was lower (p < 0.008) compared to that of healthy controls. Patients with Alzheimer disease studied present increase in biometal blood levels, especially of copper and iron, and such increase can be different according to the disease stage and can cause more impairment cognitive functions in AD.     

NR2B-Containing NMDA Receptors Expression and Their Relationship to Apoptosis in Hippocampus of Alzheimer’s Disease-Like Rats

Although studies have shown that excitotoxicity mediated by N-methyl-D-aspartate receptors (NMDARs, NR) plays a prominent role in Alzheimer's disease (AD), the precise expression patterns of NMDARs and their relationship to apoptosis in AD have not been clearly established. In this study, we used Abeta (Aβ) 1-40 and AlCl(3) to establish AD rat model. The behavioral changes were detected by morris water maze and step-down test. The hippocampal amyloid deposition and pathological changes were determined by congo red and hematoxylin-eosin staining. Immunohistochemistry was used to detect expression of NR1, NR2A and NR2B, and TUNEL staining was used to detect apoptosis. Results showed that water maze testing escape latency of AD-like rats was prolonged significantly. Reaction time, basal number of errors, and number of errors of step-down test were increased significantly; latency period of step-down test was shortened significantly in AD-like rats. Amyloid substance deposition and obvious damage changes could be seen in hippocampus of AD-like rats. These results suggested that AD rat model could be successfully established by Aβ1-40 and AlCl(3). Results also showed that expression of NR1 and NR2B were significantly increased, but expression of NR2A had no significant change, in AD-like rat hippocampus. Meanwhile, apoptotic cells were significantly increased in AD-like rat hippocampus, especially in CA1 subfield and followed by dentate gyrus and CA3 subfield. These results implied that NR2B-, not NR2A-, containing NMDARs showed pathological high expression in AD-like rat hippocampus. This pathological high expression with apoptosis and selective vulnerability of hippocampus might be exist a specific relationship.     

Treatment of Na?ve Patients with Chronic Hepatitis C Genotypes 2 and 3 with Pegylated Interferon Alpha and Ribavirin in a Real World Setting: Relevance for the New Era of DAA

Evidence based clinical guidelines are implemented to treat patients efficiently that include efficacy, tolerability but also health economic considerations. This is of particular relevance to the new direct acting antiviral agents that have revolutionized treatment of chronic hepatitis C. For hepatitis C genotypes 2/3 interferon free treatment is already available with sofosbuvir plus ribavirin. However, treatment with sofosbuvir-based regimens is 10–20 times more expensive compared to pegylated interferon alfa and ribavirin (PegIFN/RBV). It has to be discussed if PegIFN/RBV is still an option for easy to treat patients. We assessed the treatment of patients with chronic hepatitis C genotypes 2/3 with PegIFN/RBV in a real world setting according to the latest German guidelines. Overall, 1006 patients were recruited into a prospective patient registry with 959 having started treatment. The intention-to-treat analysis showed poor SVR (GT2 61%, GT3 47%) while patients with adherence had excellent SVR in the per protocol analysis (GT2 96%, GT3 90%). According to guidelines, 283 patients were candidates for shorter treatment duration, namely a treatment of 16 weeks (baseline HCV-RNA <800.000 IU/mL, no cirrhosis and RVR). However, 65% of these easy to treat patients have been treated longer than recommended that resulted in higher costs but not higher SVR rates. In conclusion, treatment with PegIFN/RBV in a real world setting can be highly effective yet similar effective than PegIFN± sofosbuvir/RBV in well-selected naïve G2/3 patients. Full adherence to guidelines could be further improved, because it would be important in the new era with DAA, especially to safe resources.     

Amino acid consumption in naïve and recombinant CHO cell cultures: producers of a monoclonal antibody

Most commercial media for mammalian cell culture are designed to satisfy the amino acid requirements for cell growth, but not necessarily those for recombinant protein production. In this study, we analyze the amino acid consumption pattern in naïve and recombinant Chinese hamster ovary (CHO) cell cultures. The recombinant model we chose was a CHO-S cell line engineered to produce a monoclonal antibody. We report the cell concentration, product concentration, and amino acid concentration profiles in naïve and recombinant cell cultures growing in CD OptiCHO™ medium with or without amino acid supplementation with a commercial supplement (CHO CD EfficientFeed™ B). We quantify and discuss the amino acid demands due to cell growth and recombinant protein production during long term fed batch cultivation protocols. We confirmed that a group of five amino acids, constituting the highest mass fraction of the product, shows the highest depletion rates and could become limiting for product expression. In our experiments, alanine, a non-important mass constituent of the product, is in high demand during recombinant protein production. Evaluation of specific amino acid demands could be of great help in the design of feeding/supplementation strategies for recombinant mammalian cell cultures.     

The Effect of “Ferrodex”, Vitamin A and “Rovimix β-Carotene” on the Iron and Copper Levels in Blood Plasma of Calves

In 8 Versuchen wurde der Gärungsverlauf bei der Silierung von nitratarmem Grünfutter von Welschem Weidelgras, Knaulgras und Gras‐Leguminosen‐Gemenge geprüft. Aus den Ergebnissen geht hervor, daß bei Fehlen von Nitrat im Grünfutter bereits zu Gärbeginn Buttersäure entsteht, parallel zur Milchsäuregärung, auch in leicht vergärbarem Grünfutter. Diese frühzeitige Buttersäurebildung ist mit dem Fehlen von Nitrat als natürlicher Clostridieninhibitor zu erklären. Die Clostridienentwicklung verläuft zu Gärbeginn demnach wesentlich schneller als bisher angenommen wurde. Offensichtlich dienen leicht lösliche Kohlenhydrate als Substrat für die Buttersäurebildung. In nitratarmem Grünfutter werden deshalb Clostridien als Nahrungskonkurrenten für die Milchsäurebakterien wirksam. Aminosäuren werden zu Gärbeginn nicht abgebaut. Trotz z.T. hoher Buttersäuregehalte sind die Ammoniakgehalte gering. Höhere Homologe der Buttersäure fehlen. Die Milchsäuregärung erreicht trotz hoher Zuckergehalte im Grünfutter meist nur ein begrenztes Ausmaß. Es werden Unterschiede in der Vergärbarkeit der Kohlen‐hydratfraktion zwischen den Gräsern angenommen.     

Cytokine and Antibody Responses to Plasmodium falciparum in Na?ve Individuals during a First Malaria Episode: Effect of Age and Malaria Exposure

Age- and exposure-dependent immune responses during a malaria episode may be key to understanding the role of these factors in the acquisition of immunity to malaria. Plasma/serum samples collected from naïve Mozambican children (n = 48), European adults (naïve travelers, n = 22; expatriates with few prior malaria exposures, n = 15) and Mozambican adults with long-life malaria exposure (n = 99) during and after a malaria episode were analyzed for IgG against merozoite proteins by Luminex and against infected erythrocytes by flow cytometry. Cytokines and chemokines were analyzed in plasmas/sera by suspension array technology. No differences were detected between children and adults with a primary infection, with the exception of higher IgG levels against 3D7 MSP-1₄₂ (P = 0.030) and a P. falciparum isolate (P = 0.002), as well as higher IL-12 (P = 0.020) in children compared to other groups. Compared to malaria-exposed adults, children, travelers and expatriates had higher concentrations of IFN-γ (P≤0.0090), IL-2 (P≤0.0379) and IL-8 (P≤0.0233). Children also had higher IL-12 (P = 0.0001), IL-4 (P = 0.003), IL-1β (P = 0.024) and TNF (P = 0.006) levels compared to malaria-exposed adults. Although IL-12 was elevated in children, overall the data do not support a role of age in immune responses to a first malaria episode. A T_H1/pro-inflammatory response was the hallmark of non-immune subjects.     

Correction to: β-Asarone Regulates ER Stress and Autophagy Via Inhibition of the PERK/CHOP/Bcl-2/Beclin-1 Pathway in 6-OHDA-Induced Parkinsonian Rats

Neurochemical Research -     

Positive and Negative Coupling of the Metabotropic Glutamate Receptors to a G Protein–activated K+ Channel,GIRK, in Xenopus Oocytes

Metabotropic glutamate receptors (mGluRs) control intracellular signaling cascades through activation of G proteins. The inwardly rectifying K⁺ channel, GIRK, is activated by the βγ subunits of G_i proteins and is widely expressed in the brain. We investigated whether an interaction between mGluRs and GIRK is possible, using Xenopus oocytes expressing mGluRs and a cardiac/brain subunit of GIRK, GIRK1, with or without another brain subunit, GIRK2. mGluRs known to inhibit adenylyl cyclase (types 2, 3, 4, 6, and 7) activated the GIRK channel. The strongest response was observed with mGluR2; it was inhibited by pertussis toxin (PTX). This is consistent with the activation of GIRK by G_i/G_o-coupled receptors. In contrast, mGluR1a and mGluR5 receptors known to activate phospholipase C, presumably via G proteins of the G_q class, inhibited the channel''s activity. The inhibition was preceded by an initial weak activation, which was more prominent at higher levels of mGluR1a expression. The inhibition of GIRK activity by mGluR1a was suppressed by a broad-specificity protein kinase inhibitor, staurosporine, and by a specific protein kinase C (PKC) inhibitor, bis-indolylmaleimide, but not by PTX, Ca²⁺ chelation, or calphostin C. Thus, mGluR1a inhibits the GIRK channel primarily via a pathway involving activation of a PTX-insensitive G protein and, eventually, of a subtype of PKC, possibly PKC-μ. In contrast, the initial activation of GIRK1 caused by mGluR1a was suppressed by PTX but not by the protein kinase inhibitors. Thus, this activation probably results from a promiscuous coupling of mGluR1a to a G_i/G_o protein. The observed modulations may be involved in the mGluRs'' effects on neuronal excitability in the brain. Inhibition of GIRK by phospholipase C–activating mGluRs bears upon the problem of specificity of G protein (GIRK interaction) helping to explain why receptors coupled to G_q are inefficient in activating GIRK.