Kinetic constants for the inhibition of camel retinal acetylcholinesterase by the carbamate insecticide lannate

We have designed this study to determine various kinetic parameters of camel retinal membrane‐bound acetylcholinesterase (AChE; EC 3.1.1.7) inhibition by carbamate insecticide lannate [methyl N‐{{(methylamino)carbonyl}oxy} ethanimidothioate]. All these kinetic constants were derived by simple graphical methods. The value of kinetic parameters was estimated as follows: 0.061 (μM)⁻¹, 1.14 (μM)⁻¹, 0.216 μM, 0.016 min⁻¹, 0.0741 (μM min)⁻¹, 0.746 μM, and 4.42 μM for velocity constant (K_v), new inhibition constant (K_nic), dissociation constant (K_d), carbamylation rate constant (k_2c), overall carbamylation rate constant (k′2 ), 50% inhibition constant (K_I50), and 99% inhibition constant (K_I99), respectively. These unique methods may be used to estimate such kinetic parameters for time‐dependent inhibition of enzymes by variety of chemicals, insecticides, herbicides, and drugs. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 13: 41–46, 1999     

The Modulatory Effect of Metabotropic Glutamate Receptor Type-1α on Spike-Wave Discharges in WAG/Rij Rats

Modulatory function of metabotropic glutamate type 1 (mGlu1) receptors plays a crucial role in the pathophysiology of some neurological disorders, including schizophrenia and epilepsy. In this study, the expression of mGlu1α receptors in the thalamic nuclei was assessed during development of absence seizures in the WAG/Rij rats, a valid genetic animal model of absence epilepsy. In addition, the effect of pharmacological modulation of mGlu1α receptors in the laterodorsal (LD) nucleus of the thalamus on the characteristic features of bioelectrical brain activities in the WAG/Rij rats was assessed. The expression of mGlu1α receptors in the LD was assessed in four experimental groups of both WAG/Rij and Wistar rats with 2 and 6 months of age. Agonist and antagonist of mGlu1α receptors were infused in LD in the six months old WAG/Rij (epileptic) rats. The protein level of mGlu1α receptors in the thalamus of the 6-month-old WAG/Rij rats was lower than non-epileptic animals. In addition, the distribution of mGlu1α receptors in different thalamic nuclei was lower in the 6-month-old WAG/Rij compared to age-matched Wistar rats. The gene expression of mGlu1α receptor was also significantly lower in 6-month-old WAG/Rij rats in the LD compared to other animal groups. The microinjection of mGlu1α receptors agonist and antagonist in the LD reduced the duration of spike-wave discharges (SWDs) and increased the amplitude and duration of SWDs, respectively, in 6-month-old WAG/Rij rats. The alterations of mGlu1α receptors expression in the thalamus of epileptic WAG/Rij rats as well as its modulatory effects in the generation of SWDs suggest the potential of mGlu1 receptors as a therapeutic target in absence epilepsy.     

Taste attractiveness of free amino acids for juveniles of Persian sturgeon <Emphasis Type="Italic">Acipenser persicus</Emphasis>

Taste preferences towards 20 free amino acids (L-isomers, 0.1–0.0001 M) were determined in juveniles of Persian sturgeon Acipenser persicus. It was found that most amino acids (16) had a positive effect on extraoral gustatory reception (increased the frequency of catching artificial pellets by fishes). The most efficient amino acids were the following: threonine, histidine, arginine, asparagine, phenylalanine, cysteine, glutamine, and glycine. Fifteen amino acids were efficient for intraoral gustatory receptors: serine, arginine, cysteine, histidine, alanine, and some others. The presence of these amino acids in pellets increased the consumption. Amino acids decreasing the frequency of catching or consumption of pellets were not found. Highly significant positive correlation was found between the amino acid ranges mediated by the extraoral and intraoral gustatory reception. The fishes demonstrated maximum sensitivity to aspartic acid, 0.01 and 0.001 M, respectively, for intraoral and extraoral gustatory systems. The threshold concentrations of arginine and glycine were higher for the intraoral gustatory system (0.01 M) than for the extraoral one (0.1 M). The comparison of Persian sturgeon with sturgeon fishes (Russian sturgeon A. gueldenstaedtii, Siberian sturgeon A, baerii, and starred sturgeon A. stellatus) studied earlier confirmed high species specificity of intraoral taste preferences in representatives of Acipenser genus. Species specificity of extraoral taste ranges was less pronounced.     

The role of caveolae and caveolin 1 in calcium handling in pacing and contraction of mouse intestine

In mouse intestine, caveolae and caveolin‐1 (Cav‐1) are present in smooth muscle (responsible for executing contractions) and in interstitial cells of Cajal (ICC; responsible for pacing contractions). We found that a number of calcium handling/dependent molecules are associated with caveolae, including L‐type Ca²⁺ channels, Na⁺‐Ca²⁺ exchanger type 1 (NCX1), plasma membrane Ca²⁺ pumps and neural nitric oxide synthase (nNOS), and that caveolae are close to the peripheral endo‐sarcoplasmic reticulum (ER‐SR). Also we found that this assemblage may account for recycling of calcium from caveolar domains to SR through L‐type Ca ⁺ channels to sustain pacing and contractions. Here we test this hypothesis further comparing pacing and contractions under various conditions in longitudinal muscle of Cav‐1 knockout mice (lacking caveolae) and in their genetic controls. We used a procedure in which pacing frequencies (indicative of functioning of ICC) and contraction amplitudes (indicative of functioning of smooth muscle) were studied in calcium‐free media with 100 mM ethylene glycol tetra‐acetic acid (EGTA). The absence of caveolae in ICC inhibited the ability of ICC to maintain frequencies of contraction in the calcium‐free medium by reducing recycling of calcium from caveolar plasma membrane to SR when the calcium stores were initially full. This recycling to ICC involved primarily L‐type Ca²⁺ channels; i.e. pacing frequencies were enhanced by opening and inhibited by closing these channels. However, when these stores were depleted by block of the sarco/endoplasmic reticulum Ca²⁺‐ATPase (SERCA) pump or calcium release was activated by carbachol, the absence of Cav‐1 or caveolae had little or no effect. The absence of caveolae had little impact on contraction amplitudes, indicative of recycling of calcium to SR in smooth muscle. However, the absence of caveolae slowed the rate of loss of calcium from SR under some conditions in both ICC and smooth muscle, which may reflect the loss of proximity to store operated Ca channels. We found evidence that these channels were associated with Cav‐1. These changes were all consistent with the hypothesis that a reduction of the extracellular calcium associated with caveolae in ICC of the myenteric plexus, the state of L‐type Ca²⁺ channels or an increase in the distance between caveolae and SR affected calcium handling.     

Comparison of Beta-value and M-value methods for quantifying methylation levels by microarray analysis

Background  

High-throughput profiling of DNA methylation status of CpG islands is crucial to understand the epigenetic regulation of genes. The microarray-based Infinium methylation assay by Illumina is one platform for low-cost high-throughput methylation profiling. Both Beta-value and M-value statistics have been used as metrics to measure methylation levels. However, there are no detailed studies of their relations and their strengths and limitations.     

Intraperitoneal Aminoguanidine Improves Sciatic Nerve Ischemia–Reperfusion Injury in Male Sprague-Dawley Rats

The present work was designed to investigate the potential protective effects of post-ischemic treatment with aminoguanidine (AG) on sciatic nerve ischemia/reperfusion (I/R) injury in rat. Seventy-two rats were divided into 12 groups (n = 6). We used ischemia model in these groups by occluding the right common iliac and femoral arteries for 3 h with a silk suture 6-0 using slipknot technique. Treatment groups (2, 4, 6, 8, 10, and 12) received 150 mg/kg AG intraperitoneally 24 h after induction of ischemia. After certain time intervals of reperfusion (2, 4, 7, 14, and 28 days), the function of the hind limb was assessed using behavioral scores based on gait, racing reflex, toe spread, pinch sensitivity, paw position, and grasp. After euthanasia, sciatic nerves were removed at the end of reperfusion times and sections were cut at 5 μm, then were stained for light microscopy studies and graded for ischemic fiber degeneration (IFD), edema, and apoptosis. Maximal behavioral deficit occurred at 7 days of reperfusion. The comparison of behavioral score pertaining to the control and AG groups revealed significant differences and showed also a better time course in recovery (P < 0.05). Other than 3 and 4 groups, the amount of edema in AG treatment groups showed significant differences compared with control groups (P < 0.05). IFD was also significantly decreased in the AG treatment groups than controls. Most importantly, I/R-induced apoptosis were improved significantly on the 4th, 7^th, and 14th days of reperfusion in AG-treated groups compared to controls. In conclusion, our findings suggest that post-ischemic administration of AG exhibits protective effect against sciatic nerve I/R injury.     

Lectins influence chondrogenesis and osteogenesis in limb bud mesenchymal cells

The role of cell surface glycoproteins in cell behavior can be characterized by their interactions with plant lectins. This study was designed to identify the effects of lectins on chondrogenesis and osteogenesis in limb bud mesenchymal cells in vitro. Limb bud mesenchymal cells from mouse embryos were cultured in high-density micromass culture. Wheat germ agglutinin (WGA), concanavalin A (ConA), peanut agglutinin (PNA), Dolichos biflorus agglutinin (DBA) and Ricinus communis agglutinin (RCA) were added separately to the culture media. Cells were cultured for 5 or 9 days, and cell viability was assayed by neutral red on day 5. The micromasses were stained with alcian blue, alizarin red S and Von Kossa stains, and alkaline phosphatase assays were also done. Dolichos biflorus agglutinin induced an increase in chondrogenesis, calcium precipitation and proteoglycan production. ConA and PNA did not affect chondrocyte differentiation but induced chondrocytes to produce more proteoglycan. Wheat germ agglutinin reduced chondrification and ossification but induced mesenchymal cells to store lipid droplets. Ricinus communis agglutinin 1 was toxic and significantly reduced cell survival. In conclusion, DBA was the most effective inducer of ossification and chondrification. Wheat germ agglutinin induced adipogenesis instead. These assays showed that lectins play important roles in limb bud development.     

Aminoguanidine Changes Hippocampal Expression of Apoptosis-Related Genes,Improves Passive Avoidance Learning and Memory in Streptozotocin-Induced Diabetic Rats

Cognitive dysfunction occurs in patients with diabetes mellitus. The objective of this study was to examine whether bilateral intrahippocampal CA1 (intra-CA1) injection of aminoguanidine (AG) can either affect the Bcl-2 family gene expression or reduce the diabetic imposing abnormalities of passive avoidance learning (PAL) and memory. Rats were divided into five groups: control (C), control treated with normal saline (CS), control treated with AG (S-AG), diabetics (D), and diabetics treated with AG (D-AG). Diabetes mellitus was induced by a single intraperitoneal injection of streptozotocin (STZ) (50 mg/kg). AG (30 μg/rat) or vehicle was administered intra-CA1 bilaterally at the onset of hyperglycemia. PAL was assessed 7 weeks later. Animals were killed, and hippocampus was dissected following the behavioral test. The expressions of Bax, Bcl-2, and Bcl-xl mRNAs were measured using semiquantitative RT-PCR technique. The result of passive avoidance task showed that AG significantly improved the cognitive performance in diabetic rats. Moreover, AG treatment decreased the levels of Bcl-2 and Bcl-x_L expressions in diabetic group. The ratio of Bax/Bcl-2 and Bax/Bcl-x_L decreased significantly in AG-treated diabetic animals. In conclusion, initial treatment with AG by intra-CA1 micro-injection improves the impaired passive avoidance task in STZ-induced diabetic rats which may be related to the decreased Bax/Bcl-2 and Bax/Bcl-x_L ratios.