Native Enzyme Mobility Shift Assay (NEMSA): a new method for monitoring carboxyl group modification of carboxymethylcellulase from Aspergillus niger

A simple, sensitive, accurate and more informative assay for determining the number of modified groups during the course of carboxyl group modification is described. Monomeric carboxymethylcellulase (CMCase) from Aspergillus niger was modified by 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) in the presence of glycinamide. The different time-course aliquots were subjected to non-denaturing PAGE and the gel stained for CMCase activity. The number of carboxyl groups modified are directly read from the ladder of the enzyme bands developed at given time. This method showed that after 75 min of modification reaction there were five major species of modified CMCases in which 6 to 10 carboxyls were modified.     

Characterisation and Regional Localisation of Pre- and Postsynaptic Glycoproteins of the Chick Forebrain Showing Changed Fucose Incorporation Following Passive Avoidance Training

To identify those glycoproteins whose synthesis or modification is necessary for memory formation, we have studied the uptake of radiolabelled fucose into synaptic plasma membranes (SPMs) and postsynaptic densities (PSDs) derived from two specific left and right forebrain loci, at two different times after training of 1-day-old chicks on a one-trial passive avoidance learning task. To increase the reliability of the comparison, a double-labelling method was used. Tissue samples from intermediate medial hyperstriatum ventrale (IMHV) and lobus parolfactorius (LPO) were isolated at 6 and 24 h after training. At both times, training resulted in region-specific changes, both increases and decreases, in incorporated radioactivity into pre- and postsynaptic glycoproteins. After 6 h, there was a relative decline in incorporation into both SPMs and PSDs of the right IMHV of trained chicks, a decline that persisted in the PSDs until 24 h. A small decline in incorporation in SPMs from the right LPO of trained chicks at 6 h was reversed by 24 h, by which time there was a 64% increase in incorporation into SPMs and a 24% increase into PSDs of the left LPO. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis of left and right hemisphere samples containing LPO revealed that 6 h after training the main effect was presynaptic, including a reduction of incorporation into high molecular mass glycoproteins, of 150-180 kDa, and an increase in a lower molecular mass (41 kDa) fraction. By 24 h after training, a left hemisphere presynaptic glycoprotein of molecular mass approximately 50 kDa showed the biggest increase in fucosylation. In addition, a wide group of postsynaptic glycoproteins of both hemispheres, in the ranges 150-180, 100-120, and 33 kDa now showed increases in incorporation. Some other fractions showed decreases. These results are in accord with previous data on incorporation obtained using the amnesic agent 2-deoxygalactose. They also support the hypothesis that memory formation involves the strengthening of connections between pre- and postsynaptic neurons of the LPO by growth or modulation of pre- and postsynaptic structures.     

Antifertility effect of Andrographis paniculata (Nees) in male albino rat

Dry leaf powder of A. paniculata, when fed orally to male albino rats, at a dose level of 20 mg powder per day for 60 days, resulted in cessation of spermatogenesis, degenerative changes in the seminiferous tubules, regression of Leydig cells and regressive and/or degenerative changes in the epididymis, seminal vesicle, ventral prostate and coagulating gland. There was reduction in the weight and fluid content of the accessory glands. The treatment also resulted in accumulation of glycogen and cholesterol in the testis, and increased activities of lactate dehydrogenase in testis and alkaline phosphatase in testis and ventral prostate. The results suggest antispermatogenic and/or antiandrogenic effect of the plant.     

Efficacy of intra-lesional injections of meglumine antimoniate once a week vs. twice a week in the treatment of cutaneous leishmaniasis caused by L. tropica in Iran: A randomized controlled clinical trial

Treatment of Cutaneous leishmaniasis (CL) is based on using antimoniate derivatives; patients’ compliance for systemic injections is low due to the pain and systemic complications. In this randomized open trial, the efficacy of intra-lesional (IL) injections of meglumine antimoniate (MA) once a week vs. twice a week in the treatment of Anthrpoponothic CL caused by L. tropica was studied. Eligible volunteer patients were selected according to inclusion/exclusion criteria. The included patients were randomly allocated to receive IL-MA injections once a week or twice a week. The primary outcome was set as complete healing of the lesion(s), and defined as complete re-epithelialization and absence of induration in the lesions. A total of 180 parasitologicaly proven CL patients caused by L. tropica were recruited, 90 patients were treated with weekly IL-MA and 90 patients received IL-MA twice a week. The complete cure was 87.9% vs. 89.2% in the group received weekly and twice a week IL-MA injections, respectively (P = 0.808). Patients’ compliance was acceptable and side effects were limited to a few local allergic reactions to MA. Median time to healing was significantly shorter in patients who received IL-MA twice a week (median ± SE) 37±3.8, (CI: 29.6–44.4) days compared to whom received IL-MA once a week 60±2.3, (CI: 55.6–64.5) days (P< 0.001), however the number of injections was higher in group who received IL-MA twice a week (12 vs. 9 injections). In conclusion, the rate of cure in the group of CL patients with IL-MA twice a week was not significantly different from the group who received IL-MA once a week shorten, but the duration of healing was shorter in the group who received IL-MA twice a week while the group received more injections so is recommended to use IL-MA once a week due to the fact the compliance is acceptable with limited side effects.Clinical Trial Registration: IRCT20081130001475N13; https://en.irct.ir/.     

Use of response surface methodology to study the effect of media composition on aflatoxin production by Aspergillus flavus

Aflatoxins are one of the most important secondary metabolites. These extrolites are produced by a number of Aspergillus fungi. In this study, we demonstrate the effect of media components and enhanced aflatoxin yield shown by A. flavus using response surface methodology in response to different nutrients. Different components of a chemically defined media that influence the aflatoxin production were monitored using Plackett–Burman experimental design and further optimized by Box–Behnken factorial design of response surface methodology in liquid culture. Interactions were studied with five variables, namely sorbitol, fructose, ammonium sulfate, KH₂PO₄, and MgSO₄.7H₂O. Maximum aflatoxin production was envisaged in medium containing 4.94 g/l sorbitol, 5.56 g/l fructose, 0.62 g/l ammonium sulfate, 1.33 g/l KH₂PO₄, and 0.65 g/l MgSO₄·7H₂O using response surface plots and the point prediction tool of the DESIGN EXPERT 8.1.0 (Stat-Ease, USA) software. However, a production of 5.25 μg/ml aflatoxin production was obtained, which was in agreement with the prediction observed in verification experiment. The other component (MgSO₄.7H₂O) was found to be an insignificant variable.     

A cyclodextrin host-guest recognition approach to an electrochemical sensor for simultaneous quantification of serotonin and dopamine

An electrochemical sensor for simultaneous quantification of serotonin (5-hydroxytryptamine, 5-HT) and dopamine (DA) using a β-cyclodextrin/poly(N-acetylaniline)/carbon nanotube composite modified carbon paste electrode has been developed. Synergistic effect of multi-walled carbon nanotube (MWCNT) in addition to the pre-concentrating effect of β-cyclodextrin (β-CD) as well as its different inclusion complex stability with 5-HT and DA was used to construct an electrochemical sensor for quantification of these important neurotransmitters. The overlapping anodic peaks of 5-HT and DA at 428 mV on bare electrode resolved in two well-defined voltammetric peaks at 202 and 363 mV vs. Ag/AgCl respectively. The oxidation mechanism of 5-HT and DA on the surface of the electrode was investigated by cyclic voltammetry and it was found that the electrode processes are pH dependent and electrochemical oxidation of 5-HT is totally irreversible while the electrode gave a more reversible process to DA. Under optimized conditions, linear calibration curves were obtained in the range of about 4-200 μM with a detection limits down to sub-μM levels (S/N=3) after 20-s accumulation, for both. The proposed sensor was shown to be remarkably selective for 5-HT and DA in matrices containing different species including ascorbic acid and uric acid. The suitability of the developed method was tested for the determination of 5-HT and DA in the Randox Synthetic Plasma samples and acceptable recoveries were obtained for a set of spiked samples.     

Decreased Expression of Transporters Reduces Folate Uptake across Renal Absorptive Surfaces in Experimental Alcoholism

In this study, we examined the mechanistic insights of folate reabsorption during alcoholism, considering enhanced renal excretion as one of the major contributing factors to alcohol-induced folate deficiency. Male Wistar rats were fed 1g/kg body weight/day ethanol (20% solution) orally for 3 months. The results on characterization of the folate transport system in renal basolateral membrane (BLM) suggested it to be a carrier-mediated, acidic pH-dependent and saturable one. Chronic ethanol feeding decreased the uptake mainly by increasing the K _m and decreasing the V _max of the transport process at the BLM surface. At the molecular level, reduced folate transport activity in renal tissue during chronic ethanol ingestion was attributable to decreased expression of reduced folate carrier (RFC) and folate binding protein (FBP). Antibodies against RFC protein revealed a parallel change in RFC expression in both brush border and BLM surfaces during chronic alcoholism. Such findings highlight the role of downregulation of RFC and FBP expression and provide mechanistic insight into the observed reduced folate transport efficiency at renal absorptive surfaces in alcoholism, which may result in low blood folate levels commonly observed in alcoholics.