Carbachol can potentiate N-methyl-D-aspartate responses in the rat hippocampus by a staurosporine and thapsigargin-insensitive mechanism

Experiments were performed to investigate the mechanism underlying the potentiation of N-methyl-D-aspartate (NMDA) responses by carbachol (CCh) in the CA1 region of rat hippocampal slices. CCh (300 nM) potentiated responses to NMDA, but not to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), in a readily reversible manner. Potentiation occurred in slices treated with 200 nM tetrodotoxin and perfused with Mg(2+)-free medium. It also occurred in slices treated with either staurosporine (1 microM), which is a potent inhibitor of a variety of protein kinases including protein kinase C (PKC), or thapsigargin (10 microM), which depletes intracellular Ca2+ stores by preventing their refilling. However, CCh-induced potentiation was abolished in slices perfused with Ca(2+)-free medium. These data suggest that low concentrations of CCh can acutely potentiate NMDA responses in the hippocampus by a Ca(2+)-sensitive process that is probably independent of both the activation of PKC and the release of Ca2+ from intracellular stores. This mechanism is similar to that underlying the potentiation of NMDA responses by the metabotropic glutamate receptor (mGluR) agonist, aminocyclopentane-1S,3R-dicarboxylic acid (1S,3R-ACPD).     

An expert PID controller uses refined ziegler and nichols rules and fuzzy logic ideas

This article proposes a scheme for the on-line adjustment of three mode controller settings based on experimental measurements of closed-loop performance. It uses a recently developed heuristic tuning procedure to identify estimated process parameters. This method may give rise to conflicting estimates. Fuzzy Set theory is applied to manage the situation in terms of a fuzzy conjunction to combine the various estimates. PID control was chosen because of its wide use in the industrial environment due to driving simplicity and robustness. The article shows design, development, and computer simulation aspects.     

Repair of DNA methylphosphotriesters through a metalloactivated cysteine nucleophile

The Escherichia coli Ada protein repairs methylphosphotriesters in DNA by direct, irreversible methyl transfer to one of its own cysteines. Upon methyl transfer, Ada acquires the ability to bind specific DNA sequences and thereby to induce genes that confer resistance to methylating agents. The amino-terminal domain of Ada, which comprises the methylphosphotriester repair and sequence-specific DNA binding elements, contains a tightly bound zinc ion. Analysis of the zinc binding site by cadmium-113 nuclear magnetic resonance and site-directed mutagenesis revealed that zinc participates in the autocatalytic activation of the active site cysteine and may also function as a conformational switch.     

Assessment of the efficacy of a low temperature hydrogen peroxide gas plasma sterilization system

The STERRAD 100 sterilization system (Johnson & Johnson Medical Ltd) uses low temperature hydrogen peroxide gas plasma for sterilization of heat labile equipment. The efficacy of the machine was tested by contaminating a standard set of instruments with different organisms and using a filtration method to assess recovery of organisms from the wash fluids of instruments post-sterilization. Experiments were performed under clean (the organism only) and dirty (organism mixed with egg protein) conditions. A parallel study conducted using a 3M STERIVAC ethylene oxide sterilizer could not be completed owing to closure of the ethylene oxide plant. For sterilization of instruments with long and narrow lumens, hydrogen peroxide adaptors are necessary. The STERRAD 100 sterilizer can achieve effective sterilization of heat labile instruments with a reduction of 6 log10 cfu/mL of organisms tested. This method has the advantages over ethylene oxide sterilization of safety, ease of maintenance and no requirement for aeration time.     

Environmental assessment of an uncontrolled sanitary landfill,Pocos de Caldas,Brazil

There are more than 20,000 uncontrolled sanitary landfill areas in Brazil. The paper reports an integrated study undertaken to develop an environmental assessment of the uncontrolled sanitary landfill area of the city of Po?os de Caldas, State of Minas Gerais, Brazil. A number of remedial alternatives were evaluated. The USGS modular 3D finite difference groundwater flow model (Modflow) and Modular 3D Finite Difference Mass Transport Model (MT3D) software were used to simulate groundwater flow and contaminant transport modelling, as well as to predict changes due to the proposed remediation measures. The remedial alternatives considered for normal conditions from 20 to 40 year periods were waste removal, vertical cut-off barriers, extraction wells and top capping. The results indicated that extraction wells and waste removal are the best alternatives because they significantly reduce both the extension and levels of the contamination plume even after 5 years. The extraction well proved a less expensive remedial alternative than waste removal.      

Prevalence of serologic markers of HBV, HDV, HCV and HIV in non-injection drug users compared to injection drug users in Gran Canaria, Spain

Von Hippel-Lindau disease (VHL) is an autosomal dominant tumour syndrome caused by germline mutations of the VHL tumour suppressor gene located on chromosome 3p25-26. In VHL tumours may occur in 14 different target organs, including the eye. Retinal angiomas are considered the first manifestation of VHL disease in 43% of cases, and the cumulative probability of developing a retinal angioma in one or both eyes rises during each decade of life, reaching 80% for patients over 80 years old. Since 1976 patients with VHL at the University Hospital of Utrecht and their at-risk relatives have been screened periodically by a multidisciplinary team. Long-term follow-up ophthalmological data were analysed with special attention to natural course and results of treatment. In addition, we looked for a genotype-phenotype correlation. Retinal angiomas were found in all families. In one large family with a missense mutation (V170D) of the VHL gene, in which the complete spectrum of visceral- and central nervous system (CNS) features of VHL is present, macular, parapapillary, optic disc and ora serrata angiomas were also found. In general, however, a clear-cut genotype-phenotype correlation could not be found. Only early detection and treatment of peripheral retinal angiomas can be expected to decrease the percentage of patients with decreased visual acuity. Therefore, early detection and treatment of these tumours is of paramount importance. Ophthalmological screening of patients and persons at risk should start as early as possible. In patients with apparently sporadic retinal angiomas it is advisable to perform germline DNA analysis, since the risk of developing VHL is high, especially if the angiomas are bilateral, or unilateral and multifocal, if the patient is young, or if there is a family history suggestive of VHL.     

Calcium additional to that bound to the transport sites is required for full activation of the sarcoplasmic reticulum Ca-ATPase from skeletal muscle

The sarcoplasmic reticulum Ca-ATPase is fully activated when approximately 1 microM [Ca2+] saturates the two transport sites; higher [Ca] inhibits the ATPase by competition of Ca-ATP with Mg-ATP as substrates. Here we describe a novel effect of EGTA and other chelators, raising the possibility of an additional activating effect of Ca in the sub- or low microM range. Sarcoplasmic reticulum membranes were isolated from rabbit skeletal muscles. The ATPase activity was measured after incubation at 37 degreesC in 3 mM ATP, 3 mM MgCl2, 50 mM MOPS-Tris (pH 7.2), 100 mM KCl, and variable CaCl2, EGTA and calcimycin. In the absence of added EGTA and Ca the ATPase activity is high due to contaminant Ca. The determination of the ATPase activity in the presence of increasing amounts of EGTA, without added Ca, yields a decreasing sigmoidal function. Ki ranged between 20 and 100 microM, depending on the enzyme concentration. Pi production is linear with time for several [EGTA] yielding suboptimal ATPase activities, which are inhibited by thapsigargin. These suboptimal Ca-ATPase activities are inhibited by preincubation of the enzyme in EGTA, at pH 7.2. This effect increases upon increasing EGTA concentration and preincubation time. The inhibitory effect of the previous exposure of the enzyme to EGTA is partially but significantly reverted by increasing [Ca2+] during incubations. Calcimycin and EDTA have similar effects as EGTA when added in preincubations. The effect of calcimycin is fully reverted by optimal [Ca2+] in incubations. The effects of EGTA, EDTA and calcimycin in preincubation are not additive. The results suggest that an additional calcium, lost during preincubations from a site with affinity near 1 microM, is necessary for full activation of the ATPase.     

Cleavage of Müllerian inhibiting substance activates antiproliferative effects in vivo

Müllerian inhibiting substance (MIS), an inhibitor of growth and development of the female reproductive ducts in male fetuses, requires precise proteolytic cleavage to yield its biologically active species. Human plasmin is now used to cleave and, thereby, activate immunoaffinity-purified recombinant human MIS at its monobasic arginine-serine site at residues 427-428. To avoid the need for exogenous enzymatic cleavage and to simplify purification, we created an arginine-arginine dibasic cleavage site (MIS RR) using site-directed mutagenesis to change the serine at position 428 (AGC) to an arginine (cGC). The mutant cDNA was then stably transfected into a MIS-responsive ocular melanoma cell line, OM431, followed by cloning for amplified expression to test its biological activity in vitro and in vivo. Media from each clone were assayed for production of MIS RR by a sensitive ELISA for holo-MIS, and high- and low-producing clones were selected for further study. Media from the highest MIS RR producer caused Müllerian duct regression in an organ culture bioassay. Other transfections were done with an empty vector (pcDNAI Neo) or a construct lacking the leader sequence and thus failing to secrete MIS, to serve as controls. The OM431 clones containing the MIS RR mutant were growth inhibited in monolayer culture. The high- and low-producing MIS RR OM431 clones, along with transfected OM431 controls, were injected into the tail veins of immunosuppressed severe combined immunodeficiency mice for in vivo analyses. Four to 6 weeks later, pulmonary metastases were counted in uniformly inflated lungs. OM431 clones containing the more easily cleaved MIS RR displayed a significant dose-dependent reduction in pulmonary metastases when compared to the lungs of animals given injections of OM431 clones containing empty vector, leaderless MIS, or wild-type MIS that requires activation by plasmin cleavage. Since the purification protocol of MIS RR is less complicated than that for wild-type MIS, which requires subsequent enzymatic activation, MIS RR can be used for scale-up production with increased yields for further therapeutic trials against MIS-sensitive tumors.     

Regional 5-hydroxyindoleacetic acid production in humans

Veno-arterial plasma concentration differences and regional organ plasma flows were used to quantify the relative amounts of 5-hydroxyindoleacetic acid (5-HIAA) contributed by various sites into the peripheral circulation. Positive venoarterial concentration gradients were found in the hepatosplanchnic, forearm, cardiac and jugular vessels in the healthy subjects. The renal circulation was determined to be the principal site of 5-HIAA clearance, extracting 18 +/- 2 nmol/min. The gut was the greatest contributor to the total 5-HIAA plasma pool with the relative contributions of the various organs being as follows: hepatosplanchnic organs 58%, skeletal muscle 26%, brain 6% and the heart 3%. The source of 5-HIAA stemming from these regional beds remains unknown, it may derive from serotonin taken up by and deaminated in ubiquitous endothelial cells, enterochromaffin cells of the gut, peripheral serotonergic nerves, serotonin turnover in platelets or perhaps the metabolism of serotonin taken up by sympathetic nerves. To test the latter hypothesis we examined 23 patients with chronic congestive heart failure and 9 patients with pure autonomic failure to investigate the possible effects of sympathetic nervous system overactivity and underactivity on peripheral 5-HIAA production and plasma 5-HIAA concentration. The resting arterial plasma 5-HIAA concentration in the heart failure patients was increased three-fold. This elevated plasma 5-HIAA concentration was attributable to an increased rate of whole body 5-HIAA production. The arterial 5-HIAA plasma concentration in the autonomic failure patients was paradoxically elevated, being 70% greater than that of the healthy subjects. The increased 5-HIAA plasma concentration in these patients was accounted for by a reduction in 5-HIAA plasma clearance. In all subjects studied there was a weak relationship only between total body norepinephrine spillover to plasma and the arterial 5-HIAA plasma concentration. We found that in healthy subjects the overflow of 5-HIAA into the hepatic vein was significantly related to the underlying degree of sympathetic activity. It can be concluded that 5-HIAA is produced at a number of sites throughout the body with the arterial plasma concentration being dependent on both the level of production and plasma clearance. By far the majority of 5-HIAA in plasma is derived from the gut with only minimal contribution from the brain.