Long-lasting synaptic potentials and the modulation of synaptic transmission.

Long-lasting postsynaptic potentials (PSPs) generated by decreases in membrane conductance (permeability) have been reported in many types of neurons. We investigated the possible role of such long-lasting decreases in membrane conductance in the modulation of synaptic transmission in the sympathetic ganglion of the bullfrog. The molecular basis by which such conductance-decrease PSPs are generated was also investigated. Synaptic activation of muscarinic cholinergic receptors on these sympathetic neurons results in the generation of a slow EPSP (excitatory postsynaptic potential), which is accompanied by a decrease in membrane conductance. We found that the conventional "fast" EPSPs were increased in amplitude and duration during the iontophoretic application of methacholine, which activates the muscarinic postsynaptic receptors. A similar result was obtained when a noncholinergic conductance-decrease PSP--the late-slow EPSP--was elicited by stimulation of a separate synaptic pathway. The enhancement of fast EPSP amplitude increased the probability of postsynaptic action potential generation, thus increasing the efficacy of impulse transmission across the synapse. Stimulation of one synaptic pathway is therefore capable of increasing the efficacy of synaptic transmission in a second synaptic pathway by a postsynaptic mechanism. Furthermore, this enhancement of synaptic efficacy is long-lasting by virtue of the long duration of the slow PSP. Biochemical and electrophysiological techniques were used to investigate whether cyclic nucleotides are intracellular second messengers mediating the membrane permeability changes underlying slow-PSP generation. Stimulation of the synaptic inputs, which lead to the generation of the slow-PSPs, increased the ganglionic content of both cyclic AMP and cyclic GMP. However, electrophysiological analysis of the actions of these cyclic nucleotides and the actions of agents that affect their metabolism does not provide support for such a second messenger role for either cyclic nucleotide.     

Preparation, application and testing of permanent antibacterial and antiviral coatings

Protocols for the synthesis of the microbicidal polycation N,N-dodecyl,methyl-polyethylenimine and coating (painting) of glass slides with this polycation's butanol solution are described. Subsequently detailed are the procedures for validating that the resultant coated slides are essentially 100% lethal to the human bacterial pathogens, Staphylococcus aureus and Escherichia coli, as well as to two common strains of influenza virus. The time required to prepare and apply the cationic polymer and to test its microbicidal efficiency is conservatively estimated to be <4 weeks.     

Modulation of 5-HT3 receptor-mediated response and trafficking by activation of protein kinase C

Modulation of neurotransmitter-gated membrane ion channels by protein kinase C (PKC) has been the subject of a number of studies. However, less is known about PKC modulation of the serotonin type 3 (5-HT3) receptor, a ligand-gated membrane ion channel that can mediate fast synaptic transmission in the central and peripheral nervous system. Here, we show that PKC potentiated 5-HT3 receptor-mediated current in Xenopus oocytes expressing 5-HT3A receptors and mouse N1E-115 neuroblastoma cells. In addition, using a specific antibody directed to the extracellular N-terminal domain of the 5-HT3A receptor, treatment with the PKC activator, 4 beta-phorbol 12-myristate 13-acetate (PMA), significantly increased surface immunofluorescence. PKC also increased the amount of 5-HT3A receptor protein in the cell membrane without affecting the amount receptor protein in the total cell extract. The magnitude of PMA potentiation of 5-HT3A receptor-mediated responses is correlated with the magnitude of PMA enhancement of the receptor abundance in the cell surface membrane. PMA potentiation is unlikely to occur via direct phosphorylation of the 5-HT3A receptor protein since the potentiation was not affected by point mutation of each of the putative sites for PKC phosphorylation. However, preapplication of phalloidin, which stabilizes the actin polymerization, significantly inhibited PMA potentiation of 5-HT-activated responses in both N1E-115 cells and oocytes expressing 5-HT3A receptors. On the other hand, latrunculin-A, which destabilizes actin cytoskeleton, enhanced the PMA potentiation of 5-HT3A receptors. The observations suggest that PKC can modulate 5-HT3A receptor function and trafficking through an F-actin-dependent mechanism.     

Distinct molecular basis for differential sensitivity of the serotonin type 3A receptor to ethanol in the absence and presence of agonist

Ethanol can potentiate serotonin type 3 (5-HT(3)) receptor-mediated responses in various neurons and in cells expressing 5-HT(3A) receptors. However, the molecular basis for alcohol modulation of 5-HT(3) receptor function has not been determined. Here we report that point mutations of the arginine at amino acid 222 in the N-terminal domain of the 5-HT(3A) receptor can alter the EC(50) value of the 5-HT concentration-response curve. Some point mutations at amino acid 222 resulted in spontaneous opening of the 5-HT(3A) receptor channel and an inward current activated by ethanol in the absence of agonist. Among these mutant receptors, the amplitude of the current activated by ethanol in the absence of agonist was correlated with the amplitude of the current resulting from spontaneous channel openings, suggesting that the sensitivity of the receptor to ethanol in the absence of agonist is, at least in part, dependent on the preexisting conformational equilibrium of the receptor protein. On the other hand, point mutations that conferred greater sensitivity to ethanol potentiation of agonist-activated responses were less sensitive or insensitive to ethanol in the absence of agonist. For these receptors, the magnitude of the potentiation of agonist-activated responses by ethanol was inversely correlated with the EC(50) values of the 5-HT concentration-response curves, suggesting that these mutations may modulate ethanol sensitivity of the receptor by altering the EC(50) value of the receptor. Thus, distinct molecular processes may determine the sensitivity of 5-HT(3A) receptors to ethanol in the absence and presence of agonist.     

Employment of a noninvasive magnetic method for evaluation of gastrointestinal transit in rats

ABSTRACT: AC Biosusceptometry (ACB) was previously employed towards recording gastrointestinal motility. Our data show a reliable and successful evaluation of gastrointestinal transit of liquid and solid meals in rats, considering the methods scarcity and number of experiments needed to endorsement of drugs and medicinal plants. ACB permits real time and simultaneous experiments using the same animal, preserving the physiological conditions employing both meals with simplicity and accuracy.     

Rapid purification of 2,4-dichlorophenol hydroxylase by biospecific desorption from 10-carboxydecylamino-sepharose

10-Carboxydecylamino-Sepharose, which bears a mixture of ionic and aliphatic substituent groups, adsorbs 2,4-dichlorophenol hydroxylase from Acinetobacter in a non-biospecific manner. The enzyme has been specifically desorbed by its substrate, 2,4-dichlorophenol, giving a 42-fold purification (to greater than 90% purity) in a single step. The enzyme contained 3.1 moles of FAD per mole and displayed a catalytic constant of 14.7 s(-1). Mixed-function adsorbents probably have wide applicability for biospecific desorption of proteins. The present report indicates that they may be useful in the purification of aromatic hydroxylases bearing flavin prosthetic groups that readily dissociate in conventional purification procedures employing conditions of high ionic strength.     

Fibrils of Prostatic Acid Phosphatase Fragments Boost Infections with XMRV (Xenotropic Murine Leukemia Virus-Related Virus), a Human Retrovirus Associated with Prostate Cancer

The xenotropic murine leukemia virus-related virus (XMRV) has recently been detected in prostate cancer tissues and may play a role in tumorigenesis. It is currently unclear how this virus is transmitted and which factors promote its spread in the prostate. We show that amyloidogenic fragments known as semen-derived enhancer of virus infection (SEVI) originating from prostatic acid phosphatase greatly increase XMRV infections of primary prostatic epithelial and stromal cells. Hybrid simian/human immunodeficiency chimeric virus particles pseudotyped with XMRV envelope protein were used to demonstrate that the enhancing effect of SEVI, or of human semen itself, was at the level of viral attachment and entry. SEVI enhanced XMRV infectivity but did not bypass the requirement for the xenotropic and polytropic retrovirus receptor 1. Furthermore, XMRV RNA was detected in prostatic secretions of some men with prostate cancer. The fact that the precursor of SEVI is produced in abundance by the prostate indicates that XMRV replication occurs in an environment that provides a natural enhancer of viral infection, and this may play a role in the spread of this virus in the human population.Viruses are etiologic agents of various human cancers, including cervical carcinoma (caused by human papillomavirus), Kaposi''s sarcoma (caused by human herpesvirus 8), hepatocellular carcinoma (caused by hepatitis B virus and hepatitis C virus), and adult T-cell leukemia (caused by human T-cell leukemia virus type 1) (6). Genetic and epidemiologic evidence suggests that prostate cancer may also have an infectious etiology, although a causative agent has not been identified (4, 12). The gammaretrovirus xenotropic murine leukemia virus-related virus (XMRV) is a candidate human tumor virus based on its association in human prostate tumors with a reduced-activity variant of the antiviral gene, RNASEL (also known as the hereditary prostate cancer 1 gene or HPC1) (17) and because it is a member of a viral family known to cause leukemias and lymphomas in different mammalian species (8). Interferon, through its effector RNase L, potently inhibits XMRV replication (5). XMRV integration sites in human prostate cancer tissues were mapped to cancer breakpoints, common fragile sites, micro-RNA genes, and cancer-related genes (11). Many of these genes are implicated directly or indirectly in prostate cancer and metabolic pathways that affect prostate cancer, including androgen signaling. XMRV has also been observed in prostate tissue from a nonfamilial prostate cancer patient and in an individual without prostate cancer (7). The possible role of XMRV in prostatic cancer raises questions about its ability to infect the prostate and the route of viral transmission.Recently, it has been shown that fragments of prostatic acid phosphatase (PAP), an abundant nonspecific protein phosphatase produced by the prostate (18) and secreted in semen in large quantities (about 2 mg/ml) (16), form amyloid fibrils that drastically enhance human immunodeficiency virus type 1 (HIV-1) infection (14). The fibrils of PAP_248-286, termed semen-derived enhancer of virus infection (SEVI), enhanced the infectious virus titer by several orders of magnitude by capturing HIV-1 virions and promoting their attachment to target cells. The ability of SEVI to promote the interaction between virions and the cell surface is independent of the viral glycoprotein and hence is not restricted to HIV-1, although subsequent fusion between the viral and cellular membranes still required gp120, CD4, and an appropriate coreceptor (14). A recent study indicates that the positive charges on SEVI (pI = 10.21) promote infectivity by neutralizing negative-charge repulsion between HIV particles and the cell surface (15).Because SEVI originates from the prostate (the organ from which XMRV infection was discovered [17]) and promotes viral attachment in a relatively nonspecific manner, we sought to determine its effect on XMRV infection. Here we demonstrate that XMRV infectivity is greatly enhanced by SEVI or human semen and that XMRV RNA is detectable in expressed prostatic secretions (EPS) from human tumor-bearing prostates.     

Further studies of the selective inhibition by ouabain of antigen-induced proliferation of human lymphocytes

Cultures of human lymphocytes incubated for 48 hr in the presence of 2 × 10^?7M solutions of the cardiotonic steroid ouabain lose the proliferative response to antigens (SL-0, SK-SD) but can still proliferate when stimulated by nonspecific mitogens (PHA, Con A, pokeweed mitogen). The two-way mixed lymphocyte reaction was also irreversibly lost if cells of both donors were subjected to ouabain pretreatment. Neither cell counts nor cell viability (determined by dye exclusion) were significantly affected by the ouabain treatment. Pretreatment of a suspension of macrophages with the cardiac glycoside did not diminish their capacity to restore the proliferative response to antigen of macrophage-depleted lymphocyte suspensions; on the other hand, untreated macrophages could not restore the proliferative response of cultures of ouabain-pretreated lymphocytes. The ouabain treatment did not change the proportion of cells able to bind fluorescent anti-immunoglobulin nor did it modify the proportion of lymphocytes forming rosettes with either untreated, or antibody coated, red cells. Increased concentration of K⁺ in the medium, either during or after the ouabain treatment, did not reduce the ouabain effect. We conclude that the selective loss of certain lymphocyte functions caused by ouabain pretreatment was due to an effect on the lymphocyte and not on the macrophage; the effect was not due to the elimination of a relatively large fraction of the cells nor to a generalized disappearance of membrane antigens and receptors.     

Predictors of long-term weight loss in adults with modest initial weight loss, by sex and race

Effective weight management interventions could reduce race-sex disparities in cardiovascular disease (CVD), yet little is known about factors associated with successful weight loss maintenance in race-sex subgroups. In the Weight Loss Maintenance trial (WLM), overweight/obese (BMI 25-45 kg/m(2)) adults who lost ≥4 kg in a 6-month behavioral weight loss intervention (phase I) were randomized into one of three 30-month maintenance interventions (phase II). To investigate predictors in subgroups, randomized groups were combined for this analysis. Of 1,685 phase I participants, 1,032 (61%) entered phase II, including 12% black men (BM), 26% black women (BW), 25% white men (WM), and 37% white women (WW). Weight change over the 36-month study ranged from -2.3% (95% confidence interval = -3.1 to -1.5%) in BW to -4.5% (95% confidence interval = -5.7 to -4.0%) in WM, the result of differential weight loss during phase I. Within race, men lost significantly more weight than women, but within sex group, weight loss did not differ significantly between races. Although participants regained weight during phase II, regain did not differ by race-sex group, and mean weight at the end of the study was significantly lower than phase I entry weight for each subgroup. In regression models, phase I weight loss predicted overall 36-month weight loss in all race-sex groups. Healthy dietary pattern at entry, improvement in dietary pattern, or both were predictive in three of four race-sex groups. Few other variables other than initial weight loss and dietary pattern were predictive. Future research should identify additional modifiable influences on long-term maintenance after a modest weight loss.