Effector contributions to G beta gamma-mediated signaling as revealed by muscarinic potassium channel gating.

Receptor-mediated activation of heterotrimeric G proteins leading to dissociation of the G alpha subunit from G beta gamma is a highly conserved signaling strategy used by numerous extracellular stimuli. Although G beta gamma subunits regulate a variety of effectors, including kinases, cyclases, phospholipases, and ion channels (Clapham, D.E., and E.J. Neer. 1993. Nature (Lond.). 365:403-406), few tools exist for probing instantaneous G beta gamma-effector interactions and little is known about the kinetic contributions of effectors to the signaling process. In this study, we used the atrial muscarinic K + channel, which is activated by direct interactions with G beta gamma subunits (Logothetis, D.E., Y. Kurachi J. Galper, E.J. Neer, and D.E. Clap. 1987. Nature (Lond.). 325:321-326; Wickman, K., J. A. Iniguez-Liuhi, P.A. Davenport, R. Taussig, G.B. Krapivinsky, M.E. Linder, A.G. Gilman, and D.E. Clapham. 1994. Nature (Lond.). 366: 654-663; Huang, C.-L., P.A. Slesinger, P.J. Casey, Y.N. Jan, and L.Y. Jan. 1995. Neuron. 15:1133-1143), as a sensitive reporter of the dynamics of G beta gamma-effector interactions. Muscarinic K+ channels exhibit bursting behavior upon G protein activation, shifting between three distinct functional modes, characterized by the frequency of channel openings during individual bursts. Acetylcholine concentration (and by inference, the concentration of activated G beta gamma) controls the fraction of time spent in each mode without changing either the burst duration or channel gating within individual modes. The picture which emerges is of a G beta gamma effector with allosteric regulation and an intrinsic "off" switch which serves to limit its own activation. These two features combine to establish exquisite channel sensitivity to changes in G beta gamma concentration, and may be indicative of the factors regulating other G beta gamma-modulated effectors.

Muscarinic K+ channel in the heart. Modal regulation by G protein beta gamma subunits.

The membrane-delimited activation of muscarinic K+ channels by G protein beta gamma subunits plays a prominent role in the inhibitory synaptic transmission in the heart. These channels are thought to be heterotetramers comprised of two homologous subunits, GIRK1 and CIR, both members of the family of inwardly rectifying K+ channels. Here, we demonstrate that muscarinic K+ channels in neonatal rat atrial myocytes exhibit four distinct gating modes. In intact myocytes, after muscarinic receptor activation, the different gating modes were distinguished by differences in both the frequency of channel opening and the mean open time of the channel, which accounted for a 76-fold increase in channel open probability from mode 1 to mode 4. Because of the tetrameric architecture of the channel, the hypothesis that each of the four gating modes reflects binding of a different number of Gbeta gamma subunits to the channel was tested, using recombinant Gbeta1 gamma5. Gbeta1 gamma5 was able to control the equilibrium between the four gating modes of the channel in a manner consistent with binding of Gbeta gamma to four equivalent and independent sites in the protein complex. Surprisingly, however, Gbeta1 gamma5 lacked the ability to stabilize the long open state of the channel that is responsible for the augmentation of the mean open time in modes 3 and 4 after muscarinic receptor stimulation. The modal regulation of muscarinic K+ channel gating by Gbeta gamma provides the atrial cells with at least two major advantages: the ability to filter out small inputs from multiple membrane receptors and yet the ability to create the gradients of information necessary to control the heart rate with great precision.

Cytogenetic analysis of peripheral lymphocytes in a population exposed to vinyl chloride through an accidental release into the environment.

On the first of June, 1996 an environmental accident occurred in Schönebeck, Germany in which free vinyl chloride was evaporated into the atmosphere. Thereby, the human population living in this area was exposed to vinyl chloride and its byproducts. Chromosomal aberrations were measured in peripheral blood lymphocytes from 29 potentially exposed and 29 non-exposed (control) individuals. Both groups were matched regarding age, gender and smoking habits. Two hundred metaphases were analysed for chromosomal aberrations per each individual. The exposed group showed a statistically significant increase in the mean frequency of aberrant cells (1.47% versus 1.07% in the control group). Chromosomal aberrations in peripheral lymphocytes have been shown to be a very sensitive biomarker of genotoxic effects not only for occupational exposure to vinyl chloride as reported several times during the last 20 years, but also for an accidental environmental exposure. A follow-up cytogenetic study is recommendable.

Adaptive and synergistic effects of a low-dose ENU pretreatment on the frequency of chromosomal aberrations induced by a challenge dose of ENU in human peripheral blood lymphocytes in vitro.

N-Ethyl-N-nitrosourea (ENU) is an alkylating agent whose mutagenic and carcinogenic potential has been extensively studied but its ability to induce cytogenetic adaptive responses in normal human cells has not been investigated so far. The aim of our present experiments was to study the effect of a pretreatment with a low concentration of ENU (2 x 10(-5) M) on the frequency of chromosomal aberrations induced by a subsequent 50 times higher concentration of ENU (10(-3) M) in human lymphocytes isolated from buffy coats of 4 donors. Two different inter treatment times and three harvesting times were applied to the lymphocytes from each donor. A cytogenetic adaptive response was shown by the lymphocytes of one donor only when the time span between the low adapting and the higher challenging concentration was 4 h. The other three donors did not respond with significant differences in the yield of cells with aberrations. The complex interaction between the ENU-induced multiple primary DNA lesions and various DNA repair mechanisms as well as the influence of cell cycle effects on the induction of clastogenic adaptive response are discussed.

Chromosomal aberrations in humans as genetic endpoints to assess the impact of pollution.

The chromosomal aberration assay with peripheral blood lymphocytes has been used routinely during the last three decades to survey exposure of humans to various genotoxic agents. A large number of biomonitoring studies are based on this genetic endpoint. A great deal of data exists on occupational, life-style or medical exposure situations but less evidence of the validity of the assay is available with regards to environmental exposure. In the present paper we report our investigations on the impact of pollution in two different populations using chromosomal aberrations in human peripheral blood lymphocytes as a biomarker of chronic exposure to heavy metals and dioxins/furans for a long period and as a biomarker of acute exposure to accidentally released vinyl chloride in the air. In order to study genotoxic effects (chromosomal aberrations) of heavy metals and dioxins/furans, 52 exposed individuals from a polluted area were compared to 51 matched controls from a distant non-industrialized area. A statistically significant increase was observed in the frequency of chromosomal aberrations in peripheral blood lymphocytes from the exposed population (1.90% aberrant cells vs. 1.11% for the controls). In the case of the vinyl chloride accident, chromosomal aberrations were analyzed in peripheral blood lymphocytes from 29 potentially exposed and 29 non-exposed individuals (matched controls). The exposed group showed a statistically significant increase in the frequency of aberrant cells (1.47% vs. 1.07% for the controls).

Plant extracts induce chromosome aberrations and sister-chromatid exchanges in Chinese hamster ovary cells and human lymphocytes.

Effects of extracts from Vicia faba were compared with those of Zea mays for the induction of sister-chromatid exchanges (SCEs) and of chromosome aberrations (CAs) in Chinese hamster ovary (CHO) cells. CA induction by the maize extract was also tested in human lymphocytes. The extracts from roots and leaves of Vicia faba induced CAs and SCEs in CHO cells. The extracts from maize leaves also induced SCEs and CAs in CHO cells, and CAs in human lymphocytes. Maize extracts were more potent in inducing SCEs than Vicia extracts and the SCE- and CA-inducing capacity of maize extracts decreased during preincubation before addition to cells.

Frequency of HPRT mutants in humans exposed to vinyl chloride via an environmental accident.

The mutant frequency (MF) in the hypoxanthine-guanine-phosphoribosyl-transferase (HPRT) locus of peripheral blood T-lymphocytes was measured in a population environmentally exposed to vinyl chloride - a toxic and carcinogenic substance through an accidental release into the atmosphere. It was compared to MF in a control group of unexposed individuals. Both groups were re-investigated in a follow-up study, 2 years later. No significant difference could be observed in MF between exposed and controls either at the accident nor in the follow-up study. Approximately the same mean HPRT mutant frequencies were observed for both groups in T-lymphocytes from blood samples obtained shortly after the accident and from the follow-up blood samples. Both groups showed a higher mean MF in the re-investigation samples which is most probably due to the significantly lower average cloning efficiency (CE) under non-selective conditions and because of the inverse relationship between CE and MF. The exposed population showed a higher mean T-cell CE at the initial blood sampling as compared to the control group. The concurrent cytogenetic analyses of peripheral lymphocytes showed a significant increase in cells with aberrations in the exposed population. Clastogenic but not mutagenic activity of vinyl chloride was observed in our study.

Contamination of sediments in Varna Lake and Varna Bay.

Varna Bay is one of the hot spots along the Black Sea coastal zone. Its ecological state is strongly influenced by the connection with the Varna Lake. Along the lake coast are located many sources of pollution such as rivers, ports, chemical industry, WWTPs. The shipping is another pressure on the environment. The study was carried out in the western part of the Varna Lake and Varna Bay. The following parameters: heavy metals, polichlorinated biphenyls, chlororganic pesticides, phenols, petroleum hydrocarbons were analysed in sediments. The high content of the last one is indicative for an extremely unfavourable long-year exploitation of the lake as a water route. The metals concentration in lakes sediments shows the influence of anthropogenic inputs. Comparative analysis of the sediments shows higher concentrations of contaminants in Varna Lake. and near the ports. As a consequence of the worsened state of the bottom a very poor benthic community characterizes a part of the investigated area in the lake.

[Influence of the storage of slaughtered birds at low temperature on the viability of some microorganisms]. / Vliianie na s'khraniavaneto na zaklanite ptitsi pri niski temperaturi v'rkhu zhiznenostta na niakoi mikroorganizmi

Studied was the effect of the period of storing slaughtered broiler birds at low temperatures on the activity of pathogenic staphylococci, enteropathogenic Escherichia coli organisms and pathogenic species of the Proteus genus. It was found that freezing at minus 34 degrees C and storage of the slaughtered birds at minus 18 degrees C contribute to the considerable decrease in the bacterial count of the investigated test organisms, this being observed to a highest extent during the first fifteen days of storage.

A follow-up study on chromosomal aberrations in lymphocytes of alcoholics during early, medium, and long-term abstinence.

The frequencies of structural chromosomal aberrations were analyzed in peripheral blood lymphocytes of 31 chronic alcoholics at the beginning of an intensive outpatient treatment program at a neuropsychiatric clinic and were compared with 31 controls matched for gender, age, smoking habits, and nondrinkers. A statistically significant difference was observed in the level of chromosomal aberrations in somatic cells from alcoholics when compared with controls (3.01% vs. 1.28%, p < or = 0.001). A follow-up study was carried out for a subset of the patients after 3 months (8 subjects) and 12 months (14 subjects) of controlled abstinence. A statistically significant increase in the mean frequency of cells with aberrations was observed in the group of 14 subjects reinvestigated after 12 months of abstinence when compared with the mean value of the first blood samples immediately after hospitalization (4.61% vs. 3.01%; p < or = 0.001). An excessive increase in cigarette consumption during alcohol abstinence, reflected by a dramatic elevation of CO-hemoglobin levels, may, at least in part, account for this finding. In conclusion, chronic alcoholism leads to genotoxic effects that, instead of recovering after 1 year of alcohol abstinence, are even enhanced, most likely due to the "shift in addictive behavior."