Extracts from Leonurus sibiricus L. increase insulin secretion and proliferation of rat INS-1E insulinoma cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Mongolian medicine (TMM) uses preparations from herbs as one form of medication for the treatment of a diversity of diseases including diabetes mellitus (DM). We evaluated the effect of extracts from the plant Leonurus sibiricus L. (LS), used in TMM to treat typical symptoms of type 2 DM, on insulin secretion, electrophysiological properties, intracellular calcium concentration and cell proliferation of INS-1E insulinoma cells under standard cell culture conditions (SCC; 11.1mM glucose). MATERIALS AND METHODS: Insulin secretion was measured by ELISA, electrical properties were assessed by whole cell patch clamping, intracellular calcium concentration (Cai) by Fluo-4 time lapse imaging, insulin receptor expression was verified by RT-PCR and cell proliferation assessed by CellTiter-Glo® cell viability assay. RESULTS: Insulin released from INS-1E cells into the culture medium over 24h was significantly increased in presence of 500 mg/L aqueous LS extract (LS OWE) as well as methanolic LS extract (LS MeOH/H2O) but not in the presence of the butanol-soluble extract (LS MeOH/BuOH). Acute application of LS OWE resulted in a depolarization of the cell membrane potential paralleled by an initial increase and subsequent decline and silencing of action potential frequency, by KATP channel inhibition, persisting depolarization and an increase in Cai. The electrophysiological effects were comparable to those of 100 µM tolbutamide, which, however failed to elevate insulin secretion under SCC. Furthermore all LS extracts stimulated INS-1E cell proliferation. CONCLUSIONS: The finding that extracts from Leonurus sibiricus L. enhance insulin secretion and/or foster cell proliferation may provide possible explanations for the underlying therapeutic principles in the empirical use of LS-containing formulations in DM and DM-related disorders as applied in TMM.

From farm to fork follow-up of thermotolerant campylobacters throughout the broiler production chain and in human cases in a Hungarian county during a ten-months period.

A study tracking thermotolerant campylobacters from the setting of the broilers throughout the whole rearing period, slaughter and sale of chicken products in five consecutive broiler rotations of the same henhouse as well as in two different other farms was conducted in a well-defined geographic area (Hajdú-Bihar county, Hungary) between March 2006 and Feb 2007. All notified cases of human campylobacteriosis in this area during the study period were also included. One hundred and one, 44, 23 and 282 Campylobacter jejuni and 13, 15, 20 and 60C. coli were isolated from broiler houses, slaughterhouses, retail shops and human samples, respectively. Sixty-two isolates collected from broilers or their environment selected from different flocks (57C. jejuni, 5C. coli), 92 isolates collected from abattoirs and retail shops (72C. jejuni, 20C. coli), as well as 85 randomly selected human isolates (74C. jejuni, 11C. coli) were subjected to PFGE analysis using restriction enzymes KpnI and SmaI. Sixty-six of the isolates produced unique Sma-Kpn profiles; the majority (46) of these were of human origin. The remaining isolates formed PFGE clusters of between 2-25 isolates with 14 (12C. jejuni and 2C. coli) main clusters comprised of five or more isolates with identical KpnI-SmaI patterns. Two genetic clones of C. jejuni (clone A, n=25; clone B, n=20) included 18% of isolates from different sources. Generally, isolates from one cluster were found in 1-3 different flocks, notably, clone B was present in three rotations including those from the two independent farms. Six of the seven investigated flocks had one or two characteristic prevalent clones. Transmission of clones between consecutive flocks was frequently seen. Spread of both C. jejuni and C. coli was traced multiple times along the food chain; eight C. jejuni, but no C. coli clones were detected both in broilers and humans. These data suggest that broilers were the major source for C. jejuni but not for C. coli in the studied area and period. For C. jejuni the carryover of strains between consecutive flocks may be a common event, but the strain is eventually replaced by another and consecutive carryover events seem to be infrequent. The majority of the human disease was due to nonepidemic strains; some clones were transmitted from more than one broiler flocks (including epidemiologically unrelated flocks) to humans multiple times.

Galanin family of peptides in skin function.

The skin, the largest organ of the body, functions as a barrier between the body proper and the external environment, as it is constantly exposed to noxious stressors. During the last few years, the concept of an interactive network involving cutaneous nerves, the neuroendocrine axis, and the immune system has emerged. The neuroendocrine system of the skin is composed of locally produced neuroendocrine mediators that interact with specific receptors. Among these mediators are neuropeptides, including members of the galanin peptide family--galanin, galanin-message associated peptide, galanin-like peptide, and alarin--which are produced in neuronal as well as nonneuronal cells in the skin. Here, we review the expression of the galanin peptides and their receptors in the skin, and the known functions of galanin peptides in different compartments of the skin. We discuss these data in light of the role of the galanin peptide family in inflammation and cell proliferation.

Galanin family of peptides in skin function.

The skin, the largest organ of the body, functions as a barrier between the body proper and the external environment, as it is constantly exposed to noxious stressors. During the last few years, the concept of an interactive network involving cutaneous nerves, the neuroendocrine axis, and the immune system has emerged. The neuroendocrine system of the skin is composed of locally produced neuroendocrine mediators that interact with specific receptors. Among these mediators are neuropeptides, including members of the galanin peptide family--galanin, galanin-message-associated peptide, galanin-like peptide, and alarin--which are produced in neuronal as well as nonneuronal cells in the skin. Here we review the expression of the galanin peptides and their receptors in the skin, and the known functions of galanin peptides in different compartments of the skin. We discuss these data in light of the role of the galanin peptide family in inflammation and cell proliferation.

Cell volume regulatory ion transport in the regulation of cell migration.

Cell migration is typically accomplished by the generation of protrusive mechanical forces and is achieved by repeated spatially and temporally coordinated cycles including the formation of a leading edge, the formation of new and disruption of older adhesions to the substratum, actomyosin based contractions and retraction of the trailing edge. Beside the well-described roles of the cytoskeleton and cell adhesions during these processes, a growing body of evidence indicates that the precise regulation of the cell volume is an indispensable prerequisite for coordinated cell migration. On the one hand during cell migration cell volume is continuously tormented by mechanical and morphological alterations, which pose changes to the intracellular hydrostatic pressure, metabolic changes and the formation or degradation of macromolecules like actin, which distort the osmotic equilibrium and the action of chemoattractants, hormones and transmitters, which frequently alter the electrical properties of a cell and thus cause cell swelling or shrinkage, respectively. On the other hand, a migrating cell actively has to govern cell volume regulatory ion transport mechanisms in order to create the appropriate micro- or even nanoenvironment in the intra- and/or extracellular space, which is necessary to guarantee the correct polarity and hence direction of movement of a migrating cell. This chapter will focus on the role of the cell volume regulatory ion transport mechanisms as they participate in the regulation of cell migration and special emphasis is given to their interplay with the cytoskeleton, their meaning for substrate adhesion and to the polarized fashion of their subcellular distribution.

The ICln interactome.

The many different functional phenotypes described in mammalian cells can only be explained by an intense interaction of the underlying proteins, substantiated by the fact that the number of independently expressed proteins in living cells seems not to exceed 25 K, a number way too small to explain the >250 K different phenotypes on a one-protein-one-function base. Therefore, the study of the interactome of the different proteins is of utmost importance. Here, we describe the present knowledge of the ICln interactome. ICln is a protein, we cloned and whose function was reported to be as divers as (i) ion permeation, (ii) cytoskeletal organization, and (iii) RNA processing. The role of ICln in these different functional modules can be described best as being a 'connector hub' with 'date hub' function.

Hypotonicity and ethanol modulate BK channel activity and chloride currents in GH4/C1 pituitary tumour cells.

AIM: Description of the effects of hypotonic cell swelling and ethanol on maxi Ca2+-activated K+ channel (BK channel) activity and Cl- channel activity in GH4/C1 pituitary tumour cells. METHODS: Whole cell-, cell attached- and outside-out patch clamp measurements, fluorescence (fluo-3) measurements of intracellular Ca2+ concentration, cell size video monitoring. RESULTS: GH4/C1 pituitary tumour cells respond to both hypotonicity and ethanol with cell swelling which is followed by a regulatory volume decrease (RVD). Tetraethylammonium and 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) induced cell swelling per se and inhibited hypotonicity induced RVD. Ethanol-induced swelling is paralleled by an increase in the intracellular Ca2+ concentration and augmented by DIDS. BK channel activation by hypotonicity and ethanol is demonstrated in patch clamp experiments both in intact cells (cell attached configuration) and a subset of excised membrane patches (outside-out configuration). Cell swelling and addition of ionomycin under isotonic conditions leads to the activation of outwardly rectifying Cl- currents with time dependent activation at positive potentials. CONCLUSIONS: In GH4/C1 cells both hypotonicity and ethanol lead to cell swelling, RVD and to activation of BK channels. The hypotonicity-induced BK channel activation can also be observed in cell free outside-out patches. Hypotonicity, but not ethanol leads to the activation of Cl- channels with features of Ca2+-activated Cl- currents.

On-demand release of corrosion-inhibiting ions from amorphous Al-Co-Ce alloys.

Controlled release technologies are often used to supply chemicals or drugs at given rates. Release often occurs on contact with solution. However, some applications, such as corrosion protection, require containment of the active species in a reservoir and their slow release when needed. Conductive polymers have been used as reservoirs for corrosion inhibitors whose triggered release occurs by galvanic reduction or ion exchange. This work shows one of the first examples of pH-controlled release of corrosion-inhibiting ions from an amorphous metallic coating where the pH change that triggers release is a consequence of the onset of corrosion. This corrosion-inhibition strategy provides further corrosion protection beyond the traditional roles of barrier and sacrificial cathodic protection using a metal coating. For instance, zinc galvanizing provides sacrificial cathodic protection and acts as a barrier, but does not supply inhibitor ions. In the coating described here, protection of an underlying structural alloy exposed at coating defects is demonstrated by inhibitor ion release in addition to barrier function and sacrificial cathodic protection.

Molecular and functional aspects of anionic channels activated during regulatory volume decrease in mammalian cells.

The ability of cells to readjust their volume after swelling, a phenomenon known as regulatory volume decrease (RVD), is a fundamental biological achievement guaranteeing survival and function of cells under osmotic stress. This article reviews the mechanisms of RVD in mammalian cells with special emphasis on the activation of ion channels during RVD.

Follow-up genotoxicological monitoring of nurses handling antineoplastic drugs.

Most of the antineoplastic drugs used in the treatment of tumors are carcinogenic to humans. Hospital nurses are often subject to possible occupational carcinogen exposure. Exposure may occur during handling and administration of infusion solutions containing cytostatics. A genotoxicological monitoring system to detect genotoxic changes was developed in our laboratory, helping to improve working conditions and subserving primary prevention. Multiple-endpoint follow-up genotoxicological monitoring was performed in peripheral blood lymphocytes (PBLs) among 4 groups of 95 nurses (152 investigations) occupationally exposed to cytostatics. The results were compared to those of historical and industrial controls. The genotoxicological endpoints were the determination of the frequency of sister chromatid exchanges (SCEs) and the cells with high-frequency SCEs (HFC), the frequency of structural and numerical chromosome aberrations. and the measurement of ultraviolet-light-induced unscheduled DNA-repair synthesis (UDS). In Hospital 1, where nurses worked without a safety cabinet, the percentage of cells with chromosome aberrations (AC) was significantly higher than that of the controls. In Hospital 2, where nurses used inadequate safety cabinets (with horizontal airflow), significantly elevated levels of AC, SCE, HFC, and UDS were detected. During follow-up, in Hospital 2 at the time of the second investigation AC was still significantly higher, although safety conditions had been improved. The results indicate the presence of genotoxic damage in hospital nurses working with no or inadequate safety equipment. In Hospitals 3 and 4 where nurses using biological safety cabinets, the results were lower than those in the previous two groups. In Hospital 3 in the first year of the study AC was as at the level of industrial controls. During follow-up in the course of the repeated investigations a fluctuation in AC above the control level and an increase in HFC in yr 4 and 6 of the study were observed. In this group, the fluctuation in AC and HFC during the study points to the possibility of genotoxic exposure with cytostatics despite of the use of suitable safety cabinets, drawing attention to other possible routes of exposure. In Hospital 4, both AC and HFC were elevated. These data corroborate the need to maintain safety measures to avoid exposure, and the necessity of intervention in the case of exposure when using and handling hazardous carcinogenic agents. The results also indicate a certain expression time for genotoxic changes, which can lead to late somatic mutations as well as to a possible higher risk of cancer.

Working condition-related improvement in genotoxicological parameters of Hungarian road pavers.

Multiple-endpoint follow-up genotoxicology monitoring was performed in a group of 22 Hungarian road pavers between 1996 and 1999. The studied endpoints were the determination of structural and numeric chromosome aberration (CA), sister chromatid exchange (SCE), high-frequency SCE and HPRT mutation frequencies, and ultraviolet (UV)-light-induced unscheduled DNA synthesis in peripheral blood lymphocytes (PBLs). The workers (8 hand pavers and 14 finishers, mean age 37 yr) used tar-free asphalt. The results were compared with those of 6 work-site controls (35 yr), 101 historical controls (38 yr), and 87 industrial controls (38 yr). The most marked changes were found in the CA frequencies. In the control, the mean CA frequency was 1.6%. In the first study, increased CA frequencies were found in the donors that either had been exposed to hot asphalt fumes or had cleaned the equipment with crude oil. The mean CA frequency of the 14 finishers working in closed cabins was 3.67% in 1996. The increased CA frequency was attributed to the high level of hot asphalt fumes due to insufficient ventilation. By 1999 the mean CA frequency decreased to 1.23%. For the 8 hand pavers working in open air the mean CA frequency was 3.6% in 1996. The obtained data suggested that the increase in CA frequencies was due to the use of petroleum and crude oil; therefore, these substances were replaced with harmless detergents. By 1999 the mean CA frequency decreased to 1%. In finishers the mean CA frequency returned to the control level 1 yr later (1999) than in the case of hand pavers. The chromosome-type aberrations remained predominant during the follow-up. The individual variations observed were attributed to smoking and inadequate personal protection. The obtained results suggest that the use of tar-free asphalt and harmless detergents with adequate personal protection does not increase the frequencies of the genotoxicological parameters compared to controls. Consequently, an improvement in working conditions can prevent further exposures and thus decrease the cancer risk of road pavers.

Regional magnetic resonance imaging lesion burden and cognitive function in multiple sclerosis: a longitudinal study.

OBJECTIVE: To investigate the relationship between magnetic resonance imaging regional lesion burden and cognitive performance in multiple sclerosis (MS) over a 4-year follow-up period. DESIGN: Twenty-eight patients with MS underwent magnetic resonance imaging and took the Brief, Repeatable Battery of Neuropsychological Tests in Multiple Sclerosis at baseline, 1-year, and 4-year follow-up. An automated 3-dimensional lesion detection method was used to identify MS lesions within anatomical regions on proton density T2-weighted images. The relationship between magnetic resonance imaging regional lesion volumes and the Brief, Repeatable Battery of Neuropsychological Tests in Multiple Sclerosis results was examined using regression analyses. RESULTS: At all time points, frontal lesion volume represented the greatest proportion of total lesion volume, and the percentage of white matter classified as lesion was also highest in frontal and parietal regions. On neuropsychological testing, when compared with age- and educational level-matched control subjects, patients with MS showed significant impairment on tests of sustained attention, processing speed, and verbal memory (P<.001). Performance on these measures was negatively correlated with MS lesion volume in frontal and parietal regions at baseline, 1-year, and 4-year follow-up (R = -0.55 to -0.73, P<.001). CONCLUSIONS: Multiple sclerosis lesions show a propensity for frontal and parietal white matter. Lesion burden in these areas was strongly associated with performance on tasks requiring sustained complex attention and working verbal memory. This relationship was consistent over a 4-year period, suggesting that disruption of frontoparietal subcortical networks may underlie the pattern of neuropsychological impairment seen in many patients with MS.

Structure and function of the ion channel ICln.

Normal function of organs and cells is tightly linked to the cytoarchitecture. Control of the cell volume is therefore vital for the organism. A widely established strategy of cells to counteract swelling is the activation of chloride and potassium channels, which leads to a net efflux of salt followed by water - a process termed regulatory volume decrease. Since there is evidence for swelling-dependent chloride channels (IClswell) being activated also during pathological processes, the identification of the molecular entity underlying IClswell is of utmost importance. Several proteins are discussed as the channel forming IClswell, i.e. phospholemman, p-glycoprotein, CLC-3 and ICln. In this review we would like to focus on the properties of ICln, a protein cloned from a Madin Darby canine kidney (MDCK) cell library whose expression in Xenopus laevis oocytes resulted in a nucleotide sensitive outwardly rectifying chloride current closely resembling the biophysical properties of IClswell.

Functional reconstitution of ICln in lipid bilayers.

Reconstitution of purified ICln in lipid bilayer leads to functional ion channels showing varying rectification. The reconstituted single channels have a conductance of approximately equal to 3 pS and their open probability is sensitive to nucleoside analogues. Mutation of a putative nucleotide binding site identified at the predicted extracellular mouth of the ICln channel protein leads to the reduction of the nucleoside-analogue sensitivity. Reconstituted ICln channels can be permeated both by cations and anions. The relative permeability of cations over anions depends on the presence of calcium. In the presence of calcium reconstituted ICln channels are more permeable to bromide than chloride, and more permeable to potassium than sodium. Similarly in NIH3T3 fibroblasts, the relative permeability of cations over anions of swelling-dependent chloride channels depends on extracellular calcium. Site-directed mutagenesis revealed the calcium-binding site responsible for the shift of the selectivity from cations towards anions of reconstituted ICln channels. Additional indirect structural information has been obtained by mutating a histidine in the predicted pore region of ICln. This histidine seems to have access to the ion-conducting tunnel of the pore. Our experiments show that ICln can act as an ionic channel, which does not exclude additional functions of the protein in regulatory mechanisms of the cell. Since knocking down the ICln protein in fibroblasts and epithelial cells leads to an impaired regulatory volume decrease (RVD) after cytoplasmic swelling and reconstituted ICln channels show several biophysical features of ion channels activated after swelling, ICln is a molecular candidate for these channels.

The somatostatin analogue peptide TT-232 induces apoptosis and chromosome breakage in cultured human lymphocytes.

Somatostatin receptors are supposed to be important in the regulation of apoptosis. In this study, we measured apoptosis occurring spontaneously, or induced by the synthetic somatostatin analogue, the peptide TT-232. We examined isolated human peripheral blood lymphocytes (PBL) from 32 nurses exposed bedside to cytostatic drugs, 12 chronic lymphoid leukaemia (CLL) patients prior to treatment, and 19 unexposed, healthy donors without anamnestic occupational exposure to genotoxic agents. Cells were stimulated by phytohaemagglutinin-P (PHA) and cultured for 69 h with or without 15 microg/ml TT-232, respectively. Cell kinetic parameters and apoptosis were determined by flow cytometry after staining with FITC-labeled anti-BrdU and propidium iodide (PI) and the results on spontaneous and peptide-induced apoptosis were compared with the obtained chromosome aberration frequencies (CA). The peptide TT-232 unexpectedly induced chromosome breakage in addition to apoptosis. The mean spontaneous apoptotic fractions were 6.65+/-0.89%, 6.46+/-0. 53%, and 3.07+/-0.57%, and the mean CA yields in the samples without TT-232 were 1.74+/-0.46%, 2.44+/-0.40%, and 4.50+/-1.05%, for healthy subjects, nurses, and CLL patients, respectively. A total of 15 microg/ml TT-232 treatment in healthy subjects increased the mean CA frequency (10.38+/-1.57%), as well as the apoptotic cell fraction (2.63+/-0.45 times higher than the corresponding untreated sample). In TT-232-treated PBLs of nurses, CA remained unchanged and the mean apoptotic cell fraction showed only a slight increase (1.24+/-0.11 times higher than the untreated). Among CLL patients, TT-232 treatment significantly increased both CA (up to 17.83+/-4.04%) and the ratio of apoptotic cells (21.78+/-11.00 times higher than the untreated). These results demonstrated significant differences in apoptosis sensitivity in controls, nurses and CLL donors, after 15 microg/ml TT-232 treatment. Data also indicate that the induced CA yields in CLL donors with high CA are in correlation with TT-232-induced apoptosis.