Low frequency electric current noise in glioma cell populations.

Measuring the electrical activity of large and defined populations of cells is currently a major technical challenge to electrophysiology, especially in the picoampere-range. For this purpose, we developed and applied a bidirectional transducer based on a chip with interdigitated gold electrodes to record the electrical response of cultured glioma cells. Recent research determined that also non-neural brain glia cells are electrically active and excitable. Their transformed counterparts, e.g. glioma cells, were suggested to partially retain these electric features. Such electrophysiological studies however are usually performed on individual cells and are limited in their predictive power for the overall electrical activity of the multicellular tumour bulk. Our extremely low-noise measuring system allowed us to detect not only prominent electrical bursts of neuronal cells but also minute, yet constantly occurring and functional, membrane capacitive current oscillations across large populations of C6 glioma cells, which we termed electric current noise. At the same time, tumour cells of non-brain origin (HeLa) proved to be electrically quiescent in comparison. Finally, we determined that the glioma cell activity is primarily caused by the opening of voltage-gated Na+ and K+ ion channels and can be efficiently abolished using specific pharmacological inhibitors. Thus, we offer here a unique approach for studying electrophysiological properties of large cancer cell populations as an in vitro reference for tumour bulks in vivo.

Systemic absorption of rifamycin SV MMX administered as modified-release tablets in healthy volunteers.

The new oral 200-mg rifamycin SV MMX modified-release tablets, designed to deliver rifamycin SV directly into the colonic lumen, offer considerable advantages over the existing immediate-release antidiarrheic formulations. In two pharmacokinetics studies of healthy volunteers, the absorption, urinary excretion, and fecal elimination of rifamycin SV after single- and multiple-dose regimens of the new formulation were investigated. Concentrations in plasma of >2 ng/ml were infrequently and randomly quantifiable after single and multiple oral doses. The systemic exposure to rifamycin SV after single and multiple oral doses of MMX tablets under fasting and fed conditions or following a four-times-a-day (q.i.d.) or a twice-a-day (b.i.d.) regimen could be considered negligible. With both oral regimens, the drug was confirmed to be very poorly absorbable systemically. The amount of systemically absorbed antibiotic excreted by the renal route is far lower than 0.01% of the administered dose after both the single- and multiple-dose regimens. The absolute bioavailability, calculated as the mean percent ratio between total urinary excretion amounts (ΣXu) after a single intravenous injection and after a single oral dose under fasting conditions, was 0.0410±0.0617. The total elimination of the unchanged rifamycin SV with feces was 87% of the administered oral dose. No significant effect of rifamycin SV on vital signs, electrocardiograms, or laboratory parameters was observed.

Virodhamine and CP55,940 modulate cAMP production and IL-8 release in human bronchial epithelial cells.

BACKGROUND AND PURPOSE: We investigated expression of cannabinoid receptors and the effects of the endogenous cannabinoid virodhamine and the synthetic agonist CP55,940 on cAMP accumulation and interleukin-8 (IL-8) release in human bronchial epithelial cells. EXPERIMENTAL APPROACH: Human bronchial epithelial (16HBE14o(-)) cells were used. Total mRNA was isolated and cannabinoid receptor mRNAs were detected by RT-PCR. Expression of CB(1) and CB(2) receptor proteins was detected with Western blotting using receptor-specific antibodies. cAMP accumulation was measured by competitive radioligand binding assay. IL-8 release was measured by ELISA. KEY RESULTS: CB(1) and CB(2) receptor mRNAs and proteins were found. Both agonists concentration-dependently decreased forskolin-induced cAMP accumulation. This effect was inhibited by the CB(2) receptor antagonist SR144528, and was sensitive to Pertussis toxin (PTX), suggesting the involvement of CB(2) receptors and G(i/o)-proteins. Cell pretreatment with PTX unmasked a stimulatory component, which was blocked by the CB(1) receptor antagonist SR141716A. CB(2) receptor-mediated inhibition of cAMP production by virodhamine and CP55,940 was paralleled by inhibition of tumor necrosis factor-alpha (TNF-alpha) induced IL-8 release. This inhibition was insensitive to SR141716A. In the absence of agonist, SR144528 by itself reduced TNF-alpha induced IL-8 release. CONCLUSIONS AND IMPLICATIONS: Our results show for the first time that 16HBE14o(-) cells respond to virodhamine and CP55,940. CB(1) and CB(2) receptor subtypes mediated activation and inhibition of adenylyl cyclase, respectively. Stimulation of the dominant CB(2) receptor signalling pathway diminished cAMP accumulation and TNF-alpha-induced IL-8 release. These observations may imply that cannabinoids exert anti-inflammatory properties in airways by modulating cytokine release.

Paralogous gene analysis reveals a highly enantioselective 1,2-O-isopropylideneglycerol caprylate esterase of Bacillus subtilis.

Carboxylesterase NP of Bacillus subtilis Thai I-8, characterized in 1992 as a very enantioselective (S)-naproxen esterase, was found to show no enantiopreference towards (S)-1,2-O-isopropylideneglycerol (IPG) esters. The ybfK gene was identified by the B. subtilis genome project as an unknown gene with homology to carboxylesterase NP. The purpose of the present study was to characterize the ybfK gene product in order to determine whether this paralogue of carboxylesterase NP had an altered or enhanced stereospecificity. The ybfK gene was cloned and expressed in B. subtilis using a combination of two strong promoters in a multicopy vector. The enzyme was purified from the cytoplasm of B. subtilis by means of anion exchange and hydrophobic interaction chromatography. The purified YbfK is an enzyme of 296 amino acids and shows an apparent molecular mass of 32 kDa (SDS/PAGE). Comparison of the activities of YbfK and carboxylesterase NP towards caprylate esters of IPG revealed that YbfK produces (S)-IPG with 99.9% enantioselectivity. Therefore, we conclude that we have isolated a paralogue of carboxylesterase NP that can be used for the enantioselective production of (S)-IPG.

Has blood glucose level measured on admission to hospital in a patient with acute pancreatitis any prognostic value?

BACKGROUND: Early detection of pancreatic necrosis allows better management of the disease. Contrast-enhanced computed tomography (CT) as the gold standard for detecting pancreatic necrosis is expensive. AIM OF THE STUDY: This study was to evaluate for the first time whether blood glucose estimation on hospital admission--a simple, cheap, readily available laboratory parameter--may detect pancreatic necrosis and have prognostic value in acute pancreatitis. METHODS: Single blood glucose estimation upon hospital admission was evaluated prospectively for detecting pancreatic necrosis and as a prognostic indicator. The study included 241 nondiabetic patients with a first attack of acute pancreatitis. All underwent CT within 72 h of admission. RESULTS: High blood glucose (> 125 mg/dl) correlated significantly with complex high clinical and biochemical prognostic scores (Ranson, Imrie), a high Balthazar score, pancreatic pseudocysts, and a long hospital stay, but not with organ failure, indication for artificial ventilation, dialysis, surgery, length of intensive care, and mortality. Pancreatic necrosis detection sensitivity of high blood glucose was 83%, specificity 49%, positive predictive value 28%, and negative predictive value 92%. CONCLUSION: A patient with normal blood glucose on admission is unlikely to have pancreatic necrosis. Contrast-enhanced CT would not be needed unless the patient fails to improve.

The genetic organization and evolution of the broad host range mercury resistance plasmid pSB102 isolated from a microbial population residing in the rhizosphere of alfalfa.

Employing the biparental exogenous plasmid isolation method, conjugative plasmids conferring mercury resistance were isolated from the microbial community of the rhizosphere of field grown alfalfa plants. Five different plasmids were identified, designated pSB101-pSB105. One of the plasmids, pSB102, displayed broad host range (bhr) properties for plasmid replication and transfer unrelated to the known incompatibility (Inc) groups of bhr plasmids IncP-1, IncW, IncN and IncA/C. Nucleotide sequence analysis of plasmid pSB102 revealed a size of 55 578 bp. The transfer region of pSB102 was predicted on the basis of sequence similarity to those of other plasmids and included a putative mating pair formation apparatus most closely related to the type IV secretion system encoded on the chromosome of the mammalian pathogen Brucella sp. The region encoding replication and maintenance functions comprised genes exhibiting different degrees of similarity to RepA, KorA, IncC and KorB of bhr plasmids pSa (IncW), pM3 (IncP-9), R751 (IncP-1beta) and RK2 (IncP-1alpha), respectively. The mercury resistance determinants were located on a transposable element of the Tn5053 family designated Tn5718. No putative functions could be assigned to a quarter of the coding capacity of pSB102 on the basis of comparisons with database entries. The genetic organization of the pSB102 transfer region revealed striking similarities to plasmid pXF51 of the plant pathogen Xylella fastidiosa.

Phenotypic and molecular characterization of conjugative antibiotic resistance plasmids isolated from bacterial communities of activated sludge.

In order to isolate antibiotic resistance plasmids from bacterial communities found in activated sludge, derivatives of the 3-chlorobenzoate-degrading strain Pseudomonas sp. B13, tagged with the green fluorescent protein as an identification marker, were used as recipients in filter crosses. Transconjugants were selected on agar plates containing 3-chlorobenzoate as the sole carbon source and the antibiotic tetracycline, streptomycin or spectinomycin, and were recovered at frequencies in the range of 10(-5) to 10(-8) per recipient. A total of 12 distinct plasmids, designated pB1-pB12, was identified. Their sizes ranged between 41 to 69 kb and they conferred various patterns of antibiotic resistance on their hosts. Two of the plasmids, pB10 and pB11, also mediated resistance to inorganic mercury. Seven of the 12 plasmids were identified as broad-host-range plasmids, displaying extremely high transfer frequencies in filter crosses, ranging from 10(-1) to 10(-2) per recipient cell. Ten of the 12 plasmids belonged to the IncP incompatibility group, based on replicon typing using IncP group-specific PCR primers. DNA sequencing of PCR amplification products further revealed that eight of the 12 plasmids belonged to the IncPbeta subgroup, whereas two plasmids were identified as IncPalpha plasmids. Analysis of the IncP-specific PCR products revealed considerable differences among the IncPbeta plasmids at the DNA sequence level. In order to characterize the gene "load" of the IncP plasmids, restriction fragments were cloned and their DNA sequences established. A remarkable diversity of putative proteins encoded by these fragments was identified. Besides transposases and proteins involved in antibiotic resistance, two putative DNA invertases belonging to the Din family, a methyltransferase of a type I restriction/modification system, a superoxide dismutase, parts of a putative efflux system belonging to the RND family, and proteins of unknown function were identified.

Horizontal gene transfer as a biosafety issue: a natural phenomenon of public concern.

The transfer of genetic information between distantly or even unrelated organisms during evolution had been inferred from nucleotide sequence comparisons. These studies provided circumstantial evidence that in rare cases genes had been laterally transmitted amongst organisms of the domains bacteria, archaea and eukarya. Laboratory-based studies confirmed that the gene pools of the various domains of organisms are linked. Amongst the bacterial gene exchange mechanisms transduction, transformation and conjugation, the latter was identified as the mechanism with potentially the broadest host range of transfer. Previously, the issue of horizontal gene transfer has become important in the context of biosafety. Gene transfer studies carried out under more natural conditions such as in model ecosystems or in the environment established that all gene transfer mechanisms worked under these conditions. Moreover, environmental hot-spots were identified where favourable conditions such as nutrient enrichment increased the probability of genetic exchange among bacteria. In particular, the phytosphere was shown to provide conducive conditions for conjugative gene exchange. Concern has been expressed that transfer of recombinant DNA (e.g. antibiotic resistance genes) from genetically modified organisms (GMOs) such as transgenic plants to phytosphere bacteria may occur and thus contribute to the undesirable spread of antibiotic resistance determinants. Studies which were performed to address this issue clearly showed that such a transfer occurs, if at all, at extremely low frequency.

Intergeneric transfer of conjugative and mobilizable plasmids harbored by Escherichia coli in the gut of the soil microarthropod Folsomia candida (Collembola).

The gut of the soil microarthropod Folsomia candida provides a habitat for a high density of bacterial cells (T. Thimm, A. Hoffmann, H. Borkott, J. C. Munch, and C. C. Tebbe, Appl. Environ. Microbiol. 64:2660-2669, 1998). We investigated whether these gut bacteria act as recipients for plasmids from Escherichia coli. Filter mating with E. coli donor cells and collected feces of F. candida revealed that the broad-host-range conjugative plasmid pRP4-luc (pRP4 with a luciferase marker gene) transferred to fecal bacteria at estimated frequencies of 5.4 x 10(-1) transconjugants per donor. The mobilizable plasmid pSUP104-luc was transferred from the IncQ mobilizing strain E. coli S17-1 and less efficiently from the IncF1 mobilizing strain NM522 but not from the nonmobilizing strain HB101. When S17-1 donor strains were fed to F. candida, transconjugants of pRP4-luc and pSUP104-luc were isolated from feces. Additionally, the narrow-host-range plasmid pSUP202-luc was transferred to indigenous bacteria, which, however, could not maintain this plasmid. Inhibition experiments with nalidixic acid indicated that pRP4-luc plasmid transfer took place in the gut rather than in the feces. A remarkable diversity of transconjugants was isolated in this study: from a total of 264 transconjugants, 15 strains belonging to the alpha, beta, or gamma subclass of the class Proteobacteria were identified by DNA sequencing of the PCR-amplified 16S rRNA genes and substrate utilization assays (Biolog). Except for Alcaligenes faecalis, which was identified by the Biolog assay, none of the isolates was identical to reference strains from data banks. This study indicates the importance of the microarthropod gut for enhanced conjugative gene transfer in soil microbial communities.

Diagnostic intervals for recognizing celiac disease.

The aim of this retrospective study was to determine the time intervals between the onset of symptoms and diagnosis of celiac disease on the basis of a questionnaire that was published in the journal of the German Celiac Society (Verbandszeitschrift der Deutschen Zöliakie-Gesellschaft). 408 adult patients in whom the diagnosis of celiac disease was made after the age of 15 responded to the questionnaire. The time interval between the onset of symptoms and diagnosis (total diagnostic interval) was 5.4 (median) and 10.1 +/- 12.3 (mean +/- SD) years, interval-1 (time interval between the onset of symptoms and the first visit to a doctor) was 0.4 (median) and 2.2 +/- 6.6 (mean +/- SD) years, and interval-2 (time interval between the first visit to a doctor and the diagnosis) was 3.9 (median) and 8.0 +/- 10.4 (mean +/- SD) years. The time intervals shortened only a little over the years. At all times, interval-2 was significantly longer than interval-1. There were no differences between female (n = 328) and male (n = 80) patients and between the age groups. Furthermore, none of the gastrointestinal and non-gastrointestinal symptoms had had a distinct influence on all diagnostic intervals and also the fact that other family members having the disease did not shorten any of the intervals. In summary, the diagnostic intervals for recognizing celiac disease are still unacceptably long. More public awareness work has to be done so that patients can recognize their symptoms and doctors especially can suspect celiac disease sooner and perform the necessary diagnostic procedures when patients present with suggestive symptoms.

Pulmonary infiltrations. Sign of severe acute pancreatitis.

CONCLUSION: Pleural infiltrates are indicative of severe acute pancreatitis and a negative prognostic parameter for the course of the disease. BACKGROUND: The purpose of this study was to determine the frequency of pulmonary infiltrates in acute pancreatitis and their importance for evaluating the severity of the disease. METHODS: Incidence and localization of pulmonary infiltrates were evaluated in 140 patients with acute pancreatitis. Chest X-ray was obtained within 24 h, and a contrast-enhanced computed tomography within 72 h after admission. Ranson's prognostic parameters were estimated within the first 48 h. RESULTS: Patients with pulmonary infiltrates (n = 36, 26%) had a significantly higher Ranson's score and pancreatic necroses more frequently than patients without this complication, plus a higher mortality rate.

Drug induced acute pancreatitis: incidence and severity.

To determine the incidence and severity of drug induced acute pancreatitis, data from 45 German centres of gastroenterology were evaluated. Among 1613 patients treated for acute pancreatitis in 1993, drug induced acute pancreatitis was diagnosed in 22 patients (incidence 1.4%). Drugs held responsible were azathioprine, mesalazine/sulfasalazine, 2',3'-dideoxyinosine (ddI), oestrogens, frusemide, hydrochlorothiazide, and rifampicin. Pancreatic necrosis not exceeding 33% of the organ was found on ultrasonography or computed tomography, or both, in three patients (14%). Pancreatic pseudocysts did not occur. A decrease of arterial PO2 reflecting respiratory insufficiency, and an increase of serum creatinine, reflecting renal insufficiency as complications of acute pancreatitis were seen in two (9%) and four (18%) patients, respectively. Artificial ventilation was not needed, and dialysis was necessary in only one (5%) case. Two patients (9%) died of AIDS and tuberculosis, respectively; pancreatitis did not seem to have contributed materially to their death. In conclusion, drugs rarely cause acute pancreatitis, and drug induced acute pancreatitis usually runs a benign course.

Pleural effusions: a new negative prognostic parameter for acute pancreatitis.

OBJECTIVE: To evaluate the incidence, localization, and size of pleural effusions in 133 patients with acute pancreatitis. METHODS: A contrast-enhanced computed tomographic scan was prospectively obtained within 72 h after admission. RESULTS: Patients with pleural effusions (66 = 50%) had significantly more severe morphological changes of the pancreas and necroses (independent of the size and localization of the effusions and etiology of the acute pancreatitis), more often had a pancreatic pseudocyst, and had a higher mortality rate than patients without this complication. CONCLUSION: Pleural effusions are indicative of severe acute pancreatitis and are a negative prognostic parameter for the course of the disease.

Glycine effects on in vitro motor pattern generation in mouse spinal cord.

Bath applications of glycine typically inhibited electromyogram (EMG) activity in the gastrocnemius (G) and tibialis anterior (TA) muscles of neonatal mice, in vitro. Although rhythmic bursting occurred in response to glycine administration, cycle alternation between individual EMG bursts in G and TA muscles was not observed. That strychnine (a glycine 1 receptor antagonist) and cycloleucine (a glycine 2 receptor antagonist) could evoke motor rhythm, when given separately or in combination, confirms that glycine transmission is not required for motor pattern generation in mice. Strychnine application resulted in synchronized EMG bursting in G and TA muscles, suggesting that glycine 1 receptor activation does provide some reciprocal inhibition between the G and TA motor nuclei.

Distribution of NMDA receptor binding in developing mouse spinal cord.

The role of NMDA receptors in spinal sensorimotor processes has been studied in neonates and adults, while NMDA binding has previously been described only in mature spinal cord. In autoradiographs of mouse lumbar cord, NMDA-sensitive [3H]glutamate binding peaked transiently around postnatal days 6-10. Differentiation increased progressively from day 10 to adulthood. Mature levels were highest in substantia gelatinosa and moderate in dorsomedial dorsal horn. The results are described in relation to functional and anatomical features of developing spinal cord.

8-OH-DPAT in the midbrain central gray inhibits lordosis behavior.

Sexually receptive female rats were infused intracranially with 500-2,000 ng 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) into the midbrain central gray (MCG), in the vicinity of the dorsal raphe nucleus (DRN), or directly into the DRN. When cannulae were located within the DRN, there was little evidence of change in lordosis behavior but a decrease in locomotor activity was commonly observed. In contrast, when cannulae were located anterior, ventromedial, or lateral to the DRN inhibition of lordosis behavior was rapid and robust. Both the lordosis-to-mount ratio (L/M) and the quality of the lordosis reflex were reduced following the infusion. The MCG receives lordosis-facilitating input from the ventromedial nucleus of the hypothalamus and from ascending sensory pathways and contributes information to descending motor systems involved in the lordosis response. Thus, the MCG is a critical link in the completion of the estrogen-dependent lordosis reflex. The present results suggest that 5-hydroxytryptamine1A receptors in the MCG prevent the completion of this reflex.

Comparison of spontaneous motor pattern generation in non-hemisected and hemisected mouse spinal cord.

Spontaneous electromyogram (EMG) patterns in the gastrocnemius (G) and tibialis anterior (TA) muscles of spinal cord-hindlimb explants from neonatal mice were investigated. Compared to non-hemisected explants, neither longitudinal hemisection of the spinal cord nor hemisection plus transection at L1 significantly altered the incidence of spontaneous motor rhythm. Therefore, not only does each half of the neonatal spinal cord contain sufficient circuitry to generate motor rhythm, but the more reduced preparations were just as likely to produce such activity. Hemisected preparations, however, exhibited slower rhythm, perhaps due to the loss of excitatory commissural connections. No correlation was found between the number of cycles in a rhythmic sequence and cycle period. In hemisected as well as non-hemisected explants, sequences of spontaneous EMG rhythm occurred in either the G or TA muscle, but not in both muscles simultaneously. Consequently, cycle-to-cycle alternation between rhythmic bursting in the G and TA muscles was not observed. The excitability in such preparations was apparently insufficient for maintained activations of both muscles (either for cycle-to-cycle alternation or for co-contraction).

Effects of the 5-HT1A agonist, 8-OH-DPAT, on sexual behaviors of the proestrous rat.

The effects of the 5-HT1A agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), were examined in intact, proestrous rats. Although this compound has been reported to inhibit sexual receptivity of hormonally primed, ovariectomized rats, this is the first report of its effect in intact females. After intraperitoneal treatment with 8-OH-DPAT (0.01 to 0.25 mg/kg), there was a dose-dependent suppression of lordosis behavior. The inhibition occurred within 10-15 min after the higher doses and lasted at least an hour after treatment. When females were treated with 1 mg/kg trifluoromethylphenyl piperazine (TFMPP) 30 min prior to treatment with 0.1 mg/kg 8-OH-DPAT, females recovered more rapidly from the inhibitory effects of 8-OH-DPAT. After bilateral, intrahypothalamic infusion of 50 to 1000 ng 8-OH-DPAT, inhibition of sexual behavior resembled that seen following systemic treatment with 0.1 mg/kg 8-OH-DPAT, females recovered more rapidly from the inhibitory effects of 8-OH-DPAT. After bilateral, intrahypothalamic infusion of 50 to 1000 ng 8-OH-DPAT, inhibition of sexual behavior resembled that seen following systemic treatment. Cannula locations in the ventromedial hypothalamus, but not the posterior hypothalamus, produced rapid inhibition of lordosis behavior. Both the frequency and the quality of lordosis behavior were reduced within 5 to 10 min after bilateral infusion of 200 to 1000 ng (but not 50 ng) 8-OH-DPAT, and females often successfully avoided attempted mounts by the male. These results suggest that activation of ventromedial hypothalamic 5-HT1A receptors reduces lordosis behavior.