Three distinct modes of exocytosis revealed by amperometry in neuroendocrine cells.

Neurotransmission requires Ca(2+)-dependent release of secretory products through fusion pores that open and reclose (partial membrane distention) or open irreversibly (complete membrane distention). It has been challenging to distinguish between these release modes; however, in the work presented here, we were able to deduce different modes of depolarization-evoked exocytosis in neuroendocrine chromaffin and PC12 cells solely by analyzing amperometric recordings. After we determined the quantal size (Q), event half-width (t(50)), event amplitude (I(peak)), and event decay time constant (τ(decay)), we fitted scatter plots of log-transformed data with a mixture of one- and two-dimensional Gaussian distributions. Our analysis revealed three distinct and differently shaped clusters of secretory events, likely corresponding to different modes of exocytosis. Complete membrane distention, through fusion pores of widely varying conductances, accounted for 70% of the total amount of released catecholamine. Two different kinds of partial membrane distention (kiss-and-run and kiss-and-stay exocytosis), characterized by mode-specific fusion pores with unitary conductances, accounted for 20% and 10%, respectively. These results show that our novel one- and two-dimensional analysis of amperometric data reveals new release properties and enables one to distinguish at least three different modes of exocytosis solely by analyzing amperometric recordings.

Use of highly discriminatory fingerprinting to analyze clusters of Clostridium difficile infection cases due to epidemic ribotype 027 strains.

We compared multilocus variable-number tandem-repeat analysis (MLVA) and macrorestriction endonuclease analysis using pulsed-field gel electrophoresis (PFGE) to determine their utility to identify clusters of Clostridium difficile infection (CDI) among 91 isolates of PCR ribotype 027 (NAP1, for North American pulsed-field type 1) from nine hospitals (and 10 general practitioners associated with one institution) in England. We also examined whether mortality in CDI cases was associated with specific MLVA subtypes. PFGE discriminated between ribotype 027 strains at >98% similarity, identifying five pulsovars (I to V) of 1 to 53 isolates. MLVA was markedly more discriminatory, identifying 23 types of 1 to 15 isolates (>71% similarity). PFGE pulsovars I and IV contained 14 and 8 MLVA types, respectively. Twenty-one of twenty-three (91%) of MLVA types were specific to individual PFGE pulsovars. Four CDI clusters were identified in institution A by conventional epidemiological analysis. MLVA typing identified two enlarged and two additional clusters. Thirty of forty-four (68%) patients in institution A with CDI caused by ribotype 027 strains were assigned to seven distinct clusters by a combination of MLVA typing and epidemiological records. Of 33 patients, comprising 14 different MLVA types, nine (27%) died by day 30 (early deaths). Eight of nine (89%) were associated with PFGE type IV C. difficile ribotype 027. Five of nine early deaths were associated with MLVA type 16, which was the dominant type in this cohort (10/33 cases); 4 other distinct MLVA types accounted for the other early deaths. MLVA was far superior to PFGE for analyzing clusters of CDI both within and between institutions. Further study is needed to examine whether subtypes of C. difficile ribotype 027 affect outcome.

Enzymatic enantioselective C-C-bond formation in microreactors.

We have demonstrated that multiple crude enzyme lysates containing a hydroxynitrile lyase can be used for the enantioselective synthesis of cyanohydrins from aldehydes in microchannels. Using a microreactor setup, two important parameters were efficiently screened consuming only minute amounts of reagents. More importantly, results from the continuous flow reaction were fully consistent with results obtained from larger batchwise processes in which a stable emulsion was formed.

Design and Synthesis of Quenched Activity-based Probes for Diacylglycerol Lipase and α,ß-Hydrolase Domain Containing Protein 6.

Diacylglycerol lipases (DAGL) are responsible for the biosynthesis of the endocannabinoid 2-arachidonoylglycerol. The fluorescent activity-based probes DH379 and HT-01 have been previously shown to label DAGLs and to cross-react with the serine hydrolase ABHD6. Here, we report the synthesis and characterization of two new quenched activity-based probes 1 and 2, the design of which was based on the structures of DH379 and HT-01, respectively. Probe 1 contains a BODIPY-FL and a 2,4-dinitroaniline moiety as a fluorophore-quencher pair, whereas probe 2 employs a Cy5-fluorophore and a cAB40-quencher. The fluorescence of both probes was quenched with relative quantum yields of 0.34 and 0.0081, respectively. The probes showed target inhibition as characterized in activity-based protein profiling assays using human cell- and mouse brain lysates, but were unfortunately not active in living cells, presumably due to limited cell permeability.

Characteristics and incidence of Clostridium difficile-associated disease in The Netherlands, 2005.

During a 2-month period in 2005, 13 laboratories participated in a surveillance study of Clostridium difficile-associated disease (CDAD) in 17 hospitals in The Netherlands. The median incidence rate of CDAD was 16/10 000 patient admissions (2.2/10 000 patient-days) and varied from 1 to 46/10 000 patient admissions according to hospital. In total, 81 patients with CDAD were reported; 49 (61%) patients had nosocomial CDAD, and 29 (36%) patients were admitted to hospital when already suffering from diarrhoea. Two (2%) deaths were attributable to CDAD; both of these patients were admitted with severe community-onset CDAD and were aged >80 years. Among 64 toxinogenic isolates, ten (16%) belonged to PCR ribotype 027 and ten (16%) to PCR ribotype 014. Type 027 was identified in ten patients from one hospital during an unrecognised outbreak. Toxinotyping of the 64 isolates revealed the presence of six different toxinogenic types, with 41 (64%) isolates of toxinotype 0, ten (16%) isolates of toxinotype III, and nine (14%) isolates of toxinotype V. Of the 64 toxinogenic isolates, seven (11%) had a 39-bp deletion in the tcdC gene, 11 (17%) had an 18-bp deletion, and one (1%) had a deletion of c. 44 bp. Genes for binary toxin were present in 21 (33%) of the 64 toxinogenic isolates, mainly associated with toxinotypes III and V. It was concluded that the median CDAD incidence rate of 16/10 000 patient admissions in The Netherlands is considerably lower than that in Canada and the USA, and that the emerging type 027 can spread unnoticed. The high proportion (36%) of CDAD cases with a community onset has important implications for future studies of the epidemiology of CDAD.

Pores in synthetic nerve conduits are beneficial to regeneration.

Current opinion holds that pores in synthetic nerve guides facilitate nerve regeneration. Solid factual support for this opinion, however, is absent; most of the relevant studies assessed only morphological parameters and results have been contradictory. To evaluate the effect of pores, the rat sciatic nerve was either autografted or grafted with nonporous, macroporous (10-230 mum), and microporous (1-10 microm) biodegradable epsilon-caprolactone grafts. Twelve weeks later, the grafted nerves were resected, and the electrophysiological properties were determined in vitro. Subsequently midgraft-level sections were inspected, and peroneal nerve sections were evaluated morphometrically. Finally, the gastrocnemic and tibial muscle morphometrical properties were quantified. The microporous nerve graft performed much better than the nonporous and macroporous grafts with respect to most parameters: it was bridged by a free floating bundle that contained myelinated nerve fibers, there were more nerve fibers present distal to the graft, the electrophysiological response rate was higher, and the decrease in muscle cross-sectional area was markedly smaller. Hence, the present study demonstrates the beneficial effect of synthetic nerve guide pores on nerve regeneration, although with the caveat that not pores per se, but only small (1-10 microm) pores were effective.

Rapid diagnosis of toxinogenic Clostridium difficile in faecal samples with internally controlled real-time PCR.

A real-time PCR assay for Clostridium difficile was developed, based on the tcdB gene, which detected all known toxinogenic reference strains (n = 45), within 30 serogroups and 24 toxinotypes. The analytical sensitivity was 1 x 10(3) CFU/mL, and the detection limit in faeces was 1 x 10(5) CFU/g. The optimal protocol for DNA extraction from faecal samples involved use of the MagnaPure system with a Stool Transport and Recovery (STAR) buffer pre-treatment. In a 1-month prospective study of 85 patients with diarrhoea, the sensitivity, specificity and positive and negative predictive values of the assay were 100%, 94%, 55% and 100%, respectively, compared with the standard cell cytotoxicity assay.

Interaction between ultraviolet A and ultraviolet B radiations in skin cancer induction in hairless mice.

The rate of tumor induction by UV-A radiation rises more slowly with time and accumulated dose than that by UV-B radiation. It has recently been shown that this difference disappears when frank papillomas are excluded from the analysis. Thus, the rate of development of "nonpapillomas" (mainly squamous cell carcinomas and precursors) can be fully characterized by a typical tumor induction time, e.g., the time until 50% of the mice bear tumors. This has opened the possibility to investigate how UV-A and UV-B exposures add up in the induction of squamous cell carcinomas, which is an important issue in risk assessments of artificial UV-A sources for cosmetic or medical purposes. We present the results of an experiment in which 6 groups of 24 albino SKH:HR1 mice were treated daily for 600 days with either effective UV-A radiation, effective UV-B radiation, or combinations of both. The observed times it took for 50% of the mice to bear tumors in the combination groups were compared with those calculated on the basis of arithmetical addition of effective UV-A and effective UV-B doses. We did not find a statistically significant (P < 0.05) deviation from additivity.

Relative susceptibilities of XPA knockout mice and their heterozygous and wild-type littermates to UVB-induced skin cancer.

Although xeroderma pigmentosum (XP) patients are rare, carriers of XP genes (heterozygotes) are much more common. Whether such carriers have an increased skin cancer risk is unknown. Recently developed mouse models for XP have opened up the possibility of determining the skin cancer risk of heterozygotes relative to wild types. Therefore, the XPA knockout trait has been crossed into hairless mice, and squamous cell carcinomas of the skin have been induced by low daily UVB exposures for 500 days in all three genotypes (-/-, +/-, and +/+). The carcinogenic response of the heterozygotes did not significantly differ from that of their wild-type littermates. Tumors in the XPA -/- animals appeared with a latency time that was decreased by a factor of 4.2. From this, we estimate that a functional XPA gene provides a "protection factor" of 60 (95% confidence interval, 15-250) against UV carcinogenesis, which is greater protection than that against acute UV effects, such as erythema and edema (protection factor between 7 and 16). Deficient nucleotide excision repair appears to have a more dramatic impact on skin cancer susceptibility than on sensitivity to acute UV effects.

Impact of global genome repair versus transcription-coupled repair on ultraviolet carcinogenesis in hairless mice.

The nucleotide excision repair (NER) system is comprised of two subpathways, i.e., transcription-coupled repair (TCR) and global genome repair (GGR). To establish the relative importance of TCR and GGR for UV effects on the skin, we have used hairless knockout mouse strain lacking either TCR (CSB -/-) or GGR (XPC -/-). In single exposure experiments, we found that CSB -/- mice have a 7-16 times higher susceptibility to sunburn than XPC -/- mice and than heterozygous (+/-) and wild-type (+/+) controls. Exposure to 80 J/m2 UV radiation (i.e., suberythemogenic in CSB -/-) on 10 consecutive days gives rise to epidermal hyperplasia in CSB -/- and XPC -/-, whereas repair-proficient controls do not show epidermal hyperplasia from these exposures. In addition, CSB -/- mice develop marked parakeratosis, whereas XPC -/- mice and controls do not. Under continued exposure to this daily dose, squamous cell carcinomas appear in CSB -/-, XPC -/-, and in the control groups, whereas only in the CSB -/- animals is a fairly high number of benign papillomas also found. The median latency time of squamous cell carcinomas (diameters > or = 1 mm) is 84 days for the XPC -/- mice, 115 days for the CSB -/- mice, and 234-238 days for the heterozygous and wild-type control groups. These results indicate that GGR is more important than TCR in protection against UV-induced carcinomas of the skin but not against other UV effects such as sunburn, epidermal thickening, scaling of the stratum corneum, and development of papillomas. These results also indicate that GGR capacity may serve as a better predictor for skin cancer susceptibility than sensitivity to sunburn. The relative cancer susceptibilities of GGR- and TCR-deficient skin could well depend on the balance between an increased mutation rate and the presence (in CSB -/-) or lack (in XPC -/-) of a compensatory apoptotic response.

Early p53-positive foci as indicators of tumor risk in ultraviolet-exposed hairless mice: kinetics of induction, effects of DNA repair deficiency, and p53 heterozygosity.

p53 mutations appear to be early events in skin carcinogenesis induced by chronic UVB irradiation. Clusters of epidermal cells that express p53 in mutant conformation ("p53 positive foci") are easily detected by immunohistochemical staining long before the appearance of skin carcinomas or their precursor lesions. In a hairless mouse model, we determined the dose-time dependency of the induction of these p53+ foci and investigated the relationship with the induction of skin carcinomas. The density of p53+ foci may be a good direct indicator of tumor risk. Hairless SKH1 mice were exposed to either of two regimens of daily UVB (500 or 250 J/m2 broadband UV from Philips TL12 lamps; 54% UVB 280-315 nm). With the high-dose regimen, the average number of p53+ foci in a dorsal skin area (7.2 cm2) increased rapidly from 9.0 +/- 2.1 (SE) at 15 days to 470 +/- 80 (SE) at 40 days. At half that daily dose, the induction of p53+ foci was slower by a factor of 1.49 +/- 0.15, very similar to a previously observed slower induction of squamous cell carcinomas by a factor of 1.54 +/- 0.02. In a double-log plot of the average number of p53 + foci versus time, the curves for the two exposure regimens ran parallel (slope, 3.7 +/- 0.7), similar to the curves for the number of tumors versus time (slope, 6.9 +/- 0.8). The difference in slopes (3.7 versus 6.9) is in line with the contention that more rate-limiting steps are needed to develop a tumor than a p53+ focus. By the time the first tumors appear (around 7-8 weeks with the high daily dose), the dorsal skin contains >100 p53+ foci/cm2. To further validate the density of p53+ foci as a direct measure of tumor risk, we carried out experiments with transgenic mice with an enhanced susceptibility to UV carcinogenesis, homozygous Xpa knockout mice (deficient in nucleotide excision repair) and heterozygousp53 knockout mice (i.a. partially deficient in apoptosis). In both of these cancer-prone strains, the p53+ foci were induced at markedly increased rates, corresponding to increased rates of carcinoma formation. Therefore, the frequency of p53+ foci appears to correlate well with UVB-induced tumor risk.

Ultraviolet-induced cyclobutane pyrimidine dimers are selectively removed from transcriptionally active genes in the epidermis of the hairless mouse.

This study describes the induction and repair of UV-induced cyclobutane pyrimidine dimers (CPD) in transcriptionally active and inactive genes in the epidermis of the hairless mouse. Mice were exposed to a single dose of 2000 J/m2 ultraviolet B and kept in darkness for up to 24 h. The CPD frequency was measured in the transcriptionally active hypoxanthine-guanine phosphoribosyltransferase gene, the adenosine deaminase gene, the inactive c-mos protooncogene, and the haptoglobin gene using the CPD-specific enzyme T4 endonuclease V. Sixty % of the CPD was removed from the active genes during the first 4 h, after which no further repair took place up to 24 h. In contrast, the inactive genes did not show any removal of CPD. Assuming that the rate of repair in the c-mos and haptoglobin genes is representative for the repair rate in the genome overall, these results suggest only marginal repair of UV-induced CPD in the mouse epidermis in vivo. The selective repair of active genes in the epidermis of mice resembles that of rodent cells in culture and shows the biological relevance of repair studies performed with cultured rodent cells in vitro.

Low incidence of p53 mutations in UVA (365-nm)-induced skin tumors in hairless mice.

Mutations with clear "UVB fingerprints" have been observed in the p53 gene of human nonmelanoma skin tumors and of experimentally UVB-induced murine skin tumors. Although UVA (315-400 nm) radiation is also a complete carcinogen, its contribution to sunlight-induced mutagenesis remains poorly characterized. There is experimental evidence that the production of reactive oxygen species plays a more dominant role with long-wave UVA than with UVB radiation. We have induced skin tumors (n = 42) in hairless SKH:HR1 mice (n = 14) by daily exposure to long-wave UVA (365-nm) radiation. The incidence of p53 alterations in these tumors is low compared to UVB-induced tumors; positive staining for the p53 protein was observed in only 50% of the tumors, and less than 15% of the tumors showed a mutation in one of the exons 5, 7, or 8 of the p53 gene. The pattern of p53 staining was more irregular and less dense compared to UVB, and the mutations (all C-->T) were mainly (six of seven) located at codon 267. Besides a general p53 hotspot, this codon is also the main hotspot for UVB-induced skin tumors in these mice. No mutations specific for UVA, ie., mutations specific for reactive oxygen species, could be detected.

Mouse model for the DNA repair/basal transcription disorder trichothiodystrophy reveals cancer predisposition.

Patients with the nucleotide excision repair (NER) disorder xeroderma pigmentosum (XP) are highly predisposed to develop sunlight-induced skin cancer, in remarkable contrast to photosensitive NER-deficient trichothiodystrophy (TTD) patients carrying mutations in the same XPD gene. XPD encodes a helicase subunit of the dually functional DNA repair/basal transcription complex TFIIH. The pleiotropic disease phenotype is hypothesized to be, in part, derived from a repair defect causing UV sensitivity and, in part, from a subtle, viable basal transcription deficiency accounting for the cutaneous, developmental, and the typical brittle hair features of TTD. To understand the relationship between deficient NER and tumor susceptibility, we used a mouse model for TTD that mimics an XPD point mutation of a TTD patient in the mouse germline. Like the fibroblasts from the patient, mouse cells exhibit a partial NER defect, evident from the reduced UV-induced DNA repair synthesis (residual repair capacity approximately 25%), limited recovery of RNA synthesis after UV exposure, and a relatively mild hypersensitivity to cell killing by UV or 7,12-dimethylbenz[a]anthracene. In accordance with the cellular studies, TTD mice exhibit a modestly increased sensitivity to UV-induced inflammation and hyperplasia of the skin. In striking contrast to the human syndrome, TTD mice manifest a dear susceptibility to UV- and 7,12-dimethylbenz[a]anthracene-induced skin carcinogenesis, albeit not as pronounced as the totally NER-deficient XPA mice. These findings open up the possibility that TTD is associated with a so far unnoticed cancer predisposition and support the notion that a NER deficiency enhances cancer susceptibility. These findings have important implications for the etiology of the human disorder and for the impact of NER on carcinogenesis.

XPA-deficiency in hairless mice causes a shift in skin tumor types and mutational target genes after exposure to low doses of U.V.B.

Xeroderma pigmentosum (XP) patients with a defect in the nucleotide excision repair gene XPA, develop tumors with a high frequency on sun-exposed areas of the skin. Here we describe that hairless XPA-deficient mice also develop skin tumors with a short latency time and a 100% prevalence after daily exposure to low doses of U.V.B. Surprisingly and in contrast to U.V.B.-exposed repair proficient hairless mice who mainly develop squamous cell carcinomas, the XPA-deficient mice developed papillomas with a high frequency (31%) at a U.V. dose of 32 J/m2 daily. At the highest daily dose of 80 J/m2 mainly squamous cell carcinomas (56%) and only 10% of papillomas were found in XPA-deficient hairless mice. p53 gene mutations were examined in exons 5, 7 and 8 and were detected in only 3 out of 37 of these skin tumors, whereas in tumors of control U.V.B.-irradiated wild type littermates this frequency was higher (45%) and more in line with our previous data. Strikingly, a high incidence of activating ras gene mutations were observed in U.V.B.-induced papillomas (in 11 out of 14 tumors analysed). In only two out of 14 squamous cell carcinomas we found similar ras gene mutations. The observed shift from squamous cell carcinomas in wild type hairless mice to papillomas in XPA-deficient hairless mice, and a corresponding shift in mutated cancer genes in these tumors, provide new clues on the pathogenesis of chemically- versus U.V.B.-induced skin carcinogenesis.

Defective global genome repair in XPC mice is associated with skin cancer susceptibility but not with sensitivity to UVB induced erythema and edema.

It is generally presumed that xeroderma pigmentosum (XP) patients are extremely sensitive to developing UV erythema, and that they have a more than 1000-fold increased skin cancer risk. Recently established mouse models for XP can be employed to investigate the mechanism of these increased susceptibilities. In line with human data, both XPA and XPC knockout mice have been shown to have an increased susceptibility to UVB induced squamous cell carcinomas. In XPA knockouts, nucleotide excision repair of UV induced DNA photolesions is completely defective (i.e., both global genome repair and transcription coupled repair are defective). We determined the strand specific removal of cyclobutane pyrimidine dimers and pyrimidine [6-4] pyrimidone photoproducts from the p53 gene in cells from XPC knockout mice and wild-type littermates. Analogous to human XPC cells, embryonic fibroblasts from XPC knockout mice are only capable of performing transcription coupled repair of DNA photolesions. We show that these XPC knockout mice, in striking contrast to XPA knockout mice, do not have a lower minimal erythema/edema dose than their wild-type littermates. Hence, defective global genome repair appears to lead to skin cancer susceptibility, but does not influence the sensitivity to acute effects of UVB radiation, such as erythema and edema. The latter phenomena thus relate to the capacity to perform transcription coupled repair, which suggests that blockage of RNA synthesis is a key event in the development of UV erythema and edema.

Detection of thymine dimers in suprabasal and basal cells of chronically UV-B exposed hairless mice.

An immunocytochemical method was developed to study induction and removal of DNA damage in specific cell populations in the epidermis of hairless mice during chronic ultraviolet (UV) exposure. Identification of mouse suprabasal cells was performed with an immunoperoxidase stain. This stain was shown not to affect the fluorescent nuclear stains, used to reveal DNA and DNA damage. In skin cells from hairless mice irradiated daily with 1500 J/m2 UV-B for 11 consecutive days, cyclobutane thymine dimers accumulated in epidermal cells and reached a maximum level after 3 d. Thereafter dimer levels dropped to a lower, more constant level. So epidermal cells in vivo, both suprabasal and basal cells, remove dimers effectively, in contrast to cultured rodent cells, which display hardly any repair in genomic DNA. Dimer content in suprabasal cells was higher than that in basal cells, but initially the patterns of induction and removal of dimers in both cell types were rather similar. At days 4-11, however, after the drop in dimer content, the amount of dimers in basal cells prior to UV exposure was almost as low as that in non-exposed cells. The results presented here suggest important roles for both UV-induced DNA repair and cell proliferation in protecting epidermal cells against the mutagenic and carcinogenic effects of UV.

Resting membrane potentials and excitability at different regions of rat dorsal root ganglion neurons in culture.

To study the role of electrical membrane processes in neuronal regeneration and growth, resting membrane potentials and action potentials of sensory (dorsal root ganglion) neurons growing in culture were measured at the soma, neurite and growth cone using the whole-cell patch-clamp technique. Our results show that resting membrane potentials measured at the soma (-56.8 +/- 8.8 mV), neurite varicosity (-55.8 +/- 5.2 mV) and growth cone (-57.2 +/- 4.1 mV) of growing neurons were not statistically different. The membrane resistance measured around the resting membrane potential at the neurite varicosity (160 +/- 70 M omega) was smaller than those at the soma (687 +/- 540 M omega) and growth cone (922 +/- 825 M omega). The resting membrane potential measured at the soma using a perforated patch (-60.3 +/- 4.4 mV) was not different from that measured in the normal whole cell. In both configurations, isotonic KCl (140 mM) depolarized the membrane potential to above 0 mV. The K+ channel blockers quinine, Cs+, 4-aminopyridine and tetraethylammonium depolarized the membrane potential by 10-40 mV, while Na(+)-free extracellular solution hyperpolarized it by about 10 mV. Extracellularly applied ouabain, intracellular Na(+)-free or low Cl(-)-containing solutions did not affect the resting membrane potential. Similar results were obtained for growth cones. Action potentials could be evoked by current pulses in 81% of somata and in all growth cones, but not in neurite varicosities. Current-induced repetitive firing was found in 19% of somata and in 65% of growth cones.(ABSTRACT TRUNCATED AT 250 WORDS)

Ionic currents in cultured rat suprachiasmatic neurons.

Whole-cell voltage-clamp recordings were made from cultured neurons obtained by dissociation of the suprachiasmatic area of rat fetuses. Neurons were held for seven to 14 days in culture. These neurons possessed several voltage-dependent ionic currents. A transient inward Na+ current was present, which could be completely blocked by tetrodotoxin. No inward Ca2+ currents were detected. Three types of outward K+ currents were recorded, which could be separated to a reasonable extent by their differences in voltage sensitivity and pharmacology. These K+ currents corresponded to the transient current IA, the delayed rectifier current IKo and a calcium-dependent current IK(Ca) as described in other neurons. The A current activated at -50 mV, reached half-maximal conductance at about -30 mV and maximum conductance between 0 and 30 mV. During depolarizing steps it inactivated completely within 100 ms and steady-state inactivation was half-maximal at -66 mV. The outward rectifier activated at -30 mV, reached half-maximal conductance close to 0 mV and maximum conductance at about 70 mV. Slow inactivation of IKo occurred with 50% reduction in amplitude at the end of 2 s depolarizations above 0 mV. The K+ channel blocker 4-amino-pyridine (4 mM) reduced the amplitude of IA by 21% and of IKo by 32%, whereas tetraethylammonium (10 mM) decreased IA by 27% and IKo by 83%. The calcium-dependent K+ component was also voltage dependent and was present at voltages more positive than 0 mV. No inward rectifying K+ current was present. Considering its voltage dependence, IA must play a role in determining the excitability of these neurons, through its probable influence on the action potential threshold and interspike interval. Both IA and IKo should take part in membrane repolarization following an action potential. The Ca(2+)-dependent current should also contribute to repolarization following any event which gives rise to an increase in intracellular Ca2+. Apart from IA, which may make a slight contribution, none of these currents appear to be involved in determining the resting membrane potential. All three outward current components will act together in suprachiasmatic neurons to control their spontaneous firing frequency, which is the major feature of the output of these neurons in vivo. Variations in properties of these conductances could contribute to the circadian rhythm in firing frequency described in suprachiasmatic hypothalamic neurons.

The urinary excretion of prostaglandins and thromboxane B2 in healthy volunteers: a study in males and females, and on the influence of seminal fluid contamination.

Radioimmunoassay measurements of prostaglandins (PGs) E2, F2 alpha, 6-keto-PGF1 alpha and thromboxane (Tx) B2 in 24 h urine specimens from a male and a female healthy volunteer on several consecutive days revealed a dramatic increase of PGE2, PGF2 alpha, 6-keto-PGF1 alpha on days, upon which they had sexual intercourse; only TxB2 remained stable. Furthermore, the PGE2/PGF2 alpha ratio rose to values greater than 0.5 on days with sexual intercourse. This was found to be due to contamination of the urine samples by seminal fluid. Two 24 h urine samples from each of 26 healthy male and female volunteers (HV) revealed higher (p less than 0.01) mean PGE2 and PGF2 alpha values in males than in females. The results show that the interpretation of the urinary PG excretion as a measure of renal PG synthesis should be considered carefully, and that a PGE2/PGF2 alpha ratio greater than 0.5 indicates probable seminal contamination of urine.