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An improved nucleic acid amplification test (NAAT) to detect Chlamydia trachomatis infections, based on PCR amplification within its cryptic plasmid (CT1/CT2 Test) was developed. DNA was extracted from urogenital swabs and a 594-bp long DNA fragment from the cryptic plasmid (pCT) was amplified. The sensitivity and specificity of the CT1/CT2 Test were determined to be 100 and 99 %, respectively, when directly compared with current amplification kit for sexually transmitted diseases (MPCR). Basic epidemiological data related to the patients attending gynecological and/or urological clinics are also provided. The overall prevalence rate in this group of patients suspected for C. trachomatis infection was determined to be about 95 per 1000 (88 and 107 per 1000 in females and males, respectively). It demonstrates that the CT1/CT2 Test is suitable for epidemiological screening and/or diagnostic practice. [ABSTRACT FROM AUTHOR]

A simple nucleic acid amplification test (NAAT) was developed for detection of Ureaplasma urealyticum infection based on the PCR amplification of the urease gene (UU1/UU2 Test). DNA was extracted from urogenital swabs and a 225-bp long DNA fragment was amplified by PCR. NAAT was compared to the commercial amplification kit for sexually transmitted disease reference assay. The sensitivity and specificity of the UU1/UU2 Test were determined to be 100 and 98.9 %, respectively. The overall prevalence rate in this group of patients was found to be about 236 per 1000 (283 and 166 per 1000 in females and males, respectively). These data demonstrate that UU1/UU2 Test is suitable for effective epidemiological screening and/or diagnostic practice. [ABSTRACT FROM AUTHOR]

To examine the effects of the activation of adenosine 5′-triphosphate (ATP)-sensitive K channels in a skeletal muscle we have applied the ATP-sensitive K channel opener SR44866 whilst recording single ion channels, voltage-clamped membrane currents, evoked action potentials and tension in sartorius muscles of the frog. In excised inside-out membrane patches SR44866 opened channels which could be inhibited by internal ATP and glibenclamide. In voltage-clamped individual muscle fibres SR44866 evoked a glibenclamide-sensitive membrane current which reversed at −70 mV. The effect of SR44866 was dose dependent with an effective concentration for 50% maximal effect (EC) of 67 μM and a slope factor of 2. SR44866 dose dependently reduced the duration of the spike after-potential, spike overshoot, V, tetrodotoxin-sensitive voltage-gated inward membrane currents and muscle twitch tension. From this evidence it can be concluded that the opening of ATP-sensitive K channels may be associated with the inhibition of contraction of skeletal muscle. [ABSTRACT FROM AUTHOR]

Mouse pancreatic fragments were loaded withCa and placed in a flow cell. The concentration ofCa in the effluent was measured. The effects of changing the tension of carbon dioxide onCa efflux were observed and compared with effects of pancreatic secretagogues. The normal control solution was equilibrated with 5% CO, 95% O. Shift to solutions equilibrated with 10, 20, 50 or 100% CO evoked a dose-dependent increase in fractionalCa efflux, with a just detectable effect at 10% and a maximal one at 50%. The CO-evoked Ca release was not due to anoxia, since a short period of exposure to a 100% N-equilibrated solution had no effect. A decrease in extracellular pH (tris buffering) had only a very modest effect onCa efflux. CO-evoked Ca release under conditions avoiding extracellular pH changes (20% CO, 100 mM NaHCO). This CO-evoked enhancedCa efflux was sustained during a 30 min stimulation period, but was abruptly terminated on return to the control solution (5% CO, 25 mM NaHCO). NH (10 mM) added to the 20% CO-equilibrated solution for a brief interval in the middle of a period of CO-evoked enhancedCa efflux evoked a rapid return of the fractional Ca efflux towards the resting level. This effect was rapidly reversible. While the CO-evoked Ca release was largely sustained, the ACh-evoked increase inCa fractional efflux was entirely transient. The CO-evoked Ca release was not inhibited by a background of sustained ACh stimulation. ACh-evoked Ca release, however, was markedly inhibited in the presence of sustained CO stimulation. 2,4 Dinitrophenol (1 mM) in combination with iodoacetate (2 mM), while markedly reducingCa uptake into the fragments during the loading period had little or no effect on the ACh-evoked increase inCa fractional efflux. The CO-evoked Ca release, however, was markedly reduced by these metabolic inhibitors. The local anaesthetic procaine (1 mM) virtually abolished ACh- or caerulein-evoked Ca release without having any influence on the CO effect. It is concluded that CO releases Ca from pancreatic acinar cells by means of intracellular acidification. This effect may in part be due to H displacement of Ca from intracellular membrane binding sites and partly due to release of Ca from compartments (organelles) into which Ca has been actively accumulated. [ABSTRACT FROM AUTHOR]

Sustained intra-acinar microiontophoretic injection of the fluorescent dye Lucifer Yellow CH in isolated fragments of mouse pancreas reveals a finite limit to the extent of intercellular communication between acinar cells. In two preparations for which complete sets of serial sections could be obtained the dye-coupled intercommunicating acinar units consisted of 110 and 230 individual exocrine acinar cells. [ABSTRACT FROM AUTHOR]

Direct cell to cell movement of the fluorescent dye Lucifer Yellow CH (457 daltons) in exocrine acinar tissue is demonstrated by direct observation of living mouse pancreatic segments. Electrical uncoupling of pancreatic acinar cells by local application of a high concentration of acetylcholine significantly restricts cell to cell passage of the fluorescent dye. This result shows that a secretagogue can control direct movement of organic molecules between cells through junctional channels. [ABSTRACT FROM AUTHOR]

Cultured mouse embryonic stem (ES) cells are used for both in vitro and in vivo studies. The uncommitted pluripotent cells provide a model system with which to study cellular differentiation and development; they can also be used as vectors to carry specific mutations into the mouse genome by homologous recombination. To ensure successful integration into the germ line, competent totipotent diploid ES cell lines are selected using a cell injection bioassay that is both time consuming and technically demanding. The prolonged in vitro culture of rapidly dividing ES cells can lead to accumulated changes and chromosomal abnormalities that will compromise the biological function and abrogate germ line transmission of chimeric mice carrying novel genetic mutations. Such in vitro conditions will vary between individual laboratories; for example, differences in the serums used for maintenance. Using a number of different criteria we attempt in this paper to define the parameters that we found to be key factors for optimization of the biological potential of established ES cell lines. The successful integration into the germ line is dependant on acquiring or deriving a competent totipotent mouse ES diploid cell line. In this paper parameters and criteria are defined which we found to be key factors for the optimization of the biological potential of established ES cell lines. [ABSTRACT FROM AUTHOR]

The single-channel current recording technique has been used to study the influences that the pyridine nucleotides NAD, NADH, NADP and NADPH have on the gating of ATP-sensitive K+ channels in an insulin-secreting cell line (RINm5F). The effects of the nucleotides were studied at the intracellular surface using either excised inside-out membrane patches or permeabilized cells. All four pyridine nucleotides were found to evoke similar effects. At low concentrations, 100 microM and less, each promoted channel opening whereas high concentrations, 500 microM and above, evoked channel closure. The degree of K+ channel activation by pyridine nucleotides (low conc.) was found to be similar to that evoked by the same concentrations of ADP or GTP, whereas the degree of K+ channel inhibition (high conc.) was less marked than that evoked by the same concentrations of ATP, and never resulted in refreshment of K+ channels following removal. The effects of NAD, NADH, NADP and NADPH seemed to interact with those of ATP and ADP. In the presence of 1 mM ADP and 4 mM ATP, 10 to 100 microM concentrations of the pyridine nucleotides could not evoke channel opening, whereas concentrations of 500 microM and above were found to evoke channel closure. In the presence of 2 mM ATP and 0.5 mM ADP, however, 10 to 100 microM concentrations of the pyridine nucleotides were able to activate K+ channels. [ABSTRACT FROM AUTHOR]

The effects of ADP upon the gating of ATP-sensitive K+ channels from rat ventricular myocytes have been investigated by patch-clamp single-channel current recording experiments. ADP was applied to the internal surface of excised inside-out membrane patches and depending upon the experimental protocol and the concentration it was found that ADP could either inhibit or stimulate openings of ATP-sensitive K+ channels. In the absence of inactivation, ATP-sensitive K+ channels were inhibited by ADP in a dose-dependent manner. Partially inactivated channels, on the other hand, were stimulated by low (10 to 250 microM) and inhibited by high (greater than 250 microM) concentrations of ADP. ATP-sensitive K+ channels which were being inhibited by ATP (less than 1 mM) could be opened by the simultaneous application of ADP (50 microM to 1 mM). ADP had no effect upon channels inhibited by mM concentrations of ATP. The situation was further complicated when it was found that inhibition evoked by ADP was strongly attenuated by the presence of Mg2+ ions whilst channel stimulation, whether of partially inactivated channels or channels inhibited by ATP, required the presence of Mg2+ ions. The analog of ADP, ADP beta S, always evoked inhibition of ATP-sensitive K+ channels which was not affected by the presence or absence of Mg2+ ions. [ABSTRACT FROM AUTHOR]

The control of K+ channels in the insulin-secreting cell line RINm5F has been investigated by patch-clamp single-channel current recording experiments. The unitary current events recorded from cell-attached patches are due to large and small inwardly rectifying ATP-sensitive K+ channels with conductance properties similar to the two channels previously identified in primary cultured rat islet cells (Findlay, I., Dunne, M.J., & Petersen, O.H. J. Membrane Biol. 88:165-172, 1985). Cell permeabilization through brief exposure to 10 microM digitonin or 0.05% saponin (outside the isolated membrane patch area) results in a dramatic increase in current through the cell-attached patch due to opening of many large and small K+-selective channels. These channels are inhibited in a dose-dependent manner by ATP applied to the bath (near-complete inhibition by 5 mM ATP). During prolonged ATP exposure (1-5 min) the initial inhibition is followed by partial recovery of channel activity, although further activation does occur when ATP is subsequently removed. From the maximal number of coincident channel openings in the permeabilized cells (in the absence of ATP), it is estimated that there are on average 12 large ATP-sensitive K+ channels per membrane patch, but in the intact cells less than 5% of the membrane patches exhibited three or more coincident K+ channel openings, indicating the degree to which the channels are inhibited in the resting condition by endogenous ATP. Stimulation of RINm5F cells to secrete insulin was carried out by challenging intact cells with 10 mM D-glyceraldehyde.(ABSTRACT TRUNCATED AT 250 WORDS) [ABSTRACT FROM AUTHOR]

K+ channels in cultured rat pancreatic islet cells have been studied using patch-clamp single-channel recording techniques in cell-attached and excised inside-out and outside-out membrane patches. Three different K+-selective channels have been found. Two inward rectifier K+ channels with slope conductances of about 4 and 17 pS recorded under quasi-physiological cation gradients (Na+ outside, K+ inside) and maximal conductances recorded in symmetrical K+-rich solutions of about 30 and 75 pS, respectively. A voltage- and calcium-activated K+ channel was recorded with a slope conductance of about 90 pS under the same conditions and a maximal conductance recorded in symmetrical K+-rich solutions of about 250 pS. Single-channel current recording in the cell-attached conformation revealed a continuous low level of activity in an apparently small number of both the inward rectifier K+ channels. But when membrane patches were excised from the intact cell a much larger number of inward rectifier K+ channels became transiently activated before showing an irreversible decline. In excised patches opening and closing of both the inward rectifier K+ channels were unaffected by voltage, internal Ca2+ or externally applied tetraethylammonium (TEA) but the probability of opening of both inward rectifier K+ channels was reduced by internally applied 1-5 mM adenosine-5'-triphosphate (ATP). The large K+ channel was not operational in cell-attached membrane patches, but in excised patches it could be activated at negative membrane potentials by 10(-7) to 10(-6) M internal Ca2+ and blocked by 5-10 mM external TEA. [ABSTRACT FROM AUTHOR]

The Ca-activated K channel in rat pancreatic islet cells has been studied using patch-clamp single-channel current recording in excised inside-out and outside-out membrane patches. In membrane patches exposed to quasi-physiological cation gradients (Na outside, K inside) large outward current steps were observed when the membrane was depolarized. The single-channel current voltage ( I/V) relationship showed outward rectification and the null potential was more negative than −40 mV. In symmetrical K-rich solutions the single-channel I/V relationship was linear, the null potential was 0 mV and the singlechannel conductance was about 250 pS. Membrane depolarization evoked channel opening also when the inside of the membrane was exposed to a Ca-free solution containing 2 mm EGTA, but large positive membrane potentials (70 to 80 mV) were required in order to obtain open-state probabilities ( P) above 0.1. Raising the free Ca concentration in contact with the membrane inside ([Ca]) to 1.5×10 m had little effect on the relationship between membrane potential and P. When [Ca] was increased to 3×10 m and 6×10 m smaller potential changes were required to open the channels. Increasing [Ca] further to 8×10 m again activated the channels, but the relationship between membrane potential and P was complex. Changing the membrane potential from −50 mV to +20 mV increased P from near 0 to 0.6 but further polarization to +50 mV decreased P to about 0.2. The pattern of voltage activation and inactivation was even more pronounced at [Ca]=1 and 2 μ m. In this situation a membrane potential change from −70 to +20 mV increased P from near 0 to about 0.7 but further polarization to +80 mV reduced P to less than 0.1. The high-conductance K channel in rat pancreatic islet cells is remarkably sensitive to changes in [Ca] within the range 0.1 to 1 μ m which suggests a physiological role for this channel in regulating the membrane potential and Ca influx through voltage-activated Ca channels. [ABSTRACT FROM AUTHOR]

Real-time recording of the kinetics of systemically administered drugs in in vivo microenvironments may accelerate the development of effective medical therapies. However, conventional methods require considerable analyte quantities, have low sampling rates and do not address how drug kinetics correlate with target function over time. Here, we describe the development and application of a drug-sensing system consisting of a glass microelectrode and a microsensor composed of boron-doped diamond with a tip of around 40 μm in diameter. We show that, in the guinea pig cochlea, the system can measure-simultaneously and in real time-changes in the concentration of bumetanide (a diuretic that is ototoxic but applicable to epilepsy treatment) and the endocochlear potential underlying hearing. In the rat brain, we tracked the kinetics of the drug and the local field potentials representing neuronal activity. We also show that the actions of the antiepileptic drug lamotrigine and the anticancer reagent doxorubicin can be monitored in vivo. Our microsensing system offers the potential to detect pharmacological and physiological responses that might otherwise remain undetected.