The complete genome of the crenarchaeon Sulfolobus solfataricus P2.

The genome of the crenarchaeon Sulfolobus solfataricus P2 contains 2,992,245 bp on a single chromosome and encodes 2,977 proteins and many RNAs. One-third of the encoded proteins have no detectable homologs in other sequenced genomes. Moreover, 40% appear to be archaeal-specific, and only 12% and 2.3% are shared exclusively with bacteria and eukarya, respectively. The genome shows a high level of plasticity with 200 diverse insertion sequence elements, many putative nonautonomous mobile elements, and evidence of integrase-mediated insertion events. There are also long clusters of regularly spaced tandem repeats. Different transfer systems are used for the uptake of inorganic and organic solutes, and a wealth of intracellular and extracellular proteases, sugar, and sulfur metabolizing enzymes are encoded, as well as enzymes of the central metabolic pathways and motility proteins. The major metabolic electron carrier is not NADH as in bacteria and eukarya but probably ferredoxin. The essential components required for DNA replication, DNA repair and recombination, the cell cycle, transcriptional initiation and translation, but not DNA folding, show a strong eukaryal character with many archaeal-specific features. The results illustrate major differences between crenarchaea and euryarchaea, especially for their DNA replication mechanism and cell cycle processes and their translational apparatus.

Surface potentials measure ion concentrations near lipid bilayers during rapid solution changes.

We describe a puffing method for changing solutions near one surface of lipid bilayers that allows simultaneous measurement of channel activity and extent of solution change at the bilayer surface. Ion adsorption to the lipid headgroups and screening of the bilayer surface charge by mobile ions provided a convenient probe for the ionic composition of the solution at the bilayer surface. Rapid ionic changes induced a shift in bilayer surface potential that generated a capacitive transient current under voltage-clamp conditions. This depended on the ion species and bilayer composition and was accurately described by the Stern-Gouy-Chapman theory. The time course of solute concentrations during solution changes could also be modeled by an exponential exchange of bath and puffing solutions with time constants ranging from 20 to 110 ms depending on the flow pressure. During changes in [Cs+] and [Ca2+] (applied separately or together) both the mixing model and capacitive currents predicted [Cs+] and [Ca2+] transients consistent with those determined experimentally from: 1) the known Cs(+)-dependent conductance of open ryanodine receptor channels and 2) the Ca(2+)-dependent gating of ryanodine receptor Ca2+ channels from cardiac and skeletal muscle.

Response of ryanodine receptor channels to Ca2+ steps produced by rapid solution exchange.

We used a flow method for Ca2+ activation of sheep cardiac and rabbit skeletal ryanodine receptor (RyR) channels in lipid bilayers, which activated RyRs in < 20 ms and maintained a steady [Ca2+] for 5 s. [Ca2+] was rapidly altered by flowing Ca(2+)-buffered solutions containing 100 or 200 microM Ca2+ from a perfusion tube inserted in the cis, myoplasmic chamber above the bilayer. During steps from 0.1 to 100 microM, [Ca2+] reached 0.3 microM (activation threshold) and 10 microM (maximum Po) in times consistent with predictions of a solution exchange model. Immediately following rapid RyR activation, Po was 0.67 (cardiac) and 0.45 (skeletal) at a holding voltage of +40 mV (cis/trans). Po then declined (at constant [Ca2+]) in 70% of channels (n = 25) with time constants ranging from .5 to 15 s. The mechanism for Po decline, whether it be adaptation or inactivation, was not determined in this study. cis, 2 mM Mg2+ reduced the initial Po for skeletal RyRs to 0.21 and marginally slowed the declining phase. During very rapid falls in [Ca2+] from mM (inhibited) to sub-microM (sub-activating) levels, skeletal RyR did not open. We conclude the RyR gates responsible for Ca(2+)-dependent activation and inhibition of skeletal RyRs can gate independently.

Effects of diltiazem upon a rapidly exchanging calcium compartment related to repriming in frog skeletal muscle.

Following spontaneous relaxation, fast skeletal muscle must first repolarize and then undergo a first-order repriming reaction before depolarization will result in maximal tension production. 45Ca exposure during repriming defined two Ca compartments during subsequent efflux, named Ca(fast) and Ca(fast). Ca(slow) had an average time constant of 112 +/- 17 min. On the basis of slow turnover and content determined by a variety of methods, I suggest Ca(slow) represents Ca within the sarcoplasmic reticulum. Ca(fast) contained 12 pmol Ca per fibre and resting exchange had a time constant of 5.1 +/- 0.4 min. A total of 12 pmol 45Ca within Ca(fast) was released during a maximal contracture. Most of the Ca released from Ca(fast) rapidly entered the extracellular space; however, 0.39 +/- 0.15 pmol Ca per fibre transferred from Ca(fast) into Ca(slow) when the muscle bundle contracted. When 1-10 microM diltiazem reduced contracture time-tension, release of Ca(fast) was reduced proportionally. When 10 microM diltiazem paralyzed excitation-contraction coupling, Ca(fast) was not released. Refilling of Ca(fast) was proportional to the extent of repriming during 45Ca exposure. Although release and refilling of Ca(fast) is related to contraction, its role in excitation-contraction coupling remains to be elucidated.

Dose-dependent antagonism and potentiation of nitrous oxide antinociception by naloxone in mice.

Administration of the anesthetic gas nitrous oxide (N2O) evoked a concentration-dependent antinociceptive effect in mice as assessed by the abdominal constriction test. Depending on the dose and route of pretreatment with the opioid receptor blocker naloxone, the N2O drug effect was either antagonized or potentiated. After s.c. pretreatment with milligram per kilogram doses of naloxone, dose-related antagonism occurred; picogram per kilogram doses potentiated N2O-induced antinociception. The i.c.v. pretreatment with microgram quantities of naloxone also antagonized N2O antinociception in a dose-related fashion; i.c.v. pretreatment with femtogram doses was without effect. On the other hand, intrathecal (i.t.) pretreatment with femtogram quantities of naloxone potentiated N2O antinociception; i.t. pretreatment with microgram quantities continued to antagonize the antinociceptive effect. The same pattern of interaction was observed in mice challenged with the kappa opioid analgesic drug trans (+- 3,4-dichlow-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl] benzeneacetamide methane sulfonate (U-50, 488H) after s.c., i.c.v. or i.t. pretreatments with high and low doses of naloxone. These results 1) demonstrate further similarities in the opioid receptor mediation of N2O and U-50, 488H antinociceptive effects and also 2) support the concept of high-affinity spinal opioid receptors, whose blockade by s.c.- or i.t.- but not i.c.v.-administered low-dose naloxone can potentiate the antinociceptive effects of both N2O and U-50,488H. These findings suggest that the antinociceptive effect of N2O might be modulated by a descending opioid system that inhibits analgesia.

Benzodiazepine receptor-mediated behavioral effects of nitrous oxide in the rat social interaction test.

The present study was conducted to ascertain whether an anxiolytic effect of nitrous oxide was demonstrable in rats using the social interaction test and whether this drug effect might be mediated by benzodiazepine receptors. Compared to behavior of vehicle-pretreated, room air-exposed rats, rat pairs exposed to nitrous oxide showed a generally inverted U-shaped dose-response curve with the maximum increase in social interaction encounters occurring at 25% and significant increase in time of active social interaction at 15-35%; higher concentrations produced a sedative effect that reduced social interaction. Treatment with 5.0 mg/kg of the anxiolytic benzodiazepine chlordiazepoxide also increased social interaction. Pretreatment with 10 mg/kg of the benzodiazepine receptor blocker flumazenil, which alone had no effect, significantly antagonized the social interaction-increasing effects of both nitrous oxide and chlordiazepoxide. In summary, these findings suggest that nitrous oxide produces a flumazenil-sensitive effect comparable to that of chlordiazepoxide and implicate central benzodiazepine mechanisms in mediation of the anxiolytic effect of nitrous oxide.

Na/Ca exchange and excitation--contraction coupling in frog fast fibres.

A 3 Na/Ca exchanger in the transverse tubular wall is modelled as the coupling mechanism between transverse tubular depolarization and Ca release from the sarcoplasmic reticulum. At rest, the Ca-occupied site faces the transverse tubular lumen. Upon depolarization, the difference in chemical potentials of Na and Ca gives a net inward force on Ca resulting in a reorientation of the exchanger so the Ca site now faces the myoplasm and releases Ca to stimulate Ca-induced Ca release from the sarcoplasmic reticulum. The rotation of the exchanger's asymmetrical charge could generate the 'charge movement' signal. As depolarization continues, the site is depleted of Ca and contraction ends spontaneously. Repolarization reorients the exchanger; the depleted Ca site now faces the transverse tubular lumen and slowly refills with Ca (repriming). A kinetic model is capable of controlling both twitch and contracture tension. The Na/Ca exchange blocker dichlorobenzamil (Merck) (10 microM), elevated external Na and low pH all slowed the rate of rise of potassium contracture tension. The ratios of rates of tension rise were dCB/control = 0.4 +/- 0.1, elevated external Na/Tris = 0.6 +/- 0.1, pH 6.3/control = 0.7 +/- 0.01. These results can be mimicked with the kinetic model by slowing the rate of 'rotation' (and hence charge movement) by 50%. Elevated internal Na increases the rate of rise of contracture tension; elevated internal Na/control 1.6 +/- 0.3. Dichlorobenzamil also slows the recovery following spontaneous relaxation; the time constant (68 s) of repriming is unchanged but shifted to longer recovery times. Reduced external Na and pH 6.3 also slow recovery in a similar manner, consistent with delayed rotation of the Ca-depleted site. These results suggest that Na/Ca exchange is a step in both the excitation contraction coupling chain and the repolarization-repriming sequence.

Calcium influx in contracting and paralyzed frog twitch muscle fibers.

Calcium uptake produced by a potassium contracture in isolated frog twitch fibers was 6.7 +/- 0.8 pmol in 0.7 cm of fiber (mean +/- SEM, 21 observations) in the presence of 30 microM D600. When potassium was applied to fibers paralyzed by the combination of 30 microM D600, cold, and a prior contracture, the calcium uptake fell to 3.0 +/- 0.7 pmol (11): the fibers were soaked in 45Ca in sodium Ringer for 3 min before 45Ca, in a potassium solution, was added for 2 min; each estimate of uptake was corrected for 5 min of resting influx, measured from the same fiber (average = 2.3 +/- 0.3 pmol). The calcium influx into paralyzed fibers is unrelated to contraction. This voltage-sensitive, slowly inactivating influx, which can be blocked by 4 mM nickel, has properties similar to the calcium current described by several laboratories. The paired difference in calcium uptake between contracting and paralyzed fibers, 2.9 +/- 0.8 pmol (16), is a component of influx related to contraction. Its size varies with contracture size and it occurs after tension production: 45Ca applied immediately after contracture is taken up in essentially the same amounts as 45Ca added before contraction. This delayed uptake is probably a "reflux" refilling a binding site on the cytoplasmic side of the T membrane, which had been emptied during the prior contracture, perhaps to initiate it. We detect no component of calcium uptake related to excitation-contraction coupling occurring before or during a contracture.

Effect of external ions on 45Ca efflux from cat intestinal muscle.

The surface-bound Ca of isolated circular smooth muscle of cat small intestine can be removed by substitution of LiCl for NaCl in Krebs solution. This substitution removed surface-bound Ca (45Ca) and allowed us to study transmembrane 45Ca efflux. Neither the resting membrane potential nor contractility changed when Li was substituted for Na. Li removed the same extracellular 45Ca store as did ethylene glycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid. The resting transmembrane 45Ca efflux was inhibited by La3+ and was unchanged in Li, tris(hydroxymethyl)aminomethane, arginine, and sucrose Krebs solution. The extra 45Ca efflux observed upon electrical stimulation was no greater in Na-Krebs than Li-Krebs, but during response to acetylcholine the extra 45Ca efflux was greater in Na-Krebs than Li-Krebs. We conclude that the surface-bound Ca is sensitive to external Na and that the transmembrane Ca efflux is not completely dependent on external Na.

Net calcium fluxes in anterior byssus retractor muscle with phasic and catch contraction.

Calcium in the artificial seawater bathing whole Mytilus anterior byssus retractor muscles (ABRM) was measured by a specific Ca electrode under various conditions of activation, catch, and catch relaxation. Activation in response to ACh was associated with uptake of Ca by the muscles. Phasic contractions produced a small Ca uptake; catch contractions produced a larger and sustained Ca uptake. After tension relaxation, the muscle lost an amount of Ca roughly equal to that gained. Catch relaxation by 5-hydroxytryptamine (5HT) was associated with Ca release. ACh at identical concentrations, applied to the muscle for increasingly longer times, produced increasing amounts of Ca uptake. Regardless of the previous gain of Ca by the muscle, 5HT applied for a constant interval caused release of the same amount of Ca. A model for the Ca control system in ABRM based on this and previously obtained 45Ca efflux data is proposed.

45Ca efflux from anterior byssus retractor muscle in phasic and catch contraction.

Phasic or catch contractions in Mytilus anterior byssus retractor muscle (ABRM) were activated by acetylcholine (ACh) and catch relaxation was initiated by 5-hydroxytryptamine (5HT). During phasic contraction and early in catch there is a brief increase in 45Ca efflux. When catch occurs, there is a subsequent drop in 45Ca efflux which then slowly recovers as catch tension declines. With catch relaxation by 5HT there is a biphasic increase in 45Ca efflux, identical to that seen when 5HT is applied to resting muscle. Compartment analyses based on the magnitude of pairs of these responses at varying times of the washout indicated that the increase in 45Ca efflux with activation originates from a compartment with the same time constant as the intermediate (80--100 min) compartment already described by previous resting efflux experiments. The decrease in 45Ca efflux during catch also involves this compartment. The increase in 45Ca efflux with 5HT originates from a more slowly exchanging Ca store with a time constant of approximately200 min.

The action of serotonin on calcium-45 efflux from the anterior byssal retractor muscle of Mytilus edulis.

(45)Ca efflux was studied in resting anterior byssal retractor muscle. The data are described by a three-compartment system. The most rapidly exchanging compartment, with an average time constant of 7 min, contains about 0.9 mM Ca/liter muscle, and probably represents extracellular space. A second compartment, with a time constant of 83 +/- 5 min, contains 1.2 mM Ca/liter, and may represent a membrane calcium store. The presence of a third, or more, compartments, probably representing sarcoplasmic reticulum and contractile proteins, is indicated by the fact that the final time constant is 10 times the 83 min time constant of the second compartment. Serotonin (5HT), on initial application, increases (45)Ca efflux from this third compartment(s). This effect has a typical dose-response relationship with a maximum response appearing at 10(-7)M5HT. In addition, removal of 5HT causes a secondary increase in (45)Ca efflux which has a maximum at a 5HT concentration of 10(-7)M and declines at both higher and lower doses.

Calcium efflux from frog twitch muscle fibers.

An apparatus is described which collects the effluent from the center 0.7 cm of a single muscle fiber or bundle of muscle fibers. It was used to study the efflux of (45)Ca from twitch muscle fibers. The efflux can be described by three time constants 18 +/- 2 min, 300 +/- 40 min, and 882 +/- 172 min. These kinetics have been interpreted as those of a three-compartment system. The fastest is thought to be on the surface membrane of the muscle and of the T system. It contains 0.07 +/- 0.03 mM Ca/liter of fiber and the Ca efflux is 0.11 +/- 0.04 pM Ca/cm(2). sec. The intermediate rate compartment is thought to represent the Ca in the longitudinal reticulum. It contains approximately 0.77 mM Ca/liter. Only the efflux from this compartment increases during stimulation. The most slowly exchanging compartment is poorly defined. Neither Ca-free nor Ni-Ringer solutions alter the rate of loss from the fastest exchanging compartment. Ni apparently alters the rate of loss from the slowest compartment.

Ca fluxes in single twitch muscle fibers.

Ca influx and efflux in single twitch muscle fibers were determined by the movement of 45Ca. The isotope was assayed by counting the center 1 cm of a fiber while it was in nonradioactive Rnger's solution. The average resting influx in 1.0 mM Ca Ringer's was 0.26 pM Ca/cm2. sec for 5 to 20 min influx periods. The average additional influx upon stimulation in 1.0 mM Ca was 0.73 pM Ca/cm2. twitch. The efflux after both resting and stimulated 45Ca influx can be described by a single exponential curve with an average time constant of 125 min. This relationship is an indication of Ca exchange with a single intracellular compartment. This compartment contains an estimated 47% of the total muscle Ca at 1.0 mM Ca. When the Ca in the Ringer was reduced to 0.5 mM Ca, both the resting and stimulated Ca fluxes decreased. When Ca was raised to 1.8 mM, the stimulated influxes increased but the resting influx did not.