Showing papers by "University of Maryland, Baltimore published in 1988"

Deficit and nondeficit forms of schizophrenia: the concept.

Abstract: The authors provide a rationale for distinguishing the primary, enduring negative symptoms of schizophrenia (termed "deficit symptoms") from the more transient negative symptoms secondary to other factors. They argue that the former are more likely to provide a basis for meaningful subtyping of the schizophrenic syndrome, while the latter are more likely to respond to currently available treatments. They describe their experience in using clinical judgment based on longitudinal observations to identify deficit and nondeficit subtypes of schizophrenic patients and propose criteria for defining schizophrenia with the deficit syndrome.

Experimental Campylobacter jejuni Infection in Humans

Abstract: Two strains of Campylobacter jejuni ingested by 111 adult volunteers, in doses ranging from 8 x 10(2) to 2 x 10(9) organisms, caused diarrheal illnesses. Rates of infection increased with dose, but development of illness did not show a clear dose relation. Resulting illnesses with strain A3249 ranged from a few loose stools to dysentery, with an average of five diarrheal stools and a volume of 509 mL. Infection with strain 81-176 was more likely to cause illness, and these illnesses were more severe, with an average of 15 stools and 1484 mL of total stool volume. All patients had fecal leukocytes. The dysenteric nature of the illness indicates that the pathogenesis of C. jejuni infection includes tissue inflammation. Ill volunteers developed a serum antibody response to the C. jejuni group antigen and were protected from subsequent illness but not infection with the same strain.

Toxin, toxin-coregulated pili, and the toxR regulon are essential for Vibrio cholerae pathogenesis in humans.

Abstract: Isogenic mutant strains of V. cholerae O1 lacking elements of a genetic regulon controlled by toxR and implicated in virulence were tested in volunteers. A deletion mutation in ctxA, the gene encoding the A subunit of cholera toxin, markedly attenuated disease symptoms without affecting intestinal colonization. Deletion of toxR, the gene encoding the cholera toxin-positive regulatory protein resulted in a diminution in colonizing capacity. A deletion mutation in tcpA, encoding the major subunit of the toxin coregulated pilus (regulated by toxR), abolished the colonizing capacity of this strain. These results show for the first time the role of a specific pilus structure in colonization of the human intestine by V. cholerae O1 and exemplify the significance of a genetic regulon in pathogenesis.

Structural and functional basis for GABAA receptor heterogeneity.

Abstract: When γ-aminobutyric acid (GABA), the major inhibitory neurotransmitter in vertebrate brain, binds to its receptor it activates a chloride channel. Neurotransmitter action at the GABAA receptor is potentiated by both benzodiazepines and barbiturates which are therapeutically useful drugs (reviewed in ref. 1). There is strong evidence that this receptor is heterogeneous1–7. We have previously isolated complementary DNAs encoding an α- and a β-submit and shown that both are needed for expression of a functional GABAA receptor8. We have now isolated cDNAs encoding two additional GABAA receptor α-subunits, confirming the heterogeneous nature of the receptor/chloride channel complex and demonstrating a molecular basis for it. These α-subunits are differentially expressed within the CNS and produce, when expressed with the β-subunit in Xenopus oocytes, receptor subtypes which can be distinguished by their apparent sensitivity to GABA. Highly homologous receptor subtypes which differ functionally seem to be a common feature of brain receptors.

A comparative study of human periodontal ligament cells and gingival fibroblasts in vitro.

Abstract: Both periodontal ligament and gingival tissue are thought to harbor cells with the ability to stimulate periodontal regeneration, i.e., formation of new bone, cementum, and connective tissue attachment. To understand further the role of these cells in the regenerative process, we compared human periodontal ligament cells and gingival fibroblasts, both derived from the same patient, same passage, in vitro. Protein and collagen production was significantly greater in periodontal ligament cells when compared with that of gingival fibroblasts. In addition, periodontal ligament cells had higher alkaline phosphatase levels when compared with those of gingival fibroblasts.

Mechanism of release of calcium from sarcoplasmic reticulum of guinea-pig cardiac cells.

Abstract: 1. The mechanisms that control release of Ca2+ from the sarcoplasmic reticulum (SR) of guinea-pig ventricular cells were studied by observing intracellular calcium concentration ([Ca2+]i transients) and membrane currents in voltage-clamped guinea-pig ventricular myocytes perfused internally with Fura-2. 2. Sarcolemmal Ca2+ current was identified through the use of tetrodotoxin (TTX) and Ca2+ channel antagonists (verapamil) and agonists (Bay K 8644). 3. Changes in [Ca2+]i attributable to release of Ca2+ from the SR were identified through the use of ryanodine, which abolishes the ability of the SR to release Ca2+. Ryanodine-sensitive increases in [Ca2+]i could be elicited either by depolarization or by repolarization (from depolarizing pulses to relatively positive membrane potentials). 4. At appropriate voltages, it is the initial fast change in [Ca2+]i elicited by either depolarization or repolarization that is abolished by ryanodine, and is defined here as ryanodine sensitive. 5. The amplitude of the ryanodine-sensitive [Ca2+]i transient elicited by depolarization had a bell-shaped dependence on membrane potential with a maximum of about 500 nM at 10 mV, and with the upper minimum between 60 and 70 mV. Verapamil-sensitive current activated over approximately the same potential range as the [Ca2+]i transient, with a peak amplitude at 10 mV, and a reversal potential of 65 mV. 6. When a holding potential of -68 mV and TTX (30 microM) were used, the most negative pulse potential at which activation of an inward current occurred was -49 mV while changes in [Ca2+]i occurred at -43 mV. 7. Ryanodine-sensitive increases in [Ca2+]i elicited by repolarization (tail transients) were maximal for repolarization to 0 mV. Smaller changes in [Ca2+]i than maximal were elicited by repolarization to both more positive and more negative potentials than 0 mV. The peak amplitude of the verapamil-sensitive tail currents elicited by repolarization increased continuously as the membrane was repolarized to potentials more negative than 60 mV. 8. Increasing depolarizing pulse duration beyond 10-20 ms did not increase the amplitude of the [Ca2+]i transient, but prolonged it. 9. The experimental results are compared to the predictions of two theories on the mechanism of excitation-contraction coupling: Ca2+-induced release of Ca2+ (CICR), as it has been formulated from data in skinned cardiac cells, and a charge-coupled release mechanism (CCRM), as it has been formulated to explain excitation-contraction coupling in skeletal muscle. 10. Some of the results are clearly not consistent with certain features of a charge-coupled release mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)

Pelvic fracture in multiple trauma: classification by mechanism is key to pattern of organ injury, resuscitative requirements, and outcome

Abstract: Three hundred forty-three multiple trauma patients with major pelvic ring disruption were studied and subdivided into four major groups by mechanism of injury: antero-posterior compression (APC), lateral compression (LC), vertical shear (VS), and combined mechanical injury (CMI). Acetabular fractures which did not disrupt the pelvic ring were excluded. The mode of injury was: MVA, 57.4%; motorcycle, 9.3%; fall, 9.3%; pedestrian, 17.8%; crush, 3.8%. The LC and APC groups were divided into Grades 1-3 of increasing severity. The pattern of organ injury: including brain, lung, liver, spleen, bowel, bladder, pelvic vascular injury (PVASI), retroperitoneal hematoma (RPH) and complications: circulatory shock, sepsis, ARDS, abnormal physiology, and 24-hr total fluid volume administration were all evaluated as a function of mortality (M). As LC grade increased from 1 to 3 there was increased % incidence of PVASI, RPH, shock, and 24-hr volume needs. However, the large incidence of brain, lung, and upper abdominal visceral injuries as causes of death in Grade 1 and 2 fell in LC3, with limitation of the LC3 injury pattern to the pelvis. As APC grade increased from 1 to 3 there was increased % injury to spleen, liver, bowel, PVASI with RPH, shock, sepsis, and ARDS, and large increases in volume needs, with important incidence of brain and lung injuries in all grades. Organ injury patterns and % M associated with vertical shear were similar to those with severe grades of APC, but CMI had an associated organ injury pattern similar to lower grades of APC and LC fractures. The pattern of injury in APC3 was correlated with the greatest 24-hour fluid requirements and with a rise in mortality as the APC grade rose. However, there were major differences in the causes of death in LC vs. APC injuries, with brain injury compounded by shock being significant contributors in LC. In contrast, in APC there were significant influences of shock, sepsis, and ARDS related to the massive torso forces delivered in APC, with large volume losses from visceral organs and pelvis of greater influence in APC, but brain injury was not a significant cause of death. These data indicate that the mechanical force type and severity of the pelvic fracture are the keys to the expected organ injury pattern, resuscitation needs, and mortality.

Significance and meaning of neurological signs in schizophrenia.

Abstract: The authors review studies of abnormal signs on clinical neurological examination of schizophrenic patients. In spite of a number of methodologic limitations, the cumulative evidence strongly argues that there are more neurological signs in schizophrenic patients than in nonpsychiatric control subjects. Although less consistent, there is considerable evidence of more neurological signs in schizophrenic patients than in patients with affective disorders or with mixed, nonpsychotic disorders. The existing literature suggests several preliminary hypotheses with respect to neuroanatomical localization of neurological signs, subtyping of schizophrenia, and utility of studies of relatives at high risk and family history studies. Directions for future research in these areas are described.

Role of pharmacokinetics in the outcome of infections.

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Characterization of enteroadherent-aggregative Escherichia coli, a putative agent of diarrheal disease.

Abstract: Escherichia coli that exhibit the aggregative pattern of adherence to HEp-2 cells (enteroadherent-aggregative E. coli [EA-AggEC]) have been epidemiologically incriminated as a cause of diarrhea. We undertook a preliminary microbiological and pathogenetic characterization of 42 isolates of this putative pathogen. The strains were negative by tests with DNA probes for enteropathogenic, enterotoxigenic, enteroinvasive, and enterohemorrhagic E. coli and, by serotype, did not fit these categories. Thirty-nine of 42 strains had a 55-65-megadalton plasmid; many shared DNA homology. With one representative strain, plasmid transfer was accompanied by transfer of smooth lipopolysaccharide, fimbriae expression, and the aggregative property. EA-AggEC caused characteristic lesions in rabbit and rat ileal loops. The intestinal lesions and (Shiga-like) limb paralysis and death in rabbits inoculated with live organisms suggest toxin involvement; assays for Shiga-like toxins were negative. These preliminary results support the contention that EA-AggEC may represent a distinct category of diarrheagenic E. coli.

Protection by milk immunoglobulin concentrate against oral challenge with enterotoxigenic Escherichia coli.

Abstract: Enterotoxigenic Escherichia coli is a common cause of traveler's diarrhea. Prophylaxis against traveler's diarrhea has been associated with side effects from bismuth subsalicylate and the development of resistance to antimicrobial agents. We undertook a double-blind controlled trial in which a bovine milk immunoglobulin concentrate with high titers of antibodies against enterotoxigenic E. coli was used as prophylaxis against E. coli challenge in volunteers. Lyophilized milk immunoglobulins were prepared from the colostrum of cows immunized with several enterotoxigenic E. coli serotypes and fimbria types, E. coli heat-labile enterotoxin, and cholera toxin. As a control, an immunoglobulin concentrate with no anti-E. coli activity was prepared. Ten volunteers received buffered immunoglobulin concentrate against enterotoxigenic E. coli, and 10 received the control immunoglobulin concentrate, dissolved in water, three times a day. No side effects were observed. On the third day of immunoglobulin proph...

Volunteer studies of deletion mutants of Vibrio cholerae O1 prepared by recombinant techniques.

Abstract: Vibrio cholerae O1 A-B- vaccine strain JBK 70 and A-B+ CVD 101 prepared by recombinant DNA techniques from pathogenic EI Tor Inaba N16961 and classical Ogawa 395, respectively, were fed to 38 volunteers in single doses of 10(4) to 10(10). Although severe diarrhea did not occur in any vaccine, more than one-half developed mild diarrhea. These attenuated strains colonized well and elicited prominent vibriocidal and antitoxic (CVD 101) antibody responses. Recipients of a single dose of JBK 70 were significantly protected when challenged with 10(6) wild-type N16961. Diarrhea occurred in 7 of 8 controls but in only 1 of 10 vaccines (P less than 0.003, 89% vaccine efficacy), demonstrating the potency of immune mechanisms that do not involve cholera antitoxin. Further derivatives were prepared to explore the pathogenesis of the residual diarrhea, considering that either intestinal colonization by the vaccine itself or accessory toxins might be responsible. CVD 102, an auxotrophic mutant of CVD 101, did not cause diarrhea but colonized poorly and elicited feeble immune responses. Derivatives of JBK 70 and CVD 101 (CVD 104 and 105) deleted of genes encoding the EI Tor hemolysin still caused mild diarrhea. Genetically engineered strains can be colonizing, highly immunogenic, and protective single-dose oral vaccines, but they must be further attenuated before they can be considered for use as public health tools.

Characterization of urease from Campylobacter pylori.

Abstract: Campylobacter pylori, a suspected agent of gastritis and peptic ulceration, rapidly hydrolyzes urea. Because urease serves as the basis of detection of the organism in gastric biopsies and may represent an important virulence factor, biochemical characteristics of the enzyme were determined. C. pylori was isolated from antral biopsies from 10 patients with complaints of abdominal pain or history of peptic ulcer disease. All isolates were urease positive, with an average rate of hydrolysis by cell lysates being 36 +/- 28 mumol of NH3 per min per mg of protein, more than twice that of Proteus mirabilis and 10 times that of other urinary tract isolates. The enzyme had an apparent molecular weight of 625,000 +/- 15,000 by column chromatography, an isoelectric point of 5.9, a Km of 0.8 +/- 0.1 mM urea, an optimal temperature of 45 degrees C, and an optimal pH of 8.2. Ten isolates tested produced ureases with identical electrophoretic mobilities on nondenaturing 5% polyacrylamide activity gels. Acetohydroxamic acid (100 micrograms/ml), hydroxyurea (85 micrograms/ml), flurofamide (0.05 micrograms/ml), and EDTA (8 mM) inhibited enzyme activity by 50%. Cell lysates retained 50% of initial urease activity after 6 days and 40% activity after 18 days when stored at 4 degrees C in 20 mM sodium phosphate, pH 6.8. At -70 degrees C for 18 days, 1 mM EDTA or 15% glycerol preserved 40 or 34%, respectively, of initial activity. The urease of C. pylori appears to be biochemically unique from the enzymes of other common urease-producing species. Images

Phorbol ester increases calcium current and simulates the effects of angiotensin II on cultured neonatal rat heart myocytes.

Abstract: The effects of increased protein kinase C activity were studied in neonatal rat myocytes grown in primary culture. The changes in mechanical and electrical behavior, as well as protein phosphorylation, that followed the apparent activation of protein kinase C by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) were examined. As spontaneous beating frequency was increased minimally by 10 nM TPA and by 100% with 85 nM TPA, shortening amplitude, shortening velocity, and relaxation velocity decreased concomitantly. In contrast, 4-alpha-phorbol-12,13-didecanoate (alpha-PDD), which does not activate protein kinase C, had no effect on beating behavior at 800 nM. In voltage-clamped single myocytes, both steady-state and transient components of the cadmium-sensitive calcium current were increased by the addition of TPA (65 nM). Neither the time constant for the inactivation of the transient component of this calcium current nor the reversal potential was altered by TPA. The phosphorylation state of a discrete set of proteins, with apparent molecular weights of 32 and 83 kDa, was enhanced when TPA was added to intact myocytes. Angiotensin II enhances the phosphorylation state of the same set of proteins as observed with TPA. We conclude that activation of protein kinase C can modify mechanical behavior and increase L-type Ca2+ channel activity in cultured neonatal rat ventricular myocytes. The remarkable similarity in mechanical, electrical, and protein phosphorylation responses of cultured neonatal myocytes following TPA or angiotensin II application indicate that protein kinase C may mediate the action of angiotensin II.

Functional recovery and medical costs of trauma: an analysis by type and severity of injury.

Abstract: This study was designed to delineate the factors that influence the extent and rate of recovery as related to the characteristics and duration of functional limitations resulting from trauma. The study population was 597 surviving trauma patients aged 16-45 years from two trauma centers in a single state system which follow similar care protocols, and included patients with extremity, abdomen, thorax, brain, and spinal cord injuries. Of 479 trauma patients (80% of the total) who were followed for a full year, 57% had no activity restrictions, 16% had some limitation with either a major or minor physical activity, but did not have any difficulty with mobility or self care, and 27% were limited in either mobility or one of the five basic self-care activities. Further analyses show that of the 262 individuals who were working full-time before the injury, 57% had actually returned to full-time employment within the year. Factors in addition to type and severity of trauma that influence return to work were higher educational level, white collar employment, higher preinjury income, and the presence of supportive individuals among family or friends. Recovery as defined by functional status and return to full-time work is analyzed by body region and severity of the principal injury sustained.

Toward a Functional Categorization of Slow Waves

Abstract: This study is concerned with slowly varying, long-duration brain event-related potential (ERP) components, referred to as Slow Wave activity. Slow Wave activity can be observed in the epoch following P3b, suggesting that it reflects further processing invoked by increased task demands, beyond the processing that underlies P3b. The present experiment was designed to distinguish Slow Wave activity related to specific types of task demands which arise during difficult perceptual (pattern recognition) and conceptual (arithmetic) mental operations. Three late ERP components that respond differentially in amplitude to manipulation of perceptual and conceptual difficulty were identified: 1) A P3b, with a topography focused about Pz, evidently related to the subjective categorization of easy and difficult conceptual operations, that increased when the subjective low-prohability operation was performed; 2) A longer latency, centroparietal positive Slow Wave that increased directly with perceptual difficulty but was not affected by conceptual difficulty; 3) A very long latency negative Slow Wave, broadly distributed over centroposterior scalp, that increased directly with conceptual difficulty while its onset was delayed when perceptual difficulty increased. DESCRIPTORS: Event-related potentials. Slow Wave, P3b, Mental arithmetic. Pattern recognition. Information processing. Several reports have described slowly varying, view). These "Slow Waves" can be observed in the long-duration brain event-related potential (ERP) epoch following P3b, suggesting that they reflect components whose amplitudes relate directly to task further processing invoked by increased task dedemands (see Ruchkin & Sutton, 1983, for a re- mands, beyond the processing that underlies P3b. Slow Wave activity has been found in a variety This research was supported in part by a U.S.P.H.S. of tasks. Comparisons of these data suggest that grant from the National Institute of Neurological and there are systematic differences in Slow Waves such Communicative Disorders and Stroke, NS 11 199. We are that they appear to be reducible to two broad catindebted to Dr. Richard Coppola for providing laboratory egories based on their onset latencies, those reflectcomputer software and Mr. Howard Canoune for editorial jng gj^jjer (1) perceptual operations or (2) concep

Angiotensin II increases spontaneous contractile frequency and stimulates calcium current in cultured neonatal rat heart myocytes: insights into the underlying biochemical mechanisms.

Abstract: The effect of angiotensin II on cultured neonatal rat heart myocytes was studied by measuring changes in cell length, the magnitude and kinetics of the calcium current, and changes in cyclic adenosine 3',5'-monophosphate (cAMP) and phosphoinositide metabolism. Spontaneous beating frequency of multicellular networks was increased by angiotensin II with a maximal increase of 100% above control values at concentrations of 5 nM or greater. The half-maximal response occurred at 0.6 nM angiotensin II. Shortening amplitude, shortening velocity, and relaxation velocity decreased concomitantly with the increasing contractile rate. In voltage-clamped single myocytes, both steady-state and transient components of the calcium current were increased by the addition of angiotensin II. Angiotensin II had no effect on either control or isoproterenol-stimulated adenylate cyclase activity in myocyte membranes. Neither the basal levels nor the isoproterenol-stimulated cAMP accumulation in intact cells was affected by addition of hormone. In myocytes labeled with [3H]inositol, angiotensin II stimulated the formation of [3H]inositol phosphates. One minute after addition of 5 nM angiotensin II, inositol monophosphate and inositol bisphosphate levels were increased to 73% and 99%, respectively, above control values and remained elevated at 10 minutes. Inositol trisphosphate levels were not significantly different from control values at either time point. Nifedipine (10 microM) had no effect on angiotensin II-induced increases in [3H]inositol phosphates. We conclude that the increases in both spontaneous beating rate and calcium current in angiotensin II-stimulated cultured neonatal heart cells are not dependent on cAMP or inositol trisphosphate levels but may involve sustained phosphoinositide hydrolysis.

Phosphorylation of neurofilaments is altered in amyotrophic lateral sclerosis

Abstract: We used a library of monoclonal antibodies (Mab) that distinguish phosphorylated (P+) and non-phosphorylated (P-) neurofilament (NF) epitopes to examine phosphorylation of NF in lower motor neurons of patients with amyotrophic lateral sclerosis (ALS), of neurologically normal controls of different ages, and of patients with central chromatolysis due to injuries to motor root axons. Monoclonal antibodies directed to P+ NF immunostained five to ten times more neuronal perikarya in ALS than in age-matched controls. Spheroids, which are NF containing axonal enlargements, found in significantly greater number in proximal axons in ALS, were also intensely immunostained with Mab to P+ NF. Moreover, anterior root axons in five of eleven cases of ALS reacted only with the Mab to P+ NF, while both P- and P+ NF were present in motor roots from controls. In control groups, the number of neuronal perikarya and spheroids that immunoreacted with the Mab to P+ NF increased moderately with age. Chromatolytic lower motor neurons were recognized by Mab to P+ NF. Our results show that the process of phosphorylation is altered in ALS. We propose that phosphorylation of NF in ALS occurs prematurely and that it is more likely to be associated with an impairment of NF transport than to be part of a chromatolytic reaction of lower motor neurons.

Na channels and two types of Ca channels in rat pancreatic B cells identified with the reverse hemolytic plaque assay.

Abstract: The reverse hemolytic plaque assay (RHPA) was used to study the secretory properties of single rat pancreatic B cells, and to identify insulin-secreting cells for patch-clamp experiments. In secretion studies using the RHPA, we find that the percentage of secreting B cells and the amount of insulin secreted per B cell increase as the glucose concentration is raised from 0 to 20 mM. Using the whole-cell variation of the patch-clamp technique, we find that identified B cells have three types of channels capable of carrying inward current: (a) tetrodotoxin-sensitive, voltage-dependent Na channels, which are nearly completely inactivated at -40 mV, (b) fast deactivating (FD) Ca channels, and (c) slowly deactivating (SD) Ca channels. We have shown that Na channels are functionally significant to the B cell, because tetrodotoxin partially inhibits glucose-induced insulin secretion. The properties of FD and SD Ca channels differ in several respects. FD channels deactivate at -80 mV, with a time constant of 129 microseconds, they are half-maximally activated near +15 mV, they do not inactivate during 100 ms, they conduct Ba2+ better than Ca2+, and they are very sensitive to washout during intracellular dialysis. SD channels, on the other hand, deactivate with a time constant of 2.8 ms, they are half-maximally activated near -5 mV, they inactivate rapidly, they conduct Ba2+ and Ca2+ equally well, and they are insensitive to washout.

Structure and synthesis of a potent glutamate receptor antagonist in wasp venom

Abstract: A low molecular weight toxin isolated from the venom of the digger wasp Philanthus triangulum, first noted by T. Piek, is a potent antagonist of transmission at quisqualate-sensitive glutamate synapses of locust leg muscle. This philanthotoxin 433 (PTX-433) has been purified, chemically characterized, and subsequently synthesized along with two closely related analogues. It has a butyryl/tyrosyl/spermine sequence and a molecular weight of 435. Its two analogues, PTX-343 and PTX-334 (the numerals denoting the number of methylenes between the amino groups of the spermine moiety), are also active on the glutamate synapse of the locust leg muscle; PTX-334 was more potent and PTX-343 was less potent than the natural toxin. Such chemicals are useful for studying, labeling, and purifying glutamate receptors and may become models for an additional class of therapeutic drugs and possibly insecticides.

3-Hydroxyanthranilate oxygenase activity is increased in the brains of Huntington disease victims

Abstract: An excess of the tryptophan metabolite quinolinic acid in the brain has been hypothetically related to the pathogenesis of Huntington disease. Quinolinate's immediate biosynthetic enzyme, 3-hydroxyanthranilate oxygenase (EC 1.13.11.6), has now been detected in human brain tissue. The activity of 3-hydroxyanthranilate oxygenase is increased in Huntington disease brains as compared to control brains. The increment is particularly pronounced in the striatum, which is known to exhibit the most prominent nerve-cell loss in Huntington disease. Thus, the Huntington disease brain has a disproportionately high capability to produce the endogenous "excitotoxin" quinolinic acid. This finding may be of relevance for clinical, neuropathologic, and biochemical features associated with Huntington disease.

Changes in the calcium current of rat heart ventricular myocytes during development.

Abstract: 1. Calcium current (ICa) was recorded in single rat heart cells at two periods during development: (1) at 2-7 days post-partum (neonatal), and (2) at 6-8 weeks (adult). 2. We measured both transient and steady-state components of ICa and could describe ICa in terms of the steady-state activation (d infinity) and inactivation (f infinity) parameters, the channel reversal potential (Echannel) and a relative conductance parameter, gr. 3. In adult single cells, the application of ryanodine (10 microM), an agent known to alter the function of the sarcoplasmic reticulum (SR), abolished contraction rapidly and increased ICa. Ryanodine also produced a 13 mV shift in f infinity towards more positive potentials and altered its slope, while producing a small increase in gr but no effect on d infinity. In neonatal single cells, ryanodine (10 microM) had no significant effect on contraction, ICa, d infinity, f infinity, or gr. Caffeine (10 mM), a less specific agent widely used to investigate sarcoplasmic reticulum function, had actions similar to those of ryanodine. 4. In adult myocytes, when EGTA (10 or 20 mM) or bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA, 10 mM) were included in the pipette solution, contractions were rapidly abolished, while a small (4 mV) shift of f infinity to more positive potentials was seen. A large additional shift of f infinity was observed when ryanodine (10 microM) was added to the superfusion solution in the continued presence of EGTA or BAPTA. The alterations of ICa in EGTA (or BAPTA) plus ryanodine were the same as those seen in ryanodine alone. In neonatal cells, in contrast, when EGTA or BAPTA were included in the pipette solution we observed only a small effect on f infinity and the application of ryanodine had no effect. 5. Electron micrographs of our preparations show that the dissociated adult cells have sharp sarcolemmal borders, fully developed sarcomeres with T-tubules and sarcoplasmic reticulum membranes. In contrast, the neonatal cells that we use have few of these intracellular structures. Our observations in these preparations are consistent with the work of others (e.g. Penefsky, 1974; Hirakow & Gotoh, 1975; Ishikawa & Yamada, 1975; Legato, 1975; Hoerter, Mazet & Vassort, 1981). 6. Our data suggest that fully developed sarcoplasmic reticulum in rat heart cells can affect ICa.(ABSTRACT TRUNCATED AT 400 WORDS)

Inactivation of calcium release from the sarcoplasmic reticulum in frog skeletal muscle.

Abstract: 1. The rate of calcium release (Rrel) from the sarcoplasmic reticulum (SR) in voltage clamped segments of frog skeletal muscle fibres was calculated from myoplasmic free calcium transients (delta[Ca2+]) measured with the calcium indicator dye Antipyrylazo III. 2. During a 100-200 ms depolarizing pulse Rrel reached an early peak and then declined markedly. The time course and extent of decline of Rrel were independent of membrane potential over a range of potentials where release activation varied severalfold. 3. For test pulses applied shortly after relatively large or long conditioning pulses Rrel completely lacked the early peak. The peak gradually recovered as the interval between the conditioning and test pulses was increased to 1 s. 4. A latency was often observed before the start of recovery of the peak in Rrel. The latency appeared to be correlated with the time for delta[Ca2+] to fall below a certain level, indicating that recovery of the peak might represent reversal of a calcium-dependent process. It was therefore proposed that the rapid decline in Rrel during a pulse was due to calcium-dependent inactivation of the SR calcium release channels. 5. Inactivation continued to develop during the interval between a relatively large 20 ms conditioning pulse and a test pulse applied 20 ms later. This was as expected for calcium-dependent inactivation of SR calcium release because of the elevated [Ca2+] between the conditioning and test pulses. It was not as expected for external membrane potential-dependent inactivation. 6. Small steady elevations in [Ca2+] due to relatively small 200 ms conditioning pulses produced marked inactivation of Rrel, indicating an apparent dissociation constant for calcium-dependent inactivation only slightly above resting [Ca2+]. 7. All observations could be well simulated by a two-step model for inactivation in which myoplasmic free calcium equilibrates rapidly with a high-affinity calcium receptor on the release channel and then the calcium-receptor complex undergoes a slower conformational change to the inactivated state of the channel. 8. An alternative model in which calcium binds to a soluble receptor (e.g. free calmodulin) and then the calcium-receptor complex binds to and directly inactivates the channel was shown to be formally identical to the preceding model. Either model could closely simulate all observations.