Proton currents through amiloride-sensitive Na channels in hamster taste cells. Role in acid transduction. The activity of taste cells maintained in the intact hamster tongue was monitored in response to acid stimulation by recording action currents from taste receptor cells with an extracellular "macro" patch pipette: a glass pipette was pressed over the taste pore of fungiform papillae and perfused with citric acid, hydrochloric acid, or NaCl. Because this technique restricted stimulus application to the small surface area of the apical membranes of the taste cells, many nonspecific, and potentially detrimental, effects of acid stimulation could be avoided. Acid stimulation reliably elicited fast transient currents (action currents of average amplitude, 9 pA) which were consistently smaller than those elicited by NaCl (29 pA). The frequency of action currents elicited by acid stimuli increased in a dose-dependent manner with decreasing pH from a threshold of about pH 5.0. Acid-elicited responses were independent of K+, Na+, Cl-, or Ca2+ at physiological (salivary) concentrations, and were unaffected by anthracene-9-carboxylic acid, tetraethylammonium bromide, diisothiocyanate-stilbene-2,2'-disulfonic acid, vanadate, or Cd2+. In contrast, amiloride (< or = 30 microM) fully and reversibly suppressed acid-evoked action currents. At submaximal amiloride concentrations, the frequency and amplitude of the action currents were reduced, indicating a reduction of the taste cell apical conductance concomitant with a decrease in cell excitation. Exposure to low pH elicited, in addition to transient currents, an amiloride-sensitive sustained d.c. current. This current is apparently carried by protons instead of Na+ through amiloride-sensitive channels. When citric acid was applied while the taste bud was stimulated by NaCl, the action currents became smaller and the response resembled that produced by acid alone. Because of the strong interdependence of the acid and salt (NaCl) responses when both stimuli are applied simultaneously, and because of the similarity in the concentration dependence of amiloride block, we conclude that amiloride-sensitive Na+ channels on hamster taste receptor cells are permeable to protons and may play a role in acid (sour) taste.


Nature of increase in quantal release by the thallous ion at frog end plates with and without nerve stimulation. The monovalent thallous ion (Tl) was evaluated at the frog end plate in vitro with intracellular microelectrodes. Recordings included end plate potentials (EPPs), and miniature end plate potentials (MEPPs). Replacement of extracellular potassium (K) by 2.5 mM Tl (a) caused increases in MEPP and EPP amplitudes, MEPP frequency, and quantal content, and (b) caused complete recovery of the EPP facilitation index at BAPTA-loaded nerve terminals. Tl's effects were reversible and concentration dependent, and persisted for > 3 h. The increase in MEPP frequency and its rate of decline due to Tl washout were more pronounced at 0 calcium (Ca)-2 mM EGTA than at 0.3 mM EGTA, suggesting that Tl's effects were not due to elevation of internal Ca. Unlike heavy metal ions reportedly capable of substituting for Ca, 0.2 mM Tl did not block, but further enhanced, elevated MEPP frequencies, occurring after nerve stimulation or in high K, to greater levels with barium (Ba) than with Ca. 200 nM omega-conotoxin (omega-CTX) blocked Tl's effect, indicating that Tl primarily entered the nerve terminal via Ca channels. A 50% reduction in sodium (Na) did not modify Tl's effect, although removal of K in the presence of 20 microM ouabain and 2.5 mM Tl caused an exaggerated increase in MEPP frequency, which decreased with a 50% reduction in Na. Based on the analysis, Tl neither substituted for Ca nor elevated internal Ca and Na, nor were its effects antagonized by ouabain; Tl increased quantal secretion, possibly by a fusogenic mechanism, after its entry into the nerve terminal.


Expression of oncogenes and tumor suppressor genes in human hepatocellular carcinoma and hepatoblastoma cell lines. The expression of nine oncogenes (c-myc, N-myc, N-ras, H-ras, k-ras, abl, fos, src, and raf) and two tumor suppressor genes (p53 and RB) were studied by northern blot hybridization in six human hepatocellular carcinoma or hepatoblastoma cell lines (PLC/PRF/5, Hep3B, Hep G2, 2.2.15, HLE, and HLF) and in a human embryonic lung fibroblast cell line (WI-38) to look for differences that might be associated with the presence (PLC/PRF/5, Hep3B, and 2.2.15) or absence (Hep G2, HLE, and HLF) of integrated hepatitis B virus (HBV) DNA. The levels of expression of the oncogenes and tumor suppressor genes were unrelated to the presence or absence of integrated HBV-DNA. Furthermore, the intensity of expression of these oncogenes was no greater in the 2.2.15 cell line (consisting of Hep G2 cells transfected with hepatitis B virus) than in untransfected Hep G2 cells.


Monitoring of patients for cytomegalovirus after organ transplantation by centrifugation culture and PCR. A modified centrifugation culture technique and a polymerase chain reaction (PCR) is described for detection of early antigen and IE antigen DNA, respectively, for rapid and sensitive monitoring of active cytomegalovirus (HCMV) infection after organ transplantation. In a preliminary study, 541 clinical specimens (blood, urine, bronchoalveolar lavage, pharyngeal wash, sputum) from 59 organ recipients were assayed for HCMV antigen by centrifugation culture; 144 samples were tested by PCR simultaneously. Antigenemia detected by centrifugation culture correlated strongly with active HCMV infection and clinical symptoms and proved useful for monitoring the efficacy of antiviral therapy. PCR was more sensitive in an earlier phase of infection when centrifugation culture was still negative. The clinical usefulness of both methods is discussed.


HCMV-DNA is detected more frequently than infectious virus in blood leucocytes of immunocompromised patients: a direct comparison of culture-immunofluorescence and PCR for detection of HCMV in clinical specimens. In two studies comparing detection of human cytomegalovirus (HCMV) in 118 patients (93 of whom were immunocompromised) by the polymerase chain reaction (PCR) and virus isolation using either early antigen detection by culture-immunofluorescence or conventional cytopathic effect, DNA-PCR was found to be the most sensitive, followed by culture-immunofluorescence, then by cytopathic effect. Urine was inhibitory to the action of Taq polymerase; this was overcome by concentration of HCMV with PEG 6000 prior to gene amplification. Without PEG treatment, HCMV-DNA in 6 of the 11 specimens positive by culture-immunofluorescence was not detectable by PCR. In healthy seropositive individuals, HCMV-DNA was not detected in leucocytes. However, in immunocompromised patients with AIDS or transplants, and therefore at high risk of HCMV infection or reactivation, blood leucocytes were usually positive for HCMV-DNA (19/20), some for as long as 20 weeks after initial detection and persisting for long after culture-immunofluorescence became negative. Neither HCMV-RNA nor infectious HCMV were detected in the follow-up blood leucocyte specimens from immunocompromised patients who had detectable HCMV-DNA in these cells. These data suggest that persistence of HCMV-DNA in blood leucocytes of immunocompromised patients after reactivation or primary infection may be due to persistence of non-viable virus, residual HCMV genomic DNA, or latent HCMV-DNA.


Replication of varicella zoster virus in primary human keratinocytes. The ability of varicella zoster virus (VZV) to infect and replicate in human keratinocytes in culture was examined. Primary human keratinocytes derived from the abdomen, breast, and foreskin were plated as monolayers and infected by co-cultivation with VZV infected fibroblasts (MRC-5 cells). Replication and spread of the virus was assayed by plaque assay and immunofluorescence of infected cells using a VZV specific monoclonal antibody. Although all three types of keratinocytes tested were capable of supporting productive VZV infection, the keratinocytes showed a 1.5 to 2 log reduction in virus yield as compared to infection of monolayer cultures of MRC-5 cells. Results from immunofluorescence studies and plaque assays indicate a slower rate of cell-to-cell spread of the virus. Testing of an anti-VZV compound in this novel assay system demonstrated an interesting sensitivity compared to that observed in conventional assay systems.


Activation of human herpesvirus-6 in children with acute measles. Virological and serological studies were carried out prospectively to evaluate the possible activation of human herpesvirus-6 (HHV-6) in 50 infants and children with acute measles by isolation of HHV-6 from peripheral blood and by determining neutralizing antibodies to the virus. All but 5 patients (90%) were seropositive to HHV-6 in the acute stage of measles and 18 (40%) had a significant increase in HHV-6 antibody titers thereafter, whereas only 2 of 27 patients who were initially seropositive to Epstein-Barr virus (EBV) viral capsid antigen (VCA) had a significant rise in antibody titers to EBV VCA. Among 18 patients with a significant increase in HHV-6 titers, the virus was isolated from the peripheral blood mononuclear cells of three patients in the early convalescent stage of measles. These results indicate that activation of HHV-6 may occur frequently a few weeks after primary infection with the measles virus.


Ligand atom partial charges assignment for complementary electrostatic potentials. The design of molecules to fit into the active site of receptors is a rapidly developing area of pharmacology and medicinal chemistry. A good ligand needs a suitable geometry and also appropriate electrostatic properties. The electrostatic properties of the ligand should complement those of the receptor. We present a method for the assignment of atom-centred point charges for a ligand, based on the electrostatic potential of the receptor. These point charges are chosen to give the best possible complementarity to the receptor electrostatic potential over the van der Waals surface of the ligand. We demonstrate that point charges can be chosen to give good electrostatic complementarity, and suggest that a molecule with similar electrostatic properties should bind well to the receptor.


Burnout in doctorally prepared nurse faculty. This correlational study examined burnout in doctorally prepared nurse faculty and its relationships to management style, collegial support, and workload. The Matthews Burnout Scale and the Maslach Burnout Inventory were used to measure burnout of 400 doctorally prepared American Nurses Association (ANA) members employed as faculty in schools of nursing. Analysis of 236 (59%) returns revealed that 39% of the faculty experienced moderate to high levels of burnout. Significant negative relationships (p < .05) were found between burnout and participative management, presence of collegial support, and time spent in research and in clinical practice. Multiple regression indicated that management style was the strongest predictor of burnout, with collegial support the second predictor.


We need to know more: nurse educators' interest and expertise in gerontology. An assessment was made of 129 faculty who teach nursing students about the care of aged clients to determine their level of interest and expertise in 21 content areas related to gerontology. Faculty rated their expertise consistently lower than the relevance of, and their interest in, each of the content areas. Areas identified as most important for their professional growth tended to be areas where faculty ranked their expertise as already being high. The mean self-rated expertise of faculty who taught a course in gerontological nursing was significantly higher than those who did not. Most respondents lacked formal education in gerontology: 9% of respondents had gerontology in their undergraduate program; 27% in their graduate program. Only 4% were certified as gerontological nurses or gerontological nurse practitioners.


The use of cognitive style mapping as a predictor for academic success of first-semester diploma nursing students. This study determined the relationship between the components of the cognitive map and the diploma nursing student's successful academic completion of the first semester. This ex post facto study applied the Modified Hill Cognitive Style Model (MHCSM) instrument; the final grade, the dependent variable, was intercorrelated with the instrument's 28 mapping elements. Data analysis indicated three positively correlated predictors: a preference for finding meaning from written words, for independent problem-solving, and for a logical deductive approach in decision-making. Four negatively correlated predictors included a preference for finding meaning from the spoken word, for finding meaning from sight, for problem-solving with peers, and for categorical reasoning. Implications applicable to nursing education are included.


Integrating experienced and novice nurses into graduate home health education. Preparing master's-level professionals in home health nursing presents challenges because nurses come to graduate programs with varying degrees of home health experience. The purpose of this study was to examine how nurses with varying experience in home health nursing are integrated into graduate clinical practicum courses. Representations of 10 home health graduate programs were surveyed regarding how they adapt their clinical practicums to meet the special needs of their novice and experienced nurses. Respondents revealed that individualization was used to meet the individual needs of students. Individualization allows for creativity in developing learning experiences that are both challenging and satisfying for the student. However, lack of consistency in the advanced practice skills of graduates, and lack of time efficiency for faculty and agency personnel can also occur. Faculty members need to share creative ways of teaching students, to identify unique opportunities for clinical experiences, and to collaborate on research.


Caring between nursing students and physically/mentally handicapped children: a phenomenological study. Advanced technology has increased the life expectancy to age 20 for most chronically ill children. Consequently, nurses will be encountering an increasing number of physically and mentally handicapped children. Nursing students' discovery of the meaning of caring with exceptional children will help prepare them for interactions with these special children during their nursing careers. A phenomenological study was undertaken to explore 36 nursing students' caring experiences with physically and mentally handicapped children. Van Kaam's phenomenological methodology was used to analyze the data. Sharing the findings of this phenomenological study with nursing students is one approach faculty can use to help reduce their anxiety regarding upcoming clinical rotations with exceptional children.


Preceptor development programs: an interpretive approach. The behavioral model commonly used in preceptor educational programs is inadequate for conveying the complexity of precepting. An interpretive approach is offered as an alternative for preceptor education. Using an interpretive approach is one way to extend our understanding of precepting as an educational process. A preceptor education program is described along with examples of narratives from preceptors. Interpretations of each narrative provide nurse educators with a model for utilizing narratives with preceptors. Themes of teaching, nursing as precepting, and timing are explored. Restoring the narrative to the nursing practice of precepting promises to recapture the richness and complexity of precepting.


Report of an innovative research program for baccalaureate nursing students. In summary, an innovative low-cost way to teach undergraduate students about research and to socialize students into attending research conferences has been developed. It is not perfect yet, but with time, critical students, and responsive research-productive faculty, each program should improve. It is not surprising that sophomore students do not achieve the objectives at the same level as older students. As students move closer to the "real" world of nursing practice and develop increasing sophistication about nursing in general and research in particular, they are, hopefully, more knowledgeable consumers of nursing research. What is particularly satisfying to the planners of those Research Days is that through the experience of attending Undergraduate Research Day at various points in their educational progress, students are socialized into discussing research. Additionally, they seemed to develop some degree of comfort with this aspect of their future nursing role. The RN and former student panel participants normalized research involvement for the student attendees. Panel member stories about their mistakes and successes made students realize that nursing investigations need not be the sole property of those with doctoral degrees. A serendipitous outcome of these programs was an increased awareness by students of the specific research project in which their teachers were engaged. Students informally reported a feeling of pride and reflected accomplishment. The importance of timing in offering such programs should not have been a surprise at this urban commuter university. Unwittingly, in scheduling the Friday afternoon program the planners ignored the initial consideration that the program not impose financial hardship on students.(ABSTRACT TRUNCATED AT 250 WORDS)


In-vivo comparisons of clot formation on titanium and hydroxyapatite-coated titanium. We investigated in vivo clot formation on the surface of hydroxyapatite (HA)-coated titanium (Ti) implants and on non-coated Ti implants. Immediately after tooth extraction implant samples were inserted into the blood clot, in the same extraction site, for 1, 30, 60, or 120 seconds. Samples were processed for scanning electron microscopy (SEM). Qualitative observations of clotting topography were made by direct SEM viewing. Neither of the implant surfaces appeared to differ markedly in the degree of clotting during the 120 seconds of implantation; they revealed very early clot formation and limited clot attachment. These results were compared to the findings obtained in a previous study using identical methods with an intact periodontal ligament (PDL), root planed roots, and roots planed and treated with pH 1 citric acid. The PDL surface had the most rapid clot formation at all time periods. By 120 seconds, all root surfaces had completed clot formation.


Tissue response to biphasic calcium phosphate ceramic with different ratios of HA/beta TCP in periodontal osseous defects. The purpose of this study was to determine the optimal ratio of calcium hydroxyapatite (HA) to beta tricalcium phosphate (beta TCP) in a biphasic porous calcium phosphate (BCP) ceramic for effective repair of periodontal osseous defects. Defects were surgically produced in beagle dogs and made chronic for 4 months to simulate periodontal disease. Mucoperiosteal periodontal flaps were reflected, followed by osseous defect debridement and root planing. Specially prepared ceramic with different HA/beta TCP ratios were implanted into the prepared defects. The sites were allowed to heal for 6 months, animals were euthanized, and site-blocks were removed for histological study. During the follow-up phase, scaling and polishing were done once a month, and standardized probing attachment levels were recorded pre- and 6-months postoperatively. The Duncan's multiple range test showed that all the treatments produced statistically significant higher gain in probing attachment levels than the control group (0HA/0 beta TCP) (P < 0.05). Among the 7 "active" treatment groups, 2 (65/35 and 85/15) had significantly higher gain in probing attachment levels than those in 3 groups (50/50, 100/0, and 0/100) (P < 0.05). Histologically, higher HA ratio (but not 100% HA) showed accelerated new bone formation and new attachment levels. Based on histological results, the 85HA/15 beta TCP ratio appears to demonstrate greater gain in attachment level and bone regeneration in the treatment of periodontal osseous defects.


Inhibition of bone DNA and collagen production by surface-associated material from bacteria implicated in the pathology of periodontal disease. Gentle extraction of oral bacteria implicated in the pathogenesis of periodontal disease, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, or Eikenella corrodens, with saline removes the extracellular components while leaving the bacteria intact. This readily-solubilized surface-associated material (SAM) has been demonstrated to significantly inhibit DNA and collagen synthesis by murine calvaria at concentrations as low as 10 ng/ml. DNA and collagen synthesis in isolated calvarial osteoblasts were also inhibited by these SAM preparations with similar dose responses. The inhibitory effect of these bacterial expolymers was blocked by 1 microM indomethacin. The potent inhibitory actions on bone synthesis of the SAM from these bacteria may contribute to the alveolar bone loss found in patients with periodontal disease.


Ca2+ current and charge movement in adult single human skeletal muscle fibres. 1. The Vaseline-gap technique was used to record calcium currents (ICa) and charge movement in single cut fibres from normal human muscle. Experiments were carried out in 2 or 10 mM-extracellular Ca2+ concentration ([Ca2+]o) and at 17 or 27 degrees C. 2. The passive electrical properties of the fibres with this technique were: membrane resistance for unit length rm = 59.4 k omega cm; longitudinal resistance per unit length ri = 4.9 M omega/cm; longitudinal resistance per unit length under the Vaseline seals re = 438 M omega/cm; specific membrane resistance Rm = 1.176 k omega cm2; input capacitance = 5.53 nF; specific membrane capacitance = 8.9 microF/cm2. 3. The maximum amplitude of ICa at 17 degrees C was: in 2 mM [Ca2+]o, -0.42 microA/microF and in 10 mM [Ca2+]o, -1.44 microA/microF. At 27 degrees C and in 10 mM [Ca2+]o, it increased to -3.04 microA/microF. The calculated temperature coefficient (Q10) for the increase in amplitude from 17 to 27 degrees C was 2.1. 4. Ca2+ permeability (PCa) was calculated using the Goldman-Katz relation; in 2 mM [Ca2+]o at 17 degrees C, PCa = 1.26 x 10(-6) cm/s; in 10 mM [Ca2+]o at 17 degrees C, PCa = 2.23 x 10(-6) cm/s; in 10 mM [Ca2+]o at 27 degrees C, PCa = 4.03 x 10(-6) cm/s. 5. The activation curve calculated from the PCa was shifted by 10 mV to positive potentials when raising [Ca2+]o from 2 to 10 mM. Increasing the temperature did not change the curve. The mid-point potentials (Va 1/2) and steepness (k) of the activation curves were: at 17 degrees C, in 2 mM [Ca2+]o, Va 1/2 = -1.53 mV and k = 6.7 mV; in 10 mM [Ca2+]o, Va 1/2 = 9.96 mV and k = 6.8 mV; at 27 degrees C and 10 mM [Ca2+]o, Va 1/2 = 11.3 mV and k = 7.7 mV. The activation time constant in 10 mM [Ca2+]o reached a plateau at potentials positive to 10 mV, with a value of 93.8 ms at 17 degrees C and 17.4 ms at 27 degrees C. The calculated Q10 was 4.5. 6. The deactivation of the current was studied from tail currents at different membrane potentials in 10 mM [Ca2+]o.(ABSTRACT TRUNCATED AT 400 WORDS)


Extracellular divalent and trivalent cation effects on sodium current kinetics in single canine cardiac Purkinje cells. 1. The effects of the extracellular divalent cations barium, calcium, cadmium, cobalt, magnesium, manganese, nickel and zinc and the trivalent cation lanthanum on macroscopic sodium current (INa) were characterized in enzymatically isolated single canine cardiac Purkinje cells under voltage clamp at 9-14 degrees C. 2. All di(tri)valent cations produced depolarizing shifts in the conductance-voltage relationship. The order of efficacy, taken as the concentration required to produce a 5 mV shift in the mid-point of peak INa conductance, from least to most effective was (mM): Ca2+ (2.97) approximately Mg2+ (2.67) approximately Ba2+ (1.93) > CO2+ (1.02) approximately Mn2+ (0.88) > Ni2+ (0.54) > La3+ (0.095) approximately Cd2+ (0.083) approximately Zn2+ (0.076). 3. Addition of di(tri)valent cations also produced depolarizing shifts in voltage-dependent availability. The order of efficacy from the least to most effective was (mM): Cd2+ (7.70) approximately Mg2+ (6.86) approximately Ba2+ (4.50) > Ca2+ (2.47) approximately CO2+ (1.87) approximately Mn2+ (1.24) approximately Ni2+ (1.20) > Zn2+ (0.300) > La3+ (0.060). 4. The Gouy-Chapman-Stern equations were used to evaluate di(tri)valent cation efficacy in binding to surface charges. Surface charge density was estimated as 0.72 sites nm-2, and it was assumed that Mg2+, the divalent cation that produced the smallest shift, screened but did not bind to surface charges. Based on voltage-dependent availability, KD from lowest to highest affinity were (mM): Ba2+ (2500) > CO2+ (1670) approximately Mn2+ (1430) approximately Ca2+ = Cd2+ = Ni2+ (1200) > Zn2+ (250) > La3+ (30). 5. All di(tri)valent cations also produced a concentration-dependent acceleration of INa tail current relaxation. The addition of Ca2+ and La3+ produced acceleration of tail current relaxations that could be accounted for by the surface charge effects predicted from the shift in voltage-dependent availability. Cd2+, which produced almost no change in voltage-dependent availability, dramatically accelerated tail current relaxation. Zn2+, Ni2+, Mn2+ and CO2+ also produced greater acceleration of tail current relaxation than could be accounted for by surface charge effects. 6. Di(tri)valent cations delayed time to peak INa in a concentration-dependent manner. The time to peak INa-voltage relationship was well described by an exponential plus a constant, and di(tri)valent cations did not affect the slope factor or constant but shifted the relationship in the depolarizing direction. Similar to their effect on tail currents, addition of some di(tri)valent cations produced larger effects on time to peak INa than expected from the shift of voltage-dependent availability.(ABSTRACT TRUNCATED AT 400 WORDS)


The giant cardiac membrane patch method: stimulation of outward Na(+)-Ca2+ exchange current by MgATP. 1. A giant patch method was used to study the stimulatory effect of cytoplasmic MgATP on outward Na(+)-Ca2+ exchange current in inside-out cardiac membrane patches (1-10 G omega seals with 14-24 microns pipette tip diameters) excised from guinea-pig, rabbit and mouse myocytes. 2. To establish the validity of the method with respect to structure, bleb formation was examined with electron microscopy and with confocal fluorescence light microscopy. The blebs, which form as the sarcolemma detaches, excluded intracellular organelles and transverse tubules. The blebbed cells contained normal sarcomeres, sarcoplasmic reticulum, triads and diads. 3. To further establish the validity of the method for ion transport studies, measurements of Na(+)-K+ pump currents and charge movements are described briefly which demonstrate (i) free access to the cytoplasmic membrane side, (ii) MgATP dependence comparable to reconstituted pump (Kd, 94 microns), (iii) fast, rigorous concentration control and (iv) Na(+)-K+ pump densities in the range of whole-cell densities. 4. Stimulation of outward Na(+)-Ca2+ exchange current by MgATP attenuated exchange current decay during step increments of cytoplasmic sodium, shifted the secondary activation of outward exchange current by cytoplasmic calcium to lower free calcium concentrations and, particularly in mouse cardiac sarcolemma, induced cytoplasmic calcium-independent current. 5. Upon removal of MgATP the stimulatory effect usually decayed with a t50 (half-time) of about 3 min. However, the reversal took place much more rapidly (t50, 5-20 s) in patches from individual guinea-pig and rabbit myocyte batches. When decay was rapid, secondary activation by cytoplasmic calcium was shifted to higher free cytoplasmic calcium concentrations (Kd, 10-65 microns-free calcium). 6. With repeated applications of MgATP the rate and magnitude of the stimulatory effect progressively decreased. 7. The Kd for MgATP of the initial rate of stimulation of outward exchange current was 3 mM or greater. When decay was rapid, the steady-state dependence of exchange current on MgATP also had a Kd of 3 mM or greater. 8. Stimulation of Na(+)-Ca2+ exchange current by MgATP occurred in the absence of cytoplasmic calcium with 9 mM-EGTA. 9. The stimulatory effect of 2 mM-MgATP was not inhibited by up to 200 microM of the protein kinase inhibitor 1-(5-isoquinoline sulphonyl)-2-methylpiperazine (H7), or by peptide inhibitors of cyclic AMP-dependent protein kinase, protein kinase C and calcium-calmodulin-dependent protein kinase II.(ABSTRACT TRUNCATED AT 400 WORDS)


Mechanisms of extracellular divalent and trivalent cation block of the sodium current in canine cardiac Purkinje cells. 1. Single canine cardiac Purkinje cells were internally perfused and voltage clamped with a large-bore perfusion pipette for measurement of sodium ionic current (INa) in the absence and presence of extracellular group IIA divalent cations (Mg2+, Ba2+ and Ca2+), transition divalent cations (CO2+, Mn2+ and Ni2+), group IIB divalent cations (Cd2+ and Zn2+), and the trivalent cation La3+. 2. Open channel block of cardiac INa by external Ca2+, assessed from instantaneous INa-voltage (I-V) relationships, has been well described by a two-barrier, one-well model with a dissociation constant at 0 mV, KB(0), of 37 mM and an electrical distance, z' = delta, of 0.34. At the most negative test potentials there was less block of INa than predicted by the model, but correction of INa for the contribution of Na+ channel gating current (Ig) to the peak current improved the fit by the model. 3. The divalent cations Ba2+, Mg2+, CO2+ and Mn2+ produced voltage-dependent, open channel block of INa, which by the two-barrier, one-well model predicted a similar z' about one-third into the membrane field. The relative efficacy for voltage-dependent block was CO2+ > Mn2+ > Ca2+ > Mg2+ > Ba2+ with respective KB(0)s of 11, 13, 37, 43 and 61 mM. 4. Cd2+, Zn2+ and La3+ produced block of INa at low concentrations that was nearly voltage independent with z' < or = 0.13. Fits of single-site binding curves to peak INa in response to step depolarizations at positive test potentials gave the following apparent KD values: Zn2+ 0.14 mM, Cd2+ 0.27 mM and La3+ 0.50 mM. 5. In the presence of Cd2+, INa tail current relaxations were much faster than could be accounted for by Cd2+ binding to and/or screening of extracellular surface charges. Fits of the data to a model that assumed voltage-dependent open channel block during the tail current relaxations estimated the KB(0) for Cd2+ to be 0.80 mM. 6. Both z' and KB(0) for Ni2+ from fits of the two-barrier, one-well model to instantaneous I-V relationships varied as a function of [Ni2+], consistent with the hypothesis that Ni2+ blocked with similar affinity at a voltage-dependent and a voltage-independent site. At [Ni2+] > or = 5 mM, KB(0) was 7.6 mM and z' was 0.21.(ABSTRACT TRUNCATED AT 400 WORDS)


Calcium transients caused by calcium entry are influenced by the sarcoplasmic reticulum in guinea-pig atrial myocytes. 1. Single atrial myocytes obtained by enzyme perfusion from hearts of adult guinea-pigs were investigated using whole-cell voltage clamp and Indo-1 micro-fluorometry. 2. In myocytes loaded with a solution containing citrate as a low-affinity, non-saturable Ca2+ chelator, two types of [Ca2+]i transients could be recorded during repetitive activation of L-type Ca2+ current. Both large and small [Ca2+]i transients occurred; large transients reached peak values of about 1 microM, and small transients were about 100 nM or less in amplitude. 3. In the case of the large transients, peak [Ca2+]i was usually reached with a variable delay after repolarization from a voltage step that activated calcium current (ICa). For the small transients the rise in [Ca2+]i paralleled ICa. Upon repolarization [Ca2+]i started to decay. 4. The small transients reflect entry of Ca2+ through Ca2+ channels (entry transients), whereas the large transients are due to entry and release from the sarcoplasmic reticulum (release transients). 5. The entry transients displayed a positive staircase pattern during trains of depolarizing voltage steps despite constant or even decreasing amplitude of ICa. The steepness of the staircase was increased by elevation of [Ca2+]o. Entry transients were always smallest immediately after a release transient. 6. After functional removal of the sarcoplasmic reticulum by caffeine (1-5 mM) the staircase pattern of the transients reflecting Ca2+ entry was abolished. 7. It is concluded that the staircase pattern is due to rapid uptake by the sarcoplasmic reticulum of Ca2+ entering the cell, resulting in an attenuation of the signal. The attenuation is strongest shortly after a release signal, when the rate of sequestration of Ca2+ by the SR should be highest. 8. Evidence is provided that a compartment of the SR is involved in attenuation of the entry transients. This compartment has been identified recently as a peripheral release compartment.


Potential-dependent inward currents in single isolated smooth muscle cells of the rat ileum. 1. Calcium (ICa) and sodium (INa) currents were studied in single smooth muscle cells freshly isolated from both the newborn (1-3 days old) and adult rat ileum, using the patch-clamp technique (whole-cell configuration). 2. Under conditions when INa was blocked, two components of ICa, low-voltage activated or ICa,low and high-voltage activated or ICa,high, were observed in the newborn rat ileal cells. ICa,high and ICa,low have differing voltage ranges of activation and steady-state inactivation and time courses of recovery from inactivation. Potential dependence of ICa,low was much steeper and shifted toward negative membrane potential than that for ICa,high (slope factors and the potential of half-maximal inactivation were 13.6 and -60.6 and 8.8 and -49 mV for ICa,low and ICa,high, correspondingly). 3. Nifedipine at the high concentration of 30 microM exerted no effect on ICa,low and only slightly suppressed ICa,high, decreasing its peak to 0.81 +/- 0.04 (n = 7) at the holding potential of -80 mV and to 0.66 +/- 0.05 (n = 3) at -50 mV. ICa,high was suppressed significantly by Cd2+ ions, while ICa,low was more sensitive to Ni2+ ions. 4. Results presented here suggest that the properties of high-voltage-activated (HVA) Ca2+ channels in the rat small intestine are quite different to those described for L-type Ca2+ channels found in other smooth muscles. It is proposed that HVA Ca2+ channels are similar to N-type Ca2+ channels. 5. Comparison of Ca2+ currents in newborn and adult rat ileal cells showed that the contribution of ICa,low to the net Ca2+ current was negligible in adults, whereas the properties of HVA Ca2+ channels were similar in the neonatal and adult animals. 6. INa, studied in nominally Ca(2+)-free physiological salt solution, activated in the voltage range between -50 and -40 mV and reached its peak at -10 mV. INa was blocked in a dose-dependent manner by TTX with an apparent dissociation constant of 4.5 nM. 7. INa decay was monoexponential in the voltage range studied and its time constant decreased monotonically with membrane depolarization from 4.7 +/- 0.2 ms (n = 6) at -30 mV to 0.51 +/- 0.03 ms (n = 7) at 20 mV.(ABSTRACT TRUNCATED AT 400 WORDS)


Sulphydryl reagents trigger Ca2+ release from the sarcoplasmic reticulum of skinned rabbit psoas fibres. 1. By analogy with studies on sarcoplasmic reticulum (SR) vesicles, Ca2+ release induced by heavy metals and mercaptans (e.g. cysteine) was investigated in rabbit skinned psoas fibres through measurements of isometric tension. 2. Heavy metals (at 2-5 microM) elicited phasic contractions by triggering Ca2+ release from the SR and had the following order of potency: Hg2+ > Cu2+ > Cd2+ > Ag+ > Ni2+. Higher concentrations produced tonic contractions due to maintained high Ca2+ permeability of SR membranes. 3. Contractions induced by heavy metals required a functional and Ca(2+)-loaded SR, were dependent on [Ca2+]free, blocked by Ruthenium Red (RR), inhibited by free Mg2+ and reduced glutathione (GSH) but not by oxidized glutathione (GSSG). Such contractions were not elicited through direct interaction(s) of heavy metals with the myofilaments. 4. In the presence of catalytic concentrations of Hg2+ or Cu2+ (2-5 microM), additions of cysteine (25-100 microM) elicited rapid twitches, producing 70% of maximal force with a time to half-peak of 2 s. Contractions induced by cysteine plus a catalyst required a functional SR network, were dependent on free [Mg2+] and were blocked by RR or GSH but not by GSSG. 5. In the presence of Hg2+ (2-5 microM), low concentrations of cysteine (10 microM) elicited tonic contractures, but subsequent or higher additions of cysteine (50-100 microM) caused further SR Ca2+ release and tension, followed by rapid and full relaxation. 6. High cysteine (200-250 microM, without Cu2+ or Hg2+) blocked contractions elicited by Cl- induced depolarization of sealed T-tubules. High cysteine probably acted as a sulphydryl reducing agent which promoted rapid relaxation of the fibres through the closure of Ca(2+)-release channels and ATP-dependent re-uptake of Ca2+ by the SR. 7. In some batches of skinned fibres (approximately 10%), cysteine (5-50 microM) alone (without Hg2+ or Cu2+ catalyst) produced rapid twitches. This implied that the catalyst(s) necessary to promote the sulphydryl oxidation reaction with exogenously added cysteine may be present in intact fibres but is usually lost by the skinning procedure. 8. The data demonstrate that skeletal fibres contain a highly reactive and accessible sulphydryl site on an SR protein which can be reversibly oxidized and reduced to respectively, open and close SR Ca(2+)-release channels. A model of sulphydryl-gated excitation-contraction coupling is proposed where the voltage sensor on the T-tubule membrane directly oxidizes sulphydryl sites on SR Ca(2+)-release channels.


Transient hyperpolarization of non-contracting muscle fibres in anaesthetized rats. 1. Following laminectomy, the L5 ventral roots of anaesthetized rats were split and approximately half of the nerve fibres were stimulated at 40 Hz. Resting membrane potentials were then measured in previously contracting and non-contracting soleus muscle fibres. 2. In the non-contracting soleus fibres there was a post-tetanic increase in mean resting potential (from -82.2 +/- 7.1 (S.D.) to -91.6 +/- 8.7 mV) which was similar to that in contracting fibres (from -83.02 +/- 6.1 to -91.3 +/- 7.3 mV). In both types of fibres the hyperpolarizing responses (HRs) were evidently due to increased sodium pump activity since they could be abolished by the addition of ouabain (1 x 10(-4) M) to the bathing fluid. 3. The beta-adrenergic antagonist, propranolol, completely suppressed HRs in the non-contracting fibres and produced moderate reductions in the contracting ones. The alpha-adrenergic blocking agent, phentolamine, had no effect on the contracting fibres and only a modest, inhibitory, one on the non-contracting fibres. 4. On the basis of the above drug actions it appeared that catecholamines were necessary for the full development of HRs in contracting and non-contracting fibres; noradrenaline, released from intramuscular sympathetic nerve endings, may have been involved. 5. The increased sodium pump activity in the non-contracting fibres would serve to moderate the rise in interstitial [K+] caused by K+ efflux from the contracting fibres. By preventing passive depolarization, due to the rise in interstitial [K+], the sodium pump would also maintain the availability of non-contracting fibres for subsequent recruitment.


Central and peripheral fatigue of human diaphragm and limb muscles assessed by twitch interpolation. 1. This study used a sensitive modification of the twitch interpolation technique to compare the extent of voluntary neural drive to the diaphragm and the elbow flexors during fatigue. For the diaphragm both inspiratory and expulsive efforts were tested, and fatigue was induced by expulsive efforts which were either maximal voluntary contractions (MVCs, 10 s duration, 50% duty cycle) or submaximal contractions (50% MVC, 3 s duration, 60% duty cycle). 2. Over the series of thirty MVCs peak elbow torque declined to 57.9 +/- 3.0% (mean +/- S.E.M.) of the initial value while maximal inspiratory pressure declined to 78.7 +/- 7.3% (P < 0.05). For the diaphragm the relative decline in voluntary peak inspiratory (and expulsive) force was similar to the decline in twitch responses to single and twin (10 ms interval) stimuli. However, for the elbow flexors the decline in twitch force was disproportionately greater than the decline in maximal voluntary force. The decline in twitch force for the diaphragm could not be attributed to failure at the neuromuscular junction. 3. At the start of the exercise, twitch potentiation (following three brief MVCs) was significantly less for the diaphragm than for the elbow flexors (20% versus 61%, P < 0.01). 4. In the unfatigued state maximal voluntary efforts by subjects activated 98.4 +/- 0.4% of the stimulated elbow flexors compared with 95.0 +/- 1.5% of the diaphragm (P < 0.05). During the exercise period there was a progressive failure in the ability to activate the limb muscle ('central fatigue'; voluntary drive declined from 98.4 +/- 0.4 to 86.8 +/- 2.2%, P < 0.01) whereas the decline in voluntary activation during inspiratory contractions was not significant (from 95.0 +/- 15 to 91.5 +/- 2.5%). 5. Voluntary activation during attempted maximal efforts was less complete for both muscles when stimuli were delivered without warning. The index of voluntary activation for unwarned stimuli was lower for the diaphragm (performing expulsive efforts, 81.0 +/- 2.8%) than for the limb muscle (89.9 +/- 1.5%, P < 0.01). 6. During repeated submaximal expulsive efforts we confirmed that subjects develop a marked inability to contract the diaphragm voluntarily, but when the diaphragm performed inspiratory manoeuvres at the same level of contractile fatigue, the index of voluntary drive was greater than 94%. 7. In conclusion, when tested with inspiratory efforts the diaphragm developed less central fatigue than the limb muscle over the same exercise period.(ABSTRACT TRUNCATED AT 400 WORDS)


Postnatal changes in T-type calcium current density in rat atrial myocytes. 1. Postnatal changes in Ca2+ current were studied in voltage clamped atrial myocytes isolated from Sprague-Dawley rats. T- and L-type Ca2+ currents were identified using standard electrophysiological and pharmacological techniques. Cells were studied from seven groups of male and six groups of female rats ranging in age from 3 to 14 weeks. 2. The density of atrial T-type Ca2+ current showed significant variation during postnatal development, with a maximum density reached at 4.5-5 weeks. At this age, T-current density was 1.44 +/- 0.11 pA/pF (n = 23) for cells isolated from male and 1.25 +/- 0.09 pA/pF (n = 25) for cells isolated from female animals in bathing solutions containing 2 mM-Ca2+. T-current density in atrial cells isolated from younger animals (3.5 weeks postnatal) averaged 1.22 +/- 0.06 (n = 18) and 1.00 +/- 0.05 pA/pF (n = 22) or 85 and 80% of the maximum seen at 4.5-5 weeks for male and female rats, respectively. For rats older than 13 weeks, the average T-current density in atrial cells was 0.50 +/- 0.03 (n = 18) and 0.51 +/- 0.02 pA/pF (n = 35) or 35 and 41% of the maximum seen at 4.5-5 weeks for male and female rats, respectively. 3. In contrast to the T-type current, the density of atrial L-type Ca2+ current remained unchanged in rats from 3 to 14 weeks old. L-type current averaged 8.2 +/- 0.2 (n = 134) in male and 7.9 +/- 0.2 pA/pF (n = 102) in female rats. 4. Fluctuation analysis was used to estimate single T-channel current levels in 4.5- and 7.5-week-old male rats. While the T-current density differed by 70% at these two postnatal ages, no significant difference (P > 0.2) in single channel current was found. Single channel current was 0.12 +/- 0.01 pA (n = 9) for cells from 4.5-week-old and 0.13 +/- 0.01 pA (n = 7) for cells from 7.5-week-old rats. Currents were stimulated by test pulses from -80 to -30 mV at 5 mM-Ca2+. 5. No postnatal changes were seen in either the kinetics of activation or inactivation of macroscopic T-current.(ABSTRACT TRUNCATED AT 400 WORDS)


Phosphorylation restores activity of L-type calcium channels after rundown in inside-out patches from rabbit cardiac cells. 1. Rundown of L-type calcium channels was studied in inside-out patches made from single isolated rabbit ventricular myocytes, using barium as the charge carrier. 2. In the cell-attached patches single-channel activity was stable for more than 15 min after the patch pipette sealed. beta-Receptor stimulation by isoprenaline caused a characteristic increase in opening probability and the appearance of prolonged openings. When the patch was excised to the inside-out configuration and exposed to a simple ionic solution, channel activity disappeared within 1-2 min and never reappeared spontaneously. 3. After rundown of L-type channel activity in the excised patch, exposure of the inside face of the patch to MgATP and the catalytic subunit of the cyclic AMP-dependent protein kinase (PKAc) resulted in recovery of Ca2+ channel activity. Under these conditions channel activity could be even greater than under control cell-attached conditions, resembling channel activity after exposure to isoprenaline. This recovery of activity persisted many minutes, usually until the patch was lost. Addition of MgATP alone caused a small transient increase in channel activity in some patches. 4. Recovery of activity by MgATP and PKAc could be prevented by prior exposure of the excised patch to protein kinase inhibitor (PKI), or it could be abruptly terminated by exposure to PKI after recovery of activity. Addition to the pipette solution of okadaic acid, a protein phosphatase inhibitor, greatly slowed rundown. These findings support the proposal that dephosphorylation is an important component of rundown, and that phosphorylation is needed for channel opening activity. 5. Single-channel conductance was not altered by patch excision, but it was reduced after exposure of the excised patch to MgATP and PKAc. Mg2+ was responsible for this effect, probably by direct channel block from the inside, and Mg2+ also caused a negative shift in the channel activation, as expected from shielding of inside fixed negative charges.


An electrophysiological study of responses evoked in isolated segments of rat tail artery during growth and maturation. 1. Intracellular recordings from the smooth muscle of isolated segments of the main caudal artery of rats at various ages between 45 and 150 days postnatal were made in order to relate the spontaneous depolarizations and responses to perivascular stimulation at different levels along the artery to the differences in vessel structure and innervation density during growth of the animals. 2. In the outermost smooth muscle cells close to the neuromuscular junctions, spontaneous depolarizations with fast time courses (spontaneous excitatory junction potentials or SEJPs) were recorded. In cells lying deeper in the media, spontaneous depolarizations had a wide range of time courses and amplitudes, but only a few of those could be attributed to electrotonic attenuation of SEJPs. 3. In arterial segments taken from animals of all ages, stimuli which evoked maximal amplitude excitatory junction potentials (EJPs) 1-2 mm caudal to a suction electrode also evoked neurogenic alpha-depolarizations (NADs) with time to peak of 15 s and duration nearly 1 min. Both responses decreased progressively in amplitude along the length of the artery. NADs were blocked by phentolamine (10(-6) M) or idazoxan (10(-7) M) which were without effects on EJPs. 4. During short trains of stimuli (5 at 1 or 10 Hz), EJPs facilitated but to a lesser extent with distance along the tail. Such trains evoked NADs of greater amplitude than those following a single stimulus; these were often preceded by contractions of the artery which were restricted to the region close to the stimulating electrode. 5. Increasing stimulus voltage led to progressive prolongation of the decay phase of the EJP. After the addition of tetrodotoxin (10(-7) M), or in the presence of reduced Ca2+ and raised Mg2+ concentration, slow depolarizing potentials (SDPs) (with time to peak of 150-300 ms and decay lasting > 2 s) were recorded which were graded in amplitude with stimulus voltage. SDPs were attenuated by increasing Ca2+ concentration to 5 mM. These responses often added to the EJP at supramaximal stimulus voltages. 6. The mean amplitudes of the EJP and NAD declined significantly with age, the former to a greater degree than the latter. These changes may be explained by changes in the electrical properties of the media related to hypertrophy of smooth muscle cells as the animals grew, and emphasize the need to allow for such growth effects in studies of young rats.(ABSTRACT TRUNCATED AT 400 WORDS)


Probabilistic secretion of quanta from visualized sympathetic nerve varicosities in mouse vas deferens. 1. Sympathetic varicosities on the surface of smooth muscle cells of the mouse vas deferens were visualized with the fluorescent dye 3-3 Diethyloxardicarbocyanine iodide (DiOC2(5)) and quantal secretion recorded from these with both small diameter (4-6 microns) and large diameter (20-50 microns) microelectrodes. Small diameter electrodes were placed over one to three varicosities and large diameter electrodes over three to seven varicosities. 2. The size and distribution of varicosities along individual terminal branches was about the same when these were fluoresced with DiOC2(5) (length 1.09 +/- 0.40 microns (mean +/- S.D.); intervaricosity distance 5.53 +/- 2.68 microns) as when they were stained for catecholamines using Faglu fluorescence (length 1.05 +/- 0.43 microns; intervaricosity distance 5.12 +/- 2.79 microns) suggesting that DiOC2(5) does allow for identification of the catecholamine-containing varicosities. 3. The spontaneous excitatory junctional currents (EJCs) recorded from visualized varicosities with small diameter electrodes (amplitudes 59-67 microV) were much larger than those recorded with large diameter electrodes (amplitudes 25-29 microV). The frequency of evoked EJCs as well as the amplitude-frequency distribution of these EJCs varied greatly between sets of visualized varicosities recorded along individual branches, either with a small or large diameter electrode. These amplitude-frequency distributions typically followed Poisson statistics, in which the mean quantal content of the EJC (m) varied by over threefold for different sets of varicosities on the same branch (m was 0.07-0.21 for small electrodes whereas m was 1-3 for large electrodes). 4. Although m varied considerably for a constant number of varicosities beneath the electrode at different sites along a single branch, there was an overall correlation between m and the number of varicosities, m increasing on average 0.25 for each additional varicosity in a [Ca2+]o of 4.0 mM. 5. The frequency of evoked EJCs at visualized sets of varicosities along some branches was sufficiently high to allow binomial statistics to predict the amplitude-frequency distributions of evoked EJCs. In these cases m was again shown to vary considerably along single terminal branches, and this was primarily due to variation in the probability of secretion (p) between sets of varicosities and not to variation in binomial parameter n. 6. In one case a relatively isolated varicosity, over 3 microns from adjacent varicosities, was recorded for 30 min with a 4 microns diameter electrode. The mean and variance of the evoked EJC was similar to that of the spontaneous EJCs suggesting that this varicosity secreted at most one quantum on arrival of the nerve impulse.(ABSTRACT TRUNCATED AT 400 WORDS)


Camptothecin inhibits both the cleavage and religation reactions of eukaryotic DNA topoisomerase I. We investigated the mode of action of the antitumor drug, camptothecin, by use of a partly double-stranded suicide DNA substrate which enables uncoupling of the cleavage and religation half-reactions of topoisomerase I. The suicide DNA substrate contains a single topoisomerase I site at which SDS cleavage is strongly enhanced by camptothecin on normal double-stranded DNA. The results show that the religation reaction of topoisomerase I per se is strongly inhibited at this site compared to site that is only marginally affected by camptothecin on double-stranded DNA. This study hereby directly demonstrates that camptothecin-mediated stability of a topoisomerase I-DNA complex is sequence-dependent. The influence of camptothecin on the suicide cleavage reaction of topoisomerase I was also investigated. Surprisingly, the cleavage reaction per se is strongly inhibited by the drug. However, reformation of a cleavable suicide DNA substrate, which is fully double-stranded downstream from the cleavage position except for a nick, completely reverses the inhibitory effect of the drug on the cleavage reaction. The results suggest that the inhibitory effect of camptothecin on cleavage is due to a general decrease in the noncovalent interaction of topoisomerase I with partly double-stranded suicide DNA substrates. Based on the findings, a plausible model for camptothecin action is discussed.


Interlocking of plasmid DNAs due to Lac repressor-operator interaction. The presence of a single lac repressor binding sequence on plasmid DNAs is shown to mediate the formation of interlocked dimers in E. coli. The presence of both homo- and hetero-interlocked dimers suggests that the lac repressor complex can bring together randomly two plasmid DNA molecules to facilitate gyrase-mediated interlocking. The exclusive formation of multiply intertwined dimers also suggest that the lac repressor complex may bind simultaneously to a pair of replicated daughter plasmid molecules prior to their segregation. The formation of interlocked plasmid DNAs can be indicative of interaction between two DNA bound proteins in vivo.


Nuclear magnetic resonance solution structure of hirudin(1-51) and comparison with corresponding three-dimensional structures determined using the complete 65-residue hirudin polypeptide chain. The three-dimensional structure of the N-terminal 51-residue domain of recombinant hirudin in aqueous solution was determined by 1H nuclear magnetic resonance (NMR) spectroscopy, and the resulting high-quality solution structure was compared with corresponding structures obtained from studies with the intact, 65-residue polypeptide chain of hirudin. On the basis of 580 distance constraints derived from nuclear Overhauser effects and 109 dihedral angle constraints, a group of 20 conformers representing the solution structure of hirudin(1-51) was computed with the program DIANA and energy-minimized with a modified version of the program AMBER. Residues 3 to 30 and 37 to 48 form a well-defined molecular core with two antiparallel beta-sheets composed of residues 14 to 16 and 20 to 22, and 27 to 31 and 36 to 40, and three reverse turns at residues 8 to 11 (type II), 17 to 20 (type II') and 23 to 26 (type II). The average root-mean-square deviation of the individual NMR conformers relative to their mean co-ordinates is 0.38 A for the backbone atoms and 0.77 A for all heavy atoms of these residues. Increased structural disorder was found for the N-terminal dipeptide segment, the loop at residues 31 to 36, and the C-terminal tripeptide segment. The solution structure of hirudin(1-51) has the same molecular architecture as the corresponding polypeptide segment in natural hirudin and recombinant desulfatohirudin. It is also closely similar to the crystal structure of the N-terminal 51-residue segment of hirudin in a hirudin-thrombin complex, with root-mean-square deviations of the crystal structure relative to the mean solution structure of 0.61 A for the backbone atoms and 0.91 A for all heavy atoms of residues 3 to 30 and 37 to 48. Further coincidence is found for the loop formed by residues 31 to 36, which shows increased structural disorder in all available solution structures of hirudin, and of which residues 32 to 35 are not observable in the electron density map of the thrombin complex. Significant local structural differences between hirudin(1-51) in solution and hirudin in the crystalline thrombin complex were identified mainly for the N-terminal tripeptide segment and residues 17 to 21. These are further analyzed in an accompanying paper.


Secondary structure changes stabilize the reactive-centre cleaved form of SERPINs. A study by 1H nuclear magnetic resonance and Fourier transform infrared spectroscopy. Proteinase inhibitor members of the SERPIN superfamily are characterized by the presence of a proteolytically sensitive reactive-site loop. Cleavage within this region results in a conformational transition from an unstable "stressed" native protein to a more stable "relaxed" cleaved molecule. In order to identify the principal molecular aspects of this transition, 1H nuclear magnetic resonance (n.m.r.) and FT-IR spectroscopy were applied to the study of four SERPINs. 1H n.m.r. spectra of approximately 20 high-field ring-current-shifted methyl signals exhibited slightly different chemical shifts in the native and cleaved forms of alpha 1-antitrypsin (alpha 1-AT), alpha 1-antichymotrypsin (alpha 1-ACT) and C1 inhibitor (C1-INH), but not ovalbumin, between 20 degrees C and 90 degrees C. Ring current calculations based on crystal co-ordinates for cleaved alpha 1-AT and alpha 1-ACT and native ovalbumin showed that these signals originate from highly localized interactions between different buried residues corresponding to alpha-helix and beta-sheet segments of the SERPIN fold. The small shift changes correspond to small relative conformational side-chain rearrangements of about 0.01 nm to 0.05 nm in the protein hydrophobic core, i.e. the tertiary structure interactions in the two forms of the SERPIN fold are well-preserved, and changes in this appear unimportant for the stabilization found after reactive centre cleavage. Fourier transform infrared (FT-IR) spectroscopic studies of the amide I band showed that the native and cleaved forms of alpha 1-AT, alpha 1-ACT and C1-INH contain 28% to 36% alpha-helix and 38% to 44% beta-sheet. Second derivative FT-IR spectra using H2O and 2H2O buffers revealed very large differences in the amide I band between the native and cleaved forms of alpha 1-AT, alpha 1-ACT and C1-INH, but not for ovalbumin. The alpha-helix band was most sensitive to 1H-2H exchange, while the beta-sheet bands were not, and greater amounts of antiparallel beta-sheet were detected in the cleaved form. 1H n.m.r. showed that polypeptide amide 1H-2H exchange was greater in the native forms of alpha 1-AT, alpha 1-ACT and C1-INH than in their cleaved forms, whereas for ovalbumin it was unchanged. The FT-IR and 1H-2H exchange data show that alterations in the secondary structure are central to the stabilization of the cleaved SERPIN structure.(ABSTRACT TRUNCATED AT 400 WORDS)


Crystallization and preliminary X-ray analysis of three serotypes of foot-and-mouth disease virus. Foot-and-mouth disease viruses from serotypes O, A and C have been crystallized. The particular strains studied include O1K, A10(61), A22 Iraq 24/64, A24 Cruzeiro and C-S8c1. In addition, crystals have been grown of G67, a monoclonal antibody neutralization escape mutant derived from O1K, and of virus R100, recovered after the establishment of a persistent infection in baby hamster kidney cells with C-S8c1. Empty particles, capsids which lack the RNA genome, have also been crystallized for subtypes A22 Iraq 24/64 and A10(61). In almost all cases, crystals suitable for high resolution structure determination were obtained from (NH4)2SO4 or mixtures of polyethylene glycol and NH4Cl.


Protective effect of methylprednisolone on vascular injury in rat spinal cord injury. High-dose methylprednisolone (MP) given to patients within 8 h of traumatic spinal cord improved neural function at 6 and 12 months, suggesting a probable secondary injury process that may be amenable to therapeutic intervention. Vascular injury plays an important role in the secondary injury process of CNS trauma. We have examined the effect of MP on vascular changes, including tissue edema, vascular permeability, and polymorphonuclear (PMN) cell infiltration in a rat model of spinal cord impact injury. MP significantly reduced extravasation of fluorescein isothiocyanate dextran (FITC-D), a macromolecular tracer, by 64.3% and 50.7% with trauma forces of 20 and 40 g-cm, respectively, when MP was administered IV immediately after trauma at a bolus of 165 mg/kg, with a subsequent continuous MP infusion at 31.5 mg/kg/h for 23 h. MP reduced the water content in the 40 g-cm traumatic cord lesion to 73.0% compared to the traumatic control (74.3%, p < 0.001) at the same schedule of large dose 24-h infusion. The same doses of MP showed a trend to decrease the extent of neutrophil infiltration as determined by myeloperoxidase (MPO) activity, but the change was not significant. MP had little effect in decreasing FITC-D extravasation and cord edema when given at a lower dose (bolus of 30 mg/kg with continued infusion of 1.3 mg/kg/h for 23 h). MP did not reduce extravasation of FITC-D and edema when administered IV as one bolus injection at high (165 mg/kg) or low (30 mg/kg) doses.(ABSTRACT TRUNCATED AT 250 WORDS)


Decreased alpha 1-adrenergic receptors after experimental brain injury. The magnitude of neuronal damage in central nervous system (CNS) injury may be related, in part, to alterations in the balance between excitatory and inhibitory neurotransmitters. Previous studies have implicated a role of central inhibitory noradrenergic mechanisms in the pathophysiologic sequelae of traumatic brain injury. In the present study, we examined alpha 1-adrenergic receptor binding after parasaggital lateral fluid percussion (FP) brain injury of moderate severity (2.3 atm) in the rat. At 30 min following injury, the specific binding of [3H]prazosin to membranes isolated from left cortex (injury site) was reduced by 37% in brain-injured animals when compared to sham-operated noninjured animals (p < 0.05). However, there were no significant differences in [3H]prazosin binding to membranes of either contralateral (right) cortex or left and right hippocampi between brain-injured and sham-operated animals. Conversely, at 24 h posttrauma, specific binding to membranes of left cortex, cortex adjacent to injury site, contralateral (right) cortex, and left hippocampus was reduced by 25%, 16%, 27%, and 24%, respectively (all p < 0.05). Scatchard analysis revealed that a reduction of [3H]prazosin binding to membranes of injured animals resulted from a decrease in alpha 1-receptor binding density (B-max) but not from changes in ligand affinity. Histopathologic assessment of neuronal damage at 24 h postinjury revealed neuronal loss within injury site cortex and left hippocampus but no clearly discernible cell loss in contralateral right cortex, suggesting that the decrease in B-max might be a consequence of early pathophysiology of trauma rather than of neuronal cell loss. We suggest that alterations in alpha 1-adrenergic receptors after brain injury may result in decreased inhibitory neurotransmitter action of norepinephrine and may thus contribute to the pathophysiology of traumatic brain injury.


Molecular and genetic characterization of cytochrome oxidase-negative Aeromonas salmonicida isolated from coho salmon (Oncorhynchus kisutch). Cytochrome oxidase variants of the bacterial fish pathogen, Aeromonas salmonicida, were characterized for genetic and molecular homology with cytochrome oxidase-positive isolates that typically induce furunculosis in salmonids. Protein and lipopolysaccharide moieties of the cytochrome oxidase-negative variants were similar to their typical counterparts, based on sodium-dodecyl-sulfate polyacrylamide gel electrophoresis. Pathogenicity of aberrant isolates to brook trout (Salvelinus fontinalis) was similar to typical cytochrome oxidase-positive isolates. Colorimetric deoxyribonucleic acid (DNA) hybridization in 96-well microplates yielded homology values greater than 82.5% for typical aberrant A. salmonicida isolates when photobiotinylated DNA for reference A. salmonicida 3.101 was used as a probe. The only variation of these isolates from typical A. salmonicida was a negative cytochrome oxidase reaction.


Probable herpesvirus infection in an eastern cottontail (Sylvilagus floridanus). One wild eastern cottontail (Sylvilagus floridanus) from Milwaukee County, Wisconsin was necropsied. The lungs contained numerous multifocal, circumscribed, tan foci; the spleen was markedly enlarged and had a mottled reddish tan color; and the brain had a red to tan friable tract in the left hemisphere. Microscopically, the lung had a severe bronchiolitis and pneumonia. The bronchiolitis was characterized by epithelial cells containing eosinophilic intranuclear inclusion bodies. The encephalomalacia of the left cerebral cortex featured tissue disruption and astrocytes or neurons containing intranuclear inclusion bodies. Herpesvirus particles were found within the bronchiolar epithelial cells. Based on histopathological and ultrastructural findings, a herpesvirus seemed the most likely etiologic agent.


Experimental bluetongue and epizootic hemorrhagic disease virus infection in California black-tailed deer. Four adult black-tailed deer (Odocoileus hemioneus columbianus) and five fawns were inoculated with bluetongue virus (BTV) and one adult deer was inoculated with epizootic hemorrhagic disease (EHD) virus to produce clinical signs and lesions of hemorrhagic disease. Serologic response was monitored using the agar gel immunodiffusion (AGID) test and the competitive enzyme-linked immunosorbent assay (C-ELISA). Embryonating chicken eggs and vero cells were used to detect viremia. No animal exhibited clinical or pathologic signs of hemorrhagic disease. Bluetongue viremia was detected as early as 2 days post-inoculation (DPI-2) and in some animals, persisted until at least DPI-12. The earliest detection of BTV antibodies using the AGID was DPI-8. Two adult deer remained seropositive for BTV antibodies for > 9 mo and 1 yr, respectively, using both the AGID and C-ELISA tests. We observed cross reactions between BT and EHD antibodies using the AGID tests. Also, the AGID test did not consistently detect exposure to BTV. Viremia was not detected in the deer inoculated with EHD although this animal was AGID positive between DPI-6 and DPI-49.


[Problems in combined resection of adjacent organ in lung cancer: significance of preoperative and intraoperative ultrasonic examination]. Aggressive combined resections were carried out, on 24 lung cancer cases which showed invasion into surrounding organs. Those cases with wide infiltration of the ribs, and into surrounding intercostal tissues, and for those with invasion to diaphragm, the outcome of the operations was rather poor but, for those with invasion in pericardium and left atrium, fairly favorable results were obtained. These present results indicated that aggressive resections of adjacent organs is to be recommended for cases without N 2 infiltration. Experiences from these operations have taught us the importance and useful of diagnosis of the status of invasion of the tumor, during the operation by ultrasonogram (IUS), with direct contact of the probe to the thoracic wall or mediastinal organs, to discriminate the areas of chest wall and mediastinal organs that require resections. This method, in combination with esophagus ultrasonic endoscopy (EUS), enabled defining the infiltrated areas with accuracy far exceeding that obtained by tactile examination. Techniques of, and observations obtained by these examination methods are presented.


Ouabain-insensitive, vanadate-sensitive K(+)-ATPase of rat distal colon is partly similar to gastric H+,K(+)-ATPase. A membrane fraction from rat distal colon contained both ouabain-sensitive and -insensitive K(+)-ATPase activities, which were measured under Na(+)-free conditions. About 38% of the ouabain-insensitive K(+)-ATPase activity was inhibited by vanadate. It was determined whether the ouabain-insensitive, vanadate-sensitive K(+)-ATPase in the colon is similar or identical to gastric H+,K(+)-ATPase. This colonic K(+)-ATPase activity was inhibited completely by monoclonal antibody HK4001, which inhibits the hog gastric H+,K(+)-ATPase activity but not Na+,K(+)-ATPase or Ca(2+)-ATPase. The colonic ATPase activity was inhibited partly by SCH 28080, which is a specific reversible inhibitor of gastric H+,K(+)-ATPase. The colonic ATPase activity was stimulated by low concentrations of K+ (its half-maximal stimulating concentration was 1 mM) and inhibited by high concentrations of K+ (its half-maximal inhibiting concentration was 10 mM), indicating that high and low K+ affinity sites are present in the colonic enzyme as in gastric H+,K(+)-ATPase and that this enzyme is not fully operative under normal physiological conditions. Two other monoclonal antibodies, which inhibit the gastric H+,K(+)-ATPase activity, did not inhibit the colonic K(+)-ATPase activity. The present results suggest that the colonic ouabain-insensitive K(+)-ATPase is partly similar but not identical to the gastric H+,K(+)-ATPase.


[A case of hepatic hydrothorax treated by pleuro-venous shunt]. We reported a successful pleuro-venous shunt operation (Pl-V shunt) for a patient of liver cirrhosis with secondary hydrothorax. A 78-year-old female was admitted to our hospital because of severe dyspnea and palpitation. Chest x-ray film revealed right sided massive pleural effusion. Over several weeks the chest tube drainaged about 1,500 ml of transudative fluid per day. We performed Pl-V shunting and pleural effusion subsequently decreased in amount and dyspnea disappeared. This Pl-V shunting is thought to be useful for such a patient with massive pleural effusion which failed to respond to medical therapy.


[Small cell carcinoma of the bladder. Small cell lung cancer-associated ganglioside (Fuc GM1) expression]. A 61-year-old male visited us with chief complaints of macroscopic hematuria and bladder irritation symptoms. Cystoscope, U/S, MRI, and CT showed an extensive non-papillary, wide-based tumor centering around the anterior wall of the bladder. Transabdominal U/S-guided full-thickness biopsy indicated a pT3a (Biopsy) primary small cell carcinoma of the bladder containing neuroendocrine granules. Immunohistochemical studies revealed Fuc GM1, an antigen related to small cell carcinoma of the lung. Neoadjuvant therapy consisted of preoperative irradiation at 50 Gy and intra-arterial infusion chemotherapy with CDDP and THP. Since a follow-up full thickness biopsy indicated pT0 (Biopsy), total cystectomy was performed. Examination of the resected specimen also indicated pathological CR.


Porins and lipopolysaccharide stimulate platelet activating factor synthesis by human mesangial cells. Porins, a family of hydrophobic proteins located in the outer membrane of the cell wall of gram-negative bacteria and lipopolysaccharide (LPS), were shown to stimulate the synthesis of platelet activating factor (PAF), a phospholipid mediator of inflammation and endotoxic shock, by cultured human glomerular mesangial cells (MC). The synthesis of PAF induced by porins was rapid (peak at 20 min) and independent either from contamination by LPS or from generation of an endotoxin-induced cytokine such as tumor necrosis factor (TNF) since it was not prevented by cycloheximide, an inhibitor of protein synthesis or anti-TNF blocking antibodies. LPS also stimulated PAF synthesis by MC. However, the kinetic of PAF synthesis induced by LPS was biphasic with an early and transient peak at 10 minutes and a second and sustained peak at three to six hours. This second peak required an intact protein synthesis and was prevented by anti-TNF antibodies, suggesting the dependency on LPS-induced synthesis of TNF. Experiments with labeled precursors demonstrated that in MC, either after stimulation with porins or LPS, PAF was synthesized via the remodeling pathway that involves acetylation of 1-0-alkyl-sn-glyceryl-3-phosphorylcholine (2-lyso-PAF) generated from 1-0-alkyl-2-acyl-sn-glyceryl-3-phosphorylcholine by phospholipase A2 (PLA2) activity. Porins and LPS, indeed, induced PLA2-dependent mobilization of [14C]-arachidonic acid that was inhibited by p-bromodiphenacylbromide (PBDB). PBDB, an inhibitor of PLA2, also blocked PAF synthesis by preventing the mobilization of 2-lyso-PAF, the substrate for PAF-specific acetyltransferase.(ABSTRACT TRUNCATED AT 250 WORDS)


A circulating inhibitor of the RBC membrane calcium pump in chronic renal failure. A humoral inhibitor of the membrane calcium pump was studied in plasma from 28 normal controls, 33 patients receiving long-term hemodialysis, and 26 with chronic renal failure (CRF; creatinine clearance range was 6 to 97 ml/min). Calcium pump activity was measured as the rate of Sr2+ efflux in normal erythrocytes (RBCs) loaded with Sr2+ (a substitute of Ca2+ in the calcium pump). Plasma, and plasma ultrafiltrates from hemodialysis patients strongly inhibited calcium pump activity compared with controls without plasma (36 +/- 18 vs. 25 +/- 12, %INHIBITION/CONTROL, P < 0.05). Inhibition markedly decreased with acute hemodialysis (16 +/- 12 vs. 5 +/- 14, %INHIBITION/NORMAL PLASMA, N = 15, P < 0.001). In CRF, degree of inhibition correlated with the serum creatinine concentration (r = 0.75, P < 0.001). A kinetic study showed that plasma decreased the maximal rate of the Ca2+ pumps (Vmax) without affecting the apparent affinity for internal cations (KSr). Moreover, the plasma inhibitory factor had a low molecular weight, and was dialyzable and heat stable. In conclusion, we found evidence for an RBC membrane calcium pump inhibitor in uremic plasma, which correlates with the degree of renal insufficiency. Possibly, it may increase calcium content in RBCs and other cells and could thus be related to uremic toxicity and/or hypertension.


Red cell sodium-proton exchange is increased in Dahl salt-sensitive hypertensive rats. To investigate the relationship between red blood cell Na+/H+ exchange (EXC) and genetic factors in hypertension, we studied the maximal rate of the antiporter (mmol/liter cell x hr; flux units = FU) in three strains of genetically hypertensive rats. Salt-resistant Dahl rats (DR) were normotensive under low (0.02%) and high (8%) NaCl diets, while salt-sensitive Dahl rats (DS) became markedly hypertensive after four weeks on the high-NaCl diet. Na+/H+ exchange did not differ between DR and DS rats when both were fed with the low-NaCl diet (mean +/- SE, 31 +/- 3, N = 15, vs. 29 +/- 3 FU, N = 14). On the high-NaCl diet, the DR strain did not exhibit significant changes in blood pressure and antiporter activity, but the DS rats significantly increased their blood pressure and Na+/H+ exchange (57 +/- 4 FU, N = 13) versus DR rats (38 +/- 3 FU, N = 15, P < 0.02). DS rats also significantly increased blood pressure and antiporter activity when fed with high-NaCl diet for one week. These data indicate that high NaCl intake per se does not increase Na+/H+ EXC because the control DR strain did not exhibit transport and blood pressure alterations as observed in the DS strain. Milan hypertensive and spontaneously hypertensive rats (Charles River substrain) had higher blood pressures than Milan and Wistar-Kyoto normotensive rats when they were maintained for four weeks on a 1.5% NaCl diet; however, no differences were seen among normotensive and hypertensive strains in Na+/H+ exchange activity.(ABSTRACT TRUNCATED AT 250 WORDS)


Apparent first-order behavior under second-order kinetic conditions: a general concept illustrated by the reversible binding of hydrogen peroxide to cytochrome c oxidase. The theory of the second-order reversible reaction, A + B<-->A.B, has been extensively discussed. Apparent first-order behavior is observed when, for example, [B] >> [A]. If the reaction exhibits second-order behavior then it is presumed that the concentrations of A and B were initially equal and that they remain equal during the reaction. However, in the case of hydrogen peroxide binding to cytochrome c oxidase, Weng & Baker (1991, Biochemistry 30, 5727-5733) showed that the observed rate was rigorously first order over a broad concentration range of ligand, including the stoichiometric case. It was further shown that kobs increased linearly with [H2O2], precluding the possibility of a rate-limiting, unimolecular pre-step. The current work examines the theoretical rate equation for the bimolecular, reversible reaction when [A] = [B]. Simulations show that this equimolar condition resulted in rigorous exponential binding as kd, the equilibrium dissociation constant for the A.B complex, approached the initial concentration of A (or B). In particular, the second-order simulation was rigorously exponential when [A]o/Kd = 0.5, and showed only minor deviations when the ratio was increased to 25. These results demonstrate that a reversible, bimolecular reaction can appear first order even under second order conditions, without the need for more complicated mechanisms.


Assessing short-term recognition memory with forced-choice psychophysical methods. The rationale and methodology for using computer-controlled forced-choice psychophysical methods to assess short-term recognition memory in human subjects are presented. Here, we use non-verbal computer-synthesized auditory and visual stimuli with an adaptive psychophysical procedure. Sequence-length thresholds (SLTs, span lengths) for randomly generated binary auditory and visual-sequential patterns and simultaneous visual-spatial patterns are determined to assess short-term memory capacity. The SLTs can also be used to equate for initial retention level for delayed matching-to-sample (DMS) or delayed matching-to-non-sample (DMNS) tasks which assess memory decay. The DMS/DMNS tasks have also been modified for use with the forced-choice paradigm. In contrast to many verbal paradigms requiring immediate ordered recall, non-verbal stimuli in a forced-choice paradigm provide a more direct measure of sensory memory because long-term memory, complex encoding/decoding processes, and motor-sequencing factors are minimized or avoided. Furthermore, the forced-choice recognition memory tasks are applicable over a broad age range, are less sensitive to socio-economic factors and educational level, and avoid complex instructions. Taken together, these factors enhance the applicability of these tasks in children and adults with CNS lesions, particularly where cognitive status may be compromised.


Insulin desensitized beta 1-adrenergic receptor-mediated stimulation of adenylyl cyclase in SK-N-MC cells. Receptor cross-talk is an emerging field which investigates cross-regulation between distinct classes of receptors. In the present work, we investigated the influence of activating the insulin receptor, a tyrosine kinase receptor, on beta-agonist activation of adenylyl cyclase, which is mediated by a G protein-linked receptor. Treatment of SK-N-MC neuroepithelioma cells with insulin generated a marked attenuation of beta 1-adrenergic receptor-mediated stimulation of adenylyl cyclase. This effect required nanomolar concentrations of insulin, occurred within minutes of exposure of these cells to insulin, and did not result from down-regulation of beta-adrenergic receptors. Insulin alone reduced the maximal isoproterenol-mediated stimulation of adenylyl cyclase by 50%, while the co-addition of the phosphatase inhibitor sodium vanadate increased the magnitude of insulin inhibition to 90%. Insulin provides an additional avenue for heterologous desensitization of beta-adrenergic receptors and their transmembranal signalling pathway.


Effect of the NK-1 receptor antagonist GR 82,334 on reflexly-induced bladder contractions. The effect of intrathecal administration of the novel tachykinin NK-1 receptor antagonist GR 82,334 has been tested in three reflexes which excite urinary bladder motility. GR 82,334 at 1 but not at 0.1 nmol/rat blocked the chemonociceptive micturition reflex induced by the topical application of capsaicin (4 micrograms/50 microliters) onto the urinary bladder. At the same dose proven effective in the chemonociceptive reflex, GR 82,334 did not affect either micturition reflex induced by bladder filling or the urinary bladder contraction induced by perineal pinching. These results suggest that, in urethane-anesthetized rats, specific stimuli applied in the periphery activate NK-1 receptors at spinal cord level facilitating urinary bladder reflex contractions.


Role of opioids in peripheral analgesia. Several pharmacological, neurophysiological and immunohistological studies indicate that exogenous or endogenous opioids can have antinociceptive effects by acting at peripheral sites. Although modulation of mu, delta and kappa receptors can mediate these effects, the nature of the noxious stimulus and the underlying pathological condition may affect the types of opioid receptors involved. Thus, it would be appropriate to develop peripherally-acting opioid analgesics that do not have the untoward central side effects often associated with conventional analgesic drugs. This paper reviews the evidence supportive of a peripheral mechanism of action for opioids.


Critical role of endogenous corticotropin-releasing factor (CRF) in the mediation of the behavioral action of cocaine in rats. The role of endogenous CRF in the locomotor hyperactivity induced by cocaine was investigated by using the immunoneutralization of endogenous CRF and an antagonist of CRF-receptors (alpha-helical CRF9-41: alpha h-CRF) in rats. Different dilutions of anti-CRF antibody (1:5, 1:20, but not 1:100) injected intracerebroventricularly (i.c.v.) 24 hours before the cocaine treatment blocked the expression of locomotor hyperactivity. Pretreatment with different doses (0.001, 0.01, 0.1, 1.0 micrograms i.c.v.) of alpha h-CRF inhibited the locomotor hyperactivity induced by cocaine dose-dependently. Neither the immunoneutralization nor the receptor blockade for CRF changed the hyperactivity induced by another locomotor stimulant caffeine. These results serve as indirect in vivo evidence of the selective role of endogenous CRF in the cocaine-induced behavioral alterations. The findings have implications as concerns the possible role of CRF in human psychopathological changes induced by cocaine.


The role of alpha 1-adrenergic stimulation in inositol phosphate metabolism during post-ischaemic reperfusion. The aim of this study was to elucidate the mechanism of enhanced inositol phosphate metabolism during reperfusion. Inositol phosphate stores were prelabelled by perfusing isolated rat hearts for 1 h with [3H]inositol (1.5 microCi/ml). LiCl (10 mM) and prazosin (0.3 microM) were subsequently added 15 min before (i) 20 min control perfusion; (ii) 20 min normothermic ischaemic cardiac arrest (NICA); (iii) 20 min NICA followed by 1 min reperfusion. The ventricles were freeze-clamped before determination of isotopical incorporation of [3H]inositol into the inositol phosphates (Dowex anion exchange chromatography) and InsP3 levels (Amersham InsP3 assay system). In addition, noradrenaline release into the perfusate was also assessed (HPLC and electrochemical detection). The results showed: (i) increased noradrenaline release into the perfusate immediately after the onset of reperfusion; (ii) significant depression of [3H]inositol incorporation into inositol phosphates and InsP3 levels after 20 min NICA; (iii) reperfusion caused an immediate significant increase in isotopical incorporation of [3H]inositol into inositol phosphates as well as InsP3 levels; (iv) the alpha 1-adrenergic blocker, prazosin (0.3 microM), completely inhibited the reperfusion-induced increase in inositol phosphate metabolism. These observations suggested that increased alpha 1-adrenergic receptor stimulation by noradrenaline might be responsible for the stimulation of ventricular inositol phosphate metabolism during postischaemic reperfusion.


Possible involvement of protein kinase C and calcium in GSH efflux from Hep G2 cells. We investigated the effects of protein kinase C modulations and calcium mobilization on GSH efflux in Hep G2 cells. GSH efflux from Hep G2 cells was increased by a phorbol ester. Staurosporine, an inhibitor of protein kinase C, diminished phorbol ester-stimulated GSH efflux from the cells. GSH efflux was negatively correlated with extracellular calcium concentrations. Verapamil enhanced GSH efflux, whereas ATP decreased GSH efflux. The latter effect was diminished in the absence of extracellular calcium. Protein kinase C and calcium mobilization may be crucial factors in GSH efflux from human hepatocytes.


Stress and ACTH increase circulating concentrations of 3 alpha-androstanediol in female rats. The present experiments were carried out to determine what physiological conditions are responsible for the acute increases in serum levels of 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-androstanediol, 3 alpha-Adiol) which are seen in the intact estrous female rat within 15-30 min after mating. Blood samples were obtained from proestrus rats immediately before and 20 min after injection of exogenous hormones or initiation of stress procedures, and plasma concentrations of 3 alpha-Adiol and/or progesterone (P) were measured in these samples by RIA. Intravenous injections of ovine LH (5, 15, or 45 micrograms) or saline resulted in equivalent significant increases in plasma 3 alpha-Adiol 20 min after injection. In contrast, dose-dependent increases in 3 alpha-Adiol were seen after intravenous injection of 0 (acidic saline vehicle), 2, 4, or 8 ng ACTH1-24 to dexamethasone-pretreated rats. The highest ACTH1-24 dose also resulted in a significant increase in plasma P concentration. In a third experiment, a significant increase in 3 alpha-Adiol concentration above baseline was seen at 20 min after onset of restraint stress; in this case plasma P concentrations did not increase significantly. Finally, blood samples were obtained after onset of ether/jugular venipuncture stress two days after ovariectomy (ovx), adrenalectomy (adx), or ovx+adx on diestrus. The plasma 3 alpha-Adiol response to stress was normal in intact sham-operated and non-operated groups of controls, but was significantly diminished in the ovx and the adx groups to 28.4% of that shown by the intact animals. Circulating 3 alpha-Adiol concentrations were undetectable in 22/26 samples obtained in the ovx+adx group. These data demonstrate that plasma concentrations of 3 alpha-Adiol increase in response to stress or ACTH but not LH, and that both the ovary and the adrenal contribute to this increase.


Acute effects of angiotensin-converting enzyme inhibitor on erythrocyte sodium ion transport in essential hypertension. The acute effects of angiotensin-converting enzyme inhibitor, captopril, on sodium ion transport systems were investigated in essential hypertensive and normotensive subjects. The passive sodium efflux through the erythrocyte membrane was significantly higher and erythrocyte sodium-potassium cotransport was lower in patients with essential hypertension when compared with normal subjects. However, sodium-potassium pump activity and sodium-lithium countertransport did not differ significantly between the hypertensive patients and the normal subjects. Immediately after captopril administration, erythrocyte passive sodium efflux and sodium-potassium cotransport returned to normal levels in the hypertensive subjects. Although the plasma renin activity and plasma aldosterone concentration were altered by captopril, they did not correlate with changes in any sodium transport system. These results suggest that the changes in sodium transport systems which occur immediately after captopril administration may contribute, at least in part, to its antihypertensive action.


Identification of variable region differences in Neisseria meningitidis class 3 protein sequences among five group B serotypes. Strains of Neisseria meningitidis express one of two porin proteins. These porins have been identified as the class 2 and class 3 proteins, and express serotype-specific epitopes. The gene for the class 3 protein was amplified by the polymerase chain reaction from the DNA of a serotype 4 strain as a 1025 bp fragment. The nucleotide sequence of this gene was determined and compared with two recently published sequences. On the basis of this comparison we have identified two major variable regions in the translated protein sequence, VR1 and VR2, that may be associated with serotype specificity. Three other variable regions were also identified. The sequences in the VR1 and VR2 regions from five additional group B N. meningitidis strains of serotypes 1, 4, 8, 12, and 15, all expressing class 3 proteins, were determined. The VR1 and VR2 regions were variable and were flanked by highly conserved regions among eight different class 3 sequences. These two variable regions of 15 and 9 amino acids are predicted to be in surface-exposed loops.


Spectrum of mutations produced by specific types of restriction enzyme-induced double-strand breaks. Rejoining of DNA double-strand breaks (DSB) plays a central role in the various processes leading to DNA rearrangements. We have analyzed DNA alterations induced by restriction enzymes that produce DSB with specific types of ends. Restriction enzymes were electroporated into a human lymphoblastoid cell line that stably maintains pHAZE, an EBV-based vector containing the lacZ gene. After allowing time for DSB repair, pHAZE DNA was rescued and screened in Escherichia coli. Mapping and sequence analysis of mutant copies of pHAZE indicated that restriction enzymes induced all classes of alterations except base substitutions (base deletions and insertions, large-scale deletions, inversions, and insertions). The spectra of alterations were distinctive for each enzyme and appear to be the consequence of specific end-modification processes.


Latent infection of SCID mice with herpes simplex virus 1 and lethal cutaneous lesions in pregnancy. Some SCID mice survived primary infection with herpes simplex virus 1 without the development of peripheral lesions but established coculture-positive ganglionic latency when a low dose of a wild-type strain was inoculated intracutaneously. The latency was also evidenced by the development of the fatal zosteriform skin lesions and the isolation of the virus during pregnancy. We consider that the viral entry into neurons without successive replication, rather than the arrest of the lytic infection within the cells, is an important mechanism in the establishment of latency.


Correlative detection of human immunodeficiency virus (HIV) antigen p24 and Epstein-Barr virus DNA in vitro: clinical influence on HIV infection. The existence of molecular transactivations between EBV and HIV-1, as well as reactivations of EBV latent infections in AIDS patients, have been recently documented. In order to shed more light on the putative association between EBV and HIV, and its role in the evolution to AIDS, we have determined simultaneously p24 protein and EBV DNA in culture supernatants of peripheral blood mononuclear cells from 47 individuals suspected of having HIV infection. The results of the in vitro assays were correlated with the clinical stage of the individuals and their serologic status to EBV. Statistical analysis showed a concordance between HIV infection and in vitro detection of EBV DNA (P < 0.002); particularly, a strong correlation between the presence of EBV DNA and p24 in culture was observed (P < 0.001). These results are consistent with the occurrence of viral interactions, manifested in vitro. However, in our series, the appearance of EBV DNA in culture was not concomitant with an elevation of anti-VCA IgG titers, anti-EA titers or the development of symptomatology, suggestive of a reactivation of a latent EBV infection or a progression of HIV infection. Therefore we conclude that, although interaction between both viruses may take place at the molecular level, there is no clear evidence of the repercussion that this event may have on the clinical course of HIV infection.


The Na+/H+ antiporter in oncology in the light of the spontaneous regression of cancer and cell metabolism. Multiple metabolic and biochemical interrelationships, as well as the most recent views on mechanisms of malignant cell growth, proliferation, and oncogen activity mediated by the Na+/H+ antiporter, can be integrated from the unitarian point of view of the dynamics of the hydrogen ion to parallel pH-related mechanisms involved in the Spontaneous Regression (SR) of some malignant tumors. Also, pH-related growth inhibitors of the amiloride series are considered as possible agents to be used in the adjuvant and co-adjuvant treatment of some human tumors as well as in the control of the metastatic process and in overcoming cancer multidrug resistance (MDR).


The role of reactive oxygen species in the pathogenesis of multiple sclerosis. Although reactive oxygen species are thought to mediate cellular damage in many disease states the role of reactive oxygen species in the pathogenesis of multiple sclerosis is unknown. Data from biochemical, histochemical and pharmacological studies have been evaluated to determine if the necessary conditions exist for the formation of reactive oxygen species during a demyelination episode of multiple sclerosis. This evaluation found that not only do the necessary conditions exist for the formation of reactive oxygen species but that these species may play a significant pathogenic role in this disease. A hypothesis describing a detailed role of reactive oxygen species in the pathogenesis of multiple sclerosis is put forth.


Association of hepatitis C virus in human sera with beta-lipoprotein. Hepatitis C virus (HCV)-RNA in sera of patients with viral hepatitis C is supposed to be included, at least partially, into HCV particles. We found that the density of HCV-RNA-carrying material was variable, as determined by sucrose gradient density centrifugation (1.03-1.20 g/cm3). In some of the sera examined HCV-RNA was restricted to low densities between 1.03 and 1.08 g/cm3. In other sera additional densities of HCV-RNA were found distributed over the whole gradient with peaks at 1.12 and 1.17 and at 1.19-1.20 g/cm3. HCV-RNA banding at low densities could be completely co-precipitated with anti-beta lipoprotein, whereas HCV-RNA fractions of higher densities were only partially precipitated or not at all. In 8 of 20 sera directly examined, HCV-RNA could be completely and in 9 sera only partially co-precipitated by anti-beta lipoprotein. In 3 sera no significant precipitation could be observed.


A prospective study of the polymerase chain reaction for detection of herpes simplex virus in cerebrospinal fluid submitted to the clinical virology laboratory. Polymerase chain reaction (PCR) was prospectively performed with cerebrospinal fluid (CSF) from 51 patients whose CSF was available for analysis and was submitted for viral culture and/or herpes simplex virus (HSV) serology and 20 patients whose CSF was submitted exclusively to the Clinical Biochemistry Laboratory. Primers were used that flanked a 92 bp segment of the HSV DNA polymerase gene (35 cycles). Amplified products were electrophoresed on agarose gel, blotted onto nylon membrane, and probed with a 32P-labelled sequence internal to the primers. For nested PCR, 1 microliter of PCR product was amplified for an additional 35 cycles before electrophoresis and Southern blot analysis. Review of the clinical records revealed that 15 patients had central nervous system (CNS) infections. Specific HSV DNA sequences were detected in CSF specimens of three of the individuals [PCR(2), nested PCR(1)]. Two of these patients had disseminated HSV infection including encephalitis and one patient had aseptic meningitis. The diagnoses of the 12 patients with CNS infection who did not have HSV DNA detected in CSF included encephalitis [varicella-zoster virus (1), cytomegalovirus (1), Mycoplasma pneumoniae (1)], meningitis [Neisseria meningitidis (1), Coccidioides immitis (1), Enterovirus (1), aseptic meningitis (1)], varicella-zoster radiculitis (2), human immunodeficiency virus dementia (2), and transverse myelitis due to Epstein-Barr virus (1). Importantly, HSV DNA was also not detected in the CSF of the 36 patients who did not have CNS infection and 20 samples submitted exclusively to the Clinical Biochemistry Laboratory. Our findings demonstrate the utility of PCR as a rapid, non-invasive method for the routine laboratory diagnosis of CNS infection due to HSV.


Specificity of molecular hybridization techniques for the detection of bluetongue virus serotypes in Culicoides variipennis. Direct blot hybridization (DBH) and sandwich hybridization (SH) were evaluated for their ability to detect bluetongue virus (BTV) RNA in the biting midge Culicoides variipennis (Coquillett). Probes were derived from the L3 RNA segment of BTV, serotype 17. RNA of the five BTV serotypes occurring in the USA (BTV-2, BTV-10, BTV-11, BTV-13, and BTV-17) was extracted from pools of varying numbers of infected and uninfected biting midges and assayed by direct blot and sandwich hybridization tests. Direct blot hybridization using an RNA transcript probe or cDNA probe was a fast, efficient and sensitive technique, detecting as few as one midge infected with any BTV serotype in a pool of 50 or 100. Sandwich hybridization was able to detect the homologous serotype, BTV-17, in pools containing a single infected midge in a total of 50 or 100. However, detection of the heterologous serotypes, BTV-10, BTV-11, and BTV-13, was limited to pools containing 5 or more infected midges in a total of 50, and BTV-2 was undetectable by SH. Hybridization techniques provide an alternative to the conventional detection methods of inoculation of cell culture or embryonated chicken eggs for detection of BTV.