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Background: Warm continuous blood cardioplegia provides excellent protection, but must be interrupted by ischemic intervals to aid visualization. We hypothesized that (1) as ischemia is prolonged, the reduced metabolic rate offered by cooling gives the advantage to hypothermic cardioplegia; and (2) prior cardioplegia mitigates the deleterious effects of normothermic ischemia., Methods: Isolated cross-perfused canine hearts underwent cardioplegic arrest followed by 45 minutes of global ischemia at 10 degrees C or 37 degrees C, or 45 minutes of normothermic ischemia without prior cardioplegia. Left ventricular function was measured at baseline and during 2 hours of recovery. Metabolism was continuously evaluated by phosphorus-31 magnetic resonance spectroscopy., Results: Adenosine triphosphate was 71% +/- 4%, 71% +/- 7%, and 38% +/- 5% of baseline at 30 minutes, and 71% +/- 4%, 48% +/- 5%, and 39% +/- 6% at 42 minutes of ischemia in the cold ischemia, warm ischemia, and normothermic ischemia without prior cardioplegia groups, respectively. Left ventricular systolic function, left ventricular relaxation, and high-energy phosphate levels recovered fully after cold cardioplegia and ischemia. Prior cardioplegia delayed the decline in intracellular pH during normothermic ischemia initially by 9 minutes, and better preserved left ventricular relaxation during recovery, but did not ameliorate the severe postischemic impairment of left ventricular systolic function, marked adenosine triphosphate depletion, and creatine phosphate increase. Left ventricular distensibility decreased in all groups., Conclusions: When cardioplegia is followed by prolonged ischemia, better protection is provided by hypothermia than by normothermia. Prior cardioplegia confers little advantage on recovery after prolonged normothermic ischemia but delays initial ischemic metabolic deterioration, which would contribute to the safety of brief interruptions of warm cardioplegia.

The sigma subunit of eubacterial RNA polymerase is required throughout initiation, but how it communicates with core polymerase (alpha(2)betabeta') is poorly understood. The present work addresses the location and function of the interface of sigma with core. Our studies suggest that this interface is extensive as mutations in six conserved regions of sigma(70) hinder the ability of sigma to bind core. Direct binding of one of these regions to core can be demonstrated using a peptide-based approach. The same regions, and even equivalent residues, in sigma(32) and sigma(70) alter core interaction, suggesting that sigma(70) family members use homologous residues, at least in part, to interact with core. Finally, the regions of sigma that we identify perform specialized functions, suggesting that different portions of the interface perform discrete roles during transcription initiation.

Hsp70 family members together with their Hsp40 cochaperones function as molecular chaperones, using an ATP-controlled cycle of polypeptide binding and release to mediate protein folding. Hsp40 plays a key role in the chaperone reaction by stimulating the ATPase activity and activating the substrate binding of Hsp70. We have explored the interaction between the Escherichia coli Hsp70 family member, DnaK, and its cochaperone partner DnaJ. Our data show that the binding of ATP, subsequent conformational changes in DnaK, and DnaJ-stimulated ATP hydrolysis are all required for the formation of a DnaK-DnaJ complex as monitored by Biacore analysis. In addition, our data imply that the interaction of the J-domain with DnaK depends on the substrate binding state of DnaK.

Chaperones of the Hsp70 family bind to unfolded or partially folded polypeptides to facilitate many cellular processes. ATP hydrolysis and substrate binding, the two key molecular activities of this chaperone, are modulated by the cochaperone DnaJ. By using both genetic and biochemical approaches, we provide evidence that DnaJ binds to at least two sites on the Escherichia coli Hsp70 family member DnaK: under the ATPase domain in a cleft between its two subdomains and at or near the pocket of substrate binding. The lower cleft of the ATPase domain is defined as a binding pocket for the J-domain because (i) a DnaK mutation located in this cleft (R167H) is an allele-specific suppressor of the binding defect of the DnaJ mutation, D35N and (ii) alanine substitution of two residues close to R167 in the crystal structure, N170A and T173A, significantly decrease DnaJ binding. A second binding determinant is likely to be in the substrate-binding domain because some DnaK mutations in the vicinity of the substrate-binding pocket are defective in either the affinity (G400D, G539D) or rate (D526N) of both peptide and DnaJ binding to DnaK. Binding of DnaJ may propagate conformational changes to the nearby ATPase catalytic center and substrate-binding sites as well as facilitate communication between these two domains to alter the molecular properties of Hsp70.

Electrophysiological studies were conducted in three groups of mice to determine the possible involvement of the antibodies to presynaptic membrane receptor (PsmR), a beta-bungarotoxin (beta-BuTX) binding protein, in the pathogenesis of myasthenia gravis (MG). Mice were untreated (untreated group, n = 8) or were injected (i.p.) with blood plasma from a MG patient, which contained antibodies to PsmR, at a dose of 1 ml per day for more than 2 months (MG plasma group, n = 12) or with plasma from healthy subjects (normal plasma group, n = 10). Prior to plasma injection, cyclophosphamide was given at 300 mg/kg (i.p.) to all three groups. About three weeks after plasma injection, most mice of the MG plasma group became less mobile in comparison with those of the two control groups. Electrophysiological recording showed three main changes in the MG plasma group: (1) the increase in the frequency of miniature endplate potentials (mEPPs) induced by Krebs solution with high K+ concentration (17.5 mM) was significantly lowered, which was confirmed in mice injected with IgG (50 mg per day) from this patient for two days; (2) the quantal content of EPP was decreased; and (3) the decrement in the amplitude of a train EPP (50 Hz) was quickened. Our results suggest that this experimental model is different from that of Lambert-Eaton myasthenic syndrome and that antibodies to PsmR may also be involved in the pathogenesis of MG.

Objective: Warm blood cardioplegia requires interruption by ischemic intervals to aid visualization. We evaluated the safety of repeated interruption of warm blood cardioplegia by normothermic ischemic periods of varying durations., Methods: In three groups of isolated cross-perfused canine hearts, left ventricular function was measured before and for 2 hours of recovery after arrest, which comprised four 15-minute periods of cardioplegia alternating with three ischemic intervals of 15, 20, or 30 minutes (I15, I20, and I30). Metabolism was continuously measured by phosphorus 31-magnetic resonance spectroscopy., Results: Adenosine triphosphate level fell progressively as ischemia was prolonged; after recovery, adenosine triphosphate was 99% +/- 6%, 90% +/- 1% (p = 0.0004 vs control), and 68% +/- 3% (p = 0.0002) of control levels in I15, I20, and I30, respectively. Intracellular acidosis with ischemia was most marked in I30. After recovery, left ventricular maximal systolic elastance at constant heart rate and coronary perfusion pressure was maintained in I15 but fell to 85% +/- 3% in I20, (p = 0.003) and to 65% +/- 6% (p = 0.003) of control values in I30, while relaxation (tau) was prolonged only in I30 (p = 0.007)., Conclusions: Hearts recover fully after three 15-minutes periods of ischemia during warm blood cardioplegia, but deterioration, significant with 20-minute periods, is profound when the ischemic periods are lengthened to 30 minutes. This suggests that in the clinical setting warm cardioplegia can be safely interrupted for short intervals, but longer interruptions require caution.

Congenital analgesia is a rare genetic disorder. We report here that a 12-year-old boy was able to recover from congenital insensitivity to pain. Neurological examinations revealed that there was a 'stocking' distribution of pain decrement on the lower extremities under the patient's knee joints. Magnetic Resonance Imaging (MRI) of his brain showed gyrus thinning with sulcus widening at both sides of the parietal lobe. Southern blot hybridization probed with cDNAs of various opioid receptors did not detect any significant abnormality. Our results suggest that this rare case may not be genetically determined.

DnaK, the bacterial homolog of the eukaryotic hsp70 proteins, is an ATP-dependent chaperone whose basal ATPase is stimulated by synthetic peptides and its cohort heat shock proteins, DnaJ and GrpE. We have used three mutant DnaK proteins, E171K, D201N, and A174T (corresponding to Glu175, Asp206, and Ala179, respectively, in bovine heat stable cognate 70) to probe the ATPase cycle. All of the mutant proteins exhibit some alteration in basal ATP hydrolysis. However, they all exhibit more severe defects in the regulated activities. D201N and E171K are completely defective in all regulated activities of the protein and also in making the conformational change exhibited by the wt protein upon binding ATP. We suggest that the inability of D201N and E171K to achieve the ATP activated conformation prevents both stimulation by all effectors and the ATP-mediated release of GrpE. In contrast, the defect of A174T is much more specific. It exhibits normal binding and release of GrpE and normal stimulation of ATPase activity by DnaJ. However, it is defective in the synergistic activation of its ATPase by DnaJ and GrpE. We suggest that this mutant protein is specifically defective in a DnaJ/GrpE mediated conformational change in DnaK necessary for the synergistic action of DnaJ+GrpE.

In Escherichia coli, the heat shock response is under the control of two alternative sigma factors: sigma 32 and sigma E. The sigma 32-regulated response is well understood, whereas little is known about that of sigma E, except that it responds to extracytoplasmic immature outer membrane proteins. To further understand this response, we located the rpoE gene at 55.5' and analyzed the role of sigma E. sigma E is required at high temperature, and controls the transcription of at least 10 genes. Some of these might contribute to the integrity of the cell since delta rpoE cells are more sensitive to SDS plus EDTA and crystal violet. sigma E controls its own transcription from a sigma E-dependent promoter, indicating that rpoE transcription plays a role in the regulation of E sigma E activity. Indeed, under steady-state conditions, the transcription from this promoter mirrors the levels of E sigma E activity in the cell. However, it is unlikely that the rapid increase in E sigma E activity following induction can be accounted for solely by increased transcription of rpoE. Based upon homology arguments, we suggest that a gene encoding a negative regulator of sigma E activity is located immediately downstream of rpoE and may function as the target of the E sigma E inducing signal.

To evaluate systemic T cell activation and reactivity against putative autoantigens in multiple sclerosis (MS), numbers of interleukin-2 (IL-2) secreting cells were determined in peripheral blood of 32 patients with MS, 7 patients with acute aseptic meningitis (AM) and 12 patients with tension headache (TH). Numbers of IL-2 secreting cells were higher in MS patients compared to patients with AM + TH after stimulation with myelin proteolipid protein (PLP), myelin associated glycoprotein (MAG) and myelin oligodendrocyte glycoprotein (MOG), but not after stimulation with myelin basic protein (MBP). In response to MAG, MOG and acetylcholine receptor (AChR) the frequencies of IL-2 secreting cells were higher in patients with MS than TH, while there were no differences between AM and TH to any of the tested antigens. Between patients with MS and AM there was no difference regarding frequency of IL-2 secreting cells in response to any of the tested antigens except MAG to which the response was higher in MS patients. Six of 10 MS patients had IL-2 secreting cells in response to all four myelin antigens (MBP + PLP + MAG + MOG) or to three antigens, while this broad reactivity was not found in any control patient. There was no correlation between numbers of IL-2 secreting cells in MS patients and clinical variables, including exacerbation versus remission, disability and duration of disease. The results suggest that the systemic T cell response in patients with MS is directed to several antigens.

Interleukin 4 (IL-4)- and interferon gamma (IFN gamma)-secreting peripheral blood cells were enumerated by immunospot assay in 13 multiple sclerosis (MS) patients during exacerbations, in 24 patients with progressive multiple sclerosis (CPMS), and in 20 controls. Cells that spontaneously secreted IFN gamma were significantly higher in MS patients experiencing an attack (P < 0.001) than in controls or in CPMS (P < 0.04). IL-4-secreting cell numbers were elevated significantly and to a comparable extent in both MS groups compared to controls. Our finding of increased numbers of IFN gamma-secreting cells is in keeping with prior work showing increased IFN gamma levels in the circulation prior to and during MS attacks and increased release of IFN gamma to the supernatant in bulk cultured blood cells from MS patients. What role an increase in IL-4-secreting cells might play in MS is unclear, but it could relate to immune system regulation. Following in vitro exposure to MBP, IFN gamma-secreting cell number rose above levels observed in the absence of stimulation in controls and in both MS groups with the rise in acutely exacerbating MS patients being significantly greater than in controls. Our results provide further evidence for reactivity to MBP in MS, the significance of which in terms of pathogenesis remains clouded.

Peripheral blood and bone marrow from seronegative and seropositive myasthenics were evaluated for antibody-secreting cells (ASC). Cells secreting antibody to acetylcholine receptor (AchR) and to presynaptic membrane receptor (prsmR) were counted using an immunospot assay. Immunoglobulin G (IgG) anti-AchR ASC were present in peripheral blood lymphocytes (PBL) from nine of 13 seronegative and nine of 12 seropositive myasthenics and in bone marrow lymphocytes (BML) from nine of 13 seronegative and eight of 12 seropositive myasthenics. The mean number of IgG anti-AchR ASC was lower for seronegative than for seropositive patients (P < 0.01 for PBL and P < 0.0001 for BML). In seropositive patients the mean number of IgG anti-AchR ASC was higher for BML than for PBL (P < 0.01); in seronegative patients it was not. IgG anti-prsmR ASC were detected in PBL from four of eight seronegative and six of eight seropositive myasthenics and in BML from three of eight seronegative and five of eight seropositive patients. The mean number of IgG-anti-prsmR ASC did not differ between seronegative and seropositive patients for PBL but for BML the value was higher for seropositive than for seronegative patients (P < 0.01). We conclude that seronegative myasthenia gravis is an autoimmune disease and that ASC to AchR and to prsmR are present both in the blood and the bone marrow in seronegative patients as in seropositive ones. A major difference between the groups lies in the significantly greater number of ASC found in the bone marrow in the seropositive cohort.

The paper reports the formation and crystal structure of dihydronitidine, expounds the reasons and conditions of easily formed oxynitidine, and discusses anticancer mechanism of nitidine (cation). The crystallographic parameters of dihydronitidine are: space group P(2)1/n, a = 12.54(1), b = 9.148(5), c = 14.748(8) A, beta = 92.12(6)degrees, Z = 4. 4108 independent reflections were collected within the range of 3 degrees < or = 2 theta < or = 54 degrees, of which 2137 intensity data with I > or = 3 sigma (I) were used in the structural determination. The crystal structure has been refined by full matrix least-square method to a final R of 0.050.

Serum anti-acetylcholine receptor antibodies (A AchR Ab) and anti-synaptic premembrane antibodies (A PrM Ab) were measured in 21 patients with absence epilepsy, 21 cases with benign childhood epilepsy with centro-temporal spikes (BCECTs) and 13 cases with atypical epilepsy. Respectively, in five (23.8%), eleven (51.2%) and eight (66.7%) of the above mentioned groups of patients both A AchR Ab and A PrM Ab were found.

Antibodies against acetylcholine receptor (AChR) can be detected in most patients with myasthenia gravis (MG) and are considered to be involved in the immunopathogenesis of this disease. AChR are isolated from crude receptor preparations by binding to alpha-bungarotoxin (alpha-BuTx). Patients with MG have also antibodies against a second protein tentatively named presynaptic membrane receptor (PsmR), which has been isolated from crude receptor utilizing beta-bungarotoxin (beta-BuTx). PsmR could represent another antigen besides AChR relevant for the development of MG. We have now evaluated the T cell reactivity to PsmR in MG and controls by analysing the frequencies of cells which in response to PsmR in short-term cultures secreted interferon-gamma (IFN-gamma). The B cell response to PsmR was analysed in parallel by counting cells secreting anti-PsmR antibodies. Most patients with MG had PsmR reactive T cell in blood with a median number of about 1 per 44,000 mononuclear cells. Cells secreting anti-PsmR antibodies belonging to the IgG and IgA isotypes, less frequently of the IgM isotype were detected in most MG patients. A positive correlation was found between T cells reactive with PsmR and anti-PsmR IgG antibody secreting cells. PsmR reactive T and B cells were also detected in control patients, but at much lower numbers. Our results indicate that MG is accompanied by T as well as B cell responses to PsmR, in addition to the previously recognized responses to AChR. It remains to be shown whether these PsmR reactivities are of pathogenetic importance in MG.

To evaluate the accuracy of Doppler technique for measuring the pressure gradient (delta P) across patent ductus arteriosus (PDA) with a simplified Bernoulli equation (delta P = 4 V2) and estimating the pulmonary artery pressure (PAP) by subtracting delta P from brachial arterial pressure (BAP) measured with cuff sphygmomanometry, Doppler echocardiography and catheterization (both left and right) were performed simultaneously in 20 patients with PDA. For the values of systolic peak delta P (SPPG), mean delta P (MPG) and end-diastolic delta P (EDPG) determined by both Doppler and catheter, the correlation was excellent (r = 0.99, 0.99, 0.99). Doppler determined SPPG also correlated well with catheter determined peak to peak delta P (P-PPG) (r = 0.97). Comparison between Doppler-derived and catheter measured systolic PAP (SPAP), mean PAP (MPAP) and diastolic PAP (DPAP) also showed high correlation (r = 0.95, 0.98, 0.97). This study demonstrates that Doppler echocardiography is valuable for measurement of delta P across PDA and non-invasive estimation of PAP in patients with PDA.

Antibodies against acetylcholine receptor (AChR), a protein that binds to alpha-bungarotoxin (alpha-BuTx), are characteristic for myasthenia gravis (MG) and are considered to be of importance in the pathogenesis of the disease. Antibodies against a beta-BuTx binding protein, presynaptic membrane receptor (PsmR), have also been reported in most MG patients. We have analysed the specificity and cross-reactivity of antibodies to bovine AChR and PsmR in sera from 11 patients with MG. More than 90% of antibodies to PsmR were adsorbed specifically by PsmR conjugated affinity chromatography. Similarly, more than 90% of anti-AChR antibodies were absorbed by AChR conjugated affinity chromatography. Specificities of antibodies from affinity chromatography were also confirmed by ELISA and agarose isoelectric focusing. However, the antibodies to PsmR and AChR from MG patients' sera showed about 45-55% cross-reactivity, and there was a high correlation between serum levels of both antibodies. The demonstration of anti-PsmR antibodies could be important in documenting presynaptic damage and understanding the pathogenetic process in MG.

Myasthenia gravis (MG) is considered as an autoimmune disease of neuromuscular junction resulting from antibodies directed to acetylcholine receptors (AChR). We describe the use of beta-bungarotoxin (beta-BuTx) and alpha-bungarotoxin (alpha-BuTx) to capture their corresponding proteins from preparation of crude human muscle receptor. beta-BuTx binds to presynaptic membrane receptor (PsmR) of the whole receptor fraction, while alpha-BuTx binds to AchR. The captured proteins were used as antigen in ELISA to detect antibodies to PsmR and to AchR in sera from 82 Chinese patients with MG and in controls. In MG, antibodies to PsmR only were detected in 13%, to AchR only in 11% and both to PsmR and AchR in 54%. Only 3 of 50 patients with other neurological diseases and none of 50 healthy subjects had these antibodies. Specificity tests for antibodies showed that the detection systems which we used are specific and confident. No correlation was found between antibody levels and clinical status. The significance of the PsmR antibodies in the pathogenesis of MG is unknown. We suggest that myasthenia gravis is not only due to damage of the postsynaptic membrane, but of presynaptic structures as well.

A nitrocellulose immunospot assay to enumerate cells secreting anti-BCG antibodies was evaluated in the diagnosis of tuberculous meningitis. Among 25 Chinese patients with this disease diagnosed on clinical and cerebrospinal fluid (CSF) criteria, 24 had anti-BCG IgG antibody secreting cells in CSF, at a median value of 56 per 10(4) CSF cells. Among 6 patients examined within one week, 5 had antibody secreting cells in CSF, and all 19 patients examined 2-8 weeks after onset had such cells in CSF. Blood examined from 5 patients contained anti-BCG IgG or IgM antibody secreting cells, but usually at much lower numbers than in corresponding CSF, indicating that the specific antibody response is largely confined to CSF. Anti-BCG antibodies measured in parallel by ELISA were slightly raised in CSF in only 1 of the 6 patients examined within a week, whereas a good correspondence was seen between numbers of antibody secreting cells and antibody concentrations from the second week onwards. Detection of anti-BCG IgG antibody secreting cells has higher sensitivity and specificity than ELISA measurement of anti-BCG IgG antibodies, and represents a new, useful method for the early diagnosis of tuberculous meningitis.

To estimate more precisely the systolic pulmonary artery pressure (PASP), continuous wave Doppler pressure gradient method and catheter measurement were performed simultaneously in 85 patients. There were 30 patients with tricuspid regurgitation (TR), 35 with ventricular septal defect (VSD), and 20 with patent ductus arterosus (PDA). For maximal transtricuspid pressure gradient (TRPG) determined by continuous wave Doppler and catheter determined PASP in TR group, the correlation coefficient (r) was as high as 0.99. For Doppler determined PASP (subtracting pressure gradient across PDA from systemic arterial pressure measured by cuff sphygmomanometer) and catheter determined PASP in PDA group, r was 0.95. Doppler determined PASP in VSD group (subtracting pressure gradient across VSD from systemic arterial pressure) also correlated well with catheter determined PASP (r = 0.97). This study demonstrates that continuous wave Doppler pressure gradient method can accurately estimate PASP in patients with TR, PDA, and VSD.

Bone marrow cells from patients with multiple sclerosis (MS) were studied regarding proliferative capacity with and without mitogenic stimulation, immunohistochemical characterization of cellular phenotypes with monoclonal antibodies and morphology and compared to bone marrow cells from healthy individuals undergoing elective orthopaedic surgery. MS patients' bone marrow mononuclear cells (BM-MNC) showed higher spontaneous proliferation both in comparison with BM-MNC from controls and peripheral blood mononuclear cells (PBL) from MS patients. Phytohaemagglutinin (PHA) response was higher in MS BM-MNC than BM-MNC from controls. MS patients' BM-MNC proliferated more on interleukin-2 (IL-2) stimulation than their corresponding PBL. There was no significant difference in proliferative response of PBL between MS patients and controls. Higher levels of undifferentiated or activated cells, as measured by OKT10, were found in peripheral blood of patients with MS. Seven of 11 MS patients showed morphological signs of activation in their bone marrow. The results indicate a role for immune reactions in bone marrow in the pathogenesis of multiple sclerosis, a disease with symptoms and signs strictly confined to the central nervous system.

Since 1973 a number of studies have been conducted on the association of HLA with myasthenia gravis (MG). (Safwenberg et al. 1973, Fritze et al. 1973, Naeim et al. 1978, Bodmer & Bodmer 1978, Behan 1980). Most of these studies involved Caucasians. In this paper we report on a study carried out in Chinese patients with M.G.

Presence of cytopathic effect and enzyme reverse transcriptase in cultures of peripheral blood mononuclear cells is described in 2 of 15 patients with multiple sclerosis and none of healthy controls. These findings might indicate: the presence of a new human retrovirus in these individuals, despite the low detection rate, and the shortcomings inherent in methodology used for detection of known human retroviruses in the study of new groups of diseases, such as those with possible autoimmune background.