Cre-lox recombination in Escherichia coli cells. Mechanistic differences from the in vitro reaction. The mechanism of the Cre recombinase of bacteriophage P1 in Escherichia coli cells was analyzed by topological methods in order to determine the important features of the in vivo reaction. Lambda infection was used to introduce the cre gene into cells containing plasmid substrates. The products of Cre resolution on substrates with directly repeated sites were predominantly free circles, even though decatenation by DNA gyrase was blocked by the drug norfloxacin. Recombination by Cre was greatly stimulated by negative supercoiling, and inversion occurred inefficiently. These results are strikingly different from those found with purified enzyme in vitro. Our data imply that Cre recombination in vivo is much more tightly controlled than it is in vitro, and that Cre acts predominantly as a resolvase in vivo. We suggest a role for Cre-mediated recombination in P1 plasmid amplification that is consistent with the selectivity of the enzyme in vivo.


Cell killing by the F plasmid CcdB protein involves poisoning of DNA-topoisomerase II complexes. In Escherichia coli, the miniF plasmid CcdB protein is responsible for cell death when its action is not prevented by polypeptide CcdA. We report the isolation, localization, sequencing and properties of a bacterial mutant resistant to the cytotoxic activity of the CcdB protein. This mutation is located in the gene encoding the A subunit of topoisomerase II and produces an Arg462----Cys substitution in the amino acid sequence of the GyrA polypeptide. Hence, the mutation was called gyrA462. We show that in the wild-type strain, the CcdB protein promotes plasmid linearization; in the gyrA462 strain, this double-stranded DNA cleavage is suppressed. This indicates that the CcdB protein is responsible for gyrase-mediated double-stranded DNA breakage. CcdB, in the absence of CcdA, induces the SOS pathway. SOS induction is a biological response to DNA-damaging agents. We show that the gyrA462 mutation suppresses this SOS activation, indicating that SOS induction is a consequence of DNA damages promoted by the CcdB protein on gyrase-DNA complexes. In addition, we observe that the CcdBS sensitive phenotype dominates over the resistant phenotype. This is better explained by the conversion, in gyrA+/gyrA462 merodiploid strains, of the wild-type gyrase into a DNA-damaging agent. These results strongly suggest that the CcdB protein, like quinolone antibiotics and a variety of antitumoral drugs, is a DNA topoisomerase II poison. This is the first proteinic poison-antipoison mechanism that has been found to act via the DNA topoisomerase II.


Crystallization of the complex of actin with gelsolin segment 1. Crystals of a 1:1 complex between human gelsolin segment 1 and actin have been grown from solutions containing polyethylene glycol 6000. The crystals are orthorhombic, space group P2(1)2(1)2(1); the axes are a = 57.4 A, b = 70.4 A, c = 184.5 A. They are moderately stable to X-rays and diffract to beyond 2.5 A. There is one molecule of complex in the asymmetric unit.


Pulmonary disease in gestational trophoblastic neoplasms. Gestational trophoblastic neoplasms can present as pulmonary nodules without significant disease of the reproductive organs. This article describes a case of metastatic gestational trophoblastic disease to the lungs. This entity must be considered in the differential diagnosis in any female of reproductive age who presents with multiple pulmonary nodules. Thoracotomy has a limited role in the initial evaluation of patients with this disease. However, it may be needed in patients who have evidence of persistent pulmonary disease, despite appropriate therapy.


Protein malnourishment: a predisposing factor in acrylamide toxicity in pregnant rats. Exposure to acrylamide (3-10 mg/kg body weight) was found to be lethal for protein-deficient pregnant rats as evidenced by their increased mortality. It had no such effect on the normal protein diet fed pregnant and nonpregnant rats and the protein-malnourished nonpregnant rats. Protein deficiency during pregnancy caused a significant decrease in the activity of brain monoamine oxidase and acetylcholinesterase and striatal [3H]spiperone binding, known to label dopamine receptors; had no significant effect on the binding of 3H-QNB (quinuclidinyl benzilate) to cerebellar and [3H]diazepam to frontocortical membranes, known to label muscarinic and benzodiazepine receptors, respectively; and had no significant effect on brain glutathione (GSH) levels in comparison with pregnant rats fed normal protein diet. Exposure to acrylamide (2 mg/kg body weight) in protein-malnourished pregnant rats caused a marked decrease in the activity of monoamine oxidase and acetylcholinesterase and also in the binding of [3H]spiperone, [3H]QNB, and [3H]diazepam to striatal, cerebellar, and frontocortical membranes, respectively. Kinetic studies revealed that decreased binding of these ligands in the specific brain regions were due to decreased receptor sites (Bmax). A reduction in the brain glutathione content was also observed in these animals in comparison with those fed a low-protein diet during pregnancy. Pregnant rats fed a normal-protein diet on acrylamide exposure, however, showed no such biochemical changes in comparison with the pregnant rats fed normal protein diet. Also, no effect on any of the parameters studied was observed in the adult nonpregnant rats fed a low-protein diet (for 18 d) and those exposed to the monomer (d 6-17) fed either a normal- or low-protein diet in comparison with respective controls. The results indicate that pregnancy under conditions of malnutrition modifies the susceptibility of pregnant rats toward acrylamide.


Difference between effects of chlorpromazine and perphenazine on microsomal phospholipids and enzyme activities in rat liver. The effects of acute administration of chlorpromazine (CPZ) and perphenazine (PPZ) on hepatic microsomal phospholipids (PLs) and enzyme activities in the male rat were examined in order to elucidate the relationship between individual PLs and drug-metabolizing activity. Cytochrome P-450 and aniline (AN) hydroxylation activity were initially decreased in CPZ-treated rats, but cytochrome P-450 subsequently recovered to a level not significantly different from the control and AN hydroxylation was markedly increased, while in PPZ-treated rats, they remained depressed. CPZ increased the activities of glycerophosphate acyltransferase (GPA) and choline phosphotransferase (CPT), while PPZ increased the activities of phosphatidate cytidylyltransferase (PCT), phosphatidate phosphohydrolase (PPH) and CPT. Concurrently, CPZ raised microsomal phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine-inositol (PSI) and sphingomyelin (SM), while PPZ increased PC and PE, but did not affect the levels of PSI and SM. Acyl components of phospholipids were also modified. CPZ significantly decreased the ratio of saturated to unsaturated fatty acids, particularly in the PC and PE fractions, while the effect of PPZ was only slight. The results imply that an increase of AN hydroxylation activity may involve the incorporation of unsaturated fatty acids into enzyme-associated PC and PE.


[Multiple resistance to drugs by MRSA].

[Multiple resistance to drugs by MRSA]. Most methicillin-resistant Staphylococcus aureus (MRSA) isolates are multiply resistant to various antimicrobial agents. Therefore, MRSA strains have become a serious problem in the clinical setting. However, it should be noted that the frequency of isolation of strains resistant to aminoglycoside and minocycline was not significantly different between low- and high-MRSA isolates and the isolation frequency of high-MRSA strains, less susceptible to not only beta-lactam antibiotics but also to macrolide antibiotics and quinolones, was significantly higher than the corresponding values for low-MRSA strains. These results suggest that high-MRSA strains were selected by antimicrobial agents for the treatment with the patients.


[Aminoglycosides resistance of methicillin-resistant Staphylococcus aureus]. In the last decade, there has been a dramatic increase in the isolation frequency of methicillin-resistant Staphylococcus aureus (MRSA) in Japan. Especially, a high incidence of multiple resistant MRSA strains has been reported. These strains are resistant not only to beta-lactams but to aminoglycosides and macrolides. About 90% of MRSA strains were resistant to gentamicin (GM) and/or tobramycin (TOB), producing an aminoglycoside modifying enzyme, mainly, APH (2")/AAC (6) and/or AAD (4',4"). Based on these modifying enzymes produced, MRSA strains were classified into three groups; group 1 produced APH (2")/AAC (6), belonging to phage group I and coagulase IV, group 2 produced AAD (4',4"), belonging to phage group III and coagulase II, and group 3 produced APH (2")/AAC (6) and AAD (4',4"), belonging to phage group III and coagulase II. The epidemiological results suggest that MRSA strains changed from group 1 to group 2, and then to group 3. Recently, arbekacin (ABK), a new anti-MRSA aminoglycoside, has been introduced into clinical practice. ABK shows a potent activity to GM-resistant strains, due to poorly modification by APH (2")/AAC (6'). However, there were few ABK- and GM-resistant strains in clinical isolates. These strains produced a higher amount of the enzyme than ABK-susceptible and GM-resistant strains. This observation suggests that ABK-resistant strains might be derived from GM-resistant strains by mutation of the gene coding APH (2")/AAC (6').


[Characterization of the Staphylococcus aureus norA gene, which confers resistance to hydrophilic quinolones]. The norA gene cloned from chromosomal DNA of a quinolone-and methicillin-resistant Staphylococcus aureus strain conferred resistance to hydrophilic quinolones such as norfloxacin, enoxacin, ofloxacin, and ciprofloxacin, but no or less resistance to hydrophobic ones such as nalidixic acid, oxolinic acid, and sparfloxacin in S. aureus and Escherichia coli. Nucleotide sequence analysis of the cloned DNA fragment revealed that the norA gene could code for a protein consisting of 388 amino acid residues with a molecular weight of 42,265, which was consistent with the experimental value of about 49,000 obtained on DNA-directed translation. The deduced NorA polypeptide has 12 hydrophobic membrane-spanning regions and is partly homologous to tetracycline resistance protein and sugar transport proteins. The uptake of a hydrophilic quinolone, enoxacin, by S. aureus harboring a plasmid carrying the norA gene was about 50% of that by the parent strain lacking the plasmid, but it increased to almost the same level as that by the latter strain with carbonyl cyanide m-chlorophenyl hydrazone. On the other hand, the uptake of a hydrophobic quinolone, sparfloxacin, was hardly affected by the norA gene. These results suggest that the NorA polypeptide may constitute a membrane-associated active efflux pump of hydrophilic quinolones.


[Recent trend and development of novel antimicrobial agents for MRSA infections]. Gram-positive organisms such as Staphylococcus aureus (including MRSA), coagulase-negative staphylococci, Enterococcus spp., and Streptococcus spp. have in recent years emerged as significant pathogens in hospitals and are now being isolated more frequently than gram-negative bacilli. These organisms are often multidrug resistant. Therefore, alternative agents with potent activity against gram-positive organisms are of considerable interest. In addition to the glycopeptide antibiotic vancomycin and the aminoglycoside antibiotic arbekacin, which can be used in MRSA infections, teicoplanin, RP 59500 and daptomycin are now under basic research in Japan. These antimicrobial agents are very active against gram-positive organisms, including MRSA and appear to be potent agents against infections due to gram-positive cocci, particularly MRSA.


Effect of dibutyryl cyclic AMP-treatment on prostaglandin F2 alpha-stimulated phosphoinositide hydrolysis in cultured rat astrocytes. Dibutyryl cyclic AMP (dBcAMP)-treatment of cultured rat astrocytes induced changes in astrocyte morphology followed by the potentiation of prostaglandin F2 alpha (PGF2 alpha)-stimulated phosphoinositide (PI) hydrolysis. The potentiation was observed in astrocytes of the cerebral cortex, cerebellum, and hippocampus. The dBcAMP-treatment induced agonists-specific changes in PI hydrolysis; e.g., the potentiation of norepinephrine-effect and the reduction of the carbachol-effect. Coincubations of carbachol or norepinephrine with PGF2 alpha produced additive responses.


Fast Na+ channels and slow Ca2+ current in smooth muscle from pregnant rat uterus. Smooth muscle cells normally do not possess fast Na+ channels, but inward current is carried through two types of Ca2+ channels: slow (L-type) Ca2+ channels and fast (T-type) Ca2+ channels. Whole-cell voltage clamp was done on single smooth muscle cells isolated from the longitudinal layer of 18-day pregnant rat uterus. Depolarizing pulses, applied from a holding potential of -90 mV, evoked two types of inward current, fast and slow. The fast inward current decayed within 30 ms, depended on [Na]o, and was inhibited by TTX (K0.5 = 27 nM). The slow inward current decayed slowly, was dependent on [Ca]o (or Ba2+), and was inhibited by nifedipine. These results suggest that the fast inward current is a fast Na+ channel current, and that the slow inward current is a Ca2+ slow channel current. A fast-inactivating Ca2+ channel current was not evident. We conclude that the ion channels which generate inward currents in pregnant rat uterine cells are TTX-sensitive fast Na+ channels and dihydropyridine-sensitive slow Ca2+ channels. The number of fast Na+ channels increased during gestation. The averaged current density increased from 0 on day 5, to 0.19 on day 9, 0.56 on day 14, 0.90 on day 18, and 0.86 pA/pF on day 21. This almost linear increase occurs because of an increase in the fraction of cells which possess fast Na+ channels. The Ca2+ channel current density also was higher during the latter half of gestation. These results indicate that the fast Na+ channels and Ca2+ slow channels in myometrium become more numerous as term approaches, and we suggest that the fast Na+ current may be involved in spread of excitation. Isoproterenol (beta-agonist) did not affect either ICa(s) or INa(f), whereas Mg2+ (K0.5 of 12 mM) and nifedipine (K0.5 of 3.3 nM) depressed ICa(s). Oxytocin had no effect on INa(f) and actually depressed ICa(s) (but not IBa) to a small extent. Therefore, the tocolytic action of beta-agonists cannot be explained by an inhibition of ICa(s), whereas that of Mg2+ can be so explained. The stimulating action of oxytocin on uterine contractions cannot be explained by a stimulation of ICa(s).


Blocking effects of OPC-21268 and OPC-31260 (vasopressin V1- and V2-receptor antagonists) on vasopressin-induced constrictions in isolated, perfused dog femoral arteries. Using a perfusion technique of isolated vessels, constrictor responses to vasopressin (VP) and norepinephrine (NE) were investigated in perfused dog femoral arteries. Both OPC-21268, a selective V1-antagonist, and OPC-31260, a selective V2-antagonist, significantly shifted the VP-induced dose response curves to the right without influencing the NE-induced ones. The blocking effects of OPC-31260 were much greater than those of OPC-21268, suggesting that there may probably be functional V1- and V2-receptors in isolated dog femoral arteries that mediate vasoconstriction.


Repeated treatment with levoprotiline, a novel antidepressant, up-regulates histamine H1 receptors and phosphoinositide hydrolysis response in vivo. The effects of repeated administration of levoprotiline, a novel type of tetracyclic antidepressant on histamine H1, muscarinic acetylcholine and alpha 1-adrenergic receptors and the response of phosphoinositide hydrolysis (PI) stimulated by histamine in the cortex of the rat brain were investigated. Histamine H1 receptors were up-regulated to 120% and PI response stimulated by histamine was enhanced to 160%-200% after repeated treatment with levoprotiline (20 mg/kg, i.p., once a day for 28 days) when compared to that of the saline-treated group. No significant alterations of muscarinic acetylcholine and alpha 1-adrenergic receptors were observed. This demonstrates that the repeated treatment with levoprotiline has prominent action on the regulation of histamine H1 receptors and PI response coupling to histamine H1 receptors in vivo.


[Prognosis of small cell lung cancer with ipsilateral pleural effusion]. A total of 184 patients with small cell lung cancer (SCLC) including 18 patients with ipsilateral pleural effusion as the only evidence of metastasis beyond the primary tumor site (PL), 84 patients with limited disease (LD), and 82 patients with extensive disease (ED) were treated at the Osaka Prefectural Habikino Hospital between December 1982 and June 1990. The median survival time for patients with PL was 51 weeks; for the patients with LD, 51 weeks; and for the patients with ED, 34 weeks. The survival of PL patients was significantly better than that of ED patients (P less than 0.05), and did not differ from that of LD patients. The response rate of PL patients was not significantly different from the response rates observed in LD- and ED-patients. There was no significant difference in survival or response rate between patients with cytologically positive and those with cytologically negative PL. Ipsilateral pleural effusion was not found to be a independent prognostic factor for survival from multivariate analysis in LD patients. These results indicate that the classification of limited disease small cell lung cancer should include patients with ipsilateral pleural effusion, as suggested by the consensus report at the International Association for the Study of Lung Cancer (IASLC) Workshop in 1989.


[Serum neuron specific enolase (NSE) levels in patients with non small cell lung cancer]. Serum NSE levels were measured in 126 patients with previously untreated NSCLC. The NSE level was greater than 10 ng/ml in 29 patients (23.0%) and this was considered to be positive. Elevation of serum NSE levels correlated closely with clinical stage except stage I and II. The effect of chemotherapy was evaluated in 74 cases included 22 NSE-positive cases. The response rate was 50% in positive cases and 34.6% in negative cases. However, the median duration of response in positive cases (2.2 months) was significantly shorter than that in negative cases (6.6 months). The median survival time of 6.0 months in positive cases was brief compared with 9.6 months in negative cases. These results indicate that elevation of serum NSE level in patients with NSCLC may be a marker of poor prognosis.


[Gianturco expandable metallic stents in the treatment of superior vena cava syndrome caused by lung cancer]. Treatment of superior vena cava syndrome (SVCS) caused by advanced lung cancer is still controversial. We inserted Gianturco expandable metallic stents (GEMS) in 5 patients with SVCS due to the extension of lung cancer. GEMSs were introduced intravenously through the catheter after intraluminal balloon dilation of the stenotic sites. SVCS was successfully and easily relieved by this method without any significant complication. GEMS placement seems to be a useful alternative to bypass grafting procedure for the treatment of SVCS.


[A case report of left upper lobectomy for lung cancer performed on an aged man of 90 year old]. A 90-year-old man was admitted to our hospital because of the dyspnea on exertion and an abnormal shadow of the chest. The examinations revealed pulmonary adenocarcinoma (bronchioloalveolar cell type) of the left S3 with clinical classification of stage I. Spirometry showed severe obstructive lung dysfunction (VC 2.05 l, % VC 75.2%, FEV1.0 0.72 l, FEV1.0% 39.3%). Walking exercise in the ward and the stairs as well as instrumental physiotherapy (Triflow) partially improved his respiratory function. Left upper lobectomy and lymphnode dissection were performed. On the 3rd postoperative day the patient developed mild delirium which disappeared in a few days. He was discharged from the hospital 62 days after the operation.


[Flow cytometric analysis of DNA content in adult testicular germ cell tumors]. Flow cytometric DNA analysis was carried out in 54 patients with testicular germ cell tumors (GCTs) experienced at our hospital, to evaluate the clinical relevance of DNA index (DI) and provide some insight into the pathogenesis of testicular GCTs. Histological types with their incidences were seminomas in 31 patients and nonseminomatous germ cell tumors (NSGCTs) in 23 adults. DNA ploidy and DI were analyzed by flow cytometry in 158 paraffin embedded samples; 2.9 samples per case on the average. This study revealed that 52 cases (96%) of evaluable 54 adult GCTs were DNA aneuploid, while DNA diploid tumors were observed in only each one case of NSGCT and seminoma. There was a significant difference (p less than 0.01) between the distribution of DIs in adult NSGCTs (median DI = 1.50) and that in pure seminomas (median DI = 1.85). Although we found no significant correlation between DI and clinical staging of Japanese Urological Association, on the basis of Indiana University staging system, the median DI in NSGCT patients of the advanced extent was lower than those of the other extents. DNA heterogeneity was observed only in 4 of 23 NSGCT patients (17%) and 3 of those 4 patients were assigned to advanced extent. These data suggest that the lower DI and the presence of DNA heterogeneity may have prognostic relevance for NSGCTs.


[Chemoembolization of blood vessels in the complex treatment of children with nephroblastoma]. The results of roentgeno-endovascular intervention in nephroblastoma in 31 child are presented. For chemoembolization, suspension of lipiodol and adriamycin was used, for control of the effectiveness of the intervention--the radiopharmaceutical preparation pyrophosphate. After embolization, the decrease in tumor size was noted. This made easier the performance of nephrectomy, the volume of blood loss reduced. Pathomorphism of neuroblastoma induced by chemoembolization was studied. No complications were noted.


[Seroepidemiologic study of human herpesvirus-6 in intravenous drug addicts, with and without infection caused by human immunodeficiency virus type 1]. The human herpesvirus-6 (HHV-6) may be a cofactor of infection by the human immunodeficiency virus type 1 (HIV-1). However, there are discrepancies with respect to the possible epidemiological relation between both viruses. The aim of the present study was to study the prevalence of infection by the HHV-6 in intravenous drug addicts (IVDA) with and without HIV-1 infection. IgG antibodies vs HHV-6 (anti-HHV-6-IgG) were determined by indirect immunofluorescence in 100 IVDA (29 seronegative and 71 seropositive for HIV-1 of which 45 were in stage II and 26 in IV-C1 of CDC) as well as in 100 healthy subjects of a similar age (control group). The prevalence of anti-HHV-6-IgG was much higher in the whole group of IVDA than in the control group and was equal in the IVDA with HIV-1 infection and in those patients without infection. There was no significant difference between the latter and the control group with the same being seen between the IVDA in different stages of HIV-1 infection. The results of this study suggest the existence of an epidemiological relation between human herpes virus-6 (HHV-6) infection and human immunodeficiency virus -1 (HIV-1). However, infection by the HHV-6 has no relation with the evolutive degree of the HIV-1 infection nor with intravenous drug addiction.


Chemically-induced chronic nerve compression in rabbits--a new experimental model for the carpal tunnel syndrome. In order to create an experimental model for the carpal tunnel syndrome without the use of the commonly applied foreign bodies (silicone or rubber tubes, tourniquets etc.), the present study tried to induce a chemically provoked compression of the median nerve in rabbits. In 9 female rabbits 1 ml of Aethoxskerol 3% (Hydrox-polyethoxy dodecan) was instilled into the carpal tunnel around the median nerve after visualisation of the nerve. The other foreleg served as the control and was treated with the same amount of saline solution. Electroneurophysiologic parameters were registered preoperatively, 1 month and 6 months post surgery and histomorphologic investigations by light and electron microscopy were performed after 6 months. 6 months after treatment with Aethoxysklerol, a statistically significant lengthening of the distal latency period as well as a significant reduction of the compound potential amplitude could be observed. In accordance with these findings, morphological investigation revealed the presence of extensive granulation tissue around the median nerve together with signs of demyelination. Our results indicate that we were able to produce the development of extensive granulation tissue in the carpal tunnel of rabbits with subsequent compression of the median nerve which was confirmed by histomorphologic investigation as well as by measurement of nerve conductive velocity.


Identification and molecular analysis of glgS, a novel growth-phase-regulated and rpoS-dependent gene involved in glycogen synthesis in Escherichia coli. The putative stationary-phase sigma factor (sigma S) encoded by rpoS is essential for glycogen synthesis, but is not required for the transcription of glgC and glgA, which encode ADP-glucose-pyrophosphorylase and glycogen synthase, respectively. Using a mini-Mu random chromosomal library and a screen for glycogen overproduction, we identified a novel gene (glgS) involved in glycogen synthesis. glgS maps at 66.6 min (3247 kb) on the chromosome and constitutes a monocistronic operon. It encodes a hydrophilic and highly charged small protein, with a molecular weight of 7886, which is strongly expressed in minicells. Experiments with single-copy chromosomal glgS::lacZ gene fusions indicated that glgS expression is controlled by sigma S as well as by cAMP. Two transcriptional start sites were mapped in the upstream regulatory region of glgS. The glgSp1 transcript was absent in a cya mutant, whereas an rpoS mutant did not synthesize the glgSp2 transcript. Although glycogen synthesis is strongly stimulated by overproduction of GlgS and is inhibited by a glgS null mutation, glgS does not affect the expression of the glgCAP operon. Its potential role in the metabolic control of glycogen synthesis is discussed. Also, evidence is presented to show that the amount of glycogen accumulated in vivo in early stationary-phase cells is mainly determined by sigma S-controlled gene expression and allosteric activation of GlgC, whereas the absolute levels of expression of glgCAP as well as the intracellular concentration of cAMP are of minor importance.


A homologue of the Escherichia coli DsbA protein involved in disulphide bond formation is required for enterotoxin biogenesis in Vibrio cholerae. A strain of Vibrio cholerae, which had been engineered to express high levels of the non-toxic B subunit (EtxB) of Escherichia coli heat-labile enterotoxin, was subjected to transposon (TnphoA) mutagenesis. Two chromosomal TnphoA insertion mutations of the strain were isolated that showed a severe defect in the amount of EtxB produced. The loci disrupted by TnphoA in the two mutant derivatives were cloned and sequenced, and this revealed that the transposon had inserted at different sites in the same gene. The open reading frame of the gene predicts a 200-amino-acid exported protein, with a Cys-X-X-Cys motif characteristic of thioredoxin, protein disulphide isomerase, and DsbA (a periplasmic protein required for disulphide bond formation in E. coli). The V. cholerae protein exhibited 40% identity with the DsbA protein of E. coli, including 90% identity in the region of the active-site motif. Introduction of a plasmid encoding E. coli DsbA into the V. cholerae TnphoA derivatives was found to restore enterotoxin formation, whilst expression of Etx or EtxB in a dsbA mutant of E. coli confirmed that DsbA is required for enterotoxin formation in E. coli. These results suggest that, since each EtxB subunit contains a single intramolecular disulphide bond, a transient intermolecular interaction with DsbA occurs during toxin subunit folding which catalyses formation of the disulphide in vivo.


IS231D, E and F, three new insertion sequences in Bacillus thuringiensis: extension of the IS231 family. IS231 constitutes a family of insertion sequences widespread among Bacillus thuringiensis subspecies. Three new IS231 variants have been isolated from B. thuringiensis subspecies finitimus (IS231 D and E) and israelensis (IS231F). Like the previously described IS231A, B and C, these 1.7 kb elements display single open reading frames encoding 477/478-amino-acid proteins which share between 72% and 88% identity with those of the other members of the family. Sequence comparisons also reveal that all the iso-IS231 terminal inverted repeats are strongly conserved 20 bp sequences. A region susceptible to forming a stable hairpin structure is found just upstream of the open reading frame. Nucleotide substitutions occurring on one strand of the hairpin stems are compensated for by complementary changes at facing positions, giving credence to the hypothesis that this secondary structure plays a role in the regulation of transposition. Examination of IS231 D, E and F flanking sequences reveals that IS231F is bordered by a 12 bp direct repeat. No direct repeats were found flanking IS231D or IS231E.


Albedos for 4-, 10-, and 18-MV bremsstrahlung x-ray beams on concrete, iron, and lead--normally incident. Backscattering of radiation incident on a solid medium has been treated with some success as a reflection phenomenon. The concept of albedo has been used in the literature to deal with this problem. A Monte Carlo program has been used in this study to simulate the generation of bremsstrahlung beams from medical linear accelerators. The accuracy of the calculated spectra have been checked indirectly by calculating TPRs and comparing them with measured values. The dose albedos for 60Co and 137Cs beams normally incident on concrete have been calculated and compared with experimentally determined values in the literature. The generated energy spectra are then used to calculate the albedo factors for 4-, 10-, and 18-MV bremsstrahlung x-ray beams normally incident on concrete, iron, and lead. The number albedo decreases with increase in the atomic number of the reflecting medium. However, for high-energy photons, the dose albedo increases with increase in the atomic number of the medium. This is an important factor that needs to be considered in the design of shielding. The calculated energy spectra can be used in other problems and the albedo factors can be applied in practical shielding design for medical linear accelerators.


[Benign lymphoepithelial cysts of the parotid in a patient with HIV infection]. We report on a HIV-positive patient presenting bilateral enlargement of the parotid gland. The ultrasound examination demonstrated multiple lesions with cystic aspect. A partial surgical excision of the parotid gland was performed, and histologic examination confirmed the diagnosis of a cystic benign lymphoepithelial lesion. The specimens which were tested for Epstein-Barr virus and cytomegalovirus by in situ hybridization proved negative. The clinical and pathologic features of this unusual salivary gland lesion which affects HIV-positive patients are discussed.


Accelerator-based epithermal neutron beam design for neutron capture therapy. Recent interest in the production of epithermal neutrons for use in boron neutron capture therapy (BNCT) has promoted an investigation into the feasibility of generating such neutrons with a high current proton accelerator. Energetic protons (2.5 MeV) on a 7Li target produce a spectrum of neutrons with maximum energy of roughly 800 keV. A number of combinations of D2O moderator, lead reflector, 6Li thermal neutron filtration, and D2O/6Li shielding will result in a useful epithermal flux of 1.6 x 10(8) n/s at the patient position. The neutron beam is capable of delivering 3000 RBE-cGy to a tumor at a depth of 7.5 cm in a total treatment time of 60-93 min (depending on RBE values used and based on a 24-cm diameter x 19-cm length D2O moderator). Treatment of deeper tumors with therapeutic advantage would also be possible. Maximum advantage depths (RBE weighted) of 8.2-9.2 (again depending on RBE values and precise moderator configuration) are obtained in a right-circular cylindrical phantom composed of brain-equivalent material with an advantage ratio of 4.7-6.3. A tandem cascade accelerator (TCA), designed and constructed at Science Research Laboratory (SRL) in Somerville MA, can provide the required proton beam parameters for BNCT of deep-seated tumors. An optimized configuration of materials required to shift the accelerator neutron spectrum down to therapeutically useful energies has been designed using Monte Carlo simulation in the Whitaker College Biomedical Imaging and Computation Laboratory at MIT. Actual construction of the moderator/reflector assembly is currently underway.


Testing for antibodies to human immunodeficiency virus type 2 in the United States. The Food and Drug Administration (FDA) has recommended that all donated blood be screened for antibodies to human immunodeficiency virus type 2 (HIV-2) beginning no later than June 1, 1992. This article provides CDC recommendations for the diagnosis of HIV-1 and HIV-2 infections in persons being tested in settings other than blood centers and CDC/FDA guidelines for serologic testing with combination HIV-1/HIV-2 screening enzyme immunoassays (EIAs). Epidemiologic data indicate that the prevalence of HIV-2 infections in persons in the United States is extremely low. Therefore, CDC does not recommend routine testing for HIV-2 in settings other than blood centers. However, when HIV testing is indicated, tests for antibodies to both HIV-1 and HIV-2 should be obtained if epidemiologic risk factors for HIV-2 infection are present, if clinical evidence exists for HIV disease in the absence of a positive test for antibodies to HIV-1, or if HIV-1 Western blot results exhibit the unusual indeterminate pattern of gag plus pol bands in the absence of env bands. The following procedures are recommended if testing for both HIV-1 and HIV-2 is performed by means of a combination HIV-1/HIV-2 EIA. A repeatedly reactive specimen by HIV-1/HIV-2 EIA should be tested by HIV-1 Western blot (or another licensed HIV-1 supplemental test). A positive result by HIV-1 Western blot confirms the presence of antibodies to HIV, and testing for HIV-2 is recommended only if HIV-2 risk factors are present. If the HIV-1 Western blot result is negative or indeterminate, an HIV-2 EIA should be performed. If the HIV-2 EIA is positive, an HIV-2 supplemental test should be performed.


[Recurrent hepatocellular carcinoma after hepatic resection]. A total of 125 patients with hepatocellular carcinoma (HCC) treated by hepatic resection in our department from 1970 to 1989 were reviewed to determine recurrent factors, recurrent modes of HCC and to assess the treatment for recurrent HCC. Seventy-five of 125 patients had recurrent tumors after the first hepatic resection. The 1-, 2-, and 3-year cumulative recurrent rates after hepatic resection were 25%, 52% and 67% respectively. The size of the tumor, intrahepatic metastasis, portal vein involvement, clinical stage and DNA ploidy pattern were judged as useful predictive factors for recurrence of HCC after hepatic resection. In the patients with intrahepatic metastases, the bilateral lobes of the remnant liver were the most frequent recurrent sites. The treatment for recurrent HCC was divided into 3 groups such as re-resection, transcatheter arterial embolization (TAE) and palliative treatment. The survival curves of patients receiving re-resection and TAE were significant better than those of patients receiving palliative treatment. Patients treated by re-resection for recurrent HCC had the longest survival. The 1-, 3- and 5-year cumulative survival rates after re-resection were 84%, 60% and 48% respectively. It is concluded that the early detection of recurrent HCC is important and re-resection or TAE is effective treatment for recurrent HCC.


Cloning and expression of the genes for xylose isomerase and xylulokinase from Klebsiella pneumoniae 1033 in Escherichia coli K12. The genes xylA and xylB were cloned together with their promoter region from the chromosome of Klebsiella pneumoniae var. aerogenes 1033 and the DNA sequence (3225 bp) was determined. The gene xylA encodes the enzyme xylose isomerase (XI or XylA) consisting of 440 amino acids (calculated M(r) of 49,793). The gene xylB encodes the enzyme xylulokinase (XK or XylB) with a calculated M(r) of 51,783 (483 amino acids). The two genes successfully complemented xyl mutants of Escherichia coli K12, but no gene dosage effect was detected. E. coli wild-type cells which harbored plasmids with the intact xylAKp 5' upstream region in high copy number (but lacking an active xylB gene on the plasmids) were phenotypically xylose-negative and xylose isomerase and xylulokinase activities were drastically diminished. Deletion of 5' upstream regions of xylA on these plasmids and their substitution by a lac promoter resulted in a xylose-positive phenotype. This also resulted in overproduction of plasmid-encoded xylose isomerase and xylulokinase activities in recombinant E. coli cells.


Role of IS1 in the conversion of virulence (Vi) antigen expression in Enterobacteriaceae. When Escherichia coli HB101 harbors pWR127, a plasmid comprising the viaB gene from Citrobacter freundii WR7004 and the ColE1-derived pACKC1, the strain produces the virulence (Vi) antigen. Vi antigen expression is abolished (Vi- phenotype), however, when an IS1 or IS1-like DNA element inserts into the viaB region. To determine the sites of IS1 insertion, pWR127 DNAs extracted from 95 independently isolated Vi- strains were analyzed by digestion with the restriction endonuclease PstI and agarose gel electrophoresis. Ten insertion sites were found distributed non-randomly in an area of about 1.3 kb. Nine Vi+ strains (two Citrobacter, two E. coli, and five Salmonella strains), four of which contain pWR127, were then tested for the presence of IS1 by DNA-DNA hybridization. Of the nine strains, five were stable Vi+ and did not contain IS1. The other four which generated Vi- strains, contained IS1. When pRR134, a plasmid that contains IS1 was transferred into a stable Vi+ Salmonella typhimurium strain carrying pWR127 (OU5140), Vi- strains were produced from which pWR127 derivatives carrying IS1 inserts could be isolated. It appears, therefore, that the presence of an IS1 or IS1-like element in a strain is required for conversion of the Vi+ expression state to the Vi- expression state.


The 5'-untranslated leader sequence of potato virus X RNA enhances the expression of a heterologous gene in vivo. The 5' untranslated leader of potato virus X (PVX) RNA is shown when contiguous to the coding sequence, to enhance the expression of the neomycin phosphotransferase II gene (NPTII) in Nicotiana tabacum protoplasts in vivo. The level of transient expression of the NPTII gene in protoplasts provided by a plasmid containing the coding sequence of the NPTII gene under the control of 35S cauliflower mosaic virus (CaMV) promoter and terminator elements served as the baseline control. Insertion of the viral 5' untranslated leader sequence upstream of the NPTII ATG codon increased the level of expression 4-fold. An 83 nucleotide (nt) leader sequence (lacking only one nucleotide of the complete PVX leader) and a truncated version with a 28 deletion at the 3' end both had similar enhancing abilities. The 28 nt CA region of the PVX leader alone had no enhancement properties.


[Tachykinins and conformational aspects of their interactions with receptors]. Several peptides of the tachykinin family are reviewed. Special attention is spared to mammalian tachykinins as peptide neurotransmitters. Conformational possibilities of the tachykinins are considered in connection with their ability to interact with specific receptors. The results of theoretical and experimental studies suggest a significant identity of spatial structures of the tachykinins and explain the absence of the strict specificity in tachykinin-receptor interactions.


Analysis of the myogenin promoter reveals an indirect pathway for positive autoregulation mediated by the muscle-specific enhancer factor MEF-2. Transcriptional cascades that specify cell fate have been well described in invertebrates. In mammalian development, however, gene hierarchies involved in determination of cell lineage are not understood. With the recent cloning of the MyoD family of myogenic regulatory factors, a model system has become available with which to study the dynamics of muscle determination in mammalian development. Myogenin, along with other members of the MyoD gene family, possesses the apparent ability to redirect nonmuscle cells into the myogenic lineage. This ability appears to be due to the direct activation of an array of subordinate or downstream genes which are responsible for formation and function of the muscle contractile apparatus. Myogenin-directed transcription has been shown to occur through interaction with a DNA consensus sequence known as an E box (CANNTG) present in the control regions of numerous downstream genes. In addition to activating the transcription of subordinate genes, members of the MyoD family positively regulate their own expression and cross-activate one another's expression. These autoregulatory interactions have been suggested as a mechanism for induction and maintenance of the myogenic phenotype, but the molecular details of the autoregulatory circuits are undefined. Here we show that the myogenin promoter contains a binding site for the myocyte-specific enhancer-binding factor, MEF-2, which can function as an intermediary of myogenin autoactivation. Since MEF-2 can be induced by myogenin, these results suggest that myogenin and MEF-2 participate in a transcriptional cascade in which MEF-2, once induced by myogenin, acts to amplify and maintain the myogenic phenotype by acting as a positive regulator of myogenin expression.


[Non-radioactive diagnosis of viral infections using "DNA-peroxidase" probes]. DNA-peroxidase probes were synthesized according to a modified method (Renzetal) for the detection of lambda phage DNA (model system), polio, potato X and M, tobacco mosaic viral RNAs by spot hybridization onto nitrocellulose membranes. cDNAs (300-1400 bases) complementary to the viral RNAs were cloned in M13 phage DNA or pTZ19. Efficacy of each step of the probe construction and the diagnostic procedure were thoroughly examined. Peroxidase activity manifested with non-toxic stain (NTS) was 3-5 fold more sensitive in comparison with alpha-Cl-naphthol or bisanisidine. It was found that HRP became much more stable to heat in diluted samples and was 2-3 fold more active after coupling with polyethylene imine spacer. Also, sodium borohydride reduction of the cDNA and PEI-HRP adduct crosslinked by the glutardialdehyde resulted in the stabilization of the probes. Target nucleic acids or diagnostic samples were efficiently fixed onto nitrocellulose membranes by a short-time UV irradiation. Diagnostics of cellular extracts with the preliminary prepared probes takes 4-5 hours due to express hybridization (1 hr) with 100-200 ng/ml of specific nucleotide sequence. Up to 20 pg (less than 10(-17) M) of the purified viral nucleic acids and 30-50 pg of them in the total fraction of the cellular nucleic acids isolated from the infected cells were identified with the probes. 50-10000 fold diluted lysate of the HeLa cells infected with poliovirus (PV1) and both crude extracts of potato tuber or potato and tobacco leaf tissues infected with PVX, PVM or TMV displayed specific signals with the respective DNA-HRP probes.


Hepatocyte nuclear factor 3 beta contains two transcriptional activation domains, one of which is novel and conserved with the Drosophila fork head protein. The hepatocyte nuclear factor 3 (HNF-3) gene family is composed of three proteins (alpha, beta, and gamma) that are transcription factors involved in the coordinate expression of several liver genes. All three proteins share strong homology in their DNA binding domains (region I) and are able to recognize the same DNA sequence. They also possess two similar stretches of amino acids at the carboxyl terminus (regions II and III) and a fourth segment of homology at the amino terminus (region IV). Furthermore, the HNF-3 proteins demonstrate homology with the Drosophila homeotic gene fork head in regions I, II, and III, suggesting that HNF-3 may be its mammalian homolog. In order to define HNF-3 beta protein domains involved in transcriptional activation, we have used a reporter gene, whose transcription is dependent on HNF-3 binding, for hepatoma cell cotransfection assays with expression vectors that produced different truncated HNF-3 beta proteins. A position-independent activation domain which contained conserved regions II and III was identified at the carboxyl terminus of the HNF-3 beta protein (amino acids 361 to 458). Moreover, site-directed mutations that altered the sequences within regions II and III demonstrated their importance to transactivation. The region II-III domain does not possess amino acid sequences in common with other transcription factors and may define a novel activation motif. HNF-3 beta amino-terminal sequences defined by conserved region IV also contributed to transactivation, but region IV activity required the participation of the region II-III domain. Region IV is abundant in serine amino acids and contains two putative casein kinase I phosphorylation sites, a feature similar to protein motifs described for the transcription factors Pit-1/GHF-1 and HNF-1.


Multiple members of the retinoic acid receptor family are capable of mediating the granulocytic differentiation of HL-60 cells. The complex and diverse biological effects of retinoic acid (RA) are mediated through specific receptors that are members of the steroid hormone family of nuclear transcription factors. The RA receptor family consists of multiple structurally distinct RA receptors, which diverge primarily at the NH2-terminal domain. The evolutionary conservation of this divergent region in individual RA receptors among different species together with their tissue-specific patterns of expression suggest that the biological function and activity of the individual RA receptors may be confined to specific tissues. To test this hypothesis in hematopoietic cells, we used retrovirus-mediated gene transduction to introduce the RA receptors RAR-alpha, RAR-beta, and RAR-gamma as well as RXR-alpha into a mutant subclone of the HL-60 promyelocytic leukemia cell line (designated HL-60R) that is relatively resistant to RA-induced granulocytic differentiation. We found that each of these structurally distinct RA receptors could restore sensitivity of the HL-60R cells to RA. A critical threshold number of transduced receptors per cell appears to be necessary to restore this functional activity. Thus, the capability to mediate granulocytic differentiation of HL-60 cells is shared among distinctly different RA receptors.


Saccharomyces cerevisiae RAD5-encoded DNA repair protein contains DNA helicase and zinc-binding sequence motifs and affects the stability of simple repetitive sequences in the genome. rad5 (rev2) mutants of Saccharomyces cerevisiae are sensitive to UV light and other DNA-damaging agents, and RAD5 is in the RAD6 epistasis group of DNA repair genes. To unambiguously define the function of RAD5, we have cloned the RAD5 gene, determined the effects of the rad5 deletion mutation on DNA repair, DNA damage-induced mutagenesis, and other cellular processes, and analyzed the sequence of RAD5-encoded protein. Our genetic studies indicate that RAD5 functions primarily with RAD18 in error-free postreplication repair. We also show that RAD5 affects the rate of instability of poly(GT) repeat sequences. Genomic poly(GT) sequences normally change length at a rate of about 10(-4); this rate is approximately 10-fold lower in the rad5 deletion mutant than in the corresponding isogenic wild-type strain. RAD5 encodes a protein of 1,169 amino acids of M(r) 134,000, and it contains several interesting sequence motifs. All seven conserved domains found associated with DNA helicases are present in RAD5. RAD5 also contains a cysteine-rich sequence motif that resembles the corresponding sequences found in 11 other proteins, including those encoded by the DNA repair gene RAD18 and the RAG1 gene required for immunoglobin gene arrangement. A leucine zipper motif preceded by a basic region is also present in RAD5. The cysteine-rich region may coordinate the binding of zinc; this region and the basic segment might constitute distinct DNA-binding domains in RAD5. Possible roles of RAD5 putative ATPase/DNA helicase activity in DNA repair and in the maintenance of wild-type rates of instability of simple repetitive sequences are discussed.


A delay in the Saccharomyces cerevisiae cell cycle that is induced by a dicentric chromosome and dependent upon mitotic checkpoints. Dicentric chromosomes are genetically unstable and depress the rate of cell division in Saccharomyces cerevisiae. We have characterized the effects of a conditionally dicentric chromosome on the cell division cycle by using microscopy, flow cytometry, and an assay for histone H1 kinase activity. Activating the dicentric chromosome induced a delay in the cell cycle after DNA replication and before anaphase. The delay occurred in the absence of RAD9, a gene required to arrest cell division in response to DNA damage. The rate of dicentric chromosome loss, however, was elevated in the rad9 mutant. A mutation in BUB2, a gene required for arrest of cell division in response to loss of microtubule function, diminished the delay. Both RAD9 and BUB2 appear to be involved in the cellular response to a dicentric chromosome, since the conditionally dicentric rad9 bub2 double mutant was highly inviable. We conclude that a dicentric chromosome results in chromosome breakage and spindle aberrations prior to nuclear division that normally activate mitotic checkpoints, thereby delaying the onset of anaphase.


Roles of insulinlike growth factor 1 (IGF-1) and the IGF-1 receptor in epidermal growth factor-stimulated growth of 3T3 cells. BALB/c3T3 cells are exquisitely growth regulated and require platelet-derived growth factor, epidermal growth factor (EGF), and insulinlike growth factor 1 (IGF-1) for growth. When BALB/c3T3 cells are transfected with plasmids constitutively expressing both EGF and the human IGF-1 receptor mRNAs, the cells are capable of growing in serum-free medium without the addition of any exogenous growth factor. These cells, called p5 cells, can grow for prolonged periods in serum-free medium. BALB/c3T3 cells transfected with only the IGF-1 receptor expression plasmid (p6 cells) do not grow in serum-free medium but do grow if IGF-1 (or insulin in supraphysiological concentrations) is added. p6 cells also grow in response to EGF, confirming that the combination of EGF and an overexpressed IGF-1 receptor is sufficient for the growth of 3T3 cells. We have found that in EGF-stimulated p6 cells there is an increase in the expression of IGF-1 mRNA, that IGF-1 is secreted into the medium, and that the growth of p5 cells and EGF-stimulated p6 cells is inhibited by exposure to antisense oligodeoxynucleotides to IGF-1 receptor RNA. Finally, while cells constitutively expressing both EGF and EGF receptor RNAs grow, albeit modestly, in serum-free medium, their growth is also inhibited by an antisense oligodeoxynucleotide to IGF-1 receptor RNA. In contrast, in cells overexpressing the IGF-1 receptor, IGF-1-mediated cell growth occurs independently of the platelet-derived growth factor and EGF receptors (Z. Pietrzkowski, R. Lammers, G. Carpenter, A. M. Soderquist, M. Limardo, P. D. Phillips, A. Ullrich, and R. Baserga, Cell Growth Differ. 3:199-205, 1992, and this paper). These data indicate that an important role for EGF is participation in the activation of an autocrine loop based on the IGF-1-IGF-1 receptor interaction, which is obligatory for the proliferation of 3T3 cells.


HeT-A, a transposable element specifically involved in "healing" broken chromosome ends in Drosophila melanogaster. Eight terminally deleted Drosophila melanogaster chromosomes have now been found to be "healed." In each case, the healed chromosome end had acquired sequence from the HeT DNA family, a complex family of repeated sequences found only in telomeric and pericentric heterochromatin. The sequences were apparently added by transposition events involving no sequence homology. We now report that the sequences transposed in healing these chromosomes identify a novel transposable element, HeT-A, which makes up a subset of the HeT DNA family. Addition of HeT-A elements to broken chromosome ends appears to be polar. The proximal junction between each element and the broken chromosome end is an oligo(A) tract beginning 54 nucleotides downstream from a conserved AATAAA sequence on the strand running 5' to 3' from the chromosome end. The distal (telomeric) ends of HeT-A elements are variably truncated; however, we have not yet been able to determine the extreme distal sequence of a complete element. Our analysis covers approximately 2,600 nucleotides of the HeT-A element, beginning with the oligo(A) tract at one end. Sequence homology is strong (greater than 75% between all elements studied). Sequence may be conserved for DNA structure rather than for protein coding; even the most recently transposed HeT-A elements lack significant open reading frames in the region studied. Instead, the elements exhibit conserved short-range sequence repeats and periodic long-range variation in base composition. These conserved features suggest that HeT-A elements, although transposable elements, may have a structural role in telomere organization or maintenance.


Substitutions in the hydrophobic core of the alpha-factor receptor of Saccharomyces cerevisiae permit response to Saccharomyces kluyveri alpha-factor and to antagonist. Mutations in the Saccharomyces cerevisiae alpha-factor receptor that lead to improved response to Saccharomyces kluyveri alpha-factor were identified and sequenced. Mutants were isolated from cells bearing randomly mutagenized receptor gene (STE2) plasmids by an in vivo screen. Five mutations lead to substitutions in hydrophobic segments in the core of the receptor (M54I, S145L, S145L-S219L, A229V, L255S-S288P). Remarkably, strains expressing these mutant receptors exhibited positive pheromone responses to desTrp1,Ala3-alpha-factor, an analog that normally blocks these responses. The M54I mutation appeared to affect only ligand specificity. The other mutations conferred additional effects on signaling or recovery. Two mutants were more sensitive to alpha-factor than wild type (S145L, A229V). One mutant was more sensitive to alpha-factor-induced cell cycle arrest initially, but then recovered more efficiently (S145L-S219L). One mutant (L255S-S288P) conferred positive pheromone responses to alpha-factor as assayed by FUS1-lacZ reporter induction, but did not display growth arrest. The hydrophobic receptor core thus appears to control activation by some ligands and to play roles in aspects of signal transduction and recovery.


Transcriptional regulation by triiodothyronine requires synergistic action of the thyroid receptor with another trans-acting factor. Human placental lactogen B (hCS-B) promoter activity is strongly stimulated by triiodothyronine (T3) in pituitary GC cells through interaction between the thyroid receptor and a thyroid receptor-binding element (TBE) spanning coordinates -67 to -41. This TBE is adjacent to the binding site for pituitary factor GHF1 (-95 to -68) which seems necessary for T3 stimulation of hCS-B promoter activity (M. L. Voz, B. Peers, A. Belayew, and J. A. Martial, J. Biol. Chem. 266:13397-13404, 1991). We here demonstrate actual synergy between the thyroid receptor and GHF1. Indeed, in placental JEG-3 cells devoid of factor GHF1, hCS promoter activity is barely stimulated by T3, while a strong response is observed in pituitary GC cells. In the latter, furthermore, neither the TBE nor the GHF1-binding site alone is sufficient to render the thymidine kinase promoter responsive to T3, while in combination they promote strong T3 stimulation. Close proximity between these sites is required for optimal synergy: T3 stimulation globally decreases with increased spacing. Furthermore, synergy occurs not only with a GHF1-binding site but also with all other factor recognition sequences tested (Sp1, NF1, CP1, Oct1, and CACCC boxes) and even with two other copies of the TBE. Nor is it specific to hCS TBE, since the palindromic sequence TCAGGTCA TGACCTGA (TREpal) also exhibits cooperativity.


Effect of ICRF-193, a novel DNA topoisomerase II inhibitor, on simian virus 40 DNA and chromosome replication in vitro. The effect of ICRF-193, a noncleavable-complex-forming topoisomerase II inhibitor, on simian virus 40 (SV40) DNA and SV40 chromosome replication was examined by using an in vitro replication system composed of HeLa cell extracts and SV40 T antigen. Unlike the topoisomerase inhibitors VP-16 and camptothecin, ICRF-193 had little effect on DNA chain elongation during SV40 DNA replication, but high-molecular-weight DNAs instead of segregated monomer DNAs accumulated as major products. Analysis of the high-molecular-weight DNAs by two-dimensional gel electrophoresis revealed that they consisted of catenated dimers and late Cairns-type DNAs. Incubation of the replicated DNA with topoisomerase II resulted in conversion of the catenated dimers to monomer DNAs. These results indicate that ICRF-193 induces accumulation of catenated dimers and late Cairns-type DNAs by blocking the decatenating and relaxing activities of topoisomerase II in the late stage of SV40 DNA replication. In contrast, DNA replication of SV40 chromosomes was severely blocked by ICRF-193 at the late stage, and no catenated dimers were synthesized. These results are consistent with the finding that topoisomerase II is required for unwinding of the final duplex DNA in the late stage of SV40 chromosome replication in vitro.


Housekeeping Na,K-ATPase alpha 1 subunit gene promoter is composed of multiple cis elements to which common and cell type-specific factors bind. Na,K-ATPase alpha 1 subunit gene (ATP1A1) is one of the housekeeping genes involved in homeostasis of Na+ and K+ in all animal cells. We identified and characterized the cis-acting elements that regulate the expression of ATP1A1. The region between -155 and -49 was determined as a positive regulatory region in five cultured cell lines of different tissue origins (MDCK, B103, L6, 3Y1, and HepG2). The region was divided into three subregions: from -120 to -106 (including the Sp1 binding site), from -102 to -61, and from -58 to -49 (including an Sp1 consensus sequence). Cell type-specific factors binding to the middle subregion (from -102 to -61) were detected by gel retardation analysis, using nuclear extracts prepared from MDCK and B103 cells. Two gel retardation complexes were formed in the B103 nuclear extract, and three were formed in the MDCK nuclear extract. DNA binding regions of these factors were located at -88 to -69 and differed from each other in DNase I footprinting experiments. These factors also showed different binding characteristics in gel retardation competition and methylation interference experiments. The identified cis element was named the ATP1A1 regulatory element. The core sequence of this element is found in several other genes involved in cellular energy metabolism, suggesting that the sequence is a common regulatory element responsive to the state of energy metabolism.


The 63-kilobase circular amplicon of tunicamycin-resistant Leishmania amazonensis contains a functional N-acetylglucosamine-1-phosphate transferase gene that can be used as a dominant selectable marker in transfection. Tunicamycin (TM)-resistant Leishmania amazonensis has been found previously to contain amplified chromosomal DNA, existing exclusively as extrachromosomal circles of 63 kb. Fragments of this DNA cloned into plasmids were functionally analyzed by transfection of wild-type cells. A clone with a 15-kb fragment of the 63-kb circle was initially found to confer TM resistance. A library of the 15-kb fragment was then prepared and used in toto to transfect wild-type cells. The transfectants that emerged after selection were found to contain a plasmid with an insert of 4.6 kb. Evidence from deletion experiments suggests that this is the minimal transfection-effective fragment. Sequencing of the 4.6-kb DNA revealed 1.4-kb homolog of N-acetylglucosamine-1-phosphate transferase genes. The L. amazonensis gene is similar to those from two other sources in their deduced peptide sequence by 65 to 70% and in hydropathic characteristics. The L. amazonensis gene is amplified by more than 128-fold over the wild type and overproduces a major transcript of 2.4 kb in all transfectants. The endogenous copy of this gene was amplified by polymerase chain reaction from the wild type and cloned into pX-NEO, a Leishmania expression vector. Amplification of this plasmid in the transfectants by selection with G418 simultaneously made them resistant to TM. Evidence provided thus indicates that the 1.4-kb DNA is an N-acetylglucosamine-1-phosphate transferase gene whose amplification is responsible for TM resistance in Leishmania variants and transfectants.


Chicken ovalbumin upstream promoter transcription factor (COUP-TF) dimers bind to different GGTCA response elements, allowing COUP-TF to repress hormonal induction of the vitamin D3, thyroid hormone, and retinoic acid receptors. Alignment of natural chicken ovalbumin upstream promoter transcription factor (COUP-TF) response elements shows that, in addition to the predominant direct repeat of the GGTCA motif with a 2-bp spacing, there are other functional COUP elements with variations in the GGTCA orientation and spacing. We systematically analyzed the binding of in vitro-synthesized COUP-TFs and showed that COUP-TF is capable of binding to oligonucleotides containing both direct repeats and palindromes and with different spacings of the GGTCA repeats. Subsequently, we analyzed four possible mechanisms proposed to explain how COUP-TF could bind to these spatial variations of the GGTCA repeat. We demonstrated that the functional DNA-binding form of COUP-TF is a dimer which requires two GGTCA half-sites to bind DNA. We demonstrated that the COUP-TF dimer undergoes a remarkable structural adaptation to accommodate binding to these spatial variants of the GGTCA repeats. A functional consequence of the promiscuous DNA binding of COUP-TF is its ability to down-regulate hormonal induction of target gene expression by other members of the steroid-thyroid hormone receptor superfamily such as the vitamin D3, thyroid hormone, and retinoic acid receptors. Our data indicate that COUP-TF may have an important role in hormonal regulation of gene expression by these receptors.


ABF1 is a phosphoprotein and plays a role in carbon source control of COX6 transcription in Saccharomyces cerevisiae. Previously, we have shown that the Saccharomyces cerevisiae DNA-binding protein ABF1 exists in at least two different electrophoretic forms (K. S. Sweder, P. R. Rhode, and J. L. Campbell, J. Biol. Chem. 263: 17270-17277, 1988). In this report, we show that these forms represent different states of phosphorylation of ABF1 and that at least four different phosphorylation states can be resolved electrophoretically. The ratios of these states to one another differ according to growth conditions and carbon source. Phosphorylation of ABF1 is therefore a regulated process. In nitrogen-starved cells or in cells grown on nonfermentable carbon sources (e.g., lactate), phosphorylated forms predominate, while in cells grown on fermentable carbon sources (e.g., glucose), dephosphorylated forms are enriched. The phosphorylation pattern is affected by mutations in the SNF1-SSN6 pathway, which is involved in glucose repression-depression. Whereas a functional SNF1 gene, which encodes a protein kinase, is not required for the phosphorylation of ABF1, a functional SSN6 gene is required for itsd ephosphorylation. The phosphorylation patterns that we have observed correlate with the regulation of a specific target gene, COX6, which encodes subunit VI of cytochrome c oxidase. Transcription of COX6 is repressed by growth in medium containing a fermentable carbon source and is derepressed by growth in medium containing a nonfermentable carbon source. COX6 repression-derepression is under the control of the SNF1-SSN6 pathway. This carbon source regulation is exerted through domain 1, a region of the upstream activation sequence UAS6 that binds ABF1 (J. D. Trawick, N. Kraut, F. Simon, and R. O. Poyton, Mol. Cell Biol. 12:2302-2314, 1992). We show that the greater the phosphorylation of ABF1, the greater the transcription of COX6. Furthermore, the ABF1-containing protein-DNA complexes formed at domain 1 differ according to the phosphorylation state of ABF1 and the carbon source on which the cells were grown. From these findings, we propose that the phosphorylation of ABF1 is involved in glucose repression-derepression of COX6 transcription.


Binding characteristics of the thyroid hormone receptor homo- and heterodimers to consensus AGGTCA repeat motifs. Previous studies have shown that thyroid hormone receptors can form homo- and heterodimeric complexes when binding to response elements. We report here the binding characteristics of thyroid hormone receptor (TR) homo- and heterodimers binding to synthetic oligonucleotides with directly and palindromically repeated consensus motifs (AGGTCA). Binding assays showed that TR homodimer formation on DNA had a low specificity and cooperativity, and very fast off rates. In contrast, TRs and retinoic acid receptors readily formed heterodimers with higher specificity and affinity on direct repeats of the AGGTCA motif spaced by four or five nucleotides, although these heterodimer/DNA complexes were only moderately stable when compared to DNA-bound TR/retinoid X receptor heterodimers. Also, TR/retinoic acid receptor heteromeric binding to other elements, including the synthetic T3RE-pal element, was of low specificity. These biochemical results suggest that TRs are unlikely to regulate transcription as homodimers in vivo, and that TR heterodimers mediate the effects of thyroid hormone.


An enhancer element responsive to ras and fms signaling pathways is composed of two distinct nuclear factor binding sites. In order to precisely define the sequences that constitute the ras-responsive enhancers element present in the murine retrotransposon NVL3, point mutations were introduced into the previously defined minimal transcriptional enhancer DNA. Analyses of the effects of these point mutations in transient transfection experiments, in gel retention assays, and by methylation interference footprinting indicated that the enhancer element was composed of two binding sites for distinct nuclear factors. Both binding sites were required for activation of the enhancer by either ras or v-fms oncogenes, and the distinct nuclear factors were found in extracts from cells that contained either oncogene. UV cross-linking analysis revealed that the AP1-related binding site, TGACTCT, was recognized by a nuclear factor of apparent molecular size of 50 kilodaltons, that is probably c-jun. The other binding site, CAGGATAT, is very similar to sites recognized by the ets-family of transcription factors, and was recognized by the 120-kilodalton ras-responsive factor-1. Activation of the NVL3 element was reconstituted in an in vitro transcription assay. The ets-related binding site was necessary for this in vitro reconstitution of activity. Thus, the NVL3 enhancer is related to the previously described oncogene-responsive enhancer element present in polyoma virus and is also related to elements identified in several cellular genes known to be ras-responsive, including the transforming growth factor-beta 1 gene.


Rapid regulation of corticotropin-releasing hormone gene transcription in vivo. Regulation of corticotropin-releasing hormone (CRH) gene expression in vivo was assessed via in situ hybridization histochemistry, using probes directed against an intronic sequence of the CRH gene. Initial characterization of the CRH intron (CRHin) probe revealed specific localization of signal to the nuclear compartment of neurons in the medial parvocellular paraventricular hypothalamus, which are known to produce CRH peptide and mRNA. Abundance of CRHin signal was low, commensurate with a low resting pool of CRH heteronuclear RNA (hnRNA), representing CRH primary transcript. Regulation of CRH hnRNA levels was assessed after acute glucocorticoid synthesis blockade by injection of metyrapone. Metyrapone inhibits the conversion of 11-deoxycorticosterone to corticosterone, thereby rapidly depleting glucocorticoids and serving as a discrete stimulus for hypothalamo-pituitary-adreno-cortical activation. Plasma hormone measurements verified the efficacy of treatment, as metyrapone-treated rats showed extremely low basal corticosterone levels at all postinjection time points, while exhibiting progressive increases in plasma ACTH release over the 60-min postinjection period. CRH hnRNA levels were markedly increased 15-30 min after metyrapone injection, consistent with a rapid induction of CRH gene transcription in response to the stimulatory event. CRH mRNA, on the other hand, did not exceed control levels until 60 min post metyrapone, illustrative of a temporal lag between transcriptional changes and detectable changes in mRNA pools. Additional sections from metyrapone-and vehicle-treated rats were hybridized with probes complementary to mRNA encoding the immediate-early gene c-fos. c-fos was not present under unstimulated conditions yet was rapidly induced upon metyrapone treatment or vehicle injection (15 min).(ABSTRACT TRUNCATED AT 250 WORDS)


A point mutation (Ala229 to Thr) in the hinge domain of the c-erbA beta thyroid hormone receptor gene in a family with generalized thyroid hormone resistance. Various point mutations in the c-erbA thyroid hormone receptor (TR) beta gene of unrelated kindreds have been reported to be responsible for different phenotypes of generalized thyroid hormone resistance. We now report a new point mutation, Td, in one of two TR beta alleles of three affected members of one family, designated family T. In contrast to the previously described point mutations, all located in the T3-binding domain of the TR beta gene, mutation Td was identified in the carboxy-terminal part of the hinge domain. Direct sequencing of the polymerase chain reaction-amplified whole coding region of the patients' fibroblast TR beta genes displayed a single guanine to adenine transition at cDNA nucleotide position 985. This altered alanine (GCC) to threonine (ACC) in codon 229. Garnier prediction of the consequence of the mutation indicated an altered secondary structure. The G----A nucleotide substitution was not present in 80 random TR beta alleles, suggesting that this point mutation is responsible for generalized thyroid hormone resistance in family T. The in vitro expressed mutant TR beta was shown to bind with high affinity to various thyroid hormone response elements. However, the affinity of the TR beta to bind to T3 was reduced 3-fold, indicating that the hinge domain of the TR beta is important for full ligand-binding activity. Moreover, it seems that multiple subdomains of the TR beta interact cooperatively to achieve optimal T3 activity.


Heterodimeric receptor complexes determine 3,5,3'-triiodothyronine and retinoid signaling specificities. Thyroid hormone receptors (TRs) and retinoic acid receptors (RARs) have been shown to interact with nuclear auxiliary proteins resulting in heteromeric complexes that bind strongly to their responsive elements. Recently the retinoid X receptors (RXRs) have been identified as one class of these nuclear proteins. RXRs strongly increase binding of TRs and RARs to a synthetic thyroid hormone (and retinoic acid) responsive element. Here results show that the binding of the heteromeric complexes to various natural response elements is highly specific and dictated by the partner of RXR in the complex. TR alpha and TR beta formed complexes with RXR alpha that strongly and selectively bound to natural thyroid hormone responsive elements, i.e. those from the rat alpha-myosin heavy chain gene and the rat malic enzyme gene. RXR alpha complexes with RAR alpha, RAR beta, and RAR gamma bound selectively to retinoic acid responsive elements from the human RAR beta 2 gene (hRAR beta 2), the gene of the rat cellular retinol binding protein I and the human apolipoprotein A1 gene. Under the conditions used here RXR alpha by itself did not bind to any of the responsive elements tested. Although TRs and RARs formed heterodimers with RXR in solution, these complexes were strongly stabilized by specific, high affinity response elements, but not by low affinity response elements. Transfection analyses showed strong synergism between receptors that formed effective heterodimers in transcriptional activation on several but not all response elements. Overall, these data demonstrate that RARs and TRs are unlikely to function as monomers or homodimers on the response elements investigated here and require RXRs or comparable proteins for effective response element activation.


Cloning and functional expression of a mouse gonadotropin-releasing hormone receptor. GnRH plays a pivotal role in the reproductive system, and GnRH analogs have wide therapeutic applications ranging from the treatment of prostatic cancer to infertility. Determination of the predicted structure of the GnRH receptor (GnRHR) would illuminate the mechanisms of receptor activation and regulation and allow directed design of improved GnRH analogs. We report the cloning of a cDNA representing the mouse GnRHR and confirm its identity using Xenopus oocyte expression. Injection of sense RNA transcript leads to the expression of a functional, high affinity GnRHR. Expression of the GnRHR using gonadotrope cell line RNA, however, is blocked by an antisense oligonucleotide. In situ hybridization in the rat anterior pituitary reveals a characteristic GnRHR distribution. The nucleotide sequence encodes a 327-amino acid protein which has the seven putative transmembrane domains characteristic of G protein-coupled receptors, but which lacks a typical intracellular C-terminus. The unusual structure and novel potential regulatory domain of the GnRHR may explain unique aspects of its signal transduction and regulation.


Neuromuscular transmission as a function of motor unit size in patients with prior poliomyelitis. We studied neuromuscular transmission in 16 patients with prior poliomyelitis by measuring single fiber electromyographic (SFEMG) jitter. This was compared with 3 indirect methods of assessing reinnervation: SFEMG fiber density, macro EMG, and the presence of fiber type grouping on muscle biopsy. In patients with acute poliomyelitis before the age of 10, there was a positive correlation between the extent of neuromuscular transmission impairment, demonstrated by increased SFEMG jitter, and the enlargement of the motor unit, as indicated by increased fiber density, increased macro EMG signals, and fiber type grouping on muscle biopsy. However, there was no correlation between any of these parameters and the presence or absence of new symptoms of weakness. These findings suggest that impaired neuromuscular transmission is most common in patients with prior poliomyelitis whose motor units have been maximally enlarged by axonal sprouting, but is independent of the presence or absence of new symptoms of weakness.


Distal small fiber neuropathy: results of tests of sweating and autonomic cardiovascular reflexes. The purpose of this study was to evaluate sweating and cardiovascular autonomic function in patients with distal small fiber neuropathy (DSFN). Sweat testing by the quantitative sudomotor axon reflex test was abnormal in 32 of 40 (80%) patients. The thermoregulatory sweat test was abnormal in 18 of 25 (72%) patients; one or both tests were abnormal in 36 of 40 (90%). Minor heart rate abnormalities were present in 11 of 40 (28%) patients. We conclude that, in patients with DSFN: (a) the sympathetic sudomotor fibers are frequently affected, and that evaluation of sweating is a useful diagnostic test; (b) that the autonomic nerves controlling heart rate are less affected.


Wallerian degeneration in human nerves: serial electrophysiological studies. After nerve transection, the distal stump undergoes Wallerian degeneration (WD). Little information is available concerning sequential changes in nerve conduction measurements during WD in humans. Five patients with nerve injuries were studied temporally. Motor-evoked amplitudes were reduced by 50% at 3 to 5 days after injury; the response was absent by day 9. Sensory-evoked amplitudes were reduced by 50% at 7 days after injury; the response was absent by day 11. Sensory and motor nerves with shorter distal stumps showed earlier loss of amplitude than did those with longer distal stumps. Denervation potentials were seen 10 to 14 days after injury. Our results suggest that WD occurs earlier if the distal stump is shorter, and that motor-evoked responses are affected earlier than sensory-evoked responses. The time-lag between the loss of the motor-evoked response and the appearance of denervation potentials, the latter coinciding with reduction of sensory evoked responses, suggests that failure of neuromuscular transmission precedes axonal loss during WD.


Early postoperative ulnar neuropathies following coronary artery bypass surgery. Ulnar neuropathies following surgery are common. However, they often go undetected during the early postoperative period, because the patient may be unaware of symptoms related to the neuropathy. Nerve conduction studies are useful in localizing the lesion, but are usually employed only in cases developing signs and symptoms. We undertook this study to determine the incidence, time of onset, and outcome of clinical and subclinical ulnar neuropathies. Electrophysiological studies were carried out preoperatively, immediately following surgery, and 4 to 6 weeks postoperatively in 20 coronary artery bypass patients. Conduction velocity across the elbow was reduced in 3 limbs (8%) postoperatively, all of which were detected immediately following surgery. One patient developed conduction block and weakness in ulnar supplied intrinsic hand muscles. Denervation was seen in 2 cases and, in 1 case (5%), a right brachial plexus injury was clinically evident 5 days following surgery. All newly developing ulnar neuropathies were asymptomatic, with most recovering to their preoperative electrophysiological status at follow-up.


Major growth reduction and minor decrease in mitochondrial enzyme activity in cultured human muscle cells after exposure to zidovudine. Zidovudine-induced mitochondrial myopathy in AIDS patients reported recently might be due to inhibition of mitochondrial DNA polymerase gamma. We investigated the effect of zidovudine on proliferation, differentiation, activity of mitochondrial- and nuclear-encoded enzymes, and mitochondrial DNA (mtDNA), in cultured human muscle cells. Marked inhibition of cell proliferation was found, even in the presence of low (10 mumol/L) zidovudine concentrations. Enzyme activity of the nuclear-encoded mitochondrial citrate synthase was not affected, and the partially mitochondrial-encoded cytochrome c oxidase was not decreased, except only after exposure to high concentrations (5 mmol/L) zidovudine. No decrease of mtDNA content and no mtDNA deletions were found in zidovudine-exposed muscle cells. We propose that the effect of zidovudine on muscle, seen in zidovudine-treated AIDS patients, results mainly from decrease in proliferation of muscle cells rather than inhibition of mtDNA replication.


Diagnostic sensitivity of the laboratory tests in myasthenia gravis. The diagnostic sensitivity of three laboratory tests [serum antiacetylcholine receptor antibody (AChR-ab) assay, the repetitive nerve stimulation (RNS) test, and, the single fiber EMG (SFEMG)] for myasthenia gravis (MG) was compared in 120 patients. In all cases, at least one of the tests was abnormal. SFEMG was the most sensitive test, being abnormal in 92% of cases, followed by the RNS test (77%) and the AChR-ab assay (73%). SFEMG was abnormal in all cases with negative AChR-ab and RNS tests, in 97% of cases with negative AChR-ab assay, in 89% of cases with negative RNS test, and in 89% of cases with mild MG. We conclude that one of these three tests is abnormal in all cases of MG, and that the SFEMG is most sensitive in the diagnosis of MG.


Modeling fiber type grouping by a binary Markov random field. A new approach to the quantification of fiber type grouping is presented, in which the distribution of histochemical type in a muscle cross section is regarded as a realization of a binary Markov random field (BMRF). Methods for the estimation of the parameters of this model are discussed. The first order BMRF, which is used in this article, contains 2 parameters: alpha and beta. The parameter beta is of prime importance, as it is an interaction parameter which governs the degree of type grouping. The value of this parameter is estimated for 9 muscle biopsies. The interpretation of the results is discussed.


Prediction of the structure of a receptor-protein complex using a binary docking method. To validate procedures of rational drug design, it is important to develop computational methods that predict binding sites between a protein and a ligand molecule. Many small molecules have been tested using such programs, but examination of protein-protein and peptide-protein interactions has been sparse. We were able to test such applications once the structures of both the maltose-binding protein (MBP) and the ligand-binding domain of the aspartate receptor, which binds MBP, became available. Here we predict the binding site of MBP to its receptor using a 'binary docking' technique in which two MBP octapeptide sequences containing mutations that eliminate maltose chemotaxis are independently docked to the receptor. The peptides in the docked solutions superimpose on their original positions in the structure of MBP and allow the formation of an MBP-receptor complex. The consistency of the computational and biological results supports this approach for predicting protein-protein and peptide-protein interactions.


Convergence of 9-cis retinoic acid and peroxisome proliferator signalling pathways through heterodimer formation of their receptors. Peroxisomes are cytoplasmic organelles which are important in mammals in modulation of lipid homeostasis, including the metabolism of long-chain fatty acids and conversion of cholesterol to bile salts (reviewed in refs 1 and 2). Amphipathic carboxylates such as clofibric acid have been used in man as hypolipidaemic agents and in rodents they stimulate the proliferation of peroxisomes. These agents, termed peroxisome proliferators, and all-trans retinoic acid activate genes involved in peroxisomal-mediated beta-oxidation of fatty acids. Here we show that the receptor activated by peroxisome proliferators and the retinoid X receptor-alpha (ref. 6) form a heterodimer that activates acyl-CoA oxidase gene expression in response to either clofibric acid or the retinoid X receptor-alpha ligand, 9-cis retinoic acid, an all-trans retinoic acid metabolite; simultaneous exposure to both activators results in a synergistic induction of gene expression. These data demonstrate the coupling of the peroxisome proliferator and retinoid signalling pathways and provide evidence for a physiological role for 9-cis retinoic acid in modulating lipid metabolism.


Effect of disulfiram (DS) on mitochondria from rat hippocampus: metabolic compartmentation of DS neurotoxicity. This experiment was designed to study the acute effects of disulfiram on mitochondrial enzymes in nonsynaptic and synaptic mitochondria from rat hippocampus. Cytochrome c oxidase, monoamine oxidase-B, glycerolphosphate acyltransferase and beta-hydroxybutyrate dehydrogenase were studied. Differences in enzyme activity were seen in controls. Cytochrome c oxidase activity was higher in synaptic mitochondria whereas glycerolphosphate acyltransferase activity was higher in nonsynaptic mitochondria. Mitochondria from disulfiram treated rats, particularly synaptic mitochondria, exhibited lower specific activities of cytochrome c oxidase and monoamine oxidase-B. These alterations were not limited to either the inner or outer mitochondrial membrane. Transmission electron microscopy revealed that mitochondria from disulfiram treated rats were severely altered in isolated preparations as well as in those from whole tissue. This study shows that disulfiram exerts a differential effect on mitochondrial subpopulations.


Aging-induced decrease in dopaminergic-stimulated phosphoinositide metabolism in rat brain. Accumulation of [3H]inositol phosphate and [3H]inositol labeling of phosphoinositides were evaluated in brain slices of 3-, 6-, 12-, and 24-month-old Fischer-344 rats. The dopamine agonist, SKF38393, stimulated significantly lower accumulations of inositol trisphosphate, inositol bisphosphate, and inositol monophosphate in the striatum, hippocampus, and frontal cortex of 24-month-old rats compared with the 6-month-old animals. No differences, however, were observed between the 3, 6, and 12-month-old groups. Furthermore there were marked decrements of 41% to 58% in the labeling of phosphoinositides in the different brain regions of the aged animals. Dose-response studies in forebrain slices of the 6-month-old and 24-month-old animals showed aging-related decrements of 53% (p less than 0.001) and 48% (p less than 0.001) in the maximal SKF38393-stimulated labeling of phosphoinositides and accumulation of inositol phosphates, respectively. These data suggest that aging of the rat brain is associated with alterations in the basal turnover of the inositol cycle and in the sensitivity of the transduction pathway to dopamine receptor stimulation.


Sigma receptors in schizophrenic cerebral cortices. Antipsychotics represent high affinity for sigma receptors and sigma-like drugs often have the psychotomimetic properties. Besides, the receptors are unevenly distributed in human brain. These findings suggest that sigma receptors might be involved in the pathophysiology of schizophrenia. Sigma receptors in rat and human brain were measured with [3H]-1, 3, di-o-tolylguanidine (DTG) and non-specific binding of [3H]DTG was determined in the presence of 10(-5)M haloperidol. Monovalent and divalent cations strongly inhibited [3H]DTG binding. Glutamate, aspartate and glycine also decreased the binding to human cerebral membranes. With post-mortem brain samples from 12 schizophrenics and 10 controls, sigma receptors were measured in 17 areas of cerebral cortex. Sigma receptors binding showed the regional differences in the cortex, but no significant differences between schizophrenics and controls were observed except the superior parietal cortex where the binding significantly increased in the schizophrenic group. These results suggest that sigma receptors in cerebral cortices might not be directly concerned with the pathophysiological role in schizophrenia.


Characteristics of endothelin receptors in the cerebral cortex and spinal cord of aged rats. Characteristics of endothelin receptors were studied in male Fischer 344 rats at 4-, 15- and 24-months of age and [125I]ET-1 binding showed a single high affinity binding site in their cerebral cortex and spinal cord membranes. The density and affinity of ET binding sites were found to be similar in rats of various age groups. To determine the affinity of ET isotypes to ET-1 binding sites in cerebral cortex and spinal cord, competition studies were performed and K(i) values of ET-1, ET-2 and ET-3 for [125I] ET-1 binding sites were determined. It was found that ET-1 had 100 and ET-2 had 25-100 times lower K(i) values as compared to ET-3, indicating that ET receptors in cerebral cortex and spinal cord are of ETA type. In spinal cord, the K(i) values of ET-1 and ET-2 for ET receptor were found to be similar. However, in cerebral cortex the K(i) values of ET-1 were found to be at least 6 times lower than ET-2. It is inferred that there are two subtypes of ET(A) receptors, ET(A1) which have higher affinity for ET-1 in comparison to ET-2, as found in cerebral cortex and ET(A2) which have higher affinity for ET-2, as found in spinal cord. The K(i) and IC50 values of ET-1, ET-2, and ET-3 for [125I]ET-1 binding sites in cerebral cortex and spinal cord were found to be similar in 4-, 15- and 24-month-old rats.(ABSTRACT TRUNCATED AT 250 WORDS)


Protein kinase C activator phorbol 12, 13-dibutyrate inhibits platelet activating factor-stimulated Ca2+ mobilization and phosphoinositide turnover in neurohybrid NG108-15 cells. The protein kinase C (PKC) activator, phorbol 12, 13-dibutyrate (PDBu) dose-dependently inhibited platelet-activating factor (PAF)-induced [Ca2+]i elevation and inositol monophosphate (IP1) accumulation in neurohybrid NG108-15 cells with IC50 values of 162 nM and 35 nM, respectively. Pretreatment of NG108-15 cells with PKC inhibitor H-7 partially prevented the inhibitory effect of PDBu on PAF-induced [Ca2+]i elevation as well as PI metabolism in NG108-15 cells. Pretreatment of the cells with pertussis toxin (PTX) resulted in a dose-dependent inhibition of PAF-induced IP1 and IP3 accumulation but only slightly affected PAF-induced [Ca2+]i elevation in NG108-15 cells. The results reveal that PAF receptor-mediated Ca2+ mobilization and PI metabolism in NG108-15 cells are regulated by PKC while a PTX-sensitive G protein is coupled to PAF receptor for inducing activation of phospholipase C.


[The use of different suture materials in thyroid surgery. A statistical evaluation of the immediate and long-term results]. Six groups of patients for a total of 120 cases were examined for cervical wound suture results. Different synthetic absorbable sutures and different suture techniques were employed. Good aesthetic results were always obtained in long-term follow-up. The patients sutured with Polydioxanone, a monofilament with prolonged breaking strength retention, had a lower incidence of local complications.


Excitatory amino acid binding sites in the trigeminal principal sensory and spinal trigeminal nuclei of the rat. Quantitative autoradiography was used to examine the density and distribution of excitatory amino acid (EAA) binding site subtypes in the principal sensory and spinal trigeminal nuclei of the rat trigeminal complex. The highest densities of N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), kainate and metabotropic receptors were found in the superficial laminae (I and II) of subnucleus caudalis, a region known to be densely innervated by primary afferent nociceptive terminals. Lower densities of EAA binding sites were observed in spinal subnuclei interpolaris and oralis and within the principal sensory nucleus. These results are consistent with the hypothesis that EAAs are involved in primary afferent nociceptive neurotransmission.


High concentrations of neutral amino acids activate NMDA receptor currents in rat hippocampal neurons. High concentrations (30 microM-5 mM) of the neutral amino acids L-serine, L-cysteine, L-alanine, L-proline and glycine elicited inward current responses when applied to hippocampal neurons patch clamped at -60 mV in the presence of 1-10 microM glycine and 1 microM strychnine. The amplitude of the response to L-serine increased in a concentration-dependent fashion within the range 0.1-10 mM (EC50, 2.6 mM). L-Serine (1 mM) currents were attenuated by Mg2+ (100 microM) and completely blocked by the competitive N-methyl-D-aspartate (NMDA) antagonist 3-(2-carboxypiperazin-4-yl)-propyl-1- phosphonic acid (CPP) (30 microM). The CPP block could be overcome by raising the concentration of L-serine. We conclude that high concentrations of some neutral amino acids activate NMDA receptor-coupled ion channels by acting as agonists at the NMDA recognition site.


Neuronal cell cultures as a model for assessing neurotoxicity induced by encephalitic viruses. Primary dispersed and organotypic cultures were prepared from selected brain areas and spinal cords of rat (Sprague-Dawley) and mouse (SJL/OLA(F) Ness-Ziona) fetuses and neonates. Following fiber regeneration, synapse formation and myelination, cultures were infected with one of the following viruses: Rabies CVS-21 strain, Sindbis Alphavirus, West-Nile Flavivirus and Theiler Murine Encephalomyelitis virus. Light and electron microscopical studies showed clear differences in the target cells for virus infection; time of viral replication and in the intensity and specificity of the cytopathic effects induced by these viruses. Thus, Sindbis and Theiler viruses induced severe cytotoxicity and demyelination due to rapid viral replication in both neurons and all glial cell types. Rabies and West-Nile viruses, on the other hand, replicated mainly in neurons and at a much slower rate, causing only mild damage to the cells and the myelin sheath. A very specific alignment of West-Nile virions was observed along the interperiod lines of the myelin sheath in several myelinated axons. This peculiar arrangement of the virions, entrapped between the myelin lamellae may lead to a novel concept in the understanding of viral infection.


Glia toxicity in dissociation cell cultures induced by cyclosporine. Intravenously applied cyclosporine is the most effective and best analyzed immunosuppressive agent to date. Most frequently, this drug shows nephrotoxic or hepatotoxic side effects, which have already been investigated in detail. In addition to these adverse effects, there is also clear clinical evidence for toxic damage to the central nervous system. On the basis of magnetic resonance tomography and computed tomography studies, the white matter seems to be primarily affected. A systematic approach to neurotoxicity has been established in the following model. Mixed in-vitro cell cultures of dorsal root ganglia (DRG) and of the central nervous system were prepared from 6 to 14 day old chick embryos (E6-E14). For cultivation of nerve and glia cells we used beta NGF, a soluble trophic factor, and NTF B 82 as a matrix factor. Differentiated cultures were incubated with cyclosporine for intravenous application. Within a period of several days up to two weeks the cultures were analyzed using phase contrast microscopy, light microscopy, scanning and transmission electron microscopy. Glia cells and fibroblasts showed the most pronounced toxic effects. Their cytoplasm was infiltrated with smaller and larger vesicles which contained neutral lipids. Control cultures remained unaffected. Because of the close correlation between the in-vitro damage of glia cells and the clinically observed alteration of the white matter, we think our in-vitro model is helpful for the investigation of the neurotoxic effects of cyclosporine and immunotherapeutic drugs.


Long-lasting impairment of neuroendocrine response to psychological stress in heroin addicts. Performance in a vigilance task and the associated neuroendocrine changes during the performance in the task were examined in healthy subjects and in three groups of male heroin-addicts both before undergoing rehabilitation programmes and at various intervals from withdrawal (5 days, 1 to 2 mon, and 3 to 48 mon, respectively). Plasma levels of ACTH, beta-endorphin (EP) and prolactin (PRL) were measured every 10 min before and during performance. In drug addicts, simple reaction times never showed any significant difference as compared to control values. Despite similar baseline levels, ACTH exhibited a markedly depressed response to psychological testing in drug-addicts as compared to controls. Whereas a three-fold increase in ACTH was observed in 'normal' subjects during the performance (from 17 to 54 ng/l), mean values from drug-addicts remained unchanged. EP levels showed a wide scatter of individual values and inconsistent time courses over performance testing: after short-term abstinence, EP showed a three-fold increase over baseline control values but, contrary to what seen in 'normal' subjects, no changes over time were recorded. After long-term abstinence, basal EP was close to control values, but its increase during the testing period was still blunted. PRL levels decreased over the testing period both in controls and in heroin addicts. Thus, despite the lack of obvious signs of neurotoxicity, drug abusers show neuroendocrine changes consistent with a long-lasting selective impairment of the hypothalamic modulation of pituitary secretion.