The behavioural and neuronal effects of the chronic administration of benzodiazepine anxiolytic and hypnotic drugs.

Benzodiazepine anxiolytic and hypnotic drugs are some of the most widely prescribed drugs in the Western world. Despite this fact, the mechanisms that underlie the development of tolerance to, and dependence upon, benzodiazepines are poorly understood. The aim of this review is to summarize and critically evaluate the experimental evidence relating to the chronic behavioural and neuronal effects of benzodiazepines. Behavioural studies in animals generally indicate that tolerance gradually develops to the muscle relaxant, ataxic, locomotor and anticonvulsant effects of benzodiazepines. The evidence relating to the development of tolerance to the anxiolytic effects of benzodiazepines is less clear. The literature on the possible mechanisms of benzodiazepine tolerance and dependence is large, highly complex and difficult to interpret. The effect of chronic benzodiazepine treatment varies enormously as a function of the benzodiazepine used and the treatment schedule employed. Many studies have demonstrated a down-regulation of benzodiazepine binding sites, although affinity is usually unchanged. The evidence relating to the number and affinity of GABAA binding sites is unclear. Some studies suggest that chronic benzodiazepine administration results in a reduction in the number of Cl- channels associated with the GABAA receptor complex, although it is not clear that the efficacy of the GABA binding site in operating the Cl- channel necessarily changes. There is, however, substantial evidence to support the hypothesis that chronic benzodiazepine treatment results in a reduction in the coupling between the GABAA and benzodiazepine binding sites (the "functional uncoupling hypothesis"). Although some electrophysiological studies suggest that chronic benzodiazepine treatment results in a subsensitivity to GABA, this effect seems to be highly area-specific.

Tolerance to the ataxic effects of diazepam in guinea pig is not associated with a reduced sensitivity of GABAA receptors in the vestibular nucleus.

Some studies have suggested that drug tolerance observed following repeated benzodiazepine exposure may be associated with the development of a subsensitivity to gamma-aminobutyric acid (GABA) in dorsal raphe and hippocampal neurons. In other areas such as the substantia nigra such subsensitivity has not been found. The aim of the present study was to determine whether tolerance develops to the ataxic effects of diazepam on the righting reflex following low (i.e. 2 mg/kg i.p.), multiple daily doses and, if so, whether it is correlated with the development of a subsensitivity of medial vestibular nucleus neurons to the selective GABAA receptor agonist, isoguvacine. Guinea pigs which received i.p. vehicle injections three times daily for 5 days, or single daily doses of 2 or 6 mg/kg diazepam, showed increased righting reflex latencies in response to a 6 mg/kg diazepam challenge dose. However, guinea pigs which received 2 mg/kg diazepam i.p., three times daily for 5 days, exhibited minimal or no ataxia when given the same diazepam challenge dose, indicating the development of tolerance. Brain stem slices including the medial vestibular nucleus were removed from guinea pigs which had received the same diazepam and vehicle three times daily injection schedules, and recordings were made from single neurons during superfusion of isoguvacine. Although medial vestibular nucleus neurons from animals which received chronic diazepam administration showed smaller decreases in firing rate in response to 10(-8) M isoguvacine, the difference was not statistically significant compared to neurons from animals which received vehicle treatment or acute diazepam treatment. Resting activity was also similar between the diazepam and vehicle groups, in contrast to a previous study which had shown hyperexcitability in medial vestibular nucleus cells from animals which had received single daily injections for up to 60 days. These results suggest that, in contrast to studies which have employed single daily doses, tolerance to the ataxic effects of diazepam on the righting reflex occurs rapidly with divided daily doses. However, this tolerance is not correlated with significant changes in the sensitivity of GABAA receptors on medial vestibular nucleus neurons.

A university outbreak of Escherichia coli O157:H7 infections associated with roast beef and an unusually benign clinical course.

An outbreak of Escherichia coli O157:H7 infections occurred after a graduation banquet at a university in Wisconsin. Sixty-one (32%) of 193 banquet attendees developed a gastrointestinal illness; 2 were hospitalized, none developed hemolytic-uremic syndrome or thrombotic thrombocytopenic purpura, and none died. The spectrum of illness was unusually mild, with 61% of ill persons reporting nonbloody diarrhea. A strain of E. coli O157:H7, indistinguishable from the outbreak strain by toxin type, plasmid profile, and pulsed-field gel electrophoresis, was isolated from an unopened package of an uncooked round of beef from the original shipment of meat. An investigation suggested that both undercooked roast beef and salad cross-contaminated with beef were vehicles of transmission. These findings demonstrate that meat from beef cattle may transmit E. coli O157:H7, and such infections among young to middle-aged adults may be mild and may often go undetected.

The effects of long-term, low-dose diazepam treatment on the guinea pig righting reflex and medial vestibular nucleus neuronal activity.

Guinea pigs received a 2 mg/kg IP injection of diazepam, or an equivalent volume of vehicle, daily for 28-60 days. To determine whether tolerance developed to the ataxic effects of diazepam on the righting reflex, daily righting reflex latency (RRL) measurements were made before and 20, 30, and 40 min following the diazepam or vehicle injection for 28 days. Analyses of the RRLs for individual animals indicated that a significant decrease in RRL over time (indicating tolerance) occurred in only one out of nine animals receiving diazepam and in none of the vehicle animals. Medial vestibular nucleus (MVN) neurons in brain stem slices from animals receiving chronic diazepam treatment had a significantly higher average firing rate than those from vehicle controls. These results suggest that: a) long-term treatment with single 2 mg/kg daily IP injections of diazepam does not result in tolerance to diazepam's ataxic effects on the righting reflex in the majority of animals; b) this form of diazepam treatment may, nonetheless, induce a hyperactivity of brain stem MVN neurons that may be consistent with the occurrence of a withdrawal syndrome.

Community-acquired Legionnaires' disease associated with a cooling tower: evidence for longer-distance transport of Legionella pneumophila.

In the period August 10-29, 1986, 29 confirmed cases of Legionnaires' disease occurred in Sheboygan, Wisconsin; two cases were fatal. No common source of indoor exposure was identified. Water specimens were obtained from all known cooling tower units in Sheboygan; Legionella pneumophila serogroup 1 was isolated at 1 x 10(6) colony-forming units per liter from a specimen obtained August 27 at plant A. This isolate was identical to the only clinical isolate by monoclonal antibody and isoenzyme subgrouping. Of 29 persons with Legionnaires' disease, 21 lived or worked within one mile (1.6 km) of plant A; seven of the remaining eight visited within one to two miles (1.6 to 3.2 km) of plant A from three to seven days before onset of illness. Attack rates were highest for persons living within 0.5 mile (0.8 km) of plant A. These findings associate a cooling tower with community-acquired Legionnaires' disease and suggest that dissemination of Legionella may occur over longer than previously recognized distances.

Rapid dot blot quantitation of viral DNA and amplified genes in less than 1,000 human cells.

The copy number of intracellular DNA sequences can be quantitated rapidly with great sensitivity in 100 to 1,000 cells as starting material. The method applies DNA from lysed cells to a charged nylon membrane that permits successive hybridizations with probes for different genes or DNA sequences. This method was tested with eight types of human cells, including leukemic cells, and has detected Epstein-Barr virus (DNA virus) in immortalized cells, integrated HTLV-I (RNA retrovirus) in infected cells, and determined copy numbers of the amplified multiple drug-resistant gene in human cells resistant to various cytotoxic agents. It could also be used for estimating copy number of transfected DNA in human or other mammalian cells. The described method is not as sensitive as polymerase chain reaction may potentially prove, but is easily quantitated for accurate clinical diagnosis where sensitive and quantitative assays must be carried out on a limited number of cells. Examples of the method's clinical application are the staging of human neuroblastomas and the evaluation of oncogene amplification, which has prognostic value for both overall survival and relapse time in breast cancer patients.

Complexes of polyoma virus medium T antigen and cellular proteins.

Antibodies against synthetic peptides corresponding to the carboxyl-terminal six amino acids, Lys-Arg-Ser-Arg-His-Phe (KF), and an internal region, Glu-Glu-Glu-Glu-Tyr-Met-Pro-Met-Glu (EE), of polyoma virus medium T antigen were used successively to purify medium T antigen by affinity chromatography. Medium T antigen from cell extracts was first bound to anti-KF antibodies and released from the immune complex with excess KF peptide; then it was bound to anti-EE antibodies and released with excess EE peptide. Two proteins, pp60c-src and a new protein of approximately equal to 61,000 Da (61-kDa protein), were copurified because they formed complexes with medium T antigen. The 61-kDa protein-medium T antigen complex was detected in extracts from wild-type-infected and transformed cells but not from cells infected with NG59 virus, which has a mutation in the medium T gene and is transformation defective. Instead, NG59 medium T antigen formed a complex with another cellular protein of approximately equal to 72,000 Da.

Structural and functional modification of pp60c-src associated with polyoma middle tumor antigen from infected or transformed cells.

The polyoma middle tumor antigen (MTAg) associates with the src proto-oncogene product pp60c-src in infected or transformed rodent cells. The tyrosine protein kinase activity of pp60c-src, as measured by in vitro phosphorylation of pp60c-src itself or the exogenous substrate enolase, was increased 10- to 20-fold in cells transformed or infected with transformation-competent polyoma virus compared with controls. pp60c-src associated with MTAg and precipitated with polyoma antitumor serum had a novel site(s) of in vitro tyrosine phosphorylation within its amino-terminal domain. These observations suggest that association of MTAg with pp60c-src alters the accessibility of pp60c-src tyrosine residues for phosphorylation in vitro and increases pp60c-src protein kinase activity. Several transformation-defective mutants of MTAg did not cause amino-terminal tyrosine phosphorylation of pp60c-src in vitro or enhance its protein kinase activity, suggesting that these properties correlate with the transforming ability of MTAg. However, one transformation-defective MTAg mutant, dl1015, did cause amino-terminal tyrosine phosphorylation of pp60c-src in vitro and did enhance its protein kinase activity. This suggests that properties of MTAg, in addition to modifying the structure and function of pp60c-src, may be important for transformation.

Evaluation of two broth disk methods for antibiotic susceptibility testing of anaerobes.

We evaluated the aerobic thioglycolate broth disk and the vaspar overlay broth disk methods for antibiotic susceptibility testing of 144 strains of anaerobes. For penicillin, carbenicillin, chloramphenicol, and metrionidazale, both broth disk methods yielded at least 95% agreement with results obtained by the National Committee for Clinical Laboratory Standards reference agar dilution procedure. For cefoxitin and clindamycin, the agreement was ca. 90%. Overall, the aerobic thioglycolate broth disk and vaspar overlay broth disk methods yielded agreements of 93.3 and 93%, respectively, with the National Committee for Clinical Laboratory Standards method.

False-positive V/Q study secondary to a central venous catheter in the pleural space.

Pulmonary ventilation perfusion imaging in a 55-year-old woman demonstrating a false-positive study secondary to injection of the perfusion agent into the patient's central venous line.

Polyoma middle-sized T antigen can be phosphorylated on tyrosine at multiple sites in vitro.

The polyoma middle-sized T antigen (MT antigen) is associated with a protein kinase activity which phosphorylates tyrosine residues in polyoma T antigens in vitro. We have studied the sites of tyrosine phosphorylation of MT antigens phosphorylated in immunoprecipitates or in soluble form after partial purification by immunoaffinity chromatography. By analyzing the amino acid sequences of tryptic peptides of MT antigen, and by analyzing deletion mutant MT antigens, we have identified two major sites of phosphorylation in MT antigen, tyrosines 250 and 315. Additional sites were phosphorylated under some conditions. A synthetic peptide (Glu.Glu.Glu.Glu.Tyr.Met.Pro.Met.Glu), corresponding to the sequence around tyrosine 315, was phosphorylated when added to immunoprecipitates containing MT antigen.

Purification of polyoma virus medium-size tumor antigen by immunoaffinity chromatography.

We have used antibodies against the synthetic peptide Lys-Arg-Ser-Arg-His-Phe, corresponding to the six COOH-terminal amino acids of the polyoma virus medium tumor (T) antigen, to purify the medium T antigen by affinity chromatography. Release of the medium T antigen from the anti-peptide antibody was achieved under mild conditions by using a large excess of the peptide in an isotonic buffer at neutral pH containing mixed detergents. This procedure yielded a 2,500-fold purification of the medium T antigen in a single step. The protein kinase activity associated with the medium T antigen was also released and was studied in this active state in solution. Sedimentation analysis showed that the bulk of the purified medium T antigen was in a monomeric form (Mr about 42,000) not associated with protein kinase activity. A small fraction of the medium T antigen was found in a rapidly sedimenting form (Mr about 200,000) that possessed protein kinase activity.

Antibodies specific for the polyoma virus middle-size tumor antigen.

We have obtained antibodies specific for the polyoma virus middle-size tumor antigen (middle T antigen) by immunizing rabbits with a synthetic peptide, Lys-Arg-Ser-Arg-His-Phe, corresponding to the six carboxy-terminal amino acids of the middle T antigen predicted from the nucleotide sequence of polyoma DNA. The antipeptide serum precipitates the polyoma middle T antigen but not the small or large tumor antigens, and precipitation is inhibited in the presence of the peptide. Two cellular proteins, 30,000 and 26,000 daltons, are also precipitated specifically by the antipeptide serum and may have amino acid sequences related to the peptide. Two other cellular proteins, 33,000 and 25,000 daltons, are precipitated only in the presence of the peptide and may associate with it in cell extracts. Antisera directed against synthetic peptides are likely to be important in various ways, including the production of antibodies directed against particular determinants and the recognition of unknown proteins whose genes have been analyzed.

An activity phosphorylating tyrosine in polyoma T antigen immunoprecipitates.

Polyoma T antigen immunoprecipitates contain a protein kinase-like activity which preferentially phosphorylates material of 50-60,000 daltons molecular weight. Phosphorylation is not diminished in extracts of polyoma tsA mutant-infected cells shifted to the nonpermissive temperature late in infection, conditions which inactivate the large T antigen. Phosphorylation is reduced or absent in cells infected with polyoma host range nontransforming (hr-t) mutants, which have defective small and medium T antigens. The major acceptor of phosphate is not the heavy chain of immunoglobulin, but appears to be the polyoma medium T antigen. The large T antigen is also phosphorylated, but usually to a lower specific activity. In terms of acid and alkali sensitivity and electrophoretic and chromatographic mobility in one and two dimensions, the phosphorylated residue behaves identically to phosphotyrosine and differently than phosphorylated serine, threonine, lysine and histidine.

Regions of the polyoma genome coding for T antigens.

The early region of the polyoma genome encodes three T antigens. We have analyzed the organization of the coding regions for the T antigens, using the nucleotide sequence of polyoma DNA and peptides derived from purified, radio-labeled T antigens, separated by two-dimensional electrophoresis and chromatography. We compared the peptides, predicted from the nucleotide sequence of the DNA, with those derived from the purified T antigens. We also compared chemically synthesized peptides, predicted from the DNA sequence, with observed peptides. The results show that the three polyoma T antigens are encoded in overlapping regions of the viral DNA, translated, in part, in two different reading frames.

Isolation and characterization of polyoma virus genomes with deletions between the origin of viral DNA replication and the site of initiation of translation in the early region.

We introduced deletions in the early region of the polyoma virus genome near the HaeII restriction enzyme cleavage site, between the origin of viral DNA replication and the site of initiation of translation of the polyoma T antigens. We analyzed the DNA of the deletion mutants by restriction enzyme digestion. Four of the mutants had deletions beginning very close to the HaeII site and extending clockwise toward the site of initiation of translation. The deletions near the HaeII site varied in size from about 10 base pairs to about 55 base pairs. The mutants containing deletions near the HaeII site were capable of lytic growth in mouse 3T6 cells and were capable of transforming rat F2408 cells, as judged by focus formation.

Prolonged apnea of an oral surgery patient after administration of succinylcholine.

To properly diagnose the presence of a genetic pseudocholinesterase variation, the family history is important. Furthermore, the DN and pseudocholinesterase tests are most useful, but if facilities for these are not available, one should be wary of patients with abnormal liver function tests. Certainly medical history, family medical history, laboratory evaluation, and knowledge of the disease processes should avoid unnecessary complications.

Translation of polyoma virus T antigens in vitro.

Polyoma virus-specific RNA isolated from the cytoplasm of lytically infected cells can be translated in vitro to yield three T antigens, of Mrs approximately 90,000, 60,000, and 22,000. The tryptic peptide patterns of the T antigens synthesized in vitro are similar or identical to the patterns of the corresponding proteins in polyoma-infected cells. All three proteins incorporate methionine donated from initiator tRNA in vitro. Polyoma cRNA codes for a protein that is slightly larger than the 22,000 T antigen and that, by other criteria, is similar to the 22,000 T antigen. Translation of cRNA does not yield the 90,000 and 60,000 T antigens, suggesting that the generation of the mRNAs for these T antigens requires the removal of intervening sequences. The mRNA for the 90,000 T antigen is smaller than the mRNAs for the 22,000 and 60,000 proteins. All three proteins share common NH2-terminal sequences, and the 60,000 T antigen may be translated partially in a different reading frame from sequences also coding for the 90,000 T antigen. The demonstration that polyoma virus codes for three different T antigens raises the possibility that all three proteins may be involved in cell transformation.

Characterization of T antigens in polyoma-infected and transformed cells.

Polyoma-infected 3T6 cells contain a number of proteins precipitable by serum from rats carrying polyoma-induced tumors. The virus codes for three species having apparent molecular weights of 90,000, 60,000 and 22,000 daltons, as determined by polyacrylamide gel electrophoresis (90K, 60K and 22k). The 90K and 22K species produced by a large plaque and a small plaque wild-type polyoma have similar mobilities, but the 60K species produced by the large plaque wild-type. In cells infected by each of seven polyoma tsA mutants, the 90K species is unstable at the nonpermissive temperature, while the 60K and 22K species are stable. In cells infected by a mutant carrying a deletion between roughly 98 and 3 map units in the early region of the viral genome, the 22K species is present, but the 90K and 60K species are absent. Tryptic peptide analysis of the isolated 90K, 60K and 22K species shows that the three species have common N terminal regions. The 60K and 22K species contain amino acid sequences not found in the 90K species , and the 60K species has several unique, methionine-containing peptides not found in either the 22K or 90K species. Two polyoma-transformed BHK cell lines do not have detectable amounts of the 90K protein.