Inactivation of the TATA-binding protein-containing complex TFIIIB contributes to the mitotic repression of RNA polymerase III transcription, both in frogs and in humans (J. M. Gottesfeld, V. J. Wolf, T. Dang, D. J. Forbes, and P. Hartl, Science 263:81-84, 1994; R. J. White, T. M. Gottlieb, C. S. Downes, and S. P. Jackson, Mol. Cell. Biol. 15:1983-1992, 1995). Using extracts of synchronized proliferating HeLa cells, we show that TFIIIB activity remains low during the early part of G1 phase and increases only gradually as cells approach S phase. As a result, the transcription of all class III genes tested is significantly less active in early G1 than it is in S or G2 phase, both in vitro and in vivo. The increased activity of TFIIIB as cells progress through interphase appears to be due to changes in the TATA-binding protein-associated components of this complex. The data suggest that TFIIIB is an important target for the cell cycle regulation of RNA polymerase III transcription during both mitosis and interphase of actively proliferating HeLa cells.
The human immunodeficiency virus (HIV) fusion inhibitor siamycin I, a 21-residue tricyclic peptide, was identified from a Streptomyces culture by using a cell fusion assay involving cocultivation of HeLa-CD4+ cells and monkey kidney (BSC-1) cells expressing the HIV envelope gp160. Siamycin I is effective against acute HIV type 1 (HIV-1) and HIV-2 infections, with 50% effective doses ranging from 0.05 to 5.7 microM, and the concentration resulting in a 50% decrease in cell viability in the absence of viral infection is 150 microM in CEM-SS cells. Siamycin I inhibits fusion between C8166 cells and CEM-SS cells chronically infected with HIV (50% effective dose of 0.08 microM) but has no effect on Sendai virus-induced fusion or murine myoblast fusion. Siamycin I does not inhibit gp120 binding to CD4 in either gp120- or CD4-based capture enzyme-linked immunosorbent assays. Inhibition of HIV-induced fusion by this compound is reversible, suggesting that siamycin I binds noncovalently. An HIV-1 resistant variant was selected by in vitro passage of virus in the presence of increasing concentrations of siamycin I. Drug susceptibility studies on a chimeric virus containing the envelope gene from the siamycin I-resistant variant indicate that resistance maps to the gp160 gene. Envelope-deficient HIV complemented with gp160 from siamycin I-resistant HIV also displayed a resistant phenotype upon infection of HeLa-CD4-LTR-beta-gal cells. A comparison of the DNA sequences of the envelope genes from the resistant and parent viruses revealed a total of six amino acid changes. Together these results indicate that siamycin I interacts with the HIV envelope protein.
Chyluria is a rare manifestation of tuberculosis. A patient in whom it was a presenting symptom is described.
Peptide deformylase (PDF), a metallohydrolase essential for bacterial growth, is an attractive target for use in the discovery of novel antibiotics. Focused chelator-based chemical libraries were constructed and screened for inhibition of enzymatic activity, inhibition of Staphylococcus aureus growth, and cytotoxicity. Positive compounds were selected based on the results of all three assays. VRC3375 [N-hydroxy-3-R-butyl-3-(2-S-(tert-butoxycarbonyl)-pyrrolidin-1-ylcarbonyl)propionamide] was identified as having the most favorable properties through an integrated combinatorial and medicinal chemistry effort. This compound is a potent PDF inhibitor with a Ki of 0.24 nM against the Escherichia coli Ni2+ enzyme, possesses activity against gram-positive and gram-negative bacterial pathogens, and has a low cytotoxicity. Mechanistic experiments demonstrate that the compound inhibits bacterial growth through PDF inhibition. Pharmacokinetic studies of this drug in mice indicate that VRC3375 is orally bioavailable and rapidly distributed among various tissues. VRC3375 has in vivo activity against S. aureus in a murine septicemia model, with 50% effective doses of 32, 17, and 21 mg/kg of body weight after dosing by intravenous (i.v.), subcutaneous (s.c.), and oral (p.o.) administration, respectively. In murine single-dose toxicity studies, no adverse effects were observed after dosing with more than 400 mg of VRC3375 per kg by i.v., p.o., or s.c. administration. The in vivo efficacy and low toxicity of VRC3375 suggest the potential for developing this class of compounds to be used in future antibacterial drugs.
Eikenella corrodens is a facultative anaerobic bacillus which is part of the normal flora of the oral cavity and has an unusual antibiotic sensitivity for an anaerobe. The case history is presented of a young man with chest wall infiltration by Eikenella corrodens.
BACKGROUND: The presence of Haemophilus influenzae in the oropharynx is correlated with the subsequent development of chest infection. The importance of colonisation of the trachea by bacteria at the time of surgery is uncertain. This study investigated the tracheal flora at the time of intubation in 24 patients undergoing elective upper abdominal surgery. METHODS: The bacterial flora of the trachea was sampled in all 24 patients immediately after intubation and immediately before extubation. Patients were assessed postoperatively for the development of chest infection. RESULTS: Bacteria, including H influenzae in five cases, were isolated from the post-intubation brushings of the trachea of 15 patients. The pre-extubation brushings from only four patients yielded growth. Three of five patients developing a chest infection were colonised by H influenzae according to the postintubation brush, compared with two of 19 without chest infections. Before extubation two of five developing chest infections had H influenzae in the trachea but none of 19 without infection. All but one of the patients from whom H influenzae was isolated were smokers. CONCLUSIONS: These results suggest that the increased risk of postoperative chest infection in cigarette smokers may be due in part to colonisation of the trachea by H influenzae at the time of operation.
The mitotic state is associated with a generalized repression of transcription. We show that mitotic repression of RNA polymerase III transcription can be reproduced by using extracts of synchronized HeLa cells. We have used this system to investigate the molecular basis of transcriptional repression during mitosis. We find a specific decrease in the activity of the TATA-binding-protein (TBP)-containing complex TFIIIB. TBP itself is hyperphosphorylated at mitosis, but this does not appear to account for the loss of TFIIIB activity. Instead, one or more TBP-associated components appear to be regulated. The data suggest that changes in the activity of TBP-associated components contribute to the coordinate repression of gene expression that occurs at mitosis.
The oropharyngeal flora was determined before and after operation in 127 patients undergoing upper abdominal surgery. Swabs of the oropharynx were obtained on the day before operation and on the first, third, and fifth postoperative days. Isolation of Haemophilus influenzae, Streptococcus pneumoniae, and coliforms was noted. In the 108 patients with the full series of throat swabs the incidence of oropharyngeal colonisation by H influenzae was 16% and was unchanged after operation. S pneumoniae was present in only 5.6 (six patients) before operation and the incidence fell to 1.9% (two patients). There was a transient rise in coliform colonisation postoperatively. Twenty four patients developed a chest infection. In eight a bacterial cause was established, in six H influenzae and in two S pneumoniae. There was a significant relation between the carriage of H influenzae before operation and development of a chest infection. H influenzae was also found more often in cigarette smokers. The presence of S pneumoniae or coliform organisms before surgery was not related to the development of infection. The high incidence of postoperative chest infection in cigarette smokers appears to be due in part to preoperative colonisation of the oropharynx by H influenzae.
The effect of upper abdominal surgery under general anaesthesia on the cough threshold was studied in 26 patients, on the basis of the concentrations of capsaicin and citric acid causing cough. Cough threshold was determined after administering doubling doses of nebulised aerosols of capsaicin and citric acid before operation and on the first and fourth postoperative days. There was an increase in cough threshold (decrease in cough sensitivity) in response to both inhaled irritants on the first postoperative day from the preoperative day and a return towards preoperative values by the fourth day after surgery. The increase in cough threshold on the first postoperative day correlated with the time since opiate administration (r = 0.7 for capsaicin, 0.52 for citric acid). These results show that the threshold concentration of chemical irritants causing cough is increased on the first postoperative day after upper abdominal surgery.
The indications for the prescription of oxygen concentrators have been assessed in 82 patients against the Department of Health guidelines, except that only one set of blood gas and spirometry measurements was required if the patient's condition was stable. Of the 49 patients recommended for this treatment by a chest physician, 41 (82%) fulfilled the requirements, compared with only 11 of 33 patients where the concentrator was recommended by a general practitioner or non-specialist physician. Eleven patients died within one month of prescription. The results show that many patients are being prescribed oxygen concentrators without assessment and underline the importance of full assessment and attention to prognosis before prescription.
The impact of the installation of an oxygen concentrator on the lifestyle of 30 patients in two health districts has been investigated using a questionnaire. Marked improvements in general well-being (83% of respondents), breathing (82%), mobility (62%) and sleep pattern (52%) were reported. The long term nature of the aims of treatment were understood by 83% of the respondents and the mean period of time the patients used the concentrator was satisfactory. However, 34% of respondents had a concentrator with only one outlet and 70% had the concentrator situated in a commonly used room with the possibility of problems with noise. Thirty one percent of the respondents were still smoking. The recommendations given to patients for the sitting of the concentrator and the number of outlets should be improved. However, the oxygen concentrator was found to be generally well tolerated and this refutes criticism that patients may find it restricting.
The antitumor antibiotics ravidomycin and desacetylravidomycin were studied by the biochemical lambda prophage induction assay. In this assay, induction of the enzyme beta-galactosidase is measured as a specific indication of the ability of an agent to directly or indirectly damage DNA. Induction was observed only when these two antibiotics were irradiated with light in the presence of the indicator organism. Drug treated with light followed by incubation with the indicator organism in the dark did not cause induction. Light in both the near UV and visible wave length ranges activated these antibiotics; near UV and visible blue wavelengths were most effective, while 597-nm light was totally ineffective. The amount of induction caused by these drugs varied directly with the dosage of light provided. Bacterial growth inhibition, as well as cytotoxicity for a human colon carcinoma cell line, was also dramatically enhanced by light. These data suggest that ravidomycin and desacetylravidomycin are potent photosensitizing, DNA-damaging agents.
Fifteen related dogs were studied for susceptibility to malignant hyperthermia using halothane challenge and caffeine contracture tests. These dogs had hypertrophied muscles, were of a nervous temperament and had rectal temperatures at the upper limit of the normal range. Clinical pathology findings were mild elevations of serum aspartate transaminase and mean corpuscular hemoglobin. In vitro caffeine contracture tests were performed on muscle biopsies from five of these dogs. The concentration of caffeine required to increase resting tension by 1 g in biopsy specimens of these dogs was significantly lower than that required for control dogs: 7.6 ± 1.38 (x̄ ± SEM) versus 15.5 ± 2.52 mM (P < 0.025), and in the presence of 1% halothane, 3.6 ± 1.44 versus 10.6 ± 2.19 mM (P < 0.05). Internal nuclei, fiber caliber variation and fiber hypertrophy were found in histological studies of muscle biopsies. Two other dogs possibly died of a canine stress syndrome analagous to the porcine stress syndrome which occurs in malignant hyperthermia susceptible swine. Eight others of this family were anesthetized with halothane or methoxyflurane. Methoxyflurane did not trigger the syndrome. The first exposure to halothane caused death from malignant hyperthermia in two dogs and a third died on the second exposure to halothane. Postmortem findings were nonspecific. The other three dogs exposed to halothane recovered uneventfully. Inheritance of the defect conforms to a multifactorial pattern, with gradations of susceptibility.
In order to assess the incidence of Legionnaires' disease in a district general hospital, 90 consecutive patients with pneumonia seen over a 3-year period were investigated. Only 2 patients were found to have Legionnaires' disease, indicating that it is not a frequent cause of pneumonia in the Bristol area.
The chest radiographs of 60 adult patients with serologically proven mycoplasma pneumonia were reviewed. Confluent or patchy consolidation was most commonly seen, and involved one lobe only in 40% of patients. Widespread nodular opacities were seen much less frequently (7%). Pleural fluid was rare. Complete resolution was almost invariable, 40% of radiographs having cleared by four weeks and 96% by eight weeks.
The effects of lymphography on ventilatory function and gas transfer factor were studied in nine patients. Serial measurements made up to one month showed no change in the forced expiratory volume in one second or vital capacity. A small but reversible fall in transfer factor was found. The greatest reduction was at 24 or 48 hours. It was concluded that patients with normal lungs are unlikely to encounter difficulties but that patients with severe lung disease require careful assessment before lymphography.
Deoxyribonucleic acid (DNA)-dependent ribonucleic acid (RNA) polymerase (EC 220.127.116.11) isolated from a rifampin-sensitive strain of Mycobacterium smegmatis was 90% inhibited by 1 μg of rifampin per ml; enzyme from a rifampin-resistant mutant was not affected by this concentration of antibiotic. Inhibition of phenylalanine-1-14C incorporation by rifampin in growing cultures was complete about 6 min after addition of antibiotic. Under the same conditions, uracil-2-14c incorporated was blocked after 1.5 to 2 min. Rifampin kills M. smegmatis very slowly. When rifampin-inhibited cultures were transferred to a rifampin-free medium, there was a partial resumption of uracil-2-14C incorporation, even in the presence of chloramphenicol. We conclude that a primary event in the inhibition of M. smegmatis by rifampin is the block of DNA-dependent RNA polymerase.
The response to a standard water load (20 ml/kg body weight) was studied in 20 patients with chronic obstructive airways disease and in 13 healthy subjects. The percentage of the water load excreted in four hours was significantly lower in the patients (mean 51%) than in the controls (mean 106%). The maximum urine flow, osmolar clearance, free water clearance, and creatinine clearance were also significantly reduced in the patients. There was a significant inverse correlation between the percentage of load passed and the arterial PCO2 (r = -0·798). Among the several possible causes of the reduced excretion of water which are discussed is a direct effect of hypercapnia.
After the oral administration of 0·5 mg of digoxin in tablet form to fasting subjects peak plasma levels were reached in 30 to 60 minutes. Levels then fell to reach a plateau at six to eight hours. When the same dose was given after food the peak plasma concentrations were significantly lower, but the concentrations reached in samples obtained from two to eight hours after the dose did not differ appreciably from corresponding samples obtained in the fasting experiments.
In a four-week cross-over study of 21 patients on maintenance therapy, digoxin taken regularly in the fasting state produced plasma concentrations similar to those obtained when the drug was taken after meals.
The rapid appearance of digoxin in the blood suggests that the oral route of administration is adequate for most patients who require rapid digitalization, and the timing of maintenance dosage in relation to meals is unimportant.
Plasma digoxin concentrations were measured by radioimmunoassay in 116 patients with atrial fibrillation on long-term oral treatment with the drug, and in 23 patients with digoxin toxicity. The mean concentrations were 1·4 ng./ml. and 3·1 ng./ml., respectively. Though an overlap occurred between the therapeutic and toxic ranges, toxicity is unlikely to occur below a level of 2 ng./ml. Plasma concentration showed a poor correlation with resting heart rate during atrial fibrillation. In patients with good renal function, however, a significant correlation was found between oral dose and plasma concentration. No evidence was obtained for increased sensitivity to therapeutic concentrations of the drug in elderly subjects, but the doses required to achieve these concentrations tended to be less than in younger patients.
A study of the effect of additional oral potassium on the low body potassium of seven patients with severe valvular heart disease showed that the potassium supplements were retained. After one month's treatment the exchangeable potassium was significantly increased, but the predicted value was not reached in any of the patients. There was no significant change in plasma potassium. These results suggest that it is of benefit to increase the dose of oral potassium for at least one month preoperatively in patients undergoing cardiac surgery.
Exchangeable sodium and potassium, total body water, and sulphate space were measured in 42 patients with severe valvular heart disease who were free of oedema. Compared with normal subjects of the same height, no increase in exchangeable sodium was found but a mean potassium depletion of 27% was shown. This depletion was not related to diuretic therapy, and no relationship between the degree of depletion and postoperative arrhythmias was found. It is concluded that the major cause of the low exchangeable potassium is the reduction in cell mass that occurs in chronic heart disease, and that there is no significant fall in the intracellular potassium concentration.