Fluorescent treponemal antibody absorption (FTA-ABS) tests using blood samples collected on filter paper. One hundred and eleven (111) blood specimens were collected from patients with known or suspected syphilis by venipuncture and on a small piece of No. 3 grade filter paper. Both specimens from each patient were tested by bluorescent treponemal antibody absorption (FTA-ABS) test. Of the 62 patients whose venipuncture sera were FTA-ABS positive, filter paper blood samples were also positive in 56 (90%) and negative in 6 (10%). Complete agreement was obtained on paired specimens from 49 patients who were FTA-ABS negative. These preliminary results suggest that finger stick blood samples, collected on filter paper, could be used for FTA-ABS testing of remote rural populations--such as in areas where yaws is endemic.


Application of the enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of human African trypanosomiasis (sleeping sickness). An enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of human African trypanosomiasis (sleeping sickness) is described. A crude extract of a Trypanosoma brucei suspension which was purified from all blood components was used as antigen. In rabbits experimentally infected with T. brucei or T. rhodesiense both homologous (anti-T. brucei) and heterologous (anti-T. rhodesiense) Trypanosoma antibodies could be detected with ELISA using T. brucei as antigen. The sensitivity of ELISA was comparable with that of the immunofluorescence (IF) technique. Sera of patients with sleeping sickness were examined with ELISA and IF. It proved possible to discriminate between groups of individuals with and without trypanosomiasis. Cross reactions were only observed with serum from a patient in which antibodies to Leishmania were detected. No cross reactions were observed in sera from patients with malaria, toxoplasmosis, schistosomiasis, or echinococcosis. ELISA represents a good alternative to IF in the serology of African trypanosomiasis, and may be particularly suitable for mass screening purposes.


The effects of Lampit (Bayer 2502) on the interaction of Trypanosoma cruzi with vertebrate cells in vitro. The effects of 3-methyl-4-(5'-nitrofurfurylidene-amino)-tetrahydro-4H-1,4-thiazine-1,1-dioxide [Lampit, Bayer 2502] on the intracellular cycle of Trypanosoma cruzi were studied with an in vitro steady-state culture system that permitted the continuous analysis of individual host cell-parasite interactions. Lampit concentrations of 10(-4) and 10(-5) M significantly affected the ability of trypomastigotes to penetrate vertebrate cells. Lampit concentrations above 10(-4) M were toxic to the host cell population and Lampit concentrations below 10(-5) M did not affect the ability of trypomastigotes to penetrate vertebrate cells. Lampit concentrations of 10(-5) M or greater completely inhibited the intracellular cycle of T. cruzi whereas Lampit concentrations of 10(-7) M or less had no effect on the intracellular cycle. Continuous perfusion with 10(-6) M Lampit resulted in a linear increase in intracellular parasite reproduction time. A stable strain of T. cruzi was produced in vitro that was resistant to a 100-fold greater concentration of Lampit that the parent strain.


Severe bronchial asthma: factors influencing intensive care management and outcome. Over a seven year period 2933 patients were admitted with asthma. Respiratory failure occurred on 106 occasions. A total of 26 patients died including 11 of the 106 who developed respiratory failure. The intra-hospital management of asthma is outlined. The experience is analysed to indicate those patients particularly at risk. These include those who develop pneumothorax, have associated chest infections or have recently ceased steroid therapy.


Measurement errors in pneumotachography due to pressure transducer design. When pneumotachography is used during intermittent positive pressure ventilation (IPPV), the design of some differential pressure transducers can cause measurement errors. These errors may be avoided by using differential pressure transducers which have small, equal chambers free from temperature sensitive electrical elements. Few of the transducers which are sensitive enough for clinical use fulfil these requirements.


Historical note: Drumine--a new Australian local anaesthetic. An article in the Australiasian Medical Gazette of October, 1886 indicates the method of extraction, experimentation and therapeutic application of an active principle, prepared from Euphorbia Drummondii. Further correspondence is noted, refining the method of extraction, reporting cases, answering criticisms, and announcing eventually, drumine's commercial preparation. Despite enthusiastic support, the drug soon disappears from the therapeutic scene.


Airway pressure changes observable with constant-flow inflation and an end-inspiratory pause. Constant-flow inflation and a prolonged end-inspiratory pause were studied, observing the airway pressures in model lungs. The technique demonstrated the changes associated with compliance, resistance and gas redistribution under varying conditions. The studies suggest that when intratracheal pressures indicate a large element of gas redistribution, constant flow inflation and end-inspiratory pause produce pendelluft, and other pressure patterns are preferable for I.P.P.V.


Sixty day trial of a new inhalant dosage form of metaproterenol. A 5% solution of metaproterenol, 10 whiffs (1.5 mg) q.i.d., was administered from a hand bulb nebulizer to 28 asthmatics for 60 days. Spirometry in double-blind, crossover tests against an 0.5% isoproterenol solution on days 0-1 and 59-60 and global evaluations of response showed that metaproterenol was more effective and longer acting. No tolerance to metaproterenol developed. Side-effects were negligible.


Terbutaline: an effective bronchodilator by inhalation. Ninety-nine patients with severe bronchial asthma were tested with two inhalations of Terbutaline. The peak expiratory flow rate of each was determined by means of the Wright peak flow meter, and compared with a baseline taken previously and recorded. The final results were compared with those obtained in the same patients who alternately had received two inhalations of Salbutamol. The average improvement with Terbutaline was 20% per patient as compared with 16% with Salbutamol, indicating no statistically significant therapeutic difference between the two drugs.


Double-blind, controlled study of clemastine fumarate, chlorpheniramine and placebo in patients with seasonal allergic rhinitis. A whole body plethysmograph (body box) equipped with a flow meter (see Figure 1) was used for objective quantification of the effects of single doses of clemastine fumarate 2.68 mg, chlorpheniramine 4 mg and placebo in a double-blind study of 48 patients with seasonal allergic rhinitis. This technique offers an objective means of assessing drug effects on nasal congestion and obstruction. Before the development of whole body plethysmography, only subjective assessments of antihistamines' effects on nasal blockage or congestion were available. These subjective reports usually noted that nasal blockage or congestion was refractory to antihistamines or minimally relieved by them. However, in this study, nasal and oral airway resistances, each measured by whole body plethysmography, were lowered by clemastine fumarate and chlorpheniramine. These results were corroborated by the patients' and physician's assessments of changes in symptom severity and the physician's evaluation of intranasal photographs taken for each patient. Oral airway resistance of patients treated with clemastine fumarate was improved to a significantly greater extent than in patients receiving placebo. At two hours post-drug, patients receiving clemastine fumarate usually showed a greater response in most assessments than those receiving chlorpheniramine, and the trend of most comparisons was clearly in favor of clemastine fumarate. Patients in all three treatment groups experienced drowsiness but both incidence and severity were lower with clemastine fumarate.


Double-blind, controlled study of clemastine fumarate, chlorpheniramine and placebo in the symptomatic treatment of seasonal allergic rhinitis in desensitized and nondesensitized patients. In double-blind trials clemastine fumarate 2.68 mg. chlorpheniramine 4 mg and placebo were randomly assigned to two groups of patients with seasonal allergic rhinitis. Thirty-nine desensitized patients were given one of the three test drugs in a parallel design; 67 nondesensitized patients each received two of the three drugs in a crossover design. Assessment of drug activity in each study was by whole body plethysmography and intranasal color photography as well as by subjective methods. Objective measurements showed clemastine fumarate was significantly superior to placebo and often better than chlorpheniramine in decreasing true nasal resistance and relieving nasal congestion. High placebo responses characterized the subjective evaluations, although the active drugs were clearly better. Responses varied somewhat between desensitized and nondesensitized patients. The number of reports of sedative effect, high in all groups, seemed to be more closely related to these antihistamine conditioned patients than to activity of the drugs themselves, based on previous reports of low sedation with clemastine fumarate. The techniques described proved very useful in distinguishing relative activity of antihistamines. Clemastine fumarate, the new antihistamine studied, appears to offer certain advantages over the older drug, chlorpheniramine.


A double-blind study of food extract injection therapy: a preliminary report. A double-blind, crossover study of food extract injection therapy is reported. There was a significantly better response to food injection therapy than to placebo. In many cases the response of lifelong, severe, intractable syndromes was rapid and dramatic, often beginning in three to four days, and symptoms often returned within three to four days after beginning a course of placebo therapy. In clinical practice this should prove to be a useful practical tool for demonstrating the etiologic role of foods and providing substantial relief in several serious syndromes for which no other satisfactory method of etiologic diagnosis or effective therapy is available.


Treatment of steroid-dependent asthma with triamcinolone acetonide aerosol. Corticosteroids, originally developed as topical agents, have proved effective in aerosol form in the treatment of asthma. Of 33 chronic bronchial asthmatic patients dependent on oral corticosteroids, 16 were randomly selected for treatment with triamcinolone acetonide aerosol and 17 for treatment with triamcinolone acetonide aerosol and 17 for treatment with placebo to determine the effectiveness of the active drug and the feasibility of eliminating or reducing oral steroids. Aerosol dosages of 800 g daily continued for 12 weeks. The patients improved about 30, 50 and 60% in mean FVC, FEV1, and FEF25 75, respectively. Their mean oral steroid dose dropped 77%, with 13 of the 16 discontinuing oral steroids entirely, and their asthma symptoms decreased about 30%. Placebo patients showed minimal or no improvement in these areas. The clinical results indicated that 14 of 16 active aerosol and 4 of 17 placebo aerosol patients improved over baseline. No side effects or fungal infections appeared and only two patients experienced oral-steroid withdrawal symptoms.


Improved methods of producing precipitating aspergillus antigens. Improved methods for producing Aspergillus fumigatus antigens capable of giving sensitive and reproducible results by counterimmunoelectrophoresis (CIEP) are described. Among the factors investigated were the source of casein hydrolysate used for N source, method of incubating the CIEP slide following a run, effect of freeze pressing and lyophilization, age of culture and ratio of C to N source in the medium.


Epidemiologic aspects of allergic aspergillosis. The incidence of allergic bronchopulmonary aspergillosis (ABA) seems much less in the United States than in Great Britain. The suggestion that there are differences in the atmospheric burden of A. fumigatus prompted the authors to evaluate skin reactivity to A. fumigatus in their clinic population. Immediate skin reactivity was found significantly less than to Alternaria, a common Midwest mold. Because of the association of A. fumigatus with decaying vegetation, compost piles in the St. Louis area were cultured. Hot or working piles were extremely rich in A. fumigatus while cold or finished piles were devoid of this organism. A group of nonatopic organic farmers showed no serum precipitating antibody to A. fumigatus. It is suggested that active asthmatics with immediate skin reactivity to A. fumigatus avoid exposure to working compost piles.


Comparisons of the complement-fixation and indirect fluorescent antibody reactions in the detection of bovine babesiasis. Complement-fixation (CF) and indirect fluorescent antibody (IFA) antigens were prepared from Babesia bigemina isolates obtained in Texas. These serologic procedures were evaluated on 130 serum samples sequentially collected from 5 B bigemina-infected mature cattle, beginning on the day of exposure and continuing for 175 day thereafter. Both tests were effective in detecting specific antibodies for the first 84 days of infection, with 57 of 60 (95%) serums tested being positive on the CF test and 57 of 57 (100%) tests being positive to the IFA test. During the interval from 98 to 175 days, 24 of 60 (40%) of the serums tested were positive with the CF test, and 53 of 56 (95%) were positive with the IFA test. During the first 84 days, a similar linear regression occurred in both CF and IFA serum titers, but after 98 days the IFA regression flattened out, whereas the CF titers decreased below the sensitivity threshold in 60% of the serums tested.


The upper airways. II. The larynx and trachea. The laryngotracheal airway is the bottleneck of the respiratory system. Whereas disease in the nose or in the bronchial airways will affect respiratory airflow in a limited way, laryngotracheal disease must always be considered potentially life threatening. The nasal airway is the normal access to the lungs. As such, it serves vital functions in modifying the condition of ambient air before its access to the lungs. Impairment of the efficacy of nasal function in that regard will inevitably cast an unaccustomed burden on the lower airways and may possibly play a role in the long-term development of small airway disease in the lungs. When the nose is bypassed, acutely, as in orotracheal intubation, or chronically, as in tracheostomy or the laryngectomee, the pulmonary airways inevitably suffer. Attempts to substitute for the nose by humidification of inspired air are essential, but our current methods are not free of hazard and are of questionable efficacy. A better understanding of upper airway function and the injury resulting from malfunction or conducting ambient air directly to the trachea is now within our grasp but is not yet fully realized.


Pulmonary aerosol deposition in chronic bronchitis: intermittent positive pressure breathing versus quiet breathing. Quiet breathing by mouth of a 3-mum aerosol was compared with intermittent positive pressure delivery of the same aerosol to the lung in a group of bronchitics. The pattern of lung deposition and the total dose delivered to the lung were measured. The differences between quiet breathing and intermittent positive pressure breathing were found not to be significant (P greater than 0.5). In addition, during similar or greater total ventilation, intermittent positive pressure breathing delivered a mean of 32% less aerosol to the lung than did quiet breathing. These findings suggest that intermittent positive pressure breathing as currently used for aerosol delivery cannot be expected to enhance peripheral deposition of inhaled aerosols in patients with airway obstruction.


Defense mechanisms of the respiratory membrane. The success or failure of pulmonary defense mechanisms largely determines the appearance of clinical lung disease. The lung is protected by interlucking systems of nonspecific and specific defenses. Inhaled substrances can be isolated by mechanical barriers or can be physically removed from the lung either by transport up the bronchial mucociliary escalator or by transport through interstitial and lymphatic channels leading to lymph nodes. Substances can be locally detoxified within the lung by interaction with secretory proteins, such as antibodies, or by neutralization and dissolution within phagocytic cells. The pulmonary alveolar macrophage is the central figure in the protection of the respiratory membrane, operating in all 3 of the nonspecific modes of defense and augmented by specific immunologic mechanisms as well. Alterations in macrophage function and physiology may be crucial in determining the effectiveness of pulmonary defense. Recent advances in the cell biology of the alveolar macrophage have led to a greater understanding of its complex funcition. The multiple origins of macrophages from local and circulating cell pools and the variability in their fate and lifespan reflect the multi-faceted role of this cell type. The importance of the interactions between macrophages, orther lung cells, and other defense mechanisms has become increasingly clear. As well as functioning as resident defender of the alveolus, the macrophage is an important effector of the pulmonary immune response and plays a key role in the pathogenesis of a wide variety of inflammatory, destructive, and fibrotic lung diseases. Humoral and cell-mediated immune responses amplify and direct lung defenses against infection and may also participate in protection against other agents. Immunoglobulin A and G, microbial neutralizing and opsonizing anti-bodies, and macrophage-stimulating T lymphocytes are the major immunospecific forms of lung defense. Infectious agents, cigarette smoke, air pollutants, industrial dusts, and a spectrum of coexistent disease states may impair pulmonary defense mechanisms and increase susceptibility to asute and chronic respiratory diseases. A thorough understanding of the ways in which the lung protects itself against the daily assault of infectious, toxic, and immunogenic materials should lead to a beter understanding of pathogenesis and consequences of lung disease and to better clinical care of the patient with respiratory disease.


Alterations in host responsiveness in patients with prostatic cancer following cryosurgery or transurethral resection. Anesthesia, stress, trauma or the operation per se have been reported to result in alterations of host resistance in a wide range of diseases. The effect of such changes on the thymolymphatic system of patients with prostatic cancer is not known. While evaluating in vitro parameters of cellular immunologic responsiveness in patients with prostatic cancer, we have observed a depression two to seven days following cryosurgery or transurethral resection (TUR) of the proliferation of phytohemagglutinin (PHA)-stimulated peripheral blood lymphocytes (PBL). Contrary to the reduced proliferation of PBL cultured in autologous and homologous serum from patients receiving TUR, patients receiving cryosurgery, while also showing reduction in autologous serum, showed increased responsiveness when cultured in homologous serum. Although transient, depression of lymphocyte proliferation, particularly if involving tumor-cloned T-cells, may provide reduced surveillance to potential metastatic tumor cells leading to an alteration of tumor-host homeostasis. The potential of reduced surveillance, at least in the case of TUR, appears to be supported by observations that patients dying from prostatic cancer at our institution had an antecedent TUR. Identifying those patients with changes in responsiveness before surgery, as well as those prone to develop or undergo further reductions in responsiveness after surgery, would appear to be relevant in the management of patient with prostatic as well as other malignancies. Pre- and/or postoperative immunotherapy in such patients may be indicated.


Renal artery occlusion in transplant recipients. Five cases of renal artery occlusion occurring in kidney transplant recipients are reported--an incidence of 1.4%. Two patients recovered moderate renal function after thrombectomy in spite of the estimated occlusion time of four hours and 12 hours, respectively, under normothermic conditions. Three cases of occlusion were associated with pre-existing renal artery stenosis. This experience reinforced the optimistic and aggressive approach to treatment of renal artery thrombosis. Renal artery occlusion may simulate acute rejection in the transplant recipient.


[Bifid sternum. a case report (author's transl)]. A newborn with bifid sternum and early surgical management is reported.


Failure of specific immunotherapy in fulminant type B hepatitis. Investigators at 30 centers evaluated an intravenous hepatitis B immune globulin preparation in the therapy of fulminant type B hepatitis. Patients with serum positive for hepatitis B surface antigen were treated at stage II to stage IV of hepatic encephalopathy. A central computer program randomized cases for treatment with hyperimmune globulin or albumin placebo. During the first 6 months, the dose of hepatitis B immune globulin was 1.32 g of immunoglobulin G protein; during the last 7 months, it was 5.28 g. Neither dose eliminated antigenemia. In the placebo group, death occurred in four of eight cases randomized at stage II, five of eight at stage III, and 10 of 12 at stage IV. In the group treated with hyperimmune globulin, death occurred in three of five patients randomized at stage II, seven of 12 at stage III, and six of eight at stage IV. The study, therefore, showed no benefit of treatment with exogenous antibody.


Methyltestosterone therapy in hereditary angioedema. In a double-blind study of four patients with hereditary angioedema, the efficacy of methyltestosterone (taken daily in 10-mg linguet form) in preventing attacks was shown. There were 19 episodes during 11.8 months of placebo administration, compared with only four attacks during the 46 months of cumulative methyltestosterone treatment (P less than 0.001). The mean serum C4 protein level was twice as high in all patients when they were taking the drug (176 +/- 36 mug/ml) as compared with the placebo (84 +/- 21 mug/ml), and rose to normal range in three of four patients.


Flucytosine.

Flucytosine. Flucytosine is a systemic antifungal drug that is readily absorbed from the gastrointestinal tract. The most clearly documented therapeutic effect has been in cryptococcosis, candidiasis, and chromomycosis. An important limitation of the use of flucytosine in all three diseases has been drug resistance arising during therapy. The addition of low-dose, intravenous amphotericin B to flucytosine therapy of cryptococcosis has appeared to decrease the frequency of secondary flucytosine resistance. In addition, the two drugs have an additive or slightly synergistic effect against flucytosine susceptible isolates of Cryptococcus and Candida. The combination is probably the treatment of choice in cryptococcal meningitis and offers promise in the therapy of systemic candidiasis and nonmeningeal cryptococcosis.


Cryocoagulation of retinal vessels: an experimental model of intravascular coagulation. Various amounts of cryothermy were applied to the posterior pole of rabbit eyes to determine changes in retinal tissue and vasculature. Four consecutive freezing and refreezing applications of -60 degrees was the most effective technique for achieving central retinal vessel occlusion. Electron microscopic observations of sequentially sectioned occlusion. Electron microscopic observations of sequentially sectioned occluded vessels showed that the vessels were occluded by a platelet plug distally and packed erythrocytes proximally. Fibrin formation was not found in the occluded retinal vessels but was seen within platelet plugs in choroidal vessels. Because cryocoagulation simulates the normal physiologic means of coagulation more closely than other vessel occluding techniques, cryocoagulation of the primary retinal vessels is suggested as an experimental model for intravascular coagulation.


A reappraisal of the use of antiseptics in surgical practice. The use of antiseptics was reappraised because of the increasing problem of antibiotic-resistant bacteria. A formaldehyde (noxythiolin) and a halogen (povidone-iodine) were investigated, these being the most appropriate antiseptic groups. Povidone-iodine solution significantly reduced the mortality of mice (P less than 0.01) and rats (P less than 0.01) with peritonitis. Noxythiolin (1% and 0.5%) did not. Antiseptic irrigation of the rat colon before and after anastomosis resulted in significantly fewer 'poor' anastomoses (P less than 0.05) without inhibiting healing. Noxythiolin 2.5% and 1% significantly reduced peritoneal adhesion formation in rats, but a newly formulated povidone-iodine solution with increased polyvinylpyrrolidone content was superior to noxythiolin 1% and 0.5%. Povidone-iodine neither inhibited rat abdominal wound healing nor induced bacterial resistance. In 3 subsequent controlled clinical trials a dry-powder povidone-iodine formulation halved wound infection after grid-iron appendicectomy (P less than 0.025), reduced infection in elective 'clean' surgery, and significantly reduced infection after a wide variety of 'potentially contaminated' abdominal procedures (P less than 0.01). A return to the principles of Lister is advocated.


[Results of an experimental study of the antitumor activity of 1-asparaginase from E. coli and Erw. carotovora]. The antitumour activity of the preparations of L-asparaginase from E. coli and Erw. carotovora with respect to lymphadenosis L-5178 and Yorker's carcinosarcoma (ascitic cariants) has been established. No difference in antitumour efficacy of the preparation of L-asparaginase obtained from E. coli and Erw. carotovora was noted.


Superficial mycoses: treatment with a new, broad-spectrum antifungal agent: 1% clotrimazole solution. A 1% clotrimazole solution was used as an antifungal for Tinea versicolor, Cutaneous Candidiasis and Dermatophytosis. All studies were double-blind and controlled, active vs vehicle. The final evaluation was made at a two-week follow-up after the treatment was stopped, which for most of the clinical conditions studied, was 28 days. The 1% clotrimazole solution was found to be effective with no local irritation noted.


Bullous disease of childhood: report of a case demonstrating antibasal cell antibody. This report describes a child with protracted bullous disease responsive only to high doses of steroids, who on immunofluorescent testing, consistently displays antibasal cell antibody. The child had been treated with penicillin a week prior to the development of the bullous disease. The etiologic possibility of this drug history in the evolution of the bullous disease is raised, with particular respect to recent reports of similar circulating antibody in drug reactions.


Rubella virus and rheumatoid arthritis.

Rubella virus and rheumatoid arthritis. A collection of synovial fibroblasts from 19 patients with rheumatoid arthritis (RA) and 12 patients with osteoarthrosis or other non-RA disease has been examined for rubella virus antigens by immunofluorescence and radioimmunoassay with negative results. Eluates of synovial membrane prepared under conditions likely to dissociate antigen-antibody complexes have shown no rubella antibody. A serological survey of RA patients and those with other forms of arthritis has shown no differences in the frequency or levels of rubella haemagglutination-inhibiting antibody. These results provide little support for various hypotheses that a persistent infection with rubella virus underlies or initiates the rheumatoid process.


Bullous tophi in gout.

Bullous tophi in gout. Two gouty patients developed bullae containing massive numbers of monosodium urate crystals. Both patients had had treatment with systemic corticosteroids. A burn precipitated one bulla, showing that local tissue injury can be a factor in tophus localization.


Bullet emboli in the pulmonary and systemic arteries. Bullet embolization into the systemic and the pulmonary circulation is a rare complication of penetrating wounds. From 1966 to 1975 10 patients with bullet embolus (5 in the systemic and 5 in the pulmonary arteries) were treated at Grady Memorial Hospital. The embolization occurred shortly after the initial injury in all cases except for two in which it occurred within two weeks. The patients with bullet embolus were either asymptomatic or some of those with systemic arterial embolization had symptoms and signs of acute arterial occlusion and some of the patients with pulmonary embolization had symptoms and signs of pulmonary embolus. The diagnosis was suspected in all cases because there was no wound of exit and because of the plain regional xray studies, the missile was not present in the expected area. The diagnosis was strengthened when, on screening xray studies of the rest of the body, the missile was found in a remote area and it was confirmed by arteriography. Embolectomy was performed in all cases of both groups except one with pulmonary embolus, and all patients did well and have no residual disability related to the embolus. This study suggests that bullet embolization to the systemic or pulmonary circulation occasionally occurs following bullet wound injury. The diagnosis should be strongly suspected when there is no exit wound and when roentgenographically the missile is not found in the suspected area. In such cases, screening xray pictures of the rest of the body should be obtained, and, if the bullet is found in a remote area, arteriography should be performed to confirm the diagnosis. Embolectomy should be done as soon as feasible and it usually affords very good results.


Lobular carcinoma of the breast: an overview. The biologic nature, diagnostic features and therapeutic management of patients with lobular carcinoma in its in situ and invasive forms are discussed. Although recorded studies emphasize that patients with lobular carcinoma in situ are "at risk" for the development of invasive cancer, it has not been unequivocally demonstrated whether such an event represents a persistence of cancer due to inadequate excision or a de novo lesion. In support of the latter is the contention that lobular carcinoma exhibits a propensity for multicentricity and bilaterality. The recognition that the histologic types of the subsequent invasive cancers are not universally lobular invasive might also be cited in this regard. This information also bears upon views purporting a stepwise development of lobular invasive carcinoma from its in situ analog. Analysis of our own material fails to confirm any significant association between invasive lobular carcinoma and multicentric lesions. The diagnostic difficulty in distinguishing lobular hyperplasia from in situ lobular carcinoma and the inadvisability of frozen sections for this purpose is noted. Although the results of some electron microscopic studies of the in situ lesion challenge the propriety of its "pure in situ" nature; this criticism does not appear valid from both a pathological as well as pragmatic standpoints. The various schemes have been proposed concerning the surgical management of patients with lobular carcinoma in situ are presented and discussed. Certain biologic principles prompt consideration of segmental mastectomy and axillary node sampling as an alternative, commodious form of treatment for such lesions. There does not appear to be any unique reason to invoke any different treatment regimen for lobular invasive carcinoma than has been utilized for other invasive breast cancers.


HBs antigenemia in renal allograft recipients. Serial HBs Ag determinations were performed on 98 renal allograft recipients with functioning grafts for 6 to 108 months, 85 of whom were followed from the initiation of dialysis. Twenty-six (27%) recipients had HBs antigenemia following transplantation. Thirteen (50%) of the 26 recipients were HBs Ag positive during the period of dialysis and 13 developed HBs antigenemia 1 to 44 months following transplantation. Seventeen (65%) of the 26 HBs Ag positive patients had hepatic dysfunction which was detected by biochemical surveillance and not associated with clinical symptomatology. There was no evidence of progressive hepatic insufficiency. HBs Ag persisted in 24 (92%) recipients for 6 to 49 months. Clearing of antigenemia occurred in only two patients, both of whom ultimately rejected their grafts. The presence of HBs Ag had no adverse effect on graft function. Temporary reduction in azathioprine dosage with hepatic dysfunction was not associated with rejection episodes. The major hazard posed by the HBs Ag positive recipient is the potential reservoir for spread to the general population because of the persistence of antigenemia.


Ultrasound in renal transplantation.

Ultrasound in renal transplantation. Because of important differences in prognosis and treatment, prompt and accurate diagnosis of fever, decreasing function, and pain and tenderness around a renal allograft is crucial for differentiation between acute rejection and other post-operative conditions which may give a similar picture. Ultrasound examinations within 24 hours have been performed on all transplant recipients exhibiting symtoms compatible on all transplant recipients exhibiting symptoms compatible with an episode of acute rejection between September 1973 and June 1976 at the Peter Bent Brigham Hospital. The patients were separated into four groups dependent upon diagnosis by ultrasound; Group I(73 patients); allograft enlargment consistent with acute rejection; Group II (14 patients); dilitation of the collecting system; Group III (14 patients); perinephric fluid collections; Group IV (6 patients); miscellaneous conditions. The accuracy of the ultrasound technique was compared within each group to the results from intravenous pyelography, retrograde pyelography, serial renograms, kidney biopsy and/or surgical exploration. This convenient, non-invasive and reproducible method has been extremely effective in the differential diagnosis of rejection in clinical transplantation.


Protracted oral chemotherapy with fluorinated pyrimidines as an adjuvant to surgical treatment for stomach cancer. Studies of oral chemotherapy of 5-fluroruracil (5-FU) combined with FT-207, a furanyl analog of 5-FU, as an adjuvant to surgical treatment of stomach cancer have been performed since 1970 in the first department of surgery of Chiba University, Japan. These studies were performed on 107 patients with curative stomach cancer and 22 patients with non-curative stomach cancer. These patients received consecutively 3.5mg/kg/day of 5-FU and 8mg/kg/day of FT-207 for 24 to 36 months and were compared with control patients who had undergone only surgical treatment. A significant higher survival was observed in the curatively resected patients receiving oral 5-FU and FT-207, as compared to the control group. Studies on non-curative stomach cancer patients treated with the same dose schedule gave encouraging results. Because of the limited number of patients treated in this study, the drawing of reliable conclusions must wait the results of further studies.


Resolution of breast pain and calcification with renal transplantation. Vascular calcification in chronic renal failure and dialysis patients is well-documented and generally considered to be a consequence of decreased phosphorus excretion, secondary hyperparathyroidism, and increased calcium-phosphorus product. Following renal transplantation or parathyroidectomy, gradual resolution of metastatic calcification in the affected areas occurs. The case presented documents the consequence of secondary hyperparathyroidism with calcification of mammary vessels leading to severe breast pain with resolution of the pain and vessel calcification after renal transplantation.


Alterations of alkaline phosphatase activity during adaptation of Escherichia coli to phosphite and hypophosphite. When Escherichia coli cells were grown in media containing either phosphite or hypophosphite as the sole source of phosphorus, the responded to this situation primarily in the same way as phosphate-limited cultures: The activity of alkaline phosphatase increased drastically, which under natural conditions would enable the cells to compensate for the shortage of phosphate. Subsequent transfers, however, resulted in a quite different response: While the phosphatase activity of phosphate-limited cells stays at a high derepressed level, its increase was followed by a gradual decline in organisms grown on phosphite of hypophosphite. After eight to ten transfers on these P-compounds, phosphatase activity was back to its initial, repressed, low level, indicating that the cells were fully adapted to these substrates. Adaptation to either PO3-3 or PO3-2 was completely abolished if the cells were again grown with PO3-3 as P-source, whereafter the entire process of adaptation had to be repeated. The observed adaptation pattern, reflected by the alterations of phosphatase activity, was qualitatively equal with PO3-3 and PO3-2, but quantitatively different, because the response to hypophosphite gave much higher values than the increase obtained with phosphite. Phosphite-adapted cells are not simultaneously adapted to hypophosphite, but their response to the latter was less intense than observed after direct transfers from PO3-4 to PO3-2. Adaptation to hypophosphite, however, led simultaneously to phosphite adaptation, so that these cells can utilize both P-compounds as a substitute for phosphate.


Reliability of psychiatric diagnosis. I. A methodological review. This article reviews some methodological aspects of studies of diagnostic reliability in psychiatry. We define and discuss the concept of interrater reliability and review some of the ways in which the design of the reliability study can influence the results. Three basic methodological issues are raised, including: importance of structured interviews and objective diagnostic criteria, the importance of a test/retest vs an interviewer/observer design, and the calculation of reliability in a way that takes chance agreement into account.


Relaxation therapy and high blood pressure. Thirty-one patients receiving medical treatment for essential hypertension were randomly distributed into three groups: (1) relaxation therapy, (2) nonspecific therapy, and (3) medical treatment only. The nonspecific therapy group spent the same amount of time with the therapists as the relaxation group but was not given a specific therapy. Blood pressures were measured at a different time and in a different place from the behavioral treatments. The relaxation therapy group showed a significant reduction in blood pressure postreatment compared with the nonspecific therapy and medical treatment only groups, even when those patients whose medication was increased were excluded from the data analysis. At follow-up six months post-treatment, the relaxation group showed a slight decrement in treatment effects, while both the nonspecific therapy and medical treatment only groups showed continued improvement; thus, there was not a significant difference between groups.


The effect of lithium carbonate on affect, mood, and personality of normal subjects. Data reflecting affect, mood, and personality attributes of 23 normal men were compared after two weeks of placebo administration and two weeks of therapeutic serum lithium levels (mean, 0.91 mEq/liter). The study was a placebo-controlled, split-half crossover, double-blind design. Affect and mood were measured by three self-rating instruments, independent rater observation, and by the subjects' "significant others." Two personality inventories were administered. Substantial affect and mood changes are induced by lithium carbonate. Lethargy, dysphoria, a loss of interest in interacting with others and the environment, and a state of increased mental confusion were reported. No generalized effects were found in the responses to ther personality inventories.


The effect of lithium carbonate on the cognitive functions of normal subjects. The responses of 24 normal male subjects were compared after weeks of placebo administration and two weeks of lithium carbonate administration (mean serum lithium level, 0.9 mEq/liter) on a series of tasks of intellectual function, aesthetic judgement, and semantic creativity. This was a placebo-controlled, split-half crossover, double-blind design. There were no significant changes on semantic creativity or aesthetic perception measures following lithium carbonate maintenance. There were lithium carbonate-related performance deficits on three of five performance tasks concerned with cognitive and/or motor functions. The deficit is probably due to a lithium carbonate-induced slowing of performance, consistent with our previous report of subjective effects in normal subjects. The implications of slowing on possible behavioral mediating mechanisms by which lithium carbonate exerts its clinical effects are discussed.


Ocular immunopathology in murine lupus.

Ocular immunopathology in murine lupus. Immunoglobulin deposits and DNA were identified by use of immunofluorescence microscopy within the ciliary process and choroid of a majority of (NZB X W)F1 mice with murine lupus. The number of animals with such deposits increased from 57% at 36 weeks of age to 90% at one year of age. Ultrastructural examination showed electron-dense deposits between the basement laminae of the endothelium and pigmented epithelium of the ciliary process, accompanied by alterations in the epithelial basement membranes. Similar electron-dense deposits were also identified in the vicinity of the lamina choroidocapillaris. The titer of antibody to DNA did not correlate with the presence of ocular deposits, suggesting that time is an important factor in the development of ocular immunopathology in these animals.


Reference accuracy in articles accepted for publication in the Archives of Physical Medicine and Rehabilitation. Error rates and types of errors found in bibliographies of 129 articles accepted for publication by the Archives of Physical Medicine and Rehabilitation were carefully checked over a 13-month period. Verification is done before publication by the Archives. Of a total of 1.867 reference citations, 1.005 (54%) were incorrect and 115 (6%) could not be verified.


Use of systemic antibiotics for prophylaxis in surgery: a critical review. We reviewed the English-language literature over a 16-year period (1960 through 1976) on the subject of prophylaxis with systemic antibiotics in surgery. Trials in genitourinary and cardiovascular surgery were not reviewed. Our definition of prophylaxis is antibiotic administration of the absence of infection or contamination. Of 131 articles reporting clinical trials using systemic antibiotics for prophylaxis, only 24 met the criterion on an appropriately designed study that generated evaluable data. In these, systemic antibiotics were shown to be of value in reducing wound infections after abdominal and vaginal hysterectomy, cesarean section, biliary surgery, total hip replacement, and microneurosurgical craniotomy. Antibiotic prophylaxis was of no value in laparotomy and groin hernia repair. Patients undergoing any of 21 different operations did not benefit from prophylactic antibiotic administration, though study groups were too small or infection rates too low to allow for firm conclusions. In certain patients at high risk of infection, systemic prophylaxis is warranted. Future clinical studies must be designed as randomized, blinded, prospective trials, with antibiotics administered by a parenteral route beginning preoperatively.


Control of lumbar arterial bleeding during aortic aneurysm surgery. Lumbar backbleeding from an opened aortic aneurysm may be very troublesome. Suturing of these ostia is occasionally difficult because of an unyielding calcified vessel wall. We describe a new technique utilizing warm bone wax to occlude these ostia.


[Ultrastructural characteristics of the lymph node cells in lympho- and reticulosarcomas]. Electron-microscopic studies of the lymphatic nodes in lympho- and reticulosarcomas of man showed that tumoral cells retained the ultrastructural signs of initial normal cells (lymphoid or reticular) in the degree sufficient for their utilization in differential diagnosis of these two forms of the disease.


[Morphology and morphogenesis of cancer of the gallbladder and extrahepatic biliary ducts]. The article presents a survey of the problems of pathological anatomy, morpho- and histogenesis of cancer of the gallbladder and extrahepatic biliary ducts. Data on the role of inflammatory epithelial hyperplasias, adenomatous polyps in the subsequent malignancy of the biliary tract mucosa are presented. Problems of squamous-cell carcinoma of this localization are discussed. Results of experimental studies of tumours of the biliary ducts are presented.


Glossopharyngeal neuralgia, asystole, and seizures. Glossopharyngeal neuralgia, asystole, and seizures occurred in a patient with an internal carotid occlusion and external carotid stenosis. Swallowing was the triggering mechanism for these events. Mechanical stimulation of the pharynx failed to reproduce the symptoms. An ischemic injury to the glossopharyngeal nerve in the region of the jugular foramen that resulted in an artificial synapse is the proposed etiology.


Acute infantile motor unit disorder. Infantile botulism? Eight infants with an acute reversible motor unit disorder are described, including two infants from whom Clostridum botulinum type A was isolated from stool specimens. The clinical spectrum includes constipation, cranial nerve deficits, pupillary involvement, and generalized hypotonic weakness. There were no deaths, and all infants have had complete clinical recovery. A characteristic electromyographic (EMG) pattern was present in part until clinical recovery. This distinctive pattern consisted of brief, small, abundant for power exerted motor unit potentials. This EMG pattern in the context of the clinical syndrome may well be diagnostic for acute infantile motor unit disorder.


The rebound phenomenon of Gordon Holmes. Contrary to a prevalent belief, the rebound phenomenon of Gordon Holmes is not a sign of cerebellar disease. It is elicited by having the patient attempt to move a limb against resistance. When the resistance is suddenly removed, the limb normally moves a short distance in the desired direction and then rebounds (jerks back in the opposite direction). Gordon Holmes pointed out that the rebound phenomenon is (1) present in normal libs, (2) exaggreated in spastic limbs, and (3) absent in limbs affected by cerebellar disease. An awareness of Holmes's observations may be helpful in the diagnosis of both cerebellar disease and spasticity.


Epithelial downgrowth following penetrating keratoplasty in the aphake. Four of approximately 1,500 patients receiving corneal grafts developed epithelial downgrowth following penetrating keratoplasty. All were aphakic prior to grafting and had complicated postoperative courses. Epithelial proliferation over the iris and endothelium was demonstrated in three cases at the time of a repeat keratoplasty. While rare, this complication may occur more frequently than has been previously recognized.


[Experience in the treatment of pressure sores in congenital and acquired paraplegia (author's transl)]. We collected experience in 326 operated skin sores. Our successful treatment depends on a special surgical technic and after care.


Autoimmune phenomena of the conjunctiva and cornea. A case report. A 20-year-old woman had erosions of the corneal and conjunctival epithelium and sterile stromal ulcerations of the cornea necessitating multiple corneal grafts, which were unsuccessful. An immunopathological basis for her disease was suggested by the demonstration of tissue-fixed antibodies and complement in her own conjunctival epithelium by direct immunofluorescence. In addition, circulating antibodies to normal corneal and conjunctival epithelium were demonstrated in the patient's serum by indirect immunofluorescent techniques and the titer of circulating antibodies was found to correlate with disease activity. The involvement of the fellow eye after evisceration of the first eye and reduction of systemic steroids in association with a greatly elevated level of circulating antibodies provided additional evidence for an autoimmune process. A background of immunological susceptibility was found in the patient and her family by history and immunological examination.


[Cortical infantile hyperostosis. Report of a case and revision of the literature]. We describe a case of infantile cortical hyperostosis or Caffey-Silverman's disease. This is the fourth case reported in the national literature and the third one in our Hospital de Pediatría of the National Medical Center. We made an extensive review of the national and international literature and we defined the characteristic findings in this pathologic entity. We analyzed the etiologic possibilities, which are not yet clear and emphasized the role of the genetic factors. We suggest to investigate intentionally the asymptomatic colaterals, on the way to establish the real frequency of the disease.


[Massive air embolism in a newborn infant treated with a positive-pressure respirator]. We report a case of gas embolism secondary to the use of positive end respriatory pressure in a newborn with ideopathic respiratory syndrome. The anatomicopathological findings lead to the hypothesis that the gas migrates from the broken alveoli through the lymphatic vessels to the venous circulation.


[Rosette-forming peripheral lymphocytes, in children with measles]. During measles infection in human beings, depression of cellular immunity appears. The authors studied several groups of children with measles complaining of various degrees of malnutrition in terms of rosette forming lymphocytes in peripheral blood; marked reduction of T lymphocytes were seen in all of the groups during the acute stage of the disease, that persisted up to two weeks after the exanthematous stage. When comparison was established of eutrophic and 1st degree malnourished children with those affected with 3rd degree malnutrition, differences were found in the recovery of the number of rosett-E forming cells.


[Treatment with human colostrum of children with prolonged infectious gastroenteritis]. The results of treatment with human colostrum in 6 babies with protracted infectious diarrhea from enteropathogenic E. coli and/or Salmonella are reported. Active diarrhea was present in 5 cases and the 6th was a carrier. Remission was obtained in cases with diarrhea and all showed increase in weight. Negativeness of stool cultures was achieved in four cases and in the other two, the bacteriologic control was not complete. One patient died from a secondary pulmonary infectious problem. As an average, all patients showed rise in secretory IgA and in IgG measured at the duodenal fluid level and in comparison with a control group, there were statistically significant differences. Serum immunoglobulins showed no changes following administration of human colostrum. Although the number of patients reported is small, the results obtained may be considered favorable.


The bronchodilator effect of NAB 365.

The bronchodilator effect of NAB 365. 1. Dose response relationships for salbutamol and a new bronchodilator drug NAB 365 have been obtained in patients with reversible airways obstruction. These show that the latter is about one hundred times more potent than the former. 2. NAB 365 has a very long half-life. The implications of this are discussed.


Observations on the antibody rsponses in molluscum contagiosum. A survey of the incidence of molluscum contagiosum virion antigens in patients with single or multiple lesions showed that all lesions contain virus antigen. The virion antigens were detected in the prickle cell, granular and keratin layers. Hyperimmune sera against vaccinia, rabbit-pox and mouse-pox failed to stain the inclusion bodies in molluscum contagiosum lesions. Virus-specific antibodies were present in 69% of the patients. Although virus-specific IgG antibody was predominant, virus-specific IgM and virus-specific IgA were found in a few patients. An IgM staining in the prickle cell, granular and keratin layers of normal skin adjoining the molluscum contagiosum lesion was observed. The virus-specific antibody response was induced in a few patients after the treatment.


Immunopathological studies in herpes gestationis. Four cases of herpes gestationis are reported and the immunopathological findings in these patients described. Complement deposition at the basement membrane zone of the patients' peri-bullous skin was seen in all patients, immunoglobulin deposition in two. A circulating factor capable of fixing complement on the basement membrane zone of normal human skin was present in three patients. These findings are discussed and compared with the immunopathological findings in bullous pemphigoid.


A separation of clinical from epidermal thinning effect in the topical glucocorticoid clobetasone butyrate. Clobetasone butyrate ointment has been shown to be more effective in treating psoriasis and eczema than flurandrenolone ointment yet to cause less epidermal thinning in a human experimental model. This is an indication that the clinical activity of topical glucocorticoids may not necessarily be inseparable from their propensity to cause atrophy of the skin.


A double-blind trial of 0-05% clobetasol proprionate in the treatment of vitiligo. 0-05% clobetasol proprionate (CP) in its proprietary cream base was significantly superior to the cream base alone in the treatment of vitiligo. Twelve of twenty-three patients partially repigmented during 4 months' treatment with CP. All patients showed dermal atrophy with the CP.


Oral non-dystrophic bullous eruption mainly limited to the gingivae: a mechano bullous response. A variant of cicatricial mucous membrane pemphigoid? Fourteen patients with recurrent blistering entirely restricted to the mouth have been observed for up to 7 years. Their average age was 52 years, and there was a predilection for females (ratio 2-5:1). Patients presented with thick-roofed blisters and denuded, red, boggy areas of mucosa. The area of maximal involvement with the labial gingiva, and whilst the blisters would arise spontaneously, mechanical trauma was the obvious provocative factor in all patients. Gentle friction on normal looking mucosa would produce a bulla which became blood filled. Re-epithelialization usually occurred within 1-3 weeks, with no scarring. Biopsies showed subepithelial bullae, and direct immunofluorescence was positive in the basement membrane zone of 2 of the 5 cases examined. The most striking feature was the extreme fragility of the epithelial attachment to the underlying corium, as shown by a useful clinical test with a probe. In 10 patients, the condition gradually remitted and the probe test became difficult to perform. The term acquired oral non-dystrophic epidermolysis bullosa was considered for the diagnosis, although a forme fruste of cicatricial mucous membrane pemphigoid remained an alternative.


Methotrexate uptake by psoriatic epidermis. Psoriatic epidermis has been shown to be more sensitive to the effects of methotrexate than normal epidermis. One possible explanation for this observation is more rapid active transport of methotrexate into psoriatic cells. This study compares the uptake of methotrexate by normal and psoriatic epidermis and demonstrates the influence of extracellular pH on this process. Methotrexate uptake was not found to be greater in psoriatic epidermis than normal. A possible explanation for this unexpected finding is discussed.


A trial of oral ritodrine for the prevention of premature labour. A double blind clinical trial of oral ritodrine for prevention of premature labour was conducted in 38 primigravidae in whom the internal os of the cervix was one or more fingerbreadths dilated at 28 to 32 weeks gestation. The results suggest that oral ritodrine may not be effective in preventing premature labour.


Selective inhibition by danazol of follicle stimulating hormone during the luteal phase. In each of six healthy, normally menstruating women, serum oestradiol, progesterone, basal and post luteinizing hormone releasing hormone (LHRH) gonadotrophin measurements were made during the luteal phase of a normal cycle and in a subsequent cycle in which 800 mg of danazol was given daily from the fifth day after the presumptive date of ovulation. No differences in the serum oestradiol, progesterone, or basal gonadotrophin levels were detected, but there was a selective impairment of the follicle stimulating hormone response to LHRH. The implications of these findings are discussed.


A randomized controlled trial of bed rest and sedation or normal activity and non-sedation in the management of non-albuminuric hypertension in late pregnancy. One hundred and thirty-five patients took part in a randomized controlled trial designed to determine whether bed rest and sedation are of any general benefit to either the mother or the baby in pregnancies complicated by mild non-albuminuric and non-symptomatic hypertension after the 28th week. The results suggest that they are not.


Simultaneous in situ carcinoma of the cervix, vulva and perineum after immunosuppressive therapy for renal transplantation. A 31-year-old woman developed multiple synchronous in situ carcinomas of the cervix uteri, vulva and perineal skin, six years after successful renal transplantation for chronic renal failure due to chronic pyelonephritis. During this time she had been continuously treated with immunosuppressive drugs (azathioprine and prednisolone). The lesions were treated by local excision and there was no evidence of recurrence nearly two years after the primary treatment. The case history is compared to others recently described and highlights the risk of epithelial as well as lymphomatous malignancies in renal transplant recipients.


Management of keratoplasty in the early stages. Frequent, if not daily, slit-lamp examination of the patient with a penetrating transplant is indicating for more than one reason: it will lead to the early detection of any defect of the host-graft junction or of the graft's posterior surface; it will permit an early diagnosis of the peripheral cornea-iris touch syndrome with all its potential consequences (acute, irreversible mydriasis, iris atrophy, and now and then secondary glaucoma); it will give an exact clue to when to remove the continuous sutures long after these have been covered by epithelium; and it will alert the surgeon in the event of a localised infection. Early and late opacification of the graft will also be correctly appraised, so that the surgeon will know when to wait and when to take the appropriate action.


Metronidazole compared with penicillin in the treatment of acute dental infections. The efficacy of metronidazole in the treatment of acute dental infections was investigated and compared with parenteral penicillin in a controlled trial. All 37 patients in the trial responded satisfactorily and metronidazole appeared to be as effective as parenteral penicillin. A further 24 patients treated with metronidazole also responded satisfactorily. Bacteriological studies of pus obtained from 25 patients revealed the presence of many species of obligate anaerobes. Since metronidazole is only active against obligate anaerobic bacteria it is concluded that these organisms are the important pathogens in acute dental infections.


Magnetic resonance studies on manganese-nucleotide complexes of phosphoglycerate kinase. Measurements of the relaxation rate of water protons (PRR) have been used to study the interaction of yeast phosphoglycerate kinase with the manganous complexes of a number of nucleotides. The results indicate that phosphoglycerate kinase belongs to the same class of enzymes as creatine kinase, adenylate kinase, formyltetrahydrofolate synthetase, and arginine kinase, with maximal binding of metal ion to tne enzyme in the presence of the nucleotide substrate. However, an analysis of titration curves for a number of nucleoside diphosphates (ADP, IDP, GDP) showed that there is a substantial synergism in binding of the metal ion and nucleotide to the enzyme in the ternary complex. The metal-substrate binds to the enzyme approximately two orders of magnitude more tightly than the free nucleotide; Other evidence for an atypical binding scheme for Mn(II)-nucleoside diphosphates was obtained by electron paramagnetic resonance (EPR) studies; the EPR spectrum for the bound Mn(II) in the enzyme-MnADP complex differed substantially from those obtained for other kinases. An identical EPR spectrum is observed with the MnADP complex with the rabbit muscle enzyme as with the yeast enzyme. In contrast, the dissociation constant for the enzyme-MnATP complex is approximately fourfold lower than that for enzyme-ATP, and there are no substantial changes in the electron paramagnetic resonance spectrum of MnATP2- when the complex is bound to phosphoglycerate kinase. A small but significant change in the PRR of water is observed on addition of 3-phosphoglycerate (but not 2-phosphoglycerate) to the MnADP-enzyme complex. However, addition of 3-phosphoglycerate to enzyme-MnADP did not influence the EPR spectrum of the enzyme-bound Mn(II).


Galactose-1-phosphate uridylyltransferase: isolation and properties of a uridylyl-enzyme intermediate. Galactose-1-P uridylyltransferase catalyzes the interconversion of UDP-galactose and galactose-1-P with UDP-galactose and glucose-1-P by a double displacement pathway involving a uridylyl-enzyme intermediate. The amount of radioactivity incorporated into the protein by uracil-labeled UDP-glucose is decreased by the presence of UDP-galactose, which completes with UDP-glucose for uridylylating the enzyme. The amount of glucose-1-P released upon reaction of the enzyme with UDP-glucose indicates that the dimeric enzyme contains more than one active site per molecule, 1.7 on the average for the most active preparation obtained. This suggests that there is one uridylylation site per subunit and that the subunits are similar or identical. The ureidylyl-enzyme is stable to mild alkaline conditions, 0.10 M NaOH at 60 degrees C for 1 h, but is very sensitive to acid, being largely hydrolyzed after 12 h at pH 3.5 and 4 degrees C. The principal radioactive product resulting from hydrolysis of [uracil-2-14C]uridylyl-ens of the uridylyl-enzyme under the latter conditions is [l]ump. The hydrolytic properties of the uridylyl-enzyme show that the uridylyl moiety is bonded to the protein through a phosphoramidate linkage. Complementary studies on the effects of group selective reagents on the activity of the enzyme suggest that the active site nucleophile to which the uridylyl group is bonded may be a histidine residue. The enzyme is rapidly inactivated by diethyl pyrocarbonate at pH 6 and 0 degrees C and reactivated by NH2OH. UDP-glucose at 0.5 mM fully protects the enzyme against diethyl pyrocarbonate while 70 mM galactose-1-P has only a slight protective effect. Uridylyl-enzyme in inactivated by diethyl pyrocarbonate at no more than 2% of the rate for free enzyme. The enzyme is not inactivated by NaBH4 or by NaBH4 in the presence of UDP-glucose. It is not inhibited by 1 mM pyridoxal phosphate or by 0.5 mM 5-nitrosalicylaldehyde at pH 8.6 and it is not inactivated by NaBH4 in the presence of pyridoxal phosphate. The enzyme is inactivated by 5 to 50 muM p-hydroxymercuribenzoate at pH 8.5, but substrates exert no detectable protective effect against this reagent. It is concluded that the enzyme contains at least one essential sulfhydryl group which is not located in the active site in such a way as to be shielded by substrates.


Editing mechanisms in protein synthesis. Rejection of valine by the isoleucyl-tRNA synthetase. Although the isoleucyl-tRNA synthetase from Escherichia coli (IRS) does not catalyze the overall mischarging of tRNAIle with valine, it does undergo the first step of the reaction, the formation of an IRS-Val-AMP complex. The addition of tRNAIle to this complex leads to its quantitative hydrolysis and the IRS acts as an ATP pyrophosphate in the presence of valine and tRNAIle (Baldwin, A.N., and Berg, P. (1966), J. Biol. Chem. 241, 839). It is shown that during the ATP pyrophosphatase reaction: (a) IRS forms an IRS-Val-AMP complex; (b) the turnover number of the ATP pyrophosphatase reaction is the same at the rate constant for the transfer of isoleucine from IRS-Ile-AMP to tRNAIle over a wide range of temperature and pH; (c) mischarged Val-tRNAIle is hydrolyzed by IRS with a turnover number of 10 s-1 at pH 7.78 and 25 degrees C, compared with a value of 1.2 s-1 for the transfer of isoleucine from IRS-Ile-AMP to tRNA or for the ATP pyrophosphatase reaction. Although this appears to be consistent with an editing mechanism in which there is a slow transfer of the valine from the IRS-Val-AMP to tRNAIle follwed by the rapid hydrolytic step, as recently found for the rejection of threonine by the valyl-tRNA synthetase, there is an inconsistency. This scheme predicts that on mixing IRS.[14C]Val-AMP with tRNAIle there should be a transient misacylation of the tRNA such that about 10% of the [14C]Val is present as [14C]Val-tRNAIle at the peak. But 0.8% or less is found. This could possibly be caused by the IRS having a higher hydrolytic activity during the mischarging reaction than is measured on mixing the unligated enzyme with performed Val-tRNAIle. Alternatively, a two-stage editing mechanism must be considered in which the majority of the Val-AMP is destroyed before the transfer to tRNA in the major editing step, while the hydrolytic activity of the IRS towards Val-tRNAIle is a second editing step to mop up any mischarged tRNA formed by the Val-AMP escaping the first editing step. It is shown that the "kinetic proofreading" mechanism of Hopfield is not consistent with the experimental data.


Conjugated polyene fatty acids as fluorescent probes: biosynthetic incorporation of parinaric acid by Escherichia coli and studies of phase transitions. The use of the fluorescent fatty acid, parinaric acid (9, 11, 13, 15-octadecatetraenoic acid) (PnA), was studied in cells of an unsaturated fatty acid auxotroph of Escherichia coli. Growth conditions were found that permitted biosynthetic incorporation of PnA (up to 3%) into membrane phospholipids during growth on oleic or elaidic acid. Fluorescence measurements of incorporated PnA revealed phase transitions in cells, membranes, and phospholipids at temperatures that reflected the fatty acid composition of the sample. Transitions had a well-defined onset from high temperature, while the lower and end point was less well defined. cis- and trans-PnA (cis, trnas, trans, cis, and all trans, respectively) gave comparable results. Similar phase transitions were detected with PnA, which was not biosynthetically incorporated. Fluorescence of tryptophan was measured in E. coli membranes as a function of concentration of PnA. Significant quenching of tryptophan fluorescence by PnA was observed.


Escherichia coli DNA-directed beta-galactosidase synthesis in presence and absence of Ca2+. DNA-dependent synthesis of beta-galactosidase was optimized in extracts made from cells lysed by a standard French pressure cell. Extracts made at 3200 psi synthesized up to 25-fold more beta-galactosidase than extracts made at 7500 psi. beta-Galactosidase synthesis was cyclic 3', 5' AMP dependent, as expected, and in optimal conditions transcription and translation proceeded at 8.6 nucleotides and 2.7 amino acids per s, respectively. The high pressure extracts were stimulated 3- to 5-fold by Ca2+, especially at low Mg2+ concentrations. In contrast, extracts prepared at low pressure were inhibited as much as 50-fold by Ca2+ ions. The inhibition by Ca2+ was analyzed further. Addition of kasugamycin, an antibiotic that acts on ribosomes, to reactions containing Ca2+ stimulated beta-galactosidase synthesis to nearly control levels. Extracts from a kasugamycin resistant mutant were neither inhibited by Ca2+ nor stimulated by the addition of kasugamycin to in vitro reactions containing Ca2+. The change in the mutant was ascribed to the ribosomes by testing combinations of soluble proteins, ribosome wash, and ribosomes from parental and mutant strains. These results suggest that Ca2+ ions inhibit translation by ribosomes, very likely at an initiation step; and that they enhance enzyme synthesis only in conditions where translation is inefficient (high-pressure extracts at low concentrations of Mg2+, for example). This latter effect is probably a consequence of increased RNA stability in the presence of Ca2+ (Cremer, K., and Schlessinger, D. (1974), J. Biol. Chem. 249,4730).


Photoreactivating enzyme from Escherichia coli: appearance of new absorption on binding to ultraviolet irradiated DNA. The photoreactivating enzyme, PRE, monomerizes pyrimidine dimers in DNA in a light requiring reaction (lambda greater than 300 nm). However, the purified PRE from E. coli has no well-defined absorption band for lambda greater than 300 nm. Using absorption difference spectroscopy, we show that when PRE is mixed with ultraviolet-irradiated DNA, new absorption appears in the spectral region required for catalysis. There is a concomitant decrease in the absorption of the mixture for wavelength less than 300 nm. The hyperchromicity for lambda greater than 300 nm is true absorption, not an artifact due to light scattering. Both the hyperchromicity (lambda greater than 300 nm) and hypochromicity (lambda less than 300 nm) can be reversed by irradiation of 365 nm with identical first-order kinetics. We estimate the molar extinction coefficient of the new absorption to be 6900 +/- 1400 at 350 nm. We conclude that the PRE from E. coli does not possess a distinct "chromophore" which by itself is entirely responsible for the absorption of photoreactivating light. Instead, new absorption results when PRE binds its substrate, dimer-containing DNA.


Isolation of amino-terminal fragment of lactose repressor necessary for DNA binding. lac repressor can be dissected by trypsin into a homogenous tetrameric core (accounting for residues 60 to 347), carrying inducer binding activity, and the monomeric amino-terminal peptides ("headpieces") accounting for residues 1 to 59 and 1 to 51, respectively. This restriction of the action of trypsin on lac repressor is obtained in 1 M Tris-HCl (pH 7.5)-30% in glycerol at 25 degrees C since only the peptide bonds at lysine-59 and to a lesser extent after at arginine-51 are cleaved under these conditions. The headpieces can be purified by gel filtration. They have ordered secondary structure as revealed by circular dichroism studies. The monomeric headpieces show the relatively weak binding to nonoperator DNA but not the highly specific and strong binding to operator DNA typical for tetrameric lac repressor.


Visualization of ribosome--single-stranded DNA complexes in the electron microscope. A procedure is described which allows ribosomes bound to single-stranded DNA to be visualized in the electron microscope. The number of bound ribosomes may be determined and the position of the bound ribosomes may be readily measured along the DNA. The distribution of ribosomes bound to separated l and r strands of lambda DNA was shown to conform to the pattern predicted for binding at specific sites. The procedure should allow mapping of ribosome binding sites for the determination of genetic maps and may also be useful for studying translational control and relative binding affinities for ribosomes.


Mechanism of action of adenosylcobalamin: glycerol and other substrate analogues as substrates and inactivators for propanediol dehydratase--kinetics, stereospecificity, and mechanism. A number of vicinal diols were found to react with propanediol dehydratase, typically resulting in the conversion of enzyme-bound adenosylcobalamin to cob(II)alamin and formation of aldehyde or ketone derives from substrate. Moreover, all are capable of effecting the irreversible inactivation of the enzyme. The kinetics and mechanism of product formation and inactivation were investigated. Glycerol, found to be a very good substrate for diol dehydratase as well as a potent inactivator, atypically, did not induce cob(II)alamin formation to any detectable extent. With glycerol, the inactivation process was accompanied by conversion of enzyme-bound adenosylcobalamin to an alkyl or thiol cobalamin, probably by substitution of an amino acid chain near the active site for the 5'-deoxy-5'-adenosyl ligand on the cobalamin. The inactivation reaction with glycerol as the inactivator exhibits a deuterium isotope effect of 14, strongly implicating hydrogen transfer as an important step in the mechanism of inactivation. The isotope effect on the rate of product formation was found to be 8.0. Experiments with isotopically substituted glycerols indicate that diol dehydrase distinguishes between "R" and "S" binding conformations, the enzyme-(R)-glycerol complex being predominately responsible for the product-forming reaction, while the enzyme-(S)-glycerol complex results primarily in the activation reaction. Mechanistic implications are discussed. A method for removing enzyme-bound hydroxycobalamin that is nondestructive to the enzyme and a technique for measuring the binding constants of (R)- and (S)-1,2-propanediols are presented.


A study of the quaternary structure of Escherichia coli RNA polymerase using bis(imido esters). The quaternary structure of Escherichia coli RNA polymerase has been studied by cross-linking with a periodate-cleavable bis(imido ester), N,N'-bis(2-carboximidoethyl)tartaramide dimethyl ester dihydrochloride (CETD). The cross-linked holoenzyme gives a characteristic five-band pattern after electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. The components of each band have been unambiguously identified by (a) molecular-weight measurements, (b) comparisons of cross-linking patterns of holoenzyme and core enzyme, and (c) periodate cleavage of cross-links followed by a second dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The bands are (1) alphabeta and alphabeta', (2) sigmabeta and sigmabeta', (3) alphasigmabeta', (4) betabeta', and (5) sigmabetabeta'. Bands 2 and 4 are the most prominent at low reagent concentrations (up to 2.5 mM) but band 5 becomes the most prominent at higher concentrations. There are no bands corresponding to alphaalpha and alphasigma; a faint band has been tentatively identified as alphabetabeta'. Shorter bis(imido esters) are much less effective cross-linking reagents than CETD and they do not give rise to any other cross-linked species. On the basis of these observations, a model for the subunit arrangement of RNA polymerase is proposed.


Stabilization by the 30S ribosomal subunit of the interaction of 50S subunits with elongation factor G and guanine nucleotide. The role of the 30S ribosomal subunit in the formation of the complex ribosome-guanine nucleotide-elongation factor G (EF-G) has been examined in a great variety of experimental conditions. Our results show that at a large molar excess of EF-G or high concentrations of GTP or GDP, 50S ribosomal subunits are as active alone as with 30S subunits in the formation of the complex, while at lower concentrations of nucleotide or lower amounts of EF-G, addition of the 30S subunit stimulates greatly the reaction. The presence of the 30S ribosomal subunit can also moderate the inhibition of the 50S subunit activity that occurs by increasing moderately the concentrations of K+ and NH4+, and extends upward the concentration range of these monovalent cations in which complex formation is at maximum. The Mg2+ requirement for complex formation with the 50S subunit appears to be slightly less than that needed for association of the 30S and 50S ribosomal subunits. Measurement of the reaction rate constants of the complex formation shows that the 30S ribosomal subunit has only little effect on the initial association of EF-G and guanine nucleotide with the 50S subunit; but once this complex is formed, the 30S subunit increases its stability from 10- to 18-fold. It is concluded that stabilization of the interaction between EF-G and ribosome is a major function of the 30S subunit in the ribosome-EF-G GTPase reaction.


Mechanistic interpretation of the influence of lipid phase transitions on transport functions. In an attempt to understand the mechanism by which a structural change of membrane lipids affects transport functions, the temperature dependence of transport rate has been measured to below the low temperature end of the fluid in equilibrium ordered phase transition of the membrane lipids. The unsaturated fatty acid requiring Escherichia coli strain T105 was supplemented with either trans-delta9-octadecenoate or trans-delta9-hexadecenoate or supplemented with and subsequently starved for cis-delta9-octadecenoate. Fluid in equilibrium ordered phase transitions measured in whole cells using the fluorescence probe N-phenyl-1-naphthylamine were compared with the temperature dependence of beta-glucoside and beta-galactoside transport. In addition to the previously observed downward "break" in the Arrhenius plot of transport rate which occurred near the middle of the phase transition temperature range, a second upward "break" was observed which could be correlated with the low-temperature end of the phase transition. These experiments are interpreted in terms of a partitioning of transport proteins between ordered and fluid domains which is described by a lateral distribution coefficient, k. This distribution coefficient varies with the membrane lipid composition as well as with the transport system. Values for k suggest a 2-20-fold preference for the partitioning of transport proteins into the fluid parts of the membrane.


Evidence for a precursor-product relationship in the biosynthesis of ribosomal protein S20. The kinetics of labeling ribosomal protein S20 of Escherichia coli strains H882 and H882 groE44 have been examined using partial reconstitution as a means of binding this and some other 30S subunit proteins selectively to 16S RNA from crude extracts prepared by acetic acid extraction of pulse-labeled whole cells. The rate of labeling of S20 during short pulses at 44 degrees C is less than 20% of that observed at 28 degrees C. S20 can be recovered from the cells labeled at the higher temperature if they are chased at 28 degrees C, but not at 44 degrees C, in the presence of excess sulfate prior to their extraction. These observations suggest that S20 is derived from a precursor whose processing is blocked at 44 degrees C. Among the proteins extracted from cells labeled at 44 degrees C capable of binding to 16S RNA is a novel polypeptide, p2, which is not normally present on the 30S subunit. The kinetics of its appearance at 44 degrees C, and its chasing at 28 degrees C, suggest a precursor-product relationship with S20. p2 contains a tryptic peptide with the chromatographic properties of the peptide Ser-Met-Met-Arg at position 25-28 in S20. A second methionine-containing peptide at positions 49-59 of S20 is missing from p2. In addition, the apparent molecular weight of p2 (8600) is less than that of S20 (9500). p2 may represent the product of degradation of a precursor to S20, yet retains the ability to bind to 16S RNA. It is much less likely that p2 is a bona fide precursor which is converted into S20 by fusion to some other polypeptide.


Nuclear magnetic resonance and fluorescence studies of substrate-induced conformational changes of histidine-binding protein J of Salmonella typhimurium. The histidine-binding protein J of Salmonella typhimurium binds L-histidine as a first step in the high-affinity active transport of this amino acid across the cytoplasmic membrane. High-resolution nuclear magnetic resonance spectroscopy has been used to monitor the conformation of histidine-binding protein J in the presence and absence of substrate. Evidence is presented to show that this binding protein undergoes a conformational change involving a substantial number of amino-acid residues (including tryptophans) in the presence of L-histidine and that this change is specific for L-histidine. In order to monitor the involvement of tryptophan residues in the substrate-induced conformational change, 5-fluorotryptophan has been incorporated biosynthetically into the histidine-binding protein J using a tryptophan autotroph of Salmonella typhimurium. There are no significant differences in the conformation and binding activity between the 5-fluorotryptophan-labeled and the normal histidine-binding protein J. Proton and fluorine-19 nuclear magnetic resonance studies of the 5-fluorotryptophan-labeled binding protein show that at least one (and possibly two) of the tryptophan residues undergo(es) a change toward a more hydrophobic environment in the presence of L-histidine. These observations are supported by fluorescence data and by differences in the reactivity of the tryptophan residues of this protein toward N-bromosuccinimide in the presence and absence of substrate. The present results are consistent with models for the action of periplasmic-binding proteins in shock-sensitive transport systems of gram-negative bacteria which require a substrate-induced conformational change prior to the energy-dependent translocation of substrates.


Positions of sea urchin (Strongylocentrotus purpuratus) histone genes relative to restriction endonuclease sites on the chimeric plasmids pSp2 and pSp17. The positions of the several sea urchin histone genes on the eukaryotic fragments of the chimeric plasmids pSp2 and pSp17 have been mapped relative to the Eco RI and Hind III restriction endonuclease sites on the plasmids. Two principal mapping methods using the electron microscope have been used: (a) the R-loop procedure and a new modification thereof to map the genes on duplex DNA; (b) the gene 32-ethidium bromide technique to visualize RNA-DNA hybrids on single strands of DNA. It is known that there are two histone genes, H3 and H2A, on pSp17. There are two Eco RI sites at the two junctions of the procaryotic segment with the eucaryotic segment on the plasmid. We show, by an electron microscope method, that for H2A, with a length of 0.52 kilobases (kb), one end of the gene is situated 0.02 to 0.03 kb from one RI site, and that there is a Hind III site within this gene at about 0.13 kb from the end phe other RI site of this plasmid. The H4 gene lies between H2B and H1. The ms the incubation temperature is raised up to a temperature just below that at which strand dissociation of the duplex DNA occurs.


The mechanism of catabolite inhibition of invertase by glucose in Saccharomyces cerevisiae. Saccharomyces cerevisiae -136ts synthesized invertase in media containing maltose and sucrose. In the presence of glucose synthesis of enzyme took place when the sugar concentration was lower than 1%. At higher concentrations enzyme formation was repressed. Analysis of the glucose effect before RNA inhibition showed that the hexose interfered with the transcription of DNA into invertase messenger RNA. Translation of invertase messenger already formed was also inhibited and the kinetics of this effect was similar to that produced by cycloheximide. Invertase activity was independent of glucose suggesting that the hexose produces no catabolite inhibition of invertase activity. Inhibition of invertase translation by glucose turned out to be reversible but the amount of enzyme produced was dependent on duration of treatment. It is suggested that the catabolite repression of invertase synthesis produced by glucose operates at the levels of transcription and translation and produces an increase in the rate of mRNA degradation. The catabolite repression has no effect on secretion and does not interfere with the catalytic activity of invertase.


Large and small invertases and the yeast cell cycle. Pattern of synthesis and sensitivity to tunicamycin. We have examined the pattern of synthesis of the glycoprotein form of invertase and of the smaller carbohydratefree from in synchronous culture to obtain further infromation concerning their biosynthetic relationship. Saccharomyces mutant 1710 was chosen since its invertase production is almost completely derepressed during growth in 0.1 M mannose medium. The large enzyme, unlike the small form, binds to concanavalin A-Sepharose, and on this basis the two types can conveniently be separated for analysis. Large invertase was produced throughout the cell cycle. Synthesis of the small invertase was periodic; the single burst occurred at or close to the budding stage. Tunicamycin, which inhibits the sypthesis of external glycoproteins, halted formation of the large enzyme but not of the small form, and there was no accumulation of invertase activity with the properties of the small enzyme. Hence, it is unlikely that the small form is a precursor of the large one. Despite marked differences in their amino acid compositions, the two enzymes have many similarities. They are probably, in part, the products of the same gene(s), and the differences between them may largely reflect differences in post-translational processing.


Mapping and length measurements of restriction enzyme fragments by electron microscopy. 1. We have mapped by electron microscopy the DNA-fragments formed by the action of the restriction endonuclease from Arthrobacter luteus of phi X 174 replicative form DNA. These fragments were separated by polyacrylamide gel electrophoresis and hybridized to phiX 174 single stranded DNA. The partial duplex molecules were inspected in the electron microscope. In this way the relative order of eleven fragments ranging in size from approximately 100 to 1000 nucleotide pairs has been established and compared with that deduced from reciprocal digestion studies. 2. The measured lengths of the fragments agreed well with the lengths found by gel electrophoresis. 3. The purity of the isolated fragments was checked. Most of the contaminating fragments derive from nearest neighbours in the preparative polyacrylamide gels.


Increased synthesis of abundant poly(A)-containing RNA in a DNA defective mutant of Saccharomyces cerevisiae containing the "killer character". A Saccharomyces cerevisiae strain which contains both the "killer character" and a ts mutation in the initiation of nuclear DNA synthesis (cdc4) was studied. Incubation of this strain at the restrictive temperature caused a 3--4 fold increase in the relative rate of synthesis of abundant RNA which contains poly(A) and a 2--3-fold increase in the relative rate of synthesis of killer dsRNA. Thus, the amount of killer dsRNA found in these cells seems to be correlated to the amount of abundant poly(A)-RNA.


On the control of ribosomal protein biosynthesis in Escherichia coli. I. Studies on ribosomal protein biosynthesis in amino acid-starved cells. The rate of individual ribosomal protein synthesis relative to total protein synthesis has been determined in Escherichia coli rel+ and rel- cells, under valyltRNA deprivation. These strains have a temperature-sensitive valyl-tRNA synthetase. Starvation was obtained following transfer to the cells to non-permissive temperature. Ribosomal proteins were obtained by treatment of either total lysates of freeze-thawed lysozyme spheroplasts or ammonium sulphate precipitate of ribosomes, with acetic acid. Differential labelling of the ribosomal proteins was observed in both strains: proteins from the rel+ strain appear more labelled than those from the rel- strain, the rate of labelling of individual proteins being about the same in both strains. Moreover ribosomal proteins were found as stable during starvation as total protein. It is thus concluded that in starving cells individual ribosomal proteins are not synthesized at equal rates. This indicates that the synthesis of ribosomal proteins is not only under the control of the rel gene.


On the control of ribosomal protein biosynthesis in Escherichia coli. II. Studies during recovery from amino acid starvation. The rate of synthesis of ribosomal proteins relative to that of total protein was measured at various times during recovery from arginine starvation in isogenic re+ and rel- strains of Escherichia coli K 12. Total ribosomal proteins are preferentially synthesized early during recovery. Higher rates of synthesis are obtained in the rel+ strain than in the rel- strain. Differential rates of synthesis of individual ribosomal proteins are observed at the various times studied. The rate of synthesis of individual proteins increases with time up to maximum values then the rates come down to values similar to those found in exponentially growing cells. The time of restart of synthesis of each protein has been estimated (1) by the time at which the maximum value is reached, and (2) by measuring the rate of synthesis at early time (3 min). Most ribosomal proteins behave similarlly in rel- and rel+ strains. Proteins have been listed from highly labelled (early proteins) to poorly labelled (late proteins). The significance of the order of restart is considered.


Partial derepression of the isoleucine-valine enzymes during methionine starvation is Salmonella typhimurium. Methionine starvation of methionine auxotrophs in the presence of excess branched-chain amino acids results in a partial derepression of the isoleucine and valine enzymes. Reversed-phase chromatography indicated that isoleucine, valine and leucine tRNA were altered during methionine starvation. In addition, the total tRNA isolated from cells under these conditions were undermethylated. The observed derepression may be caused by the inability of methyl-deficient tRNA's to participate adequately in normal regulatory functions.


Inactivation of infectivity of phiX174 DNA by menadione and reduced menadione. Interaction of menadione and reduced menadione with phage phiX174 DNA was investigated. A concentration of 2-10(-4) M menadione inactivated 60% of the infectivity of phiX174 DNA to spheroplasts of Escherichia coli, while reduced menadione inactivated 97% of the infectivity of phiX174 DNA at the same concentration. Alkaline sucrose gradient centrifugation revealed 2-10(-5) M reduced menadione caused approximately 24% of phiX174 DNA to produce strand break under the condition of 80% lethanlity. DNA strand break was not observed even at 4 - 10(-4) M menadione. These results indicated that there were different mechanisms for inactivation of phiX174 DNA between menadione and reduced menadione.


Peptide substrates for chymosin (rennin). Kinetic studies with bovine kappa-casein-(103-108)-hexapeptide analogues. Kinetic parameters have been determined for the reaction between chymosin (EC 3.4.23.4) and synthetic peptide analogues of the sequence Leu-Ser-Phe-Met-Ala-Ile around the chymosin-sensitive Phe(105)-Met(106) bond of bovine kappa-casein. From the present and earlier results it is concluded that a minimum length of the molecular backbone with three amino acid units on both sides of the scissile bond is required to make the peptide a good substrate for the enzyme. In addition, hydrophobic side chains in the positions 103 and 108, and particularly the hydroxyl group of Ser-104 contribute to the effectiveness of the enzyme-substrate interactions. The substrate properties are markedly influenced by changes in the steric and/or polar character of the amino acid side chains in the positions 105 and 106.


Quasi-elastic light scattering studies on pyruvate oxidase. Quasi-elastic or dynamic light scattering has been used to examine the translational diffusion properties of the enzyme pyruvate oxidase (pyruvate: ferricytochrome beta 1 oxidoreductase, EC 1.2.2.2.). Controlled proteolysis of the enzyme converts the native form of the enzyme to a protease-activated form which has a specific activity about 20-fold greater than the native oxidase. Light scattering studies indicate no significant change in the size or shape of pyruvate oxidase as a result of this proteolytic activation. In both cases the enzyme may be characterized as a hydrated sphere with a Stokes radius of about 53A. The sedimentation velocity-diffusion technique was used to obtain the molecular weight of this tetrameric enzyme, about 252 000 with a value of f/f0 of 1.25.


Regulation of aspartate carbamoyltransferase of Escherichia coli by the interrelationship of magnesium and nucleotides. Purified aspartate carbamoyltransferase from Escherichia coli K12 (carbamoylphosphate: L-aspartate carbamyltransferase, EC 2.1.3.2) shows greater activity with nucleotide effectors as the magnesium nucleotide complex than with similar amounts of the sodium nucleotide. Regulation of aspartate carbamoyltransferase activity in vivo may occur by changes in the total concentration of regulatory nucleotides or, under conditions of magnesium-limited growth, by variation of the saturation of the nucleotides with magnesium.


The solubilization of bone and dentin collagens by pepsin. Effect of cross-linkages and non-collagen components. Bone and dentin collagen are less susceptible to solubilization by pepsin digestion then is skin collagen. Digestion at 4 degrees C for 72 h solubilized only 35.3% of bovine cortical bone and 5.6% of bovine dentin compared with nearly 100% dissolution of bovine skin. Sodium dodecyl sulfate-acrylamide gel electrophoresis and molecular sieve chromatography showed that, for bone and dentin, intact alpha chains and cross-linked aggregates of beta, gamma and higher weight remained intact after pepsin solubilization but lower molecular weight fragments also were prevalent indicating chain scission in helical regions. Electron microscopic examination of segment long spacing precipitates of the soluble collagens confirmed the presence of solubilized polymerized collagen. The principal reducible cross-link in both bone and dentin was the precursor of dihydroxylsinonorleucine and this cross-link was also present in the solubilized collagens. Small amounts of non-collagenous proteins and glycosaminoglycans of different compositions in dentin and bone resisted extraction before pepsin digestion. However, the differences in solubilization of the collagens have been related to differences in cross-linkage placement.


Penicillin-binding proteins of Escherichia coli. Comparison of a strain carrying an R-factor and the parent strain. Both from Escherichia coli K12 W3630 carrying an R-factor, R+75, and from the parent strain at least six penicillin- and cephalosporin-binding proteins were obtained as soluble forms. The molecular weights of the binding proteins of the strain carrying an R-factor were similar to those of the parent strain and not affected by the presence of an R-factor which specified the production of a beta-lactamase. Gel filtration with [14C]benzylpenicillin suggested the equimolar binding of benzylpenicillin to each binding protein. Three binding proteins of E. coli carrying R+75 and two binding proteins of the parent strain were purified by affinity chromatography followed by gel filtration. In fluorescence titration, various penicillins and cephalosporins were shown to bind to the purified binding proteins and their association constants were in the range of 0.4 to 21-10(3) M-1. The binding proteins of both strains did not react with the antibody against the beta-lactamase specified by R+75.


Selectively deuterated amino acid analogues. Synthesis, incorporation into proteins and NMR properties. Selectively deuterated analogues of histidine, tyrosine, phenylalanine and tryptophan have been synthesized by chemical exchange. These analogues have been characterized by NMR spectrometry and used for growth of bacteria. Active lactose repressor protein has been isolated from cells grown on the deuterated amino acids, and denatured 1H and 2H NMR spectra have been determined for the protein.


The origin of the sulfur atom in thiamine. The mode of biosynthesis of the thiazole moiety of thiamine, 4-methyl-5beta-hydroxyethyl thiazole (MHET) was studied using Salmonella typhimurium as test organism. It was shown by isotope incorporation experiments, that the sulfur atom, but not carbon-3, of cysteine is incorporated into MHET, indicating a separation of the sulfur atom of cysteine from the carbon chain during incorporation. Isotope competition experiments revealed that the incorporation of [35S]cysteine is not significantly diluted by the presence of methionine, homocysteine, and glutathione. No incorporation of label from [14C]glutamate and [14C]formate was observed, leaving the origin of the five-carbon unit still in doubt.