Cyclocryotherapy for glaucoma after penetrating keratoplasty. Thirty-six eyes underwent cyclocryotherapy after penetrating keratoplasty for medically unmanageable elevated intraocular pressures. Thirty were controlled with one procedure, five required three procedures. Eighty-two percent of the transplants remained clear after cryotherapy and 19 of 23 with clear grafts obtained useful visual acuity. Our results including the number of patients whose glaucoma was controlled by this procedure and the percent of clear grafts following cryotherapy are far better than results of other forms of glaucoma surgery after keratoplasty. The complication rate was 14% for all procedures. Most procedures were performed within 12 weeks of the corneal transplant. Cyclocryotherapy is necessary for the successful management of glaucoma after penetrating keratoplasty to avoid graft failure and must be performed if acceptable visual acuity is to be attained.


Endogenous Aspergillus endophthalmitis occurring after kidney transplant. Two months after renal transplantation, a 26-year-old man developed pneumonia that was recalcitrant to antibiotic therapy and proved by biopsy to be due to cytomegalovirus and Aspergillus fumigatus. Ten days later while on amphotericin B therapy, he developed an endophthalmitis proved by smear and culture of a vitreous aspiration to be caused by A. fumigatus. Despite intravitreous and systemic amphotericin B the vision deteriorated and the eye was enucleated. Microscopic examination disclosed an intense endophthalmitis with vitreous and retinal abscesses. The second patient was a 29-year-old woman who developed severe hypertension and graft rejection one month after renal transplant, despite massive immunosuppressive therapy with prednisone, azathioprine, and cobalt 60 irradiation. She developed pneumonia, meningitis, and died. A postmortem examination revealed disseminated aspergillosis. A single choroidal abscess due to Aspergillus with an associated retinal hemorrhage was observed in the left eye.


The pulmonary inflammatory response. Cellular events in experimental pulmonary arterial hypersensitivity disease. Horseradish peroxidase (HRP) or bovine serum albumin (BSA) were covalently linked to polyacrylamide or agarose beads and were injected into control Syrian hamsters and hamsters previously immunized with either HRP or BSA. Animals sensitized to soluble antigen and subsequently challenged intravenously with the same antigen immobilized on beads developed an acute focal inflammatory response within 2 to 6 hours after injection. The acute response involved local deposition of IgG and complement (beta1A/beta1C globulin), polymorphonuclear leukocyte exudation, and variable amounts of hemorrhage. A focal vasculitis was occasionally present. Within 72 hours the reaction had become largely mononuclear or granulomatous in nature, and giant cell formation was seen within 4 days after immobilized antigen injection. Severe reactions developed only upon recognition of specific antigenic determinants; thus hamsters immunized against soluble HRP developed characteristic lesions only upon intravenous challenge with HRP-coated beads but not with beads coated with unrelated antigen (BSA). The beads elicited only a mild foreign body reaction in the control hamsters at 5 to 7 days after injection which was temporally and histopathologically distinct from the lesions in immunized hamsters. Thus, the state of existing immunity can influence the character and severity of the local pulmonary inflammatory response.


A primate model of hyperacute renal allograft rejection. Hyperacute renal allograft rejection is initiated by primary immune injury to vascular endothelium and is propagated by secondary vasoconstriction, platelet aggregation and intravascular coagulation. Previous dissociation of these primary and secondary events, with graft survival in one human, suggested that the more usual graft failure was due to secondary injury. As a basis for further modification studies, this primate model most closely resembled its counterpart in man, as the onset and intensity of functional, morphologic and biochemical alterations were similar. Unmodified allografts failed within 5 minutes. The earliest and most abnormal finding was marked reduction in renal blood flow affecting all compartments. By 5 minutes, histologic changes of hyperacute rejection as well as antibody and faint C3 deposits were noted, but biopsies suggested that the initial flow reduction was more likely due to vasoconstriction, which was then followed by vascular obstruction. Glomeruli appeared most damaged, but at the highest antibody titer arterial injury was more prominent. Early red cell sequestration and stasis was marked, followed by progressive platelet clumping and neutrophil infiltration. While the decline in renal venous C3 levels was prompt, as in man, early intrarenal activation of the coagulation, fibrinolytic and kinin-forming systems could not be demonstrated, and fibrin formation was sparse by light and fluorescence microscopy. Qualitatively similar histologic and functional alterations were noted in autograft controls. While the initiating event was unclear and may have been accentuated by the arteriovenous shunts utilized, the final mechanism was probably marked vasoconstriction with renal ischemia. Intrarenal C3 consumption was an important finding and was not associated with tissue deposits of antibody or complement; it may provide a parallel with the progressive complement-mediated injury associated with acute myocardial ischemia noted by others. Endothelial injury was not seen in arteries, and all alterations were delayed in onset and progressed more slowly than in allografts. These findings may elucidate the mechanism of early malfunction of most autografts. Treatment of additional autografts with increasing doses of heparin progressively reversed these changes and even prevented the initial reduction in blood flow. Therefore, many alterations consistent with hyperacute rejection which are probably immediately responsible for graft failure can also be initiated by nonspecific, nonimmunologic events and, where injury is less intense, can be prevented pharmacologically. This model provides a means of dissecting the injurious events and subsequent evaluation of the effectiveness and interaction of various agents on the damaging secondary alterations which occur during hyperacute rejection.


Medical practice without antianxiety drugs. The benzodiazepines and hydroxyzine were removed from a statewide Medicaid pharmacopeia. Barbiturate sedatives, hypnotic and antipsychotic medication, and tricyclic antidepressants remained available, but no substitution was made for about two-thirds of the deleted ataractic medications. The authors conclide that most physicans regard ataractics as unique and do not use them interchangeably with the other drugs.


Reoperation after esophagomyotomy for achalasia of the esophagus. Eleven patients who required reoperation for persistent or recurrent symptoms after esophagomyotomy are reported on. Failure of the original operation was attributed to inadequate myotomy in three, healed myotomy in four, and reflux esophagitis in four. The myotomy was extended or a new myotomy created in the first two categories of patients. Three of the four patients with reflux esophagitis were treated by antrectomy and Roux-en-Y esophagojejunostomy; one of these also required concomitant exision of the esophagogastric junction because of stricture. The fourth patient with esophagitis ultimately required colonic interposition for relief of symptoms. Ten of the eleven patients were improved after reoperation, but results were less good than those achieved by a properly performed primary esophagomyotomy. This -re-emphasizes the need for attention to the technical details of esophagomyotomy if good results are to be achieved.


Clinical applications of thermodilution cardiac output determinations. The pulmonary arterial catheter was shown to be safe and easily positioned in a group of 200 patients. Thermodilution cardiac output measurement was shown to be simple, rapid, and safe. The technic was as accurate as the simultaneously measured indocyanine green cardiac output, and more reproducible, particularly at high and low flows. The construction of myocardial performance curves during a fluid challenge is described. The value of these curves is guiding fluid, drug, and respirator therapy is illustrated by case presentations.


[Experimental erysipelas in different species as a model for systemic connective tissue disease. I. Systemic vascular processes during organ manifestation (author's transl)]. The similarities between erysipelas in animals and rheumatic diseases in man have been discussed since the work of Nieberle (1931). The present work sets out to investigate the course of organ manifestations in pigs, rats, and mice using germ-free or specific pathogen-free experimental animals. Particular consideration will be given to the initial systemic vascular processes as well as to the significance of the erysipelas antigen. In several experiments, a total of 166 pigs--partly gnotobiotic or specific pathogen-free animals--37 specific pathogen free Wistar rats and 57 albino mice were orally and/or parenterally infected with standardized erysipelas strains of serotype B. Clinical examination post infection were carried out with the EKG and by x-raying the joints of the extremities. All large parenchymatous organs, as well as heart valves, aorta and synovia were examined histologically in paraffin sections. In mice and rats, joints of the extremities were embedded in toto in metracrylate. Besides various histological staining methods, histochemical reactions were used to demonstrate mucopolysaccharides and fibrin. The myocardium, central nervous system and synovia of several joints were examined with the electron microscope. In the pig, immunohistological methods demonstrating the presence of fibrin, complement and IgG, as described by Seidler et al. (1971) and Trautwein et al. (1972), were used. The most important changes in joints, heart valves heart musculature and blood vessels occur during the early bacteriemic phase. A distinct sticking effect develops in the mouse 3.5 hours p.i., in the rat 24 hours p.i., and in the pig 36 hours p.i. Simultaneously, hyaline thrombi occur in capillaries and venules; these are seen as parallel, loosely-packed fibrin fibers in the electron microscope. With the aid of immunofluorescence fibrin, IgG and complement C3 can also be demonstrated here. Exudates rich in fibrin develop parallel to the microthrombosis. In pigs and rats vascular and myocardial necroses develop to 3  days p.i. The mice do not survive the 3rd p.i. 39% of the pigs showed edema and mesenchymal activation of varying intensity in the heart valves between the 3rd and 8th day p.i. Besides the insudation of the valves, endocardial thromboses developed in 80% of the mice. Endocarditis, aand in addition large aortic thromboses were recognized in more than 50% of the rats. As early as the 4th day p.i., coagulopathy, angionecrosis and exudation led to acute arthritic symptoms..


[Corticosteroid cataracts following kidney transplantation. Investigations on the influence of additional factors upon the development of opacities (author's transl)]. 15 patients with kidney transplants were followed up for a period of 3 to 45 months. All but one developed lenticular opacities in the posterior subsapsular region. The opacities were classified according to their severity. It could be shown that the cataract index was correlated to the total amount of corticosteroids given. There is probably an additional effect of age and azathioprin therapy. Possibly, the time of treatment with intermittent hemodialysis also influences cataract development.


[Clinical studies on the narcotic effects of nitrous oxide (author's transl)]. In 340 male and 450 female patients of various age the depth of anaesthesia acquired during induction with nitrous oxide (75%) and oxygen (25%) was investigated by means of clinical methods. Premedication consisted of atropine (0,1 mg/1o kg body weight) in 26% and atropine plus pethidine (or morphine) in 74% of the patients, respectively of the total, 2.4% of the patients failed to enter the analgesic-amnestic level, determined as I-3, while 97.6% occupied and more than 50% of the patients surpassed it. Thus the MAC of nitrous oxide in man seems to be 75 volume per cent or less. The depth of anaesthesia varied significantly with duration of inhalation (p smaller than 0.001). Up to five minutes of inhalation 5% of the patients failed to arrive at I-3, while 95% reached this plane and 46% surpassed it. After more than five minutes of inhalation the corresponding values were 1%, 99% and 56%, respectively. Significantly deeper levels of anaesthesia were acquired by females than by males (p smaller than 0.001) during the initial 10 minutes of inhalation. After that time sexual differences disappeared. Patients of good physical condition reached deeper planes of anaesthesia than bad risk cases (p smaller than 0.001). The use of analgesics as premedicants reduced the effectiveness of nitrous oxide, as compared to premedicated patients with atropine alone (p smaller than 0.01 in men and p smaller than 0.05 in women). Adults below 30 years of age entered the stages II and III-1 significantly less frequently than older patients (p smaller than 0.01), whereas children (up to 14 years of age) reached those stages significantly more frequently than adult patients in general (p smaller than 0.01).


Attention, learning, and personality during ketamine emergence: a pilot study. To analyze the memory, learning, attention, and personality effects associated with emergence from partial ketamine induction, 11 resident physicians were tested. Ketamine was found to be truly dissociative in that it seemed to impair learning of and memory for unusual word associations, at least in young physicians.


Alupent syrup: results of a six-month trial in asthmatic children. A six-month clinical trial of Alupent Syrup was conducted in 23 school-age children with bronchial asthma to assess the long-term efficacy and safety of this dosage form of metaproterenol sulfate. At three intervals during the study period, double-bind crossover tests against placebo were performed. Pulmonary function measurements revealed significant improvement in PEFR and FEV, after metaproterenol, and the results obtained at the end of ths study demonstrated that no tolerance to the drug had developed. The absence of adverse reactions attested to the safety of the drug in long-term management of childhood asthma.


The adult respiratory distress syndrome and pancuronium bromide. The institution and maintenance of artificial ventilation for a patient with the adult respiratory distress syndrome (ARDS) is frequently difficult because the hypoxic patient is often confused, agitated, or combative. Pancuronium bromide, a recently introduced neuromuscular blocking agent, was used to facilitate artificial ventilation in 6 patients with ARDS. As compared with morphine sulfate and diazepam, pancuronium bromide has fewer cardiovascular and central-nervous system effects. It also has fewer cardiovascular effects than does curare, and its prolonged administration does not lead to a desensitization neuromuscular block as is seen with succinylcholine. However, continuous high-quality nursing care is necessary if this drug is used.


Binding of anti-human immunoglobulins to human epidermal cells. According to this study human skin scrapings bind anti-human IgE and IgM immunoglobulins as demonstrated by fluorescent miscroscopy; this is not dependent on the atopy of the subject.


Immune response to Escherichia coli.

Immune response to Escherichia coli. Chickens from a line artifically selected for lew level of plasma corticosterone (LPC) produced higher antibody titers, produced antibody earlier, continued the production of antibody longer, responded to lower dosages of antigen, and had greater body weight gain than those from a line slected for a high level of plasma corticosterone (hpc). the HPC line resisted Escherichia coli challenge and antigen more effectively than the LPC line. It appears that, although the HPC line is more effective in its defense against bacterial infections, the LPC line can be aided more by vaccines.


Properties of free endotoxin from Pasteurella multocida. Free endotoxin (FET) from virulent encapsulated Pasteurella multocida or from an avirulent nonencapsulated mutant is capable of inducing active immunity, but the lipopolysaccharide (LPS) moiety of the endotoxin is not. These results suggest that a protein of P multocida is involved in the stimulation of active immunity. The serologic specificity of the FET is associated with the LPS moiety, which is related to a heat extracted antigen that is used for serotyping P multocida. The FET is capable of producing widespread vascular alteration and death. It is present in the vascular system of turkeys with acute fowl cholera, and it can be detected with the "Limulus" test for endotoxins and with the gel diffusion precipitin test.


Avian salmonellosis: detection and characterization of serologic response to major serogroup infections. Serologic procedures for the diagnosis of salmonella infections in poultry flocks have evolved from laboratory and field macrotest procedures to recently reported microtests with savings of cost, time, and space. Microtest methods when used as the sensitive and specific microantiglobulin test provide a means of effectively detecting birds exposed to salmonella serogroup B, C, and D infections. Progress will be made in the better detection and control salmonella infections in poultry when attention is given to all major salmonella serogroups rather than a few selected serotypes.


Progress in the control of coccidiosis with anticoccidials and planned immunization. New anticoccidials continue to be introduced to the poultry industry on an average of about one every 2 years. Revised requirements have increased costs of discovery, development, and approval by the Food and Drug Administration to several million dollars for each product. Gross sales of anticoccidials are now estimated to be in excess of $30,000,000 annually. Anticoccidials are almost universally used in starter rations for meat-type birds being raised under floor-pen management. Protection is more important with these fast-growing birds than with egg-producing types where immunity and cage layer management modify demands for anticoccidials. Coccidiosis mortality has generally been eliminated by all 25 approved anticoccidials if properly used. Selection of specific anticoccidials is based on the ability of the anticoccidial to: (1) improve weights and (2) feed conversion, and (3) to suppress development of lesions. Costs of the product may influence decisions on which one to use. With some anticoccidials the rapid emergence of drug resistant strains has been the biggest problem. "Switching" among the 13 classes of anticoccidials and the so-called "shuttle program" are common methods used to avoid development of resistance. Preventive medication will probably continue to constitute the major method of coccidiosis control with meat-type poultry if attempts to develop economic cage-type management do not supplant current litter and floor-pen management. Immunity acquired through incidental or planned immunization is more important in control of coccidiosis with layer and breeder flocks raised in floor-pens. Some progress in development of avirulent immunogenic strains of different species of coccidia has been reported and further research efforts in this direction should be encouraged.


Pathogenesis of Trypanosoma equiperdum in rabbits. Seventeen rabbits experimentally infected with Trypanosoma equiperdum were examined for lesions at different times during a 5-month period. A chancroid developed at the inoculation site in the skin, and later, similar lesions appeared in skin of the ears, eyelids, and nose. The inflammatory reaction was primarily granulomatous, and viable trypanosomes were present in all skin lesions. The rabbits had reticuloendothelial hyperplasia of spleen and lymph nodes. In 3 rabbits, there was amyloid deposition in splenic lymph nodules and renal glomeruli. Amyloid had typical fibrillar appearance by electron microscopy (EM) and was reactive for immunoglobulin M (IgM) and immunoglobulin G (IgG) with fluorescent antibody (FA). With time, there was lessening capability of T equiperdum to agglutinate in antiserums of infected rabbits, suggesting that antigenic variation occurred during infection. The serum concentrations of IgM and IgG increased significantly in the infected rabbits and then remained elevated or they decreased, though fluctuating widely. Three of 5 infected rabbits demonstrated depressed antibody response to injected ovine erythrocytes.


Evaluation of an indirect method for estimating myofiber number in transverse sections of skeletal muscle. The total number of myofibers contained in the transverse sections of 6 canine pectineus muscles was counted, using direct- and indirect-counting methods. For the direct-counting method, the transverse section of each muscle was photographed, from which a montage of each section was constructed. Then, the total number of myofibers contained in each montage was counted. The number obtained by the direct-counting method was used to determine the accuracy of the indirect-counting method. For the indirect-counting method, 40 randomly selected areas in each section were photographed. Then, the total number of myofibers contained in the randomly selected areas of each section was counted. From these counts, the total number of myofibers contained in each transverse section was calculated after the entire cross-sectional area of each section was determined by planimetry. The indirect-counting method was performed 3 times for each section. By direct count, the number of myofibers in a section was between 12,368 and 163,223. Generally, the indirect-counting method tended to overestimate the number of myofibers per section. The mean percentage error of estimates plus or minus standard error for all muscles and trials was 3.89 plus or minus 1.17; the range was minus 6.17 to +11.97. The percentage errors of the indirect counts were significantly correlated with the number of myofibers/sq mm and total number of myofibers/section; for every 1,000 myofibers/sq mm or 100,000 myofibers/section, an error of 2 or 5%, respectively, can be anticipated. The indirect-counting method utilized 10 to 20% of the man-hour effort required by the direct-counting method.


Immunofluorescent studies on chronic pneumonia in swine with experimentally induced African swine fever. Chronic pneumonia experimentally produced in 14 pigs with African swine fever (ASF) virus was studied by immunofluorescene (IF) and histopathologic techniques. Frozen sections prepared from pulmonary tissues of the infected pigs were stained with fluorescein-conjugated antiserums against ASF viral antigen, porcine immunoglobulin G (IgG), procine complement (C), and porcine fibrinogen. The viral antigen(s) was mainly seen in macrophages and cell debris in alveolar walls and lumens. This finding indicates that the virus replicated in the cytoplasm of alveolar macrophages that subsequently degenerated and released the viral antigen. Diffuse immunoglobulin (Ig) deposition was found in necrotic cells and debris. Immunoglobulin also was seen bound to intracytoplasmic inclusion bodies in some degenerating alveolar macrophages. This finding indicates that antibody against ASF viral antigen(s) excluded from blood circulation or produced by local immunocytes (or both) reacted with viral antigen at intramacrophage and extramacrophage levels and resulted in the formation of insoluble antigen-antibody (Ag-Ab) complexes. The participation of C in the immune complex was evident in the early stage of the pneumonia, but was less evident in the subsequent extensive, progressive necrotic processes. Fibrin deposits were visible only in the early necrotic area of alveolar walls and lumens. Possible mechanisms inducing extensive necrosis are discussed.


Development of specific in vitro lymphocyte responses in cattle infected with Mycobacterium bovis and with Mycobacterium avium. Two calves infected with Mycobacterium avium and another 2 with Mycobacterium bovis were studied for development and specificity of lymphocyte activation responses and skin reactivity to purified protein derivatives (PPD). Lymphocytes cultured in vitro from infected calves manifested significant uptake of tritiated thymidine in response to the PPD prepared from the homologous organisms, whereas heterologous PPD produced little or no stimulation. The time of appearance of specifically sensitized lymphocytes was studied, as was the specificity of skin reactivity.


Interacinar pathways in the human lung.

Interacinar pathways in the human lung. Normal lung specimens from patients 18 to 86 years of age were inflated, fixed, and cleared. After micropuncture of the distal airspaces and injection of silicone rubber, the dissemination pattern was studied by cinematography. Free interacinar flow was commonly observed. The major pathways of spread among adjacent acini were the interacinar ducts. These were short, tubular structures 200 mum in diameter that were continuous with respiratory bronchioles and alveolar ducts. The flow of silicone rubber was impeded only by the septa of the secondary lobule of Miller. Our finding support the view that the smallest morphologic unit of airspace disease is more likely to be the secondary lobule than the acinus.


Cavitary pulmonary cryptococcosis complicated by aspergilloma. A roentgenogram of a 31-year-old office worker who had developed pleuritic pain showed multiple abscess cavities in the left lower lobe, and a clinical diagnosis of cryptococcosis was considered because of cryptococci recovered from a sputum smear and a positive indirect fluorescent antibody test. Lobectomy (after unsuccessful amphotericin therapy) proved the presence of a destructive cryptococcosis with Aspergillus proliferating in the cavities.


Effect of continuous positive pressure ventilation on cardiac output. Ten mongrel dogs were placed on 5 and 10 cm. H20 of CPPV. Cardiac output, pulmonary vascular resistance and pleural pressures were measured. At 5 cm. H20/CPPV there was no significant change in cardiac output or pulmonary vascular resistance. At 10 cm. H20 CPPV cardiac output significantly decreased and pulmonary vascular resistance increased. The physiologic basis for these changes is discussed and related to the clinical situation.


Changing surgical indications in azotemic secondary hyperparathyroidism. It would appear that the initial enthusiasm for surgery in azotemic secondary hyperparathyroidism was unsupported by conclusive data, but that a therapeutic alternative recommending surgical abstinence represents an equally extreme position, particularly since the long-term effects of sustained mild hyperparathyroidism are unknown. The proper course at present for the patient's best interests might best be described as selective. Final resolution of conditional therapy in azotemic hyperparathyroidism awaits the completion of objective studies.


Penetrating keratoplasty in a patient with 75% third degree burns. A 25-year-old woman was severely burned in a gasoline explosion in her home. One month after her admission to the hospital a mycotic corneal ulcer, caused by Candida albicans, developed and progressed to a descemetocele in spite of medical therapy. A therapeutic penetrating keratoplasty was performed and the graft remained clear until the present time (10 months of follow-up). The one episode of clinical corneal graft rejection occurred at the same time the first evidence of return of immune competence was seen in her skin grafts. The corneal rejection episode was successfully treated by topical corticosteroids.


A simple levator resection operation.

A simple levator resection operation. A simple procedure for identification and isolation of the levator muscle is presented. With this technique the conjunctiva need not be cut from its tarsal connection. No extra sutures are used to create a lid fold. It is emphasized again that the criterion for the amount of levator resection is not the degree of ptosis but the amount of levator action.


[Right ventricle with double outlet associated to total anomalous pulmonary venous drainage to innominate vein]. The clinical findings of 2 patients are studied with an association of malformations not previously reported: double outlet right ventricle and total anomalous pulmonary venous connection to the innominate vein. The following features are worth while to be underlined: 1. Both patients showed a discordance between viscero-atrial situs and direction of the apex; 2. In both cases, particularly in case 2, there existed a very unusual spacial relationship between ventricles, the right ventricle being superior with respect to the left ventricular cavity; 3. From the embryological viewpoint, this ventricular arrangement is supposed to be due to an arrest of the development of the heart in the straight tube stage, there being, however, an upwards expansion of the atrioventricular canal and common atrium; 4. An accurate diagnosis of the common or divided nature of the atrioventricular valves has been impossible in these cases; 5. These combination of lesions constitutes a challenge to cardiologists and surgeons as far as the surgical indication is concerned. In that sense, the simple anastomosis of the common pulmonary vein to the left atrium, although is not a radical cure, may be a feasible and beneficial procedure.


Benign paroxysmal positonal vertigo: A clinical study. Summary--Ru-Vert, a combination product, containing in each tablet, 25 mg of pentylenetetrazol, 12.5 mg of pheniramine maleate, and 50 mg of nictonic acid, was evaluated in the treatment of seventeen patients with benign paroxysmal positional vertigo. The study was double-blind with crossover. Ru-Vert at a dosage of two tablets t.i.d. was found to reduce significantly the nystagmus and the vertigo induced by Hallpike maneuvers in these patients.


Serum and urine polyamines in cancer.

Serum and urine polyamines in cancer. Polyamines in serum and urine have been found to be elevated in patients with cancer. A variety of methods employed for these measurements are discussed. Normal values obtained by the most recent methods are presented, and a survey has been made of polyamine levels in cancer patients.


McArdle's disease: a review.

McArdle's disease: a review. The clinical features of McArdle's disease (inherited deficiency of skeletal muscle phosphorylase) and the histological and biochemical changes are described. Their possible causes are discussed in the light of recent knowledge of the biochemistry of muscular contraction. Diagnostic tests are detailed. Attention is drawn to the possibility that the disease may be due to primary defect of motoneurons.


Current status of zinc deficiency in the pathogenesis of neurological, dermatological and musculoskeletal disorders. Recent clinical and experimental studies suggest that zinc deficiency may play an important role in the pathogenesis of (1) acrodermatitis enteropathica, and in certain cases of (2) hypogonadal dwarfism, (3) congenital malformations, (4) hypogeusia and hyposmia, (5) nyctalopia and (6) impaired wound healing. Distrubances of zinc metabolism also occur in a broad spectrum of other clinical disorders. The pathophysiological factors which are responsible for hypozincemia include: (1) nutritional deficiency and/or intestinal malabsorption of zinc; (2) hyperzincuria secondary to aminoaciduria; (3) hormonal effects (cortisol, growth hormone, estrogens); (4) hypoalbuminemia; and (5) effects of leukocytic endogenous mediator. The clinical diagnosis of zinc deficiency in patients with specific neurological, dermatological and musculoskeletal disorders is complicated by the complex interactions of these pathophysiological factors and by the need for more dependable laboratory indices of zinc deprivation.


The measurement of erythropoietin.

The measurement of erythropoietin. The bioassay of erythropoietin in animals is reviewed. The techniques employed are those of hypertransfusion and hypoxia using polycythemic mice.


[Effect of antibiotics on the indices on nonspecific immunity]. Multiple (for 14 and 20 days) administrations of antibiotics, such as tetraolean, tetracycline and penicillin from the beginning of the animal immunization (rabbits and albino rats) with typhoid fever and candidosis vaccines inhibited activity of both the non-specific and the specific immunity.


[Caffeine as an inhibitor of the conjugation transfer of R-factors. A study of the quantitative effect of caffeine on the conjugation transfer of R-factors]. The effect of caffeine on the conjugation transfer of R-factors was studied using a standartized kinetic conjugation system described earlier. It was shown that caffeine had a pronounced inhibitory effect on R-factor transfer. The inhibition coefficients at a caffeine concentration of 2000 gamma/ml were from 2.5 (when using strain CSH-2 R222 of E. coli as the donor) to 13.8 (when using strain J5-3 RI drd of E. coli as the donor). Higher concentrations of caffeine induced a significant increase in the coefficient. Since R-transfer inhibition was induced by rather high concentrations of caffeine, the effect of equivalent concentrations of some normal metabolites of the purine and pyrimidine series, such as ATP, guanine, cytosine and thymine was studied. It was shown that the substances had no inhibitory effect on R-factor transfer. Inhibition of P-transfer by caffeine did not depend on either the type of R-factor or the microbial host.


[Study of the conditions for mass transfer in the nystatin biosynthetic process]. The kinetics of the biomass growth, nystatin production and carbohydrate consumption in cultures of act. noursei in 180 liter fermenters was studied. Under conditions of intensive agitation (Klas greater than 6) dependence of the specific productivity on the specific rates of growth and carbohydrate consumption was found. No such dependence between the above parameters were observed under conditions of poor agitation (Klas smaller than 3.5). It was shown that at the beginning of the cultivation process the coefficient of oxygen transfer Kla rapidly decreased. Later the value of Kla decreased slightly. Dependence of the maximum activity of the culture fluid on the average coefficient of masstransfer with respect to oxygen was observed.


[Study of the effect of nystatin and mycoheptin on the intrauterine development of the rat fetus]. The effect of nystatin and mycoheptyne on pregnancy and fetus of rats of Wister line was studied after single and multiple oral administrations of the drugs. Nystatin was used in single doses of 3000, 500 and 100 mg/kg. In chronic experiments the dose was 500 mg/kg. It was found that nystatin had a slight abortive effect when used during the whole period of pregnancy. Mycoheptyne was used in single doses of 1000, 100 and 20 mg/kg. In chronic experiments the dose was 100 mg/kg. It was noted that mycoheptyne had a higher toxic effect on the pregnant rats than nystatin. The animal death and abortus were observed more often. The level of the changes depended on the dose, time and period of the antibiotic use. No defects in the rat fetus were found.


["Typical" and "atypical" damages of the organ of hearing from the administration of streptomycin]. Two areas of the damage localization in the organs of hearing after streptomycin use were found in the experiments with guinea pigs, i.e. (1) the Corti's organ of the lower part of the basal helix, the zone of perception of the maximum frequency sounds and (2) the tympanic muscles. Administration of streptomycin subcutaneously in a daily dose of 300 mg/kg for 50 days resulted in pronounced atrophic changes in the fibres of m. tensor tympani and some atony of m. stapedius. On the basis of the histological examination of the tympanic muscles after prolonged use of streptomycin and comparison of the periods of the tonus reduction in the skeletal muscles and Preier reflex after a single administration of streptomycin in maximum tolerating doses, it was concluded that streptomycin affected the state of the tympanic muscles as a myorelaxant. The atrophic changes in the tensor as a result of prolonged streptomycin use was due to chronic atony of the muscle. The decrease in the contraction capacity of the tensor must result in loosening of the drum membrane tension, impairement of the muscle activity coordination, decreased ability for elimation of the auditory ossicle fluctuation. Increased audibility limits within middle and low frequencies and noise in the ears during prolonged treatment of patients with streptomycin may be associated with chronic atony of the tympanic muscles and mainly tensor.


Toxigenic thermophilic and thermotolerant fungi. Twenty-three isolates of fungi, representing 13 thermophilic and thermotolerant species, were bioassayed for toxigenicity to brine shrimp, chicken embryos, and rats. Thirteen isolates representing nine genera were highly toxic to at least two of the three systems. Seven additional isolates of five genera were slightly toxic.


Rapid determination of the amount of cetylpyridinium chloride bound by bacteria. A modification of the Colowick and Womack procedure for measuring ligand binding by macromolecules is described for drug binding by bacteria. This technique is based on the determination of drug concentration in the dialysate from a bacteria-drug mixture at equilibrium. The dialysis cell of the original procedure was replaced by a Minibeaker (Bio-Rad), which has a larger membrane surface area, and the dialysate was continuously monitored with a spectrophotometer equipped with a flow cell. With this system, only 3 min was required to determine the amount of cetylpyridinium chloride bound by Escherichia coli K-12 strain P678. Possible applications of the technique are discussed.


Improved membrane filter method for fecal coliform analysis. A two-layer agar method has been developed which consistently yields higher recovery of fecal coliforms on membrane filters when compared to the existing membrane fecal coliform procedure. This method has been evaluated by three laboratories using samples of raw and chlorinated waste water, and reservoir, river, and marine waters. Verification of 1,013 fecal coliform colonies isolated from 61 water samples averaged 92% on this proposed procedure. Comparison with the Standard Methods membrane fecal coliform procedure revealed the two-layer agar method had an overall increased sensitivity to fecal coliform detection in these waters. It is therefore proposed that this procedure be evaluated as an alternative to the Standard Methods fecal coliform membrane Filter test in the examination of chlorinated secondary effluents, marine waters, and any natural waters that may contain pollutants with heavy metal ions.


Violet red bile 2 agar for stressed coliforms. Counts on a new, autoclave-sterilizable violet red bile (VRB-2) agar were compared with counts on freshly boiled VRB agar. Yields on VRB-2 agar averaged 217, 180, 130, and 112% of counts obtained on the control medium for samples of water, cottage cheese, frozen vegetables, and raw milk, respectively. The general principle used for the development of VRB-2 agar could be applied to many other kinds of selective plating media.


Repair and enumeration of injured coliforms by a plating procedure. Surface plating of coliforms on Trypticase soy agar, followed by 1 to 2 h of incubation at 25 C and subsequent overlay with violet red bile agar, was found to be a useful method for the repair and enumeration of coliforms injured by freezing.


Jejunal microflora in malnourished Gambian children. Growth of bacteria greater than 10-5 organisms/ml was found in 22 children, of whom 17 gave a histroy of chronic diarrhoea. The other 8 children had either no diarrhoea or where having an acute attack lasting for a few days. In those with chronic diarrhoea, Esch. coli, bacteroides, and enterococci tended to occur more frequently, whereas streptococci occurred more frequently in those with acute diarrhoea. Bacilli, staphylococci, micrococci, klebsiellas, pseudomonads, and candidas often occurred in both groups and in large numbers in those with chronic diarrhoea. This confirms previous reports in other parts of the world that some children with malnutrition have considerable bacterial contamination of the jejunum, and that this may be of aetiological significance as a cause of much of the diarrhoea seen in malnourished children. It is possible too that this may be important in the pathogenesis of malnutrition. The presence of intestinal parasites in these malnourished children is also noted. A double-blind trial in the use of antibiotics in this condition is advocated to determine whether it is possible to break the diarrhoea-malabsorption-malnutrition cycle. At the same time the effect of simply removing the child to a more sanitary environment, together with an estimate of the natural clearance of bacteria from the upper intestine, should be evaluated.


Phagocytosis and killing ability of Candida albicans by blood leucocytes of healthy term and preterm babies. The phagocytosis and killing ability of leucocytes of 24 term and 22 preterm babies against Candida albicans were studies during the first 20 days of life because of the increased incidence of monilia infection at this time. The leucocytes of 14 adults aged 20 to 30 years served as controls. The phagocytosis ability of the leucocytes in adults, term, and preterm babies was not significantly different, mean values being respectively 66-7%, 57%, and 56-9%. The killing ability of the leycocytes in term and preterm babies was lower when compared with that of adult leucocytes (P less than 0-001 for term and P less than 0-01 for preterm infants). The mean value in adults was 27-5%, in term infants 9-7%, and in preterm infants 9-5%. It is suggested that as the addition of adult serum did not improve the candidacidal ability of newborn leucocytes, the killing defect should be sought in the leucocyte itself and not in serum factors.


Macrophage-lymphocyte clustering in rheumatoid arthritis. The cells in synovial fluid from patients with rheumatoid arthritis contain a small percentage of macrophages. Such macrophages were isolated and cultured alone and with homologous and heterologous lymphocytes for 24 hours, in an attempt to identify possible contact between living lymphocytes and macrophages. Such contact was found, with clustering of lymphocytes around macrophages, and was particularly well shown by scanning electron microscopy.


Suppression of malaria with monthly administration of combined sulphadoxine and pyrimethamine. A trial of suppression of malaria by administration of combined sulphadoxine-pyrimethamine tablets every 28 days was undertaken in West Malaysia during 1972. One thousand subjects were followed over a 10-month period, including control groups on placebo and on weekly chloroquine. Subjects were examined monthly for parasitaemia, drug reactions, leucopenia, teratogenicity and haemolysis among the subjects deficient in glucose-6-phosphate dehydrogenase. Rates of new infections in the placebo group were 8.0% with Plasmodium falciparum and 6.2% with P. vivax; in the group receiving weekly chloroquine, 5.1% P. falciparum and 0.3% P. vivax; and in the group receiving monthly sulphadoxine-pyrimethamine, 0.3% P. Falciparum and 1.0% P. vivax. The effective rate of cure of new infections with P. falciparum by a single suppressive dose of combined sulphadoxine-pyrimethamine given the following month was 88.7%. No serious side effects were observed.


Liver disease in rheumatoid arthritis and Sjøgren's syndrome. Prospective study using biochemical and serological markers of hepatic dysfunction. Inter-relationships of biochemical and immunological tests of liver function have been studied in a prospective study of 216 patients with rheumatoid arthritis (RA), 32 patients with Sjogren's syndrome, and 27 patients with the sicca syndrome, and these results have been compared with those obtained 289 patients with osteoarthrosis or with a form of seronegative polyarthropathy. In general the prevalence of abnormalities in serum alkaline phosphatase, bromsulphthalein excretion, smooth muscle antibody, and mitochondrial antibody in the former three groups was higher than in patients with osteoarthrosis. Patients with Sjogren's syndrome with RA had a higher prevalence of abnormalities of bromsulphthalein excretion, salivary duct antibody than patients with the sicca syndrome. Patients with RA had a higher pervalence of rheumatoid factor than those with the sicca syndrome. Patients with a positive smooth muscle or mitochondrial antibody were found to have a higher prevalence of hepatomegaly and splenomegaly, of abnormal liver function tests, of other autoantibodies, and of histological abnromalitis of liver than those in whom these tests were negative.


Metrifonate in urinary schistosomiasis. A field trial in northern Nigeria. A field trial of metrifonate in the form of Bilarcil tablets (Bayer) was carried out in 39 northern Nigerian schoolchildren heavily infected with Schistosoma haematobium. Three oral doses of 7.5 mg/kg were given at four-week intervals. The drug proved effective (mean reduction in egg counts eight weeks after completion of treatment 94.5%) and was excellently tolerated. Its use on a larger scale is suggested.


Management of acute aortic dissection.

Management of acute aortic dissection. The therapy for acute dissecting aneurysm of the aorta remains a difficult problem for thoracic surgeons. Because of an excessive operative mortality in patients with acute dissection who were operated on within 24 hours of hospital admission, we have utilized intensive medical management to delay surgical intervention. Even patients with acute aortic insufficiency can be supported medically, allowing their operations to be delayed at least 3 weeks or longer. Since this policy has been implemented, there has been no operative mortality in our last 13 patients with acute dissection. Medical therapy as the definitive treatment is now reserved solely for Type III dissections or for patients who cannot be operated on for other reasons. This report outlines our rationale for therapy and our current method of managing acute dissection.


Calcified postoperative epicardial granuloma. A sterile calcified granuloma on the external surface of a right ventriculotomy in an 8-year-old boy was identified four years after closure of a ventricular septal defect; He had no symptoms, but a systolic ejection murmur was identified and disappeared after resection of the lesion. Sensitivity to silk suture material is suggested by the operative findings and a history of multiple silk suture sinuses developing in his uninfected original thoracotomy incision.


Overview of recent research in depression. Integration of ten conceptual models into a comprehensive clinical frame. Disciplinary fragmentation and nosological and semantic controversies have obscured the impressive advances made in the area of depressive disorders during the past decade. This article is an attempt to translate data derived from psychodynamic, sociobehavioral, and neurobiologic research into a clinically meaningful framework. We review ten models of depression with special emphasis on newer models supported by empirical and experimental studies, and present a new model, which incorporates and synthesizes findings from different schools. Depressive illness is conceptualized as the feedback interaction of three sets of variables at chemical, experiential, and behavioral levels with the diencephalon serving as the field of action.


Interaction of drug therapy with marital therapy in depressive patients. Comparative effects of the antidepressant drug, amitriptyline hydrochloride, marital therapy, and the drug-psychotherapy interaction effects were studied in a 12-week course of treatment of outpatient depressives. Patients were assigned randomly to four treatment groups, in a 2 times 2 factorial design: (1) drug-marital therapy; (2) drug-minimal contact; (3) placebo-marital therapy; and (4) placebo-minimal contact. Both drug and marital therapy showed substantial beneficial advantages over their control conditions, but drug therapy was faster and generally superior in symptom relief and clinical improvement. Marital therapy was superior in family role task performance and perception of the marital relationship. For reducing hostility and enhancing the perception of the marital relationship, drug therapy had a better early effect, but marital therapy had superior effects by the end of treatment.


Antidepressants in black and white inpatients. Differential response to a controlled trial of chlorpromazine and imipramine. Differential effects of chlorpromazine, imipramine hydrochloride, and a placebo were examined in 159 black and 555 white depressed patients in a multihospital collaborative study. In making these comparisons, the effects of age and social class were controlled. The major study findings were the differential effects of the active drugs for the black men and women. Chlorpromazine was the most efficacious treatment for black women, whereas imipramine was most efficacious for black men. These differences occurred on global ratings of improvement as well as on specific symptoms such as depression, anxiety, guilt-worthlessness, sleep disturbances, and social participation. Black patients also evidenced a higher improvement rate at one week, irrespective of treatment, than did the white patients.


An adrenergic drug in depression.

An adrenergic drug in depression. It has been suggested that depression is a disease of cholinergic dominance and since the beta-adrenergic blocking drug propranolol hydrochloride can cause depression, there arises the possibility that a beta-adrenergic stimulant could benefit the condition. For ethical reasons, the adrenergic drug metaproterenol sulfate was combined with chlordiazepoxide hydrochloride and compared to placebo and chlordiazepoxide in a formal double-blind trial. However, the results did not show any advantage for the addition of metaproterenol either in respect of enhanced antidepressant effect or a reduced incidence, nature, or severity of side-effects.


Double-blind comparison of lithium carbonate and imipramine in treatment of depression. The effect of lithium carbonate therapy on patients with depression is still unconfirmed. Our past studies have shown a favorable response to the drug in patients with depression of mild or moderate severity. Therefore, we performed a controlled double-blind study of lithium carbonate and imipramine hydrochloride in 64 patients with depression. No significant differences were noted in the overall therapeutic response, depression scale scores, or clinical effects between the two drug groups.


[The alumina oxide ceramic-metal composite endoprosthesis. A new hip endoprosthesis for partially cement free implantation (author's transl)]. The favorable combination of properties of dense, pure Al2O3-ceramics as a material for endoprostheses of the high load bearing joints have been confirmed in more than 4 years of experimental work with different types of animals. During this period Al2O3-ceramic parts for total hip replacement have already been developed and tested as well as the special instrumentation and the technique of the surgical procedure concerned. Based on these experiences a new total hip endoprostheses together with its complete technique for the operation is presented with which an improved alternative for replacements of Weber-Huggler-prostheses is offered and the application of total hip replacements can be extended to patients below 60 years of age.


Isolation of the spirochetes in the perilymph despite prior antisyphilitic therapy: a case report. For the congenital syphilitic patient to develop sensorineural deafness is uncommon. For the congenital syphilitic patient to have persistence of treponemes after treatment is rare. To demonstrate viable organisms in the inner ear perilymph of the congenital syphilitic patient, despite treatment with penicillin, has not been recorded to date. This is a case report of such a patient.


Misadventures with injectable polytef (Teflon). The intracordal injection of polytef (Teflon)-glycerine suspension has proved most effective in relieving dysphonia due to unilateral recurrent nerve paralysis. However, if errors in the technique of injection are committed or if patients are not properly selected, a potentially good result will not be achieved or the voice will be made worse.


Accuracy of the Bing and Rinne tuning fork tests. Fifty children seen in an ENT clinic received the Bing and Rinne tuning fork tests, otoscopic examination, and puretone audiometry to determine the accuracy of the tuning fork tests in predicting otoscopic abnormality or conductive hearing loss. Neither tuning fork test showed high accuracy. The Bing test overidentified by yielding negative results (conductive loss) for many ears with normal hearing or sensorineural loss. It correctly identified conductive loss or otoscopic abnormality only slightly better than chance57% to 66% of the time. The Rinne test identified with high accuracy those ears with a 40 dB or greater air-bone gap. For air-bone gaps of 10-35 dB, only 27% of the ears gave negative results, meaning correct identification. However, when a negative Rinne occurred, the result was in error only 2% of the time.


Effects of injected deposteroid on posttonsillectomy morbidity: a double-blind study. There are numerous studies using various agents to reduce posttonsillectomy morbidity. Due to lack of conclusive results a double-blind study using methylprednisolone acetate (Depo Medrol) was conducted. A total of 165 patients were randomly divided into two groups. Following tonsillectomy 2.5 ml of solution was injected into the base of each tonsillar fossa. The study group received 20 mg of deposteroid to each fossa while the control group got normal saline. The deposteroid reduced postoperative pain but did not significantly alter other factors contributing to morbidity such as difficulty in swallowing or resumption of a normal diet. The deposteroid appeared to have no effect on the rate of healing of the tonsillar fossa.


Silicone elastomer sheeting in the development of pedicle flaps. A "delay" of a pedicle flap stimulates improvement in the flap circulation, probably through partial ischemia. The degree of stimulation prsent at any given time is inversely proportional to the length of time that has passed after the delay because progressive revascularization across the delay lines reduces the ischemis stimulus. This revascularization can ve prevented by placing silicone elastomers (Silastic) sheeting between the flap and its bed.


[Influence of hypothyreodism on the quantitative Karyo- and cytoarchitecture of rat hepatocytes. (A morphometrical and cytophotometrical study on rat hepatocytes 3 weeks after thyreoidectomy) (author's transl)]. Thyroid hormoned influence the quantitative cytoarchitecture of hepatocytes by stimulating the membrane synthesis of mitochondria and ergastoplasm. The cellular and nuclear changes produced by thyroidectomy were analysed cytophotometrically and morphometrically. 21 days after thyroidectomy the liver tissues of 10 male Wistar rats were examined. 5 sham-operated animals served as controls. All rats received Altromin-R-standard diet. The cytophotometric analysis is based on the method described by Sandritter. The morphometric study was carried out following Weibel's methods. As a consequence of the changed cellular metabolism, smaller hepatocytes and nuclei (but larger nucleoli) are found in the hypothyroid rats. As compared with controls, hepatocytes hardly show tetraploid nuclei, which is due to inhibited polyploidisation. The mitochondrial content has become considerably smaller in the hypothyroid hepatocytes. The cristal membranes, the mitochondrial and peroxysomal volume per hepatocyte show a similar reaction pattern, which is considered to be a sign of reduced cellular metabolism. The volume loss of the ergastoplasm confirms this assumption.


Further investigations on M. leprae. Growth promotion and inhibition by organic substances and observations on antagonistic and syntergistic effects. The multiplication of 2 strains of M. leprae on a medium containing a sonic extract (SE), prepared from M. smegmatis, was promoted by cysteine, tryptophane and dimethylasulfoxide (DMSO), while glutamic acid, glutamine and histidine exerted variable effects.  The final effects of glutamic acid and glutamine were determined by the total concentration of both compounds together.  The presence of cysteine and glutamic acid alone or together with DMSO abolished all inhibitory effects.  Desferal did not enable the multiplication of M. leprae on media devoid of SE prepared from M. smegmatis.  However with SE and 0.005 per cent and 0.002 per cent concentrations of Desferal its initial growth was accelerated.  Its final counts, noted after an 8-month incubation, did not exceed those observed without Desferal.  Puring and pyrimidine compounds promoted markekly the multiplication of M. leprae (counts greater than 3 times 10-7/ml).  The highest counts were observed with pyrimidines (thymine, thymidine, cytosine) applied single or combined.


Effect of propranolol on left ventricular function, segmental wall motion, and diastolic pressure-volume relation in man. Precise quantitation of the effects of the non-selective beta adrenergic blocking drug propranolol (3.15 mg/kg body weight) on left ventricular function, segmental wall motion, and diastolic pressure-volume relation in man has been performed. High fidelity left ventricular pressure measurements and simultaneous single-plane angiocardiograms were recorded on a video disc and volumes calculated by a light-pen computer system. Systolic segmental wall motion was computer analysed using the long axis-quadrasection method. Patients were transvenously atrially paced to maintain a constant heart rate. The haemodynamic effects of propranolol may vary depending upon the extent of pre-existing myocardial disease. In some patients ventricular function, as measured by ejection fraction, may be reduced. This reduction in ejection fraction appears to result from overall reduction in segmental wall motion, but also from accentuation of segmental wall abnormalities. These results are consistent with the thesis that beta adrenergic blocking drugs may inhibit compensatory sympathetic mechanisms. The diastolic effects of propranolol may include quite substantial increases in ventricular volumes in those patients with impaired cardiac function. With respect to the intact human ventricle, propranolol may increase diastolic volume for a given level of ventricular pressure. Thus, in a static sense, the ventricle in these patients could be viewed as being more compliant after propranolol administration. However, the fact that the length-tension relation, as measured by the slope of the logarithmic pressure versus volume plot is unaltered by propranolol, suggests that the muscle comprising the ventricle itself exhibits no alteration in its passive elastic properties.


Functional and post-oral contraceptive amenorrhoea: response to luteinizing hormone releasing hormone (LH-RH). Basal levels of luteinizing hormone (LH) and follicle stimulating hormone (FSH) in serum and their response to the luteinizing hormone releasing hormone (LH-RH) were measured in a group of patients with functional amenorrhoea. The results in this group were compared to those obtained in a second group of patients with secondary amenorrhoea which had followed cessation of a combined oral contraceptive. No difference either in the basal levels or in the response to LH-RH could be found. These data indicate that there is no detectable difference in pituitary gonadotrophin status between the two groups and that the two types of amenorrhoea have ghe same aetiological background.


Pathological mechanisms to explain some cases of amenorrhoea without organic disease. A test is described to define the site of the lesion causing amenorrhoea in women of child-bearing age who have normal ovaries and a normal response to luteinizing hormone-releasing hormone (LH-RH). Patients able to produce a release of LH following the administration of 1 mg. of oestradiol benzoate (EB) have normal hypothalamic function and thus the site of the defect must lie more centrally in the brain. Patients unable to show these LH surges have either a primary or secondary abnormality affecting their hypothalamic oestrogen "receptor" mechanisms or their ability to produce LH-RH.


Translation of poly(riboadenylic acid)-enriched messenger RNAs from the yeast, Saccharomyces cerevisiae, in heterologous cell-free systems. Poly(riboadenylic acid) [poly(A)] enriched messenger RNAs from yeast have been used to direct the synthesis of yeast polypeptides in mouse Krebs II ascites and wheat embryo extracts. Both cell-free systems, synthesize polypeptides over a molecular weight range of 10,000-100,000. Autoradiograms of sodium dodecyl sulfate-polyacrylamide slab gels used to fractionate [35S]methionine-labeled in vitro products reveal that about 25 major bands (each of them possibly representing multiple polypeptides) are produced by each cell-free system. Each of these coelectrophoreses with a major polypeptide labeled in vivo or in a yeast lysate. These results suggest that cell-free translational machinery from eukaryotes is not able to discriminate in an all or none fashion against messenger RNAs which are available to it. While yeast poly(A)-enriched messenger RNA directs the synthesis polypeptides over approximately the same molecular weight range in both cell-free systems, the wheat germ system directs the incorporation of 45 times the amount of [3H]serine into Cl3CCOOH-precipitable polypeptides. This is in contrast to the 2.5-fold more efficient translation of hemoglobin mRNA in the wheat embryo extract. Thus, the extract from mammalian cells is able to translate mRNA from a lower plant with a much lower efficiency than it translates hemoglobin mRNA, and at a lower efficiency than is observed using a cell-free system from wheat embryos. This indicates that the wheat embryo system is the one of choice for translation of yeast messenger RNA.


The irreversible step in formation of initiation complexes of Escherichia coli. At some stage during initiation the ribosomal subunits of Escherichia coli must become irreversibly coupled, since polysomal ribosomes, in contrast to free ribosomes, are not dissociated by initiation factor IF-3. To determine when irreversibility develops we have compared the response to IF-3 of mature, puromycin-reactive initiation complexes, made with GTP, and of intermediate, puromycin-unreactive complexes, made with GMPPCP. The latter complexes initially appeared to be dissociated by the factor but this effect was found to be due to artificial loss of the ligands at the Mg2+ concentration customary in the test for dissociation. At a slightly higher Mg2+ concentration (4 mM), sufficient to retain the ligands, the GMPPCP complexes were not significantly dissociated by IF-3, at concentrations that caused complex dissociation of free ribosomes. It thus appears that the intermediate 70S initiation complex, though less stable to ionic dissociation than the mature complex, is in effect irreversible under physiological conditions.


The predicted secondary structure of the N-terminal sequence of the lac repressor and proposed models for its complexation to the lac operator. Rules for the prediction of protein conformation (Chou, P. Y., and Fasman, G. D. (1974), Biochemistry 13, 211, 222) have been applied to the N-terminal sequence 1-60 of the lac repressor. This analysis predicts beta structure at sequences 4-9 and 15-20, helices at 26-32, 38-45, and 52-57, and beta turns at 48-51 and 14-17. Repressor mutants lacking operator binding capacity in which Pro replaces Ser-16 and Ala replaces Thr-19 (Weber, K., Platt, T., Ganem, D., and Miller, J. H. (1972), Proc. Natl. Acad. Sci. U.S.A. 69, 3624) have no effect on the prediction of beta structure at residues 15 to 20, which suggests that the polar side chains of Ser-16, Tyr-17, Gln-18, and Thr-19 participate in intermolecular hydrogen bonding with conplementary polar groups on the lac operator. The loss of operator binding capacity on replacement of Ala by Val at position 53 in the repressor results from a predicted secondary structural change from helix to beta structure for residues 52-57 which can be transmitted to the N-terminal sequence via a beta turn at residues 48-51. The basic residues at positions 33, 35, and 37 between the helical regions 26-32 and 38-45 probably bind to the phosphate groups on the operator on complexation. It is proposed that complex formation involves the interaction of either a beta structure (residues 15-20) or a right-hand twisted antiparallel beta-pleated sheet (residues 4-9 and 12-20) with operator DNA.


Binding of Met-tRNA-f to native and derived 40S ribosomal subunits. Our previous work has shown that the native 40S ribosomal subunits (those found free in the cell sap) but not polyribosomal 40S subunits have additional associated proteins that are removed by 0.5 M KCl. In this communication we present evidence that in the Ehrlich cell one of the native subunit associated proteins is the mammalian initiation factor that forms a Met-tRNA-f-factor-GTP complex, and is required for the binding of Met-tRNA-f to the 40S subunit. Initial examination of the KCl wash of the Ehrlich cell total ribosomal pellet revealed a factor which (1) shifted the elution of Met-tRNA-f and of GTP from the included to the excluded volume on Sephadex G-100 chromatography, (2) stimulated the binding of Met-tRNA-f to Millipore filters, and (3) stimulated the binding of Met-tRNA-f to salt-washed 40S subunits. These activities were dependent upon or enhanced by GTP; were inhibited by GDP; were much greater for Met-tRNA-f than for Met-tRNA-m or for lysyl-tRNA; and were concentrated in the KCl ribosomal wash and were not detected in the cell soluble fraction. Met-tRNA-f bound in conjunction with a specific amount of KCl wash protein, to form a distinctive particle of bouyant density 1.40 g cm minus 3 in CsCl, identical in density to one form of the native 40S subunit. Native 40S subunits, but no other subunits, contained a factor which was eluted by 0.5 M KCl and which (1) stimulated the binding of Met-tRNA-f to Millipore filters, and (2) stimulated the binding of Met-tRNA-f to salt-washed 40S subunits. The factor appeared to be localized on the native 40S subunit of density 1.40 g cm minus 3.


Physical properties of some ribosomal proteins in solution and evidence for molecular interactions between isolated ribosomal proteins. Many previous studies have been directed toward obtaining a physical visualization of the relationship between the protein and RNA in the ribosomal subunits isolated from Escherichia coli. The current study is the first report where an attempt has been made to directly assess interactions between a pair of isolated ribosomal proteins separate from the intact system by means of sedimentation equilibrium analysis. The molecular weights of the proteins S3, S4, S5, S6, S7, S8, and S20 from the 30S subunit of the E. coli ribosome were determined under conditions of assembly of the subunit by sedimentation equilibrium. All of the proteins exhibited molecular weights consistent with monomeric behavior (i.e., in agreement with the measurement of the ultimate molecular weight in denaturing solvents as reported in other studies as well as in the current study) except S8 which indicates a tendency to self-associate. Hydrodynamic measurements on the proteins indicate that these proteins are not completely disorganized in solution such as a random coil, although not as compact as globular proteins. The frictional coefficient ratios found for these ribosomal proteins range from 1.4 to 1.9. The hydrodynamic data are discussed as containing some evidence that stable interaction sites could exist in the proteins. The molecular weight data are considered pertinent to a sedimentation equilibrium study of protein-protein interactions that may be occurring in the ribosomal subunits. Two proteins, S3 and S5, considered in this investigation were found to exhibit no tendency to self-associate under conditions of reassembly. When the two proteins are mixed under those same conditions, however, a species with a molecular weight greater than that of either S3 or S5 is observed to be formed. The interpretation is presented that a molecular interaction between S3 and S5 is the cause. The system is described as containing S3, S5, and a complex between S3 and S5 with a stoichiometry of 1:1 and an association equilibrium constant of 5.7 times 10-5 l./mol (delta G-o equals minus 7.25 kcal/mol). Since the association appears to be specific and of moderate strength, it is concluded that the interaction could have some pertinence with respect to conferring a structural arrangement in the ribosomal subunit. Moreover, it is concluded that protein-protein interactions, in general, must be considered in addition to the well documented significant RNA-protein relationships when models for ribosome structure and assembly are formulated.


Interaction of steroids with nucleic acids. 17beta-Estradiol and testosterone bind to both native and denatured DNA, and to RNA and poly(A)-poly(U). Binding affinity depends on the conformation of nucleic acid. Lowering the electrolyte concentration and raising the temperature increase the binding of 17beta-estradiol to native DNA and decrease that to denatured DNA. In 0.01 M NaCl and at 37 degrees, more 17beta-estradiol is bound to native DNA than to denatured DNA. Higher binding of steroid to denatured DNA relative to native DNA at low temperature and high ionic strength is related to larger fraction of binding sites per unit nucleotide in denatured DNA. In addition to 17beta-estradiol and testosterone, 17alpha-estradiol, 17beta-estradiol-3-methyl ether and 19-nortestosterone also stabilize the structure of nucleic acids and poly(A)-poly(U) against thermal denaturation. The 17beta-estradiol induced elevation of the T-m of DNA is diminished by methanol or high NaCl concentration. These results indicate the involvement of hydrogen bonding and hydrophobic interactions between steroids and nucleic acids. The results of binding isotherms and optical studies suggest a conformational dependence of the binding of steroids to nucleic acids.


Nucleotide specificity of stringent factor and the synthesis of analogs of guanosine 5'-diphosphate 3'-diphosphate and guanosine 5'-triphosphate 3'-diphosphate. The ribosome-dependent stringent factor reaction was found to be nonspecific with regard to the number of phosphate groups linked to the 5' position of guanosine nucleotides. Both GMP and guanosine 5'-tetraphosphate could accept a pyrophosphoryl group from ATP although at a much lower rate than GDP or GTP. Guanosine 5'-monophosphate 3'-diphosphate and guanosine 5'-tetraphosphate 3'-diphosphate were the products of these reactions. Furthermore, 3'-linked analogs of guanosine 5'-diphosphate 3'-diphosphate and guanosine 5'-triphosphate 3'-diphosphate were synthesized from the corresponding ATP analog, adenosine 5'-O-(3-thiotriphosphate). The stringent factor catalyzed reverse reaction was found to be specific for guanosine 5'-diphosphate 3'-diphosphate, and was essentially inactive to the isomeric form, guanosine 5'-diphosphate 2'-diphosphate.


The regulation of transcription in bacteriophage T5-infected Escherichia coli. The expression of bacteriophage T5-specific RNA and protein in infected cells is temporally separated into three classes: class I (preearly), class II (early), and class III (late). By immunoprecipitation techniques we have shown that T5 infection of cells leads to the synthesis of one class I polypeptide (11,000 daltons) and two class II polypeptides (90,000 and 15,000 daltons) capable of binding to the RNA polymerase of the host Escherichia coli cell. One of the class II polypeptides (90,000 daltons) is the product of gene C2, which is an essential gene product required for the initiation of class III RNA synthesis. The colicinogenic factor, ColIb, is a plasmid which prevents the normal synthesis of class II and the III bacteriophage T5-specific RNA in infected colicinogenic (ColIb+) cells. In T5-infected colicinogenic cells, only the T5 class I polypeptide is found associated with the RNA polymerase. Mutants of T5, designated T5h minus, are capable of growth on both noncolicinogenic and ColIb+ hosts. Extracts of T5h minus infected ColIb+ cells were shown to lack a small class I polypeptide (12,000 daltons) as compared to T5-infected cells. The h minus mutation, however, has no effect on the levels of the class I T5 polypeptide of similar molecular weight which is bound to the RNA polymerase. One effect of the h minus mutation is to enhance the quantities of the two class II polypeptides bound to the enzyme.


Amino acid sequence of dogfish trypsin.

Amino acid sequence of dogfish trypsin. The amino acid sequence of pancreatic trypsin from the spiny Pacific dogfish (Squalus acanthias) has been determined and compared with the sequences of bovine and porcine trypsin. Dogfish trypsin contains one less amino acid residue (222) than the other two enzymes. Two-thirds of the residues in corresponding positions in dogfish and bovine trypsin are identical and the sequences ofall three enzymes are homologous. Of the 223 amino acid residues of bovine trypsin, 77 are replaced without significant changes in function. Seven replacements, all conservative, occur in the interior of the protein; the remainder are on the surface. All residues known to be components of the active site of bovine trypsin are present in corresponding positions in dogfish trypsin. Comparison of the three enzymes suggests calcium binding sites in dogfish trypsin. A corrected sequence of bovine trypsin identifies residue 67 as Asn and residues 84-87 as Ser-Asn-Thr-Leu.


The nature of protein association with chromatin. The reality of the nonrandom distribution of histones along the chromatin strand was investigated in several ways. It does not appear to derive from histone exchange during shearing and is evident in chromatin fixed with formaldehyde prior to shearing. Endogenous or Escherichia coli polymerase are preferentially associated with regions of chromatin with a low protein/DNAratio. Although RNA polymerase and histones are fixed to chromatin after formaldehyde treatment with high efficiency, only a minor fraction of non-histone protein is fixed under similar conditions. Even after washing in high salt to minimize adventitious association, most remaining non-histone protein fails to be fixed. The utility of this approach for defining chromosomal proteins is discussed.


Comparative ability of RNA and DNA to prime DNA synthesis in vitro: role of sequence, sugar, and structure of template-primer. The priming efficiency of oligo(RNA) vs. oligo(DNA) in a homopolymer template-homooligomer primer system was compared using four DNA polymerases. The templates included (dT)n, (dA)n, (dC)n, and (dI)n. Primers were the oligomers of the complementary DNA OR RNA with chain lengths of 6 to 23. The DNA polymerases used were from Micrococcus luteus, avian myeloblastosis virus (AMV), and Escherichia coli (polymerase I and polymerase III). The polymerases demonstrated a preference for the DNA primers with (dC)n, (dA)n, and (dI)n templates. However, when (dT)n was the template, all but the AMV polymerase preferred (rA)11 more than 200-fold better than (dA)12. This preference was due to the physical structure of the initiation complex. The structures of the oligo-polymer complexes were characterized by mixing curves, melting curves, and analytical bouyant density analyses. (rA)11 + (dT)n formed predominatly a duplex structure, whereas (dA)12 + (dT)n formed the three- stranded structure, (dA12-2(dT)n. The Km of the duplex with E. coli Pol III was 2.9 mugM (rA)11. The Ki of the triplex was 2.2 mugM (dA)12, indicating that Pol III could bind to the triplex but would not elongate the (dA)12 primer. The influence of structure on priming also was demonstrated with longer oligomers, (dA)23 and (rA)23, where the (dA)23 formed more duplex-like structures and primed more than the (dA)12.(dT)10 + (dA)n complexes also were shown to form triplex structures that inhibited priming. These results show that template-primer structure has more influence on priming than the sugar moiety or the sequence of the nucleic acid.


The effect of modification of T7 DNA by the carcinogen N-1-acetylaminofluorene: termination of transcription in vitro. To study the effects of N-2-acetylaminofluorene (AAF) modification of DNA on transcription, purified DNA from bacteriophage T7 was modified in vitro to varying extent with AAF and transcribed by DNA-dependent RNA polymerase from Escherichia coli. The main effects of AAF modification on transcription are a marked inhibition of the rate and extent of trna synthesis with relatively little effect on initiation except at very high AAF doses. Calibration of the percent modification with [14-C]AAF and analysis of the size of the RNA product by double isotope labeling and polyacrylamide gel electrophoresis support the following mechanism of transcription inhibition: most of the AAF residues bound to the coding strand of the DNA cause premature termination of transcription, at or near the site of modification, with release of RNA polymerase. This results in the production of shorter RNA chains with increasing amounts of bound carcinogen. The data are consistent with there being no reinitiation and/or synthesis of RNA distal to the AAF-modification site.


Europium as a fluorescent probe of transfer RNA structure. The binding of europium(III) to Escherichia coli tRNA-fMet,Glu and to unfractionated E. coli tRNA has been investigated by using the 4-thiouridine sensitization of europium 5-Do yields 7-F1 emission and changes in the lifetime of the 5-Do state of europium reported earlier (J. M. Wolfson and D. R. Kearns (1974), J. Am. Chem. Soc. 96, 3653). Binding of the first 3-4 europium ions is independent and sequential, approximately 600 times stronger than the magnesium binding, and the binding sites are located near the 4-thiouridine residue found at position 8 in a number of E. coli tRNA. Competition experiments suggest the strong binding sites are the same for magnesium and europium. The europium binding properties of both unfractionated E. coli tRNA and purified tRNA-fMet are quite similar, indicating that the location of the strong binding sites and their binding constants are nearly the same for a large group of tRNA. The europium binding properties of native and denatured tRNA are quite different, however.


Kinetic studies on coenzyme binding and coenzyme dissociation in tryptophanase immobilized on sepharose. The binding rate of pyridoxal '5-phosphate (Pxa-P) to apotryptophanase and the dissociation rate of the coenzyme from holotryptophanase were able to be determined by following the enzyme activity in continuous flow reactions on a column of immobilized tryptophanase. When the enzyme activity was assayed continuously in the flow system in the absence of coenzyme added to the reaction mixture, immobilized holotryptophanase lost gradually its initial activity owing to dissociation of coenzyme. The coenzyme dissociation at a given concentration of substrate (tryptophan) followed first-order kineticsmin a low substrate concentration range below the Km value, a more decreased rate constant was obtained for the coenzyme dissociation. This indicates that the coenzyme is more dissociable from the apoenzyme-coenzyme-substrate complex (ECS complex) rather than from the apoenzyme-coenzymecomplex (holoenzyme). Immobilized tryptophanase freed of coenzyme restored rapidly its original activity, when the assay mixture containing a given concentration of substrate and Pxa-P was passed through the immobilized enzyme column. The coenzyme binding at a given coenzyme concentration followed first-order kinetics, but the rate was not first order in regard to the coenzyme concentration. A plot of the reciprocal of the first-order rate constant obtained vs. the reciprocal of the coenzyme binding occurs in a two-step fashion; the first step is rapid and the second step is rate determining. Both the dissociation constant for the first step and the rate constant for the second step were shown to be independent of the substrate concentration. This means that Schiff base formation between Pxa-P and tryptophan in the assay mixture has no effect on the binding of Pxa-P to apoenzyme. The coenzyme dissociation constant at a given substrate concentration was calculated from both the rate constant of the coenzyme binding and the rate constant of the coenzyme dissociation. The values obtained by this method at different substrate concentrations were almost identical with those measured at the corresponding substrate concentrations directly by an ordinary method.


Isolation and partial characterization of anglefish proglucagon. Evidence is presented that proglucagon from anglefish islets is a single chain polypeptide with 78 amino acid residues and that the glucagon portion of it is liberated after tryptic cleavage. The most striking characteristic in the conversion of the anglerfish proglucagon to glucagon is that the cleaved peptide bonds display enormous sensitivity toward trypsin. Thus, conversion of the prohormone to glucagon occurs very rapidly within 3-10 min with a 1:500-1:1000 molar ratio of enzyme to substrate. Further, trypic cleavage of the anglerfish glucagon requires higher concentrations of trypsin (molar ratio 1:25 enzyme to substrate) and longer incubation time. The behavior of proglucagon and glucagon toward trypsin shows striking similarities with the tryptic conversion of anglerfish proinsulin to insulin.


Ribosomal subunit interaction as studied by light scattering. Evidence of different classes of ribosome preparations. The Mg2+-dependent equilibrium of ribosomal subunits was studied by light scattering in the absence of any other factor of protein synthesis. The difference of intensity in the light scattered by dissociated 30S-50S couples and by 70S particles was used as a method to measure the percentage of association as a function of Mg2+ under conditions of nonperturbation of the equilibrium. We found that pressure-resistant (type A) and non-pressure-resistant (type B) ribosomes may be characterized by the different behavior of their association equilibrium curves. The thermodynamic parameters of this equilibrium and their comparison for the two classes of ribosomes were calculated from experiments at different temperatures.


Identification of the collagenous proteins synthesized by cultured cells from human skin. Cells cultured from human skin synthesize precursor forms of types I and III collagens. After denaturation and reduction, the polypeptide chains obtained from these molecules had molecular weights estimated to be 140,000 and 120, 000. The larger chains, the pro alpha chains, are believed to be derived from the original precursor molecules. The smaller chains arise from altered forms, p-collagens, that may be normal intermediates in the conversion of both procollagens to collagens I and III.


Some characteristics of and structural requirements for the interaction of 24,25-dihydrofusidic acid with ribosome - elongation factor g Complexes. Fusidic acid inhibits polypeptide chain elongation by binding to the ribosome - elongation factor-G - GDP complex and thereby preventing its dissociation. The experiments reported here quantitate the interaction of the antibiotic [3H]-24,25-dihydrofusidic acid, an active analog of fusidic acid, with the ribosome - elongation factor-G - GDP comples. All components of the complex are essential for [3H]-24,25-dihydrofusidic acid binding. The stoichiometry of the interaction is ca. 1:1, and the Ka apparent, as determined by equilibrium dialysis, is 2.6 times 10-6 M-minus 1. It is further shown that GTP and GDP are equally effective in forming complexes to which the antibiotic may bind, whereas GMP and beta,gamma-methyleneguanosine triphosphate will not form complexes to which the antibiotic may bind. In order to examine the structural basis of the mode of antibiotic action shown by fusidic acid, we have considered two activities of 21 structural analogs of this antibiotic: ability to bind to the aforementioned ternary complex and ability to stabilize this complex. The comparative binding capability of the analogs were extablished through competition experiments with [3H]-24,25-dihydrofusidic acid. The data obtained from these experiments can be summarized as follows. (1) The C17-20 double bond of fusidic acid appears to be critical for both binding and complex stabilization activities. (2) A carboxyl group in the vicinity of the C20 carbon is also essential for both activities. (3) Modifications of other functional groups in the molecule can lead to significantly decreased stabilization of the ternary ribosome complex and/or ability to compete with [3H]-24,25-dihydrofusidic acid for binding to the complex, but do not demonstrate absolute structural requirements for either activity.


A comparative study of the 50S ribosomal subunit and several 50S subparticles in EF-T-and EF-G-dependent activities. A series of ribosomal subparticles derived from the 50S subunit has been studied and compared in EF-T- and EF-G-dependent reactions. Three different 50S cores were prepared by CsC1 isophycnic centrifugation and one by NH(4)Cl-ethanol extractionm the 50S CsCl core a had lost proteins L1, L7, L8, L10, L12, L16, L25, L33, and some L6 and L11. The 50S CsCl core b additionally lacked protein L6, and 50S CsCl core c also lacked protein L5, L15, L18, L27, L28, L30, and most of L9, L14, L19, and L21. The 50S NH(4)Cl-ethanol core had lost up to 90 percent of proteins L7, L12 and 30-60 percent of proteins L8, L10, and L29. The 50S CsCl core a had much reduced activity in EF-G and none in EF-T GTPase reactions while 50S CsCl cores b and c were inactive. Addition of proteins L7, L12 restored the activity for both the EF-T- and EF-G-dependent GTPase with all of the three 50S CsCl cores, increasing stepwise from core c to core a; The 50S NH(4)Cl-ethanol core was partially active in the EF-G GTPase over the 2-30 mM MG-2+ range tested, while EF-T only showed some activity inthe upper portion of this range...


Polyfluoroalkylamine derivatives of luteinizing hormone-releasing hormone. A series of C-terminally fluorinated analogs of luteinizing hormone-releasing hormone (LH-RH) was synthesized by a combination of solid-phase and classical techniques and found to have interesting properties when assayed for LH- and FSH-releasing activities over a prolonged period of time in immature male rats. One of the peptides, desGly-10-LH-RH-2,2,2-trifluoroethylamide, was found to be about nine times more effective than LH-RH in releasing LH, or approximately twice as active as the corresponding alkylamide peptide, desGly-10-LH-RH-ethylamide. However, desGly-10-LH-RH-2,2,3,3,3-pentafluoropropylamide was only slightly more active than LH-RH and considerably less active than the corresponding propylamide analog. A family of peptides was also prepared containing D-alanine in position six of the chain in conjunction with the C-terminal modifications. D-Ala-6,desGly-10-LH-RH-2,2,2-trifluoroethylamide, surprisingly, gave patterns of gonadotropin release which were only as intense and virtually identical with those obtained with D-Ala-6,desGly-10-LH-RH-ethylamide which in turn releases about 20 times more LH than a similar dose of LH-RH. D-Ala-6,desGly-10-LH-RH-propylamide also gave almost identical patterns of gonadotropin release. D-Ala-6,desGly-10,LH-RH-2,2,3,3,3-pentafluoropropylamide was considerably less potent, being only five times more effective than LH-RH.


Evidence for fidelity of chromatin reconstitution. Several lines of evidence are presented which support the contention that chromatin may be dissociated, fractionated, and reconstituted without altering the compositional, structural, or transcriptional integrity of the genome. The similar compositions of native and reconstituted chromatins are suggested by the absence of significant differences in their protein/DNA ratios and in the polyacrylamide gel electrophoretic profiles of their histones and nonhistone chromosomal proteins. Criteria for fidelity of genome structure in reconstituted chromatin include binding of reporter molecules with specificity for the minor groove of DNA, binding of histones, number of sites available for addition of nucleotides, and circular dichroism spectra. When the transcriptional activities of native and reconstituted chromatins were compared under conditions where reinitiation is prohibited, significant changes were not observed. Taken together, the present results strongly suggest, but do not conclusively establish, fidelity of chromatin reconstitution.


Conformations and structural transitions in polydeoxynucleotides. Polydeoxynucleotides of different base sequence, the alternating poly[d(A-T)]-poly[d(A-T)], crab satellite DNA, on the one hand, and double-stranded homopolymer complexes poly[d(A)]-poly[d(T)], poly[d(I)]-poly[d(C)], on the other, display significant differences in their conformation and conformational transitions. Infrared linear dichroism investigations indicate that the alternating poly[d(A-T)]-poly[d(A-T)], enzymatically synthesized, adopts a lower humidity a well-expressed A* form in which stability is relatively small,i.e., restricted to limited relative humidity. This A form is characterized by the orientation of the bisector of the phosphate OPO group at 34 degrees with respect to the helical axis, which is slightly lower than that of DNA. In contrast, for the homopolynucleotide double-stranded complex poly(dA)-poly(dT) and also for poly(dI)-poly(dC), the B yields A conformational change is not observed. Instead poly(dA)-poly(dT) exists at lower humidity in a stable modified B form. Thus the present results indicate that homo(dA)-homo(dT) double-stranded sequences prevent the B yields A structural transition. All AT-containing polydeoxynucleotides and crab satellite DNA adopt a high humidity a modified B form characterized by the orientation of the bisector of the phosphate group OPO at 64 degrees with respect to the helical axis which is significantly lower than 68-74 degrees observed in DNAs. The base pairing geometry in poly(dA)-poly(dT), poly[d(A-T)]-poly[d(A-T)], and also in poly(dI)-poly(dC) is apparently a Watson and Crick type. Thus the observed differences in conformation are not due to different base pairing scheme. It is suggested that in DNAs of high AT content the presence of homo(dT)-homo(dA) sequences and the relatively low stability of the A form of d(A-T) alternating sequences may inhibit the change to the A form. A possible role of these sequences in DNA recognition by protein is suggested.


Amino acid sequence of two functional sites in yeast glycogen phosphorylase. The structure of two functional sites in baker's yeast (Saccharomyces cerevisiae) glycogen phosphorylase (EC 2.4 1.1) was determined as part of a study on the evolution of regulatory enzymes. S-Carboxymethylated, MaBH4-reduced 32-P-labeled yeast phosphorylase a was cleaved with CNBr, thermolysin, and pepsin. Peptides labeled with 32-P or carrying the fluorescent pyridoxyl marker were isolated and purified using ion-exchange chromatography and gel filtration. CNBr cleavage yielded a single radioactive phosphopeptide (42 residues long) and one small fluorescent peptide with the unique sequence epsilon-Pxy-Lys-Phe-Val-Met. Thermolysin digestion gave rise to one radioactive octapeptide and two fluorescent peptides, 15 and 2 residues long, respectively. From a combination of substractive Edman degradations and digestion with yeast protease C, the sequence of the 32-P-labeled octapeptide was established. Phosphothreonine was identified as the sole phosphorylated amino acid, giving the following structure for the site involved in the covalent regulation of yeast phosphorylase: Leu-Thr(P) -Gly-Phe-Leu-Pro-Gln-Glu. The two fluorescent thermolytic peptides, together with two additional pyridoxyl peptides isolated after peptic digestion of the enzyme yielded the following sequence around the site binding pyridoxal-5'-P, the cofactor essential for phosphorylase activity: Ile-Ser-Thr-Ala-Gly-Thr-Glu-Ala-Ser-Gly-Thr-Ser-Asn-Met-Lys(P Pxy)-Phe-Val-Met. While the phosphorylated site bears no resemblance to the site of covalent control in vertebrate phosphorylases, the pyridoxal-P binding site in the yeast enayme displays remarkable homologies with its animal counterparts; the finding that 14 out of 18 amino acids are identical strongly suggests that the cofactor must be directly involved in catalysis.


Red cell surface structure. Stabilization by cholesterol sulfate as evidenced by scanning electron microscopy. Scanning electron microscopic studies demonstrate that the normal biconcave shape of the human erythrocyte is maintained in hypotonic saline when physiological levels (10 minus 5M) of cholesterol sulfate are added to the solutions. Cholesterol sulfate is a naturally occurring sterol conjugate in plasma and erythrocyte membranes and we propose that it may belong to that class of amphipathic molecules responsible for the maintenance of structure of the erythrocyte by interaction with membrane components.


Redox properties of beta-type cytochromes in Escherichia coli and rat liver mitochondria and techniques for their analysis. We describe here apparatus and procedures for conducting potentiometric titrations and for analyzing the collected data in terms of the number of components present, their amounts and their midpoint potentials. Using these procedures we have determined the presence of three forms of cytochrome b1 in Escherichia coli with midpoint potentials at pH 7.1 OF about minus 50, plus 110 and plus 220 mV. We were not able to demonstrate a change in any of these potentials by the addition of phosphate, ATP, or 2, 4-dinitrophenol. We have been able to confirm the presence of two forms of cytochrome b in non-energized mitochondria and the apparent conversion of the low-potential component to a new high potential component upon energization of the mitochondria. However we cite further experimental data that question the actual conversion of one form of cytochrome b to another. An alternative interpretation based on our analysis suggests that the high voltage component may be present in a masked form in the non-energized mitochondria.


Sequence homology of nuclear and mitochondrial DNAs of different yeasts. 1. Both nuclear and mtDNA of four different yeasts show approximately 10% homology as measured by DNA-DNA filter hybridization. These homologous sequences are mainly attributable to the ribosomal cistrons. 2. Melting curve analysis shows that the heterologous mitochondrial DNA-DNA hybrids contain several times more mismatching than the nuclear DNA-DNA hybrids. 3. DNA-rRNA hybridization shows that the sequences of the ribosomal cistrons in both the nuclear and the mitochondrial genome have been conserved during evolution. 4. However, melting curve analysis of the DNA-RNA hybrids shows that the sequence of the nuclear ribosomal cistrons have undergone considerable fewer nucleotide substitutions than their mitochondrial counterparts. 5. The results suggest that the mitochondrial ribosomal cistrons have evolved more rapidly than the nuclear cistrons. This is discussed in the light of theories on the rat of molecular evolutin.


Size, conformation and purity of chloroplast DNA of some higher plants. 1. Chloroplast DNA of Antirrhinum majus, Oenothera hookeri, Beta vulgaris and Spinacia oleracea band at the same buoyant density of 1.697 g-cm-3 in neutral CsCl equilibrium gradients. The corresponding nuclear DNAs band at 1.691, 1.703, 1.695 and 1.695 g-cm-3, respectively. The purity of chloroplast and nuclear DNA can be assessed objectively only in the cases of Antirrhinum and Oenothera. 2. Electron microscopic analysis of chloroplast DNA, purified in CsCl or CsCl/ethidium bromide gradients, revealed up to 80% circular molecules. Of these about 15% were of supertwisted conformation. Best yields of circular molecules were recovered when populations of unbroken chloroplasts were subjected to DNAase and phosphodiesterase treatment, and when the DNA was purified from viscous lysates by centrifugation into a CsCl cushion. Treatment of plastids with DNAase alone did not guarantee complete degradation of nuclear DNA. 3. The average contour length of the open circular chloroplast DNA molecules was basically similar for all four plants. They were 45.9 plus or minus 2.1 mum for Antirrhinum, 45.7 plus or minus 1.9 mum for Spinacia, 44.9 plus or minus 1.7 mum for Beta and 45.2 mum for Oenothera. This is comparable to the size derived for the coding capacity of chloroplast DNA from reassociation experiments. As much as 15% of the total population of circles in chloroplast DNA of Spinacia were circular dimers.


The 5'-termini of bacterial RNA. I. The monophosphorylated termini. A two-dimensional fingerprint technique is described which separated oligonucleotides by length and number of phosphates present at the termini. The diversity of terminal nucleotide sequences was found to be quite restricted in bacterial 4-5 S RNA: in the strain used here the predominant dinucleotide species was pG-C, and the only trinucleotides produced were pG-G-Up and pG-G-Cp. Examination of total pulse-labeled RNA revealed the same restricted set of terminal trinucleotides.