Beta-adrenergic (isoproterenol) regulation of antidiuretic hormone. Antidiuretic hormone (ADH) and isoproterenol (ISO) both cause renal retention of water when infused iv into animals. It has been suggested that the agents share a common mechanism of action but the current work reveals marked differences in the effect of ISO and ADH on urinary excretion of sodium (ISO produces fall from 50 plus or minus 14 to 3 plus or minus 0.6 mu Eq per 20 min, P greater than 0.0005; ADH causes a fall from 62 plus or minus 14 to 45 plus or minus 9, P greater than 0.05) and creatinine (ISO causes a fall from 157 plus or minus 14 to 71 lus or minus 13, P greater than 0.0005; ADH produces fall from 155 plus or minus 12 to 145 plus or minus 14, P greater than 0.05), which suggest that the actions of the agents may differ. To study that possibility, ISO was infused into normal rats and rats with hereditary diabetes insipidus (DI rats) due to a genetic defect in ADH production. Doses previously used (0.2 mug per kg per min, and doses 10 and 100 fold less were infused. At 0.002 mug per kg per min, urine osmolality in both groups of rats declined (DI rats from 177 plus or minus 22 to 141 plus or minus 13 MOsm per kg, P greater than 0.01, and normals from 162 plus or minus 17 to 142 plus or minus 15 MOsm per kg, P greater than 0.05). At 0.02 mug per kig per min, there was no significant change is osmolality. At 0.02 mug per kig per min, osmolality rose in both groups but the response was significantly blunted in the DI rats (normals: 351 plus or minus 32 to 626 plus or minus 79 mOsm per kg, P greater than 0.005; DI rat: 204 plus or minus 23 to 241 plus or minus 30 P greater than 0.05). It is concluded that intravenous ISO has a dual effect on water excretion; at low doses it is associated with reduced urinary osmolality possibly by direct renal action and at high doses it is associated with antidiuresis by stimulating release of pituitary ADH.


Progressive renal artery stenosis. An experimental model. A method of inducing slow gradual constriction of a renal artery is described. A metal constrictor is partially filled with ameroid, a hydroscopic material. Over a period of weeks, the ameroid expands, causing increasing obstruction to the renal artery. Data revealed a progressive decrease in function as measured by p-aminohippurate, inulin, and creatinine clearance on the affected side with an increase in function in the contralateral side.


Direct effect of prostaglandins in renal function and renin release in the presence of renal ischemia in the dog. Prostaglandins PGE-1 or PGA-1 (0.5 to 1 mug per min) were infused into the stenosed renal artery of anesthetized hypertensive dogs. Increased urine volume, sodium and potassium excretion, and p-aminohippurate clearance were found during the prostaglandin infusion period in the infused kidney as compared to the control periods before infusion. Creatinine clearance was increased during infusion of PGE-1. The noninfused, nonischemic kidney showed no effect at the time of infusion with PGE-1 but in the case of PGA-1, the p-aminohippurate and creatinine clearances and urine diuresis were decreased. As a result, the mean aortic blood pressure decreased. Both prostaglandins increased the renal vein renin in the infused kidney. PGA-1 did affect renin release of the noninfused kidney, but PGE-1, which is rapidly inactivated by the lung, did not have this effect. Renin release seems to be influenced by electrolyte diuresis operating through the macula densa mechanism. However, the lowering of blood pressure seen in this study cannot exclude the involvement of the stretch receptors (the juxtaglomerular cells) for renin release. The increased renin release after prostaglandin administration seems to be a protective renal mechanism against the drug-induced hypotension. It seems to be induced by the direct sodium and water diuretic effects of prostaglandins.


Ureteral response to partial obstruction. Smooth muscle hyperplasia and connective tissue proliferation. A technique for producing a reversible form of partial ureteral obstruction in the experimental animal has been described. The response of the rabbit ureteral muscularis to partial obstruction was characterized by hypertrophy of the smooth muscle cells within the first 3 days followed by hyperplasia of the leiomyocytes which appeared to continue as long as the obstruction was present. Deposition of connective tissue was noted between the smooth muscle cells after 2 weeks of partial obstruction increasing to a marked extent by 8 weeks.


A comparative evaluation of Avitene and gelfoam for hemostasis in experimental canine prostatic wounds. Microcrystalline collagen has been reported to be an effective hemostatic agent in brain, liver, kidney, etc. This experimental study in dogs and rats shows that microcrystalline collagen is a more effective hemostat in prostatic hemorrhage than is purified gelatin solution. No gross or histologic evidence of tissue damage or calcification was induced by microcrystalline collagen or purified gelatin solution. Wafers of microcrystalline collagen that are placed in rat bladders dissolve and do not induce calculogenesis.


A highly basic small protein associated with spermatogenesis in the human testis. A very basic and acid-soluble small protein was isolated in the pure form from specimens of human testes that displayed histologic evidence of normal spermatogenesis. The protein is virtually indistinguishable in its electrophoretic properties from a testis-specific basic protein that has been previously described in a number of other eutherian mammalian species. The homogeneous human testicular basic protein is rich in arginine, lysine, and serine, and is devoid of cyst(e)ine, phenylalanine, glutamate, glutamine, isoleucine, and tryptophan. Its amino acid composition is extremely close to that of a testis-specific basic protein previously described in the rat. The basic protein from human testis is electrophoretically distinct from the principal basic chromosomal protein of human spermatozoa and could not be detected in ejaculated human spermatozoa by the procedure used to extract the chromosomal basic protein.


Viscoelastic properties of bladder wall strips. Ninety-five stress-decrease curves of dog bladder strips, selected from 204 experimentally determined curves, were analyzed in terms of a model which can be described by an equation containing two or three exponential terms and a constant. As might be expected, the three-exponential equation gave the better fit. The results are discussed and compared with those derived from measurements on whole bladders. The relaxation constants determined for bladder strips agree remarkably well with those for whole bladders, indicating that the value of these constants is independent of geometry.


Therapy questionably infarcted testis.

Therapy questionably infarcted testis. Observations made upon dogs and humans with apparent infarction of the testis secondary to torsion of the spermatic cord suggest that the treatment of choice for questionably viable testis is detorsion of the cord, replacement of the testis in its hemiscrotum, and fixation of the opposite testis. This form of therapy may be applicable to all "torsed testes" regardless of viability.


Ultrastructural changes in human prepubertal prostatic epithelium grown in vitro. Ultrastructural changes in the prepubertal human prostatic epithelium maintained in vitro are described and are compared with the ultrastructure of the same tissue before culture. In cultured cells, rough endoplasmic reticulum and Golgi complex are poorly represented and the latter also loses its polarity. Increase in cytoplasmic microfilaments is discussed in relation to possible vitamin A deficiency. Primary and secondary lysosomes, arising from autophagy and endocytosis, occur in large numbers as autophagosomes, myelin figures, residual bodies, and multivesicular bodies. Prostatic acid phosphatase activity, an important secondary sex characteristic, is influenced by sex hormones and malignancy; since this enzyme is lysosome-associated, special emphasis is placed on lysosomal changes. Some ultrastructural changes in rough endoplasmic reticulum, Golgi complex, and the lysosomal system are similar to those observed after castration. This study presents ultrastructure of cultured cells which form the basis for studies involving neoplastic transformation, aging, and hormonal manipulation using an in vitro model. This is necessitated by the absence of an in vivo animal model for prostatic neoplasia; hence studies on prostatic oncogenesis, and age-related phenomenon, must be done on cells in vitro. Significance of this study is enhanced by the fact that normal human prepubertal prostate has not been studied before and normal viable prostate is generally not available for investigations.


Uric acid dihydrate as urinary calculus component. The analysis of uric acid dihydrate was performed using infrared spectroscopy and X-ray diffraction. Both methods permit differentiation with regard to uric acid. Pure uric acid dihydrate was prepared according to a modified technique and the dA values (lattice distances (of A) were determined from several recordings. The extreme instability of the synthetic uric acid dihydrate is stressed, and the conversion speed is determined. During the evaluation of 7,750 analyses of urinary calculi in the course of the past 3 years, 1,126 (14.5 per cent) uric acid calculi and 283 (3.7 per cent) with a mixed portion of uric acid dihydrate were found.


A new concept of the anatomy of the anal sphincter mechanism and the physiology of defecation. The external anal sphincter: a triple-loop system. Since anal sphincters are used as the continent sphincters in some urologic operations, a study of their anatomic structure and function seems necessary. The anatomy of the external anal sphincter has been studied in 18 cadavers by dissection and serial histologic sections. The muscle has been found to consist of a series of U-shaped loops which are distinguishible as three main "loops": top, intermediate, and base. The puborectalis and the deep portion of the external sphincter have been found to be one muscle which is given the name "top loop." No concentric circular muscle bundles could be detected at any level of the external sphincter except in the base loop. A new concept of the mechanism of action of the external sphincter in anal continence and during defecation is presented. An air-tight occlusion of the anal canal could be achieved by the "triple-loop system" of the external sphincter which compresses opposed alternating anal segments. An incomplete anal occlusion by a single loop contraction is completed and potentiated by the succeeding loop action. The last fecal portion is dispelled from the anal canal by a process of "vermicular contractions" which is the result of the loop arrangement of the muscle bundles. Single-loop continence has been discussed. It is suggested that unless all three of the loops are destroyed, any single loop can act as a sphincter which maintains continence to solid stools but not to fluid ones or flatus.


Removal of pulmonary artery band.

Removal of pulmonary artery band. From 1963 to September 1973, 60 patients underwent definitive repair of cardiac lesions for which pulmonary artery banding had been performed previously. Most bandings had been performed for intractable heart failure secondary to large pulmonary flow. The pulmonary artery band resulted in a fibrous reaction around the pulmonary artery or its branches in all instances. Angioplastic procedures on the main pulmonary artery or the left and right pulmonary arteries were usually necessary to relieve the stenosis produced by the band and the resultant scarring. The operative mortality in this group of patients was 28% and pulmonary artery banding was considered to be a factor in 4 of the 17 deaths. The absence of absolute protection from the development of progressive pulmonary vascular disease, the mortality associated with banding, and the mortality and increased technical difficulties associated with removing the band at the time of the repair support one-stage definitive procedures rather than palliative banding of the pulmonary arteries in all but the most exceptional situations.


Long-term results after closed mitral valvotomy. This paper is an analysis of the experiences of the author with patients under-going closed mitral valvotomy between 1948 and December 1963, and thus represents a follow-up of at least 10 years. Five hundred and seventy-one of 618 patients survived operation; the mortality rate of 7.6% is much higher than pertains at present. It includes the early pioneer days when many very ill patients were operated on. There were only five deaths in the most recent 400 consecutive cases. The mortality should be low today. A detailed follow-up shows that many patients are completely cured of their mitral stenosis: of 168 patients alive longer than 11 years, 41 survived for 11 to 15 years, 67 for 16 to 20 years and 60 for over 20 years. Restenosis was diagnosed in 128 patients (22.4%) and 93 of them had a second operation. When the valve is severely damaged, especially when it is regurgitant, the results are not as good and valve excision and replacement are often needed, but valvotomy for pure mitral stenosis can give excellent and lasting results.


An approach to the surgery of mitral valve disease in children. Isolated mitral valve surgery was carried out in 182 children under the age of 16 years. Closed valvulotomy was performed in 72, annuloplasty or reconstruction in 10 and valve replacement in 100 of the children. Preoperative atrial fibrillation implied severe disease. Acute rheumatic carditis was not a contraindication to operation. Two patients died after closed mitral commissurotomy and the results were unsatisfactory in nine children with valve calcification and in 12 with a small jet of incompetence. Restenosis occurred in 10 patients during a review period of five years. The long-term results of annuloplasty were disappointing, and this procedure is not recommended for children with mitral incompetence. Ten of the 100 patients who underwent mitral valve replacement died in hospital and there were two late deaths. Seventy-seven patients are completely well and 11 have grade II disability. Severe mitral valve disease in children is associated with high morbidity and mortality, unless normal hemodynamics are restored. Since the results of operation are so satisfactory, we believe that the natural history of this condition is most favorably altered by surgical treatment.


Long-term results of valve replacement in children suffering from rheumatic heart disease. Severe pathological changes in the cardiac valves are often observed at an early age in children in the developing countries. Mitral stenosis is best managed by closed commissurotomy. However, mitral insufficiency, aortic insufficiency and tricuspid lesions may lead to life-threatening hemodynamic effects which necessitate valve replacement. This differs from experience in the developed countries where surgery for rheumatic valvular disease is limited to the adult. Our experience includes 33 children aged 5 to 16 years. Twenty-four children underwent single valve replacement, eight had two valves replaced and one had triple valve replacement. All were classified Grade IV or late Grade III (New York Heart Association). Four children had to be operated on despite known rheumatic activity. Two children in shock and pulmonary edema underwent emergency operation. There were two hospital deaths and eight late deaths. Patients have been followed for up to eight years. Twenty children are now classified as Grade I and lead completely normal lives and the remaining three are classified as Grade II. Postoperative catheterization studies have documented improvement from severe preoperative hemodynamic changes to near normal values at rest after operation. The cardiothoracic ratio has decreased impressively. We conclude that the natural history of rheumatic heart disease in children with severely damaged heart valves is favorably modified by valve replacement.


Isolated aortic valve replacement. A six-year follow-up. A selected group of 259 patients who underwent aortic valve replacement was analyzed retrospectively for early and late mortality and late valve failures up to six years following operation. In this group, 100 patients received aortic valve homografts, 100 pulmonary valve autografts and 59 Starr-Edwards prosthetic valves. The two tissue valve groups, composed of younger, healthier individuals, had a lower early mortality rate than did the group receiving prostheses; mortality decreased even further with additional surgical experience. The homograft group also showed a lower long-term mortality and complication rate. The incidence and significance of the appearance of a diastolic murmur in the tissue valve group were also studied and seemed to play a major prognostic role for these patients.


The late fate of autologous fascia lata valve grafts in the aortic position. Fascia lata has been used for aortic valve replacement in 206 patients over a period of 10 years. Important advantages of this method are easy availability of the fascia tissue, excellent valve function in the early follow-up, and the absence of thromboembolic complications and of anticoagulant therapy. Long-term follow-up revealed a 10% incidence of infective endocarditis. Late deterioration of valve function occurred in 60% of patients after an average interval of five years. Electron microscopic study of fascia lata valve cusps removed one to seven years after implantation showed no endothelial layer, but an amorphous surface coating. The tissue remained viable, but showed necrotic foci and areas of proliferation. These morphologic alterations caused thickening and sclerosis of the valve cusps, which were responsible for the late deterioration of valvular function.


Surgery for tricuspid valve disease.

Surgery for tricuspid valve disease. Sixty patients underwent repair or replacement of the tricuspid valve, 58 for acquired and two for congenital lesions. In 59 of the cases, the tricuspid surgery formed part of multiple valve surgery. Preoperatively, 33 patients (55%) were in New York Heart Association class IV and 25 in class III functional capacity. The tricuspid valve was replaced in 42 patients and repaired in 18. Forty-seven patients (78%) survived operation, but 10 of these died during a follow-up period of two to seven years. Thirty-four of the 37 long-term survivors show marked improvement. Operative mortality was 26% for tricuspid valve replacement and 11% for repair; late mortality was 14% and 22%, respectively. It should be stressed that the tricuspid valve replacement group consisted of patients whose preoperative condition was worse and whose valvular pathology was more severe than that of those who had tricuspid repair. In view of this, and the better long-term results of tricuspid valve replacement, it is concluded that the procedure of choice for serious organic disease of the tricuspid valve is valve replacement.


Experience with tissue heart valves.

Experience with tissue heart valves. Experience with tissue valves for heart valve replacement over the past seven years is presented and analyzed. Between February 1967 and March 1969, 87 patients had heart valves replaced with preserved heterologous aortic valves. Valve failure occurred in 19 of the 70 operative survivors. There are 45 long-term survivors with heterologous aortic valves; 10 have regurgitant murmurs and 35 have valves functioning normally. Microscopic examination of failed valves has shown that the heterologous aortic valve gradually becomes a mechanically vulnerable structure in a state of rejection. During the past five years. autologous and homologous fascia lata and heterologous pericardium, mounted on a support frame, were used in 241 patients (131 aortic, 103 mitral and seven tricuspid). The follow-up period for the fascia lata group was 40 to 62 months and for the pericardial group 7 to 39 months. There have been significant differences in valve function with regard to both the site of valve insertion and the type of tissue used. Pericardial valves in the aortic position have produced the best results and autologous fascial valves in the mitral position, the worst. Valve failure occurred in only six patients, all with autologous fascia in the mitral position. The incidence of thromboembolism in the entire series was very low even though anticoagulants were not used. Analysis of clinical and hemodynamic results has shown that preserved heterologous pericardium has the potential for an adequate heart valve substitute.


Late results of mitral and aortic valve replacement. Experience with isolated mitral and aortic valve replacement at the University of Oregon Medical School since 1965 is presented. Results with non-cloth-covered and cloth-covered valves were analyzed with actuarial techniques and were compared in terms of late survival, thromboembolic complication rate, reoperation rate and the influence of anticoagulation therapy. The cloth-covered prostheses have substantially lowered the incidence of emboli after mitral replacement, and have thus far eliminated emboli after aortic replacement in patients receiving warfarin. Although anticoagulation therapy is still necessary, a clinical trial is currently in progress to assess the adequacy of antiplatelet drugs. The choice of a cloth-covered or non-cloth-covered valve should be individualized for each patient, based on the different risks and benefits of each prosthesis.


Present indications for direct myocardial revascularization. The present indications for direct myocardial revascularization in coronary arteriosclerosis are discussed. It is important to establish a universally acceptable classification of myocardial ischemia and such a classification is proposed. Without this classification, the advantages and disadvantages of medical and surgical treatment cannot be analyzed. Ergometric studies are of great value in deciding between medical and surgical treatment. Cine coronary angiography is essential in the accurate diagnosis and selection of patients for surgery. It is a safe procedure, even when undertaken in an emergency situation. The success of surgical therapy for coronary arteriosclerosis is emphasized and its superiority over medical treatment for most forms of angina pectoris is demonstrated.


Postinfarction ventricular aneurysm.

Postinfarction ventricular aneurysm. Postinfarction ventricular aneurysm may present as a dyskinetic or as an akinetic segment, depending upon the presence or absence of intraluminal thrombus. The overwhelming majority of aneurysms are localized to the anteroseptal portion of the left ventricle, while relatively few involve the diaphragmatic surface of the left ventricle. The introduction of extracorporeal circulation has offered a realistic form of surgical therapy for this serious complication of occlusive coronary disease. Proper surgical treatment involves excision of the aneurysmal wall and reconstruction of the ventriculotomy in such a way as to foreshorten the fibrosed portion of the interventricular septum. The technique described greatly alleviates residual paradox, which may be significant after conventional aneurysmectomy. The most impressive feature of ventricular aneurysmectomy concerns long-term survival. The natural history of the disease suggests that less than 20% of patients with ventricular aneurysm live five years after the initial infarction. In contrast, 76% of the first 400 consecutive aneurysmectomy patients operated on by the Cleveland Clinic team were alive four years after operation.


Applications of intra-aortic balloon counterpulsation. Intra-aortic balloon counterpulsation (IABC) is the form of mechanical circulatory assistance in widest clinical use today. The clinical results with IABC employed in 63 patients over a four-year period are presented. The clinical conditions necessitating mechanical circulatory assistance were: cardiogenic shock following acute myocardial infarction; myocardial infarction complicated by mitral valyular regurgitation or ventricular septal defect; preinfarction angina syndrome; postcardiotomy cardiogenic shock with pump oxygenator dependence; postcardiotomy cardiogenic shock during the postoperative recovery period; and septic shock. Survival and discharge from hospital occurred with 32 of 63 patients (51%). Evaluations of left ventricular function were studied in 20 patients on IABC by construction of Frank Starling curves, with cardiac output determined by thermodilution techniques. In general, IABC shifted the curves to the left and increased their slope.


Thoracic injuries in the Yom Kippur war. Experience in a base hospital. Forty-two patients were treated for combat injuries of the chest, caused by shrapnel fragments, bullet wounds, blunt trauma and blast injury. Twenty-three required only intercostal tube drainage, while 19 underwent thoracotomy. There were two deaths, both unrelated to the thoracic injury. Our indications for emergency thoracotomy were hemorrhage of 1,000 ml with the initial insertion of the chest tube or 500 ml of fresh bleeding during the first 2 hr after insertion of the chest tube, massive air leak, extensive chest wall injury, the location of metal fragments in the mediastinum, evidence of cardiac tamponade or significant mediastinal shift. The positive results obtained in this series of cases were due to the effective first aid treatment on the battlefield and in field hospitals, and to an aggressive policy of operative intervention where indicated, together with intensive postoperative care.


Ischemic myocardial contracture ("stone heart"). A complication of cardiac surgery. Stone heart or ischemic myocardial contracture is a complication of cardiopulmonary bypass characterized by contracture of the myocardium and inability to obtain cardiac output on manual massage. Stone heart has occurred only during aortic valve replacement and/or coronary artery bypass procedures. Predisposing factors are chronic congestive heart failure and myocardial hypertrophy with fibrosis secondary to longstanding aortic valve disease and/or coronary artery occlusive disease. Although the condition is rare, the outcome is usually fatal. Because of the biochemical and physiological implications of this syndrome, its prevention has been challenging. On the basis of our current concepts, prevention has been afforded by topical and general hypothermia and pretreatment with a small i.v. bolus of propranolol just prior to aortic occlusion. These measures have been utilized in potentially high-risk patients with the predisposition for stone heart and, thus far, the results have been satisfactory. The stone heart syndrome is an entity which should be recognized by all cardiac surgeons. Preventive measures must be aimed at protection of the myocardium during anoxic cardiac arrest.


Results of surgery for mitral insufficiency due to coronary artery disease. Twenty-six patients with combined mitral and coronary artery disease were divided into two groups: in group 1, only mitral valve surgery was performed, while in group 2, mitral valve surgery was performed along with coronary artery revascularization. Depressed ventricular function was not a contraindication to surgery; only 2 of 10 patients in group 2 with ejection fractions below 0.4 failed to survive operation. The hospital mortality was similar in both groups, and was higher for mitral valve replacement than mitral valve repair. Follow-up data revealed a higher incidence of late mortality in patients not undergoing revascularization, the main cause of death being myocardial infarction. The addition of revascularization also improved functional capacity.


An operation for the treatment of intractable peptic stricture of the esophagus. The current management of severe strictures of the esophagus resulting from reflux esophagitis is unsatisfactory. A new operation comprising esophagoplasty and intrathoracic fundoplication is described. This preliminary report records the results of this operation in 10 patients. There was one operative death. Of the nine survivors, followed for six months to three years, seven are completely free of symptoms. The remaining two have mild residual symptoms, but no dysphagia.


The surgical treatment of total anomalous pulmonary venous connection. Thirty-five patients had complete repair of total anomalous pulmonary venous connection between 1 January 1968 and July 1974. The mean age of the patients was 5.8 months. The hospital mortality rate was 43%; six of seven patients less than one month of age died. There were no deaths in the nine patients over six months of age at the time of operation. Inadequate cardiac performance was responsible for eight of the nine deaths in patients operated on since 1 September 1971. Thirteen percent of the patients who left the operating room alive were reoperated on for bleeding early in the postoperative period. No patient has required tracheostomy since 1 September 1971. Three of the 20 long-term survivors have required reoperation. The late results are good. Ways of improving results in the future are discussed.


Combined use of surface and perfusion hypothermia in intracardiac surgery in infants under one year of age. Deep hypothermia has recently gained popularity as an adjunct to intracardiac surgery for neonates and infants. A number of Japanese investigators played a role in the development and clinical application of both simple surface-induced hypothermia and the combined use of surface and perfusion cooling. The developmental history of both methods is described in detail. At the Heart Institute of Japan, 71 infants under one year of age underwent intracardiac surgery over the past six years. There has been a notable decrease in operative mortality since the employment of circulatory arrest under deep hypothermia and limited pump-perfusion. This trend is marked in infants with ventricular septal defect and pulmonary hypertension, and in those with total anomalous pulmonary venous connection. The Landé-Edwards membrane oxygenator proved helpful in prevention of postoperative pulmonary complications.


A comparison between partial and complete ligation of the inferior vena cava for the prevention of recurrent pulmonary embolism. Interruption of the inferior vena cava is the definitive surgical measure for the control of pulmonary emboli. There is, however, some controversy as to whether the preferred treatment is complete or partial interruption. This report summarizes our experience with 49 patients who underwent partial ligation, using a technique developed in our Department, and 66 patients, who underwent complete ligation of the inferior vena cava. The incidence and severity of sequelae due to venous stasis in the legs were less in the group that underwent partial interruption.


Aerosol vaccination against feline panleukopenia. Forty-seven cats were vaccinated with modified live-virus feline panleukopenia (FPL) vaccine administered by a nebulizer connected to a metal vaccination chamber. All 29 cats that dif not have FPL serum-neutralizing (SN) antibodies at the time of vaccination developed high SN titers. Five were then exposed to virulent FPL virus. Clinical signs of illness were not observed. Of 18 cats with maternally derived antibody, 14 failed to develop an immune response after vaccination. Three of these 14 cats were exposed virulent virus and all developed typical signs of FPL. It was concluded that aerosol FPL vaccination is an effective and practicable method of immunizing large number of cats.


Phaeohyphomycosis caused by Drechslera spicifera in a cat. A slowly evolving subcutaneous mycosis in a 10-year-old domestic shorthair cat was found to be caused by Drechslera spicifera, the imperfect state of ascomycete Cochliobolus spicifer. The cat had circular, nodular, granulomatous lesions over its sternum. Scattered individual and small groups of septate hyphae and chlamydospores were found in histologic sections. Many of the hyphae also had bizarre dilatations. Most of the fungal elements were hyaline; a few, however, were dematiacious. Because the fungus was not organized into granules in tissue, the disease could not be classified as a mycetoma. The preferred name for infections of this type is phaeohyphomycosis.


Some effects of posture on the radiographic appearance of the kidneys of the dog. Kidneys move in response to postural changes. In laterally recumbent dogs the lower kidney glides craniad, whereas the upper kidney tends to droop, yielding radiographs in which the upper kidney is often clearer and more bean-shaped than the lower. Routine lateral projections of the kidneys are best made in right lateral recumbency.


Hepatic arteriovenous fistula in two Saint Bernard pups. Two saint bernard pups, 7 and 5 months old respectively, were examined because of anorexia, vomiting, and ascites. Exploratory laparotomy disclosed arteriovenous fistula of the right medial lobe of the liver in one dog, and in the right medial and quadrate lobes of the other one. Surgical removal of the affected lobes resulted in cessation of presenting signs. Both dogs remained healthy but had poor weight gain and vomited occasionally. After more than 2 years of apparently good health, both dogs were euthanatized and necropsied because of presumably unrelated gastrointestinal disturbances.


Isolation in Illinois of a foreign strain of Ehrlichia canis, the causative agent of canine ehrlichiosis (tropical canine pancytopenia). Ehrlichiosis (tropical canine pancytopenia) was diagnosed in Illinois in a 4 1/2-year-old mixed breed dog that had spent the previous 2 years in India. The dog had periodic epistaxis, anemia, leukopenia, hypoalbuminemia, and hypergammaglobulinemia. A titer of 1:160 for Ehrlichia canis was detected by the indirect fluorescent antibody test (IFAT). Subinoculation of blood into a clinically normal dgo resulted in typical signs of canine ehrlichiosis, and E canis was identified in circulating mononuclear cells. The recipient died 23 days after inoculation, and typical gross and microscopic lesions of ehrlichiosis were found at necropsy. Ecchymoses and petechiae were found on the gallbladder, kidneys, small intestinal mucosa, cerebrum, and cerebellum. Plasmacytosis and perivascular cuffing was evident in various organs, especially the cerebrum, cerebellum, and meninges.


An epornitic of fatal chlamydiosis (ornithosis) in South Carolina turkeys. An unusual epornitic of fatal chlamydiosis occurred in a flock of 10,283 domestic turkeys in South Carolina. Total mortality over a 2-week period was 483 birds (4.7% of the flock). The principal gross lesion was severe pericarditis, but there was little or no airsacculitis, an observation at variance with many previous reports of chlamydiosis. Furthermore, an unusually heavy infestation of the turkeys with sanguivorous black flies (Simulium slossonae and S congareenarum) was observed at the time of the epornitic, an occurrence that may have permitted rapid transmission of chlamydiae between turkeys in the affected flock. The strains of Chlamydia psittaci isolated from naturally infected turkeys caused pericarditis and heptopathy as well as occasional airsacculitis, with a 41% mortality in intravenously (IV) inoculated turkeys. Turkeys inoculated intramuscularly (IM) or intraperitoneally (IP) did not die; however, airsacculitis was observed in more than 85% of turkeys inoculated intraperitoneally or exposed to infection by pen contact with inoculated turkeys. The strain was highly infectious but not lethal by the latter method of transmission. The strain was similar to other virulent chlamydiae isolated from turkeys, in that small numbers of the organism caused fatal infection in guinea pigs when inoculated IP.


Experimentally induced feline calicivirus infection: clinical signs and lesions. Sixty-six specific-pathogen-free cats were allotted to 10 groups and exposed by aerosol to 10 feline calicivirus (FCV) isolates. Viruses of different virulence were identified. The more virulent FCV caused pyrexia, depression, dyspnea, pneumonia, vesicles, or ulcers of the tongue and ulceration of the hard palate and nostrils. The FCV of low virulence caused similar lesions of the tongue, palate, and nostrils but little or no malaise, pyrexia, or pneumonia. Lesions produced by FCV usually were confined to the oral mucosa, tonsils, and lungs. Lesions in the nasal or trachea were associated with 2 of the 10 FCV tested.


Acetaminophen toxicosis in the cat.

Acetaminophen toxicosis in the cat. Administration by the owner of three 325-mg (5-gr) tablets of acetaminophen (N-acetyl-p-aminophenol) to each of 2 adult Burmese cats was associated with severe illness of both cats and death of one. Administration of two 325-mg tablets to each of 2 experimental adult cats resulted in severe illness. Marked cyanosis was observed in experimental cats within 4 hours after administration of one 325-mg tablet. Cyanosis was apparently due to anoxia associated with conversion of hemoglobin to methemoglobin by acetaminophen or its metabolites. Anemia, hemoglobinuria, and icterus were subsequently observed in the cats. Anemia and hemoglobinuria were caused by intravascular hemolysis of red blood cells (RBC). Icterus was due to both lysis of RBC and hepatic necrosis. Facial edema developed in 3 of 4 cats, but the pathogenesis of this lesion was not determined. The doses of acetaminophen were extremely large; however, administration of comparable doses to cats by their owners is a potential hazard because the drug is available without prescription as a 325-mg tablet. From information available at present, it seems that acetaminophen administration to the cat causes more dramatic clinical signs and is more likely to be fatal than the same doses of salicylates. Because phenacetin is metabolized to acetaminophen, similar clinical signs may occur in cats given phenacetin.


Use of a flutter valve in treatment of pneumothorax in dogs and cats. The use of a catheter and flutter valve to accomplish continuous thoracic drainage was evaluated in 12 dogs and 2 cats over a 2-year period. The system was found to be safe, reliable, and well tolerated. Compared with other drainage systems, this system requires less care from the veterinary staff.


An immunologic approach to population control in dogs. Testicular, thyroidal, and adrenal activities were measured in male Beagles immunized against bovine luteinizing hormone for 15 or 52 weeks. Circulating antibodies were associated with atrophy of the testes, epididymides, and prostate gland. The effects of immunization were apparent by high amounts of antibodies and by reproductive dysfunction for as long as a year in 3 of 4 dogs. The thyroid and adrenal glands were not detectably affected. A single dose of human chorionic gonadotropin stimulated production of antibodies that failed to cross react with canine gonadotropins or to cause reproductive failure in immunized dogs. A single dose of ovine gonadotropin stimulated production of antibodies that were associated with inability to ejaculate for more than 20 weeks.


Survey of canine and feline populations: Alameda and Contra Costa Counties, California, 1970. Age, sex, breed, and reproductive data were obtained on canine and feline populations in Alameda and Contra Costa counties, California. The demographic data were determined as of Dec 31, 1970, the reproductive data for all of 1970. The total of 27,076 households interviewed represented 5% of all households in the area. The canine and feline populations were estimated as 224,815 and 151,176, respectively. There was 1 dog for 7.3 persons and 1 cat for 10.8 persons. Almost half of the households included at least 1 dog or 1 cat. From the proportional distribution by age, it appeared that growth in the canine population may have peaked in 1968, with subsequent declines in pups entering households in 1969 and 1970. It was found that 47.8% of bitches and 64.6% of queens were neutered. Intact bitches of all ages averaged 0.2 litters each, those of queens, 0.9 litters each. The highest reproducing ages for both bitches and queens was between 1 and 3 years of age. In that age range, 62.9% of all canine litters and 74.4% of all feline litters were born; there were 0.4 litters per intact bitch and 1.6 litters per intact queen.


Nature and use of commercial dog foods.

Nature and use of commercial dog foods. Twenty-six commercial dog foods were analyzed. They represented the 4 main types of products intended to support the growth and maintenance of normal dogs, and the special dietary products intended primarily for use in dogs with chronic renal failure. None of the products contained extremes of protein, fat, or carbohydrate that could cause nutritional problems. Several products contained concentrations of calcium that might be too high if fed ad libitum to pups. The renal diets tended to be marginal in sodium and potassium contents, especially for use during renal or cardiac disease. Estimates of available energy were used as the basis for recommending amounts of the 4 main types of dog food to be fed daily to dogs of various body weights. These deviated widely from comparable recommendations in the 1972 NRC Nutrient Requirements of Dogs but agreed almost exactly with those in the 1974 Gaines Baisc Guide to Canine Nutrition.


Primary tumors of the spinal cord and meninges in six dogs. Primary spinal cord tumors were detected in 6 mature dogs. Two of the tumors were astrocytomas, 2 were meningiomas, and 2 were neurilemmomas. Five tumors were in the cervical portion and 1 was in the lumbar portion of the spinal cord.


Lysocellin, a new polyether antibiotic. I. Isolation, purification, physico-chemical and biological properties. A new antibiotic, lysocellin (K-5610), WAS ISOLATED FROM Streptomyces cacaoi var. asoensis K-9 Met-. Lysocellin was obtained as a colorless crystalline needles from both the cultural filtrate and the mycelium of the organism. The antibiotic melted at 158 similar to 160 degrees C and had a molecular formula C34H59O10Na-1/2H2O. It had antimicrobial activity against gram-positive bacteria, antibiotic-resistant Staphyloccus aureus and some fungi, but not against gram-negative bacteria. Based on its physico-chemical and biological properties lysocellin was identified as a new polyether antibiotic.


Isolation of galantins I and II, water-soluble basic peptides. Studies on antibiotics from the genus Bacillus. III. Two water-soluble basic antibiotics named galantins I and II were isolated from a strain resembling Bacillus pulvifaciens. Both antibiotics are peptides containing glycine, alanine, ornithine, lysine and some unknown ninhydrin-positive components. An approximate empirical formula C50 plus and minus H98plus and minus 2O17N16 is indicated for galantin I. These are active against some gram-positive, acid-fast and gram-negative bacteria.


Isolation of a new peptide antibiotic TL-119. Studies on antibiotics from the genus Bacillus. IV. A new antibiotic TL-119 active against gram-positive bacteria was isolated from a strain resembling Bacillus subtilis. The antibiotic is a neutral substance, soluble in a mixture of chloroform and methanol, and is a peptide with an empirical formula of C42H57N7O9, containing threonine (1), alanine (1), valine (1), leucine (1) and phenylalanine (2).


Isolation of a new peptide antibiotic complex 61-26. Studies on antibiotics from the geneus Bacillus. V. A new antibiotic named 61-26 active against gram-positive bacteria and some fungi was isolated from a Bacillus strain. The antibiotic is a weakly basic peptide slightly soluble in aqueous alcohols. An approximate empirical formula of C50H93N11O17 and constituent amino acids of aspartic acid (1 mole), serine(2 moles), alanine (2moles), and sum of valine and isoleucine (2 moles) are indicated.


Neocarzinostatin-induced breakdown of deoxyribonucleic acid in HeLa-S3 cells. Degradation of DNA in HeLa-S3 cells mediated by an acidic antitumor protein, neocarzinostatin (NCS), was examined. The concentration of NCS required for induction of DNA degradation was considerably higher than that which caused inhibition of DNA synthesis. Sedimentation analysis of DNA revealed that HeLa-S3 cell DNA first received single-strand nicks within 60 minutes after exposure to the antibiotic, whereas detectable double-strand scissions eventually gave rise to the accumulation of double-stranded DNA fragments of heterogeneous size. When the cells exposed to NCS were transferred to NCS-free medium at early stages of the degradation, the single-strand nicks caused in DNA were repaired by a process which was sensititive to puromycin.


Isolation of covalently closed circular deoxyribonucleic acid from Streptomyces coelicolor A3(2). Covalently closed circular deoxyribonucleic acid (DNA) was isolated from two strains of Streptomyces coelicolor A3(2), representing two of the known fertility types. In each of the two strains circular DNA of about 20 times 10-6 daltons could be detected, amounting to about 1.5% of the total cellular DNA. The possible function of this DNA is discussed.


Isolation and properties of a recombination-deficient mutant of Micrococcus radiodurans. A mutant of micrococcus radiodurans which is deficient in recombination has been isolated after treatment of the wild type with N-methyl-N'-nitro-N-nitrosoguanidine. We have called this mutant Micrococcus radiodurans rec30. The efficiency of recombination in this mutant, as measured by transformation, is less than 0.01% that of the wild type. It is 15 times more sensitive to the lethal action of ultraviolet radiation, 120 times more sensitive to ionizing radiation, and 300 times more sensitive to mitomycin C (MMC) than the wild type. It is probably inactivated by a single MMC-induced deoxyribonucleic acid cross-link per genome. The excision of ultraviolet-induced pyrimidine dimers is normal. There is no radiation-induced degradation of deoxyribonucleic acid. All spontaneous revertants selected for resistance to low levels of MMC had wild-type resistance to radiation and MMC, and the same efficiency of recombination as the wild type, suggesting that the recombination deficiency of the strain is due to a single mutation. Deoxyribonucleic acid from this mutant can transform M. radiodurans UV17 presumed deficient in an exr type gene to wild type.


Regulation of the Thiobacillus intermedius glucose uptake system by thiosulfate. Cells of the mixotrophic chemolithotroph (facultative autotroph) Thiobacillus intermedius which have been grown on a glucose-yeast extract medium, a condition in which glucose is used as a source of energy, accumulate the non-metabolizable analogue 2-deoxy-d-glucose against a concentration gradient in a predominantly unchanged state. On the other hand, cells grown mixotrophically on a thiosulfate-glucose medium, a condition in which glucose provides cell carbon but is not used extensively for energy, and in which enzymes of the Entner-Doudoroff pathway are repressed, do not accumulate 2-deoxy-d-glucose significantly. Similarly, cells grown chemolithotrophically on thiosulfate-carbonate do not take up this sugar. Transfer of thiosulfate-yeast extract-grown cells, which lack the capacity to accumulate 2-deoxy-d-glucose, to a glucose-yeast extract medium results in the induction of the concentrative sugar uptake system. The capacity of induced cells to take up 2-deoxy-d-glucose is inhibited by thiosulfate. Thus, the transport system for glucose appears to be regulated in this organism so that the sugar is accumulated only under conditions where it is utilized as a source of energy, and the presence of the preferred energy source leads to both repression and inhibition of the uptake system.


Role of vitamin K2 in the organization and function of Staphylococcus aureua membranes. A mutant of Staphylococcus aureus auxotrophic for menadione (a vitamin K2 precursor) was used to study the effects of menadione deprivation on the structure and function of the cell membrane. The phospholipid composition and metabolism was essentially unaltered by menadione deprivation. Removal of this percursor caused cellular levels of the cytochromes, protoheme, vitamin K2, and several membrane-bound flavoprotein dehydrogenase activities to decrease as a function of growth dilution. The cytochromes were enzymatically reducible and maintained in the same proportions as menadione-supplemented cells. Oxidative phosphorylation, however, was reduced more than 10-fold and membrane vesicles obtained from menadione-deprived cells were unable to couple glycine transport to L-lactate oxidation. Succinic dehydrogenase and adenosine 5' triphosphate hydrolysis appeared unaffected by menadione deprivation. These data suggest that menadione deprivation in the mutant stops the synthesis of vitamin K2 and other electron transport chain components and prosthetic groups. Although individual electron transport chain members remained fully functional during menadione deprivation, the overall efficiency of the chain, measured in terms of its function in electron transport, oxidative phosphorylation, and electron transport chain-linked transport, dropped greatly. This suggests that the synthesis of vitamin K2 is modulated to the synthesis of other components of the electron transport system, and that their organization into a functional system requires a specific concentration of vitamin K2 with respect to total membrane lipid.


Exocellular glycopeptide from a Penicillium charlesii mutant incapable of growth on D-galactose. The compositions of exocellular saccharide-containing polymers from six mutants of Penicillium charlesii incapable of growing on galactose were investigated. The polymers from the mutants contain a much smaller percentage of galactose than that reported for the peptidophosphogalactomannan (PPGM) from the wild-type organism (Gander et al. 1974). A polymer containing only one galactosyl residue per 49 mannosyl residues was investigated in detail. This polymer is a glycopeptide (peptidomannan) with an amino acid composition similar to that of peptidophospogalactomannan and a mass of about 23,000 daltons. Treatment of peptidomannan with 0.4 N NaOH releases mannan, mannopentaose, mannotetraose, mannotriose, mannobiose, and mannose residues, which are attached to the peptide by O-glycosidic linkage to seryl and threonyl groups. The quantity of glycerol and threitol, derived from mannosyl and internal galactofuranosyl residues, respectively, following Smith degradation, showed that peptidomannan contains 2 mol of internal galactofuranosyl residues per mol of polymer. The polymer contains only 3 mol of (1 yields 5)-linked galactofuranosyl residues per mol of polymer, as described by analysis of the methylation products. Methylation analysis also indicates that the polysaccharide contains primarily (1 yields 2)-linked (67.5%) and (1 yields 6)-linked (20.2%) mannopyranosyl residues. However, acetolysis of the polymer suggests that 37% of the residues are (1 yields 6)-linked. Mannopentaose, mannotetraose, mannotriose, mannobiose, and mannose in a molar ratio of 0.30:0.11:0.15:0.39:0.06, respectively, are released by acetolysis. This result is similar to that obtained with peptidophosphogalactomannan. We conclude that the occurrence of large numbers of galactofuranosyl residues in the major extracellular glycopeptide is not an obligatory requirement for glycopeptide formation.


Citrate uptake in membrane vesicles of Klebsiella aerogenes. In whole cells of Klebsiella aerogenes grown anaerobically on citrate as sole carbon source, citrate uptake is followed by rapid catabolism of the substrate via the inducible citrate fermentation pathway. Membrane vesicles prepared from such cells take up citrate but do not catabolize it. Vesicles process d-lactate dehydrogenase and the Na+-requiring oxalacetate decarboxylase. Citrate is taken up in the presence of Na+, and other monovalent cations, such as NH4+, Rb+, Cs+, or K+, do not substitute for Na+. Li+ appears to act synergistically with Na+. Citrate uptake is inhibited by N-2, cyanide, azide, sulfhydryl reagents, dinitrophenol, fluorcitrate, and hydroxycitrate.


Characterization of chromatin-bound erythrocyte histone V (f2c). Synthesis, acetylation, and phosphorylation. Synthesis and enzymatic modification of histone V was 1 order of magnitude lower in mature gander erythrocytes as compared with immature enriched cells hwich were capable of DNA synthesis. Application of shallow, linear gradient chromatography was used to demonstrate qualitative changes as well. This technique permitted the separation of newly synthesized and phosphorylated histone V from older, less phosphorylated molecules but did not discriminate between acetylated species. The most easily eluted fractions were those most recently synthesized, acetylated, and phosphorylated. While lysine chased into the other subfractions of histone V, phosphate did not, indicating a dephosphorylation step in the immature cells. Acetylation of histone V which occurs at a very low level was closely related to its synthesis. No differences in molecular weights or amino acid compositions were apparent, and behavior on polyacrylamide gels was similar to whole histone V. It is proposed that phosphorylation of histone V may play an important role in the modulation of the effect of histone V in immature cells on condensation and template restriction of chromatin which occurs in the terminal stages of differentiation of the avian erythroid cells.


Deoxyribonucleic acid-dependent ribonucleic acid polymerases in the dimorphic fungus Mucor rouxii. Three forms of DNA-dependent RNA polymerase have been separated by chromatography of extracts of yeast-like cells and mycelium of the dimorphic fungus Mucor rouxii. Each of the three eznymes has been purified by means of protamine sulfate precipitation, ion exchange chromatography, affinity chromatography, and velocity sedimentation. Electrophoresis under denaturing conditions showed differences in the subunit compositions of all three purified enzymes. The properties of the enzymes from M. rouxii were similar to those of polymerases from other eukaryotic organisms. Denatured DNA was a better template than native DNA for all three enzymes but each enzyme had a distinct pattern of activities with different templates. Enzymes I and III displayed optimal activity with Mn-2gs the divalent cation and were stimulated significantly by Kcl and (NH4)2S04. Enzyme II had a greater activity with Mg-2gnd was only slightly stimulated by KCl and (NH4)2SO4. None of the enzymes were inhibited by cycloheximide or by rifampicin: all were inhibited by actinomycin C and rifampin AF/018: only enzyme II was inhibited by alpha-amanitin. No differences could be found in the properties of the same enzymes isolated from yeast-like cells or mycelium.


Identification of functional arginine residues in ribonuclease A and lysozyme. A specific color reaction has been developed for the detection of N-7, N-8-(1,2-dihydroxycyclohex-1,2-ylene)-L-arginine-containing peptides. The reaction is based on the fact that hydroxylamine converts the blocking group to cyclohexanedione dioxime, which forms a red nickel complex. N-7, N-8-(1,2-dihydroxycyclohex-1,2-ylene)-L-arginine-containing peptides can also be detected by diagonal electrophoresis from the change of electrophoretic mobility of these peptides on interaction of the blocking group with borate. Since the modified arginine residues are resistant to tryptic cleavate, changes in tryptic peptide patterns can also be utilized to identify the presence of modified arginine residues. A combination of these approaches was used to identify the arginine residues modified by cyclohexanedione treatment. Bovine panctreatic RNase A loses approximately 90% of its activity on cyclohexanedione treatment with the modification of 2 to 3 arginine residues. Arginine-39 reacts most rapidly and its modification contributes most to inactivation of the enzyme. Arginine-85 also reacts rapidly with cyclohexanedione. Arginine-10 reacts slowly and no reaction was observed with arginine-33. Removal of the blocking groups by hydroxylamine treatment resulted in complete recovery of enzyme activity in samples where arginine-39 and arginine-85 had been modified, whereas 80% of activity was regained from samples where arginine-10 had also been modified. With egg white lysozyme, all 11 arginine residues react with cyclohexanedione, resulting in partial inactivation of the enzyme. The fully modified enzyme retains 35% of its activity. Since arginine residues are important for electrostatic interaction between the enzyme and the negatively charges cell surface, even the modified, basic residues can provide the necessary positive charges. In the presence of borate, activity is almost completely abolished, since the modified arginine-borate complex has a reduced net positive charge. Upon removal of the blocking groups by hydroxylamine, even the fully modified lysozyme regains complete activity. With the exception of the most reactive arginine (residue 5), modification of all other arginine residues contributes equally to inactivation of the enzyme. The possible reason for the importance of arginine-5 in maintaining activity is discussed. Advantages of the present method for the selective reversible modification of arginine residues of proteins and for the identification of reactive arginine residues are evaluated.


Mechanism of inactivation of the flavoenzyme lactate oxidase by oxalate. Lactate oxidase from Mycobacterium smegmatic is strongly inhibited by oxalate. The inhibition is reversible in the absence and irreversible in the presence of light. Oxalate is bound to the enzyme in a two-step process with an over-all Kd of 1.6 times 10--5 M. The first step is a fast second order reaction with k-1/k1 equals 8.3 times 10--3 M, leading to a Complex I. This complex is then reversibly converted to a different Complex II in a slow first order reaction (k2 equals 40 min--1; k-2 equals 0.07 min--1), which is accompanied by major spectral perturbations of the flavin spectrum. With oxamate, two steps could not be demonstrated, and its binding is described by a single step reversible process, which is second order in oxamate (k1 equals 6.8 times 10-3 M--1 s--1, k-1 equals 28 s--1). Upon illumination the enzyme-oxalate Complex II is converted very rapidly at position N(5) of the flavin. Slow hydrolysis in the dark under anaerobic conditions subsequently yields free reduced enzyme. The light reaction of the oxamate comples is, in contrast, very slow and yields a stable N(5) urea adduct of the reduced flavin.


Partial purification and characterization of beta-galactosidase from rat brain hydrolyzing glycosphingolipids. Adult rat brain beta-galactosidase was partially purified with the use of lactosylceramide, galactosyl-N-acetylgalactosaminyl-galactosyl-glucosylceramide, galactosyl-N-acetylgalactosaminyl-(N-acetylneuraminyl)galactosyl-glucosylceramide, and 4-methylumbelliferyl theta-galactoside as substrates. Approximately 50-fold purification was achieved by solubilization, ammonium sulfate fractionation, Sephadex G-200 and DEAE-cellulose column chromatography. Activities toward the above four substrates behaved essentially identically throughout the pruification procedure. Considerable differences were noted between the apparent properties determined with whole homogenate and those of the purer enzyme preparations. Based on these studies, assay procedures for the purified preparation for the three glycosphingolipid substrates were standardized. Inhibition studies with the use of varieties of simple sugars, oligosaccharide chains prepared from glycosphingolipids, and intact sphingolipids suggested that the enzyme which cleaves lactosylceramide may be different from the enzyme(s) which is active toward the other two glycosphingolipids. The oligosaccharide chains of the glycosphingolipids were much poorer inhibitors for the respective glycosphingolipid beta-galactosidases than the original intact glycosphingolipids or ceramide, and in some instances, even unrelated sphingolipids. These findings indicated the importance of the lipophilic groups and perhaps of the entire molecular configuration of glycosphingolipids in determining the specificity of these glycosphingolipid theta-galactosidases.


The exterior surface of the chicken erythrocyte. The exterior surface of mature chicken erythrocytes has been labeled with a cationic membrane impermeable reagent, rho-nitrophenyl-N,N,N-trimethy[125I]iodotyrosinate. This reagent forms stable covalent bonds with the amino groups of exposed protein and lipid companents. Two major protein components with subunit molecular weights of 100,000 and 55,000 are found on the outer surface of mature chicken erythrocytes. Both of the labeled components coincide were periodic acid-Schiff base-stained areas on sodium dodecyl sulfate polyacrylamide gel electrophoresis indicating that they may be glycoproteins.


A membrane protein from human erythrocytes involved in anion exchange. Phosphate transport across the membrane of human erythrocytes, at pH 6.42, is a saturable process with an external Km of 80 mM and a Vmax of 2.8 mmol per liter of red cells per min. Transport is inhibited in a reversible manner by the sulfanilate anion and in an irreversible manner by the isothiocyanate derivative of the sulfanilate anion. This reagent reacts with only one membrane protein. When 300,000 molecules of reagent per cell are covalently attached to protein, transport is completely inhibited. This transport protein is a glycoprotein, it belongs to the general class designated as component a by Bretscher, and it therefore spans the bilayer. Transport does not involve movement of the protein around an axis parallel to the plane of the membrane.


Biphasic inactivation of procine heart mitochondrial malate dehydrogenase by pyridoxal 5'-phosphate. Temperature studies have indicated that from 0 to 37 degrees, the time-dependent inactivation of mitochondrial malate dehydrogenase from porcine heart by pyridoxal 5-phosphate (pyridoxal-5-P) is biphasic. The initial phase of the inactivation is reversible but can be made irreversible by reduction with sodium borohydride. The reduced pryidoxal-5-P-enzyme adduct exhibits a new absorbance maximum at 325 nm and a fluorescence emission at 392 nm when excited at 325. The irreversible second phase of the inactivation is accompanied by the appearance of a new 325-nm absorbance maximum, in the absence of reduction, and a fluorescence emission centered about 390 to 400 nm when excited at 325. The evidence presented suggests the formation of a Schiff base between pyridoxal-5-P and a nucleophilic residue, most likely lysine, of malate dehydrogenase during the first phase of inactivation. An X-azolidine-like structure, a further derivative of the Schiff base, possessing spectral properties consistent with the reported data, may be formed during the second phase; this presumably involves a second nucleophilic residue of the enzyme, implicating the action of pyridoxal-5-P as a bifunctional reagent in this instance. The presence of the coenzyme, NADH, protects the enzyme from inactivation, suggesting that pyridoxal-5-P interacts at or near the malate dehydrogenase active center. Simultaneous binding studies using pyridoxal-5-P with known malate dehydrogenase competitive inhibitors AMP, ADP, and nicotinamide indicate that the pyridoxal-5-P modification occurs in the general area of the ADP portion of the coenzyme binging site. Furthermore, the presence of nicotinamide enhances pyridoxal-5-P binding to and inactivation of malate dehydrogenase.


Interaction of guanosine nucleotides with elongation factor 2. I. Equilibrium dialysis studies. Binding of the guanosine nucleotides, GDP and GTP, to elongation factor 2 (EF-2) from rat liver was studied by equilibrium dialysis. It was found that the enzyme has one binding site for GDP with a dissociation constant of 4 times 10--7 M. The examination of GTP binding was difficult due to the simultaneous presence of GDP and GTP even in purified GTP preparations. This problem was further magnified by traces of GTPase in the enzyme preparation. However, by analyzing the incubation mixtures by thin layer chromatography the fraction of the total nucleotide binding to EF-2 which was due to GDP could be determined and corrected for. A GTP binding curve, corrected for GDP binding, and GTP hydrolysis extrapolated to one binding site with a dissociation constant of approximately 2 times 10--6 M. The nonhydrolyzable GTP analogue, theta, gamma-methylene-guanosine-5-triphosphate, also bound to EF-2 in a 1:1 ratio. During the studies of GTP binding to EF-2 it was observed that the enzyme preparation contained a GTP-GDP transphosphorylase activity. It was initially thought that this was a novel property of EF-2, but when the activity was followed during purification of EF-2 it was whown that it was an impurity in the EF-2 preparation. ATP as well as GTP can serve as a phosphate donor in the transphosphorylation reaction; this might suggest that regeneration of GTP from GDP can take place via this pathway.


Interaction of guanosine nucleotides with elongation factor 2. II. Effect of ribosomes and magnesium ions on guanosine diphosphate and guanosine triphosphate binding to the enzyme. The effects of ribosomes and Mg-2plus on the binding of GDP and GTP to elongation factor 2 (EF-2) have been studied by an improved filter-binding assay. Both ribosomes and Mg-2plus strongly inhibit the binding of GDP but have apparently no effect on the GTP binding to the enzyme. An apparent stimulation by ribosomes of GTP binding to EF-2 is time-dependent and parallels a concomitant increase of the GDP concentration in the incubation mixture. Based on these results and evidence obtained by other investigators it is suggested that changes in the GTP:GDP ratio associated with the elongation and termination reactions of protein synthesis cause conformational changes of the respective factors which consequently will modulate the binding and dissociation of the enzymes from ribosomes. Further evidence of the role GDP may play as a modulator of protein synthesis might possibly be provided by studies of the GTP-GDP transphosphorylase activity which is present as an impurity in highly purified preparations of EF-2 as well as in ribosome preparations. It is demonstrated that relatively high concentrations of GDP in the presence of GTP completely block the ribosome-dependent GTPase activity of EF-2. Instead, the transphosphorylase enzyme(s) catalyzes an exchange reaction between GTP and GDP during which GDP remains bound to EF-2 and the relative concentrations of the two nucleotides do not change.


Isolation and characterization of acetylcholinesterase and other particulate proteins in the hemolymph of Aplysia californica. Hemolymph of the marine mollusc, Aplysia californica, contains four large particles: acetylcholinesterase, hemocyanin, a hemagglutinin, and a structure tentatively identified as erythrocurorin. We purified the acetylcholinesterase 20-fold by differential centrifugation and filtration through a column of 4% agarose. The freshly isolated esterase complex was found to have a sedimentation coefficient of 69, but the negatively stained enzyme lacked a definite structure in the electron microscope, and appeared as irregular aggregates of a 60 A subunit. The complex was unstable below pH 5 or during storage at 7 degrees. Under these conditions, enzymatic activity remained essentially unchanged. Treatment of the purified enzyme with trichloroacetic acid, organic solvents, and sodium dodecyl sulfate broke the complex down into two major subunits with molecular weights of about 70,000. Exposure of the enzyme to [3H]diisopropylfluorophosphate resulted in the labeling of one of these subunits. Although similar in specificity, the cholinesterase of the blood differed from the enzyme in Aplysia nervous tissue, which is associated with membrane. Treatment with sodium deoxycholate activated the membrane-associated enzyme but inhibited slightly that of the hemolymph; tyrocidine inhibited the hemolymph enzyme but not the enzyme of nervous tissue; and mild digestion with trypsin released the membrane-bound enzyme in an active, soluble form, but inactivated the enzyme of hemolymph. The other particulates of Aplysia hemolymph were partially characterized. Aplysia hemocyanin was similar in structure to other molluscan hemocyanins. When negatively stained, the unit particle appeared to be a disc with a diameter of 280 A and a width of 45 A. These discs were stacked to form long cylindrical arrays. The purified hemocyanin was found to contain 0.26% copper (dry weight). Using differential centrifugation and gel filtration we also obtained a 9-fold purification of Aplysia hemagglutinin. This particle was 120 A in diameter with a dark staining central core of 40 A consisting of 6 subunits. The particle tentatively identified as erythrocurorin appeared as a structure 200 A in diameter consisting of 5 V-shaped subunits.


The sialic acids. XVIII. Subcellular distribution of seven glycosyltransferases in embryonic chicken brain. The subcellular distribution of seven glycosyltransferases was studied in embryonic chicken brain. Four of the transferases are required for the synthesis of gangliosides, and three for the synthesis of the terminal trisaccharide units of serum type glycoproteins. Six of the transferases were found only in the particulate fraction of homogenates, while one (UDP-galactose:glycoprotein galactosyltransferase) was found both in the paritculate and soluble fractions of young embryonic chicken brain, but only in the particulate fraction obtained from older embryos. The source of the soluble galactosyltransferase was found to be the fluid surrounding the embryonic brain. Fractionation studies by the Whittaker technique showed that the seven activities were located primarily in the nerve ending (synaptosome)-rich fraction.


Stimulation of the uptake of soluble proteins into isolated HeLa nuclei by pancreatic deoxyribonuclease. The uptake into isolated HeLa nuclei of radioactive cytosol proteins and purified Escherichia coli ribosomal protein L7 is stimulated up to 4-fold by pancreatic deoxyribonuclease (DNase I). Similar effects are not observed with pancreatic ribonuclease A or phospholipase C. The results reported suggest that there is a general stimulatory effect of DNase on protein uptake by nuclei.


Hemolysis of human erythrocyte induced by a rapid temperature jump. When an isotonic suspension of human erythrocytes was subjected to a rapid temperature jump (heating time of 2 mus), the following phenomena were observed. Although a jump of 0.05 degrees caused no visible damage to the cell membranes, it facilitated the permeation of [3H]glucose into the cells. A jump of 0.5 degrees induced a hemolysis of the cells. The hemolysis was accompanied by extensive alterations of membrane morphology possibly caused by ruptures of the membranes. Turbidity changes of the suspension after the temperature jump were consistent with a swelling of erythrocytes followed by a shrinking of the swollen cells. The phenomena of the water transport and the hemolysis induced by the temperature jump may be understood by the theory of thermal osmosis (Spanner, D. C. (1954) Symp. Soc. Exp. Biol. 8, 76). Slow heating of the suspension shows no such effects. Thermal osmosis may be an important factor in the water transport and bioenergetics of the living cells.


Quaternary structure of partially liganded intermediates of sheep carbon monoxide hemoglobin at alkaline pH. A rapid change in absorbance was observed in the Soret region during the interval between photolysis of sheep carbon monoxide hemoglobin and the subsequent reassociation of CO in the dark. The rate constant for this spectral change was about 4000 s--1 at 20 degrees in 0.05 M solium borate, pH 9.3. The wavelength dependence of the amplitude of the absorbance change is similar to that observed when deoxygenated alpha and theta chains are allowed to recombine (Brunori, M., Antonini, E., Wyman, J., and Anderson, S. R. (1968) J. Mol. Biol. 34, 357-359), and therefore reflects changes in the quanternary structure of the hemoglobin tetramer induced by ligand displacement. The amplitude of this conformation-dependent spectral change was not a linear function of the fraction of bound CO removed by photolysis. The results suggest that of the possible intermediate species present after partial photolysis, only Hb4 and Hb4(CO) change from the ligand-bound to the ligand-free sturcture prior to CO reassociation under these alkaline conditions.


Topographical arrangement of membrane proteins in the intact myelin sheath. Lactoperoxidase incorproation of iodine into myelin surface proteins. The lactoperoxidase-catalyzed iodination technique was utilized to incorporate radioactive iodine into membrane proteins which lie on the outer surface of the myelin sheath.  An intact, myelinated nerve bundle, the dorsal column of the cat spinal cord, was employed. The enzymatically iodinated proteins were identified by polyacrylamide gel electrophoresis, and the specific radioactivity was determined. Results indicated that several high molecular weight proteins were predominantly labeled by the nonpenetrating lactopreoxidase. Proteolipid protein was also labeled, although to a lesser extent; basic protein was not labeled under these conditions. The data suggest that several high molecular weight proteins are exposed on the outer surface of the myelin sheath. Proteolipid protein is at least partially exposed on the outer surface, although it could be present at both membrane surfaces. Evidence is presented which suggests that the basic protein is located at the inner surface of the membrane, corresponding to the major dense line of myelin.


Egasyn, a protein complexed with microsomal beta-glucuronidase. Two polypeptide chains are present in murine beta-glucuronidase precipitated with a specific anti-beta-glucuronidase antibody F(ab)2 fragment. One is the catalytic subunit of beta-glucuronidase and the other has the properties predicted for the hypothetical beta-gluronidase membrane anchor protein. The new protein, named egasyn, is associated with microsomal, but no lysosomal beta-glucuronidase. It is released from the microsomal beta-glucuronidase complex by heat treatment. The YBR strain of mice carrying the Eg degrees mutation does not form an egasyn-beta-glucuronidase complex and is unable to retain beta-glucuronidase on microsomal membranes.


Characterization of globin messenger ribonucleic acids in membrane polysomes of mouse reticulocytes. Between 20 and 30% of the polysomes in mouse reticulocytes are associated with the cell membrane fraction. These polysomes are not liberated by washing with 0.5 m KCl and are therefore thought to be attached to membranes. They have the same percentage of polyadenylic acid-containing RNA, as determined by oligo(dT)-cellulose affinity chromatography, as cytoplasmic polysomes. Analysis of the RNA by polyacrylamide gel electrophoresis in aqueous solutions shows that at least 95% of the RNA migrates identically with the cytoplasmic polysomal globin mRNAs. Electrophoresis in 99% formamide resolves the membrane mRNAs into two bands which migrate identically with the cytoplasmic alpha- and beta-globin mRNAs. The molar ratio of the alpha- and beta-globin mRNAs, as determined by quantitating the bands on the formamide gels, is similar to that of the cytoplasmic mRNAs. There is also no difference in biological activity between the two messenger preparations. The polyadenylic acid in the membrane mRNAs, isolated after labeling mice in vivo for 20 hours with (32P)orthophosphate, migrates with the three broad size classes previously shown to be present in the cytoplasmic globin mRNAs. The 32P specific activity of the membrane mRNAs and ribosomal RNA fractions after different times of labeling with 32Pi are similar to those from cytoplasmic polysomes. These observations show that the reticulocyte membranes contain approximately 20% of the cellular globin nRNAs, and that these mRNAs are similar to those from cytoplasmic polysomes.


Protein synthesis in rabbit reticulocytes. A study of Met-tRNA f Met binding factor(s) and Met-tRNA f Met binding to ribosomes and AUG codon. The effects of additions of Mg-2+, ribosomes, and AUG codon on the Met-tRNAf Met-initiation factor-GTP complex were studied using a Millipore filtration method (J. Biol. Chem. 248, 4500 (1973)). Upon addition of increasing concentration of Mg-2+, the Met-tRNAf Met-initiation factor-GTP complex dissociates into free Met-tRNAf Met and initiation factor (GTP), with an infection around 1.5 to 2 mM Mg-2+. The Mg-2+-induced dissociation of Met-tRNAf Met-initiation factor-GTP complex was enhanced at ice bath temperature. At 37 degrees and in the presence of 1.5 to 2mM Mg-2+, the Met-tRNAf Met-initiation factor-GTP complex catalyzes the transfer of Met-tRNAf Met to ribosomes and AUG codon. Ribosome bound Met-tRNAf Met is stable to Mg-2+ and low temperature. A Millipore filtration assay for studies of (35S)Met-tRNAf Met binding to ribosomes and Aug codon has been developed. The assay procedure is carried out in three stages. In Stage I, the Met-tRNAf Met is bound to initiation factor in the presence of GTP, AUG codon (required for Stage II reaction), and 3.7 times 10-5 M aurintricarboxylic acid. The incubation is carried out at 37 degrees for 5 min. In Stage II, ribosomes and Mg-2+ (1.5 to 2mM final concentration) are added and the incubation is continued at 37 degrees for 10 min. In Stage III, more Mg-2+ is added to make the final Mg-2+ concentration of the incubation mixture 5 mM, and the reactions are further incubated at ice bath temperature for 10 min. The reactions are then terminated by addition of excess cold wash buffer and filtered through Millipore filters. Under the standard assay conditions, the radioactivity bound to Millipore filters in the absence of ribosomes and AUG codon is markedly reduced. Addition of ribosomes alone gave a significant increase in the radioactivity bound to Millipore filters. A further 2- to 3-fold stimulation of binding of (35S)Met-tRNAf Met to Millipore filters was observed when both ribosomes and AUG codon were added. The Met-tRNAf Met bound to ribosomes under the assay condition was reactive with puromycin. Upon DEAE-cellulose chromatography of a partially purified mixture of initiation factors (IF), Met-tRNAf Met binding activities separate into two forms, and are designated as IF-1A and IF-1B. These two forms can be distinguished by the stabilities of their respective Met-tRNAf Met-IF-1-GTP complexes to Mg-2+. The Met-tRNAf Met-IF-1A-GTP complex is distinctly more stable in the presence of Mg-2+ than Met-tRNAf Met-IF-1B-GTP complex. Continue.


A synthetic 70-amino acid residue analog of ribonuclease S-protein with enzymic activity. A 70-residue analog of RNase S-protein was synthesized by the solid phase method. It was obtained by omitting the NH2 terminus from positions 21 to 25 and the segments 36 to 40, 58 to 73, 87 to 96, and 113 to 114. Four residues were inserted to link the ends formed by the deletions. Half-cystine residues that had not been part of the deletions were replaced by alanine or leucine residues. The synthetic polypeptide was separated by gel filtration into a dimer and a monomer. Both fractions were purified further by ion exchange chromatography. The dimeric 70-residue S-protein analog had a specific activity of approximately 4% using RNA as substrate. It also cleaved other substrates of RNase A such as 5'-(3'-cytidylyl)-guanosine, 5'-(3'-uridylyl)-guanosine, and polycytidylic acid. The monomer of the 70-residue analog was less active but showed the same substrate specificity as the dimer. It was found that both fractions of the synthetic S-protein analog catalyzed only the transphosphorylation step of the RNase A mechanism and had very little if any activity in the hydrolysis step. Addition of natural S-peptide or S-protein did not increase the activity in the transphosphorylation reaction but greatly enhanced the reaction rate of the hydrolysis step. IN THE PRESENCE OF S-peptide, both monomeric and dimeric 70-residue S-protein, both monomeric and dimeric 70- residue S-protein analog had approximately 8% activity using cyclic cytidine 2':3'-monophosphate as substrate. The mixtures of monomer and dimer of the synthetic S-protein analog with natural S-protein generated even higher activities (151 and 74%, respectively) against this substrate despite the fact that the NH2-terminal portion of the natural enzyme (including His 12) was missing in both components of the two complexes. The 70-residue S-protein analog was completely inactive against DNA and (with one exception) against substrates for RNase T1. The close agreement of the substrate specificity of the synthetic analog with that of native RNase A in the transphosphorylation step suggested a remarkable conservation of the configuration of the active site despite drastic changes of the primary structure of the parent molecule. Possible implications of these results for the mechanism of action of RNase A are discussed.


Sequential degradation of keratan sulfate by bacterial enzymes and purification of a sulfatase in the enzymatic system. Pseudomonas sp. IFO-13309 and Actinobacillus sp. IFO-13310, bacteria which exhibit a symbiotic growth in a medium containing keratin sulfate as a sole carbon source, were isolated from soil. Extracts of these organisms were shown to contain an endoglycosidase, a sulfatase, and exo-beta-D-galactosidase, and an exo-beta-D-N-acetylglucosaminidase which, together, catalyze an extensive cleavage of corneal keratan sulfate. The Pseudomonas extract was particularly rich in the endoglycosidase activity and poor in the exoglycosidase activities. The Actinobacillus extract, in sharp contrast, contained principally the exoglycosidases. The sulfatase activity did not show this marked difference in distribution. A sulfatase was purified from the crude extract of Actinobacillus. The purified sulfatase reacted little or not at all with keratan sulfate, but acted on 2-acetamido-2-deoxy-6-O-sulfo-D-glucose, 2-acetamido-2-deoxy-6-O-sulfo-beta-D-glucosyl-(1 leads to 3)-D-galactose, and a tetrasaccharide trisulfate having 2-acetamido-2-deoxy-6-O-sulfo-D-glucose at the nonreducing end (prepared from keratan sulfate with an endogalactosidase). The enzyme removed one sulfate group from the tetrasaccharide trisulfate, producing an oligosaccharide which, unlike the parent oligosaccharide, was susceptible to hydrolysis with exo-beta-D-N-acetylglucosaminidase. The data suggest that the nonreducing end is the only site at wich enzymatic desulfation is carried out.


Purification of the subunits of transcarboxylase by affinity chromatography on avidin-sepharose. Transcarboxylase consists of a central 12 SH subunits each of which is linked to the central subunit by two similar to 1.3 SE biotin carboxyl carrier proteins. The subunits from dissociated transcarboxylase have been difficult to isolate because conditions which stabilize them also promote their reassociation to the intact enzyme. In this paper, we describe the use of avidin-Sepharose to adsorb the enzyme from crude extracts or partially purified transcarboxylase of propionibacteria. After removing impurities by washing the column with phosphate buffer at pH 6.5, in which the transcarboxylase is stable, the enzyme is dissociated first by elution at pH 8 yielding a fraction containing mostly 12 SH subunit which can be rapidly stabilized against dissociation to 6 SH without the problem of reconstitution because the 1.3 SE and most of the 5 SE subunits are not eluted. The second elution is at pH 9 which yields the 5 SE subunit by dissociation from the 1.3 SE biotin subunit and the 1.3 SE subunit remains bound to the avidin. The 12 SH and 5 SE subunits are further purified by glycerol density gradient centrifugation or by chromatography on Bio-Gel. Very active enzyme can be reconstituted from these subunits upon the addition of the 1.3 SE subunit.


Synthesis of membrane protein in slices of rat cerebral cortex. Protein synthesis was studied in slices from rat cerebral cortex and localized in various purified subcellular membrane fractions isolated after incubation with L-leucine. A synaptosomal fraction isolated from a several times washed crude mitochondrial pellet showed very little contamination by free membranes, and the synaptic membrane fraction isolated from it was estimated to c +ain about 50% of this component. Leucine incorporation into all fractions was highly sensitive (greater than 95%) to emetine except for the cell and synaptic mitochondrial subfractions. They were only 60 to 70% inhibited by emetine and showed 10 to 20% inhibition by chloramphenicol which probably was due to those proteins synthesized in situ by the mitochondria. The net rate of incorporation of labeled protein into the synaptosomal and synaptic membrane subfractions was low, and axonal flow was excluded as a significant source of this label. On the basis of autoradiography it was concluded that the majority of the protein label was contributed by free and membrane-enclosed ribosome-containing contaminants in these fractions. Unlabeled and labeled subcellular fractions were also analyzed by electrophoresis in the presence of sodium dodecyl sulfate. The staining profiles of the microsomal and synaptic membrane subfractions were nearly identical, whereas those of synaptic mitochondria and the soluble proteins of the cell bodies were unique. The labeling of all proteins was blocked by emetine, except for the synaptic mitochondria (cell mitochondria were not examined). These products of mitochondrial protein synthesis exhibited three labeling peaks, the major one with a molecular weight of approximately 38,000. In the absence of emetine, slices incorporated amino acids into soluble and microsomal proteins with high specific activity. The labeled proteins of the soluble fraction were more highly concentrated in the range of molecular weights smaller than 50,000 than were those in the microsomal fraction, and it was concluded that a considerable portion of the protein-synthesizing machinery of the brain is geared to form soluble proteins. The gel patterns of labeled microsomal and synaptic membranes were consistent with contamination by the former contributing most of the protein label in the latter.


Effects of extracellular cations and ouabain on catecholamine-stimulated sodium and potassium fluxes in turkey erythrocytes. In turkey erythrocytes, potassium influx can be inhibited by several cations whose order of effectiveness is Rb greater than Cs greater than Li greater than Mg = Ca = Ba. Extracellular sodium does not alter potassium influx. Sodium influx is not altered by any of these monovalent cations but magnesium, calcium, or barium reduced sodium influx by 30 to 40%. Potassium outflux is not influenced by extracellular sodium or potassium while sodium outflux is not influenced by extracellular potassium but increases progressively with increasing extracellular sodium. Isoproterenol stimulates potassium influx only when sodium or lithium is present in the medium and catecholamine stimulation increases progressively with increasing extracellular sodium. Isoproterenol-stimulated sodium influx is enhanced by extracellular potassium, rubidium or cesium, and catecholamine stimulation increases progressively with increasing extracellular potassium. Isoproterenol inhibits potassium outflux in a solution free of sodium and potassium, and this inhibition can be abolished by adding sodium but not by adding potassium. In solutions containing both sodium and potassium, isoproterenol stimulates potassium outflux, and this stimulation increases progressively with increasing extracellular sodium or potassium. Isoproterenol-stimulated sodium outflux is not influenced by extracellular sodium or potassium. Isoproterenol-stimulated cellular cyclic adenosine 3':5'-monophosphate (cyclic AMP) is reduced slightly (25%) by removing extracellular sodium and potassium from the incubation solution, but this effect is not of sufficient magnitude to account for the effects of these cations on isoproterenol-stimulated cation transport. Ouabain does not alter the effect of extracellular sodium or potassium on isoproterenol-stimulated potassium influx; however, the glycoside potentiates (by 20 to 40%) the effects of these two cations on isoproterenol-stimulated sodium influx. Ouabain does not alter potassium outflux when the incubation solution contains both sodium and potassium; however, ouabain stimulates potassium outflux in an incubation solution free of sodium or potassium.


Purification and characterization of homogeneous protein synthesis initiation factor M1 from rabbit reticulocytes. An eight-step procedure has been devised for the preparation of homogeneous rabbit reticulocyte IF-M1. Molecular weight determinations based on IF-M1 activity (gel filtration and sucrose density gradient sedimentation) and based on IF-M1 protein (low speed equilibrium sedimentation and sodium dodecyl sulfate gel electrophoresis) indicate that IF-M1 is active as a single polypeptide chain of 65,000 molecular weight. The amino acid composition of IF-M1 has been determined. There appears to be no unique features in the amino acid composition of IF-M1, except perhaps an elevated proline content (6.9 mol %). The catalytic properties of purified IF-M1 were similar to those previously reported by this laboratory for crude preparations of IF-M1. The sensitivity of IF-M1 activity to N-ethylmaleimide and heat (45 degrees) inactivation was tested in two model reactions requiring minimal complementary factors: (a) AUG-directed fMet-tRNAf binding to ribosomes; and (b) poly(U)-directed polyphenylalanine synthesis at 4 mM Mg2+ (IF-M2A, IF-M2B, EF-1, and EF-2 also required). IF-M1 activity proved to be sensitive to both N-ethylmaleimide and temperature (45 degrees). In addition, a contaminant of partially purified IF-M1 preparations has been found which is capable of fMet-tRNAf binding but is inactive in poly(U)-directed polyphenylalanine synthesis at low Mg2+ concentration.


Complex formation between mycobacterial polysaccharides or cyclodextrins and palmitoyl coenzyme A. The mycobacterial polysaccharides MMP (3-O-methyl-mannose-containing polysaccharide), MGLP (lipolysaccharide containing 6-O-methylglucose and glucose), and the cyclodextrins (cyclohexaamylose and cycloheptaamylose) form stoichiometric complexes with palmitoyl-CoA (Machida, Y., Bergeron, R., Flick, P., and Bloch, K. (1973) J. Biol. Chem. 248, 6246-6247). Complex formation is presumed to result from hydrophobic interactions. In order to enhance the hydrophobic character of the cyclodextrins the following derivatives have been synthesized: heptakis (2,di-O-propyl)-, heptakis (2,6-di-O-methyl)-, pentakis (6-O-methyl)-, heptakis (3-O-methyl)-, and permethylated beta-cyclo-dextrin. These compounds stimulate fatty acid synthesis catalyzed by the Mycobacterium smegmatis fatty acid synthetase, the magnitude of the effect decreasing in the order in which the alkylated cyclodextrins are listed above. MMP or MGLP are qualitatively indistinguishable from alkylated cyclodextrins both with respect to palmitoyl-CoA binding and with respect to effects on enzyme systems, suggesting that they form inclusion complexes of the same type. On the basis of model building it is postulated that MMP in solution assumes a helical conformation with a hydrophobic channel about 6 A in diameter and approximately 29 A long, dimensions appropriate for accommodating the paraffinic chain of palmitoyl-CoA in the form of an inclusion complex. Since palmitoyl-CoA binds to polysaccharide much more tightly than free palmitate it is further postulated that ionized groups of the CoA moiety of acyl CoA participate in the binding and do so by hydrogen bonding to the hydrophilic exterior of helical MMP. Palmitoyl-CoA, and to a lesser extent palmitate, affect the optical rotation of MMP and also of the alkylated cyclodextrins indicating that complex formation induces conformational changes in the polysaccharides.


The role of serine-123 in the activity and specificity of ribonuclease. Reactivation of ribonuclease 1-118 by the synthetic COOH-terminal tetradecapeptide, ribonuclease 111-124, and its O-methylserine and alanine analogs. The COOH-terminal tetradecapeptide of ribonuclease A, Glu-Gly-Asn-Pro-Tyr-Val-Pro-Val-His-Phe-Asp-Ala-Ser-Val, and two analogs, [Ser(Me)-123]-RNase 111-124 and [Ala-123]-RNase 111-124, were synthesized by the solid phase method and were purified to chromatographic and electrophoretic homogeneity. Methods are described for the hydrolysis and quantitative amino acid analysis of peptides containing O-methylserine. The peptides were combined noncovalently with RNase 1-118 and examined for ability to regenerate enzymatic activity in the presence of the substrates C greater than p, U greater than p, poly(C) poly(U), and poly(AF). The dissociation constants of the peptide-protein complexes, and the Michaelis constants for C greater than p and U greater than p with the reconstituted enzymes were determined. The data were used to test hypotheses, drawn from x-ray crystallographic and other studies, for the role of serine-123 in the binding of substrates by ribonuclease. It was found that Ser-123- and Ala-123-containing peptides were equally active for the hydrolysis step when measured with C greater than p as substrate and for the transphosphorylation step as measured in the assays with poly(C). The serine and alanine analogs were also equally active for the transphosphorylation step when poly AF was the substrate. With U greater than p as substrate the alanine analog was 4 times less active than the serine derivative and with poly U it was 2 times less active. The semisynthetic enzyme composed of RNase 1-118 and [Ala-123]-RNase 111-124, therefore, shows appreciable selectivity for substrates containing cytosine. It was concluded that a hydrogen bond between the hydroxyl of serine-123 and the C4 amino group of cytidine or the C-7 amino group of formycin is not important for substrate binding and catalytic activity. In contrast, the hydrogen bond between the hydroxyl of serine 123 and the C-4 carbonyl oxygen of uridine contributes significantly to substrate binding and catalytic activity. The data with serine-O-methyl ether at position 123 in the tetradecapeptide were less clear because it was difficult to separate steric effects from the contributions of hydrogen bonding. Substrate binding to ribonuclease was rationalized in terms of a binding energy equivalent to a total of two hydrogen bonds per pyrimidine.


Differences in spectra of alpha and beta chains of hemoglobin between isolated state and in tetramer. The absorption spectrum of deoxygenated human hemoglobin differs from that of the isolated alpha and beta chains, and the difference has been ascribed to the conformational changes on which depends the cooperative ligand binding. Studies on hybrid-heme hemoglobins, in which one chain contains protoheme and the other chain mesoheme, showed that the prominent peak in the Soret region of the difference spectrum for deoxyhemoglobin is due to the spectral change in alpha chains and that beta chains show a small trough in the Soret region at the wavelength 10 nm longer than that for the absorption peak. Difference spectra of oxygenated hybrid-heme hemoglobin showed only small peaks which seem to be due to the spectral changes in beta chains. Difference spectra of alpha-NO-beta-unliganded hemoglobin showed a large negative band in the Soret region at the wavelength of the absorption peak for alpha-NO chains, and beta-unliganded chains in this half-liganded hemoglobin showed a small trough corresponding to that of beta chains in fully unliganded hemoglobin. Alpha-Unliganded-beta-NO chains, however, showed only small peaks in the difference spectra. The peaks in difference spectra ascribed to each chain did not completely correlate with the kinetic results reported for hybrid-heme hemoglobin (Nakamura, T., Sugita, Y., Bannai, S. (1973) J. Biol. Chem. 248, 4119-4122) and for half-liganded hemoglobin (Antonini, E., Brunori, H., Wyman, J., and Noble, R.W. (1966) J. Biol. Chem. 241, 3236-3238). Spectral change when alpha and beta chains were mixed was a second order reaction for deoxygenated hemoglobin and was a first order reaction for oxygenated and alpha-NO-beta-unliganded hemoglobin, suggesting the different rate-limiting steps.