Incidence of ischaemic heart disease and prognostic importance of risk factors in agricultural and industrial populations. A longitudinal epidemiological study of ischaemic heart disease (IHD) in men aged 40-59 years showed that the five-year incidence of the manifest form was 7.8% in agricultural workers and 9.7% in industrial employees. The corresponding incidence of the latent form was 9.0% and 12.2%, respectively. The differences in the incidence of both forms between the population samples studied was at the border of statistical significance. The importance of risk factors was evaluated on the basis of the score represented by the difference between the respective five-year incidence of IHD in subjects with and without the risk factors concerned. The highest relative scores were found with systolic and diastolic hypertension, positive family history and obesity. For prognostic purposes, the total relative risk, represented by the sum of the scores of the risk factors proved to be useful. Autopsies confirmed that a total relative risk exceeding 40% was a sensitive indicator of morphological substrate of coronary heart disease.


The problem of unstable angina pectoris: myocardial infarction register data from Kaunas, Lithuanian SSR. Prodromal symptoms and signs of acute myocardial infarction (AMI) and clinical course of unstable angina pectoris were studied in 405 patients with AMI and 165 patients with unstable angina, respectively. The latter group consisted of patients admitted to a coronary care department. The study showed that 81.2% of patients with AMI had had a number of symptoms during the 28-day period preceding the onset of AMI. Neither the patients nor the physicians had given proper and timely attention to them. The hospitalization of patients with unstable angina was, in some cases, effective in preventing development of AMI or in eliminating life-threatening complications of AMI. This resulted in a decline of the AMI mortality.


Population study of the early phase of acute myocardial infarction and sudden coronary death. A two-year population study in the district of Prague 4 (population 190 000) showed that patients with acute myocardial infarction [AMI] came to the hospital with a considerable delay (median 10.25 hours), whose substantial part was caused by the patient's lingering in calling for medical aid (a half of them called as late as within 3 hours since the appearance of symptoms). On the other hand, out of 226 cases of sudden death caused by ischaemic heart disease (IHD), 92% of the victims died as early as within the first hour. Within the first hour, 27% of all patients with coronary attacks in the community died, and as a few as 2% only were hospitalized within this period. No prevention of sudden coronary death is possible for the time being. Nevertheless, an attempt can be made at least to reduce the risk of early death in the following two ways. The public shoudl be avised of the gravity of characteristic symptoms of AMI, danger in the case of delay, and necessity of prompt medical aid. Because a majority of victims of sudden death have positive histories of IHD, and most deaths occur in the patients' homes in the presence of their family members, these next relatives of patients with manifest IHD should be trained in the performance of cardiopulmonary resuscitation.


Present state of cardiovascular community control programme in the Czech Socialist Republic. The high incidence, great import, and long duration of cardiovascular diseases are reflected in high demands placed on the health services. Experience shows that utilization of the results of research in general practice is lagging behind. The application of any improvement in the diagnosis, therapy, and prevention in health care waits several years for its accomplishment. In order to improve this situation, the Ministry of Health of the CSR constituted, in line with WHO recommendations, a Department for Cardiovascular Diseases Control. The Department has worked out a programme of prevention and control of the major cardiovascular diseases, in particular, ischaemic heart disease, systemic hypertension and its complications, rheumatic heart disease, congenital cardiac and vascular defects, and cor pulmonale. New diagnostic, therapeutic, and preventive procedures are first tried out in so-called model areas and are only after this introduced into the national health care of people suffering from or endangered by cardiovascular diseases. In parallel, organizational measures necessary for comprehensive care are implemented. The authors report on the experience gained so far with the realization of the programme of care of people suffering  from IHD and acute myocardial infarction. They emphasize the importance of continual schooling of medical personnel and of health education of the entire population. They describe the implementation on a national scale of postgraduate cardiological courses intended especially for first-line doctors.


Foot blood-flow at isometric handgrip contraction in patients with an ischaemic disease of the lower extremities and in control subjects. At isometric contraction of forearm muscles, the mean systemic pressure and the foot blood flow are statistically significantly increased. These changes are encountered both in controls and in patients with an ischaemic disease of the lower extremities.


Community cardiovascular disease control programmes. A comprehensive approach to the control of cardiovascular diseases on community level is a new venture. There is not yet enough experience in any country with this approach. However, the attempts to develop new approaches are fully justified, since the present stress on hospital care and treatment of the patients has only little changed the incidence of cardiovascular diseases among our populations.


Community control of cardiovascular diseases. The pro and cons. The need for an effective control of cardiovascular diseases is evident. Possibilities to achieve this both in the curative and the preventive field are restricted mainly due to the insufficient knowledge of their etiopathogenesis. Although the cause is not known, the discovery to risk factors provides a sound evidence for prevention. Attempts to reverse elevated risk factor levels are all directed towards a more healthy way of life. Group oriented prevention is one of the most frequently applied approaches to test the hypothesis of preventability. While it offers many advantages, the delayed timing of the intervention and the restricted coverage limits the possibility of inference from its results. The community approach implies acting on the whole population by changing their behaviour towards a more healthy way of life. Apart from the beneficial "side effects" on other chronic diseases, their outcome in the pilot areas may be inferred to the total population. The final proof of any scientific evidence from the public health point of view is, after all, its applicability and effectiveness in real life situation.


Dose-related dilatation of airways after inhalation of metaproterenol sulfate. Studies concerning the ability of an inhaled beta2-agonist to produce dose-related bronchodilatation are conflicting. In five asthmatic, five bronchitic, and five normal subjects, specific airway conductance (Gaw/VL), flow-volume curves, and single-breath nitrogen washout were recorded after noncummulative inhalation of 0.65 mg, 1.30 mg, 1.95 mg, and, in some subjects, 2.60 mg of metaproterenol sulfate. Bronchodilatation appeared to be dose-related and was best assessed by using Gaw/VL; in all but one subject with chronic bronchitis, there was a significant linear relationship between log dose and precent change in Gaw/VL. Measurements of flow rate could demonstrate significant log dose-responses in eight subjects, maximum midexpiratory flow being the most sensitive measurement of flow rate, followed in order by forced expiratory volume in one second, flow at 50 percent of forced vital capacity, peak expiratory flow rate, and flow at 75 percent of forced vital capacity. No log dose-respone curve could be observed by using the single-breath nitrogen-washout technique. This demonstration of significant log dose-responses to inhaled metaproterenol is consistent with the response to drugs acting upon receptors and suggests that patients may benefit from increasing doses of bronchodilators.


Evaluation of the sensitivity of commercially available digoxin radioimmunoassay kits. We have evaluated the technical minimum detection limits of 12 different commercially available kits and the practical minimum detection limits of 11 kits used for the determination of the serum digoxin level by radioimmunoassay. The poor technical ability of many of the kits to distinguish between digoxin levels of 0.1 ng/ml intervals between 0 and 0.5 ng/ml and, additionally, the detection of a digoxin level up to 0.3 ng/ml with some of the kits in glycoside-free sera impose a practical working minimum detection limit of 0.5 ng/ml for the majority of these kits. We, therefore, recommend caution with interpreting or evaluating serum digoxin levels below 0.5 ng/ml.


Echocardiographic and phonocardiographic confirmation of suspected caged mitral valve malfunction. Seven patients studied by echocardiography with and without simultaneous phonocardiography for suspected malfunction of a caged mitral valve  prosthesis are presented. In case 1, with inaudible prosthetic clicks, thrombosis of the cage and immobility of the ball were suggested by echocardiographic studies and confirmed at surgery. In case 2, simultaneous echocardiographic and phonocardiographic studies demonstrated wide and variable intervals between the aortic second sound the the opening click and also "sticking" of the ball. In case 3 a thrombus prevented full motion of the ball to the apex of the cage, which was seen on the echocardiogram, while in case 4, with a thrombus within the ventricle and prosthesis, the prosthetic opening click was present intermittently and was associated with only subtle echocardiographic changes. In case 5, echocardiographic studies demonstrated abnormal rocking of the cage secondary to severe prosthetic dehiscence. In case 6, dul prosthetic clicks were to be secondary to a low cardiac-output state. In case 7, with multiple valve prostheses, simultaneous echocardiographic and phonocardiographic studies allowed identification of individual valve sounds and abnormal timing of valve opening. Based on these studies, we believe that echocardiography and simultaneous phonocardiography can yield very useful information in the evaluation of patients with suspected malfunction of a caged mitral valve prosthesis.


A consideration of the differences between a Janeway's lesion and an Osler's node in infectious endocarditis. Janeway's lesions and Osler's nodes are regarded as excellent clues to the diagnosis of infectious endocarditis; however, very few physicians have actually witnessed these findings, and there is some confusion in distingushing between the two. This article concerns a patient with infectious endocarditis due to Diplococcus pneumoniae, who had tender vesicular lesions thought to be Osler's nodes and a nontender erythematous nodule on the foot compatible with a Janeway's lesion. The original comments by William Osler and Edward Janeway are presented, and the literature following their descriptions is reviewed. It is concluded that the only essential diagnostic difference between the two is the tenderness that is associated with an Osler's node but not with a Janeway's lesion.


Evaluation of two spirometers: a comparative study of the Stead-Wells and the vitalograph spirometers. The characterisitcs and performance of the Vitalograph dry spirometer were studied, and a direct comparison was made with the Stead-Wells spirometer, as recommended by the Committees on Environmental Health and Respiratory Physiology of the American College of Chest Physicians. The Vitalograph calibrates well against the Stead-Wells spirometer for measurements of vital capacity and forced expiratory volume in one second in normal subjects and in patients with various pulmonary conditions. Both spirometers failed to satisfy the committees' recommendations for resistance and inertia, the Stead-Wells showing the better values. Nevertheless, it is suggested that the Vitalograph is very suitable for the routine assessment of ventilatory function in the laboratory or in population surveys. Its light weight, portability, and robustness give it an added advantage.


Effects of ouabain on early ventricular relaxation in patients with acute myocardial infarction. The effects of the administration of ouabain on the peak negative left ventriculat (LV) dP/dt were studied in 14 patients with acute myocardial infarction. Prior to administration of ouabain, negative LV dP/at was lower than previously reported for patients without coronary arterial disease. One hour after administration of ouabain, significant (P less than 0.05) increases in peak positive LV dP/dt and maximal velocity of contractile-element shortening and decreases in LV diastolic pressure were present. These changes were associated with small and insignificant increases in negative LV dP/dt. Early LV relaxation rate is impaired in patients with acute myocardial infarction; however, the reduction in LV diastolic pressures which occurs after administration of ouabain does not appear to be due to an increase in the rate of early LV relaxation but rather is probably related to improved contractility.


The heritable syndrome of prolonged Q-T interval, syncope, and sudden death. Electron microscopic observation. A family with the heritable syndrome of prolonged Q-T interval is reported. Three members died suddenly. Six had a prolonged Q-T interval. One had syncopal episodes for four years but has had no syncope since therapy with propranolol was initiated two years ago. The inheritance appears to be autosomal dominant. There was no evidence of hearing defects. Histologic and electron microscopic studies were performed on cardiac tissue from one patient. This is the first report of electron microscopic studies, and the findings suggest a possible defect of calcium metabolism in the myofiber.


Bacterial endocarditis after cardiac catheterization. Bacterial endocarditis is a rare complication of cardiac catheterization. We present the history of a patient who developed fatal, acute bacterial endocarditis following diagnostic cardiac catheterization. The implications regarding antibiotic prophylaxis and catheterization technique are discussed.


Mediastinal thymic cyst after open heart surgery. A benign thymic cyst seen six years after valvular heart surgery is described, and the question of the possible role of previous surgical trauma in the development of the cyst is raised.


The efficacy of trapezoidal wave forms for ventricular defibrillation. The purpose of this study was to evaluate the efficacy of a trapezoidal wave form for ventricular difibrillation. Overall efficacy showed the trapezoidal waveform to be effective for defibrillation, including patients weighing over 100 kg (220 lb). We concluded that (1) the trapezoidal waveform is an effective defibrillatory pulse and (2) the trapezoidal waveform offers pulse characteristics less deleterious than other established waveforms.


Echocardiographic findings in a patient with Candida endocarditis of the aortic valve. The echocardiographic appearance of fungal endocarditis of the aortic valve is described in a patient who subsequently died from this disease. In addition, the progressive growth of the vegetation on serial echocardiograms was recorded, and premature closure of the mitral valve was absent, notwithstanding perforation of two aortic cusps.


Echocardiographic oscillating flap in aortic root dissecting aneurysm. A patient had a history, physical findings, and chest x-ray film suggesting type I aortic dissection. This diagnosis could not be confirmed angiographically. Echocardiographic studies predicted both the presence of dissection and the anatomic findings at surgery. A regularly oscillating echo corresponding to the intimal flap was found in the false lumen. This is suggested as a new echocardiographic finding in dissecting aneurysm.


Mediastinal lipomatosis in simple obesity. A case of superior mediastinal widening due to unusual amounts of fat accumulations is reported in a patient with simple obesity, not associated with iatrogenic or primary Cushing's syndrome. The radiographic features included a smooth bilateral widening of the superior mediastinum, relative lucency, no definable mass in the lateral view, and no pressure effects on the trachea. Such mediastinal lipomatosis is a benign condition and may be related to general obesity. although rare, it should be considered in the differential diagnosis of mediastinal masses, particularly in obese individuals.


Ruptured aneurysm of aortic sinus of Valsalva into right atrium. Associated atrioventricular block presumable caused by aneurysmal compression of His bundle. We have seen a case of rupture of an aneurysm of the noncoronary sinus into the right atrium. Surgery revealed an aneurysmal mass the size of the tip of an index finger extending through the inter-atrial septum down to just above the tricuspid valvular ostium. An electrocardiogram showed first-degree atrioventricular block, while the His bundle electrogram demonstrated the presence of disturbances in the intra-atrial as well as His bundle conduction. The disturbance in His bundle conduction was interpreted as being due to compression of the His bundle by the aneurysm.


Correlation of in vitro resistance of Staphylococcus aureus to tetracycline, doxycycline, and minocycline with in vivo use. Susceptibility of 983 isolates of Staphyloccus aureus to tetracycline, doxycycline and minocycline was determined in vitro. Minocycline was shown to be more active than doxycycline, which in turn was shown to be slightly more active than tetracycline. 77% of the isolates which were resistant to tetracycline were also resistant to doxycycline, whereas only 4% of the tetracycline-resistant isolates were resistant to minocycline. The in vivo use of tetracycline correlated with increased in vitro resistance of S. aureus to tetracycline and doxycycline. A correlation between use of tetracycline and in vitro resistance to minocycline was not demonstrated.


In vitro and in vivo activity of 3-(1-methyl 1-5-nitro-2-imidazolylmethylideneamino)-2-oxazolidinone against Pasteurella. Furazolidone was more active than 3-(1-methyl)-5-nitro-2-imidazolyl-methylideneamino)-2-oxazolidinone (MABN) against a series of 34 isolates of Pasteurella and 11 of Yersinia (formerly designated Pasteurella). However, the nitroimidazole was superior to furazolidone by both subcutaneous and oral routes against a series of mouse infections incited by strains of Pasteurella. It also was superior to furazolidone and sulfaquinoxaline when administered in the diet against two Pasteurella strains in a fowl cholera model infection in chickens. The good in vitro activity of MABN plus its low toxicity suggest its further study as an agent for fowl cholera and the shipping fever complex of cattle.


Treatment of chronic urinary tract infections with cephalexin (Ceporexine) orally administered at 12-hourly intervals. 19 patients with chronic urinary tract infections were treated, after culture and sensitivity testing, with cephalexin (Ceporexine) 1 g twice daily for 10 days. All patients were free from bacteria after 2 days treatment and 18 after the treatment was finished. After 30 days, 7 had a recurrence of the urinary tract infection. The result shows that cephalexin given twice a day in the dosage of 1 g has as good an effect in the treatment of urinary tract infections as when the dosage is given 4 times a day.


[Myocardosis - myocardia - cardiomyopathy (author's transl)]. "Cardiomyopathy", a term formerly used for heart muscle disease of unknown aetiology, is at present applied to all myocardial diseases other than coronary-heart disease. This definition includes many different forms of metabolic disorders of heart muscle. Four manifestations of metabolic myocardial disease, differing in their aetiology and pathogenesis are distinguished: (1) "transport" myocardosis, (2) "arthrocytotic" myocardosis, (3) myocardia, and (4) idiopathic cardiomyopathy. They are differentiated by combined morphological and quantitative mineral examination. "Transport" myocardosis is the most common form and its particular aetiology can be further specified. Cause of death in patients with metabolic disorders often remains undiscovered even at autopsy. Combination of histological and quantitative mineral analysis of myocardium can further define the particular form.


[Plasminogen estimation as a routine test (author's transl)]. A simple method for the routine determination of plasminogen using the coagulation time is described. 0.2 ml of plasminogen reagent and 0.1 ml of sample plasma, prediluted 1:200, are incubated for 3 minutes at 37 degrees C. 0.05 ml of diluted test thrombin are then added and the coagulation time is measured. It is converted into percent of normal using a standard curve. The test correlates with an immunological method in healthy probands (r = 0.96). Initial measurements in 160 patients show that knowledge of the plasminogen titre may also be of interest outside the problems of haemostasis.


[Cholestatic jaundice after azathioprine treatment (author's transl)]. Severe cholestatic jaundice occurred in two patients receiving prednisolone and azathioprine, one for chronic progressive hepatitis, the other for Wegener's granulomatosis. Reversible abnormal liver function, involving both elimination and synthesis, was in the foreground clinically. Signs of parenchymal-cell damage were only moderately severe. When azathioprine had been discontinued all previously abnormal values returned to normal within six to eight weeks. Azathioprine is an indirect optional hepatotoxin. If given over long periods there should be regular controls of blood count and liver functions.


[Oral contraceptives, hypertension and nephrosclerosis (author's transl)]. Five personal cases and many reports in the literature demonstrate the not rare development of hypertension after oral contraceptives or an increase of an existing hypertension. In addition, the drug may also cause vascular changes in the kidneys with development of benign or malignant nephrosclerosis, similar to the increased risk of thrombo-embolic complications of the venous-arterial system. It is, therefore, necessary to check the blood pressure of women on oral contraceptive and, if hypertension exists or develops, discontinue these drugs.


[Treatment of haemophilia with factor VIII inhibitor by giving isoagglutinin-free factor VIII concentrate (author's transl)]. Two nine-year-old boys with haemophilia A and circulating inhibitor against factor VIII were given large doses of commercial AHG concentrates to control bleeding. But the isoagglutinins in the AHG concentrates caused haemolysis in both patients. When AHF (Immuno) from AB donors - a cryoprecipate without isoagglutinins - was used no further inhibitor activity was detectable, previously observed anaphylactoid reactions no longer occurred and usual doses of AHG were now sufficient to control bleeding.


[Serial biopsies in healthy carriers of hepatitis Bs antigen (author's transl)]. Serial biopsies were taken over four to five years from 15 healthy hepatitis-Bs antigen-positive persons with normal biochemical findings ("healthy carriers"). The antigen persisted throughout, but the histological picture did not worsen in any way: there was certainly no sign of chronic progressive liver disease.


Carbenoxolone sodium: its role in ulcer healing. In summary, carbenoxolone is a useful drug in accelerating short-term ulcer healing during ambulatory treatments. Provided treatment is responsibly controlled the adverse effects should not present frequent or severe problems; however, there is a clear need for an analogue which is substantially side-effect free.


A comparison of EEG seizure patterns recorded with surface and depth electrodes in patients with temporal lobe epilepsy. Surface and depth EEG seizure patterns were compared in 34 patients with intractable temporal lobe epilepsy in whom depth EEG electrodes had been chronically implanted in order to localize epileptogenic sites with a view to surgery. EEG records accompanied by clinical seizures, auras, no behavioral changes, as well as records for which no behavioral observations had been made, were judged with respect to the manner in which seizure activity originating unilaterally in the depth of one of the temporal lobes spread to the surface. For each EEG record, the onset of seizure activity in depth was classified as being focal or regional in form, and seizure activity was judged as: (1) not spreading to the surface, (2) spreading bilaterally and synchronously to the surface, (3) spreading initially to the surface ipsilateral to the depth site(s) in which the electrographic seizure first appeared, or (4) spreading initially to the surface contralateral to the depth site(s) in which the seizure activity initially occurred. EEG seizure activity was found to be less likely to propagate to the surface for those records that were either unaccompanied by behavior changes or accompanied only by auras than for those records accompanied by clinical seizures. In records accompanied by clinical seizures, seizure activity commonly propagated to the surface in a bilateral and synchronous fashion and was also found to spread initially to the ipsilateral but not to the contralateral surface. Anatomical and electrophysiological data accounting for the occurrence of ipsilateral spread were discussed. Diagnostic usefulness of surface recordings during clinical seizures in temporal lobe epilepsy was discussed.


Rolandic spikes in children with and without epilepsy. (20 subjects polygraphically studied during sleep). The authors polygraphically studied the nocturnal sleep of 20 neurologically normal children with typical centrotemporal spikes. The children were divided into two groups: (A) 10 children with centrotemporal spikes and benign epilepsy; and (B) 10 children with centrotemporal spikes without epilepsy. The mean age when the average period of sleep record was performed was the same for the two groups, 8.5 years. The cyclic organization of sleep and the percentages of the different stages were normal in all 20 subjects. The number of spikes was counted in all the subjects during wakefulness and during different stages of sleep. All subjects of both groups showed an important increase in the frequency of spikes going from drowsiness until slow sleep; on the other hand, the activity remained high during REM sleep only for subjects of Group A. Other differences between the two groups concerned the appearnce during sleep of generalized spike waves and independent Rolandic spikes. The authors conclude with the affirmation that the polygraphic study of nocturnal sleep in children with Rolandic spikes can be an additional parameter for differential diagnosis between children with and those without epilepsy.


Effects of acute cerebellectomy on maximal electroshock seizures and anticonvulsant efficacy of diazepam in the rat. Rats were cerebellectomized 72-96 hr prior to evaluation (1) during maximum electroshock seizures and (2) for their capacity to respond to pentylenetetrazol-induced clonic seizures. Cerebellectomized rats failed to exhibit tonic hindlimb extension, an endpoint characteristic of maximal electroshock seizures. The dose of pentylenetetrazol required to produce clonic seizures or death was not different in cerebellectomized and sham-operated controls. The anticonvulsant efficacy of diazepam, when assessed as a pentylenetetrazol antagonist, was not influenced by removal of the cerebellum. These data indicate that whereas cerebellar influences may suppress seizure activity which is largely focal, seizures of more diffuse origin are not markedly influenced by cerebellar activity. It is, therefore, essential that the role of the cerebellum in suppressing seizures be characterized for each kind of experimentally induced seizure process.


Kindling-related changes in afterdischarge "thresholds". Amygdaloid stimulations were applied to rats at gradually increasing current intensities once every 60 sec until and afterdischarge (AD) was recorded through the stimulation electrode. These initial AD "thresholds" (x = 75 muA) were reduced over a 4-day period by a series of 12 subthreshold stimulations. In contrast, stimulations administered on the same regimen but at a higher intensity (400 muA) had raised the AD threshold. The decreases in the AD threshold were relatively permanent, whereas the elevated thresholds were subsequently reduced to control levels by a series of subthreshold stimulations or by a 7-day stimulation-free period. Two days following the completion of Experiment 1, all of the rats were injected with a low dose of pentylenetetrazol. The incidence of pentylenetetrazol-induced epileptic symptoms was much higher in the experimental animals than in the control subjects previously subjected only to the threshold estimation procedure.


Studies on chromatin. Free DNA in sheared chromatin. Chromatin which has been hydrodynamically sheared in a low-ionic-strength buffer lacking divalent cations (I = 0.0005 M) contains a heterogeneous set of deoxyribonucleoprotein particles but no molecules of free DNA. The main finding is that a transference of sheared chromatin to 1--2 mM MgCl2 or to 0.1--0.2 M NaCl results in appearance of completely free DNA molecules. A salt-induced rearrangement of DNA-bound histones but not a partial loss of them is responsible for the observed phenomenon. Formation of free DNA molecules is accompanied by aggregation of the majority of remaining deoxyribonucleoprotein particles. The percentage of free DNA molecules in the chromatin which was sheared to an average DNA length of about 400 base pairs is increased from zero in the initial sample to 8-9% in 1 mM MgCl2 and further to approximately 25% of the total DNA in 0.15 M NaCl, 2 mM MgCl2. Free DNA molecules in the sheared chromatin are observed not only upon isopycnic banding of formaldehyde-fixed deoxyribonucleoproteins in CsCl gradients but also in non-ionic metrizamide gradients with either fixed or unfixed deoxyribonucleoprotein samples. The process of free DNA formation is a reversible one; its direction and the equilibrium state depend in particular on the ionic conditions of the medium.


DNA-dependent RNA polymerase C from Xenopus laevis ovaries. Ability to transcribe intact double-stranded DNA. DNA-dependent RNA polymerase C, partially purified from Xenopus laevis ovaries, has been resolved by DEAE-Sephadex chromatography in two forms, eluting at 0.2 M and 0.3 M ammonium sulfate, respectively. Both are sensitive to high concentrations of alpha-amanitin (200 mug/ml). Their ionic strength dependence and divalent cation requirements are indistinguishable. Quantitatively, RNA polymerase C represents the major form of RNA polymerase activity solubilized from the ovaries. Both RNA polymerases C are able to transcribe efficiently either high-molecular-weight Xenopus DNA or intact adenovirus DNA, as compared to nicked DNA. In contrast, RNA polymerase A has little activity on an intact DNA template. The salt dependence of the RNA polymerases C activity is different on the two kinds of template. Nicked DNA is efficiently transcribed up to a salt concentration of 100 mM ammonium sulfate. On intact DNA, optimal transcription is obtained at 40 mM ammonium sulfate and is inhibited by higher salt concentrations.


The purification and characterisation of the human-serum binding protein for the 25-hydroxycholecalciferol (transcalciferin). Identity with group-specific component. The binding protein for 25-hydroxyvitamin D3 has been isolated from human serum by monitoring the recovery of 3H-labeled 25-hydroxyvitamin D3. After a 500-fold purification a pure protein was obtained as judged from the constant specific activity (ratio of absorbance versus radioactivity) on agarose and DEAE-Sephadex chromatography and on the presence of a single band on both cellulose acetate and polyacrylamide gel electrophoresis. The molecular weight of the purified protein was measured by gel filtration on agarose (56000), Sephadex G-75 (58000) and dodecylsulfate-polyacrylamide gel electrophoresis (56000). On sucrose gradient ultracentrifugation a sedimentation coefficient of 4.1 S was found. The isoelectric point was 4.89 S on isoelectric focusing. The stability of the protein at 60 degrees C was enhanced by the presence of excess 25-hydroxyvitamin D3. On tandem crossed immunoelectrophoresis the purified binding protein was found to be identical to the one present in whole serum. The activity of the isolated protein was demonstrated by a Ka at 4 degrees C of 1.2 X 10(10) l-mol-1. A binding capacity of 0.8 binding site/molecule was measured on a Sephadex G-25 column. During immunological studies with this protein it became evident that the binding protein is identical with another serum protein known as group-specific component (Gc). In analogy to other serum binding proteins we propose to call this group-specific component/25-hydroxyvitamin-D3-binding protein transcalciferin.


The specificity requirements of bacteriophage T4 lysozyme. A series of bacterial cell wall glycopeptides of low  molecular weight and cell wall nucleotide precursors have been tested for their inhibitory action on the digestion by T4 lysozyme of a radioactively labeled linear uncrosslinked peptidoglycan. The disaccharide-peptides GlcNAc-MurNAc-l-Ala-D-Glu(A2pm) (C5) and GlcNAc-MurNAc-L-Ala-D-Glu(A2pm-D-Ala) (C6) as well as the monosaccharide-peptide MurNAc-L-Ala-D-Glu(A2pm) were found to be good competitive inhibitors (with similar Ki values) whereas the disaccharide-pentapeptide GlcNAcMurNAc-L-Ala-DGlu-Gly-L-Lys-D-Ala was a poor inhibitor. T4 lysozyme did not catalyse transglycosylation reactions from Escherichia coli B peptidoglycan to the disaccharide-peptide C6. No changes were seen in the circular dichroism spectra (200-250 nm) or fluorescence emmission spectra upon binding of the good inhibitors. The results obtained indicate that T4 lysozyme has a small active site capable of recognizing a unit consisting of MurNAc-L-Ala-D-Glu(A2pm).


Immunochemical isolation and characterization of vitellogenin mRNA from liver of estradiol-treated chicks. Vitellogenin mRNA was purified through three steps. A heavy polysome fraction was obtained by discontinuous sucrose density gradient centrifugation, vitellogenin polysomes were immunoprecipitated with affinity-chromatography-purified anti-lipovitellin IgG and goat anti-rabbit IgG, the enriched mRNA was isolated on a poly(U)-Sepharose column. As judged by its specific activity in a reticulocyte lysate system, vitellogenin mRNA has been enriched a 1000-fold with a recovery of 30%. On 99% formamide 3.4% polyacrylamide gels vitellogenin mRNA has an Mr of 2.4-2.5 X 10(6) and codes for a peptide of Mr 240000, which under our incubation conditions is partially degraded to smaller peptides.


Preferential stimulation of the plant mRNA synthesis by gibberellic acid. 1. Treatment of the etiolated maize seedlings with the plant hormone, gibberellic acid results in a significant enhancement of heavy polyribosome formation. 2. This is accompanied by highly increased incorporation of the labelled RNA precursors into RNA engaged in the polyribosomal complex, as well as by an increased rate of protein synthesis in vivo. 3. Determination of the specific radioactivity of particular RNA classes isolated from polyribosomes reveals that gibberellic acid stimulates mostly the synthesis of the rapidly labelled, non-ribosomal RNA fraction. 4. A considerable amount of this rapidly labelled RNA fraction, whose synthesis is preferentially stimulated by exogenous gibberellic acid contains poly(A) sequences, as shown by affinity chromatography on oligo (dT)-cellulose indicating that phytohormone causes an increased transcription of mRNA in etiolated maize seedlings. 5. When [3H]adenosine served as the RNA precursor it was found that the ratio between the heteropolymeric and polyadenylic parts of the poly(A)-RNA chain markedly changed under gibberellin treatment, suggesting that, in addition to an increased rate of mRNA synthesis, the plant hormone also affects the process of post-transcriptional polyadenylation of the newly made mRNA precursors. Possible extension of the polyadenylate segment in the presence of gibberellin may account for a longer functional half-life of the mRNA synthesized in plants treated with the phytohormone, and may explain significantly enhanced heavy polyribosome formation, as well as a higher efficiency of protein synthesis in plants treated with gibberellic acid.


Functional relationships between a reticulocyte polypeptide-chain-initiation factor (IF-MP) and the translational inhibitor involved in regulation of protein synthesis by haemin. The rate of initiation of protein synthesis in rabbit reticulocyte lysates is regulated by a translational inhibitor protein which is activated in the absence of added haemin. The effects of this inhibitor on amino acid incorporation are overcome by the protein synthesis initiation factor IF-MP which binds Met-tRNAf in a ternary complex with GTP and which can transfer this complex to small ribosomal subunits. Addition of this factor to haemin-deficient lysates prevents loss of polysomes and regenerates polysomes from 80-S single ribosomes, thus confirming an effect at the level of polypeptide initiation. The ability of the initiation factor to overcome the effects of various concentrations of the translational inhibitor suggests that the inhibitor inactivates the factor catalytically rather than stoichiometrically. In a system in vitro consisting of salt-washed 40-S ribosomal subunits, initiator Met-tRNAf and GTP, the initiation factor IF-MP transfers Met-tRNAf to the subunits in the absence of any other factor or mRNA. Equilibrium buoyant density gradient analysis in CsCl shows that formaldehyde-fixed subunits carrying Met-tRNAf bound under these conditions have a buoyant density approximately 0.02 g/cm3 lower than the bulk of salt-washed subunits, suggesting that approximately 100000 daltons of additional protein are associated with these subunits. This is in marked contrast to the amounts of protein bound to subunits incubated with Met-tRNAf and GTP in the presence of a crude ribosomal salt-wash fraction. The translational inhibitor has no effect on formation of the ternary complex IF-MP-Met-tRNAf-GTP but does impair the factor-catalysed transfer of Met-tRNAf to washed subunits. The possible mechanisms of action of the inhibitor on polypeptide chain initiation are reviewed in the light of these results.


Critical review of the use of carotid bovine grafts for hemodialysis. Review of experience at the Mayo Clinic with carotid bovine grafts in patients who undergo hemodialysis with a follow-up of of as long as 28 months revealed that the graft provides vascular access for hemodialysis when other vascular access sources have been exhausted. The graft is particularly helpful in the diabetic patient whose peripheral arteries have inadequate flow. The location in the thigh seems to be preferable to the forearm location. Clean puncture technique is important because infections usually mean graft loss and possibly severe complications, for example, hemorrhage or septicemia.


Effects of stroma-free haemoglobin solution on the blood-clotting system in dogs. The effect of transfusions of 500 ml of 5-6% stroma-free haemoglobin solutions on the blood-clotting system of dogs was studied. Slight lowering of the fibrinogen level and of the activity of clotting factors V, VII, VIII, IX and X was observed, together with some prolongation of the thrombin time. In vitro investigations indicate that prolongation of the thrombin time may depend on disturbed polymerization of fibrin monomers. After transfusion of stroma-free haemoglobin preparation, no signs of haemorrhagic diathesis were observed.


Effects of tham, isoprenaline and propranolol on blood flow and vascular resistances of the liver after in- and outflow occlusion. Relation with the splanchnic shock. The responsibility of the portal and the hepatic artery circulations during shock states has been established by studying the effects of a 15-min occlusion of two of the following blood vessels on 23 dogs: inferior vena cava below the diaphragm, portal vein and hepatic artery. Intrahepatic vascular resistances were computed from blood pressure records in these vessels and transhepatic blood flow studies using the 133Xe clearance method. The animals were treated with THAM, plasmagel, isoprenaline, and propranolol. The tolerance of the occlusion is significantly improved when the animals are treated with the association of the four drugs. The portal and the systemic arterial blood pressures return to normal more promptly. Sinusoid and peribiliary resistances are remarkably stable if compared to the changes occurring in the control animals. The well-known benefit of THAM is improved by the apparently paradoxical association of isoprenaline and propranolol. In fact, at the doses which have been used, they counterbalance their mutual disadvantages. Finally, the analysis of the hepatic blood flow rates and vascular resistances suggests that the splanchnic shock has two components: hepatic and visceral.


Effect of fasting on mucosal blood flow in antrum and corpus of the stomach. Gastric mucosal blood flow in the rat was determined by the indicator fractionation technique 86RbCl. Antral and corpus flow rates were determined after increasing periods of fasting, and the effect of vagotomy was assessed. The physiological stimulus of food in the stomach markedly increased antral as well as corpus blood flow, and after vagotomy lower flow rates were recorded in all parts of the stomach. With progressive starvation, the antral:corpus flow ratio decreased. The findings suggest that the antrum plays an active role in response to a physiological stimulus and may, as has been suggested by others, contribute to the control of parietal cell blood flow and secretion.


Synthesis of 1-hydroxy and 1-acyloxypyrrolidin-2-ones. A series of 1-hydroxypyrrolidin-2-ones (v) has been prepared by reduction of gamma-nitrocarboxylic acid esters with Zn dust in the presence of ammonium chloride. Acylation of compounds (v) gave the corresponding 1-acyloxypyrrolidin-2-ones (VI). None of the synthesized compounds showed any significant antiflammatory activity.


[Research on substances with psychotropic activity. IV. Synthesis of 2,3-diphenyl-4-methyl-4H-1,4-benzothiazin-1,1-dioxide by thermal cyclization of N-(2-desylsolfonylphenyl) glycine]. Treatment of the ethyl ester of N-benzoyl-N-(2-benzylsolfonylphenyl)glycine with potassium in benzene gave, by rearrangement, N-(2-desylsolfonylphenyl)glycine, which via thermal intramolecular cyclization and simultaneous decarboxylation gave 2,3-diphenyl-4-methyl-4H-1,4-benzothiazin-1,1-dioxide. The structure of the latter compound was confirmed chemically by oxidising the known 2,3-diphenyl-4-methyl-4H-1,4-benzothiazine with hydrogen peroxide in formic acid and by I;R. and N.M,R; spectral data.


Circular dichroism studies of imide derivatives of amines. Circular dichroism curves have been measured for phthaloyl-, maleyl- and itaconyl-derivatives of a number of amines having a general formula R--CH(NH2)--R' (in which R and R' are alkyl-, aryl- or cyclohexyl-groups) and of 1,2,3,4-tetrahydro-1-naphthylamine and 1,2,3,4-tetrahydro-2-naphthylamine. We conclude that the determination of absolute configuration of amines examined can be made on the basis of C.D. curves of their phthaloyl-, maleyl- and itaconyl-derivatives, referring to that portion of the curve relating to the imide chromophore absorption.


[Heteropolycyclic systems. V. 4-alkyl-2,3,3a,4-tetrahydro-1H-pyrro1/2,1-c/ /1,2,4/benzothiadiazin-5,5-dioxide and their bz-substituted derivatives]. The preparation of some 4-alkyl derivatives of 2,3,3a,4--tetrahydro-1H-pyrrolo(2,1-c)(1,2,4)benzothiadiazin-5,5-dioxode and bz-substituted derivatives by the action of alkyl iodides on the LiAlH4 reduction products of the corresponding 2,3-dihydro-1H-pyrrolo(2,1-c)(1,2,4)benzothiadiazin-5,5-dioxide is described. It was found that some of the substances synthesized exist in polymorphous forms and it was possible to isolate these.


[Research on monoamine oxidase inhibitors: synthesis of N-alkyl and N-aralkyl substituted ethylsyringoylhydrazides]. In continuation of previous work on compounds with anti-MAO activity, some alkyl and aralkyl-hydrazides of ethylsyringic acid (3,5-dimethoxy-4-ethoxybenzoic acid) were synthesized. Pharmacological examination showed that branching of the alkyl radical on the beta nitrogen to the carbonyl group increases anti-MAO activity which appears to be linked with the molecular dimensions of the alkyl group.


Alkaloids and procyanidins of an Uncaria sp. from Peru. The alkaloid and procyanidin composition of Uncaria sp. from eastern Peru, used in folk medicine was studied. Five alkaloids have been separated and identified as pteropodine, speciophylline, isopteropodine, uncarine F and isomytraphylline, all belonging to the oxindole group characteristic of the Rubiaceae. Moreover (--) epicatechin and four dimeric procyanidins A1, B1, B2 and B4 have been shown to constitute the polyphenolic fraction of the plant extract.


Diamides of cyclobutane-1,1-dicarbocylic acid. II. A group of nine diamides of cyclobutane-1,1-dicarboxylic acid has been prepared and examined for general depressant and analgesic activity. Several showed activity in both respects but with little overlap. Several compounds were evaluated for anticonvulsant and myorelaxant activity. No activity was found.


The theory of neutral and weakly selected genes. The controversy over whether a major fraction of evolutionary allele substitutions and protein polymorphisms in natural populations is due to random drift of neutral mutations remains unresolved; but the discussion has led to extensions of population genetic theory, particularly in stochastic models. The work of Maruyama in seeking quantities that are not strongly dependent on assumptions about population structure is particularly discussed. Under some circumstances, and perhaps quite generally, the probability of eventual fixation of a mutant gene, the total number of heterozygotes in which the gene is involved before its fixation or less, and the total number of heterozygotes during the time the gene has a specified frequency in the entire population have this desirable property. Whether there are many mutants that are so nearly neutral as to be unaffected by selective forces or not, it is clear that there are many loci at which selection is very weak and some of the evolutionary consequences of this are discussed briefly.


The evolution of function in enzymes.

The evolution of function in enzymes. The evolution of function appears to have proceeded from a primordial catalyst with little specificity to a highly specific one. As specificity increased, the need to exclude water created a need for flexibility, and flexibility in time led to regulation through induced conformational changes. Induced conformational changes in multisubunit proteins then produced cooperativity. Each of these properties is subject ot fine tuning by modification of the protein structure. The function of the enzyme is itself tuned to high efficiency by the evolution of these interrelated functions.


Origin of periodic proteins.

Origin of periodic proteins. Certain proteins, of which collagen is the best known example, have an approximately periodic sequence of amino acids. Evidence is available that the nucleic acid templates of at least some of these proteins have a large amount of internal self-complementarity. It is difficult to explain the origin of such proteins from nonperiodic proteins by accumulation of single amino acid replacements. A saltatory origin is suggested; the process would be a repetitive replication of a small nucleic acid segment. Internal self-complementarity occurs because the repeating segment incorporates sequences from both strands of DNA. The same process could produce RNA with a large amount of internal self-complementarity; tRNA may be an example.


Structural origins of mammalian albumin. The amino acid sequence and disulfide bridges of bovine serum albumin reveal nine double loops formed by the bridges. The pattern and size of loops and connecting segments between loops indicate that a basic repeating unit (domain) consists of "large double loop-short connecting segment-small double loop-long connecting segment-large double loop-connecting segment between "domains" and repeats exactly three times. Thus, albumin arose by duplication of the primordial single domain gene, followed some time later by a half-gene duplication to give the ancestor of the present triple domain structure. These duplications are estimated to have occurred about 700 million years ago, based on the differences between domain (75 to 82%), the difference between bovine and human albumins (20%),and reported time estimates for globin and immunoglobulin evolution. Still farther back in time, the single domain arose by triplication of a gene of a primordial subdomain of about 77 amino acid residues, consisting of one large double loop with a segment on each end. The structure resulting from this triplication consisted of three large loops and three long connecting segments. Later, a gene deletion for part of a connecting segment and part of the middle large loop occurred to produce the large-small-large loop pattern. A proposed three dimensional structure for the "subdomain" shows spatial and sequence similarity with the G-H helical regions of myoglobin or hemoglobin, suggesting that it arose by duplication and separation of a gene for a C-terminal segment of a primitive globin. The CYS residues were introduced as an adaptive, convergent evolutionary event after separation from globin, but before duplication of the subdomain gene.


The evolution of vertebrate fibrinogen.

The evolution of vertebrate fibrinogen. The origins of the vertebrate fibrinogen molecule and its constituents chains have been considered, first by examining certain features of the molecule as it exists in a primitive vertebrate (the lamprey), and then by comparing the amino acid sequences of certain portions of the three nonidentical chains as they exist in the human molecule. Although the lamprey protein is distinctly homologous to mammalian fibrinogens, at the present stage of characterization its three nonidentical chains appear to be even more different one from another than are the three mammalian chain types. On the other hand, certain sequence resemblances in the three human chains clearly indicate a common ancestry for the alpha-, beta-, and gamma-chains. It is concluded that the ancient fibrinogen molecule was composed of all identical chains and that its differentiation into three chain types occurred long before the divergence of lampreys and higher vertebrates.


The evolution of genes in the major histocompatibility complex. The mammalian major histocompatibility system (MHS) includes genes determining the structure of the classical major transplantation antigens (H-2K and H-2D), the I region-associated (Ia) antigens, and genes determining the structure level or both of the first four components of complement. In addition, the I region incudes a series of genes determining specific immune responsiveness to a wide variety of antigens - the Ir genes. The available evidence indicates that the K, D, and I gene products are cell surface glycoproteins that are structurally and perhaps functionally related. The multiple genes in this complex region apparently arose by a process of tandem gene duplication. There is some reason to believe that the murine MHS may have originated from genes in the T/t complex - a "supergene" near the centromere of the 17th mouse chromosome determining a series of steps in early embryonic development. Other evidence has led to the postulate that genes in the MHS have given rise to immunoglobulin structural genes by a process of translocation and further gene duplication. While these evolutionary relationships are speculative, it seems clear that the MHS determines a series of cell surface proteins that are intimately involved in cellular recognition and interaction, and in regulation of immune responsiveness by a new, nonimmunoglobulin recognition system.


Transition from the polikilotherm to the homeotherm: possible role of sodium transport and thyroid hormone. The evolutionary transition from poikilothermy to homeothermy required the recruitment of complex mechanisms that provided both higher rates of heat production and regulation of heat loss in the presence of wide variations in ambient temperature. Two pathways that have been considered to be contributors to the transition to homeothermy are thyroid-regulated thermogenesis and nonshivering thermogenesis in the adaptation to cold. These pathways may yield higher rates of heat production in homeotherms by two processes that need not be mutually exclusive: a) hydrolysis of ATP and consequent generation of the Pi acceptor--ADP, or b) decreased coupling of mitochondrial oxidative phosphorylation. A significant fraction of both thyroid and catecholamine thermogenesis has been ascribed to enhanced energy expenditure in active transmembrane Na+ transport (i.e., the Na+ pump). By extension these findings implicate the Na+ pump as a heat source in the evolution of the homeotherms. The thermogenic response to thyroid hormone is both rapid and profound in homeotherms (mammals), and more slowly evident and probably lesser in magnitude in poikilotherms (amphibia and reptiles). One crucial pathway in the evolution to homeothermy may be nonshivering thermogenesis in response to cold. In mice, most of the elevated rate of oxygen consumption of liver and skeletal muscle elicited by cold exposure is ouabain-sensitive. Thus, the Na+ pump appears to be an important heat source in the transition to homeothermy. The underlying biochemical mechanisms that mediate the contribution of the Na+ pump to heat production, including responsiveness to thyroid hormone and catecholamines, however, may not be unique to the homeotherms.


Mineral Nutrition of Aspergillus niger for citric acid production. The mineral requirements of a strain of Aspergillus niger for the production of citric acid in a synthetic medium were studied. It was observed that K2HPO4 and MgSO4. 7 H2O were required at concentrations of 0.1% and 0.02% respectively. The optimum level of each of the trace elements Fe, Mn and Zn was 1.0 mug/ml. NaC1 and CaC12 at lower concentrations had no effect on citric acid production. Trace elements, Cu, Co and Mo, had an adverse effect on the production of citric acid while Ni and V were without effect.


Steroid derivatives. LXXIX. Microbial 15 beta-hydroxylation of 17 alpha-acetoxy-16-methylene derivatives of the pregnane series by the action of Cunninghamella blakesleeana. Hydroxylation of 17 alpha-acetoxy-6-chloro-16-methylene-4,6-pregnadiene-3,20-dione (Chlorosuperlutin, I) by Cunninghamella blakesleeana yielded a 15 beta-hydroxyderivative II. Analogous transformation of 17 alpha-acetoxy-16-methylene-4,6-pregnadiene-3,20-dione (Superlutin, IV) included a hydroxylation in position 15 beta and probably also in 11 beta with a concomitant reduction of the 6,7-double bond.


Physiological characteristics of chemostatically grown Citrobacter freundii as a function of the specific growth rate and type of nutrient limitation. Citrobacter freundii was grown aerobically in a chemostat on a mineral medium with galactose or glucose as carbon and energy sources under limitation by carbon or nitrogen source respectively. At various specific growth rates ranging from 7 to 95% mumax the culture in steady state was analysed and growth yield, specific metabolic rate of substrate utilization, intracellular concentration of pyruvate, ATP, ADP, AMP and energy charge were determined and plotted as functions of dilution rate. In all four types of experiments the physiological state of cells remained practically independent of dilution rate up to D=0.6 mumax, and at a given specific growth rate nearly independent on mumax and type of limitation. At approximately D=0.6 mumax, which is close to the maximum output dilution rate Dm, the physiological state of the cells changed: growth yields decreased and intr cellular pyruvate and adenylates concentrations increased. Consequently, in a given medium two dilution rates exist at which growth rate dx/dt is the same but the physiology of the population is quite different.


Amidase activity of some bacteria.

Amidase activity of some bacteria. The amidase activity of bacteria possessing a high nitrilase activity was found to display the same spectrum although the bacteria may belong to different taxonomic groups, Bacillus, Bacteridium, Micrococcus, Brevibacteriun. The spectrum of amidase activity, although very broad, is more restricted than that of nitrilase activity. Internal amides as well as vinyl-bound amides are not hydrolyzed.


Some paradoxical effects of inhibitors of protein synthesis on protein turnover in cultured human cells. Low concentrations of cycloheximide, sufficient to block net protein synthesis in growing normal and cancer cells, had no effect on protein turnover, i.e. either the incorporation of labeled amino acids from media lacking other amino acids essential for growth, or the loss to the medium of amino acids from prelabeled cells. At the concentrations that blocked growth, the rate of amino acid incorporation from complete medium was reduced to the "turnover level" i.e. the rate of incorporation seen in amino acid-deficient media. Protein turnover was inhibited only at higher concentrations of the inhibitor. Qualitatively similar results have been obtained with puromycin, anisomycin, emetin and tylocerebrine.


Synchronization of some human cell strains by serum and calcium starvation. A technique was investigated for producing parasynchronous growth of some established, aneuploid human cell strains. Removal of both serum and calcium from exponentially growing monolayer cells tended to inhibit their growth. After 20 hr, a high percentage of the cell population was arrested in or near mitosis. Readdition of serum and calcium caused parasynchronous growth of the cells of three human strains studied. All three strains incorporated tritiated thymidine maximally 10 to 15 hr after serum and calcium were added, and cell numbers increased rapidly 17 to 25 hr after the growth medium was reconstituted. Population-doubling ranged from 80% to 100% of the theoretical. The yield of parasynchronous cells is high with this technique and may produce a significant amount of nontemporally distorted biological material upon which direct biochemical analysis can be performed at various times within the generation cycle.


Inactivation of the biological activities of the thermostable direct hemolysin of Vibrio parahaemolyticus by ganglioside Gt1. Biological activities of the thermostable direct hemolysin produced by Vibrio parahaemolyticus, such as its hemolytic activity and lethal activity, were inhibited by neuraminidase-sensitive gangliosides, of which GT1 ganglioside was the most inhibitory. Neuraminidase-resistant gangliosides did not affect the activities of the hemolysin. Results showed that horse erythrocytes, which are resistant to the hemolysin, do not contain the neuraminidase-sensitive gangliosides GT1 and GD1a. Therefore, we propose that neuraminidase-sensitive gangliosides, and especially GT1 ganglioside, may be the receptor sites on the membranes for the thermostable direct hemolysin of V. parahaemolyticus.


Regulation and extracellular glucosyltransferase production and the relationship between extracellular and cell-associated activities in Streptococcus mutans. The regulation of extracellular glucosyltransferase production in Streptococcus mutans GS-5 has been studied using a chemically defined medium. Most of the glucosyltransferase activity produced by cells grown in the chemically defined medium was extracellular, in contrast with the distribution between cell-associated and extracellular glucosyltransferase activity when cells were grown in complex medium. The production of extracellular glucosyltransferase activity coincided with the logarithmic growth phase, and further accumulation ceased when glucose was exhausted from the medium. Accumulation of extracellular glucosyltransferase activity was inhibited immediately by chloramphenicol and rifamycin, added either at the beginning of growth or during mid-logarithmic growth. Low concentrations of chloramphenicol inhibited both cellular protein synthesis and the accumulation of extracellular glucosyltransferase activity to the same extent, indicating a close coupling between glucosyltransferase synthesis and secretion. Experiments using cell lysates showed that no intracellular accumulation of glucosyltransferase activity occurred in the presence of the inhibitors and that the intracellular activity is very low relative to the cell-surface activity. The utilization of cells depleted of cell-associated glucosyltransferase activity indicated that most of the cell-associated glucosyltransferase activity does not act as a precursor for the extracellular enzyme. Sugar analogue inhibitors of glycoprotein synthesis did not have any specific effects on the synthesis or secretion of extracellular glucosyltransferase activity.


In vitro activity of guinea pig transfer factor released into plasma. Plasma fractions and plasma dialysate from 2,4-dinitrochlorobenzene- and tuberculin-sensitive guinea pigs that had been treated with either antilymphocytic serum or normal control serum were analyzed for their ability to transfer lymphocyte transformation, passive cutaneous anaphylaxis, and macrophage migration inhibition, as well as delayed hypersensitivity in vivo. Antilymphocytic serum caused rapid release of material, which has characteristics of transfer factor, into the plasma. It was dialyzable, migrated electrophoretically with the alpha globulins and albumin, possessed a 280/260 (nm) optical density ratio of 0.7, and caused in vitro lymphocyte transformation in the presence of the specific antigen. Passive cutaneous anaphylaxis antibodies were also present in the plasma of sensitive animals, but they were isolated in electrophoretic or dialysis fractions separate from those containing transfer activity.


Isolation of Brucella melitensis phage of broad biotype and species specificity. A Brucella melitensis phage was isolated. Stocks of phage were established which produced early large plaques. The phage host range included all smooth Brucella species.


Inhibition of human neutrophil chemotaxis and chemiluminescence by amphotericin B. The effect of the antifungal drugs amphotericin B, fluocytosine, miconazole, griseofulvin, and nystatin on the chemotactic responsiveness of human neutrophils was studied. Amphotericin B in a concentration of 2 mug/ml inhibited chemotatic responsiveness, and in a concentration of 5 mug/ml it also inhibited chemiluminescence. The inhibition of chemotaxis could be reversed by washing the cells. The other antifungal drugs did not inhibit chemotaxis even in concentrations much higher than those obtained in human serum during treatment.


Is the pathogenesis of Crohn's disease similar to that juvenile recurrent pyelonephritis? A study of lipid A antibody titers in Crohn's disease, ulcerative colitis and acute enteritis. In 18 patients with Crohn's disease, 28 patients with ulcerative colitis, 24 patients with enteritis and in 68 healthy adults lipid A antibody titers were determined by the passive hemolysis test. In addition, 0 antibody titers to polyvalent E. coli, Klebisella, Proteus, and Ps. aeruginosa antigens were measured by indirect hemagglutination. The patients with Crohn's disease showed a statistically significant elevation of the lipid A antibody titers compared with each of the three other groups investigated. The 0 antibody titers for the four polyvalent antigens were also higher in the patients with Crohn's disease than in the other groups. The results indicate that Enterobacteriaceae are involved in the pathogenesis of Crohn's disease. Thus long-term treatment with wide spectrum antibiotics seems to be justified. The determination of lipid A antibody titers may be a useful diagnostic tool in differentiating between Crohn's disease and ulcerative colitis.


Septicaemia and meningitis with campylobacter fetus subspecies intestinalis. Two cases of infection with Campylobacter fetus subspecies intestinalis are reported. In a 46-year-old farmer the development of septicaemic campylobacteriosis with acute gastroenterocolitis was favoured by malnutrition due to chronic alcoholism. After treatment with erythromycin the patient recovered completely. The second patient, a 40-year-old worker with meningitis, recovered after combined streptomycin-erytro-mycin therapy. In both patients antibodies against homologous and reference strains of Campylobycter fetus subspecies intestinalis, serotype 1 (Mitscherlich) were demonstrated in titres ranging from 1:16 to 1:64 by type the complement-fixation test.


The long-term outcome of hepatitis B.

The long-term outcome of hepatitis B. Among 466 hospitalized patients with serologically verified acute hepatitis B, 440 individuals (94.4%) could be followed up until normalization of liver function had occured, or for at least one year. In 90.2% of the patients followed-up liver function including galactose tolerance) returned to normal within four months after onset of illness. Chronic persistent hepatitis B surface antigen (HB Ag) for at least one year in 14 patients (50%). Liver biopsy was performed in consistent with CPH in all cases. Histological signs of chronic aggressive hepatitis developed in 15 patients  (3.4%) and persistence of HB Ag was observed in 11 of these patients (73%). No histological follow-up was performed in patients with normal liver function within four months after onset of illness. Cprticosteroid treatment in 56 patients with prolonged symptoms did not seem to predispose to persistence of HbsaG in the serum.


[A new influenza subunit vaccine: reactogenicity and antigenicity in comparison to split and whole virus vaccines (author's transl)]. The reactogenicity and immunogenicity of a new influenza subunit vaccine essentially containing only haemagglutinin and neuraminidase was studied in man. The vaccine was compared to commercially available vaccines, an adjuvant containing tween-ether split vaccine (800 IU per dose), and a fluid whole-virus vaccine (2100 IU per dose). Two dosages (700 and 2100 IU) of the fluid subunit vaccine were compared. All vaccines contained the virus strains recommended by the WHO for the 1975/76 season. In a double-blind study 399 volunteers were randomly selected to receive one of the four vaccines. The volunteers were examined for side-effects 24 and 48 hr after vaccination. Antibodies inhibiting haemagglutination were determined prior to and four weeks after vaccination. The sudunit vaccine at 700 IU per dose caused significantly fewer local side effects than the comparable split vaccine, and resulted in significantly higher antibody titers against both influenza A strains. A comparison of the subunit and whole virus vaccines containing high dosages (2100 IU) showed striking differences in reactogenicity. Subunit vaccine was very well tolerated. whereas whole virus vaccine caused systemic reactions, including fever and headache, in 15% of the volunteers. No significant reactogenicity was seen with a high dosage of subunit vaccine (2100 IU) although this is a three-fold increase on the currently used European dosage. Antibody titers were significantly enhanced however.


[Humoral and cell-mediated immune reactions in chronic posttraumatic osteomyelitis (author's transl)]. The humoral and cell mediated immune response was investigated in 90 patients suffering from chronic posttraumatic osteomyelitis. In 10-20% of the patients the serum IgG and IgM was increased, in 5-10% the levels were decreased. The values were compared with those of a control group of 35 normal healthy volunteers. Of 55 patients with staphylococcal infection only 25 had a positive antistaphylolysin titer. Eleven out of 25 patients with staphylococcal osteomyelitis showed a positive in vitro lymphocyte transformation after stimulation with alpha-staphylolysin (mean transformation rate 5.4 equal to or less than 0.9). The phytohemagglutinin and the pokeweed-mitogen response were normal. A comparison of the immunological reactivity of the patients and the clinical severity of the disease showed that patients with established delayed-type hypersensitivity phenomena suffered a significantly worse clinical course of the disease.


A cell line (SCABER) derived from squamous cell carcinoma of the human urinary bladder. An established cell line derived from a documented squamous cell carcinoma of human urinary bladder is described. The cultured cells retained the characteristic morphology of the tumor of origin for 40 in vitro passages. Numerous desmosomes were found between cultured cells. Chromosome analysis showed hypotetraploidy with no obvious modal number, while distinctive marker chromosomes and a male karyotype were present.


The induction of immunity against tumor cell transplantation with a complex of tumor cell surface substance and a serum component found in the tumor-regressor animals. Immunity against transplantation of MM2 cells, an ascites cell line of mouse mammary carcinoma, was induced in syngeneic C3H/He mice by intraperitoneal injection of a complex formed between a MM2-specific surface component isolated from the cells and a serum factor found in the serum from MM2-regressor and MM2-bearing mice. The induced immunity was transferable to other mice with the spleen cells or with the serum from the immunized animals. This cell surface component was not immunogenic by itself. A new immunogenicity was induced in the protein moiety of this substance when the serum factor was bound to the polysaccharide-containing part of the molecule. On the basis of these results, a biphasic mechanism for the induction of the humoral immunity against tumor antigens was proposed. The first phase takes place in tumor-bearing animals and gives rise to the serum factor. The second phase, which is blocked in tumor-bearing animals, consists of the induction of the humoral immunity by the complex in tumor-free animals.


Humoral reactions in syngeneic hosts against tumor cell surface incomplete saccharide moieties. Components on the surface of MM2 ascites mammary carcinoma cells induce agglutination factors in the serum of syngeneic host C3H/He mice, and bind the factors in vitro. These components have been classified into three groups: MM2-specific substances, mammary tumor virus (MTV)-associated substances and tumor-associated embryonic materials. The substances contained saccharide moieties and their terminal sugar residues were essential for the binding of the serum factors. These terminal saccharides were exposed during cell proliferation,but masked in stationary cells, at least partly, due to elongation of the saccahride moieits. The terminal structures of these polysaccharide moities of growing and stationary cells were studied by semiquantitative tumor cell agglutination using the agglutinating activities against MM2 cells of MM2-regressor serum and of FMA/R- and Ehrlich-regressor sera which had partial cross-agglutination activities. Agglutination by phytohemagglutinins, inhibition of the agglutination by saccharides or with isolated cell surface components and treatment of the cells with glycosidases were also used for this purpose.


Specific sensitization of lymphocytes to tumor antigens by co-cultivation with peritoneal cells exposed to such antigens. Peritoneal cells from BALB/c mice were seeded into 50-mm plastic Petri dishes and exposed either to antigen extracts prepared from individual BALB/c sarcomas having unique tumor-specific antigens, to similar extracts prepared from 14- to 18-day-old mouse embryos, or to culture medium only. Lymphoid cells from BALB/c lymph nodes were than added to the peritoneal cells. Following 4-5 days of co-cultivation, the lymphoid cells were harvested and tested in a 30 h microcytotoxicity assay for reactivity against tumor cells carrying the respective sensitizing antigens as well as against control cells which consisted of either tumor cells lacking the sensitizing antigens or normal skin fibroblasts. By this approach sensitization to individually unique tumor antigens, as well as to antigens shared by mouse embryos and mouse sarcoma cells, was achieved.


Stimulation of transplanted 3-methylcholanthrene-induced sarcomas in mice by specific immune ahd by normal serum. Serum from Bl x C3H mice carrying syngeneic, progressively growing, highly immunogenic 3-methylcholanthrene-induced tumors when admixed with the specific tumor and inoculated into immunologically crippled syngeneic recipients, stimulated growth as compared with serum from control normal mice. It appears that this acceleration of tumor growth is an immune effect since it is not present when a non-immunogenic (spontaneous tissue culture) tumor is used; and the active factor can be absorbed from the serum by the specific tumor but not by an immunologically unrelated tumor. Normal serum per se also stimulated tumor growth, but to a smaller extent.


The involvement of a diol-epoxide in the metabolic activation of benzo(a)pyrene in human bronchial mucosa and in mouse skin. DNA has been isolated from human bronchial segments that have been treated in short-term organ culture with 3H-labelled benzo (a) pyrene. DNA has also been isolated from mouse skin treated with 3H-labelled samples of benzo (a) pyrene, with the related radioactive 4,5-, 7,8- and 9,10-dihydrodiols and with 3H-3-hydroxybenzo (a) pyrene. Sephadex LH20 column chromatography of hydrolysates of these DNA samples showed that the hydrocarbondeoxyribonucleoside products formed in benzo (a)-pyrene-treated human bronchial mucosa and mouse skin are indistinguishable from those that are formed when 7,8-dihydro-7,8-dihydroxybenzo (a) pyrene 9,10-oxide reacts with DNA in solution. These same hydrocarbon-deoxyribonucleoside products were also found in hydrolysates of DNA from mouse skin treated with 7,8-dihydro-7,8-dihydroxybenzo (a)-pyrene but products of this type were not detected in hydrolysates of DNA following treatment of mouse skin either with the 4,5- or 9,10-dihydrodiols or with 3-hydroxybenzo (a) pyrene. This results show that the metabolic activation of benzo (a) pyrene to a diolepoxide, 7,8-dihydro-7,8-dihydroxybenzo (a) pyrene 9,10-oxide, which reacts with DNA, is the same in human bronchial mucosa, a tissue in which this hydrocarbon is suspected of being carcinogenic, as it is in mouse skin and in hamster embryo cells, two situations in which benzo(a)pyrene is known to induce malignancy.


Tumor bound immunoglobulins: the relationship between the in vivo coating of tumor cells by potentially cytotoxic anti-tumor antibodies, and the expression of immune complex receptors. In this study we investigated the relationship between binding of anti-tumor antibodies by polyoma-virus-induced SEYF- a tumor cells and the expression of immune-complex receptors on these cells. It was shown that in vivo propagated cells became progressively coated with IgG. The increase in the IgG coating of SEYF-a cells, occurring during the second week of propagation, was directly correlated with an increase in the coating of cells with potentially cytotoxic anti-tumor antibodies. The activity of these antibodies was demonstrated by cell lysis following addition of exogenous complement. Cell populations propagated in vivo for longer periods (3 weeks or more) became less sensitive to exogenous complement although their IgG coating remained high, and although higher titers of anti-tumor antibodies could be eluted from them. This indicated that antibodies coating young tumor cell populations have the capacity to activate complement whereas those coating older tumor cell populations are incapable of complement activation. Previous findings that SEYF-a cell populations are able to bind unrelated immune complexes were confirmed in the present study. We also found that the capacity of these populations to bind such unrelated complexes decreased with propagation time in vivo. The involvement of anti-tumor antibodies in this phenomenon was indicated by the finding that such antibodies inhibited binding of unrelated immune complexes by young cells but not by old cells. Furthermore, treatments causing dissociation of Ig from the cell surface restored, to some extent, the capacity of anti-tumor antibodies to inhibit immune complex binding by old SEYF-a populations.


Tube leukocyte adherence inhibition assay for the detection of anti-tumour immunity. II. Monocyte reacts with tumour antigen via cytophilic anti-tumour antibody. The peripheral blood monocyte is the reactive cell in the tube LAI assay. The monocyte loses its properties of adherence to glass upon exposure to specific antigen. Two different experiments to determine if lymphocytes, when they reacted with tumour, released mediators that were responsible for inhibiting monocyte glass adherence, gave negative results. The mechanism wherby the specific tumour antigen appeared to be recognized was the binding of cytophilic IgG antitumour antibody to receptors on the cell surface of the monocyte. The results of the experiments indicate that normal peripheral blood monocytes could be made specifically reactive ("armed") to the tumour extract by incubating normal peripheral blood leukocytes with serum from a reactive cancer patient. IgG isolated from "arming" sera was shown to have the capacity to sensitize normal leukocytes. Patients with breast cancer or malignant melanoma with limited tumour burdens had free cytophilic anti-tumour antibody in their serum, whereas the serum of patients with large tumour burdens (metastatic cancer), whose leukocytes did not react in the tube LAI assay, did not "arm".


Serum dependence of expression of the transformed phenotype: experiments with subline of mouse L fibroblasts adapted to growth in serum-free medium. Characteristics of LSF cells grown in serum-containing and serum-free medium were compared. LSF is a subline of the L-strain of mouse transformed fibroblasts adapted to continuous growth is serum-free medium. Proliferation of LSF cells in monolayer on solid substratum was almost similar in serum-containing and in serum-free media. However, several other characters were found to be altered by the addition of serum to the serum-free medium: ability of cells to form colonies in semi-solid medium increased considerably; agglutinability of cells by Concanavalin A increased; uptake of 2-deoxy-D-glucose by the cells increased considerably; ability of cells to metabolize benzo (a) pyrene was inhibited; cell morphology was altered and, in particular, the cells became less spread on the substratum and density of microvilli on the cell surface increased. All these changes induced by serum were reversed by transfer of the cells back into serum-free medium. Thus, addition of serum increased the expression of a number of cellular traits characteristic of transformed phenotype, while in serum-free medium partial phenotypic reversion of transformation was observed. A possible role of serum in the expression of the transformed phenotype is discussed. It is pointed out that cell lines adapted to growth in serum-free medium provide an experimental system convenient for analysis of the effects of different serum components on the cell phenotype.


[Value of carcinoembryonic antigen (CEA) in the diagnosis of malignant tumors of the urogenital tract]. Both in a control group and in 154 patients with malignant tumors of the urogenital tract in the serum- and urinary levels of the carcinoembryonic antigen (CEA) have been ascertained by a radioimmunoassay. Serum-CEA-values lay above normal range in 71%, and urinary CEA-levels exceeded it in 46%. Only in the case of the carcinoma of the bladder a correlation was found between the CEA-curves and the clinical findings following tumor therapy. Up to the present, the CEA-test cannot be recommended as a universal screening method, but may be useful means for diagnostics and case controls, above all, with regard to the carcinoma of the bladder.


[Preoperative irradiation in hypernephroid carcinoma]. Since 1967 we have been using preoperative radiation therapy for hypernephroma as proposed by RICHES. Radiation therapy, to include the para-aortic lymph nodes, is given in 250 rad increments 4 times weekly to a total dose of 3000 rads. After an interval of 3 weeks following the radiation therapy, we are performing the radical nephrectomy. 100 patients were treated by this method in the years 1967-1975: 32 patients were in stage I, 7 in stage II, 50 in stage III and 11 patients in stage IV. In 26 patients more than 5 years have passed since the beginning of the treatment: 46% od these survived. The survival for 3 years is 63, for 1 year after the surgery 80%. The surgical mortality rate is 2%. The object od the preoperative treatment is: 1. Devitalization of growing cells in the periphery of the tumor, thus preventing metastases and local recurrence. 2. Decreasing the size of the tumor and thereby facilitating surgery. In one-third of the cases there is radiologically demonstrable decrease in the size of the tumor mass, probably secondary to obliteration of the dilated veins in the capsule. The delay of six weeks has had no adverse effect on the outcome of the disease.


[Xanthogranulomatous pyelonephritis].

[Xanthogranulomatous pyelonephritis]. Based on literature and own case the entity of xanthogranulomatous pyelonephritis is reviewed. In particular are discussed the difficulties of the preoperative diagnostic procedures and the differential diagnosis.


Inheritance of nocardiophage phi EC in matings of nocardial lysogens. Lysogens of Nocardia erythropolis were mated with nonlysogenic strains to study the inheritance of the phi EC prophage. Crosses between lysogenic strains of the Mat-Ce mating type and nonlysogenic Mat-cE strains produced Mat-cE lysogens at a recovery rate of 17%, whereas recombination frequencies between chromosomal traits were about 2.3 x 10(-5). Crosses of lysogenic Mat-cE mating types with nonlysogenic Mat-Ce produced Mat-Ce lysogens at a recovery rate of 19%, whereas recombinants for chromosomal traits were recovered at only 1.8 x 10(-5). Crosses of homologous mating types, lysogenic Mat-Ce with nonlysogenic Mat-Ce or lysogenic Mat-cE with nonlysogenic Mat-cE, failed to transfer the prophage. It was concluded that the phi EC prophage exists as a plasmid and can be transferred at high frequencies with patterns of transfer controlled like typical nocardial fertility. Evidence that the prophage may also exist as an integrated element was observed from recombination analyses.


Compartmentation and control of arginine metabolism in Neurospora. The fate of [14-C]arginine derived from the medium or from biosynthesis has been examined in Neurospora growing in arginine-supplemented medium. In both cases the label enters the cytosol, where it is used efficiently for both protein synthesis and catabolism before mixing with the majority of the endogenous [12C]arginine pool. Both metabolic processes appear to use the same cytosolic arginine pool. It is calculated that the nonorganellar cytoplasm contains approximately 20% of the intracellular arginine pool when the cells are growing in arginine-supplemented medium. The results suggest that compartmentation of arginine is a significant factor in controlling arginine metabolism in Neurospora. The significance of these results for studies of amino acid metabolism in other eukaryotic systems is discussed.


Purification and properties of nitroalkane-oxidizing enzyme from Hansenula mrakii. A nitroalkane-oxidizing enzyme was purified about 1,300-fold from a cell extract of Hansenula mrakii grown in a medium containing nitroethane as the sole nitrogen source by ammonium sulfate fractionation, diethylaminoethyl-cellulose column chromatography, hydroxyapatite column chromatography, and Bio-Gel P-150 column chromatography. The enzyme was shown to be homogeneous upon acrylamide gel electrophoresis and ultracentrifugation. The enzyme exhibits absorption maxima at 274, 370, 415, and 440 nm and a shoulder at 470 nm. Balance studies showed that 2 mol of 2-nitropropane is converted into an equimolar amount of acetone and nitrite with the consumption of 1 mol of oxygen. Hydrogen peroxide is not formed in the enzyme reaction. In addition to 2-nitropropane, 1-nitropropane and nitroethane are oxidatively dentrified by the enzyme, but nitromethane is inert to the enzyme. The nitroalkanes are not oxidized under anaerobic conditions.


Volatile factor involved in the dimorphism of Mucor racemosus. Both hyphal and yeastlike development of Mucor racemosus and M. rouxii were demonstrated under 100% N2. Under standardized conditions in yeast extract-peptone-glucose medium, the morphology depended on the N2 flow rate and not on the glucose concentration. The effect was related to the rate of flushing of the atmosphere over the culture medium. The results indicate that a volatile compound produced by Mucor is involved in morphogenesis.


Molecular and catalytic properties of ribulose 1,5-bisphosphate carboxylase from the photosynthetic extreme halophile Ectothiorhodospira halophila. D-Ribulose 1,5-bisphosphate (RuBP) carboxylase has been purified from the photosynthetic extreme halophile Ectothiorhodospira halophila. Despite a growth requirement for almost saturating sodium chloride in the medium, both crude and homogeneous preparations of RuBP carboxylase obtained from this organism were inhibited by salts. Sedimentation equilibrium analyses showed the enzyme to be large (molecular weight: 601,000). The protein was composed of two types of polypeptide chains of 56,000 and of 18,000 daltons. The small subunit appeared to be considerably larger than the small subunit obtained from the RuBP carboxylase isolated from Chromatium, an organism related to E. halophila. Amino acid analyses of hydrolysates of both E. halophilia and Chromatium RuBP carboxylases were very similar. Initial velocity experiments showed that the E. halophila RuBP carboxylase had a Km for ribulose diphosphate of 0.07 mM and a Km for HCO3- of 10 mM. Moreover, 6-phospho-D-gluconate was found to markedly inhibit the E. halophila carboxylase; a Ki for phosphogluconate of 0.14 mM was determined.


Structure of Caulobacter deoxyribonucleic acid. The deoxyribonucleic acid of the dimorphic bacterium Caulobacter crescentus contains a component that renatures with rapid, unimolecular kinetics. This component was present in both swarmer and stalked cells and exhibited the sensitivity to endonuclease S1 expected for hairpin loops. Double-stranded side branches between 100 and 600 nucleotide pairs in length were visible in electron micrographs of rapidly reassociating deoxyribonucleic acid isolated by hydroxyapatite chromatography. No extrachromosomal elements were found in spite of systematic attempts to detect their presence. These results indicate that the rapidly reassociating fraction derives from inverted repeat sequences within the chromosome and not from cross-links or plasmids. We estimate that there are approximately 350 inverted repeat regions per Caulobacter genome. The kinetic complexity of Caulobacter deoxyribonucleic acid, however, is no greater than that of other bacteria.


Uptake and metabolism of gangliosides in transformed mouse fibroblasts. Relationship of ganglioside structure to choleragen response. NCTC 2071 cells, transformed mouse fibroblasts, when grown in chemically defined medium, are deficient in gangliosides and do not respond to choleragen. The cells lack two biosynthetic enzymes, CMP-sialic acid:lactosylceramide sialyltransferase and UDP-galactose:GM2 (GalNAc-[AcNeu]-Gal-Glc-ceramide) galactosyltransferase, which are required for ganglioside synthesis. Following uptake of ganglioside GM1 (Gal-GalNAc-[AcNeu]-Gal-Glc-ceramide) from the medium, the cells respond to choleragen; however, they remain unresponsive following uptake of gangliosides GM2 (approximately 6 X 10(6) molecules/cell) and GM3 (AcNeu-Gal-Glc-ceramide) (approximately 2 X 10(6) molecules/cell). A response was observed when the cells had bound approximately 2 X 10(7) molecules of GM2/cell. After binding GD1a (AcNeu-Gal-GalNAc-[AcNeu]-Gal-Glc ceramide) (approximately 2 X 10(5) molecules/cell), cells exhibit some response to the toxin which can be attributed to enzymatic conversion of GD1a to GM1. A second line of NCTC 2071 cells which have 2.5 X 10(7) molecules of endogenous GM2/cell is slightly responsive to choleragen; adenosine 3':5'-monophosphate (cyclic AMP) levels rise 150%. However, when these cells have bound 4.4 X 10(4) molecules of GM1 per cell, cyclic AMP levels rise 7-fold following toxin treatment. GM1, which becomes functionally integrated into the cells, appears to be the natural receptor for choleragen and is 50 to 1000 times more effective than other gangliosides in eliciting a choleragen response.


A study on the autoactivation of rabbit muscle phosphorylase kinase. Under conditions favoring its autocatalytic reaction, phosphorylase kinase may be activated and phosphorylated in 2-(N-morpholino)ethanesulfonate (Mes) buffer to a much higher level than in beta-glycerophosphate buffer. The fact that the reaction is autocatalytic is supported by several observations: (a) the progress curve of the reaction exhibits a pronounced lag phase, (b) the reaction is strongly inhibited by ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetate, which inhibits phosphorylase kinase, (c) the pH profile of the reaction resembles that of the phosphorylase b to a reaction as catalyzed by nonactivated phosphorylase kinase, and (d) the reaction is not significantly affected by adenosine 3':5'-monophosphate (cAMP) nor by the heat-stable protein inhibitor of cAMP-dependent protein kinases. When fully autoactivated, phosphorylase kinase possesses an activity that is 100% higher than that of the protein kinase-activated form. The results suggest that autophosphorylation of phosphorylase kinase may be an important regulatory mechanism. The autocatalytic reaction involves phosphorylation of the two larger subunits of phosphorylase kinase, i.e. subunits A and B, with a combined total of 7 to 9 phosphates incorporated per mol of enzyme. Although the cAMP-dependent protein kinase also catalyzes the phosphorylation of subunits A and B, the two mechanisms of phosphorylation appear to involve different sites. Prior phosphorylation of phosphorylase kinase by the protein kinase has little effect on the level of autophosphorylation. Thus activation of phosphorylase kinase may be brought about by phosphorylation of the enzyme at different sites.


Triacylglycerol synthesis in isolated fat cells. Studies on the microsomal diacylglycerol acyltransferase activity using ethanol-dispersed diacylglycerols. The acyl-CoA:1,2-diacylglycerol acyltransferase (EC 2.3.1.20) activity of isolated fat cells was predominantly (89%) localized to the microsomal subcellular fraction by assays based on the conversion of 1,2-[3H]diacyl-sn-glycerol to triacylglycerol using 1 to 4 mug of protein. A complementary assay based on the conversion of [3H]palmitoyl-CoA to triacylglycerol was developed. These methods, 100 to 1000 times more sensitive than those previously employed, were used to characterize the microsomal activity. The choice of dispersing agent for addition of diacylglycerol to the reaction mixture was crucial. Addition of diacylglycerol in ethanol resulted in the highest diacylglycerol acyltransferase activity of the methods tested. Tween 20, which has previously been employed as the dispersing agent, severely inhibited the activity. A broad pH optimum from 7.4 to 8.0 was noted and several salts stimulated the activity more than 2-fold. The activity was unstable at temperatures of 28 degrees and above. Dependences on acyl-CoAs containing 6 to 18 carbon atoms were investigated using bacterial diacylglycerol. Acetyl- and butyryl-CoA were not substrates. Highest diacylglycerol acyltransferase activities were observed with decanoyl-CoA and lower activities were noted with longer and shorter saturated chains. Maximal activity with oleoyl-CoA was only 34% of that seen with stearoyl-CoA. No simple relationship between the critical micellar concentrations of the acyl-CoAs employed and diacylglycerol acyltransferase activity was observed. The dependences on diacylglycerols containing fatty acids 6 to 18 carbon atoms in length were investigated with [3H]palmitoyl-CoA. While all the 1,2-diacyl-sn-glycerols tested were substrates, diacylglycerol acyltransferase activity was highest with 1,2-dioleoylglycerol. Maximum activity with the bacterial diacylglycerol was 86% that with 1,2-dioleoylglycerol. The diacylglycerol concentrations required for half-maximal velocity were 20 to 40 muM for long chain diacylglycerols and 2 to 3 muM for short chain diacylglycerols; these were 75- to 750-fold lower than previously reported. Microsomal diacylglycerol acyltransferase specific activities from isolated cells around 50 nmol/min/mg, 10- to 50-fold higher than previously reported from adipose tissue, were typical using dioleoylglycerol and palmitoyl-CoA as substrates. Diacylglycerol acyltransferase specific activities were 17-fold higher in microsomes from isolated fat cells than any other tissue examined. The diacylglycerol acyltransferase appears to have specificity with respect to acyl-CoAs and diacylglycerols, but a definitive interpretation is limited by the lack of data on the physical properties of these substrates in solution under the conditions employed.


Human correxonuclease. Purification and properties of a DNA repair exonuclease from placenta. An exonuclease, which hydrolyzes single-stranded DNA, has been purified from human placenta. It initiates hydrolysis at both the 3' and 5' termini of such DNA with equal facility, yielding 5'-phosphorylated oligonucleotides averaging 4 nucleotides in length. These oligonucleotides are released from both termini at equal rates with the same size distribution. Although not detectably active against intact native DNA, this enzyme can initiate hydrolysis at single-stranded breaks, creating a gap 30 to 40 nucleotides long. If a pyrimidine dimer is adjacent to this break, the enzyme, by virtue of its action at internal phosphodiester bonds, can excise such dimers. We refer to this exonuclease as human correxonuclease because of its excision capability.


A respiration-deficient Chinese hamster cell line with a defect in NADH-coenzyme Q reductase. We have recently described a Chinese hamster cell line with a greatly reduced rate of respiration. In this report we conclude that the defects is in NADH-coenzyme Q reductase (NADH oxidase), the first part of the electron transport chain. The conclusion is based on the following observations. (a) In this and in the earlier report we determined that the relevant enzymes of the Krebs cycle are present and active. (b) Oxygen consumption by isolated mitochondria is normal when driven by succinate and alpha-glycerolphosphate. (c) Difference spectra between reduced and oxidized forms indicate that all cytochromes are present and functional. (d) In contrast, substrates such as malate, glutamate, alpha-ketoglutarate, and isocitrate which generate NADH do not stimulate oxygen consumption in mutant mitochondria. (e) A direct assay of the rotenone-sensitive NADH oxidase in Lubrol-treated mitochondria from mutant cells revealed less than one-tenth of the activity when compared with wild type mitochondria. (f) The treatment of wild type cells with rotenone, a specific inhibitor of NADH-CoQ reductase, yielded an exact phenocopy of the mutant by several criteria. This is the first report of a respiration-deficient mammalian cell mutant in tissue culture.


Antibody as an immunological probe for studying the refolding of bovine serum albumin. I. The catalysis of reoxidation of reduced bovine serum albumin by glutathione and a disulfide interchange enzyme. We have used an immunochemical approach to study the refolding of bovine serum albumin. Using antibody as a probe for return of native structure, we have been able to demonstrate the regeneration of native structure at several sites on the surface of the molecule. Using this technique, we have shown that the rate of refolding of reduced bovine serum albumin catalyzed by either glutathione or rat liver disulfide interchange enzyme is greater than the rate of air reoxidation of albumin. The half-regeneration times for albumin, however, are substantially greater than those obtained with smaller proteins that have fewer disulfide bonds. We have also demonstrated that the reoxidized monomers isolated at the end of the refolding process are immunologically identical to native monomers. In addition, the tryptophan fluorescence emission maxima were the same as that of the native monomers.


Antibody as an immunological probe for studying the refolding of bovine serum albumin. II. Evidence for the independent refolding of the domains of the molecule. Antiserum to bovine serum albumin were used as a probe for native structure to study the process of refolding of denatured bovine serum albumin. Different antigenically active fragments that represent domains and subdomains of the molecule were isolated. Restricted populations of antibody to albumin were obtained by fractionating antisera to the native intact molecule on immunoadsorbents bearing these fragments. The restricted antibody populations were then used to probe the surface of the molecule during the refolding process. Some regions of the molecule refolded more rapidly than other regions. Isolated domains of albumin also refolded to native antigenic structure demonstrating that the entire polypeptide chain was not necessary for reformation of native structure. However, there does seem to be some interdomain influence on the rate of refolding of a particular domain within the intact protein.