Beta hemolytic streptococcal endocarditis: predominance of non-group A organisms. Identification of beta hemolytic streptococci (BHS) as group A or non-group A on the basis of bacitracin sensitivity rather than Lancefield serogrouping may lead to misidentification of the pathogen. In four recent cases of endocarditis due to BHS, the pathogens initially were identified as group A; Lancefield serogrouping showed all four to be group G. All four patients presented with a syndrome characteristic of acute bacterial endocarditis. Review of all cases of endocarditis at our institution for the past five years showed none due to group A BHS. In 166 previously reported cases of endocarditis due to BHS, 68 pathogens were serogrouped: 53 belonged to groups B, C, G, or H; only five were group A. Our four cases and review of the literature demonstrate the need for Lancefield serogrouping in serious beta hemolytic streptococcal infections, rather than reliance on bacitracin sensitivity.


[Prevention and therapy of arteriosclerosis (author's transl)]. Arteriosclerosis is caused by many factors. These pathogenic factors especially over-nutrition, nicotinabusus, deficiency of muscular exercise, muscular overstrain, emotional stress and concomitant basic diseases, especially arterial hypertension, diabetes mellitus and dyslipidemia are the most important points for preventive and therapeutical action. When possible the risk factors has to be eliminated, arterial hypertension, diabetes mellitus and dyslipidemia have to be treated orderly. In the pathogenesis of arteriosclerosis and atherosclerosis are known disturbances of the lipid metabolism, the blood coagulation and the metabolism of the arterial wall cells most important. Application of anticoagulants and lipid lowering medicaments did not come up to our expectations. Experiences with animal models and a double blind study (secondary prevention of myocardial infarction) have given good reason for recommending antirheumatic or as we like to say, mesenchyme suppressive drugs.


[The concept of the subculture of the aging reconsidered (author's transl)]. This article reviews the concept of a "subculture of aging" (Rose 1965) and describes the conditions necessary for the development of a subculture. The discussion proves this concept to be ambivalent. On the one hand the term "subculture of aging" covers the acquisition of new age-appropriate roles, on the other hand it stresses segregation of old people from society as a whole. The results of some preliminary studies suggest that intensified age-homogenous interaction may indeed facilitate new patterns of behaviour among older people although they are relatively well integrated. Instead of the label "subculture" the more appropriate term of "partial culture" is proposed.


The influence of aging on the myosin type of the rabbit soleus and longissimus dorsi muscles. The influence of aging on the myosin type, and on the fibre composition of both the slowly contracting ("red") M. soleus and the fast ("white") M. longissimus dorsi was examined in the rabbit. For myosin characterization isolated myofibrils were electrophoresed on SDS-polyacrylamide gels, and the fibre pattern within the respective muscles was analyzed with an immunocytochemical method. Antisera against either fast or slow rabbit myosin were collected from guinea pigs after longterm immunization. After incubation of the paraformaldehyde-fixed muscle thin sections the fibres containing either fast or slow myosin could be distinguished from each other by indirect immunofluorescence. The soleus muscles of 1 day old rabbits were composed of 25% slow and 75% fast fibres. In young-adult (5--8 mo.) rabbits the fibres were mostly slow (over 90%), while in old age (4--7 y.) again up to 50% of the soleus fibres contained fast myosin. In contrast, in the longissimus dorsi muscle constantly around 95% of the fibres contained fast myosin. In accordance with the immunocytochemical finding of an increase of fast fibres in the aging soleus muscle, the presence of fast myosin could also be demonstrated electrophoretically. With this method, soleus myofibrils from young-adult animals were observed to contain virtually slow myosin only. No slow, but only fast myosin was identified in SDS-gels of longissimus dorsi myofibrils at all ages. These results are discussed in relation to the well known metabolic alterations occurring in the mammalian skeletal muscle during aging.


Age-dependent structural changes in human neuronal chromatin. After partial digestion with micrococcal nuclease, DNA was extracted from nuclei of cerebral cortex neurons from young (23--36 y.) and old (78--85 y.) humans. The DNA fragments were subjected to gel electrophoresis, and their base-pair content determined. The nucleosomal DNA repeat length was found to increase from 170 (+/- 18) base-pairs in the young group to 199 (+/- 8) base-pairs in the old group. This increase of 29 base-pairs appears to be confined to the linker region of the nucleosomal DNA, since the core-DNA was always found to contain approx. 140 base-pairs. In addition, the amount of nuclear DNA digested by the micrococcal nuclease was observed to vary with age: after 30 min. of incubation at 37 degrees C hydrolysis of up to 80% of the nuclear DNA in the young but only up to 60% in the old neuronal nuclei was achieved. The age-dependent increase in chromosomal DNA repeat length is a direct proof of alterations in the basic chromatin structure with aging. It cannot be decided, however, whether the change in DNA digestibility is dependent on alterations of the chromatin basic structure, its superstructure, or both.


[Possibilities and contraindications of manual therapy in geriatrics (author's transl)]. Complaints of the locomotor apparatus increase in the old age. The arising of these disturbances is discussed and their effect to the locomotor apparatus with the clinical findings are described. The clinical findings are diagnosed by the methods of the Manual Medicine. The manual therapy enables the treatment of the reflectoric expression of these disturbances. The contraindications are mentioned. The "soft Tissue Technics" and the "Technics of Mobilisation" are of great importance for the treatment of complaints of the locomotor apparatus in the old age. Examples of those technics are described.


[Cardiac surgery in patients older than 60 years (author's transl)]. If indicated cardiac surgery can be performed in geriatric patients with success. Among 525 own patients there were 63 older than 60 years (12%), especially patients with stenosis and/or incompetence of aortic or mitral valve, instable angina pectoris, and certain congenital malformations (atrial septal defect). The poor prognosis of these diseases in the elderly must be compared with the higher early mortality in surgically treated aged patients.


[Treatment of leukemia in the elderly (author's transl)]. The treatment of elderly patients, who suffer from leukemia must not be standardized. Impaired bone marrow function, cardiovascular disease and other organopathias require an individually adapted therapy. The aim of treatment should be a good quality of life and not a remission at any price. Aggressive therapy in cases of acute leukemia with little progress should be avoided in favour of symptomatic treatment. CLL are treated in the progressive state of disease. Haemolytic anaemia and recurrent infections may complicate the course of CLL. CML is not a disease of old age but when it occurs intermittent therapy with cautious dosage is preferable to a continuous therapy.


[Psychosocial aspects of institutional dying in the elderly (author's transl)]. Especially in the elderly dying is less a somatic than a psychosocial act, which results from the relationship of institution, structur, professional staff, and patient. During the psychosocial dying occurs a continual death of identity, integrity, and relationship, which has the origin in station ideologies, insufficient training of the staff, psychiatric injudiciousness, and in the fail in death-contact. During a some years lasting research-work the different connections are inquired and the dangers of an over-subscribed theory of activity are represented.


[New aspects in the treatment of elderly people with carcinoma of the urinary bladder (author's transl)]. 50% of the patients with carcinoma of the bladder are older than 70 years. During the first examination a deep muscle invasion was found in about 60%. (Stages T3/T4. This situation makes forced surgical intervention in most cases impossible (e.g. radical cystectomy and segmental resection), therefore the transuretheral resection has to be the leading therapeutical procedure. During the past 10 years 80% of the bladder cancer patients were treated by TUR most ofter followed by external irradiation. The 3-year-survival rate now shows better results than before 1968 when TUR was performed in only 10% of the cases. During the period between 1968-1974 90% of the patients with superficial bladder carcinoma were still alive after 3 years, in comparison with only 52% between 1953-1967. The figures for patients with deeply invasive bladder carcinoma now shows 18% in comparison with 12%. The 3-year-survival rate for all stages shows an increase from 24% to 44%. Discussion of these results.


[New aspects pertaining to the Surgical Treatment of genital cancer in elderly women (author's transl)]. Between 1960 and 1969, 7150 major operations have been performed in the gynecological departements of West-Berlin on female patients of 60 years of age or more. Of 6658 cases the case-history was at our disposal. In contrast to reports in literature, the largest group was that of 3111 malignant tumors (46.7%). The postoperative mortality of the entire group was 7.7%, in cases of malignant tumors 12.1%.  Discussion on surgical methods for the most frequent gynecological malignomas, and on possibilities to reduce the postoperative mortality, as well as on some aspects pertaining to the gynecological geroprophylaxis.


[Diagnosis of breast cancer by contact thermography (author's transl)]. Epidemiological studies performed in cooperation with the Austrian National Institute for Statistic show an increase of breast cancer in the age-group of women older than 50 years. Especially in this age-group screening for breast cancer is very important and successful. Mammography in this age-group is the most useful and accurate method. In the group of women aged less than 50 years and particularly less than 45 years the use of mammography for screening is limited because of its risks for the induction of breast cancer in young women. Therefore the combination of clinical examination and contact-thermography is a very promising alternative for the detection of breast cancer. In 133 cases of histologically proved breast cancer the high accuracy of this combination of clinical examination and contact-thermography in comparison with mammography is shown. Based on this good results a recommendation for a detection-scheme for breast cancer with a reduction of radiation exposure in young women by the use of contact-thermography is presented and discussed.


[Disablement and needs among the elderly of several population groups in Switzerland (author's transl)]. A preliminary communication is made about an epidemiolocial survey in Switzerland concerning the habits and problems of several groups of elderly under different life conditions. Special interest is given as well the aspects of health and competence in the activities of daily life as the question of disablement and need for personal aid.


[Guttman scales for impairment due to old age in average daily activity (author's transl)]. On the basis of 100 interviews with subjects aged over 65 Guttman scales have been calculated which make it possible to quantitate the degree of competence in two activities, namely house work and mobility.


[Assessing functional health and sociability of aged persons: the development and validation of two guttman-scales (author's transl)]. Studies to develop and validate two Guttman-scales are reported, which aim at measuring level of functional health and inclination to engage in social contacts in aged persons. A first scale analysis, based on the answers of 150 old persons, resulted in two, 12-item scales, which according to Guttman's criterion were fully satisfying. In two consecutive studies Guttman-selfratings of two other samples of old people were validated against ratings of experts, who disposed of profound knowledge of the sampled persons (a physician of a nursing home, managers of homes for the aged). A second scale analysis was performed, too. The results, especially the very significant correlations between expert ratings and selfassessments, suggest a broader application of at least the functional health scale. It is proposed to use the scales, if one aims at increasing the fit between individual needs and amount of services provided, and as instruments to control for sample characteristics, if different care programs are evaluated.


[Socialpsychological aspects of sexuality in old age (author's transl)]. In contrast to other existing studies, which mostly discuss the physical changes of human sexuality during age, this socialpsychological survey analyzes the existence and relevance of negative attitudes towards sexuality in old age and their relations to the sexual activity of older people. Though - according to the results of medicinal surveys - there is no biological limit for a satisfactory sexuality during old age, after personal interviews with 500 random sampled people the hypothesis was confirmed, that older people are often thought to be asexual. Especially older women, who in most cases have no more sexual relations, often believed sexuality in age to be inappropriate, indecent, perverse or even sinful. More than that it could be shown, that sexual inactivity of older people cannot be explained by detoriation of old age only - like illness or loss of partner -, but that the normative evaluation of sexuality is probably an important determinant also.


Bacterial allergy in allergic rhinitis and bronchial asthma. Nineteen patients suffering from allergic rhinitis and bronchial asthma were studied for bacterial allergy with Staphylococcus aureus, Kelbsiella pneumoniae and Diplococcus pneumoniae. Allergy skin tests, provocative tests and the Migratory Inhibition Factor were employed. The correlation indicates to the authors that bacterial allergy is more important than bacterial "infection" as a cause of allergic rhinitis and asthma in many instances. This is often overlooked by practicing allergists.


Fundamental basis of tissue pH measurement. The first tissue pH electrode is described and illustrated and notable features are commented on. Calibration procedure is discussed particularly concerning the need for temperature control. Experience with early examples of the tissue electrode is described and its performance with an independent pH monitoring and recording system assessed. Attention is drawn to the fact that the electrode tip is immersed in a tissue fluid of uncertain composition and that the buffer power of the fluid must be of importance with regard to the reaction of the electrode to an acid-base disturbance. Acid-base status in man is still best described by the Henderson-Hasselbalch relationship and several features of this relationship are neglected by obstetricians intent on measuring only fetal pH. The need for careful animal experiments to determine the fundamental behaviour of the tissue pH electrode is emphasised.


Practical experience in the usage of a pH electrode. A total of 57 continuous pH measurements have been carried out at the University Clinic of Gynecology in Graz since September 1976. In 41 cases diagrams were suitable for evaluation. In fetal distress the pH curves showed increasing waves synchronous with labor activity. A linear pH curve seems to preclude imminent danger to the fetus. A decline in the pH curve prior to delivery may be caused either mechanically or by fetal distress.


An evaluation of the continuous tissue pH electrode (tpH) during labor in the human fetus. The subcutaneous tissue pH (tpH) of 31 fetuses has been measured continuously during labor using the tissue electrode designed by Stamm and adapted for fetal use by ROCHE Medical and Laboratory Systems. There were seven failures, 16 reliable readings, and eight erratic and low readings. Comparison of the reliable tpH readings with scalp capillary blood pH showed a correlation coefficient of 0.64 (p less than 0.001). Comparison of the last intrapartum tissue pH value and cord arterial blood pH showed a correlation coefficient of 0.86 (p less than 0.001). Difficulty in application of the electrode to the fetal scalp appears to be the biggest problem and this is also responsible for unreliable readings. Technical improvements and a substantial increase in the number of the available electrodes and accessory instruments are necessary before this procedure becomes a routine clinical tool in the delivery suite.


Continuous recording of fetal pH during labor. 58 fetuses were monitored during labor, recording simultaneously F.H.R. U.P. and subcutaneous pH using an electrode by Stamm. The values of subcutaneous pH were correlated with 98 samples of capillary blood and 46 of umbilical vein blood. The correlation indices were 0.649 and 0.39, respectively. A selection of the 44 good quality records correlated with the corresponding 68 samples of capillary blood, gave an correlation coefficient of 0.95. The possible clinical use of this technique to monitor the fetus during labor is presently under discussion.


Clinical aspects of continuous tissular pH measurements of the newborn and the fetus. The first results of continuous tissular pH readings in 58 neonates are presented. These results are compared with blood pH values and technical problems are discussed. The positive results made us proceed to sub-partu measurements. We finally give our opinion on the new ROCHE equipment and insist that it is not yet ready for routine use, but a promising tool for clinical research.


Continuous tissue pH measurement on the fetus during delivery. Tissue pH values of 70 fetuses have been measured continuously during labor using the micro-electrode designed by Stamm. Significant correlation between continuous tissue pH and corresponding micro blood values can be shown. However, the relationship between patterns of tissue pH and capillary pH values seems not to be very constant. Therefore, the reliability of individual tissue pH measurements is still questionable.


First experience with continuous pH measurements on fetus during delivery. First experience with continuous pH measurement is reported. It was possible to apply well calibrated electrodes to the fetal scalp in 22 cases. Of these 22 cases eight must be regarded as failures because of unstable values. Of the remaining 14 measurements, seven were of good (or acceptable) quality and seven of poor quality. The size of the electrodes and the excessive depth of penetration are probable reasons for these failures. An important condition for obtaining reliable pH values is a perpendicular position of the electrode in the fetal scalp. An indicator for correct position of the electrode appears to be the irregularity in the recording of the pH curve. Experience so far indicates that the pH electrode in its present form is not suitable for routine use.


Evaluation of continuous fetal scalp pH during labor. Continuous monitoring of fetal scalp tissue pH was technically acceptable in 79.4% of 35 patients sequentially studied. Overall, the correlation coefficient (r) between the continuous pH readings and the intermittent pH values was 0.75. The accuracy improved greatly in the latter 23 patients. This was probably due to improved skills of the operators but also to a revision of the calibration technique. The correlation coefficient in these 23 patients was 0.82, and 87% of the records were accurate. The mean monitoring time was 2.39 h and eight infants were monitored throughout delivery and post natal. Although good results were obtained in this study, the method still demands technical skill in order to achieve clinically useful results.


Clinical evaluation of tissue pH monitoring during labor. Second series of 61 cases. A newly developed tissue pH electrode designed for continuous pH monitoring was evaluated in 61 fetuses during labor. The results obtained in this series confirm the reliability of this method. The correlation coefficient between tissular pH and capillary blood pH is 0.94. The correlation coefficient between the last intrapartum tissue pH and the umbilical arterial blood pH is 0.84.


Continuous fetal pH monitoring. Preliminary results. Preliminary results obtained with the new subcutaneous pH electrode confirm the reliability of this method as far as the purely technical aspects are concerned. The routine clinical use is still burdened with several unsolved problems which should be worked out as soon as possible.


Analysis of sixty consecutive cases of continuous fetal pH measurement. 60 consecutive fetal CTG-pH-registrations (cardiotocography with continuous pH registration) were analyzed in order to find the causes of the nonsuccessful tracings. Of the 60 tracings, 21 were of good quality, 16 partly successful, and 23 nonsuccessful. The monitoring for each patient had lasted for an average of 154 min with the range of 5--530 min. The quality of the pH recordings were not related to the duration of the pH observation, the maternal age and parity, or to the weight of the infant. If the electrode was applied too early (cervical orifice less than 5 cm) there was a greater risk of a nonsuccessful tracing. The drift of the electrode was less than 0.06 pH-units in 90% of the cases. The rate of nonsuccessful tracings is still too high and the difficulty in sterilization, calibration, and application of the electrode is still rather pronounced. At this moment the pH-electrode should be reserved for scientific use.


Continuous fetal tissue pH monitoring in labor with high risk pregnancies. Continuous tissue pH monitoring was performed in 61 high risk pregnancies in labor. Useful data was obtained in 48 cases, and compared with fetal scalp blood pH and umbilical artery and vein blood pH. The relationshiip of tissue pH and fetal heart rate changes was analyzed. The tissue pH changes occurred within 2--3 min of the fetal heart rate change, and conformed to the expected fetal physiology of their heart rate pattern.


The effect of fibrin deposition on the sensitivity of the continuous monitoring pH electrode and on the recorded pH value: an in vitro study. One of the problems concerning the continuous pH-monitoring technique is whether the relationship between pH measured by the electrode and pH in central and/or capillary fetal blood is constant. To test to what extent a fibrin clot deposited on the pH-electrode influenced the recorded value and the sensitivity of the electrode, the following in vitro study was performed. Fibrin was deposited on the pH-electrode by means of thromboplastin and fibrinogen, or by thromboplastin and whole blood. The deposition of a clot was verified by inspection of the electrode in a microscope. The time for stabilization of the recorded pH-value and the recorded pH-value was measured in standard calibration solutions before and after deposition of the fibrin clot and after decomposition of the fibrin clot by plasmin. FDP was measured in the decomposition solution. From the study it was obvious that the stabilization time of the electrode was considerably influenced by deposition of an "unphysiological" fibrin clot, less so if the clot was deposited by means of whole blood. The recorded pH-value was not influenced.


Experience with continuous tissue pH electrode in the human fetus during labor. In our experience the method was reliable in about 80% of the cases. The unsatisfactory reliability in the remaining 20% was partly due to the electrode instability. This problem was solved by using the new type of electrode. It proved suitable for getting stable tracings for longer periods in all the cases we examined. We are unable to give an answer to the other problem regarding the absence of correlation in some cases between the tissue and the blood pH values, got through blood samples taken according to the Saling technique. In these cases, however, the tracing can be used for the recording of pH variations regardless of its absolute values. From our experience the main clinical indication of the method has been found to be in the variable decelerations with normal values of the blood pH. Using this method the frequent repetition of separate blood samplings would be avoided.


Clinical experience with fetal continuous monitoring by pH electrode. Experiences about continuous measurement with the pH electrode are reported. Difficulties with the use of the electrode noticed when sterilizing, calibrating, and fixing it on the fetal head are discussed separately.


Continuous measurement of tissue pH in the human fetus. The ROCHE pH electrode was tested clinically in 20 fetuses and one nonviable newborn with severe malformations. The proper fixation of the electrode in the fetal scalp is difficult. In the physiologic pH range a good correlation between capillary blood pH and tissue pH exists. The situation is less clear for the acidotic range.


Influence of sterilization and temperature changes on the in vitro characteristics of the pH electrode. 1. When chemical sterilization (with Korsolin, Cidex) is performed, a minimal duration of sterilization of 3 h is required for asepsis. 2. Duration of Korsolin sterilization up to 3 h does not influence the in vitro characteristics of the pH-electrode. 3. Sterilization with formaldehyde (24 h, 40 degrees C) is safe, but needs another 24 h restabilization time of the electrode. 4. The temperature dependence of the pH-electrode is minimal. With increasing temperature a slight decrease of pH measurement can be found (-0.02 pH units from 20--40 degrees C).


The relationship between tissue and arterial pH in hypercarbic rabbits. Continuous tissue pH and intermittent central arterial pH were measured in six rabbits during 10-min exposures to a mixture of 10% CO2 and 90% O2. In control and recovery situations tissue pH was more acidic than arterial pH by a mean value of 0.07 pH units. During periods of rapidly increasing pCO2, the steady state relationship was inverted with tissue pH being more alkaline than arterial pH. After a second exposure to CO2, mean tissue pH values did not recover to baseline. It is concluded that in the rabbit during acute hypercarbia, the relationship of tissue to central pH is variable. The possible implications of these results in human fetuses during labor are discussed.


A study of the pH monitor in cats.

A study of the pH monitor in cats. A study of the accuracy of the tpH electrode was performed in 10 cats. Hypoxia and acidosis were produced by ventilatory means and bicarbonate infusions were used to elevate the pH . Tissue pH correlated well with arterial (r = 0.9) within the physiological rates of change of pH. Prolonged tissue acidosis lead to eventual death. The correct and stable mechanical fixation of the pH electrode is a critical factor in assuring accurate results. The ROCHE tpH electrode accurately reflects arterial pH, and a correlation coefficient of at least 0.85 should be attainable in the human fetus.


In vivo performance of the pH tissue electrode during acute acid-base changes in the dog. The ROCHE tissue pH-electrode was tested in acute animal experiments under various metabolic conditions. In anesthetized dogs an electrode was placed in the subcutaneous tissue of the scalp, neck, and axilla. Simultaneous with the continuous measurement of tissue pH, the overall acid-base balance of the animal was measured discontinuously at 5-min intervals by means of indwelling arterial and venous catheters. Various forms of acidosis and hypoxia were produced in the experimental animal and the sensitivity of the electrodes in relation to their site was tested. A high correlation between the continuously measured tissue pH values and the corresponding arterial and venous actual blood pH values were found in the physiologic pH range. With acute changes of acid-base balance no correlation could be found in the acidotic range.


Continuous pH-measurement on anesthetized patients. 43 patients under general anesthesia were monitored by continuous tissue pH measurement and intermittent micro-blood-samples. The values obtained for the capillary pH and tissue pH were correlated. A highly significant correlation was found between tissular pH and capillary pH. Different methods of general anesthesia apparently do not influence the continuous tissue pH measurement.


Application technique of tissue pH electrode on human fetuses. The application technique of a newly developed tissue pH electrode designed for continuous pH monitoring is described. Some parameters are described as criteria of technically accurate records.


Clinical experience in the application of the tissue pH electrode. Only after placing the pH electrode through an incision could its point enter deep enough into the tissue. The storage of the pH electrode in a reference buffer considerably improved the stability of the electrode. The ECG electrode is at present the weakest link in the realization of continuous pH measurements. Improvements should be effected mainly with the ECG electrode, the electrode cable and the application instruments.


A new application tool for the ROCHE pH electrode. Using this tool the application of the pH electrode is nearly as easy as the attachment of a common ECG-scalp-electrode [3]. Visual control and lithotomy position is not necessary any more. The different tools are adapted to each other so that incision place and the tip of the pH electrode are automatically centered perpendicularly. The first results by using this tool promise to be successful. Time of application is short, the maintenance of the pH electrode is good and the application for the patient no longer disagreeable.


New instrument developments for fetal pH monitoring. Recent technical improvements in the ROCHE fetal pH monitor have simplified its use and increased the proportion of useful and accurate recordings. These improvements include electronic changes to reduce static electricity, a new spiral electrode which stabilizes the pH electrode in the fetal scalp, improved incision tools, simplified applicator tools, and a total system for reducing pH electrode handling during calibration.


Improvements in the results with the continuous pH electrode due to technical progress: a comparison between two series of cases. Our experience is based on 139 attempts of tissular pH electrode applications in the fetal scalp during labor and includes 119 records comprising 86 tracings of good quality and 33 tracings of bad quality. Technical progress in the electrode and application material allowed an increase in the good quality tracings rate (from 54.5--66%) and a decrease of the complications, particularly the numbers of broken electrodes (from 18 to 2.4%).


Principle and characteristics of the ROCHE tissue pH electrode. A miniaturized probe for continuous measurement of tissue (interstitial fluid) pH is described. The electrode design follows closely the concept described by Stamm and co-workers. The pH sensitive tip has a length of 1 mm and is made of a Li-Ba-Si glass of low electrical resistivity. The reference electrode with a capillary type liquid junction is an integrated part of the electrode assembly. This paper summarizes the important design features of the electrode and describes its in vitro characteristics.


Multicenter clinical trials of fetal pH monitoring in the U.S.A. A multicenter collaborative study of the safety and clinical efficacy of the ROCHE fetal pH monitor has been started. A common protocol and reporting forms are used. As of April, 1978, 115 case reports have been received describing results with the latest hardware. The tissue pH monitor can be relied upon to monitor pH if the values are in the normal range. Comparison of tissue pH (tpH) with 146 capillary pH samples revealed 98% accuracy of tpH in classifying normal (greater than or equal to 7.20), but false tpH acidosis occurred in 6% of normals. Therefore, acidotic tpH values must be verified. Although six infants had some degree of trauma (small lacerations), there were no serious or permanent injuries.


Effects of pH on alpha 1-antitrypsin activity in premature infants with idiopathic respiratory distress syndrome. The inhibitory and specific activities of serum alpha 1-antitrypsin (1-AT) at pH levels of 8.20, 7.45, 7.35, 7.25 and 7.15, were studied in 66 neonates with idiopathic respiratory distress syndrome (IRDS) and in 82 healthy neonates. In vivo pH values were also correlated in both groups with the specific and inhibitory activities of 1-AT. Comparisons between healthy neonates and neonates with IRDS showed that in the latter, the inhibitory and specific activities of 1-AT were lower, with every pH assayed, together with twice a percentual decrease from pH 8.20 to pH 7.15 in both activities. No statistical correlation was established in the in vivo pH of infants in both groups with the inhibitory and specific activities of 1-AT. Results suggest two alternatives. The first one related to a molecular structure different from 1-AT in infants with IRDS, and the second one regarding changes in 1-AT produced by partial breakdown or modified by hypoxia, acidosis or both.


[Myocardiopathies during pregnancy. Possible role of beta-mimetics]. The authors report two cases of severe non-obstructive cardiomyopathy presenting at the end of pregnancy in young women treated for long periods and at high dosage with beta 2-adrenoceptor stimulants, prescribed for threatened abortion or premature labour. The circumstances of the presentation, the clinical prescribed for threatened abortion or premature labour. The circumstances of the presentation, the clinical picture and the course towards complete recovery suggest that this might be a pathological process different from "cardiomyopathy of pregnancy" and be an "adrenergic myocarditis" induced or made worse by these drugs. The authors deduce some simple preventive measures.


Telemetered EEG-EOG during psychotic behaviors of schizophrenia. In an effort to establish correlations between abnormal behaviors characteristic of schizophrenia and simultaneous cerebral electrical activity, EEGs and electro-oculograms (EOGs) were continuously recorded for 2 to 24 hours by radiotelemetry from 40 patients with schizophrenia and 12 normal control subjects. Trained observers recorded specific behavior patterns permitting visual and computer analysis of EEG during hallucinations, stereotypy, catatonia, psychomotor blocking, and other characteristic manifestations of schizophrenia. Electroencephalographic abnormalities consisting of focal slow or spike activity over either temporal region were found in nearly half of the patients so recorded. In contrast to the EEG during ictal episodes of epilepsy, the abnormal wave forms of schizophrenic patients seldom coincided with episodes of blocking, stereotypy, or other abnormal behaviors. Increased extraocular activity or blinking were recorded in a majority of patients, but were not consistently associated with the abnormal behavior or perceptual events.


Alterations in cerebral laterality during acute psychotic illness. A dichotic listening test was used to assess cerebral laterality in 26 right-handed patients with schizophrenic, schizoaffective, or primary major depressive illness and in 23 controls. Clinical state was assessed by twice-daily nurses' ratings and patient self-ratings. Ratings of psychotic thought and behavior were lower during the week of highest laterality than during the week of lowest laterality (P less than .01). Similarly, when most improved, patients had higher laterality than when most ill (P less than .01). Changes in laterality were not specific to diagnostic group, were not present in control subjects, could not be related to direct drug effects, and were independent of changes in accuracy of performance. There were large, stable, interindividual differences in degree of lateralization, but no differences between patients and controls. These results are consistent with the hypothesis that in acute psychotic illness there is a breakdown in the interhemisphere inhibition that normally mediates cerebral laterality.


Bodily reactions to people and events in schizophrenics. Schizophrenic patients were tested on psychophysiologic measures within their homes and in the laboratory. Sweat gland activity and heart rate changes when patients encounter novel situations such as life events, together with the home atmosphere generated by a critical or overinvolved relative, confirmed objectively the importance of previous social measures of these factors in determining relapse. Drug effects were also found to be modified by these social factors. Environmental and socially induced changes found in the home were undetectable in the laboratory situation.


Low-dose neuroleptic regimens in the treatment of borderline patients. The use of pharmacologic agents in treating patients described as borderline generally has been accorded an insignificant, or at best, minor, role. We discuss this observation and review the literature that has dealt with this aspect of treatment. Diagnostic criteria are presented that appear to define a specific population of borderline patients who have been observed to be responsive to low doses of neuroleptic drugs. Five case histories of patients with conditions diagnosed and treated in this manner are presented, followed by a discussion of the implications of this approach in terms of clarifying the nosologic issues that have arisen around the "borderline" concept.


[Effect of food deprivation on various parameters of rumen juice and blood of sheep]. In order to find out how short-time denutrition changes the concentration of some substances in the rumen fluid and the blood, tests with full-grown sheep were carried out. Fodder was withheld from sheep with inserted Jarrett fistulae for 48 hours after normal feeding. After 48 hours the animals were given concentrated fodder, after 52 hours exclusively hay. From the 72nd hour onwards the animals were provided with fodder as usual. Samples of the rumen fluid and blood samples were taken at the beginning of the test, after the last normal feeding and then in the 24th, 32nd, 48th, 52nd, 56th, 72nd and 96th hour. We could find out that, during the 48-hour denutrition, the pH-value of the rumen fluid turned alkaline and the concentrations of ammonia, volatile fatty acids and lactic acid decreased. The protein metabolism underwent a rapid change in the organism. The protein content of the blood plasma decreased, above all the albumin content, as well as the concentration of glycoproteins and volatile amino acids. Among the various amino acids, the concentration of glycine increased highly, that of alanine and valine just slightly. The concentration of most amino acids decreased or--of some of them remained the same. Among the paramters that are characteristic of lipid and carbohydrate metabolism, the total content of lipids and cholesterin decreased, and so did the concentration of blood sugar, lactic acid and pyruvic acid in the blood plasma. The results indicate that short-time denutrition has a considerable influence on the rumen fermentation and the intermediary metabolism of ruminants. The quickly arising lack of energy of ruminants slows down the protein synthesis and increases the glyconeogenesis from amino acids. The tissue is supplied with energy by the mobilisation of lipids.


Clinical and technical aspects of hemofiltration. The removal of uremic substances in hemofiltration, in contrast to hemodialysis, is achieved by means of a convective transport across membranes of high porosity. Since 1974, more than 30 patients with chronic renal insufficiency have been treated with regular hemofiltration three times weekly for four to five hours each. After completing a pilot study, a controlled study to compare hemodialysis and hemofiltration was initiated during January,  1978. A normalization of blood pressure in patients with severe hypertension, and remarkable stability of the circulatory system, even after dehydration in patients who had hypotension in spite of fluid overload, could be demonstrated. Hemofiltration is preferred, especially in older patients with cardiovascular or cerebrovascular problems, because of its lower frequency of hypotensive episodes compared to dialysis. An important aim--the miniaturization of the artificial kidney--has not yet been achieved, however, because of the necessity for an extensive monitoring system for the exact proportioning of the sterile substitution fluid. First results in the application of a fluid regeneration system consising of a charcoal cartridge and a bioelectric cell, for degradation of urea, are presented.


Drug intervention in the management of aggression and explosive behaviour. Disturbed patients provoke fear in those around them. The disturbance that I wish to discuss is the disturbance of the acute psychotic decompensation. I do not wish to deal with the drug intervention used in the management of people prone to aggressive outbursts as a chronic phenomenon. Rather, I wish to deal with those people who are in constant danger of acting on distorted perceptions or delusional ideas and may impulsively commit homicide or suicide, or mutilate themselves despite restraint. People in this psychotic state experience severe discomfort, and optimal treatment begins with the most rapid alleviation of symptoms possible.


Colonic and oesophageal disturbance in a patient with multiple endocrine neoplasia, type 2b. We report the illness of a 32-year-old male with the syndrome of multiple endocrine neoplasia, type 2b, which comprises medullary carcinoma of the thyroid, phaeochromocytoma, oral mucosal neuromas and skeletal deformity. The patient also had evidence of ganglioneuromatosis of the alimentary tract, a finding which is held to be responsible for constipation and a number of the other gastrointestinal manifestations of the MEN, 2b. Dysphagia, which has not been previously reported in the syndrome was also present. Investigations of the oesophagus which included endoscopy, radiology and a motility study support the diagnosis of achalasia as the cause of the dysphagia.


Keratoconjunctivitis sicca in dogs associated with sulphonamide administration. This report describes the clinical characteristics of 14 cases of sulphonamide-induced keratoconjuctivitis sicca in dogs. Two drugs, sulphadiazine and salicylazosulphapyridine, were associated with the disease. It is recommended that Schirmer Tear Test be performed regularly when sulphonamides are administered long term to dogs. The hypothesis that a nitrogen-containing pyridine ring is responsible for the lacrimotoxic effect is advanced.


Loose associations: straight and crooked thinking and the group of schizophrenias. In this paper I hope to provide the basis for a discussion on logic, logical thinking and creativity in medicine, particularly in psychiatry. By way of illustration we will examine schizophrenia with particular reference to biochemistry, but the discussion will focus on semantic issues and philosophical concepts. These issues are of fundamental importance in the development of psychiatry both as art and craft. Thus the paper deals both with the study of madness and madness itself. The title of the address illustrates some forms of communication which occur in the schizophrenias. It includes references to other ideas, to formal thought disorder and illogical thinking, private puns, vague links, and pointers to other sources and authors, namely, Bleuler (1950), Ernest Jones (1959) and Thouless (1974). These are all contained in a loose framework, wherein reference is made to a key anomaly always found in, but by no means unique to schizophrenia.


A comparative approach to protein- and ligand-dependence of the Root effect for fish haemoglobins. Ligand-binding equilibria, kinetics and (13)C n.m.r. spectra of bound (13)CO, of the haemoglobins from two fishes that are very distant on the evolutionary scale, i.e. the fourth haemoglobin component from Salmo irideus and the single component from Osteoglossum bicirrhosum, were studied. The C-terminal sequence was also determined for the haemoglobin from Osteoglossum. The results show that (i) the C-terminal residues of both chains are not directly responsible for the characteristic heterotropic effect known as Root effect, since for both fish haemoglobins these residues are identical with those of human haemoglobins. (ii) In all haemoglobins characterized by the Root effect a dependence of the (13)CO n.m.r. resonances on pH is observed. However, the extent of the shift(s) depends on the particular protein, and is probably the result of a combination of both tertiary and quaternary conformational changes. (iii) Both haemoglobins from trout and Osteoglossum manifest a functional heterogeneity between the two types of chains in the tetramer, which increases with proton activity. For CO, the effect is very small for trout haemoglobin IV, and very marked for Osteoglossum haemoglobin; for O(2) strongly heterogeneous binding curves were obtained at approx. pH6.2 with both haemoglobins. (iv) Estimations of the relative values of the affinity constants for the alpha and beta chains in the tetramer were obtained for both haemoglobins from (13)CO n.m.r. spectra at low fractional saturation. On the basis of these findings the molecular mechanism underlying the Root effect is discussed.


Histidine residues of zinc ligands in beta-lactamase II. 1. The Zn(II)-requiring beta-lactamase from Bacillus cereus 569/H/9, which has two zinc-binding sites, was examined by 270 MHz 1H n.m.r. spectroscopy. Resonances were assigned to five histidine residues. 2. Resonances attributed to three of the histidine residues in the apoenzyme shift on the addition of one equivalent of Zn(II). 3. Although these three histidine residues are free to titrate in the apoenzyme, none of them titrates over the pH range 6.0--9.0 in the mono-zinc enzyme. 4. The ability of the C-2 protons of these three histidine residues to exchange with solvent (2H2O) is markedly decreased on Zn(II) binding. 5. It is proposed that these three histidine residues act as zinc ligands at the tighter zinc-binding site. 6. Resonances attributed to a fourth histidine residue shift on addition of further zinc to the mono-zinc enzyme. It is proposed that this histidine residue acts as a Zn(II) ligand at the second zinc-binding site.


Interaction of glutathione analogues with Hydra attenuata gamma-glutamyltransferase. gamma-Glutamyltransferase activity was studied in extracts of the cnidarian Hydra attenuata. The binding of gamma-glutamyl peptide analogues to the enzyme was studied by observing their effects on heat denaturation and their inhibition of p-nitroaniline release from gamma-glutamyl p-nitroanilide. Neither position-1 analogues, in which the gamma-glutamyl moiety was changed to a beta-aspartyl (beta-Asp-Abu-Gly) or an alpha-glutamyl (Glu-Abu-Gly) linkage, nor glutamate protected the enzyme against inactivation at 58 degrees C. GSH (reduced glutathione), gamma-Glu-Abu-Gly and gamma-Glu-Met on the other hand did prevent heat denaturation. GSH and analogues of GSH were competitive inhibitors of p-nitroaniline release, but those analogues in which glycine was replaced by 2-aminoisobutyrate, phenylalanine, leucine or tyrosine had Ki values that were approximately five times those of analogues with the cysteine residue replaced.


Microbial metabolism of amino alcohols. Purification and properties of coenzyme B12-dependent ethanolamine ammonia-lyase of Escherichia coli. 1. The 120-fold purification of ethanolamine ammonia-lyase from Escherichia coli extracts, to apparent homogeneity, is described. Ethanolamine, dithiothreitol, glycerol and KCl protected the apoenzyme from inactivation. 2. At the optimum pH7.5, K(m) values for ethanolamine and coenzyme B(12) were 44mum and 0.42mum respectively. The K(m) for ethanolamine was markedly affected by pH, transitions occurring at pH7.0 and 8.35. 3. The enzyme was specific for ethanolamine as substrate, none of the 18 analogues tested being active. l-2-Aminopropan-l-ol (K(i) 0.86mum), dl-1-aminopropan-2-ol (K(i) 2.2mum) and dl-1,3-diaminopropan-2-ol (K(i) 88.0mum) inhibited competitively. 4. Enzyme activity was inhibited, irreversibly and non-competitively, by the coenzyme analogues methylcobalamin (K(i) 1.4nm), hydroxocobalamin (K(i) 2.1nm) and cyanocobalamin (K(i) 4.8nm). 5. Iodoacetamide inhibited in the absence of ethanolamine, but only slightly in its presence. p-Hydroxymercuribenzoate inhibited markedly even in the presence of ethanolamine. Dithiothreitol and 2-mercaptoethanol (less effectively) restored activity to the enzyme dialysed against buffer containing ethanolamine. 6. Although K(+) ions stabilized the enzyme during dialysis or storage, they were not necessary for activity. 7. Gel filtration showed the enzyme to be of high molecular weight, ultracentrifugal studies giving s(20,w) of 16.4 and an estimated mol.wt. 560400. The isoelectric point for the apoenzyme was approx. pH5.0. inhibited enzyme activity at concentrations above 1m (95% inhibition at 3m) and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated protein subunits of mol.wt. 61400. 8. Immunological studies showed that the E.coli enzyme was closely related to those of other enterobacteria, but only distantly to that of Clostridium sp. A double precipitin band suggested that the apoenzyme may be made up of two protein components.


Enhancement of the viscosity of mucin by serum albumin. The interaction of serum albumin with a model epithelial mucin from pig stomach was explored by rotary viscometry. During 30 min of incubation of human serum albumin(20mg/ml) and pig gastric mucin (8mg/ml) in iso-osmotic buffers at 37 degrees C, the solution became markedly viscous. Viscosity enhancement was proportional to albumin concentration (2-40mg/ml), was most pronounced under conditions of low shear rate (less than 45S-1), and was considerably greater than the additive or multiplicative viscosity values calculated from albumin or mucin solutions measured separately. The viscous mucin-albumin complex was destroyed by high shear rates (greater than 90S-1), but slowly re-formed under zero shear conditions. Elevation of pH (7 to 9), ionic strength (0.1 to 1.0), and addition of disodium EDTA (5mM) did not cause marked or specific alterations in the viscosity of the mixture, suggesting that electrostatic interactions probably do not stabilize mucin-albumin complexes. Urea (7M) and heating (35 to 55 degrees C) caused a major increase in the viscosity of mucin and mucin-albumin mixtures, suggesting that rupture of hydrogen bonds, unfolding and partial denaturation of mucin promotes greater intertangling (possibly hydrophobic interactions) between mucin and albumin molecules. The implications of mucin-albumin interaction in diseases associated with mucus obstruction are briefly discussed.


Subcellular localization of gamma-glutamyltransferase in calf thymocytes. The subcellular localization of gamma-glutamyltransferase in calf thymocytes was investigated and compared with that of alkaline phosphodiesterase I, alkaline nitrophenyl phosphatase, succinate-tetrazolium oxidoreductase (succinate-INT reductase) and lactate dehydrogenase after two different methods of cell disruption and differential centrifugation. Most of the activity was recovered in the crude membrane fractions (43.0%), but significant amounts co-pelleted with the large-granule (mitochondria) fractions (31%). The specific activity of the gamma-glutamyltransferase in the purified plasma membrane was 30-50 times that of the enzyme in the cell homogenate and had a similar subcellular distribution to the plasma-membrane markers, alkaline phosphodiesterase I and alkaline nitrophenyl phosphatase. It was concluded that gamma-glutamyltransferase was primary a plasma-membrane-bound enzyme, and that its location in other subcellular fractions was probably due to their contamination with plasma-membrane vesicles.


Purification and properties of beta-N-acetylhexosaminidase from the mollusc Helicella ericetorum Müller. 1. A beta-N-acetylhexosaminidase was purified 330-fold from the digestive gland of the terrestrial mollusc Helicella ericetorum Müller. 2. Its pH optimum is 4.5 for both beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase activities in two buffer solutions; it is fully stable at 37 degrees C for 2h in the pH range 3.8--4.6 and shows one isoelectric point (pH 4.83). 3. The estimated mol.wt. is between 120,000 and 145,000. 4. The enzyme shows an endo-beta-N-acetylhexosaminidase activity on natural substrates such as ovalbumin, ovomucoid, chondroitin 4-sulphate, chitin and hyaluronic acid. 5. Two forms of the enzyme were separated by preparative polyacrylamide-gel electrophoresis. 6. Km and Vmax. for p-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside and p-nitrophenyl 2-acetamide-2-deoxy-beta-D-galactopyranoside are 0.43 mM, 30.1 micronmol of p-nitrophenol/min per mg and 0.19 mM, 8.6 micronmol of p-nitrophenol/min per mg respectively. 7. It is inhibited by Hg2+, Fe3+, acetate, some lactones, N-acetylgalactosamine, N-acetylglucosamine and mannose. 8. Mixed-substrates analysis and Ki values for competitive inhibitors indicated that beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase activities are catalysed by the enzyme at the same active site.


Rabbit liver acetyl-CoA synthetase.

Rabbit liver acetyl-CoA synthetase. Acetyl-CoA synthetase (EC 6.2.1.1) was assayed in subcellular fractions of rabbit liver homogenates. The activity was located almost exclusively in the cytosol. There was no decrease in activity when butyrate or propionate (each at 5--20 mM) were added to the assay medium.


The structure of C-polysaccharide from the walls of Streptococcus pneumoniae. The well-known immologically active component of pneumococci, C-polysaccharide, is a teichoic acid that can be isolated from the cell walls and purified by Sephadex and ion-exchange chromatography. Further details of the structure of C-teichoic acid were established by chemical degradation, including hydrolysis in acid and alkali, treatment with HF, periodate oxidation and methylation. In addition, the use of 13C n.m.r. has confirmed some of these structural features and resulted in a proposal for the order of substituents, the location of positions of substitution and the configuration of anomeric centres in the repeating unit of the polymer.


alpha-Galactosidases II, III and IV from seeds of Trifolium repens. Purification, physicochemical properties and mode of galactomannan hydrolysis in vitro. Five alpha-galactosidases (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) were identified by chromatography and by their different electrophoretic mobilities, in the germinated seeds of Trifolium repens (white clover). alpha-Galactosidases II, III and IV were purified to homogeneity, with increases in specific activity of approx. 4600-, 4900- and 2800-fold respectively. The enzymes were purified by a procedure that included (NH4)2SO4 precipitation, hydroxyapatite, Sephadex G-75 and DEAE-cellulose chromatography, and preparative polyacrylamide-gel disc electrophoresis. The purified enzymes showed a single protein band, corresponding to the alpha-galactosidase activity, when examined by polyacrylamide-gel electrophoresis. The pH optimum was determined with o-nitrophenyl alpha-D-galactoside and the galactomannan of T. repens To as substrate. All three enzymes are highly thermolabile. Hydrolysis of oligosaccharides and galactomannans was examined, including two galactomannans from the germinated seed of T. repens (T24 and T36). By sodium dodecyl sulphate/polyacrylamide-gel electrophoresis the mol.wts. of the multiple forms of enzyme were found to be identical (41 000).


Purification and properties of beta-mannanases I and II from the germinated seeds of Trifolium repens. Mode of galactomannan degradation in vitro. Two beta-mannanases (beta-mannosidases, EC 3.2.1.25) purified from the germinated seeds of Trifolium repens by a procedure that included chromatography on hydroxyapatite, gel filtration on acrylamide/agarose (Ultragel 5/4) and preparative polyacrylamide-gel-electrophoresis. The final purification step completely resolved two beta-mannanases with distinct specificities, which were termed beta-mannanase I and beta-mannanase II. beta-Mannanase I was purified 1400-fold and beta-mannanase II 1000-fold. The purified enzymes showed a single protein band when examined by polyacrylamide-gel disc electrophoresis. beta-Mannanase I, apparent mol.wt. 43 000, accounted for 49% of the total activity recovered from the final step of purification. beta-Mannanase II, apparent mol.wt. 38 000, accounted for the remaining 51% of activity. Molecular-weight determinations were by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and by the electrophoretic method of Hendrick & Smith [(1968) Arch. Biochem. Biophys. 126, 155-164]. The substrate specificities of both enzymes were examined with the galactomannans of T. repens and of Medicago sativa, as well as with manno-oligosaccharides. The pH optimum was between pH 5.1 and 5.6 for both enzymes.


Subunit ratios of separated hybrid hexamers of Neurospora NADP-specific glutamate dehydrogenase containing complementing mutationally modified monomers. The am1 and am3 mutational variants of the Neurospora crassa NADP-specific glutamate dehydrogenase show complementation activity in hybrid hexamers. A freeze-thaw hybridization method was used to construct hybrids from purified enzymes and the products were separated into species of different monomer ratio by affinity chromatography. Hexamers with am1:am3 ratios of 1:5, 2:4, 3:3, 4:2 and 5:1 were all recovered as resolved or partially resolved peaks in quantities approximating to a binomial distribution. Reassociation of monomers during the hybridization process was random, except for some differential loss of am3 protein by precipitation and an apparent absence of reassociated am1 homohexamers. Complementation activity was shown by hybrids of all five monomer ratios, owing to activation of am3 monomers by conformational constraints arising from the intrinsically inactive am1 monomers. The activating effect of such constraints was greatest in hexamers containing only a single am1 monomer and least in the 5 am1:1am3 species. When fully activated by L-glutamate all am3 monomers were equivalent in intrinsic catalytic activity, irrespective of the number of am1 monomers per hexamer.


pH-jump studies at subzero temperatures on an intermediate in the reaction of xanthine oxidase with xanthine. Xanthine oxidase is stable and active in aqueous dimethyl sulphoxide solutions of up to at least 57% (w/w). Simple techniques are described for mixing the enzyme in this solvent at--82 degrees C, with its substrate, xanthine. When working at high pH values under such conditions, no reaction occurred, as judged by the absence of e.p.r. signals. On warming to--60 degrees C, for 10 min, however, the Very Rapid molybdenum(V) e.p.r. signal was obtained. This signal did not change on decreasing the pH, while maintaining the sample in liquid nitrate reductase, caused its molybdenum(V) e.p.r. signal to change from the high-pH to the low-pH form. These findings are not compatible with the conclusions of Edmondson, Ballou, Van Heuvelen, Palmer & Massey [J. Biol. Chem. (1973) 248, 6135-6144], that the Very Rapid signal is in prototropic equilibrium with the Rapid signal, and should be important in understanding the mechanism of action of the enzyme. They emphasize the unique nature of the intermediate represented by the Very Rapid e.p.r. signal. The possible value of the pK for loss of an exchangeable proton from the Rapid signal is discussed.


Synthesis of chloromethyl ketone derivatives of fatty acids. Their use as specific inhibitors of acetoacetyl-coenzyme A thiolase, cholesterol biosynthesis and fatty acid synthesis. A general route for the synthesis of chloromethyl ketone derivatives of fatty acids is described. 5-Chloro-4-oxopentanoic acid, 7-chloro-6-oxoheptanoic acid, 9-chloro-8-oxononanoic acid and 11-chloro-10-oxoundecanoic acid were synthesized by this method and tested as covalent inhibitors of pig heart acetoacetyl-CoA thiolase. The K1 decreased by approx. 20-fold for each pair of methylenes added to the chain length, showing that the initial stage in inhibitor binding occurs at a non-polar region of the protein. This region is probably located at the enzyme active site, since inhibition was prevented by acetoacetyl-CoA or acetyl-CoA but not by CoA. The site of modification by chloromethyl ketone derivatives of fatty acids is restricted to a thiol group, since inactivation of the enzyme was prevented by reversible thiomethylation of the active-site thiol. In contrast, an amino-directed reagent, citraconic anhydride, still inactivated the enzyme, even when the active-site thiol was protected. Evidence that the enzyme thiol was particularly reactive came from studies on the pH-dependence of the alkylation reaction and thiol-competition experiments. Inhibition of the enzyme proceeded suprisingly well at acidic pH values and a 10(5) molar excess of external thiol over active-site thiol was required to prevent inhibition by 0.3 mM-9-chloro-8-oxononanoic acid. In addition to inhibiting isolated acetoacetyl-CoA thiolase, in hepatocytes the chloromethyl ketone derivatives of fatty acids also inhibited chloresterol synthesis, which uses this enzyme as an early step in the biosynthetic pathway. In isolated cells, the chloromethyl ketone derivatives of fatty acids were considerably less specific in their inhibitory action compared with 3-acetylenic derivatives of fatty acids, which act as suicide inhibitors of acetoacetyl-CoA thiolase. However, 9-chloro-8-oxononanoic acid was also an effective inhibitor of both hepatic cholesterol and fatty acid synthesis in mice in vivo, whereas the acetylenic fatty acid derivative, dec-3-ynoic acid, was completely ineffective. The effective inhibitory  dose of 9-chloro-8-oxononanoic acid (2.5-5 mg/kg) was substantially lower than the estimated LD50 for the inhibitor (100 mg/kg).


Ovalbumin digestion by human pepsins 1, 3 and 5. 1. Of the three major human pepsins, pepsin 1 has greater proteolytic activity towards ovalbumin than has pepsin 3. Pepsin 5 has low activity towards this substrate. 2. Proteolytic pH-activity curves show only on pH maximum, about pH 1.4 for pepsin 1, pH 1.4--1.5 for pepsin 3 and pH 1.2--1.4 for pepsin 5. The curve for pepsin 3 has a shoulder between pH 2.4 and 3.4. 3. The rate of digestion of ovalbumin by pepsin 1 is approximately three times slower than are those of bovine haemoglobin or human globin. 4. The results suggest that there may be a physiological advantage in having more than one pepsin.


Participation of cysteine and cystine in inactivation of tyrosine aminotransferase in rat liver homogenates. 1. Inactivation of tyrosine aminotransferase was studied in rat liver homogenates. Under an O2 atmosphere with cysteine added, inactivation was rapid after a lag period of approx. 1h, whereas a N2 atmosphere extended the lag period to approx. 3h. 2. Replacement of cysteine with cystine resulted in rapid inactivation both aerobically and anaerobically. 3. Removal of the particulate fraction by centrifuging rat liver homogenates at 13,000g for 9min resulted in an aerobic lag period of 0.5h in the presence of cystine and approx. 3h in the presence of cysteine. 4. It is proposed that the stimulatory effect of cysteine on tyrosine aminotransferase inactivation occurs largely as a result of oxidation to cystine, which appears to be a more directly effective agent.


The regulation of extramitochondrial free calcium ion concentration by rat liver mitochondria. The mechanism whereby rat liver mitochondria regulate the extramitochondrial concentration of free Ca(2+) was investigated. At 30 degrees C and pH7.0, mitochondria can maintain a steady-state pCa(2+) (0) (the negative logarithm of the free extramitochondrial Ca(2+) concentration) of 6.1 (0.8mum). This represents a true steady state, as slight displacements in pCa(2+) (0) away from 6.1 result in net Ca(2+) uptake or efflux in order to restore pCa(2+) (0) to its original value. In the absence of added permeant weak acid, the steady-state pCa(2+) (0) is virtually independent of the Ca(2+) accumulated in the matrix until 60nmol of Ca(2+)/mg of protein has been taken up. The steady-state pCa(2+) (0) is also independent of the membrane potential, as long as the latter parameter is above a critical value. When the membrane potential is below this value, pCa(2+) (0) is variable and appears to be governed by thermodynamic equilibration of Ca(2+) across a Ca(2+) uniport. Permeant weak acids increase, and N-ethylmaleimide decreases, the capacity of mitochondria to buffer pCa(2+) (0) in the region of 6 (1mum-free Ca(2+)) while accumulating Ca(2+). Permeant acids delay the build-up of the transmembrane pH gradient as Ca(2+) is accumulated, and consequently delay the fall in membrane potential to values insufficient to maintain a pCa(2+) (0) of 6. The steady-state pCa(2+) (0) is affected by temperature, incubation pH and Mg(2+). The activity of the Ca(2+) uniport, rather than that of the respiratory chain, is rate-limiting when pCa(2+) (0) is greater than 5.3 (free Ca(2+) less than 5mum). When the Ca(2+) electrochemical gradient is in excess, the activity of the uniport decreases by 2-fold for every 0.12 increase in pCa(2+) (0) (fall in free Ca(2+)). At pCa(2+) (0) 6.1, the activity of the Ca(2+) uniport is kinetically limited to 5nmol of Ca(2+)/min per mg of protein, even when the Ca(2+) electrochemical gradient is large. A steady-state cycling of Ca(2+) through independent influx and efflux pathways provides a model which is kinetically and thermodynamically consistent with the present observations, and which predicts an extremely precise regulation of pCa(2+) (0) by liver mitochondria in vivo.


Utlization of D-3-hydroxy[3-14C]butyrate for lipogenesis in vivo in lactating rat mammary gland. Incorporation of D-3-hydroxy[3-14C]butyrate into lipid in vivo suggests that lactating mammary gland is a major site of ketone-body utilization. The incorporation decreases in short-term insulin deficiency (2h) and on starvation (24h), but increases again on refeeding (2h). The activity of cytosolic acetoacetyl-CoA synthetase parallels the changes in nutritional state, but is not affected by short-term insulin deficiency.


Ribavirin treatment in murine autoimmune disease. I. Therapeutic efficacy and effect on the immune response. NZB/W F1 female mice were treated from 20 weeks of age with ribavirin (a broad spectrum antiviral drug), cyclophosphamide, or saline. Treatment with ribavirin (250 mg/kg twice weekly) prolonged survival from 9.8 to 18.5 months, reduced anti-DNA antibodies, and prevented proteinuria. Ability of ribavirin to prolong survival was dose related when given on a twice weekly schedule. However, daily ribavirin (25 mg/kg/day) was as effective as higher intermittent doses. Optimal ribavirin therapy was equal to cyclophosphamide treatment with regard to prolongation of survival. Ribavirin treatment did not significantly alter the body weight, hematocrit, WBC count, serum immunoglobulins, or Coombs reactivity. No alterations in either cellular or humoral immune responses were noted in NZB/W F1 or BALB/c mice treated for prolonged periods with ribavirin. The impressive therapeutic response to a broad spectrum antiviral agent seen in mice already manifesting immune complex nephritis provides a new therapeutic approach to the treatment of autoimmunity.


Alteration of lymphocyte function in NZB/NZW mice. IV. Response to levamisole. NZB/NZW F1 female mice received levamisole in an attempt to increase suppressor T cell function. Responses varied with dosage of drug and age of mice. When treatment with 25 microgram/gm doses (intermittent or daily) was begun at 12 weeks of age, nephritis was accelerated in spite of transient reduction of anti-DNA antibodies. When treatment was begun at 4 weeks, or when doses of 250 microgram/gm were given, no clinical effects were observed. In the mice with accelerated disease, delayed hypersensitivity and antibody responses to sheep erythrocytes increased; antibody responses to pneumococcal polysaccharide were unaffected. It is possible that some increased immune responses associated with levamisole are undesirable in murine lupus.


[Action of some gastric antisecretory drugs on histamine H2-receptors (author's transl)]. A series of compounds endowed with gastric antisecretory activity were examined for their possible effects on histamine H2 receptors in different experimental conditions. The data obtained suggest that, apart from cimetidine, all the other compounds are devoid of antagonistic properties on H2 receptors. The inhibitory effect observed in some of the examined preparations is probably connected with unspecific actions.


Isolation and characterization of Ca2+-dependent modulator protein from the marine invertebrate Renilla reniformis. An acidic, low molecular weight (18 400--19 100) protein capable of activating porcine brain phosphodiesterase in the presence of calcium has been purified 2700-fold from the anthozoan coelenterate, Renilla reniformis. The protein has physical, spectral, and chemical properties similar to those of modulator proteins isolated from mammalian species. Amino acid composition studies reveal no significant differences between the Renilla and mammalian modulator proteins. For example, we observed 1 mol of epsilon-N-trimethyllysine per mol of protein, no tryptophan or cysteine, and high levels of glutamic and aspartic acid residues. The protein from Renilla complexes with troponin I and T subunits in the presence of calcium and quantitatively replaces porcine brain modulator in the calcium-dependent activation of porcine brain phosphodiesterase. The protein has a high affinity for calcium as judged by the low levels of free calcium required for modulator-dependent activation of phosphodiesterase. The similarities in physical and chemical properties, high affinity for calcium, and identical calcium-dependent activities of this protein from Renilla (as compared with modulator protein purified from mammalian systems) suggest that a high degree of structural conservation has been retained in modulator proteins isolated from these diverse evolutionary forms.


Refolding behavior of a kinetic intermediate observed in the low pH unfolding of ribonuclease A. A transient intermediate (I3) observed previously in the unfolding of ribonuclease A has been studied by employing a sequential mixing instrument to populate selectively this species. This approach has made it possible both to determine the refolding behavior of this species and to characterize further the kinetics of its formation. (1) Formation of I3 represents the earliest detectable change in unfolding. (2) The loss of the 2'CMP binding site occurs in parallel with the exposure of the interior of the protein to solvent. (3) I3 is distinct from previously described intermediates in refolding. (4) Overall condensation of the protein to exclude solvent from the interior, as well as the formation of a substrate binding site, takes place in approximately 30 ms (pH 5.8, 47 degrees C), indicating that the formation of native structure can take place faster than had previously been supposed.


Reactivity of the imino acids formed in the amino acid oxidase reaction. The reactivity of the imino acids formed in the D- or L-amino acid oxidase reaction was studied. It was found that: (1) When imino acids reacted with the alpha-amino group of glycine or other amino acids, transimination yielded derivatives less stable to hydrolysis than the parent imino acids. In contrast, when imino acids reacted with the epsilon-amino group of lysine or other primary amines, transimination yielded derivatives more stable to hydrolysis than the parent imino acids. (2) Imino acids react rapidly with hydrazine and semicarbazide, forming stable hydrazones and semicarbazones. At pH 7.7, the rate of reaction of the imino acid analogue of leucine with semicarbazide was 10(4) times greater than that of the corresponding keto acid. The reaction of imino acids with these reagents is rapid enough to permit one to follow spectrophotometrically the amino acid oxidase reaction. Imino acids also reacted with cyanide to yield stable adducts. (3) The rate of hydrolysis of the imino acid analogue of leucine was independent of pH above pH 8.5. At lower pH values, the rate of hydrolysis increased with decreasing pH. At 25 degrees C  and in the absence of added amino compounds, this imino acid had a half-life of 22 s at pH 8.5. Its half-life was 9.9 s at pH 7.9.


1H NMR and ESR studies of oxidized cytochrome c551 from Pseudomonas aeruginosa. Near neutral pH, Fe(III) cytochrome c551 exhibits an ESR absorption due primarily to a single species with g values of 3.24, 2.06, and 1.48. These g values are somewhat different from those of horse heart cytochrome c and can be interpreted by the generalizations of Brautigan et al. [(1977) J. Biol. Chem. 252, 574] to be due to Fe binding by the imidazole anion of histidine rather than by neutral imidazole. The NMR spectrum of Fe(III) cytochrome c551 exhibits a number of hyperfine-shifted peaks whose pattern shows similarities to but many differences from that of horse heart cytochrome c. Variation in shifts of some of the peaks in the pH range 5--9 is ascribed to ionization of a somewhat buried propionic acid side chain (pK = 5.8) and to ionization of the N-terminal NH3+ group (pK = 7.7). At alkaline pH greater than 9.4, as shown by a variety of optical and ESR spectral changes, the Met-61 S ligand is replaced by other ligands.


Investigation of the pre-steady-state kinetics of fructose bisphosphatase by employment of an indicator method. The pre-steady-state kinetics for the hydrolysis of fructose 1,6-bisphosphate by rabbit liver fructose bis-phosphatase have been investigated by stopped-flow kinetics utilizing an acid-base indicator method that permits the continuous monitoring of the inorganic phosphate product. The reaction sequence is characterized by two successive first-order steps followed by establishment of the steady-state rate. The first exponential process results from a conformational change in the protein that is dye sensitive owing to a perturbation of an acidic residue on the protein. A second process reflects the rapid initial turnover of all four subunits of the enzyme with the concomitant release of inorganic phosphate followed by the rate-limiting step of the catalytic cycle. This latter step may involve a product release (fructose 6-phosphate) or a second conformational change. The catalytic cycle ends with decay of the enzyme to its initial unreactive resting state.


Application of the pH-jump method to the titration of tyrosine residues in bovine alpha-lactalbumin. A stopped-flow technique has been developed for the zero-time spectrophotometric titration of tyrosine residues in the purely native or in the purely alkaline denatured state of alpha-lactalbumin that undergoes an alkaline conformational transition in the pH region of tyrosine ionization. The progressive absorption change at 298 nm caused by a pH jump from neutral pH is shown to result from the change in ionization of the tyrosine residues brought about by a first-order process of the conformational transition. Extrapolation to zero time gives the titration curve for purely native alpha-lactalbumin. Similarly, the pH jump from highly alkaline pH gives the titration curve for the purely alkaline denatured protein. The method should be generally applicable to other proteins that contain tyrosines. Analysis of the titration curves suggests that the four tyrosines in native alpha-lactalbumin have pK values of 10.5, 11.8, 11.8, and 12.7, respectively. After the alkaline transconformation, all of them become titrated normally with a pK value of 10.3. A comparison of these results with the ionization behavior of tyrosines in hen egg white and human lysozymes is presented and discussed in terms of differences in the sequences of the proteins.


Conformations of denatured and renatured ovotransferrin. Conformational properties of native, denatured, and renatured ovotransferrin were studied. The samples were denatured either in 7.2 M urea or in acidic (pH 3.0) conditions for periods up to a few hours. Combined data from quasielastic light scattering and transient electric birefringence were used to estimate the molecular dimensions under the various conditions. The native ovotransferrin is best described as a prolate ellipsoid with a major axis a = 68 A and a minor axis b = 21 A. Such an ellipsoidal shape is consistent with a globular particle where the solvation factor is approximately 0.28 mg/mg of solute. The urea-denatured sample was more expanded and more globular than the native sample. This observation was supported by a decrease in helical content, which was shown using circular dichroism data. Complete recovery of conformation and capacity to form a colored complex with Fe3+ seemed to occur with the simple dilution of urea or by adjustment of the low pH sample to pH 7.3.


Reduction of methemerythrin by deoxymyoglobin: a protein-protein redox reaction not involving electron-transfer proteins. The stoichiometry and kinetics of reaction of methemerythrin with the deoxy forms of myoglobin and hemoglobin have been examined at I = 0.2 M and 25 degrees C. One mole of methemerythrin (on the basis of the monomer unit containing two irons) reacts with 2 mol of deoxymyoglobin and with 0.5 mol of deoxyhemoglobin. All reactions are second order. Rate constants for reaction with deoxymyoglobin are 0.25 M-1s-1 (Phascolopsis gouldii) and 5.6 M-1s-1 (Themiste pyroides) at pH 6.3. There is little effect of raising the ionic strength to 1.35 M and only a small decrease in rate when the pH is adjusted to 8.2. The rate constant for reaction of deoxyhemoglobin with P. gouldii methemerythrin is approximately 0.1 M-1s-1 at pH 6.3. Metmyohemerythrin from T. pyroides reacts slightly slower than the octamer form (k = 2.0 M-1s-1 at pH 6.3 and 7.0). Oxymyoglobin is converted to metmyoglobin by methemerythrin. The electron-transfer path is discussed and a self-exchange rate constant for hemerythrin assessed as 10(-3) M-1s-1 on the basis of Marcus's theory.


Sugar transport in Coprinus cinereus.

Sugar transport in Coprinus cinereus. Two transport systems for glucose were detected: a high affinity system with a Km of 27 muM, and a low affinity system with a Km of 3.3 mM. The high affinity system transported glucose, 2-deoxy-D-glucose (Km = 26 muM), 3-O-methylglucose (Km = 19 muM), D-glucosamine (Km = 652 muM), D-fructose (Km = 2.3 mM) and L-sorbose (Km = 2.2 mM). All sugars were accumulated against concentration gradients. The high affinity system was strongly or completely inhibited by N-ethylmaleimide, quercetin, 2,4-dinitrophenol and sodium azide. The system had a distinct pH optimum (7.4) and optimum temperature (45 degrees C). The low affinity system transported glucose, 2-deoxy-D-glucose (Km = 7.5 mM), and 3-O-methylglucose (Km = 1.5 mM). Accumulation again occurred against a concentration gradient. The low affinity system was inhibited by N-ethylmaleimide, quercetin and 2,4-dinitrophenol, but not by sodium azide. The rate of uptake by the low affinity system was constant over a wide temperature range (30--50 degrees C) and was not much affected by pH; but as the pH of the medium was altered from 4.5 to 8.9 a co-ordinated increase in affinity for 2-deoxy-D-glucose (from 52.1 mM to 0.3 mM) and decrease in maximum velocity (by a factor of five) occurred. Both uptake systems were present insporelings germinated in media containing sodium acetate as sole carbon source. Only the low affinity system could initially be demonstrated in glucose-grown tissue, although the high affinity system was restored by starvation inglucose-free medium. The half-ti me for restoration of high affinity activity was 3.5 min and the process was unaffected by cycloheximide. Addition of glucose to an acetate-grown culture inactivated the high affinity system with a half-life of 5--7.5 s. Addition of cycloheximide to an acetate-grown culture caused decay of the high affinity system with a half-life of 80 min. Regulation is thus thought to depend on modulation of protein activity rather than synthesis, and the kinetics of glucose, 2-deoxy-D-glucose and 3-O-methylglucose uptake would be consistent with there being a single carrier showing negative co-operativity. Analysis of transport defective mutants revealed defects in both transport systems although the mutants used were alleles of a single gene. It is concluded that this gene (the ftr cistron) is the structural gene for an allosteric molecule which serves both transport systems.


How do ionic channel properties depend on the structure of polyene antibiotic molecules? A study has been made of the properties of ionic channels formed in phospholipid-cholesterol bilayers by polyene antibiotics of various molecular structures. Properties of channels created by natural antibiotics with different structures of the lactone ring (amphotericin B-nystatin-mycoheptin) as well as by some derivatives of amphotericin B modified with respect to the amino and carboxyl groups are compared. Neutralization of one or both charges of the amphotericin B molecule (both by chemical modification and by pH shift) increases the probability of the channel to be in a nonconducting state. An increase of cholesterol concentration in the membrane produces an opposite effect. It is assumed that the electrostatic interaction of the amino group of an antibiotic molecule with the carboxyl group of an adjacent one stabilized the channel. Conductance and selectivity of an open channel are not influenced by changes in the charged groups. These properties strongly depend on the structure of the polar chain of the lactone ring. For example, the appearance of one more carbonyl group in the mycoheptin molecule results in a sharply decreasing anion permeability of channels. An antibiotic concentration which is necessary to observe single channels depends on the polyene chain structure: this is about 10(-7) M for tetraene nystatin and 2.10(-8) M for heptaene amphotericin B an mycoheptin.


Identification of gamma-like DNA polymerase from sea urchin embryos. A gamma-like DNA polymerase devoid of DNA polymerase-alpha and -beta activities was prepared from the nuclear fraction of blastulae of the sea urchin, Hemicentrotus pulcherrimus. The enzyme sedimented at the position of an approximate sedimentation coefficient of 3.3 S under high salt conditions by sucrose gradient centrifugation. An isoelectric point was determined to be pH 5.8. The enzyme activity was sensitive to sulfhydryl blocking reagents. Poly(rA) . oligo(dT)12--18 followed by poly(dA) . oligo(dT)12--18 was effectively utilized as a template-primer. From the above results, this polymerase seems to resemble the vertebrate DNA polymerase-gamma.


Amidination of amino groups of aldehyde reductase from human liver. Amidination of human liver aldehyde reductase (alcohol:NADP+ oxidoreductase, EC 1.1.1.2) with monofunctional n-alkane methylimidates increased the enzymic activity by 10--30%, whereas analogous bifunctional imidoesters caused a loss of activity of about 80%. Both effects were prevented in the presence of the coenzyme NADPH or NADP+, but not of the substrate 4-nitrobenzaldehyde. Amidination increased the apparent Michaelis constant of both the coenzyme (up to 20-fold) and the substrate (about 5-fold). Bifunctional imidoesters with at least 4 carbon atoms between the functional groups (approx. 0.7 nm) crosslinked the enzyme intramolecularly. This reaction was retarded in the presence of the coenzyme, whereas 4-nitrobenzaldehyde had no effect. The results suggest the presence of reactive amino groups at the coenzyme binding site of aldehyde reductase.


Exchange of free and bound coenzyme of flavin enzymes studied with [14C]FAD. The exchange of bound FAD for free FAD was studied with D-amino acid oxidase (D-amino acid:oxygen oxidoreductase (deaminating), EC 1.4.3.3) and beta-D-glucose oxidase (beta-D-glucose:oxygen 1-oxidoreductase, EC 1.1.3.4). For a simple measurement of the reaction rate, equimolar amounts of the enzyme and [14C]FAD were mixed. The exchange occurred very rapidly in the holoenzyme of D-amino acid oxidase at 25 degrees C, pH 8.3 (half life of the exchange: 0.8 min), but slowly in the presence of the substrate or a competitive inhibitor, benzoate. It also occurred slowly in the purple complex of D-amino acid oxidase. In the case of beta-D-glucose oxidase, however, the exchange occurred very slowly at 25 degrees C, pH 5.6, regardless of the presence of the substrate or p-chloromercuribenzoate. On the basis of these findings, the turnover of the coenzymes of flavin enzymes in mammals is discussed.


Glycosylation of kappa-casein. I. Localization and characterization of sialyltransferase in bovine mammary gland. A sialyltransferase (CMP-N-acetylneuraminate:D-galactosyl-glycoprotein N-acetylneuraminyltransferase, EC 2.4.99.1) which attaches N-acetylneuraminic acid to the terminal end of the carbohydrate chain of kappa-casein was found to be concentrated in Golgi apparatus-enriched fractions of bovine mammary gland. Maximum sialyltransferase activity was obtained at pH 5.5 and 37 degrees C in the presence of 1 mM dithiothreitol and Triton X-100. A Km of 0.19 mg asialo-kappa-casein/ml (0.01 mM) was obtained for the sialyltransferase. Native kappa-casein also served as acceptor for N-acetylneuraminic acid transferase of Golgi apparatus-enriched fractions although at a slower rate than did asialo-kappa-casein. The sialyltransferase has a divalent cation requirement for maximum activity which was best satisfied by the presence of 10 mM Mn2+.


Purification and properties of troponin T kinase from rabbit skeletal muscle. A protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) which catalyzes the phosphorylation of troponin T, phosvitin and casein has been purified over 2000 fold from rabbit skeletal muscle. The partial purification of this new enzyme, designated troponin T kinase, involves precipitation of contaminating proteins at pH 6.1, fractionation of the supernatant with (NH4)2SO4 and successive column chromatographies on DEAE-cellulose, hydroxyapatite and Sepharose 6B. The chromatographic patterns on DEAE-cellulose and hydroxyapatite columns show two peaks of troponin T kinase activity. Gel filtration experiments indicate the existence of multiple, possibly aggregated, forms of the enzyme. The purified enzyme does not catalyze the phosphorylation of phosphorylase b, troponin I, troponin C, tropomyosin, protamine, or myosin light chain 2 nor does it catalyze the interconversion of glycogen synthase I into the D form. Troponin T kinase is not affected by the addition of cyclic nucleotides or AMP to the reaction mixture. Divalent cations (other than Mg2+, required for the reaction) do not stimulate the enzyme, and several are inhibitory. Other characteristics of the reaction catalyzed by troponin T kinase, such as Km values for ATP and substrate proteins, pH optima, effect of the concentration of Mg2+, substitution of ATP for GTP have also been studied.


The activity of arylsulfatase A and B on tyrosine O-sulfates. L-Tyrosine O-sulfate was hydrolyzed by pure human arylsulfatase A (arylsufate sulfohydrolase, EC 3.1.6.1). The rate of hydrolysis was 1/20 of the rate with nitrocatechol sulfate, but was comparable to the rate with cerebroside sulfate. The reaction was optimal at pH 5.3--5.5 and displayed zero order kinetics with time and enzyme concentration. The Km was about 35 mM. The enzyme showed no stereospecificity and hydrolyzed D-tyrosine O-sulfate with Km and V similar to those for the L-isomer. Arylsulfatase B was less than 5% as effective as arylsulfatase A in catalyzing the hydrolysis of the tyrosine sulfates. The daily urinary excretion of tyrosine sulfate by a patient with metachromatic leukodystrophy (arylsulfatase A deficiency) was comparable to the excretion by control subjects. The biological relevance of the tyrosine sulfatase activity of arylsulfatase A remains uncertain.


Affinity chromatography and separation of the molecular forms of monkey brain alpha-L-fucosidase on fucose-linked sepharose. A simple affinity system which required coupling of alpha-L-fucose to Sepharose 4B by epichlorohydrin treatment of Sepharose 4B in the presence of alpha-L-fucose under alkaline conditions has been described. A partially purified preparation of monkey brain alpha-L-fucosidase (alpha-L-fucoside fucohydrolase, EC 3.2.1.51) was resolved at pH 5.0 into two major fractions: one bound and one retarded. The enzyme bound to the affinity column and specifically eluted by 2 mM alpha-L-fucose at pH 5.0 appeared to be homogeneous by polyacrylamide gel electrophoresis and was constituted mainly by the tetrameric form of the enzyme. The enzyme fraction retarded by the affinity column was found to contain mainly the monomeric form of the enzyme. Additional evidence for the different molecular forms of the enzyme in the bound and retarded fractions came from pH activity profiles and heat inactivation studies. The fucose-Sepharose appeared to bind the tetrameric form of the enzyme specifically and, further, alpha-L-fucose helped to retain the molecular integrity of the tetrameric enzyme.


Interaction of rat muscle AMP deaminase with myosin. I. Biochemical study of the interaction of AMP deaminase and myosin in rat muscle. AMP deaminase was completely solubilized from rat skeletal muscle with 50 mM Tris-HCl buffer (pH 7.0) containing KCl at a concentration of 0.3 M or more. The purified enzyme was found to be bound to rat muscle myosin or actomyosin, but not to F-actin at KCl concentrations of less than 0.3 M. Kinetic analysis indicated that 1 mol of AMP deaminase was bound to 3 mol of myosin and that the dissociation constant (Kd) of this binding was 0.06 micrometer. It was also shown that AMP deaminase from muscle interacted mainly with the light meromyosin portion of the myosin molecule. This finding differs from that of Ashby and coworkers on rabbit muscle AMP deaminase, probably due to a difference in the properties of rat and rabbit muscle AMP deaminase. AMP deaminase isozymes from rat liver, kidney and cardiac muscle did not interact with rat muscle myosin. The physiological significance of this binding of AMP deaminase to myosin is discussed.


Comparison of AMP deaminase from skeletal muscle of acidotic and normal rats. The deamination of AMP by AMP aminohydrolase (EC 3.5.4.6.) serves as the major source of ammonia production in skeletal muscle. It has been suggested that the ammonia may serve either in a buffering capacity to combat acidosis due to the accumulation of lactic acid produced during prolonged muscular activity, or as a substrate for glutamine formation which can ultimately be utilized by the kidney in adapting to metabolic acidosis. In view of this proposal, the properties of the enzyme obtained from skeletal muscle of acidotic rats have been compared with the enzyme from normal muscle. The specific activity of AMP deaminase in crude homogenates of acidotic muscle was not significantly different from normal levels. The enzyme from acidotic muscle was purified to homogeneity and was found to be identical to the enzyme obtained from normal muscle by the criteria of electrophoretic mobility, pH optimum, molecular weight, sedimentation coefficient, subunit composition, amino acid composition, monovalent cation requirement, substrate saturation, and inhibition by ATP, Pi and creatine-P. Thus, if the enzyme functions to prevent acidosis, the ability to respond to changes in the intracellular environment which accompany acidosis must be built into the structure of the enzyme normally found in skeletal muscle. Three lines of evidence strongly support this viewpoint: (a) the rate of deamination is approximately 2-fold higher at pH 6.5 than at pH 7.0, (b) the activity increases linearly with a decrease in the adenylate energy charge, and (c) within the normal physiological range of the adenylate energy charge, the enzyme is operating at only 10--20% of its maximum capacity.


Destruction and resynthesis of mouse beta-glucosidases. 1. Injection of a single dose of conduritol B epoxide into mice produced almost complete destruction of glucocerebrosidase (D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2.1.45) in liver, spleen, brain, and kidney within 5 h. Restoration of activity became noticeable within 1 day (2 days in the case of brain) and was about 80% of normal within 16 days. 2. The same injection produced less destruction of aryl beta-glucosidase (EC 3.2.1.21), measured at pH 5.4 with methylumbelliferyl glucoside in the absence of taurocholate. Brain showed the least amount of destruction, about 50%, but measurements of activity at lower pH values revealed complete loss of activity. This suggests that brain contains two different aryl glucosidases with differing sensitivity to the inhibitor. Liver, on the other hand, did not show differential destruction when assayed at different pH values. Resynthesis of the enzyme activities was almost complete by 16 days. 3. Injection of phenylhydrazine produced hemolysis and spleen enlargement, with concomitant increases in specific activities of glucocerebrosidase and aryl glucosidase in liver and spleen (but not in kidney). When this experiment was done in mice previously treated with conduritol B expoxide, the reappearance of cerebrosidase was found to be accelerated. This is interpreted to mean that the increased load of glucolipids from the erythrocytes had induced an enhanced synthesis of the glucohydrolase. A similar explanation may apply to aryl glucosidase and glucopeptides in the cells.


Calcium-dependent cyclic nucleotide phosphodiesterase. Inhibition of basal activity by heat-stable factors from rat cerebrum. The boiled supernatant fraction from rat cerebrum contained factors which inhibited the basal activity of a Ca2+-dependent phosphodiesterase from rat cerebrum. Two inhibitory fractions were isolated by DEAE-cellulose or Sephadex chromatography and were deemed proteins, based on their sensitivity to trypsin digestion. The inhibitory fractions eluted from DEAE-cellulose columns prior to the Ca2+-dependent activator protein. The inhibitory factors, unlike the activator protein, were stable to heat treatment under alkaline conditions. The inhibitory factors caused both an increase in Km for cyclic GMP and a decrease in V. In the presence of calcium ions and purified activator protein, the Ca2+-dependent phosphodiesterase was not inhibited by the factors, but instead was slightly stimulated. The inhibitory factors caused a slight apparent stimulation of a Ca2+-independent phosphodiesterase from rat cerebrum but this proved instead to be a nonspecific stabilizing effect which was minimicked by bovine serum albumin. After prolonged alkaline treatment, the purified activator protein caused a modest Ca2+-independent activation of Ca2+-dependent phosphodiesterase. The inhibitory factors antagonized the activation of Ca2+-dependent phosphodiesterase by alkaline treated activator protein or by lysophosphatidylcholine. The inhibitory factors had no effect on activity of trypsinized Ca2+-dependent phosphodiesterase. Of various other proteins, only casein mimicked the effects of the inhibitory factors on phoshodiesterase activity.


The hydration of a zwitterion, glycine, as a function of pH. 1. The hydration numbers of glycine (concentrations 1--3 M), as a function of concentration, were determined by surface tension measurements, using octan-1-ol as a 'reference' substance. 2. The hydration number of glycine at the isoelectric point decreased from 17.7 to 10.7 upon increasing the concentration from 1 to 3 M. 3. The changes in hydration of glycine as a function of the pH are due to the difference between hydrations of the ionized functional groups (NH+3, COO-) and the added ions (Me+ A-).


Studies on lectins. XLIV. The pH dependence of lectin interactions with sugars as determined by affinity electrophoresis. The pH dependence of association constants of the lectin-sugar complexes was determined by means of affinity electrophoresis. All the lectins studied (from the seeds of Dolichos biflorus, Glycine soja, Lens esculenta and Vicia cracca and of the fruiting body of Marasmius oreades) were characterized by a similar course of pH dependence of the association constants, with the maximum values at pH 7--9. For concanavalin A and the L-fucose binding Ulex europaeus lectin only the association constants at three selected pH values were determined. Concanavalin A does not interact with immobilized alpha-D-mannosyl residues at pH 2.3. The association constants vs. pH curves measured for lectins isolated from two different lentil varieties slightly differ in accordance with the differences observed in the interaction of these lectins with the Sephadex gel.


[Comparative biochemical studies of polyhedral proteins of nuclear polyhedrosis viruses]. Using disc polyacrylamide gel electrophoresis, the molecular weights of polyhedral proteins of nuclear polyhedrosis viruses (NPV) of Porthetria dispar, Mamestra brassicae, and Aporia crataegi were found to be 28000 +/- 3000. It was shown that NPV polyhedra of Bombyx mori, Galleria mellonella, P. dispar, and M. brassicae contain a protease. During dissolution of the polyhedra at pH 10,5 this protease specifically cleaves the matrix protein into 2--5 fragments. The amino acid compositions of NPV polyhedral proteins of P. dispar, M. brassicae, A. crataegi, Hyphantria cunae were shown to be very similar. It was found that tyrosine is a C-terminal amino acid of NPV polyhedral proteins of P. dispar, M. brassicae, and A. crataegi.