Quantitative variations of three different lectin receptors as a function of establishment and metabolism of normal and leukaemic human cell lines. Lectin binding on the cell surface was measured by the method of Kornfeld [16] using three tritiated lectins: Robinia pseudo acacia, Concanavalin A and Ricinus. It has been shown that the number of binding sites for Robinia and Con A decreases after the establishment of a leukaemic cell line, whereas the affinity constant increases for Robinia. The relationship between lectin binding and cell growth was carried out on 5 cell lines, 3 of leukaemic and 2 of normal origin. In all cases the maximum number of sites was determined at the time of doubling for the cell population; the affinity constant reciprocally decreases at this moment. For Robinia, a difference was found in both cell growth and the number of sites between an Epstein-Barr virus (EBV) negative leukaemic cell line and the other EBV positive cell lines. Lectins offer a quantitative method for measuring membrane structure variations during and after establishment of cell lines.


New concepts on the action of oestrogens in the uterus and the role of the eosinophil receptor system. Two receptor systems for oestrogens have been demonstrated in the uterus: the cytosol-nuclear receptor system and the eosinophil receptor system. It has been proposed that the cytosol-nuclear receptor system mediates the genomic response to oestrogens in the uterus, while the eosinophil receptor system is thought to mediate the uterine edema and other early oestrogenic responses in the uterus. Cortisol is known to decrease drastically the number of eosinophils in the blood and therefore to limit their availability for migration to the uterus. The present results show that cortisol also drastically reduces both the oestrogen-induced uterine eosinophilia and the uterine wet weight responses, but does not interfere with the oestrogen-induced uterine RNA and protein increases. Oestradiol-17 beta has a higher affinity than oestriol for the cytosol-nuclear receptors and is now found to be the more potent oestrogen in inducing the genomic activation in the uterus. Estriol has a higher affinity than oestradiol-17 beta for the eosinophil receptors, and therefore, oestriol is the stronger oestrogen in inducing those oestrogenic effects which are mediated by the eosinophil receptor system. We conclude that the eosinophil receptor system for oestrogens is a new system, independent of Jensen's cytosol-nuclear receptor system, and this eosinophil receptor system is involved in the mechanism of oestrogen action in the uterus.


Dynamics of eosinophils in the uterus after oestrogen administration. To investigase the role of the eosinophil leukocytes in the early oestrogenic responses in the uterus, the kinetics of oestrogen-induced uterine eosinophilia and other parameters of oestrogen stimulation were studied at very early times. Uterine eosinophils increase as early as 5 min after an intravenous injection of oestradiol to immature rats, much earlier than several other changes in the early parameters of oestrogen stimulation. Large number of uterine eosinophils are found attached to the wall of small uterine blood vessels at early times. To elucidate the mechanisms involved in the specific attraction of eosinophils to the uterus in the presence of oestrogens, the in vivo localisation of oestrogens in the rat uterus at early times was studied using a radioautographic technique. Oestrogen receptors were found in the surface of eosinophils and in the wall of small uterine blood vessels. This simultaneous presence of both oestrogen receptors is proposed to explain the specific attachment of eosinophils to uterine blood vessels in the presence of oestrogens, which is the initial step toward eosinophil penetration into the uterus.


Effect of Sarkosyl on chromatin and viral RNA synthesis. The isolation of SV40 transcription complex. The endogenous RNA polymerase activity of mouse nucleic is enhanced several-fold by the anionic detergent Sarkosyl. The action of Sarkosyl is exerted primarily on the alpha-amanitin sensitive form of the enzyme. This detergent causes the release of nearly all the protein associated with cellular DNA but does not release initiated RNA polymerase. Sarkosyl was also able to activate the RNA polymerase activity from mitotic cells, in which transcription of the highly condensed chromatin is minimal. The use of this anionic detergent has also permitted the extraction of a nucleoprotein complex from Simian Virus 40 (SV40) infected monkey cells. Molecular hybridization experiments have established the viral specificity of the RNA synthesized in vitro by the endogenous polymerase present in this complex.


Acceleration of hepatocarcinogenesis of 2,7-bis(acetamido)fluorene by carbon tetrachloride and time relation of treatment. The role of period between administration of CCl4 and feeding of 2,7-bis-(acetamido)fluorene in the hepatocarcinogenesis of mice was analyzed by using 14 groups of different feeding schedules. Simultaneous treatment with CCl4 and either continuous or intermittent feeding of 2,7-bis(acetamido)fluorene increased the average number of hepatic nodules and the incidence of hepatomas, while separation of 8-week feeding of 2,7-bis(acetamido)fluorene from 8 week treatment with CCl4 did not accelerate the hepatocarcinogenesis. Alternate treatment with CCl4 and 2,7-bis(acetamido)fluorene increased the average number of hepatic nodules but failed to induce hepatomas. Allyl alcohol, which is a hepatotoxic substance as is CCl4, also failed to accelerate hepatocarcinogenesis of 2,7-bis(acetamido)fluorene. Concerning the feeding schedule of 2,7-bis(acetamido)fluorene alone, the intermittent one-week feeding of the carcinogen with 3-week intervals decreased the average number of hepatic nodules as compared with the continuous feeding of the carcinogen.


Mixed mesodermal tumor of the uterine body: relationship between histology and survival. A clinico-pathological study of 20 cases of mixed mesodermal tumor of the uterine body is reported. Fifteen tumors were found after menopause. The mean age of all cases was 55.5 years. Histologically, mixed mesodermal tumors consisted of two different components; epithelial and sarcomatous patterns, in 18 cases. Mixed mesodermal tumors in four of the 18 cases had no adenocarcinomatous elements but showed a benign glandular structure. In the remaining two tumors, epithelial patterns were absent and only heterologous sarcomatous elements were found. Ten of the patients had received hysterectomy more than five years ago. Three of these patients, having no malignant but benign epithelial component, showed a relatively good prognosis. Adenocarcinomatous elements were found in six cases, and four patients with carcinomatous elements had poor prognosis. It can be stated that the prognosis of mixed mesodermal tumor chiefly depends on the malignancy of epithelial components, when comparing the prognosis of the tumors at the same clinical stage. On the other hand, sarcomatous elements are not likely to reflect the prognosis of the tumors. Mixed mesodermal tumor is monodermal in origin, namely, mesodermal origin, and might arise from the Muellerian duct or more primitive mesodermal tissue.


Effect of Clostridium toxoids, especially of Clostridium perfringens toxoid, on mouse transplanted tumors. The antitumor activities of toxoids of Clostridium perfringens, C. nouyi, C. septicum, and C. tetani were tested against sarcoma-180 and Ehrlich carcinoma. Among them, C. perfringens toxoid showed a high antitumor activity against the growth of the implanted sarcoma-180 ascites form. The results of the inhibiting effect of C. perfringens toxoid on Nakahara-Tokuzen-Fukuoka (NTF) reticulum cell sarcoma and methylcholanthrene-induced fibrosarcoma were also described.


Beta-glucuronidase isozyme patterns of experimental hepatomas of rats. Beta-Glucuronidase isozymes in rat tissues were separated by electrophoresis on cellulose acetate membranes. Using this method, beta-glucuronidase isozyme patterns were studied in normal rat liver and experimental hepatomas with different growth rates. Changes of isozyme patterns during postnatal development were also studied in rat liver. Normal rat liver contained six types of beta-glucuronidase, numbered I to VI in order to decreasing mobility to the cathode. Type II beta-glucuronidase stained most intensely and was detected in all the cells examined. The isozyme patterns of Morris hepatomas, which are slowly growing and not highly deviated, resembled that of normal rat liver but lacked definite Type III beta-glucuronidase. Yoshida ascites hepatomas, which are rapidly growing and highly deviated, contained only Type II, but some had Types II, V, and VI beta-glucuronidase. Embryonal liver just before birth contained only Type II, but with increase in activity during development after birth, minor bands of Type I and Types III  to VI beta-glucuronidase appeared successively to complete the adult pattern.


Increased susceptibility to carcinoma of the liver in rats with one kidney ingesting N-4-(4'-fluorobiphenyl) acetamide. The role of the kidneys in hepatic carcinogenesis was studied in inbred Buffalo strain male rats ingesting 0.04% N-4-(4'-flourobiphenyl) acetamide in the diet. Experimental groups were made up of male rats with both kidneys intact and male rats with the left kidney removed. The incidence of carcinomas of the liver and the number of rats with large carcinomas, multiple carcinomas, poorly differentiated and undifferentiated carcinomas, and metastases were greater in rats with a uninephrectomy. Apparently the animals with one kidney removed were unable to secrete the metabolites of N-4-(4'-fluorobiphenyl) acetamide as readily as the rats with both kidneys.


Protection by propylthiouracil against carbon tetrachloride-induced liver damage. Rats given a single intragastric dose of carbon tetrachloride (CCl4), 0.25, 0.50, or 1.0 ml per kg) showed a dose-dependent increase in SGOT, serum ornithine carbamyltransferase, and liver necrosis (graded histologically as 0 to 4+) 24 hr after the treatment. Daily intubation with propylthiouracil (PTU) for 10 days in doses of 5 to 50 mg per kg significantly reduced the elevation of SGOT activity, completely suppressed the serum ornithine carbamyltransferase changes, and reduced the degree of necrosis found 24 hr after the intragastric administration of CCl4. Similar protection was found when CCl4 was given intraperitoneally. When PTU was given in liguid diets for 6 days, protection against CCl4 was increased. PTU did not affect the absorption or covalent binding of 14CCl4 to lipids or proteins. Also, control and PTU-treated rats did not differ with respect to glucose-6-phosphatase activity and conjugated diene production after CCl4. Thus, it has been observed that PTU affords partial protection against some end-stage consequences of CCl4 liver injury such as cell necrosis and release of intracellular enzymes. However, PTU afforded no protection against early chemical effects such as covalent binding of CCl4 carbon, lipid peroxidation, or loss of glucose-6-phosphatase. Therefore, it is concluded that the mechanism of the PTU effect comes into play after the initial effects of CCl4 are exerted and in some unknown manner modulates the expression of these early effects.


Male pseudohermaphroditism consistent with 17,20-desmolase deficiency. A 16-year-old phenotypic female with XY genotype presented an unusual form of nonfamilial male pseudohermaphroditism. Seemingly a normal girl during childhood, the patient failed to undergo pubertal changes presenting with scant pubic hair, absent axillary hair, lack of breast development, retarded bone age and primary amenorrhea. Neither uterus nor adnexa were palpable above the blind-ending vagina. Serum testosterone and estradiol were barely detectable by radioimmunoassay, while LH and FSH reached castrate levels. Two small testes were removed from the pelvic sidewalls which, on biopsy, showed atrophy and hyalinization of seminiferous tubules, but clusters of Leydig cells without signs of hypertrophy or hyperplasia. Administration of testosterone resulted in urinary nitrogen retention and a decrease in serum LH and FSH. Radioimmunoassay of various serum or plasma steroids and gas chromatographic determination of urinary steroids prior to and following ACTH stimulation yielded results which permitted to rule out 20,22-desmolase, 3beta-hydroxysteroid dehydrogenase, 17-hydroxylase and 17beta-hydroxysteroid dehydrogenase deficiency. Low plasma dehydroepiandrosterone sulfate (DHEA-S) and androstenedione (delta4 A) concentrations, low urinary 17-ketosteroid and particularly low dehydroepiandrosterone (DHEA) excretion and the minimal rise of plasma DHEA-S and delta4 A and of urinary DHEA in response to ACTH in conjunction with a normal response of other serum and urinary C-21 steroids are consistent with 17,20-desmolase deficiency. Direct confirmation of this defect, however, seems impossible in the absence of in vitro studies of testicular steroidogenesis.


[Treatment of acne vulgaris in practice]. The aim of all acne therapy is sebostasis and treatment of Corynebacterium acnes as well as secondary infected postular changes. After discussion of Tretinoin (Retinoic acid)-treatment it is reported about 35 cases of acne vulgaris which were treated with the combination of Hexachlorophenspiritus 1%,Silicatgel locally and Tetracycline orally. The good results of this treatment are reported. Side effects have not been observed.


[Studies on the strain on drivers during high-speed driving]. In modern industrial countries the technological progress has shifted the occupational stress to the psychomental sphere. In the course of this alteration the number of work places with driving, controlling, and monitoring increased considerably. On 10 engine-drivers during high-speed driving from 210 to 250 km/h heart rate and electrocardiogram were monitored continuously. The blood pressure was measured intermittently. The elimination rate of catecholamines in urine, and the levels of cyclic AMP and catechol-O-methyltransferase in plasma were determined in reference to the trial to quantify the psychomental strain by biochemical parameters. In consequence of the individual affective conditions of the engine-drivers it has been attempted to scale the subjective strain.


[Climacteric disturbances. 2. Therapy of climacteric disturbances]. After defining the terms climacterium and menopause the causes of climacteric disturbances are explained. During the premenopausal stage disturbances of the cycle are prevailing, caused by an insufficiency of the corpus luteum. Of climacteric disturbances should be spoken only after menopause. They are divided into: vegetative disturbances, troubles of metabolism, cardiovascular dysregulation, psychic deviations, sexual troubles and changes of the skin. The therapy of disturbances during the premenopausal stage mainly consists of the substitution of progesterone or in a cycle-like estrogen-progesterone-therapy. In the premenopausal stage estrogens are the therapy of choice. Among orally efficient estrogens the conjugated estrogen and the estradiol-valerianat are preferred. Side-effects and contraindications are discussed in detail. Among gynecologists there exists no disagreement about the necessity of therapy of serious climacteric disturbances, the opinions about prophylactic estrogen-therapy in women differ.


[Experiences with depot-ACTH (Tetracosactide) in bronchial obstruction]. The difficulties of long term treatment of chronic obstructive lung disease demand knowledge and use of every reasonable therapeutic regimen. In this study effectivity and side effects of Depot-ACTH (Tetracosactid) are presented. The main indications for the use of Depot-ACTH seem to be the uneffectiveness of usual therapy and the attempt of avoiding or reducing corticosteroids. The study confirms the excellent usefulness of Depot-ACTH which did not diminish during long-term application. The side effects are rare and mostly inferior. Only allergic reactions may be serious and must be accounted for.


[Quantitative analysis of characteristics of the negative feedback regulation of ACTH secretion under stress (author's transl)]. Quantitative characteristics and a plausible mechanism of rapid rate-sensitive feedback inhibition of the CRF-ACTH-corticoid system were studied using Nembutal-anesthetized rats. The plasma corticosterone levels were varied by continuous infusion of corticosterone through a lateral caudal vein. The response to an intravenous injection of histamine (as a stressor) was distinctly inhibited in two phases of the dynamics of plasma corticosterone; one in its rapidly increasing phase (rapid rate-sensitive feedback inhibition) and the other, two hours or more after a high plasma steroid level has been attained (late level-sensitive feedback inhibition). On the other hand, there was no such inhibition between these two phases. A quantitative approach made by repeated blood sampling from single rats revealed that the critical rate of elevation of plasma corticosterone at which the rate-sensitive feedback inhibition). On the other hand, there was no such inhibition between these two phases. A quantitative approach made by repeated blood sampling from single rats revealed that the critical rate of elevation of plasma corticosterone at which the rate-sensitive inhibition begins to obviously occur was 4-6 mug%/min. Even with increasing strength of stress, the absolute size of inhibition at a fixed rate of elevation in plasma corticosterone remained almost the same. The increase in the median eminence-CRF activity, known to occur after stress, was effectively suppressed during the operation of rapid feedback inhibition. In addition, in brain noradrenaline-depleted rats, the rapid component in the feedback inhibition was thoroughly abolished, leaving the late component intact, while the opposite way in sorotonin-reduced rats. This finding suggests an involvement of brain biogenic amines in the negative feedback regulation of ACTH secretion in a rather specific way. Finally, on the basis of these results, a plausible model for the rapid rate-sensitive feedback regulation of ACTH secretion under stress was considered.


[Analysis of dynamics of corticotropin-releasing factor (CRF) activity in the rat hypothalamus under stress]. Obviously, the analysis of dynamic changes in the hypothalamic activity of corticotropin-releasing factor (CRF) is essential for understanding of the central regulatory mechanism of ACTH secretion. However, the significance of the changes in CRF activity will be extremely lessened if the effect of CRF per se be modified at the pituitary level. In fact, Yates et al. (1971) reported that the CRF effect was potentiated by the presence of vasopressin. Therefore, in this study we attempted first to determine if vasopressin does potentiate the CRF action. Next, we analyzed some aspects of CRF dynamics in the rat hypothalamus under prolonged stress. (1) Potentiation of CRF action by vasopressin. This possibility was examined by the following approaches: i) Adrenocortical responses to various mild stressors (exposure to sound, i.p. injection of saline solution, tail cut) were greater in dehydrated rats than in normal controls. ii) Similarly, the adrenocortical response to intravenous injection of stalk-median eminence extract (SME) through the tail vein under Nembutal anesthesia was larger in the dehydrated rat than in control. iii) Prior to SME administration, vasopressin in a subthreshold dose was injected intravenously to assay rats pretreated with chlorpromazine (CPZ)-morphine (M)-Nembutal (Nb). The adrenocortical response to SME, injected into the carotid artery 1 min later, was found significantly to increase due to prior administration of vasopressin. iv) However, no potentiating effect of vasopressin  was observed when SME and vasopressin (4 mU) were placed stimultaneously into the anterior pituitary tissue by the intrapituitary injection technique. v) In addition, no potentiating effect was observed in vitro incubation experiments under varying incubation conditions. Thus, it was shown that vasopressin has some potentiating effect on the stress response in vivo, but the effect is not at the pituitary level.  (II) Analysis of dynamic changes in hypothalamic CRF activity. CRF activity was estimated by the intrapituitary injection method of Hiroshige, the plasma ACTH and corticosterone levels being followed simultaneously. Plasma ACTH was determined by radioimmunoassay partly with RCC-RIA Kit, and partly with ACTH antisera (kindly supplied by Dr. W.F. Ganong) by the method of Berson and Yalow. i) In intact normal rats, the response pattern of hypothalamic CRF activity under etherlaparotomy stress was characteristically biphasic, i.e., composed of rapid and slow phases, while the plasma ACTH and corticosterone showed a sustained high level over a 2 hr of observation period.


The binding by an NAD-affinity matrix of contaminating dehydrogenases in cytochrome c oxidase preparations. An NAD-matrix is capable of the separation of three contaminant reductase activities from highly purified beef heart cytochrome c oxidase. Non-specific binding of cytochrome c oxidase occurs as well. When the matrix is saturated with respect to the non-specific binding, continued binding of the reductases can be observed. As a consequence of equilibration with the matrix, the turnover numbers of the unbound cytochrome c oxidase are increased.


[Concanavalin A induced changes of lymphocyte p-nitrophenylphosphatase (author's transl)]. The effect of K+, Na+, Mg2+ and ATP on the p-nitrophenylphosphatase activity was investigated. As an enzyme preparation a microsomal fraction of sheep lymphocytes was used. Low concentrations of Mg2+, K+ and Na+ increased, whereas high concentrations decreased the enzyme activity. There was an inhibition of activity by ATP without Na+ in the incubation medium and an increase of enzyme activity at low K:Na-ratio. By concanavalin A in a concentration of 15 mug/ml the p-nitrophenylphosphatase activity was increased in intact cells and the microsomal fraction for 30-40%. The activation was not Na+, K+, Mg2+, p-nitrophenylphosphate or ATP dependent.


Inactivation of yeast enzymes by proteinase A and B and carboxypeptidase Y from yeast. Changes in the activities of 15 different enzymes during incubation of a crude yeast extract with the purified yeast proteinases A and B, and carboxypeptidase Y, respectively, have been measured. The spectrum of action of the three proteinases on the enzymes measured differs significantly, increasing or decreasing their activities or having no effect. Incubation of purified cytoplasmic malate dehydrogenase or purified mitochondrial malate dehydrogenase with proteinases A and B results in selective inactivation of the cytoplasmic enzyme, whereas the mitochondrial activity is not affected. Carboxypeptidase Y has no effect on the activity of either dehydrogenase. The results support the idea of selective proteolysis as the mechanism of the earlier observed inactivation of cytoplasmic malate dehydrogenase, initiated by the addition of glucose to intact yeast cells grown on acetate as carbon source ("glucose effect").


A model for program evaluation in a unitized setting. The unitized psychiatric hospital poses unique problems for the design of successful program evaluation systems. Using the model of program evaluation in operation at the Harlem Valley Psychiatric Center in New York, the authors describe what they have found to be essential components of a successful evaluation system, including methods of data input, processing, and feedback. Also discussed are the roles of key personnel and the utilization of data by clinical and administrative staff. The reasons for success of the system are discussed, as well as the potential role of program evaluation in integrating services, training, and administration of the facility.


Localisation of the human ABO: Np-1: AK-1 linkage group by regional assignment of AK-1 to 9q34. Quantitative red cell adenylate kinase (AK-1) assay has been used in 8 patients with partial duplication or deletion of chromosome 9 in an attempt to find the precise intrachromosomal location of the structural gene locus. All regions of chromosome 9 are represented in abnormal dosage in at least one patient. A 43% increase in AK-1 activity was found to be associated with duplication of the terminal band of the long arm of chromosome 9. Duplication of all other parts of chromosome 9 were associated with normal enzyme activity. These findings not only confirm the assignment of the AK-1 locus to chromosome 9 made previously in somatic cell hybrids, but suggest a more precise assignment to region 9q33 leads to qter. This places the ABO:Np-1:AK-1 linkage group at the distal end of the long arm of chromosome 9.


Indophenol-oxidase in patients with Down's syndrome due to simple trisomy and to translocation 21/22. The indophenol-oxidase of erythrocytes was studied in three groups (trisomy G 21, unbalanced translocation 21/22, and control group). The increase of IPO activity in patients with trisomy  G 21 was observed, whereas in those with unbalanced translocation 21/22 the levels of IPO were slightly lower than in control group.


Hormonal studies of uridine utilization in an insect cell line CP-1268 derived from the codling moth Laspeyresia pomonella. Ecdysterone decreased cellular growth and the incorporation of uridine into RNA following 4 days of hormone exposure. This hormone did not affect uridine incorporation following short-term exposure up to 25 hours. Juvenile hormone and farnesol both significantly decreased uridine uptake and incorporation into RNA; however, uridine uptake was inhibited to a greater extent than uridine incorporation. Cyclic AMP increased the incorporation of uridine into RNA but had no demonstrable effect on the uptake process. This stimulation was not the result of cAMP degradation products. Cyclic AMP and ecdysterone together produced a significant increase in uridine incorporation into RNA. These studies demonstrate the potential utilization of insect cell lines for studying the mode of action of insect developmental hormones.


Characterization of a spontaneous undifferentiated carcinoma from an African green monkey (Cercopithecus aethiops). An adult male African green monkey (Cercopithecus aethiops) with an undifferentiated carcinoma, probably originating from the nasal mucosa, was received from the Akron, Ohio zoo. Cultivation of this tumor in vitro resulted in a mixture of fibroblastic and epithelial cells which was subsequently separated using differential trypsinization. The neoplastic nature of the cultured epithelial cells was verified by their ability to transplant into athymic nude, or antithymocyte serum-treated mice, where poorly differentiated carcinomas were produced, and cultures of the tumors that arose in nude mice were morphologically similar to pretransplantation cultures. Early cultures showed a normal male karyotype characteristic of the species; however, in long-term cultures, a clearly defined, small submetacentric Y chromosome was not observed. Electron microscopic examination of tumor tissue and cultured tumor cells revealed desmosomes and the presence of cytoplasmic (keratin-type) fibrils, which tended to be organized around the nucleus. In addition to the keratin-type fibrils, the cultured tumor cells also contained a large amount of cytoplasmic inclusion material that may represent keratohyalin granules. There was no evidence of a viral association with tumor material or cultured cells. The cultures were susceptible to infection by vesicular stomatitis virus, Herpesvirus hominis type 1, and H. saimiri, but were resistant to the Epstein-Barr virus.


Control of immune responses by cyclic AMP and lymphocytes that adhere to histamine columns. Mixed lymphocytes from human peripheral blood, murine spleens, lymph nodes or thymus glands have pharmacologically specific receptors for histamine, beta mimetic catecholamines and prostaglandins. When these cells are exposed to the panoply of drugs mentioned above, their intracellular cyclic AMP concentrations increase. The biologic consequences of such an increase were at first elusive. Now we know that the immune potential of some murine spleen cells may be modulated and the release of lysosomal enzymes and histamine from human leukocytes may be inhibited. This paper concentrates on the effects that manipulation of cells with amine receptors has on their immune function. Recent studies have revealed that a subpopulation of splenic suppressor T cells responds to increases in its cyclic AMP content by reversing its suppressive effects on the humoral antibody response. When these T cells are removed from the murine cell population by their differential adherence to insolubilized conjugates of histamine with albumin, the remainder of the cells are more responsive to sheep cell antigen, as tested by transferring the spleen cells together with the antigen into lethally irradiated recipient animals. The suppressor T cells that adhere to the insolubilized conjugates of histamine-albumin (called histamine-rabbit serum albumin-Sepharose, or HRS) are Ia positive, they appear to have receptors for histamine, beta adrenergic amines and prostaglandins of the E series, and when stimulated by these agents their in vivo and in vitro suppressor actions are reversed. The reversal seems quantitatively dependent on cyclic AMP accumulation. Receptors for the amines and prostaglandins are found on the T cell precursors of cell mediated immunity. They develop on some T effector cells in selected models of allogeneic target cell lysis. The receptors also appear to develop on selected B cells once these cells become committed to antibody production. The distribution of receptors on all leukocytes has not been adequately studied nor has their full potential in the immune response been studied in detail.


Cell-mediated immunity to varicella-zoster virus demonstrated by viral inactivation with human leukocytes. Cell-mediated immunity to varicella-zoster (V-Z) virus in persons immune to varicella has been demonstrated, using a tissue culture technique. Cell-mediated immunity was reflected by the ability of peripheral leukocytes (lymphocytes and monocytes) from human donors to inactivate V-Z virus. Leukocytes were stimulated by the addition of noninfectious V-Z antigen to cultures newly infected with V-Z virus. Several days leter, the V-Z virus in these cultures was titered. When leukocytes from donors immune to caricella were used, a significant decrease in V-Z titer, compared with controls, was noted. When leukocytes from donors susceptible to varicella were tested, no decrease in V-Z virus titer was found. A mixed population of lymphocytes and monocytes from immune donors was required to demonstrate inactivation of V-Z virus. The development of specific cell-mediated immunity to V-Z virus may play a role in termination of varicella and in prevention of second attacks of this disease.


Serological studies with reovirus-like enteritis agent. The age distribution of antibody to the human reovirus-like enteritis agent, and to the antigenically related Nebraska calf diarrhea virus (NCDV) was studied in serum specimens obtained from 592 children hospitalized in Boston. Sera were examined for complement-fixation (CF) antibody to the human agent, and for CF and indirect immunofluorescence-staining antibodies to NCDV. The curve of antibody frequency was similar in each of the three assays, showing a steep rise in the 6- to 18-month-old age groups; these results indicate the early acquisition of antibody to the reovirus-like enteritis agent. The majority of children and young adults possessed CF- and immunofluorescence-stainable antibodies. There was a significant association between antibody prevalence data obtained with the human CF antigen and with the two NCDV antigens; this association was closest between the human and NCDV CF antigens.


Cytomegalovirus infection and immunity in renal allograft recipients: assessment of the competence of humoral immunity. Cytomegalovirus (CMV) infections are prevalent in renal allograft recipients. The purpose of this ongoing study is to attempt to elucidate the mechanism(s) responsible for the enhanced susceptibility to CMV infections on the part of transplant patients and for their apparent inability to eradicate the infection once it starts. The present report assesses the competence of humoral immunity to CMV in renal allograft recipients. The total study population was comprised of 41 renal allograft recipients (10 followed prospectively) and 38 age-matched control subjects. The overall CMV infection rate in renal allograft recipients was 90.2%, and in 11 cohort control subjects it was 45.5%. Active infection was present in 61.0% of transplant patients (24.0% of these had CMV disease) and in 18.2% of the cohorts. THESE DIFFERENCES ARE SIGNIFICANT. CMV complement fixation and neutralization antibody prevalence was similar in 10 patients with renal failure undergoing hemodialysis before transplantation and in 23 control subjects. There was similarly no difference in antibody response between allograft recipients in whom the infection was primary (eight subjects) or secondary (17 subjects). We conclude that despite immunosuppressant therapy (with azathioprine and corticosteroids), humoral immunity to CMV is not impaired in transplant patients with either a primary or secondary infection.


Bacillus pumilus polysaccharide cross-reactive with meningococcal group A polysaccharide. A polysaccharide, antigenically and structurally related to meningococcal group A polysaccharide, was isolated from Bacillus pumilus Sh-17. This enteric bacterium has been implicated as a source of natural meningococcal group A immunity (Myerowitz et al., 1973). The B. pumilus polysaccharide was composed of a homopolymer of (1-6)-N-acetyl-manosamine-1-phosphate, glycerol phosphate teichoic acid-containing N-acetylglucosamine and alkali-labile alanine esters, and a mucopeptide. The cross-reaction was due to the poly-(1-6)-N-acetyl-mannosamine-1-phosphate in the B. pumilus and the meningococcal group A polysaccharides, based on the following evidence. Both polysaccharides contained N-acetyl-mannosamine phosphate. Periodate oxidized the mannosamine phosphate residues of the polysaccharide and destroyed their precipitating activity with meningococcal group A antiserum. Mild acid treatment released phosphomonoesters and destroyed the meningococcal group A precipitating activity of both polysaccharides. N-acetyl-mannosamine-6-phosphate inhibited the precipitation reaction between strain Sh-17 and meningococcal group A antisera. Only mannosamine phosphate was detected in trichloroacetic acid extracts of Sh-17 polysaccharide and meningococcal group A antigen-antibody precipitates.


Antibodies to BK virus structural and tumor antigens in human sera from normal persons and from patients with various diseases, including neoplasia. Sera from 1,279 patients with various diseases were examined for the presence of antibodies to BK virus (BKV) capsid antigens. The percentage of positive sera was comparable in all the diseases except rheumatoid arthritis and chronic nephropathies, where a slightly higher prevalence was found. Sera from 952 patients with tumors were examined for the presence of antibodies to BKV tumor and capsid antigens in comparison with a matched control group of 501 blood donors. Sera from 11 tumor patients (1.15%) and from 4 normal controls (0.80%) had antibodies to BKV tumor antigen. No higher prevalence of antibodies to BKV capsid antigens was found in any cancer type except in carcinomas of the urinary bladder, where the percentage of positive sera and of sera with high titers was higher than in other groups. BKV infection is discussed in relation to its possible connection with human non-neoplastic diseases as well as with human tumors and to its activation under conditions of immunosuppressive therapy.


Antigenic analysis of isolated polypeptides from Visna virus. The antigenic activity of 10 Visna polypeptides separated by gel filtration in the presence of 6 M guanidine hydrochloride (GuHCl) was examined with rabbit antisera made specific for Visna virus. The results showed that the first (GuHCl 1) and the ninth (GuHCl 9) polypeptide peak reacted with the antisera when examined in immunodiffusion, passive hemagglutination, and complement fixation tests. Whole virus, GuHCl 1, and GuHCl 9, when tested with the antisera, appeared to be immunologically identical in the immunodiffusion test. However, GuHCl 1 reacted weakly with the antisera by all three techniques as compared with GuHCl 9 and whole virus. GuHCl 9, when subjected to polyacrylamide gel electrophoresis containing 0.1% sodium lauryl sulfate, revealed the presence of one polypeptide with a molecular weight of 25,000. By the same method, GuHCl 1 was found to contain an aggregate of four different polypeptides, the major one having a molecular weight of 25,000. The results indicate that the antigenic activity of both GuHCl 1 and GuHCl 9 was associated with a single polypeptide having a molecular weight of 25,000.


In vitro studies on Rickettsia-host cell interactions: lag phase in intracellular growth cycle as a function of stage of growth of infecting Rickettsia prowazeki, with preliminary observations on inhibition of rickettsial uptake by host cell fragments. Two Rickettsia prowazeki seeds, an "early" seed in the logarithmic or exponential growth phase and a "late" seed in the stationary or possibly early decline phase, were prepared in chicken embryo (CE) cell cultures and compared with respect to morphology and infection cycle in CE cells in culture. Differences in size and ultrastructure of the organisms in the two seeds were similar to those seen in other gram-negative bacteria at comparable stages to growth. Vacuolar structures, rare in log-phase organisms, were common in stationary-phase organisms. Minute spherical forms reminiscent of minicells were seen in the stationary-phase preparations. In quantitative uptake experiments, organisms, typical in size and morphology of each preparation, had comparable capacity per plaque-forming unit to penetrate into CE cells in suspension when the seeds had been depleted of host cell membrane fragments and other debris. This suggests that host cell fragments, presumably of membrane origin, competitively inhibit rickettsial uptake by intact CE cells. Organisms of the log-phase organisms displayed a lag phase of about 7.5 h, during which they enlarged and increased in intensity of staining, before entering the log phase of growth.


Diarrhea in gnotobiotic calves caused by the reovirus-like agent of human infantile gastroenteritis. Gnotobiotic newborn calves were found to be susceptible to infection with the reovirus-like agent of human infantile gastroenteritis (HRVL). Infection was based on (i) seroresponse using immunofluorescence and (ii) fecal shedding of virus particles using electron microscopy. Virus was detected in fecal samples for at least 2 to as long as 7 days after inoculation, although peak virus concentrations were observed on days 1 to 4. Diarrheal illness was observed in seven calves on second to fourth serial passage of HRVL in calves but in none of four animals studied on first passage. Diarrhea began 15 to 30.5 h (mean = 22.3 h) post-inoculation and lasted less than 24 h; three of the seven animals that developed diarrhea were also depressed or anorectic.


Pathogenesis of herpes simplex virus types 1 and 2 in mice after various routes of inoculation. The pathogenesis of herpes simplex virus (HSV) types 1 and 2 was compared after inoculation of mice by different routes. Intravaginal inoculation of HSV-1 and HSV-2 produced a local infection, with virus recovery from the vagina through 5 days. Virus was recovered from the spinal cords 4 to 5 days after inoculation but not from liver, kidney, lung, spleen, or blood. Intravenous or intraperitoneal inoculation of HSV-2 produced a focal necrotic hepatitis similar to that described previously (S. C. Mogenson, B. Teisner, and H.K. Andersen, 1974). The viral etiology of the liver lesions was confirmed by virus isolation (through 4 days) and electron microscopy. No evidence of infection of the kidney, lung, blood, or spleen was observed, although virus was isolated from spinal cord homogenates 7 days after inoculation. HSV-1 inoculation by the intraperitoneal or intravenous route resulted in virus isolation from the kidney during the 7-day harvest period, without producing overt pathological changes. Virus was isolated from spinal cord homogenates 2 to 3 days after HSV-1 inoculation but not from homogenates prepared from spleen, lung, or blood. Increases in serum transaminase activity were observed after systemic (intravenous) inoculation of HSV-2 but not after HSV-1 inoculation.


Assay of bovine interferons in cultures of the porcine cell line IB-RS-2. An assay for bovine interferons has been developed using the porcine cell line IB-RS-2 and a bovine enterovirus, CBV-D, as challenge virus. The method is based on estimation of cytopathic effect measured by uptake of neutral red. Teh assay is simple, sensitive, and reproducible. A comparative test of different viruses in IB-RS-2 cells and secondary calf kidney cells revealed that the sensitivity of a virus to interferon can vary up to 1,000-fold in the two cell systems. Vesicular stomatitis virus was found to be rather insensitive to interferon in IB-RS-2 cells.


Fetal and adult bovine interferon production during bovine viral diarrhea virus infection. Levels of interferon in adult bovine serum and in fetal bovine serum and tissues were examined during the course of transplacental bovine viral diarrhea virus infection. The cows produced circulating interferon between 2 and 9 days after viral inoculation, with mean peak levels in the serum on day 4. Interferon could be routinely detected in fetal tissues (e.g., thymus, spleen, and kidney) between days 4 and 21 after viral inoculation of the cows at 149 to 150 days of gestation (mid-second trimester) and in fetal serum from day 13 through day 21. Interferon was also detectable in the serum and tissues of fetuses from dams infected at day 95 of gestation (the beginning of the second trimester). In general, no differences were found between the ability of the adult and fetus to produce interferon. Fetal lamb kidney cells were more sensitive to the antiviral effects of bovine interferon than were fetal bovine kidney cells. The antiviral substance from the fetal and adult animals was characterized as interferon by standard criteria.


Improved yields of cell-free varicella-zoster virus. Systematic studies on the replication of varicella-zoster virus in infected human fetal diploid lung cells have defined more optimal conditions for infection and harvesting of cultures and have led to the production of cell-free virus preparations with infectivity titers of greater than or equal to 10(6) plaque-forming units per ml. The highest yields of cell-free virus were obtained by (i) sonic treatment of the cellular phase of cultures inoculated with trypsin-dispersed infected cells at ratios of 1 infected cell to 6 to 10 uninfected cells in the monolayer and (ii) harvesting cells after 24 to 36 h of incubation at 36 degrees C. At this time the cultures showed minimal viral cytopathic effect. Spread of infectivity occurred much more rapidly in cultures inoculated with whole infected cells than in those infected with cell-free virus. Complement-fixing antigens with improved titers of greater than or equal to 1:128 were prepared from varicella-zoster virus-infected cell cultures in the same manner as cell-free virus, but harvested after 3 to 4 days of incubation when the cultures showed an advanced cytopathic effect.


Rhesus monkeys kidney cells persistently infected with Simian Virus 40: production of defective interfering virus and acquisition of the transformed phenotype. Monolayer cultures of LLC-MK2 rhesus monkey kidney cells became persistently infected with simian virus 40 (SV40) when infected at a multiplicity of infection of 100 plaque-forming units/cell. A stable carrier state developed characterized by extensive viral proliferation without obvious cytopathic effect other than the slow growth of these cultures. By 11 weeks all cells produced the SV40 T antigen. In contrast, less than 5% of the cells produced V antigen. Virus-free clonal isolates were obtained by cloning in SV40 antiserum. Continuous cultivation in antiserum resulted in a temporary cure of unclone cultures. When virus did eventually reappear in the "cured" cultures the titers remained low. The virus produced by the carrier culture was defective at both 31 and 37% c, and it interfered with the growth of standard s40 during mixed infection of CV-1 green monkey kidney cells. All of the interfering activity in carrier culture homogenates could be sedimented by centrifugation at 109,000 x g for 3 h. These cultures were completely susceptible to vesicular stomatitis virus. Extensive viral deoxyribonucleic acid synthesis occurred in CV-1 cells infected with carrier culture virus. Carrier culture homogenates are only slightly less cytopathic to CV-1 cells than standard SV40. The carrier culture express several properties of SV40 transformation.


Analysis and modification of opiate reinforcement. The authors describe a research protocol for the evaluation of narcotic antagonists which examines the heroin-seeking behavior of hard-core heroin addicts on a research ward under blocked and unblocked conditions. Each patient served as his own control. This paper serves as an introduction to a series of papers which follow dealing with behavioral, psychiatric, and aftercare results. It describes detailed methods and preliminary results for the first 21 subjects admitted to the study. More specific results are reported in the papers that follow.


Opiate antagonists and the modification of heroin self-administration behavior in man: an experimental study. The heroin self-administration behavior of 8 inpatient heroin addicts was examined for 10 days under blocked (i.e., following ingestion of narcotic antagonists--naloxone or naltrexone) and unblocked (no antagonist) conditions. In the unblocked state, subjects injected all the available heroin, but they ceased heroin use almost completely following antagonist administration. Possible explanations for these results are discussed along with their implications for treatment.


Aftercare on narcotic antagonists: prospects and problems. This study examines the problems and prospects of aftercare of the multiple treatment failure opiate addict who is being treated with narcotic antagonists (primarily naltrexone). At the time of this paper, 21 patients had been admitted to the Drug Addiction Research Project at McLean Hospital, with a retention rate of 42%. "Completers," "noncompleters," and "dropouts" could not be differentiated on the basis of demographic or psychiatric variables, thus making it impossible to define the population most suited for antagonist treatment. The degree of carry-over into the first month in the community, for those who successfully completed the inpatient segment of the program, disappeared over time. The nonreinforcing (lack of euphoric effect) properties of the antagonists and the resultant high level of motivation necessary for the continuation of its use by the patient make this form of treatment suitable for only a small percentage of the "hard core" addict population.


Free radicals in pyrimidines: 5-thymyl radical nitrogen splitting in irradiated thymidine. Single crystals of thymidine irradiated with 4.0 MeV electrons exhibit the well-known eight-line e.s.r. spectrum due to the 5-thymyl radical. A careful analysis of these lines revealed an extra hyperfine splitting, which by a combination of INDO-MO calculations and experiments, was attributed to an interaction between the unpaired electron and the N3-H group.


Cytomegalovirus hepatitis in an artificial kidney unit. Serum hepatitis is a dreaded risk in connection with regular dialysis treatment (RDT). Liver damage, however, can be cuased by other diseases, such as infection with cytomegalovirus (CMV). Two cases in our artificial kidney unit revealed signs of liver damage with increased liver enzyme activity. Case 1, a woman, was on RDT after an unsuccessful renal transplantation, and Case 2, a man, belonged to the staff. Serum hepatitis was initially suspected in both cases, but repeated examinations of the sera revealed no hepatitis B antigen or antibodies (HbAg and HbAb). Later on, both showed a significant increase in antibodies  in complement fixations reaction (CF) to CMV-antigen. CMV could be isolated from urine in Case 2. Case 1 had been bilaterally nephrectomized. The symptoms (tiredness, muscle pain and headache) and the course of the disease were mild in both cases and liver enzymes became normal within 1-2 weeks. Twenty out of 31 examined patients and staff had antibodies in CF to CMV-antigen, but in none was there any significant increase. The source of infection may have been transfusion of fresh blood in Case 1, but in Case 2 no particular source could be suspected. Thus, in liver damage CMV-infection may be an etiological alternative. In routine work at artficial kidney unite patients and personnel are regularly examined in respect of bilirubin, liver enzymes, HbAg and HbAb in serum. We recommend also examination of serum for antibodies in CF to CMV-antigen. Until a firm differential diagnosis has been established the patient should be isolated and the dialysis equipments used only by that patient.


Ultrastructural cytology of prostate carcinoma cells from Wistar rats. Cells from spontaneous adenocarcinomas of the prostate (Pollard, M.: J. Natl. Cancer Inst. 51: 1235, 1973) in aged Lobund Wistar rats were examined by electron microscopy. Cytologic structures of the rat prostate tumor cells resembled analogous structures from human prostate tumor cells. These findings support the prospect that the rat prostate tumor will provide a model system of prostate cancer in man.


[Virology of acute upper respiratory tract infections (author's transl)]. The virology of acute upper respiratory tract infections is presented in a survey. The first part includes general properties of viruses (structure, replication, maturation, classification), aetiological and immunological problems of viral infections. The different respiratory viruses and their clinical picture in particular are summarized in the second part.


The new program of the World Health Organization in medical virology. The World Health Organization (WHO) convened a Scientific Group to adapt its program in virus diseases to recent progress in virology. The program consists of (a) general activities, such as reference services and the supplying of reagents by the WHO Collaborating Centres and (b) specific activities to solve problems-including the promotion of necessary research-caused by certain diseases of public health importance. The Group reviewed problems caused by influenza and other respiratory viruses, enteroviruses, gastroenteritis viruses (for which types A and B have been proposed as a convenient nomenclature), viral hepatitis, viruses in water and sewage, arboviruses, arenaviruses and Marburg virus, measles and rubella vaccination, smallpox, rabies, chronic infections, herpesviruses, oncogenic viruses, congenital infections, nosocomial infections, chlamydial and rickettsial infections, and mycoplasma infections.


Characterization of single-cell clonal lines derived from HSV-2-transformed mouse cells. A BALB/c continuous cell line, 238, was transformed following infection with UV-inactivated herpes simplex virus type 2 (HSV-2) was designated H238. Several cell lines were derived from single cells of H238 by cloning in soft agar. The clonal lines express cytoplasmic antigens by immunofluorescence when reacted with rabbit HSV-2 antiserum and mouse tumor-bearer serum. The cloned cell lines induce tumors in immunocompetent mice by the subcutaneous route with a 100% incidence at an inoculum of 10(6) cells. These properties are consistent with those of the H238 parent cell line. In contrast to the parent line, however, the cloned cell lines do not elicit HSV neutralizing antibody in the tumor-bearing host.


Infiltrative corneal lesions resembling fibrous histiocytoma: clinical and pathologic findings in six dogs and one cat. Infiltrating corneal lesions developed in 6 dogs and 1 cat. In each case, the site of origin appeared to be the corneal limbus. The lesions were characterized by continuous growth, a benign appearance, and a tendency to recur following excision keratoplasty. Each lesion was of a proliferative, inflammatory nature, histologically resembling fibrous histiocytoma. Of the 6 dogs in the series, 4 were Collies.


Test method for determination of virucidal efficacy of liquid surface disinfectants. A test method was developed incorporating AOAC use-dilution stainless steel Penicylinders and H.Ep.-2 tissue culture cells for the evaluation of disinfectants as virucides. The subsequent collaborative study using Herpes simplex and poliovirus type 1 in evaluation against a quaternary ammonium compound, phenolic, and iodiphor disinfectants indicated that the basic test procedure is sound. However, an improved virus concentration technique and a modified method for drying virus-contaminated cylinders are recommended. In addition, other types of disinfectants and test viruses should be included in order to demonstrate that the procedure is versatile as well as reproducible.


Silicic acid chromatographic separation of polychlorinated biphenyls and pesticides: some contaminants and limitations. The Armour and Burke method for separating polychlorinated biphenyls (PCBs) from pesticides involves the use of silicic acid. However, we detected di-2-ethylhexyl phthalate, PCBs, and other impurities as interfering contaminants in several batches of silicic acid. The presence of H2SO4 in some batches of silicic acid is inferred. The acid may be responsible for the production of contaminants which interfere in gas-liquid chromatographic analyses. Contaminants in silicic acid are reduced by extracting the adsorbent with solvent, and/or partitioning the concentrated pesticide fraction with 1N NaOH. These purification procedures provide separations relatively free of impurities, but result in reduced adsorbent activity. PCB-pesticide separations are reproducible only within a given batch lot of silicic acid because of the varying adsorbent characteristics of each lot. Alternative adsorbents should be explored for most PCB-pesticide separations.


High-pressure liquid chromatography of benzo(a) pyrene and benzo (ghi) perylene in oil-contaminated shellfish. A high-pressure liquid chromatographic procedure is described for the determination of benzo(a) pyrene and benzo(ghi) perylene. These polynuclear aromatics are extracted with acetonitrile and partitioned into petroleum ether, the petroleum ether is removed, and the residue is saponified. The compounds are purified and isolated by passing the residue through a silica gel column and a high-pressure liquid chromatographic column, and detected by their ultraviolet absorption. Recoveries of standards through the procedure averaged 104%.


Ca2+-induced conformational changes of spin-labeled g2 chain bound to myosin and the effect of phosphorylation. One of the low molecular weight components of myosin, g2, was isolated by alkali treatment of myosin and was chemically modified with a spin label reagent, 4-maleimido-2,2,6,6-tetramethylpiperidinooxyl. The label on g2 showed a rather weakly immobilized ESR spectrum and it was clearly affected by Ca2+; the half-maximal change was at around pCa 4. The spin-labeled g2 was incorporated into myosin by exchange with the intrinsic g2 of myosin in 0.6 M KSCN or 4 M LiC1. The label on g2 became strongly immobilized on association with myosin. Under the conditions used, ESR spectral change due to Ca2+ occurred at two different concentration ranges, which were as low as pCa 8 and at around pCa 4. Phosphorylated g2 was isolated from myosin after the protein kinase [EC 2.1.1.37]-catalyzed phosphorylation of myosin and it was also modified with the maleimide label. Dephosphorylation of the phosphorylated g2 was performed using E. coli alkaline phosphatase [EC 3.1.3.1]. The effects of Ca2+ on the ESR spectra of phosphorylated and dephosphorylated g2 were investigated on the state associated with myosin. A change in the ESR spectrum from strongly immobilized to weakly immobilized states was observed with both g2 chains on the addition of Ca2+. However, the effective concentration  ranges of Ca2+ were quite different; around pCa 4 for the phosphorylated g2 and around pCa 8 for the dephosphorylated g2. The results indicate that g2 undergoes a conformational change at physiological levels of Ca2+ sufficient to saturate troponin, but it does not do so after phosphorylation.


In vitro polymerization of flagellar and ciliary outer fiber tubulin into microtubules. About 10--20% of the total protein in the outer fiber fraction was solubilized by sonication in a solution containing 5 mM MES, 0.5 mM MgSO4, 1.0 mM EGTA, 1.0 mM GTP, and 0 or 50 mM KC1 at pH 6.7. The sonicated extract was shown by analytical centrifugation to consist largely of a 6 S component (tubulin dimer), having a molecular weight of 103,000, as determined by gel filtration, and possessing a colchicine-binding activity of 0.8 mole per tubulin dimer. The tubulin fraction failed to polymerize into microtubules by itself. Addition of a small amount of the ciliary outer fiber fragments or reconstituted short brain microtubules, however, induced polymerization, as demonstrated by viscosity of flow birefringence changes as well as light or electron microscopic observations. The growth of heterogeneous microtubules upon mixing outer fiber tubulin with DEAE-dextran-decorated brain microtubules was observed by electron microscopy. Microtubules were reconstituted from outer fiber tubulin without addition of any nuclei fraction when a concentrated tubulin fraction was warmed at 35degree. A few doublet-like microtubules or pairs of parallel singlet microtubules that were closely aligned longitudinally could be observed among many singlet microtubules. Unlike other fiber microtubules, the reconstituted polymers were depolymerized by exposure to Ca2+ ions, high or low ionic strength, colchicine, low temperature or SH reagents. No microtubules were assembled under these conditions.


Synthesis of various phosphodiesters and phosphomonoesters with ribonuclease N. 1. 3'-Guanylyl-ethanol, 3'-guanylyl-propanol, and 3'-guanylyl-alpha-glycerol were synthesized by ribonuclease N1 [EC 3.1.4.8] using guanosine 2',3'-cyclic phosphate as a phosphate donor and various alcohols as phosphate acceptors. The yields of these phosphodiesters were 15%, 13.5%, 38.2%, respectively, with respect to phosphate donor under the optimum conditions. No phosphodiester was synthesized when 2-propanol was used as a phosphate acceptor. Thus, primary alcoholic hydroxyl groups may be regarded as the preferred phosphate acceptor. 2. 3'-Guanylyl-glucose and 3'-guanylyl-ribose were synthesized using glucose and ribose as phosphate acceptors. Under the optimum conditions, the yields of guanylyl-glucose amounted to 52.0%, while that of guanylyl-ribose was much lower. The guanylyl-glucose can be regarded as 3'-guanylyl-6-glucopyranose, based on the results of periodate oxidation. 3. Neither hydroxyamino acids (serine and threonine) nor N-acetylserinamide could be phosphorylated under the conditions used for the above phosphorylations. 4. 3'-Guanylyl-glycerol obtained as above was hydrolyzed by snake venon phosphodiesterase to produce glycerol 3-phosphate. The latter consisted of L-glycerol 3-phosphate (ca 17%) and the D-isomer (ca. 83%). Ribonuclease N1 thus catalyzes an asymmetric synthesis.


Physical and chemical studies on bacterial superoxide dismutases. Purification and some anion binding properties of the iron-containing protein of Escherichia coli B. Highly purified iron superoxide dismutase was obtained from Escherichia coli B using a modification of the procedure of Yost and Jridovich (Yost, F. J., Jr., and Fridovich, I. (1973) J. Biol. Chem. 248, 4905-4908). The protein contained 1.8 +/- 0.2 atoms of iron per 38,700 g of protein. We have found that cyanide does not bind to the Fe3+ ion of iron dismutase but fluoride and azide have moderately large binding constants. Optical and electron paramagnetic resonance (EPR) measurements suggested that 2 fluoride ions could associate with each iron atom with the first having an association constant of approximately 520 M-1 and the second with an estimated value of 24 M-1. Activity measurements yielded an inhibition constant for fluoride of 30 M-1. At room temperature only one azide binds to the Fe3+ (K = 760 M-1) and this does not interfere with superoxide dismutase activity. Upon freezing solutions of iron superoxide dismutase in the presence of excess azide their color changes from yellow to pink. Combined EPR and optical titrations with azide suggest the presence of two binding sites on Fe3+ with only the first being occupied at room temperature and the second binding azide only upon freezing the solution. The results suggest that each Fe3+ ion of this superoxide dismutase has two coordination positions available for interaction with solute molecules but only one is necessary for catalysis of the superoxide dismutation reaction. The EPR, optical, and circular dichroism spectra of the native protein and the various fluoride and azide complexes are presented.


Studies on the effects of coenzyme A-SH: acetyl coenzyme A, nicotinamide adenine dinucleotide: reduced nicotinamide adenine dinucleotide, and adenosine diphosphate: adenosine triphosphate ratios on the interconversion of active and inactive pyruvate dehydrogenase in isolated rat heart mitochondria. The content of coenzyme A-SH (CoASH) and acetyl-CoA of suspensions of rat heart mitochondria was stabilized by the addition of DL-carnitine and acetyl-DL-carnitine, in the presence of the respiratory inhibitor rotenone. The mitochondrial content of NAD+ and NADH was similarly stabilized by the addition of acetoacetate and DL-3-hydroxybutyrate, and the content of ADP and ATP was imposed by the addition of these nucleotides to the mitochondrial suspension, in the presence of uncoupling agent and oligomycin, to inhibit ATPase. Under these conditions, mitochondrial CoASH/acetyl-CoA, NAD+/ NADH, and ADP/ATP ratios could be varied independently, and the effect on the interconversion of active and inactive pyruvate dehydrogenase could be studied. Decreases in both CoASH/acetyl-CoA and NAD+/NADH ratios were shown to be inhibitory to the steady state activity of pyruvate dehydrogenase, and this effect is described at three different ADP/ATP ratios and different concentrations of added MgCl2. A new steady state level of activity was achieved within 10 min of a change in either CoASH/acetyl-CoA or NAD+/NADH ratio; the rate of inactivation was much higher than the rate of reactivation under these conditions. Effects of CoASH/acetyl-CoA and NAD+/NADH may be additive but are still quantitatively lesser than the changes in activity of pyruvate dehydrogenase induced by changes in ADP/ATP ratio. The variation in activity of pyruvate dehydrogenase with ADP/ATP ratio is described in the absence of changes in the other two ratios, conditions which were not met in earlier studies which employed the oxidation of different substrates to generate changes in all three ratios.


Regulation of carbohydrate permeases and adenylate cyclase in Escherichia coli. Studies with mutant strains in which enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system is thermolabile. Carbohydrate uptake and cyclic adenosine 3':5'-monophosphate (cyclic AMP) synthesis were studied employing mutant strains of Escherichia coli in which Enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system was heat-labile. Partial loss of Enzyme I activity, which resulted from incubation of cells at the nonpermissive temperature, depressed the rate and extent of methyl alpha-glucoside uptake. Temperature inactivation of Enzyme I also rendered cyclic AMP synthesis and the uptake of several carbohydrates (glycerol, maltose, melibiose, and lactose) hypersensitive to inhibition by methyl alpha-glucoside. Protein synthesis did not appear to be required for these effects. The parental strains and "revertant" strains in which Enzyme I was less sensitive to temperature did not exhibit heat-enhanced regulation. Inhibition was abolished by the crr mutation. The results suggest that Enzyme I functions as a catalytic component of the regulatory system. Simple positive selection procedures are described for the isolation of bacterial mutants which are deficient for either Enzyme I or the heat-stable protein of the phosphotransferase system.


Binding of progesterone receptor by nuclear preparations of rabbit and guinea pig uterus. Guinea pig and rabbit uterine nuclei bound [3H] progesterone in vitro only in the presence of cytosol from estrogen-stimulated uteri. Nuclei from unstimulated and estrogen-stimulated uteri bound progesterone equally well. Nuclei of nontarget tissues also bound progesterone, but to a lesser extent. The rate of nuclear bindins increased with temperature from 0-30 degrees. At 25 degrees nuclear binding remained stable for at least 3 h, but at temperatures of 30 degrees and greater, nuclear binding decreased rapidly after 15 min. Activation of the progesterone-cytoplasmic receptor complex (the change in the complex that enables it to bind quickly to nuclei at 0 degrees) took place slowly at temperatures from 0-5 degrees and rapidly at 10-25 degrees. Activation was facilitated by dilution of the cytosol. Some activation occurred in diluted cytosol in the absence of added progesterone. The cytoplasmic progesterone receptor had a sedimentation coefficient of 7 S when concentrated cytosol (20 mg of protein/ml) was incubated with progesterone at 0 degrees in 5 mM phosphate buffer. Diluting the cytosol and increasing the temperature to 20 degrees caused the sedimentation coefficient to decrease to 5.5 S. Gel filtration of guinea pig uterine cytosol on Sephadex G-100, in the absence of progesterone, yielded a progesterone-binding fraction in the void volume, with a sedimentation coefficient of 5.5 S. The complex of progesterone with the material in the void volume was taken up by nuclei at 0 degrees more rapidly than the complex of progesterone and crude cytosol. The nuclear uptake of progesterone was decreased in phosphate buffer of concentrations greater than 80 mM. Under conditions that favor the nuclear binding of progesterone, the sedimentation coefficient of the cytoplasmic progesterone receptor was 5.5 S. This may be the form of the preceptor which is taken up by nuclei. In decreasing order of effectiveness, unlabeled progesterone, 5 alpha-pregnane-3,20-dione, corticosterone 20 alpha-hydroxy-4-pregnen-3-one, testosterone, estradiol-17 beta, and cortisol competed with [3H] progesterone for binding to nuclei.


Binding properties of androgen receptors. Evidence for identical receptors in rat testis, epididymis, and prostate. Androgen receptors in crude and partially purified 105,000 X g supernatant fractions from rat testis, epididymis, and prostate were studied in vitro using a charcoal adsorption assay and sucrose gradient centrifugation. Androgen metabolism was eliminated during receptor purification allowing determination of the kinetics of [3H]-androgen-receptor complex formation. In all three tissues, receptors were found to have essentially identical capabilities to bind androgen, with the affinity for [3H] dihydrotestosterone being somewhat higher than for [3H] testosterone. Equilibrium dissociation constants for [3H] dihydrotestosterone and [3H] testosterone (KD = 2 to 5 X 10(-10) M) were estimated from independently determined rates of association (ka congruent to 6 X 10(7) M-1 h-1 for [3H] dihydrotestosterone and 2 X 10(8) M-1 h-1 for [3H] testosterone) and dissociation (t 1/2 congruent to 40 hr for [3H] dihydrotestosterone and 15 h [3H] testosterone). Evaluation of the effect of temperature on androgen receptor binding of [3H]testosterone allowed estimation of several thermodynamic parameters, including activation energies of association and dissociation (delta H congruent to 14 kcal/mol), the apparent free energy (delta G congruent to -12 kcal/mol), enthalpy (delta H congruent to -2.5 kcal/mol), and entropy (delta S congruent to 35 cal col-1 K-1). Optimum receptor binding occurred at a pH of 8. Receptor stability was greatly enhanced when bound with androgen. Receptor specificity for testosterone and dihydrotestosterone was demonstrated by competitive binding assays. The potent synthetic androgen, 7 alpha, 17 alpha-dimethyl-19-nortestosterone, inhibited binding of [3H] testosterone or [3H] dihydrotesterone nearly as well as testosterone and dihydrotestosterone while larger amounts of 5 alpha-androstane-3alpha, 17 beta-diol and nonandrogenic steroids were required. Sedimentation coefficients of androgen receptors in all unfractionated supernatants were 4 and 5 to 8 S. Differences in sedimentation coefficients were observed following (NH4)2SO4 precipitation which did not influence the binding properties of the receptors. These results, together with measurements of3alpha/beta-hydroxysteroid oxidoreductase activity in vitro, suggest that organ differences in receptor binding of [3H] dihydrotestosterone and [3H] testosterone in vivo result from relative differences in intracellular concentrations of these androgens rather than from differences in receptor affinities.


Cyclid 3':5'-nucleotide phosphodiesterase. Interconvertible multiple forms and their effects on enzyme activity and kinetics. An extract of rat liver or human platelet displayed three cyclic 3':5'-nucleotide phosphodiesterase activity peaks (I, II, and III) in a continuous sucrose density gradient when assayed with millimolar adenosine 3':5'-monophosphate (cAMP) or guanosine 3':5'-monophosphate (cGMP). The three fractions obtained from each nucleotide were not superimposable. The molecular weights corresponding to the three activity peaks of cAMP phosphodiesterase in rat liver were approximately: I, 22,000; II, 75,000; and III, 140,000. In both tissues, fraction I was barely detectable when assayed with micromolar concentrations of either nucleotide, presumably because fraction I has low affinity for cAMP and cGMP. Any one of the three forms upon recentrifugation on the gradient generated the others, indicating that they were interconvertible. The multiple forms appear to represent different aggregated states of the enzyme. The ratio of the three forms of cAMP phosphodiesterase in the platelet was shifted by dibutyryl cAMP (B2cAMP) and by the enzyme concentration. B2cAMP enhanced the formation of fraction I. Low enzyme concentration favored the equilibrium towards fraction I, while high enzyme concentration favored fraction III. When phosphodiesterase activities in the extract of rat liver, human platelets, or bovine brain were examined as a function of enzyme concentration, rectilinear rates were observed with micromolar, but not with millimolar cAMP or cGMP. The specific activity with millimolar cAMP was higher with low than with high protein concentrations, suggesting that the dissociated form catalyzed the hydrolysis of cAMP faster than that of the associated form. In contrast, the specific activity with millimolar cGMP was lower with low than with high protein concentrations. Supplementing the reaction mixture with bovine serum albumin to a final constant protein concentration did not affect the activity, suggesting that the concentration of the enzyme rather than that of extraneous proteins affected the enzyme activity. A change in enzyme concentration affected the kinetic properties of phosphodiesterase. A low enzyme concentration of cAMP phosphodiesterase yielded a linear Lineweaver-Burk plot, and a Km of 1.2 X 10(-4) M (bovine), 3 X 10(-5) M (platelet), or 5 X 10(-4) M (liver), while a high enzyme concentration yielded a nonlinear plot, and apparent Km values of 1.4 X 10(-4) M and 2 X 10(-5) M (brain), 4 X 10(-5) M and 3 X 10(-6) M (platelet), or 4 X 10(-5) M and 3 X 10(-6) (liver). Since a low enzyme concentration favored fraction I, the dissociated form, whereas a high enzyme concentration favored fraction III, the associated form, these kinetic constants suggest that the dissociated form exhibits a high Km and the associated form exhibits a low Km. In contrast, a high enzyme concentration gave a linear kinetic plot for cGMP phosphodiesterase, while a low enzyme concentration gave a nonlinear plot...


Conformational stability of the polypeptide components of human high density serum lipoprotein. An investigation of the denaturation of the major polypeptides from human serum high density lipoprotein by guanidine hydrochloride reveals that the lipid-free, water-soluble states are minimally stable relative to the random coil states. These findings are in direct contrast to the resistance of intrinsic membrane proteins (in the absence of ligand) to complete unfolding by the same denaturant. Consideration of the denaturation data for the apolipoproteins together with previously published data from this laboratory on ligand-induced conformational changes indicate that these polypeptides possess several similar conformational states which are readily interconvertible.


Amino acid transport in isolated rat thymocytes. Effects of divalent cations and ethanol. In dispersed rat thymocytes neither basal alpha-aminoisobutyric acid influx nor influx stimulated by insulin, prostaglandin theophylline, or butyryl adenosine 3':5'-monophosphate (cyclic AMP) depended on extracellular calcium or magnesium. The divalent cation ionophore A23187 inhibited both basal and stimulated alpha-aminoisobutyric acid influx. The extent to which influx was inhibited depended on ionophore concentration, extracellular calcium concentration, and time but did not depend on extracellular magnesium. Significant inhibition could be detected at an ionophore concentration of 1 muM and maximal inhibition occurred with 6 muM A23187. A23187 increased cellular uptake of calcium and there was good agred calcium uptake and that for ionophore inhibition of alpha-aminoisobutyric acid influx. Incubating cells with A23187 and then adding ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N',-tetraacetic acid completely reversed ionophore-stimulated cellular calcum uptake but did not reverse inhibition of alpha-aminoisobutyric acid influx. Thus, A23187 produces irreversible inhibition of alpha-aminoisobutyric acid transport in dispersed rat thymocytes. Ethanol abolished insulin-stimulated alpha-aminoisobutyric acid influx but did not alter basal influx or that stimulated by prostaglandin E1, theophylline, or N6,O2'-dibutyryl adenosine 3':5'-monophosphate. Inhibition could be detected with 0.2% (v/v) ethanol and insulin-stimulated alpha-aminoisobutyric influx was abolished with 1% ethanol. The effect of ethanol occurred immediately and could be reversed completely. This ability of ethanol to inhibit selectively insulin-stimulated alpha-aminoisobutyric acid influx indicates that the mechanism through which insulin stimulates alpha-aminoisobutyric acid influx is functionally distinct from the stimulation produced by cyclic AMP.


Hormone secretion and glucose metabolism in islets of Langerhans of the isolated perfused pancreas from normal and streptozotocin diabetic rats. The glucose responsiveness of alpha- and beta-cells of normal as well as untreated and insulin-treated streptozotocin diabetic rats was tested in the extracorporeal perfusion system. Also assessed was the possible in vitro effect of added insulin on the glucose sensitivity of islets from untreated diabetic animals. Insulin and glucose responsiveness of the two cell types. The rate of glucose entry islet tissue was estimated, and the effect of glucose on the tissue supply of ATP and lactate and the cyclic 3':5'-AMP level of islets was measured under the above in vitro conditions. It was demonstrated that beta-cells are more accessible to glucose than alpha-cells, that glucose entry into islet cells is not significantly modified by insulin and that glucose had no effect on ATP, lactate and cyclic 3':5'-AMP levels of islet tissue under any of the conditions investigated. High insulin in vitro elevated ATP levels of alpha-cell islets independent of extracellular glucose. Glucose caused insulin release from normal but not from diabetic islets and rapidly and efficiently suppressed stimulated glucagon secretion of the pancreas from normal and insulin treated diabetic rats. Glucose was less effective in inhibiting stimulated glucagon secretion by the pancreas from untreated diabetic rats whether insulin was added to the perfusion media or not. Therefore, profound differences of glucose responsiveness of alpha-cells fail to manifest themselves in alterations of basic parameters of glucose and energy metabolism in contrast to what had been postulated in the literature. It is however, apparent that the glucose responsiveness of alpha-cells is modified by insuling by an as yet undefined mechanism.


Purification and characterization of adrenal cortex mitochondrial cytochrome P-450 specific for cholesterol side chain cleavage activity. Cytochrome P-450 was purified from bovine adrenal cortex mitochondria by affinity chromatography using an octylamine-substituted Sepharose column. The resulting optically clear preparation was stable at -20 degrees for months. The specific concentration of cytochrome P-450 in the preparation was about 5 nmol of heme per mg of protein. The preparations were free of adrenodoxin, adrenodoxin reductase, phospholipids, and other heme contaminations. Polyacrylamide gel electrophoresis of the purified cytochrome P-450 preparation treated with sodium dodecyl sulfate and mercaptoethanol showed a single major band with a molecular weight of about 60,000. The optical absorption spectra of the preparation exhibited Soret maxima at 416, 416, and 448 nm for the Fe3+, Fe2+ and the C.Fe2+ complex, respectively. The EPR spectrum showed the characteristic features of the low spin form of ferric cytochrome P-450 with principal components 1.914, 2.241, and 2.415 of the g-tensor. The circular dichroism spectrum revealed two large negative ellipticities at 412 and 350 nm. Fluorescence spectra showed an excitation maximum at 285 nm and an emission maximum at 305 nm with a shoulder at 330 nm as the cytochrome P-450 molecule is excited at 285 nm, or an emission maximum at 335 nm when the cytochrome molecule is excited at 305 nm. After reconstitution with adrenodoxin and its reductase, this cytochrome P-450 was highly active for cholesterol desmolase with an NADPH-generating system as electron donor but was not active for steroid 11beta-hydroxylase.


Biochemical aspects of cardiac muscle differentiation. Possible control of deoxyribonucleic acid synthesis and cell differentiation by adrenergic innervation and cyclic adenosine 3':5'-monophosphate. A single injection of either isoproternol or N6, O2'-dibutyryl adenosine 3':5'-monophosphate (dibutyryl cyclic AMP) results in an inhibition in the rate of [3H]thymidine incorporation into DNA of differentiating cardiac muscle of the neonatal rat. This inhibition is not due to substantially altered cellular uptake or catabolism of [3H]thymidine. Inhibition of [3H]thymidine incorporation by isoproterenol or dibutyryl cyclic AMP is potentiated by theophylline. Maximal inhibition (95%) is observed 24 h after administration of isoproterenol, and the rate of incorporation returns to a value 80% of control by 72 h. Norepinephrine also inhibits [3H]thymidine incorporation whereas cyclic GMP, N2, 02-Dibutyryl guanosine 3':5'-monophosphate (dibutyryl cyclic GMP), and phenylephrine have little effect. Equilibrium sedimentation analysis of cardiac muscle DNA in neutral and alkaline cesium chloride gradients using bromodeoxyuridine as a density label indicate that isoproterenol and dibutyryl cyclic AMP inhibit [3H]thymidine incorporation into DNA that is replicating semiconservatively. Administration of isoproterenol or dibutyryl cyclic AMP to neonatal rats inhibits by approximately 60% the incorporation of [3H]thymidine into DNA of tissue slices of cardiac muscle prepared 16 h later. [3H]Thymidine incorporation into DNA of tissue slices is into chains that were growing in vivo. This incorporation is linear for at least 4 h of incubation and is inhibited by isoproterenol and dibutyryl cyclic AMP. Inhibition is not due to altered cellular uptake of [3H]thymidine nor is it due to a cytotoxic action. Several other compounds which elevate intracellular levels of cyclic AMP (epinephrine, norepinephrine, glucagon, and prostaglandin E1) also inhibit [3H]thymidine incorporation into DNA or cardiac muscle tissue slices. Cyclic GMP, dibutyryl cyclic GMP, sodium butyrate, and phenylephrine have little effect. Isoproterenol administered together with theophylline to neonatal rats signficantly stimulates the in corporation of [3H]phenylalanine into total cardiac muscle protein and into myosin. This enhanced incorporation may be due in part to an increase in the cellular uptake of [3H]phenylalanine. DNA synthesis  decreases progressively in differentiating cardiac muscle of the rat during postnatal development and essentially ceases by the middle of the third week (Claycomb, W. C. (1975) J. Biol. Chem. 250, 3229-3235). In reviewing the literature it was found that this decline in synthetic activity correlates temporally with a progressive increase in tissue concentrations of norepinephrine and cyclic AMP and with the anatomical and physiological development of the adrenergic nerves in this tissue. Because of these facts and data presented in this report it is proposed that cell proliferation and cell differentiation in cardiac muscle may be controlled by adrenergic innervation with norepinephrine and cyclic AMP serving as chemical mediators.


Leghemoglobin. An electron paramagnetic resonance and optical spectral study of the free protein and its complexes with nicotinate and acetate. Electron paramagnetic resonance (EPR) and optical spectra are used as probes of the heme and its ligands in ferric and ferrous leghemoglobin. The proximal ligand to the heme iron atom of ferric soybean leghemoglobin is identified as imidazole by comparison of the EPR of leghemoglobin hydroxide, azide, and cyanide with the corresponding derivatives of human hemoglobin. Optical spectra show that ferric soybean leghemoglobin near room temperature is almost entirely in the high spin state. At 77 K the optical spectrum is that of a low spin compound, while at 1.6 K the EPR is that of a low spin form resembling bis-imidazole heme. Acetate binds to ferric leghemoglobin to form a high spin complex as judged from the optical spectrum. The EPR of this complex is that of high spin ferric heme in a nearly axial environment. The complexes of ferrous leghemoglobin with substituted pyridines exhibit optical absorption maxima near 685 nm, whose absorption maxima and extinctions are strongly dependent on the nature of the substitutents of the pyridine ring; electron withdrawing groups on the pyridine ring shift the absorption maxima to lower energy. A crystal field analysis of the EPR of nicotinate derivatives of ferric leghemoblobin demonstrates that the pyridine nitrogen is also bound to the heme iron in the ferric state. These findings lead us to picture leghemoglobin as a somewhat flexible molecule in which the transition region between the E and F helices may act as a hinge, opening a small amount at higher temperature to a stable configuration in which the protein is high spin and can accommodate exogenous ligand molecules and closing at low temperature to a second stable configuration in which the protein is low spin and in which close approach of the E helix permits the distal histidine to become the principal sixth ligand.


Synthesis of the mitochondrial inner membrane in cultured Xenopus laevis oocytes. The purpose of this study was to investigate the contribution of mitochondrial and cytoplasmic protein synthesis to the biogenesis of cytochrome oxidase (ferrocytochrome c:oxygen oxidoreductase EC 1.9.3.1) and rutamycin-sensitive adenosine triphosphatase (ATP phosphohydrolase EC 3.6.1.3) in cultured oocytes of the toad, Xenopus laevis. X. laevis cytochrome oxidase was purified over 23-fold with respect to specific activity and over 29-fold with respect to specific heme a content from oocyte submitochondrial particles. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate separated the enzyme into six subunits with molecular weights of 44,000, 33,000, 23,000, 17,000, 12,000 and 9,500. the synthesis of the three larger subunits is sensitive to chloramphenicol (an inhibitor of mitochondrial protein synthesis), indicating that these subunits are made on mitochondrial ribosomes; the synthesis of the three smaller subunits is sensitive to cycloheximide (an inhibitor of cytoplasmic protein synthesis) and therefore occurs on cytoplasmic ribosomes. X. laevis rutamycin-sensitive ATPase, purified over 19-fold from oocyte submitochondrial pparticles, consists of 10 subunits with molecular weights of 56,000, 53,000, 41,000, 32,000, 29,000, 24,000, 21,000, 17,500 (2), and 11,500 on sodium dodecyl sulfate-polyacrylamide gels. The 29,000, 21,000, and one of the 17,500-dalton polypeptides are synthesized in the presence of cycloheximide and are, therefore, products of mitochondrial protein synthesis; the synthesis of the remaining seven subunits occurs in the presence of chloramphenicol, indicating that these subunits are made on cytoplasmic ribosomes. The synthesis of protein by mitochondria in cultured oocytes appears to be dependent upon cytoplasmic protein synthesis. In the presence of cycloheximide, the mitoribosomal synthesis of the subunits of cytochrome oxidase and rutamycin-sensitive ATPase is detectable only after a prior inhibition of mitochondrial protein synthesis by chloramphenicol. Oocyte mitochondrial ribosomes synthesize at least nine polypeptides after chloramphenicol treatment, three of which are components of neither cytochrome oxidase nor rutamycin-sensitive ATPase.


Structure of glycogen phosphorylase a at 3.0 A resolution and its ligand binding sites at 6 A. A model of the polypeptide backbone of the dimer of glycogen phosphorylase a (EC 2.4.1.1) was built from a 3 A resolution electron density map derived from x-ray diffraction analysis of native tetragonal crystals and two heavy atom isomorphous replacement derivatives. Each identical subunit of the dimer has a compact shape with overall dimensions of 85 X 75 X 55 A and is tightly associated with its 2-fold symmetry related subunit. There are three major excursions of the polypeptide chain of one monomer across the 2-fold axis to make extensive contacts with the other subunit. The active site, of which there are two per dimer, is shared between the two subunits at their interface and comprises a pocket-like region within a "V"-shaped framework of two alpha helices. Within this region are found the binding sites for the substrates, glucose-1-P and arsenate, a competitive inhibitor, UDP-glucose, and the allosteric effector, AMP. The site of metabolic control, Ser-14 phosphate, is hydrogen-bonded to a side chain on the outside of one of the alpha helices forming the active site and is 15 A from the AMP binding site. Maltoheptaose, a glycogen analogue and substrate for these enzymatically active crystals, binds in a second region of interest. Even at concentrations above its Km, when binding is sufficiently tight that all seven glucose moieties may be discerned, the closest of these is 25 A from the glucose-1-P binding site. We suggest that this polysaccharide binding site may represent a storage site whereby phosphorylase is bound to the glycogen particle in the muscle cell. The polypeptide chain in a third region has the same topological structure as has been observed for the nucleotide binding domains in the dehydrogenases. Adenine or adenosine (but not AMP) bind here in a position similar to the adenine ring of NAD in the dehydrogenases while glucose binds 17 A away in an interior crevice near the center of the monomer.


Population density and regulation of cell division in 3T3 cells. I. Inorganic phosphate levels, uptake and release. Triggering mechanisms for initiating density dependent inhibition of cell division in 3T3 cell monolayers are activated approximately two to three population doublings prior to cessation of cell division at monolayer confluency. This activation occurs at a critical contact cell density of approximately 8 X 10(3) cells/cm2. During this period there are selective controls on transport and storage of required low molecular weight nutrients. A possible correlation between orthophosphate and rates of cell division has been investigated. We have demonstrated a relationship between cellular concentrations of orthophosphate and initiation of density dependent inhibition of cell division. Prior to critical intercellular contact, the [Pi] in 3T3 is 10 mM. During critical contact, this concentration is quickly reduced to approximately 2 mM and remains at this concentration to confluency. Similar alterations do not occur in Py 3T3 cells, which maintain a concentration of approximately 2 mM Pi regardless of cell density. After confluent 3T3 cells are released from inhibition of cell division the [Pi] must increase several-fold before DNA synthesis commences. These are physiological changes in 3T3 cellular [Pi] as a function of cell density, and cannot be attributed to nutrient depletion, altered transport of Pi into the cell, increased [ATP], or increased [PPi] levels. The controlled modulation of [Pi] may regulate glycolysis and coordinate counter-ion changes (Ca++) may regulate mitochondrial activity.


Species-specific aggregation factor in sponges. VI. Aggregation receptor from the cell surface. An aggregation receptor from the siliceous sponge Geodia cydonium has been isolated and purified in an almost pure form. It sediments at about 2-6s, has a buoyant density of 1-51 g/ml in CsCl and elutes from Sephadex G-50 at a Ve/V0 value of 1-311. Chemical analysis revealed that the receptor consists of 81% neutral carbohydrate and 7-5% protein. The activity of the receptor is rapidly destroyed by Na-periodate. The receptor is released from the cell surface after removal of Ca2+ from the medium or after incubation of the cells with trypsin. The depleted cells can be charged again with isolated receptor molecules. The binding of the receptor molecules on the cell surface is prevented in the presence of trypsin. For optimal binding, physiological salt concentrations with respect to NaCl (540 mM NaCl) and Ca2+ ions are necessary. The receptor whose isolation is described in this report, is involved in secondary aggregation processes, which are initiated by a soluble aggregation factor. The primary aggregation of the cells is not influenced by the receptor. Time-course studies with receptor-depleted cells revealed that new aggregation receptor molecules are formed during the aggregation process. By competition experiments it could be shown that high concentrations of soluble aggregation receptor molecules inhibit secondary aggregation. The soluble receptor molecules can complete with surface-bound receptor molecules only if these are not linked with the aggregation factor.


Synthesis of the inner mitochondrial membrane and the intercalation of respiratory enzymes during the cell cycle of Chlorella. The ratio of inner to outer mitochondrial membrane area remains close to 1-8 throughout the cell cycle in synchronized cells of Chlorella fusca var, vacuolata 211-8p. Using estimates of this ratio, together with our previous estimates of mitochondrial surface area, to calculate the absolute area of inner mitochondrial membrane, it is demonstrated that growth of the inner mitochondrial membrane during the cell cycle occupies an extended period and parallels the growth of the whole cell. In contrast, the synthesis of succinate dehydrogenase and cytochrome oxidase is restricted to the last third of the cell cycle. It is concluded that mitochondrial growth involves the intercalation of periodically synthesized respiratory enzymes into membranes made earlier in the cycle, with consequent 5-fold changes in the density of active enzyme molecules in the membrane. These observations are discussed in relation to the control of mitochondiral membrane synthesis, membrane assembly and respiration rate during the cell cycle.


Neutral protease activity of Rous sarcoma (RSV) transformed chick embryo fibroblasts. The proteolytic activities of normal, Schmidt-Ruppin Rous sarcoma virus (SR-RSV) transformed, and infected (RAV) chick embryo fibroblasts (CEF) have been measured by a highly sensitive technique using 3H-acetylated haemoglobin as a substrate. When all 3 types of CEF cells were maintained in serumless media, no differences were detected in the amount of pH 3-4 protease activity released into the media over a 24-h period, and only negligible amounts of pH 7-6 proteolytic activity were found. When normal, transformed, and infected cells were maintained in serumless media and later incubated with 3H-acetylate haemoglobin, a significant proteolysis of the haemoglobin, a 6-fold increase compared to the normal CEF cells, was associated only with plates containing SR-RSV-CEF cells. A fluorescent assay for peptides confirmed that SR-RSV-CEF cells have increased cell-associated proteolytic activity. The net surface charge of the transformed CEF cells was unchanged by maintenance in serumless media but the net surface negativity of the normal and RAV-CEF cells was significantly increased by incubation in media minus serum for 24 h. This suggests that normal CEF cells, maintained in media plus serum, have a substance masking their surface charge which is absent from the surface of transformed cells, possibly because of proteolytic degradation.


Cytochalasin B-induced pseudo-cleavage of mouse oocytes in vitro: asymmetric localization of mitochondria and microvilli associated with a stage-specific response. Mouse oocytes are induced by cytochalasin B to undergo 'pseudo-cleavage' in vitro into 2 equally sized and separable compartments. This response to the drug is dependent upon the meiotic state of the oocytes, as well as upon the presence of an intact zona pellucida. The resulting 2 cellular compartments can be completely separated from another and cultured in vitro. Each of the compartments possesses characteristic structural features. The most pronounced structural differences include: (i) the presence of a nucleus (germinal vesicle) and nucleolus in one compartment; (ii) the presence of microvilli on the surface of the anucleate, but not the nucleate, compartment; and (iii) the localization (segregation) of mitochondria at the periphery of the anucleate, but not the nucleate, compartment. The results presented suggest that pseudo-cleavage induced by cytochalasin B arises as a consequence of a limited interaction of the drug with the oocyte surface and/or cortex and that it may represent a topographical dissociation of transporting and non-transporting regions of the membrane. These and other features of mouse oocytes treated with cytochalasin B are of interest in view of the involvement of the oocyte zona pellucida and plasma membrane during meiotic maturation, fertilization, and early embryogenesis.


Detection and determination of N-nitrosamines by thin-layer chromatography using fluorescamine. A novel procedure is described for the detection and determination of N-nitrosamines (NAs) on thin-layer chromatographic plates. Ultraviolet irradiation of NAs on activated plates (silica gel or aluminum oxide) yields primary or secondary amines, which after spraying with fluorescamine reagent give fluorescent or non-fluorescent products, respectively. The majority of the 24 NAs examined afforded NAs, although the volatile dimethyl-, diethyl- and pyrrolidine-N-nitrosamines gave limits of 500, 40, and 40 ng, respectively. Spectrophotometric determinations of the relative fluorescence of 0.1-40 nmoles of NAs gave rise to nearly linear calibration curves when plotted on a log-log scale.


High-performance liquid chromatography of organophosphorus insecticides. The high-performance liquid chromatographic behaviour of 23 organophosphorus insecticides has been studied on a stainless-steel column packed with silica gel. It has been stated that the usual classification of organophosphorus compounds into phosphonic, phosphoric, thiophosphoric and dithiophosphoric acid ester types gives some information about their adsorption properties. The chromatographic conditions of the analyses and a method for separation of the stereoisomers of tetrachlorvinphos are presented.


Simplified low-pressure high-resolution nucleotide analyser. A simple nucleotide analyser was constructed from commercially available components. A 20 cm X 0.4 cm I.D. column of pellicular anion exchanger operating at approximately 20 p.s.i. was found to perform identically with published methods using high-pressure equipment. The eluant was monitored using a variable-wavelength UV monitor. A very simple device for producing variable gradients was employed. The nucleotide analyser was trouble free to operate and the results were both linear and reproducible. Applications to the rapid analysis of nucleotides and the high-sensitivity analysis (approx. 10 pmoles) of cyclic 3',5'-adenosine monophosphate and cyclic 2',3'-guanosine monophosphate and tissue nucleotides are described.


Improvements of a radioimmunoassay for measurement of viral antibody in human sera. Modifications were made which increased the sensitivity of a solid-phase radioimmunoassay for serum antibody to human herpesviruses. Quantitation of antibody can be accomplished by referring to standard curve titrations.