Replication of M13 duplex DNA in soluble extracts of Escherichia coli. Effect of helix-destabilising proteins. Soluble extracts of M13-am5-infected Escherichia coli cells can carry out multiple rounds of M13 duplex DNA replication when supplemented with helix-destabilising protein of E. coli. Similarly addition of the helix-destabilising M13 gene 5 protein in low concentrations (up to 30 micrograms/ml) stimulates the replication of double-stranded M13 DNA. In contrast, higher concentrations of gene 5 protein (but not of E. coli helix-destabilising protein) cause a preferential inhibition of complementary strand synthesis resulting in a switch from double-strand replication to single-strand synthesis. Depending on the addition of the appropriate amounts of these two helix-destabilising proteins either stage of M13 DNA replication can now be studied with cell-free preparations.


Spin transition of camphor-bound cytochrome P-450. 1. local paH and electrostatic interactions. Variations of the spin state in camphor-bound cytochrome P-450 are interpreted in the light of the polyelectrolyte theory and its implications on the microenvironment of the heme. The ratio of high-spin to low-spin iron can serve as a tool to determine the local paH in the microenvironment of a group (pK0, app approximately 5.4) which governs the spin state. The local paH depends on the electrostatic potential created by negatively charged groups (pKa = 5.6), modulated in turn by paH and by the screening effect of ionic strength. A model is given for the proton-coupled spin state change.


Effect of triamcinolone on renal and hepatic phosphoenolpyruvate carboxykinase in the newborn rat. Changes in the rate of synthesis of the enzyme and in the activity of its translatable messenger RNA. The effects of triamcinolone on renal and hepatic phosphoenolpyruvate carboxykinase activity in the developing rat were investigated. The hormone induced increases in pre-existing enzyme activity of both tissues in fetal and neonatal rats, yet did not cause the primary appearance of phosphoenolpyruvate carboxykinase activity in utero. Neonatal hepatic phosphoenolpyruvate carboxykinase activity was increased 2--3 fold by triamcinolone form the 3rd to the 15th postnatal day. This was shown to be additive to the effect of Bt2cAMP on enzyme activity. The increases in phosphoenolpyruvate carboxykinase activity were demonstrated to be due to increased synthesis of the enzyme, which was accompanied by a proportionate increase in the amount of functional phosphoenolpyruvate carboxykinase mRNA, as measured by the polyribosomal and poly(A)-containing RNA directed cell-free synthesis of the enzyme. The demonstration of a triamcinolone effect on kidney and liver phosphoenolpyruvate carboxykinase activity in fetal and neonatal rats provides support for a possible role of glucocorticoids in the regulation of phosphoenolpyruvate carboxykinase activity during development.


Congenital mesoblastic nephroma.

Congenital mesoblastic nephroma. A case of mesoblastic nephroma in an infant with a 3-year, disease-free, postoperative survival period is reported. The histological aspects, with emphasis on differential diagnosis from Wilm's tumour are described.


(7-Ile) angiotensin III: a relatively selective antagonist of angiotensin steroidogenesis. The effects of (7Ile) angiotensin III (AIII) and (1-Sar-8-Ile) angiotensin II (AII) on the pressor and steroidogenic effects of angiotensin were compared in conscious rats. (1-Sar-8-Ile) AII (500 ng/kg/min) equally inhibited the pressor responses of AII, AIII and (des-1-Asp) AI by 99% (P less than 0.0001) while (7-Ile) AIII (500 ng/kg/min) was without effect. The steroidogenic effects of AII, AIII and (des-1-Asp) AI (300 ng/kg/min) were inhibited by (1-Sar-8-Ile) AII by 83%, 69% and 50%, respectively, whereas (7-Ile) AIII inhibited their steroidogenic effects by 35%, 62% and 25%. respectively. In contrast, ACTH or potassium stimulated aldosterone release was not altered by either antagonist. In sodium depleted rats, (7-Ile) AII reduced the elevated serum aldosterone levels by 64% without altering the blood pressure, while (1-Sar-8-Ile) AII lowered the blood pressure without altering the concentration of aldosterone present in the serum. Thus, (7-Ile) AIII is relatively selective in its ability to antagonize the adrenal actions of endogenous and exogenous angiontensins when compared to the pressor actions of these peptides. Furthermore, these angiotensin antagonists appear to be useful as pharmacologic tools in assessing the characteristics of the angiotensin receptors in a particular tissue.


The effects of kininase II inhibition by SQ 14225 on kidney kallikrein-kinin and prostaglandin systems in conscious dogs. The novel kininase II inhibitor SQ 14225 was intravenously administered to normal conscious dogs at the dose of 0.1 mg/kg. Urine kinin excretion increased from 14 +/- 6 ng/h to 163 +/- 18 ng/h. Separation by column chromatography showed that the increased urine kinin excretion was due to increased excretion of bradykinin. The enhanced urinary kinin excretion was associated by increased sodium (70%) and decreased kallikrein (27%) excretion. Urine volume and urinary prostaglandin excretion were not significantly affected by SQ 14225 treatment.


Effects of calcium on a cyclic nucleotide system in soleus motor nerve terminals. The effects of increased extracellular calcium levels on the responses of the motor nerve terminal to agents that increased intracellular levels of cyclic AMP was studied on the in vivo cat soleus muscle preparation. The neural and muscle responses to intra-arterially administered NaF, an activator of adenylate cyclase, and dbcAMP progressively increased as blood calcium levels rose. These results provide additional support for the hypothesis of an interaction between calcium and cyclic AMP in motor nerve endings.


Maturation process of secondary ossification centers in the rat and assessment of bone age. The maturation process from the appearance to the fusion of the secondary ossification centers of extremities was studied in Wistar rats aged 0 to 134 weeks. The examination of the secondary ossification centers made by radiography. The assessment of the stage of development was made in accordance with the criteria proposed by Ohwada and Sutow. The secondary ossification center was found to be take one of the following three types of maturation processes : (1) the acute ossification, (2) the delayed ossification, and (3) the incomplete ossification. No fusion was observed up to 134 weeks in certain epiphyses of the rat. This type of ossification designated as the incomplete ossification may be specific to the mouse and rat. The relative lengths of time required for appearance and fusion in the average prospective life were obtained for the rat. They were compared with those of the mouse and man. The relative length of time necessary for maturity of the secondary ossification centers was shown to be the shortest in the rat and the longest in man. The results suggested that the rat may reach maturity in the bone age at 17 to 21 weeks of age. The rat at this age may be regarded as being adult corresponding to age 17 weeks in mice and 18 to 24 years in man.


Examination of latent infection in germfree mice for some murine viruses. Sera from some groups of germfree mice were examined for reactivity against Sendai, reo 3, Theiler's GD VII, ectromelia, and mouse hepatitis virus antigens. Of 51 sera collected in September and October 1976 and in August and September 1977 from one germfree mouse breeding colony, there was no positive reaction against all the antigens tested. Moreover, the attempt of virus isolation from homogenates of various organs including lungs, liver, kidneys, and intestines of 6 germfree mice was unsuccessful.


Serum lipoprotein inhibitors of granulopoiesis in human bone marrow cultures. Ammonium sulphate fractionation of human serum showed that two fractions contained an inhibitor of cluster and colony formation in agar cultures of human bone marrow cells. Further investigations demonstrated this inhibition to be caused by lipoproteins. When freshly prepared, only light density lipoproteins (LDL) were found to be inhibitory. During storage, very light density lipoproteins (VLDL) acquired inhibitory activity, while high density lipoproteins (HDL) did not show any inhibition of human bone marrow cultures. The possibility of toxic degradation of lipoproteins as an explanation for the inhibition was investigated by preincubation of human marrow cells with lipoproteins for 6 hours (37 degrees C) after storage of the lipoproteins under nitrogen (4 degrees C) for various intervals up to 48 days. Preincubation was found to be non-toxic (viable cell counts) and without colony/cluster-reducing ability for incubated marrow cells compared to controls. Addition of lipoproteins to mouse marrow cells and Ehrlich ascites tumour cells resulted in no change in colony formation by Ehrlich cells, whereas mouse marrow cells were inhibited, but to a lesser degree than human marrow cells.


Hormone receptor changes during adulthood and senescence: significance for aging research. Receptors for various hormones have been examined throughout the adult life span. In most of these studies receptor concentrations were found to decrease with increasing age, either during senescence or earlier in adulthood. In contrast, binding affinities did not change with age. Tissues and cells examined have included liver, brain, adipose tissue, skeletal muscle, prostate, fibroblasts, and lymphocytes. Receptors for steroids as well as membrane-active hormones such as insulin, glucagon, catecholamines, and prolactin were studied. Rats, mice, dogs, and humans have been employed. Changes in hormone receptor levels appear to constitute a common manifestation of the aging process, and offer interesting model systems in which to study the control of genetic expression during senescence.


Age-related alterations in the development of adrenergic denervation supersensitivity. The density of beta-adrenergic receptors in the central nervous system exhibits marked age-related changes. In general, there is an initial increase in receptors soon after birth followed by a decline with advancing age; the specific pattern of the development and loss of receptors is dependent upon the brain area. The ontogenetic increase in the density of adrenergic receptors coincides temporally with the development of responsiveness to catecholamines but can proceed without an adrenergic innervation. This suggests that the biosynthesis of receptors is genetically predetermined and does not require an adrenergic input for initiation. Decreasing adrenergic activity produces an increased number of beta-receptors and a supersensitive response to adrenergic agonists. The decline in beta-receptors with advanced age appears to be related to this phenomenon of denervation supersensitivity since certain aged tissues have a diminished capacity to develop an increased number of receptors in response to a reduced sympathetic input. We conclude that the decline in beta-adrenergic receptors with age may explain the age-related decrease in the sensitivity of adenylate cyclase to catecholamines, and, consequently, the reduced physiological response to adrenergic stimuli. The mechanism for this loss of receptors may be the inability of aged tissue to develop a supersensitivity response in reaction to diminished sympathetic activity.


Aging and monoamine receptors in brain.

Aging and monoamine receptors in brain. Biochemical evidence is presented for selective decreases in biogenic amine receptor systems with age in the rabbit. Dopamine-stimulated adenylate cyclase activity in striatum, hypothalamus, frontal cortex, and anterior limbic cortex declined by about 50% as rabbits aged from less than 1 to 5 years of age. Similar decreases were found for histamine-stimulated activity in hypothalamus and the cortical regions. These changes were in maximal response rather than in affinity for amine. In contrast, dopamine-stimulated adenylate cyclase of retina and both basal and Gpp(NH)p-stimulated activity in these regions were not altered with age. In addition, with age the number of binding sites for [3H]spiroperidol, a dopamine antagonist, decreased by 30--40% without change in ligand affinity in striatum and limbic cortex. These changes in striatum and  cortex occurred in the absence of decreases in either dopamine concentration or choline acetylase activity. It is proposed that selective age-dependent decreases in the functional number of biogenic amine receptors occur in the absence of, or independent from neuronal cell loss, possibly by a mechanism of desensitization. These changes occurred in brain regions that in man are thought to be of importance in the age-related loss of cerebral function.


Lipoprotein secretion by rat liver Golgi apparatus. Lipoprotein particles and lipase activity. Lipoprotein particles of the size range of very low density lipoproteins in smooth endoplasmic reticulum, peripheral elements of the Golgi apparatus, and secretory vesicles of the immature Golgi apparatus face are 55 to 80 nm in diameter. Particles in mature secretory vesicles are smaller (45 nm). Concomitant with the change in particle size, the lumina of mature vesicles increase in electron density. A technique to fractionate immature and mature secretory vesicles was based on precipitation of a cupric-ferrocyanide complex (Hatchett's brown) through the action of a NADH-ferricyanide oxido-reductase resistant to glutaraldehyde which is characteristic of the membranes of mature secretory vesicles and of the plasma membrane of liver. Mature secretory vesicle fractions so isolated were enriched in cholesterol and depleted in triglycerides relative to immature vesicles on a phospholipid basis. Lipase activity was present in secretory vesicle fractions of the Golgi apparatus as shown by biochemical analysis and by cytochemistry. Cytochemical studies showed lipase to be present in both mature and immature vesicles but most evident in immature vesicles. The findings suggest that some very low density lipoprotein particles are converted to particles of smaller diameter during transit through Golgi apparatus. A lipase-mediated hydrolysis of triglycerides may relate to the transformation.


Dependence of intracellular alkali-ion concentrations of 3T3 and SV 40-3T3 cells on growth density. Intracellular contents of potassium and of sodium are determined for 3T3 and SV 40-3T3 cells in dependence of growth density. In parallel, total cell volume and volume of intracellular water is determined for these cells suspended in physiological buffer. Intracellular potassium concentration thus evaluated for suspended 3T3 cells exhibits a sharp decrease at cellular growth densities which lead to density dependent inhibition of cell proliferation. In the case of SV 40-3T3 cells, this drop of potassium concentration with increasing cellular growth density is not observed, which correlates well with the absence of cell density dependent inhibition of cell growth in the transformed cell line. These results support the notion that processes of stimulation of quiescent 3T3 cells or of cell density dependent inhibition of their proliferation are mediated by processes including changes of potassium transport characteristics leading to increase or decrease respectively of their intracellular potassium concentration. Furthermore, these and other results suggest, that a difference between normal and transformed cells most relevant to their different proliferation behaviour might reside in different transport characteristics for potassium of the plasma membranes of these cells.


Hepatocellular transplantation --morphological study on hepatocytes transplanted into rat spleen--. Hepatocellular transplantation into the spleen was investigated as a new attempt in utilizing isolated hepatocytes to compensate for impaired liver function. Present study was undertaken to evaluate morphological and histochemical alterations up to 6 weeks following transplantation in hepatocytes transplanted into the splenic parenchyma. Light microscopic studies revealed viable hepatocellular islets in the splenic parenchyma up to 6 weeks, although minimal cytoplasmic changes were observed. Electron microscopic studies demonstrated moderate changes in organelles, which developed gradually as the time after transplantation proceeded. Distortion and fragmentation of the membranes around the cytoplasm and organelles were not recognized. Moreover, newly formed bile canaliculi and tight junctions which indicate reconstruction of hepatic plates were observed between adjacent cell membranes, and enzyme activities were detected by cytochemical determination of glucose-6-phosphatase in the hepatocytes even 6 weeks after transplantation. The transplanted hepatocytes preserved their characteristic enzyme and fine structures as hepatocytes up to 6 weeks. Our present study based on the persistence of cellular viability suggests that inoculated hepatocytes do maintain their hepatocellular functions after transplantation.


Small intestinal morphology and epithelial cell kinetics in lamb rotavirus infections. Morphologic changes in the small intestine of rotavirus-infected gnotobiotic lambs were investigated by measurement of villi and crypts in histologic sections of jejunum, midgut, and posterior ileum. In midgut, villus atrophy developed within 12 hr of infection and was apparent until 72 hr after infection. Crypt hypertrophy was evident from 42 hr after infection until the end of the observation period (6 days after infection). Changes in posterior ileum were similar in extent, but jejunal changes were much less marked. The relatively mild effect in the jejunum is in accord with reports from other species, and provides a basis for questioning the assumption that human rotavirus affects mainly the foregut. Studies of epithelial cell kinetics were made on midgut using a microdissection and metaphase accumulation technique on sequential samples from anesthetized lambs. An increase in the cell production rate per crypt per hour from the overall control level of 5.8 was detected by 48 hr after infection. The maximum level of 21.2 was reached 8 days after infection, and this had returned to near normal by 15 days after infection. This large and sustained increase in crypt cell production probably underlies other previously described functional abnormalities.


Lethal herpes simplex virus type 1 hepatitis in a normal adult. A fatal case of human herpes simplex type 1 hepatitis is presented and compared with cases previously reported. The patient presented with fulminant hepatic necrosis, coagulopathy, leukopenia, thrombocytopenia, and absence of typical herpetic mucocutaneous lesions. Liver biopsy helped to establish a presumptive diagnosis in this patient and in one previously reported case. Theoretically, early diagnosis might enable application of specific antiviral therapy in future cases of this usually fatal infection.


[Differential diagnosis of pigmented tumors]. Some frequent diagnostic problems and the most important clinical and histologic criteria in differential diagnosis of malignant melanomas, benign pigment cell nevi, melanotic epithelial neoplastic lesions and frequent haemosiderotic tumors are delineated in a condensed survey. Considering the variety of diagnostic errors, one should never treat respectively destroy pigmented tumors of the skin without histologic investigation.


[Hormonal regulation and hormone therapy in childhood and adolescence. Part 1: development of endocrine glands, hormone production, menarche and secondary sex characteristics]. The growth curves of the endocrine glands in female puberty are described. Adrenarche with an elevated production of adrenal dehydroepiandrosterone marks the beginning of prepuberty. By decrease of hypothalamic sensitivity to sexual steroids begins the deviation of FSH and LH levels. Significant increases of gonadotrophins and prolactin occur during sleep. There is a good correlation between bone age, Tanner-stages of breast and pubic hair development and steroid hormone levels. Criteria of maturity for young girls including menarche and ovulation are given.


[Clinical and urological after care in gynecologic neoplasms]. Patients after operative and/or radiological therapy for cervical cancer should have gynecological follow-up examinations every two months in the first year. In cases of urological complications after irradiation therapy, radionephrography, infusion urogram, blood count, cystoscopy, and cystometry should also be carried out after two months. Depending on the clinical picture, radionephrography, blood count and urinalysis are to be repeated in regular intervals. After lymphography with positive findings, x-ray controls are necessary after two and four months. In cases of increasing hydronephrosis and hydroureter due to fibrostenosis of the ureter plastic surgery is recommended. Most urological complications are to be expected after radical operation and postoperative irradiation. In patients with endometrial uterine cancer, metastases mostly occur paraurethrally and in the lungs. Chest x-ray is to be taken every six months. Suspicious paraurethral hardening is cytologically diagnosed by needle biopsy. In ovarian carcinoma stage III and IV repeated x-ray controls of the lungs during longterm polychemotherapy are advisable. Prior to second look operations after combined treatment a second look laparoscopy is particularly important. Urological complications are more frequent after therapy of cervical cancer while ovarian carcinoma is more likely to develop complications of the small bowel.


Multihormonal pituitary adenomas.

Multihormonal pituitary adenomas. 66 pituitary tumors detected at autopsy were investigated for the presence of corticotropin, beta-lipotrophin, growth hormone, prolactin, thyrotropin and gonadotropins by immunocytochemistry. 56 tumors contained hormone-producing cells; 45 were found to contain 2 or more hormones. This finding confirms and extends previous morphologic and clinical observations. The majority of pituitary tumors are mixed and they probably arise from impaired regulation at the hypothalamic and/or pituitary level.


Preservation of the effects of pregnancy on rat islets of Langerhans in tissue culture. Islets of Langerhans, isolated from normal or 19-day pregnant rats, were cultured for 20 h at 37 degrees C in tissue culture medium 199. When islets were cultured in medium containing low glucose (5.5 mM), the higher adenylate cyclase activity and insulin secretory responses characteristic of islets from pregnant rats were maintained during the test period of 29 h. Islets from normal and pregnant rats were also cultured for 20 h in medium containing a very high glucose concentration (83.3 mM) in order to load the B cells with glycogen. It was found, after glycogen loading, that, while adenylate cyclase activity increased to a greater extent in islets from pregnant rats than controls, this activity was not increased in proportion to the striking changes in insulin release rate observed in pregnant rat islets. The results show that the difference in insulin secretory response between islets from normal and pregnant rats may be preserved when the islets are cultured for 20 h, and that these differences are enhanced for a variety of reasons after culture of islets in 83.3 mM glucose.


Cytomegalovirus vaccine: a realistic appraisal. The possible benefits and dangers of a vaccine program for CMV infection, especially congenital infection, cannot be rationally analyzed until more is known about the virus itself, including its manner of spread and the nature of host responses. Until then, trials with the experimental vaccines under development should be limited to patients at unusually high risk, such as those receiving renal transplants.


Neurofibrillary tangles induced by a sclerosing angioma. A sclerosing angioma in the frontal cortex of a 40 year old woman produced local pressure, with resulting alteration of the blood-brain barrier. In the narrow cortical zone affected by chronic pressure, the level of aluminum was significantly elevated; and all neurons had neurofibrillary tangles of the Alzheimer type.


Thorotrast induced hepatic cholangiocarcinoma and angiosarcoma. A 49 year old woman developed hepatic cholangiocarcinoma and angiosarcoma 22 years after the administration of Thorotrast. The etiologic association between Thorotrast and a variety of malignant hepatic neoplasms is well known, but the simultaneous occurrence of two different hepatic neoplasms has not been previously reported.


Malignant fibrous histiocytoma of bone: a study of 35 cases. Thirty-five cases of primary malignant fibrous histiocytoma of bone are reported. Twenty of these cases were collected from a retrospective analysis of other malignant bone tumors. The age range was from 11 to 69 years; the average age was 34 years. The tumor occurred most commonly in the distal femur and proximal tibia. The distinguishing histologic feature was a storiform arrangement of spindle cells. The differential diagnosis included fibrosarcoma, osteogenic sarcoma, malignant giant cell tumor, malignant lymphoma, and metastatic carcinoma. Follow-up of at least three years was available in 21 cases. Of these, nine patients were alive and free of metastases three and one-half to 12 years after treatment. Two were alive with solitary metastases at three years, and 10 patients died between three months and three years after treatment. In four cases the lesions were multicentric at the time of diagnosis and in four cases were associated with bone infarction. This tumor must be recognized as an important complication of bone infarction and should be suspected when a patient with a known history of bone infarction develops a change in symptoms. Because the prognosis of this tumor is significantly better than that in those tumors with which it had been previously grouped, and in view of its association with bone infarction, it deserves to be maintained as a distinct clinicopathologic entity. Amputation is the treatment of choice.


Enzymatic methyl esterification of pituitary polypeptides. Methyl esterification of pituitary polypeptides by protein methylase II (S-adenosylmethionine:protein-carboxyl O-methyltransferase, EC. 2.1.1.24) has been investigated. Ovine lutropin and adrenocorticotropin (alpha1-39-ACTH) were found to be good methyl acceptor substrates, followed by beta-lipotropin. While the alpha-subunit of lutropin had nearly equal the methyl accepting activity of lutropin, the beta-subunit was devoid of accepting activity. The maximum amount of esterification occurred between 15 and 30 min at 37 degrees C) depending on the methyl acceptor molecule. The rate of the methyl esterification of adrenocorticotropin fragments was also studied. While alpha7-38-ACTH had less than half of alpha1-37-ACTH methyl accepting capacity, alpha1-17-ACTH did not serve as methyl acceptor. However, when a mixture of the two fragments was preincubated, the resulting mixture had full alpha1-39-ACTH activity.


Adrenocorticotropin. 52 Synthesis and biological activity of adrenocorticotropic peptides with cystine bridges. Three analogs of the amino terminal nonadecopeptide of adrenocorticotropin which incorporate cystine cystine bridges between positions (5, 10), (3, 10), or (2, 10) have been synthesized. All of the peptide analogs showed reduced biological activity; however, the peptide with the 5, 10 cystine bridge was shown to possess significantly higher lipolytic activity in rat fat cells than the peptides with a 3, 10 or 2, 10 cystine bridge.


Intratypic serodifferentiation of poliomyelitis virus strains by strain-specific antisera. Strain-specific antisera against non-Sabin-like and Sabin-like poliovirus strains were prepared by cross-absorption of the antisera with the heterologous strain. Isolates from different parts of the world were differentiated intratypically as non-Sabin-like and Sabin-like strains with the aid of these sera in the immunodiffusion test and neutralization test. In comparison with other marker tests, intratypic differentiation can be performed more easily. Further, fewer intermediate strains were found and, in our opinion, the results are more reliable.


Analysis of the transformation of human lymphocytes by Epstein-Barr virus. I. Sequential occurrence from the virus-determined nuclear antigen synthesis, to blastogenesis, to DNA synthesis. A B-cell population of human cord blood lymphocytes was exposed to the B95-8 strain of Epstein-Barr virus (EBV), and simultaneous observations of immunofluorescence, cellular morphology and autoradiography were carried out in each individual cell. It was evident that EBV-determined nuclear antigen (EBNA) synthesis occurred prior to blastogenic response and DNA synthesis. EBNA-positive cells could be observed as early as 12 h after infection and reached a maximum of 17% at 24 h, followed by a plateau for a subsequent 12 h. The positive cells were seen exclusively as morphologically normal lymphocytes until 18 h; at 24 h, blastogenesis became evident, without cell division. DNA synthesis was detected at 36 h in EBNA-positive blast cells, after which these cells increased rapidly. EBNA synthesis was similarly evident in the presence of cytosine arabinoside, but was significantly inhibited by a short-term exposure to cycloheximide immediately after infection. These findings suggest that the early events in EBV-induced transformation of human lymphocytes occur sequentially from EBNA synthesis, to blastogenesis, to DNA synthesis and that the crucial step of such transformation is probably involved in protein synthesis occurring in the very early stage of EBV infection.


Replication of herpes simplex virus in human B lymphocytes stimulated by Epstein-Barr virus. Human B lymphocytes prepared from adult blood or cord blood by the use of neuraminidase-treated sheep red blood cells which did not respond mitogenically to phytohemagglutinin or pokeweed mitogen, responded well to the B95-8 strain of Epstein-Barr virus, but not to the P3HR-1 strain, with increased incorporation of tritiated thymidine. Replication of herpes simplex virus could be demonstrated in cultures of B lymphocytes preincubated with the B95-8 strain.


Geographical distribution of swine (Hsw1N1) and Hong Kong (H3N2) influenza virus variants in pigs in Southeast Asia. Influenza viruses of the Hsw1N1 and H3N2 subtypes were isolated in Hong Kong from pigs originating from Hong Kong, People's Republic of China (PRC), Singapore, and Taiwan. The H3N2 isolates were from pigs from Hong Kong and PRC, whereas the Hsw1N1 isolates were from pigs from Hong Kong, PRC, Singapore and Taiwan. The hemagglutinins of the H3N2 isolates were similar to those of the earlier A/Hong Kong/1/68 and A/Port Chalmers/1/73 variants from man. The Hsw1N1 isolates were similar to the human A/New Jersey/8/76 virus. These studies extend the geographical range of Hsw1N1 influenza virus and suggest that pigs in southeast Asia, like those in North America, harbor Hsw1N1 influenza viruses. These findings underline the importance of pigs as potential reservoirs for future human pandemics by the continued isolation (in Asia) of H3N2 and Hsw1N1 influenza viruses.


Presence of viral particles in the salivary gland of Calomys musculinus infected with Junin virus by a natural route. Calomys musculinus, a wild cricetid rodent, is one of the main reservoirs of Junin virus. Six of these animals were infected by being placed in close contact with animals that had been experimentally infected with the virus. They were sacrificed at 10, 15 and 20 months after contact, and their salivary glands were studied by ultrastructural, immunohistochemical and virological methods. Two animals developed chronic viremia and low titers of complement-fixing antibodies. These animals were the only ones that had high viral titers in salivary glands and blood and viral antigen and particles in salivary glands. Although some of the other animals had viremia at the beginning of the experiment, it was absent 5 months later. Complement-fixing antibodies developed in all animals. On the basis of these findings, we assumed that the salivary gland is an important site of viral synthesis and excretion. This type of chronic infection, with persistent viremia and virus shedding, is possibly important for virus perpetuation in nature and transmission to man.


Growth of type 2 herpes simplex virus in newborn and adult mononuclear leukocytes. Growth of type 2 herpes simplex virus (HSV) in newborn and adult human mononuclear leukocytes (MNL) was compared. Phytohemagglutinin stimulation of cultures for 3 days yielded comparable peak titers in newborn (10(5.3) PFU) and adult (10(5.1) PFU) MNL. Unexpectedly, 3-day cultures of unstimulated newborn MNL also substantially replicated HSV (10(4.7) PFU), whereas similarly treated unstimulated adult cells did not. Growth of HSV in freshly isolated human MNL was next investigated. MNL from 4 mothers and 6 nonpregnant adults showed no evidence of virus growth; however, leukocytes from 11 of 24 newborns (46%) supported replication. Newborn MNL manifested an increased ability to replicate HSV within 1 day of culture, whereas comparable growth in adult MNL was not achieved until the 4th day of culture. The significance of the above observations as it relates to visceral dissemination of HSV in the neonate is discussed.


Induction of lytic plaques by murine leukemia virus in murine sarcoma virus-transformed nonproducer mouse cells persistently infected with mouse hepatitis virus MHV-S. Kirsten murine sarcoma virus-transformed, nonproducer BALB3T3 (K-BALB) cells were persistently infected with mouse hepatitis virus, MHV-S. The cultures developed plaques after infection with murine leukemia viruses. If the murine leukemia virus-infected cultures were further submitted to the UV-XC assay, comparable numbers of XC plaques were obtained. The sensitivity to murine leukemia viruses, as determined by the UV-XC assay, was higher in MHV-S-infected cells as compared to uninfected K-BALB cells.


Reduction of 51Cr-permeability of tissue culture cells by infection with herpes simplex virus type 1. Infection of different strains of tissue culture cells with herpes simplex virus type 1 (HSV-1) resulted in a reduced 51Cr-permeability. A stability of the cellular membrane to Triton X-100, toxic sera and HSV-specific complement-mediated immune-cytolysis could be observed simultaneously. The results differed with respect to the cell strain used in the experiments.


Poly(adenosine diphosphate ribose) synthesis during herpes simplex virus infection. Immediately after infection of baby hamster kidney cells with herpes simplex virus (HSV), cellular DNA synthesis was blocked, while extensive HSV DNA synthesis began. These dramatic alterations of the control mechanisms for these two DNA synthesizing systems were not accompanied by a change in the poly(adenosine diphosphate ribose) polymerase activity.


Effect of glucosamine on the replication of the arenavirus Junín in Vero cells. When Vero cells infected with Junín virus are cultured in the presence of 16 mM glucosamine, no infectious virus is produced and no surface antigens or virions are detected. Cytoplasmic viral antigen, however, is synthesized normally.


Absence of interferon production in a newly established human cell line. A cell line established from human embryonic lung, HEL-R66, was demonstrated to be highly susceptible to herpes simplex virus types 1 and 2, vaccinia virus, Newcastle disease virus (NDV), Japanese encephalitis virus (JEV), western equine encephalitis (WEE) virus, Sindbis virus, vesicular stomatitis virus (VSV), and rabies virus. The maximal yields of NDV, JEV, WEE virus, and rabies virus in this cell line exceeded by 2--4 logs those in control human embryonic lung cells. Inability of this cell line to produce interferon upon treatment with native and UV-irradiated forms of virogenic and lentogenic strains of NDV and with poly I:C was revealed. A refractory state to challenging VSV did not develop in HEL-R66 cells treated with the inducers. Furthermore, pretreatment of HEL-R66 cells with interferon did not potentiate the capacity to produce interferon in response to the addition of poly I:C, whereas the same treatment enhanced the production of interferon in normal human embryonic lung cells.


Hybridization of bovine papilloma virus type 1 and type 2 DNA to DNA from virus-induced hamster tumors and naturally occurring equine tumors. DNAs from bovine papilloma virus(BPV)-induced hamster tumors and from equine connective tissue tumors of unknown etiology were examined for BPV DNA sequences by molecular hybridization. DNA from two distinct classes of BPV (type 1 and type 2) was labeled in vitro and used as probes. Analysis of DNA-DNA reassociation kinetics indicated that both virus types were capable of tumor induction in the hamster. DNA isolated from 6 of 7 equine tumors accelerated the reassociation of the BPV DNA probes. BPV type 1 or type 2 DNA hybridized extensively to DNA from 3 tumors, while 3 other tumors contained DNA sequences to which only a portion of the probes hybridized. Partial hybridization of probe DNAs to tumor DNA suggested the possible existence of a third BPV class.


Bovine enterovirus CPE at different multiplicities of infection in the absence of viral RNA synthesis. When bovine enterovirus (BEV) RNA synthesis was inhibited by 2-(alpha-hydroxybenzyl)-benzimidazole (HBB), an increase in MOI was accompanied by an enhancement of cytopathology. Since BEV RNA synthesis is inhibited and BEV protein synthesis is allowed to proceed in the presence of HBB, these results support the concept that proteins play a role in the development of cytopathology independently of viral double-stranded RNA.


A case of McCune-Albright syndrome with hyperthyroidism and vitamin D-resistant rickets. A girl aged 1 11/12 year with the unusual combination of McCune-Albright syndrome (polyostotic fibrous dysplasia, cutaneous pigmentation and precocious puberty), hyperthyroidism and vitamin D-resistant rickets is described. Urinary estrogens are increased, while serum LH and FSH responses to LH-RH are subnormal. Thyroid hormone levels in serum and 131I thyroidal uptake are increased, while TSH response to TRH is decreased. Serum phosphorus level is low and phosphate clearance high, while PTH is within normal range. Basal GH level and GH responses to various stimulations are high, but high basal GH is suppressed by oral glucose. The oral glucose tolerance test shows normal blood glucose with hypersecretion of insulin. These data suggest that the common pathogenesis of the various aspects of the syndrome is a hypersensitivity of the target organs, which include the pituitary, the thyroid, the gonads, the pancreatic islet cells and the proximal tubules of the kidney.


Electron microscopic and cytochemical studies of rat aorta. Intracellular vesicles containing elastin- and collagen-like material. Small, rounded vesicles with a dense core of amorphous material were observed in all cell types in the young rat aorta, that is, endothelial cells, smooth muscle cells and fibroblasts. They were particularly numerous in the Golgi complex but were also found in the cell periphery. The content of the vesicles had staining characteristics identical to those of elastin. Material of the same type was also found in cisternae on the maturing side of the dictyosomes and in vesicles budding from them. Reaction product for thiamine pyrophosphatase was present in both these structures, indicating that the Golgi complex is responsible for the formation of the dense-cored vesicles. This was further supported by the absence of reaction product for acid phosphatase in the cisternae and in the vesicles. Moreover, no uptake of exogenous markers was noted in the latter. On the basis of these findings it is suggested that the dense-cored vesicles have a secretory function and contain precursors of elastin. Elongated vesicles or profiles containing collagen fibrils were observed in smooth muscle cells and fibroblasts. In the cell periphery, these vesicles were often found to communicate with the extracellular space. Further inside the cells, they showed a close spatial relationship to the Golgi complex. Neither thiamine pyrophosphatase nor acid phosphatase activity was demonstrated in the elongated vesicles. Like the plasma membrane, their limiting membrane was positively stained for alkaline phosphatase. On the basis of these findings and the absence of uptake of exogenous markers in them, it is suggested that the elongated vesicles represent a means for collagen secretion in the growing aortic wall. The Golgi complex is believed to be involved in the transfer of collagen to these vesicles.


Phosphatases in the central nervous system of spiders (Arachnida, Araneae). The central nervous systems of web-building spiders (Araneidae, Agelenidae) and hunting spiders (Lycosidae, Salticidae) were tested for non-specific and specific phosphatases. Acid phosphatase exhibited weakly to moderately positive reactions in the neuronal cell bodies and in the neuropile fibre mass of all species investigated. Alkaline phosphatase could only be demonstrated in the external and internal neural lamellae of the brain and ventral cord of several specimens of the araneid species investigated. Tests for thiamine pyrophosphatase were negative with both the lead and calcium-cobalt methods. Distinctive positive reactions for adenosine triphosphatase were visible in the nervous system of all the species used, being especially strong in the optic ganglia of the hunting spiders. The demonstration of adenosine triphosphatase was only possible when applying the calcium-cobalt method after Padykula and Herman, while the lead method after Wachstein and Meisel did not produce any staining reaction at all. Controls of the histochemical reaction showed that the enzyme was activated by Ca2+ and inhibited by sulphydryl destroying reagents (e.g. PCMB), but was insensitive to ouabain. It could be probably classified as a mitochondrial proton-translocating adenosine triphosphatase.


ACTH radioimmunocytochemistry (RICH) on rat anterior pituitary cells. Radioimmunocytochemistry (RICH) was applied to detect corticotrophs in adult rat pituitaries and 8-day-old anterior pituitary monolayers by incubating sections and cultures with 125I-ACTH-anti ACTH immune complexes. After incubations autoradiography was made. In comparison, "conventional" immunostaining was carried out on adjacent sections and parallel cultures. It has been established that RICH is suitable for detection of corticotrophs.


Cytochemical analysis of organelle degradation in phagosomes and apoptotic cells of the mucoid epithelium of mice. The activity of mitochondrial cytochrome oxidase and peroxisomal catalase in the phagolysosomes and apoptotic bodies of mucoid epithelial cells was analysed. Tissue from 2-6 day old mice was used. The activity of acid phosphatase in lysosomes was also estimated. Cytochrome oxidase was demonstrated in well-preserved mitochondria inside phagosomes. Mitochondria in cells exhibiting apoptotic death also show activity of cytochrome oxidase. The enzyme activity in swollen mitochondria ceases before the membranes of the cristae disappear completely. Apoptotic bodies are phagocytosed by sister mucoid cells and, later on, they are digested inside the cell. Phagosomes which contain already degraded mitochondria show still active catalase in sequestered peroxisomes. The acid phosphatase involved in degradation of phagocytosed material originates from endocytosed lysosomes and primary and secondary lysosomes which fuse with the membranes of phagosomes.


Isolation of intramitochondrial bodies in bovine adrenocortical cells by density gradient centrifugation. Two method for isolating the intramitochondrial bodies from bovine adrenocortical cells are proposed. Electron microscopic examination shows that discontinuous sucrose density gradient centrifugation can separate the fraction rich in intramitochondrial bodies, but some indistinguishable fragments remain among them. Continuous sucrose density gradient centrifugation is probably superior to the former method in obtaining a highly purified fraction of the bodies. The amido black positive granules, presumed to be intramitochondrial bodies, are collected in the fractions of the sucrose density of around 1.27 (1.23--1.30), which lack cytochrome c oxidase activity.


Histochemical demonstration of mercury induced changes in rat neurons. A histochemical method modified for ultrastructural studies of mercury induced changes is described. Rat neurons from areas known to be influenced by mercury are used as examples. The histochemical reaction, suggested to be caused by polymercury sulphide complexes, is localized to "dense bodies" where it is visible 14 days after initiation of peroral mercury treatment (20 mg HgCl2/l drinking water).


Activities of malate dehydrogenase, 3-hydroxyacyl-CoA dehydrogenase and fructose-1,6-diphosphatase with regard to metabolic subpopulations of fast- and slow-twitch fibres in rabbit muscles. Activities of malate dehydrogenase (MDH), 3-hydroxyacyl-CoA dehydrogenase (HAD) and fructose-1,6-diphosphatase (FDPase) were determined in single fibres dissected from freeze-dried rabbit psoas and soleus muscles. Slow-twitch fibres as determined by qualitative ATPase reaction represent a rather uniform population with regard to HAD and MDH activities. In these fibres the two enzymes are in constant proportions. FDPase is found at extremely low activities in slow-twitch fibres and because of its relatively high activity in fast-twitch fibres of soleus and psoas muscle it might be used as a marker enzyme. Fast-twitch fibres in psoas muscle represent a heterogeneous population with regard to activities of MDH as well as of HAD. The two enzyme activities are not proportional in fast-twitch psoas fibres. These findings suggest the existence of metabolic subpopulations of fast-twitch fibres having a wide range of aerobic oxidative capacities and having differences in their capacity to oxidizing fatty acids.


Bent-limb syndrome in lambs raised in total confinement. A bent-limb syndrome in lambs raised in total confinement was characterized by curvature of the forelimbs. Radiographic findings included flaring of the affected long bone and thinning of the growth plate. The main histologic change was endochondral dysplasia of the long bone. In feed samples, all trace minerals analyzed were within recommended concentrations except iron, which was much higher (400 ppm dry matter) than the normal requirement of lambs (70 ppm). All mineral concentrations in serum were normal except those of inorganic phosphorus and iron, which were higher. Results of soft tissue and bone mineral analyses were normal. Altering the ratio of calcium and phosphorus did not affect the incidence of disease, but intramuscular administration of massive doses of vitamin D3 and reducing the amount of dietary iron had a prophylactic effect. The increase in serum phosphorus was probably related to the dietary excess of iron, which probably decreased vitamin D metabolite formation in the kidney, which in turn could be prevented by massive doses of vitamin D3.


Serologic survey of prevalence of ovine progressive pneumonia in Idaho range sheep. Blood samples from 2,310 mature sheep in 3 Idaho range flocks were examined by agar gel immunodiffusion to determine the prevalence of ovine progressive pneumonia. The prevalence ranged from 58% for all ages combined in one flock to 90% of cull ewes in another flock. Age-specific prevalence rates increased from 16% in yearlings to 83% in ewes greater than or equal to 7 years old. Rambouillet sheep had a significantly (P less than 0.01) lower prevalence than sheep of 5 other breeds, whereas one-half Finnsheep crosses had a significantly (P less than 0.01) higher prevalence than sheep of other breeds. Within breed and age, there was no significant difference in reproductive performance between seropositive and seronegative ewes.


Diagnostic features of ovine progressive pneumonia. Ovine progressive pneumonia, a chronic, insidious disease of adult sheep, has a relentless course leading to dyspnea, emaciation, and death. Clinical observations and serologic tests are adequate for making a tentative diagnosis. The agar gel immunodiffusion test seems to be the best serologic procedure for indicating infection with the virus but cannot be used to predict morphologic changes or clinical disease, inasmuch as many clinically unaffected animals carry the virus. A definitive diagnosis is based on finding lesions and isolating virus. Affected lungs are large and heavy as a result of interstitial accumulation of lymphoid cells and fibromuscular tissue. Frequently, interstitial lesions are accompanied by bronchopneumonia from secondary bacterial infection. The causal virus can be isolated from infected lungs by cocultivation with primary ovine or bovine cells.


Pancreatic islet cell adenocarcinoma: clinical and diagnostic features of six cases. The clinicopathologic aspects of pancreatic islet cell adenocarcinoma in 6 dogs were compared. Diagnosis was based on insulin-glucose ratios in 5 dogs. Surgical excision of the tumor resulted in absence of clinical signs for at least 1 year in 3 dogs.


Structure of bonito heart ferricytochrome c and some remarks on molecular interaction in its crystalline state. The structure analysis of bonito heart ferricytochrome c was carried out at 2.8 A resolution by X-ray diffraction. The overall features of the molecule are virtually identical with those of bonito ferrocytochromes c and other cytochromes c. In the present work, the modes of molecular packing among cytochromes c were also compared by means of intermolecular distance maps. Some differences in the structures of ferro- and ferricytochrome c may exist on the surface of the molecules.


Phosphodiesterase-phosphomonesterases from Fusarium moniliforme. Separation and properties of four isozymes. Purified phosphodiesterase-phosphomonoesterase was found to be composed of four isozymes with different isoelectric points. These isozymes, phosphodiesterase-phosphomonoesterases 1-4, were separated from one another by repeated isoelectric focusing. Very little difference in amino acid composition, enzymic properties or circular dichroism spectra was detected among the isozymes. Far-ultraviolet circular dichroism spectra showed that the enzyme contained about 10% alpha-helix and 40% beta-structure. Phosphodiesterase-phosphomonesterase is a glycoprotein, because it was adsorbed on concanavalin A-Sepharose 4B and gave a band of carbohydrate coincident with that of protein or enzymic activity on polyacrylamide disc gel electrophoresis. Carbohydrate analyses revealed that the enzyme contained 37 micron of N-acetylglucosamine and 358 micron of mannose per mg of protein. The carbohydrate contents of the four isozymes were almost the same.


Studies on nitrate reductase of Clostridium perfringens. II. Purification and some properties of ferredoxin. A ferredoxin was purified from Clostridium perfringens by DEAE-cellulose chromatography and Sephadex G-50 gel filtration. It had absorption maxima at 390 and 280 nm. The molecular weight was estimated to be 6,000 by Sephadex gel filtration and from the results of amino acid analysis. The isoelectric point was 3.0. It contained four atoms of iron, four atoms of labile sulfur, and six cysteine residues. This ferredoxin as well as ferredoxin from C. pasteurianum acted as an electron donor for nitrate reductase from C. perfringens. The ferredoxin could also act as an electron donor for the hydrogenase from C. pasteurianum in hydrogen evolution.


ADP ribosylation of rat liver nucleosomal core histones. When nucleosomal core histones were isolated from rat liver nuclei incubated with [14C]NAD+ and fractionated into the individual components (H2A, H2B, H3, and H4), [14C]adenosine diphosphate ribose (ADP-Rib) was found to be associated with all of them. However, while about 15% of the H2B molecules were modified, less than 2% of the other fractions contained radioactive ADP-Rib. The nucleotide attached to H2B was identified as a single monomer of ADP-Rib. On subjectint H2B to electrophoresis in polyacrylamide gels containing 2.5 M urea and 0.9 N acetic acid, one single band of H2B with 5% less mobility than the unomdified control was obtained. The linkage between H2B and ADP-Rib was rapidly hydrolyzed with 0.1 N NaOH or with 1 M neutral hydroxylamine. Hydrolysis of ADP-ribosylated H2B with trypsin generated a single peptide linked to ADP-Rib, which corresponded to the sequence Pro-Glu-Pro-Ala-Lys. We were able to dansylate the NH2-terminal proline, which proved that the imino group of this amino acid was not substituted. These findings, together with the chemical properties of the linkage, which were typical of those of an ester-like bond, strongly suggest that the ADP-Rib residue was linked to the gamma-COOH group of the glutamic acid in position 2 of H2B.


Cell-free synthesis of mouse corticotropin. Evidence for two high molecular weight gene products. Polysomes or mRNA prepared from cultured AtT-20/D16v mouse pituitary adenocarcinoma cells direct the efficient incorporation of amino acid into newly synthesized material in the presence of wheat germ translational factors. A significant franction of the total cell-free product is specifically immunoprecipitable with corticotropin antibody purified by immune affinity chromatography. Analysis of the cell-free synthesized immunoreactive products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals that two high molecular weight corticotropin species (Mr congruent to 32,500 and 28,000) are synthesized in an approximate 2:1 ratio. Neither product contains carbohydrate based upon concanavalin A chromatography or exposure to polysaccharidases. The smaller molecular weight product does not appear to arise from proteolytic processing since both species are synthesized in approximately the same ratio in cell-free reaction mixtures directed by either polysomes or mRNA. These results suggest that AtT-20/D16v cells contain two distinct mRNA poluations specifying the synthesis of two different high molecular weight forms of mouse corticotropin.


Deoxyguanosine kinase. Distinct molecular forms in mitochondria and cytosol. Two distinct deoxyguanosine kinase activities have been identified in calf thymus tissue. They can be differentiated by subcellular location, electrophoretic mobility, chromatographic behavior, nucleoside specificity, apparent Km values, and end product inhibition. After a 130-fold purification from mitochondrial extract, the newly discovered kinase was specific primarily for deoxyguanosine and deoxyinosine. Unlike the cytosol enzyme, which proved to be the broadly specific deoxycytidine kinase studied previously, the mitochondrial enzyme does not phosphorylate deoxycytidine. Its apparent Km for deoxyguanosine, 6 micromolar, is 2 orders of magnitude lower than that of the cytosol enzyme. The mitochondrial enzyme is strongly inhibited by dGTP and dITP and activated up to 6-fold by dTDP and UDP, whereas neither dCTP nor dATP had much effect.


Modification of human placental lactogen with plasmin. Preparation and characterization of a modified hormone with increased biologic activity. To determine the structure needed for the biologic activity of human placental lactogen (hPL), we have cleaved hPL with the proteolytic enzyme plasmin. Plasmin modified hPL (PL-hPL) was purified by gel chromatography. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis before and after reduction showed that cleavage had occurred within the Cys53-Cys165 loop and tryptic peptide maps revealed that a single peptide consisting of residues 135 to 140 had been removed. 5-Dimethylaminonaphthalene-1-sulfonyl end group analysis and digestion with carboxypeptidase B confirmed that cleavage was complete and only the single hexapeptide was removed. In a membrane binding assay for lactogenic activity PL-hPL was 2- to 3-fold more potent than hPL. Using growth hormone receptors from rabbit liver membranes, PL-hPL was also more potent than hPL, but still much less potent than growth hormone. The lactogenic activity of PL-hPL in an in vitro bioassay was 75% above that of unmodified hormone. It is concluded that plasmin cleaves homologous peptides from hPL and growth hormone and that removal of the hexapeptide from hPL results in enhanced biologic activity.


Regulatory interactions between mitochondrial genes. I. Genetic and biochemical characterization of some mutant types affecting apocytochrome b and cytochrome oxidase. The region of the mtDNA containing the structural gene for apocytochrome b is called the cob or box region. There is evidence that the same region is also involved in the regulation of cytochrome oxidase. We have isolated eight mit- mutants in this region and have ordered them using petite deletion mapping. Four of these mutants appear to map outside the boxII region on the oli2-proximal end. Analysis of restriction endonuclease fragments of the mtDNA from peptides used in the deletion mapping suggests a minimum size of 3.1 x 10(3) base pairs for the whole cob region. Although none of our mutants contained any cytochrome b or cytochrome b-linked activities, polypeptides apparently related to apocytochrome b were present in some but not all mutants. Additional regulatory effects (both positive and negative) on cytochrome oxidase by virtue of control of its subunit I were also observed. In addition to these phenotypic traits, some of the mutants accumulated novel, mitochondrially translated polypeptides not seen in wild type.


Regulatory interaction between mitochondrial genes. II. Detailed characterization of novel mutants mapping within one cluster in the cob2 region. These studies describe the properties of three mit- mutants designated EM17, EM25, and PZ1, all mapping at two closely linked sites near one of the boundaries of the region of the mitochondrial genome concerned with the specification of cytochrome b. They all exhibit complex phenotypes affecting cytochrome b, cytochrome aa3, and additional polypeptides not found in the wild type. In the case of EM 17 this complexity can be ascribed to the presence of two mutations induced in the course of the initial mutagenic treatment: one, the cob2 mutation proper, is responsible for the loss of cytochrome b which is replaced by an altered, functionally inactive polypeptide, cytochrome b. This polypeptide can be further modified, or even eliminated, by the controlled introduction of another mutation in the cob1 segment of the cob region. The reduction in cytochrome oxidase subunit I, responsible for the effects on cytochrome aa3 and enzymatic activity in EM17, is due to a second (not mit-) mutation that has been located in the par1-proximal segment of the oxi3 region. This second mutation as well as the cob mutation can be overcome, and the respective aspect of wild type function restored to EM17, by recombination with rho- strains retaining the appropriate segment(s) of the wild type genome. The phenotype of the other two mutants is due to a single mutagenic event. This conclusion is confirmed by their ability to restore wild type functions by reversion. The mutation in EM25 appears to be due to a frameshift, which has led to premature chain termination, producing a polypeptide of Mr = 15,000 related to apocytochrome b. This change is accompanied by a decrease in the amount of subunit I of cytochrome oxidase. Revertants fall into three classes: on galactose two produce a polypeptide indistinguishable from apocytochrome b, but vary in its amount, while the third fails to increase apocytochrome b above mutant levels. Production of subunit I is increased but fails to reach wild type levels. Complete restoration of wild type functions can, however, be obtained by recombination of EM25 with rho- (cob2+) strains. Mutation PZ1 results in a complete absence of any polypeptide related to apocytochrome b and of cytochrome oxidase subunit I. These cells produce a novel polypeptide with a Mr = 45,000 not found in the wild type, and unrelated to all its normal polypeptides. Reversion or recombination with rho- (cob2+) strains results in virtually complete restoration of all wild type functions and the elimination of the novel polypeptide.


Resolution of the phosphorylated and dephosphorylated cAMP-binding proteins of bovine cardiac muscle by affinity labeling and two-dimensional electrophoresis. The photoaffinity label 8-azido[32P]adenosine 3':5'-monophosphate (8-azido-cyclic [32P]AMP) was used to analyze both the cAMP-binding component of the purified cAMP-dependent protein kinase, and the cAMP-binding proteins present in crude tissue extracts of bovine cardiac muscle. 8-Azido-cyclic [32P]AMP reacted specifically and in stoichiometric amounts with the cAMP-binding proteins of bovine cardiac muscle. Upon phosphorylation, the purified cAMP-binding protein from bovine cardiac muscle changed its electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels from an apparent molecular weight of 54,000 to an apparent molecular weight of 56,000. In tissue extracts of bovine cardiac muscle, most of the 8-azido-cyclic [32P]AMP was incorporated into a protein band with an apparent molecular weight of 56,000 which shifted to 54,000 upon treatment with a phosphoprotein phosphatase. Thus a substantial amount of the cAMP-binding protein appeared to be in the phosphorylated form. Autoradiograms following sodium dodecyl sulfate-polyacrylamide gel electrophoresis of both the pure and impure cAMP-binding proteins labeled with 8-azido-cyclic [32P]AMP revealed another binding component with a molecular weight of 52,000 which incorporated 32P from [gamma-32P]ATP without changing its electrophoretic mobility. Limited proteolysis of the 56,000- and 52,000-dalton proteins labeled with 32P from either [gamma-32P]ATP.Mg2+ or 8-azido-cyclic [32P]AMP showed patterns indicating homology. On the other hand, peptide maps of the major 8-azido-cyclic [32P]AMP-labeled proteins from tissue extracts of bovine cardiac muscle (Mr = 56,000) and rabbit skeletal muscle (Mr = 48,000) displayed completely different patterns as expected for the cAMP-binding components of types II and I protein kinases. Both phospho- and dephospho-cAMP-binding components from the purified bovine cardiac muscle protein kinase were also resolved by isoelectric focusing on polyacrylamide slab gels containing 8 M urea. The phosphorylated forms labeled with 32P from either [gamma-32P]ATP or 8-azido-cyclic [32P]AMP migrated as a doublet with a pI of 5.35. The 8-azido-cyclic [32P]AMP-labeled dephosphorylated form also migrated as a doublet with a pI of 5.40. The phosphorylated and dephosphorylated cAMP-binding proteins migrated with molecular weights of 56,000 and 54,000, respectively, following a second dimension electrophoresis in sodium dodecyl sulfate. The lower molecular weight cAMP-binding component (Mr = 52,000) was also apparent in these gels. Similar experiments with the cAMP-binding proteins present in tissue extracts of bovine cardiac muscle indicate that they are predominantly in the phosphorylated form.


Quantitation of the various termini generated by type II restriction endonucleases using the polynucleotide kinase exchange reaction. Parameters of the polynucleotide kinase-catalyzed exchange reaction between [gamma-32P]ATP and 5'-phosphoryl DNAs have been measured with the termini generated by the following endonucleases: EcoRI (Berkner, K. L., and Folk, W. R. (1977) J. Biol. Chem. 252, 3176-3184), Hpa II, BamHI, and HindIII (external termini); HindII and Hpa I (blunt terminal); Hae II and Hha I (internal termini). In every case, exchange is reproducible and proportional to the number of termini. However, in most cases, the exchange reaction does not proceed to the theoretical maximum. External termini and single-stranded DNAs are labeled more rapidly and to approximately 5-fold higher levels than blunt or internal termini. Concentrations of 100 to 200 microns ADP and 12 microns ATP are optimal for labeling all types of termini with the exchange reaction.


The effect of beta-xylosides on heparan sulfate synthesis by SV40-transformed Swiss mouse 3T3 cells. The medium and cell surface heparan sulfates isolated from SV40-transformed Swiss mouse 3T3 cells were examined in the presence and absence of 1.0 mM p-nitrophenyl-beta-D-xyloside. Incubation of the SV3T3 cells with this beta-xyloside resulted in: (a) a 4- to 5-fold reduction in the molecular weight distribution of medium heparan sulfate, (b) a 10-fold increase in the total synthesis of medium heparan sulfate, and (c) a small reduction in cell growth. There was little, if any, change in either the total level of synthesis or the molecular weight distribution of cell surface heparan sulfate. The covalent association of the beta-xyloside to the medium heparan sulfate was demonstrated by an analysis of the medium heparan sulfate produced by cells grown in the presence of [35S]sulfate and the fluorogenic beta-xyloside, 4-methylumbelliferyl-beta-D-xyloside. Treatment of the purified radiolabeled and fluorogenic heparan sulfate with either nitrous acid or heparitinase resulted in a decrease in the molecular weight of both radiolabeled and fluorogenic material. The data presented in this paper are discussed with respect to both the structure of heparan sulfate and the putative role of heparan sulfate in cell social behavior.


Spectral evidence for tyrosine ionization linked to a conformational change in liver alcohol dehydrogenase ternary complexes. Resonance energy transfer from Trp-314 to ionized Tyr-286 was proposed (Laws, W. R., and Shore, J. D. (1978) J. Biol. Chem. 253, 8593-8597) as the mechanism for the observed decrease in protein fluorescence of liver alcohol dehydrogenase seen with alkaline pH, or with the formation of a ternary complex with NAD+ and trifluoroethanol. In the present study, ultraviolet difference spectra confirm the presence of ionized tyrosine not only in these two cases but also in the ternary complex with NADH and isobutyramide. Our results indicate that ternary complex formation, with either oxidized or reduced coenzyme, causes a conformational change leading to partial ionization of tyrosine residues in regions of the enzyme far from the active site.


Use of heme spin-labeling to probe heme environments of alpha and beta chains of hemoglobin. A spin label attached to a propionic acid group of the heme has been used to probe the heme environment of the alpha and beta chains of hemoglobin in both the subunit and tetrameric forms. The electron paramagnetic resonance (EPR) studies of hemoglobin hybrids in which the spin label is attached to either the alpha- or beta-heme (alpha2SLbeta 2 or alpha2beta2SL) and spin-labeled isolated chains (alphaSL and betaSL) show that: 1) alpha- and beta-hemes have different environments in the tetrameric forms of oxy-, deoxy-, and methemoglobins as well as in isolated single chains; 2) when isolated subunits associate to form hemoglobin tetramers, the environment of the alpha-heme changes more drastically than that of the beta-heme; 3) upon deoxygenation of hemoglobin, the structure in the vicinity of the alpha-heme changes more drastically than that of the beta-heme; and 4) upon the addition of organic phosphates to methemoglobin, the change in the spin state of the heme irons mainly arises from beta-heme. The results demonstrate conclusively that the alpha and the beta subunits of hemoglobin are structurally nonequivalent as are their structural changes as the result of ligation. The relationship of EPR spectrum and structure of hemoglobin is discussed.


Phosphorylation of synthetic peptide analogs of rabbit cardiac troponin inhibitory subunit by the cyclic AMP-dependent protein kinase. A series of synthetic peptide analogs of the cardiac troponin inhibitory subunit (TN-1) phosphorylation site sequence, Arg12-Pro-Ala-Pro-Ala-Val-Arg18-Arg19-Ser20-Asp21-Arg22-Ala, have been tested as substrates for the catalytic subunit of the cyclic AMP-dependent protein kinase (EC 2.7.1.37, ATP:protein phosphotransferase). As substrates, these peptides were generally inferior to the pyruvate kinase analog peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly or its COOH-terminal amide analog. Replacing Arg-19 with alanine had only a minor effect on the kinetics of phosphorylation of the TN-1 peptide analog. In contrast, replacement of Arg-22 and Arg-18 with alanine resulted in marked enhancement and reduction of the Vmax, respectively. The results of this study have demonstrated that synthetic peptide analogs of the local phosphorylation site sequences of natural substrates may differ widely in their capacity to act as substrates for this protein kinase. In the case of the TN-1 peptide analogs, the contribution of the 4 arginine residues can be distinguished in terms of their influence on the kinetics of phosphorylation.


Inhibition of erythrocyte membrane (Na+ + K+)-activated ATPase by ozone-treated phospholipids. Ozone-treated aqueous suspensions of natural phospholipids yield at least two types of inhibitors of human erythrocyte membrane (Na+ + K+)-ATPase. The more labile ones appear to be carbonyl-containing substances whose inhibitory properties are enhanced if ozonolysis takes place in the presence of putrescine or glycine. Other amines of similar structure are much less effective as potentiators. Semicarbazide destroys the inhibitory properties of the more labile substances and can release putrescine from the complexes it forms with the carbonyl products of ozonolysis. 3the more stable inhibitors are unaffected by putrescine, glycine, or semicarbazide. Synthetic, saturated phospholipids do not produce these inhibitors during ozonolysis.


Biosynthesis of lipid-linked oligosaccharides. Isolation and structure of a second lipid-linked oligosaccharide in Chinese hamster ovary cells. Previous work has shown that vesicular stomatitis virus-infected Chinese hamster ovary cells contain a major high molecular weight lipid-linked oligosaccharide which is transferred en bloc to protein during the formation of the asparagine-linked complex-type oligosaccharides of the vesicular stomatitis virus G protein (Tabas, I., Schlesinger, S., and Kornfeld, S. (1978) J. Biol. Chem. 253, 716-722). We now report the characterization of a second, lower molecular weight lipid-linked oligosaccharide. The oligosaccharide portion of this molecule was isolated and its structure was determined by methylation analysis, digestion with exoglycosidases, acetolysis and Smith periodate degradation to be: (formula: see text). Several lines of evidence are presented which indicate that this lipid-linked oligosaccharide is primarily involved in the assembly of the major lipid-linked oligosaccharide rather than in the direct glycosylation of proteins.


Modulation of glucose uptake in animal cells. Studies using plasma membrane vesicles isolated from nontransformed and simian virus 40-transformed mouse fibroblast cultures. Plasma membrane vesicles isolated from nontransformed and Simian virus 40-transformed mouse fibroblast cultures catalyzed carrier-mediated D-glucose transport without detectable metabolic conversion to glucose 6-phosphate. Glucose transport activity was stereospecific, temperature-dependent, sensitive to inactivation by p-chloromercuriphenylsulfonate, and accompanied plasma membrane material during subcellular fractionation. D-Glucose efflux from vesicles was inhibited by phloretin, an inhibitor of glucose uptake in intact cells. Cytochalasin B, a potent inhibitor of glucose uptake when tested with the intact cells used for vesicle isolation did not inhibit glucose transport in vesicles despite the presence of high affinity cytochalasin binding sites in isolated membranes. The enhanced glucose uptake observed in intact cells after viral transformation was not expressed in vesicles: no significant differences in glucose transport specific activity could be detected in vesicle preparations from nontransformed and transformed mouse fibroblast cultures. These findings indicate that cellular components distinct from glucose carriers can mediate changes in glucose uptake in mouse fibroblast cultures in at least two cases: sensitivity to inhibition by cytochalasin B and the enhanced cellular sugar uptake observed after viral transformation.


ADP ribosylation of rat liver lysine-rich histone in vitro. Purified rat liver nuclei were incubated with [14C]-NAD+ and the various nuclear protein fractions were separated. Forty per cent of the total radioactivity incorporated was associated with the histone fraction. Of this, about 50% was extracted with H1, in 0.5 N perchloric acid. When crude H1 was purified and fractionated into five different subfractions by chromatography on Bio-Rex 70, it was found that all the H1 subfractions contained radioactivity. This radioactive material was identified as oligomers of adenosine diphosphate ribose (ADP-Rib) with an average chain length which corresponded to trimers. The extent of the modification was dependent on the concentration of NAD+. About 60% of the H1 molecules were modified with a concentration of 1 mM NAD+. The presence of these oligomers of ADP-Rib introduced a large degree of microheterogeneity to H1 as detected by electrophoresis in polyacrylamide gels containing 2.5 M urea and 0.9 N acetic acid. Bands of H1 with 10 to 20% less mobility than the unmodified H1 were present. Also, as a consequence of large content of ADP-Rib, the absorption maximum shifted from 275 to 259 nm. The half-life of the bond between the oligomers of ADP-Rib and H1 was about 3 min at 37 degrees C in the presence of 0.1 N NaOH, and 10 m1 were modified. The site of ADP ribosylation in the NH2-terminal half was localized in the tryptic peptide extending from the NH2-terminal end to lysine 15. The site of modification of the COOH-erminal half was localized in the tryptic peptide which contained the only glutamic acid residue in this fragment of H1...


Inhibitor of human collagenase from cultures of human tendon. A potent inhibitor of human collagenases, released from human tendon explants in culture, has been purified and partially characterized. The tendon inhibitor has an estimated molecular weight of 25,000. It is relatively heat-stable but undergoes loss of activity following exposure to trypsin. It inhibits trypsin-activated rheumatoid synovial collagenase as well as the enzyme obtained from polymorphonuclear leukocytes. No inhibition of collagenase from Clostridium histolyticum (clostridiopeptidase A, EC 3.4.24.3) was noted. This collagenase inhibitor may be a factor in the regulation of extracellular connective tissue catabolism.


Single strand DNA binding of simian virus 40 tumor antigen. Simian virus 40 T antigen binds to both single and double strand DNA. The single and double strand DNA binding activity of crude T antigen preparations was evaluated by chromatography of the antigen on DNA-cellulose columns. Crude T antigen was retained on both native and denatured DNA-cellulos columns and was eluted from both columns under similar conditions. The interaction of highly purified T antigen with single and double strand DNA was evaluated by competition experiments using a DNA filter binding assay. These experiments showed that T antigen binds preferentially to single strand calf thymus DNA by more than an order of magnitude when compared to double strand calf thymus DNA.


In vitro synthesis of a putative precursor to the mitochondrial enzyme, carbamyl phosphate synthetase. A simple and rapid procedure is described for purification of carbamyl phosphate synthetase from the matrix fraction of rat liver mitochondria. Antibodies to the enzyme were raised in sheep and purified from antiserum by affinity chromatography on enzyme-bound Sepharose columns. When membrane-free polyribosomes, isolated from a cytosolic fraction of rat liver, were incubated in a messenger-dependent rabbit reticulocyte protein-synthesizing system in the presence of [35S]methionine, the purified antibody precipitated a product of translation representing 0.2% of total trichloroacetic acid-insoluble radioactivity. It demonstrated mobility characteristics in sodium dodecyl sulfate-polyacrylamide gels expected for a polypeptide of molecular mass approximately 5500 daltons larger than the mature mitochondrial form of the enzyme (160,000 daltons). Proteolysis of both the mature and presumptive in vitro precursor forms of the enzyme yielded respective sets of peptide fragments which gave similar patterns upon gel electrophoresis. Excess mitochondrial enzyme effectively competed with the in vitro product for interaction with anti-carbamyl phosphate synthetase antibody.


No effect of cAMP on protein synthesis in reticulocyte lysates. The results of a series of experiments are interpreted to indicate that protein synthesis in reticulocyte lysates is not affected by the reticulocyte cAMP-dependent protein kinase. The catalytic subunit of this enzyme was isolated to apparent homogeneity. Also, the protein inhibitor of this protein kinase was isolated from muscle. Neither physiological concentrations of cAMP nor any of these protein components had a detectable effect on protein synthesis in reticulocyte lysates in the presence or absence of exogenous heme. Phosphorylation of the smallest subunit of eukaryotic initiation factor 2 or the 90,000 to 100,000-dalton peptide associated with eukaryotic initiation factor 2 kinase activity were not affected by the activity of the cAMP-dependent protein kinase under conditions in which exogenous heme has a pronounced effect on these reactions.


Identification of histidine-119 as the target in the site-specific inactivation of ribonuclease A by ferrate ion. Ferrate ion, a powerful oxidant which is an analog of orthophosphate ion, has previously shown some promise as a site-specific probe of enzymes which interact with phosphate compounds. In order to explore the general applicability of this reagent, it has been tested against ribonuclease A, an enzyme whose structure and active center have been well described. Treatment with a molar ratio of ferrate to enzyme of less than 20 leads to a loss of 87% of the activity. The known competitive inhibitors, 2'-cytidylic acid, inorganic pyrophosphate, and orthophosphate all protect the enzyme from inactivation. Inactivation is accompanied by a loss of the capacity to bind 2'-cytidylic acid. Ferrate inactivation at pH 5.0 is accompanied by the modification of only one amino acid. The amino acid which was identified by amino acid and sequence analyses of peptide fragments obtained by cyanogen bromide treatment and selective proteolysis proved to be histidine-119, whose essential role at the active center has long been established.


Structural differences between apo- and holoenzyme of horse liver alcohol dehydrogenase. The three-dimensional structure of a ternary complex of horse liver alcohol dehydrogenase with reduced nicotinamide adenine dinucleotide and the inhibitor dimethyl sulfoxide has been determined to 4.5 A resolution independently of the apoenzyme structure. The electron density maps of both structures have been compared. The two coenzyme binding domains which form the center of the dimer molecular have retained their conformation and orientation within the molecule whereas the catalytic domains rotate and narrow the cleft between the domains. The active site becomes shielded from the solution by a combination of this rotation, local movements of a loop from residues 53 to 57 and coenzyme and substrate binding. Both subunits bind coenzyme and inhibitor to the same extent. The nicotinamide ring of the coenzyme is positioned close to the active zinc atom and the inhibitor is bound to this zinc atom. The difference between the two crystallographically independent subunits is small. The proposed mechanisms of action for the enzyme based on the apoenzyme structure are confirmed by the present investigation.


25-Azavitamin D3, an inhibitor of vitamin D metabolism and action. 25-Azavitamin D3 inhibited both the bone calcium mobilization and intestinal calcium transport responses of rats to vitamin D3 but not to 25-hydroxyvitamin D3. Although 25-azavitamin D3 had no effect on the response of bone to 1alpha,25-dihydroxyvitamin D3, it did diminish the response of the intestine to that metabolite. 25-Azavitamin D3 increased liver vitamin D content and reduced the concentration of 25-hydroxyvitamin D3 required to inhibit the metabolism of vitamin D3 (75 and 200 microgram) were similar to the doses of 25-azavitamin D3 required to inhibit the action of vitamin D3 in vivo (50 and 150 microgram). 25-Azavitamin D3 is thus a vitamin D antagonist, acting for the most part via inhibition of the liver 25-hydroxylation of vitamin D3.


Lifetimes and NADH quenching of tryptophan fluorescence in pig heart cytoplasmic malate dehydrogenase. The time-resolved and steady state fluorescence properties were measured for pig heart cytoplasmic malate dehydrogenase at pH 6.0 and 8.0. The fluorescence decay can be described by two rate processes, according to the functions: I(t) = 0.7e(-t/1.0) + 0.3e(-t/4.4) for the free enzyme and I(t) = 0.7e(-t/0.8) + 0.3e(-t/2.0 for the enzyme . NADH complex. Quenching by NADH of the tryptophan fluorescence is linear. The only effect of pH is to change the association constant for NADH binding; the fluorescence of the free enzyme and the fluorescence quenching by NADH, I-, and acrylamide are unaffected by pH. Thus there are no changes in conformation of the free enzyme or of the NADH complex over the range of pH 6 to 8.


Structure of simian virus 40 recombinants that contain both host and viral DNA sequences. I. The structure of variant CVPS/1/P2 (EcoRI res). The entire nucleotide sequence (1210-base-pair repeating units) of a defective variant of simian virus 40 is presented. Within this variant there are deletions of large portions of the wild type genome and an inversion within the remaining wild type viral sequences. In addition, the defective variant contains DNA sequences derived from the permissive monkey cells in which the virus was propagated. The monkey sequences include a portion that is homologous to sequences within highly repeated monkey DNA (alpha component) as well as portions derived from sequences that are infrequently repeated in the monkey genome. One out of every three to four of the tandem 1210-base-pair repeat units contains in addition, a duplication of a part of the monkey sequences. The sequence information defines the structures of a number of recombinational joints which result from deletions, inversions, duplications, and insertions of host sequences into the viral genome. The data demonstrate that the various recombinational events that resulted in the formation of this defective variant did not depend on extensive homology between recombining segments.


Structure of simian virus 40 recombinants that contain both host and viral DNA sequences. II. The structure of variant 1103 and its comparison to variant CVPS/1P2 (EcoRI res). In an effort to characterize sites of recombination between SV40 and monkey DNA, we have determined the primary sequence of a large portion of the SV40 variant, designated 1103. This virus contains DNA sequences derived both from the wild type SV40 genome and from the permissive monkey cell in which the virus was propagated. Further, the monkey sequences included in the defective genome are homologous to both highly repeated monkey DNA (alpha component) and sequences that are infrequently repeated in the monkey genome. The regions of the 1103 genome where DNA sequences were determined include 1) the segments of the variant that surround joints connecting SV40 and monkey sequences, 2) the segment that contains the joint between monkey sequences of high and low reiteration frequency, and 3) the DNA segment of the variant that is homologous to monkey alpha component DNA. Comparison of the data obtained from the sequences analysis of the SV40 variants 1103 and CVP8/1/P2 (EcoRI res) (described in Wakamiya, T., McCutchan, T., Rosenberg, M., and Singer, M. (1979) J. Biol. Chem 254, 3584-3591) reveals certain similarities between the two that may be involved in eukaryotic recombination and defective variant formation.


Purification and characterization of poly(ADP-ribose) synthetase from calf thymus. Poly(ADP-ribose) synthetase from calf thymus has been purified to apparent homogeneity by a simple and rapid method with a recovery of 10 to 20%. The enzyme activity absolutely requires the presence of DNA. Histone further stimulates the reaction. The Km for NAD and the maximal velocity at 25 degrees C and pH 8.0 in the presence of both compounds are 55 micron and 1,400 nmol/min/mg, respectively. The sedimentation coefficient (s020,w) of the enzyme is 5.80 S. The molecular weight is calculated to be 108,000 by sedimentation equilibrium method using a partial specific volume of 0.736 ml/g. This value is in good agreement with the molecular weight values of 115,000 and 120,000 determined by gel filtration on Sephadex G-200 and gel electrophoresis in the presence of sodium dodecyl sulfate, respectively. The enzyme is colorless and its absorption spectrum shows a maximum at 280 nm. From a CD spectrum, alpha helical content is estimated to be approximately 30%. The enzyme is a basic protein having a pI value of 9.8 and is rich in lysine rather than arginine. Neutral sugar, phospholipid, and DNA are not detected in the final preparation. These data indicate that the purified enzyme is a simple globular protein composed of a single polypeptide having an approximate molecular weight of 110,000.


Alveolar cell carcinoma on CT scanning. The value of the air bronchogram sign. The computed tomography findings in alveolar disease are described in a patient with alveolar cell carcinoma. The recognition of an air bronchogram on CT scanning may be useful in establishing the alveolar nature of diffuse or focal lung disease.


An analysis of Con A-mediated agglutination in a Chinese hamster ovary subclone which responds morphologically to growth in dibutyryl cyclic AMP. III. The role of microvilli in the agglutination process. We have used the H-7w subclone of a Chinese hamster ovary cell line (K1) to investigate the role of cell surface architecture (specifically microvilli, blebs, and sheets) in determining the relative agglutinability of a cell line with Con A. Our evidence clearly demonstrates that no specific, immediately recognizable surface architecture is associated with the agglutinable or non-agglutinable phenotype. Our data suggest that the expression of microvilli on the cell surface is neither necessary to nor sufficient for the phenotype described by enhanced agglutinability with Con A. Furthermore our work demonstrates that cells covered with blebs are as agglutinable as cells covered with microvilli thereby suggesting that the intertwining of microvilli may not be an essential facet of the agglutination phenomenon.


The pattern of appearance of enzymic activity during the development of the Golgi apparatus in amoebae. The appearance of enzymic activity during the development of the Golgi apparatus was studied by cytochemical staining of renucleated amoebae. In cells enucleated for 4 days, there was a great decline in size and number of Golgi bodies, or dictyosomes. Subsequent renucleation by nuclear transplantation resulted in a regeneration of Golgi bodies. Samples of amoebae were fixed and incubated for cytochemical staining at intervals of 1, 6, or 24 h after renucleation. Enzymes selected for study were guanosine diphosphatase (GDPase), esterase, and thiamine pyrophosphatase (TPPase). All three were found in the Golgi apparatus of normal amoebae but they differed in their overall intracellular distribution. GDPase was normally present at the convex pole of the Golgi apparatus, in rough endoplasmic reticulum, and in the nuclear envelope. In amoebae renucleated for 1 h, light reaction product for GDPase was present throughout the small stacks of cisternae that represented the forming Golgi apparatus. By 6 h following the operation GDPase reaction product was concentrated at the convex pole of the Golgi apparatus. Esterase, which was distributed throughout the stacks of normal Golgi cisternae, displayed a similar distribution in the forming Golgi bodies as soon as they were visible. TPPase was normally present in the Golgi apparatus but was not found in the endoplasmic reticulum. In contrast to the other enzymes, TPPase reaction product was absent from the forming Golgi apparatus 1 and 6 h after renucleation, and did not appear in the Golgi apparatus until 24 h after operation. Thus, enzymes held in common between the rough endoplasmic reticulum and the Golgi apparatus were present in the forming Golgi apparatus as soon as it was detectable, but an enzyme cytochemically localized to the Golgi apparatus only appeared later in development of the organelle. It is suggested that Golgi membranes might be derived from the endoplasmic reticulum and thus immediately contain endoplasmic reticulum enzymes, while Golgi-specific enzymes are added later in development.


Pseudohypoaldosteronism: multiple target organ unresponsiveness to mineralocorticoid hormones. The first report of a 7-month-old male with pseudohypoaldosteronism in which unresponsiveness to mineralocorticoids has been demonstrated in the kidney, colon, and sweat and salivary glands is presented here. This is documented by urinary, salivary, and sweat sodium wasting in the presence of elevated urinary aldosterone excretion, plasma aldosterone concentration, and PRA. There was no mineralocorticoid response in the kidney or salivary or sweat glands to the administration of high doses of 9 alpha-flurocortisol. Furthermore, in this patient, the colonic mucosal cells failed to respond to exogenous aldosterone administration. Repeat evaluation at 25 months of age showed persistence of the sodium wasting and multiple target organ insensitivity to administered mineralocorticoid. Since this patient has defective mineralocorticoid response in the major sodium-conserving organs, the only therapy possible was administration of sodium to compensate for total sodium loss.


Immunoreactive somatomedin A in human serum. A RIA has been developed for somatomedin A (SM-A) utilizing Sepharose-bound antibodies. This assay, measuring SM-A, the insulin-like growth factors 1 and 2, and somatomedin C, allows determination in serum samples. In comparison with a serum standard, the mean serum levels in patients with acromegaly or GH deficiency and healthy subjects were 8.7 %/- 0.7 (n = 25), 0.24 +/- 0.02 (n = 25), and 1.15 +/- 0.11 U/ml, respectively. The correlation coefficient between immunoreactive SM-A and SM-A by radioreceptor assay was highly significant (r = 0.93), although the potency ratio of SM-A between the two groups of patients was higher in the RIA than in the radioreceptor assay. Gel chromatography revealed that SM-A in acromegalic serum is bound to a carrier protein which is absent in patients with GH deficiency. After gel chromatography at low pH, 90% of applied immunoreactive SM-A was recovered in the low molecular weight fraction and consisted mainly of neutral polypeptides.


Plasma corticotropin and cortisol in patients with pituitary adenomas. Plasma ACTH and cortisol were measured during insulin-induced hypoglycemia in 39 patients with a pituitary adenoma or after hypophysectomy and in 26 healthy persons. Adrenocortical insufficiency was defined as failure to increase plasma cortisol to at least 500 nmol/liter (18 microgram/dl) during hypoglycemia. No proper distinction was possible between patients with normal and impaired cortisol response to hypoglycemia on the basis of the ACTH results. When all 39 patients were compared to the 26 controls, the cortisol results differed significantly, but those of ACTH did not. These results demonstrate a limited value of ACTH measurements in secondary adrenocortical failure.


"Acquired" adrenal hyperplasia with 21-hydroxylase deficiency is not the same genetic disorders as congenital adrenal hyperplasia. Hormonal studies and HLA genotyping were performed on the family of a patient with "acquired" adrenal hyperplasia (AAH) due to 21-hydroxylase deficiency. The results of these studies suggest that "AAH", is not the same genetic disease as CAH.


Serum somatomedin A in chronic renal failure. The serum levels of somatomedin A, determined by radioreceptor assay, were significantly higher in 57 adult patients with chronic renal failure (X = 2.57 +/- 0.12 U/ml) than in healthy subjects (n = 131; X = 0.77 +/- 0.02 U/ml). A positive correlation was found between somatomedin A and creatinine levels (r = 0.33), but somatomedin A levels did not decrease after hemodialysis. A reduction was only observed after successful renal allografting. Immunoreactive somatomedin A was also found to be increased in patients with chronic renal failure (X = 2.61 +/- 0.21 U/ml), whereas low levels were obtained by the chick bioassay. However, after separating the inhibitory factors from the stimulatory factors by gel chromatography at neutral pH, uremic serum was shown to contain increased levels of somatomedin activity. Although all somatomedin A, determined by different techniques, was present in high molecular forms, the elution pattern differed from that seen in patients with acromegaly.


Correlation between thyrotropin-displacing activity and human thyroid-stimulating activity by immunoglobulins from patients with Graves' disease and other thyroid disorders. Several reports have been published on the anti-TSH receptor antibody in putative autoimmune thyroid disorders using a radioreceptor assay. We have carried out correlative studies between the ability of serum immunoglobulins to displace radiolabeled TSH from the thyroid plasma membrane receptor [TSH-displacing activity (TDA)] and that of actual stimulation of the human thyroid gland [human thyroid-stimulating activity (hTSA)] in Graves' and other thyroid diseases and in control subjects. TDA was assayed by the use of a radioligand technique, while the activation of adenylate cyclase in human thyroid slices was measured as an index of hTSA. The same immunoglobulins were employed for both assays. In this series, positive TDA and hTSA values were found in 70.4% and 81.5% of the samples in active untreated Graves' disease, respectively. Samples from normal persons and from several patients with toxic nodular goiter gave generally negative results in both assays; in a small proportion of patients with either subacute thyroiditis or Hashimoto's thyroiditis, the TDA was positive but hTSA proved to be negative. In Graves' disease (including those patients on propylthiouracil) in remission and treated with 131I, the correlation between TDA and hTSA was not significant (r = 0.309; P greater than 0.1); even when the procedures were compared in the untreated group alone, there was no significant correlation between the two activities (r = 0.309, P greater than 0.1). These studies indicate that 1) significant TDA and hTSA are observed in Graves' disease; nevertheless, the correlation between them is not significant; 2) the hTSA assay appears to be more sensitive and specific than the TDA assay; and 3) TDA may not be synonymous with thyroid stimulation.


The effects of L-dopa on in vitro and in vivo calcitonin release from medullary thyroid carcinoma. The in vivo and in vitro effects of the dopamine precursor L-dopa on basal and stimulated calcitonin release from medullary thyroid carcinoma have been studied. In six studies of five patients, including 7- to 8-h control and test periods, oral L-dopa depressed basal calcitonin secretion by an average of 35%; the peak effects occurred within 30 min of drug administration and lasted for as long as 4 h. In seven of eight patients with medullary thyroid carcinoma (three infused with calcium and five with pentagastrin), L-dopa inhibited to varying degrees peak levels of stimulated calcitonin release and total calcitonin secretion; basal calcitonin levels, where directly tested, also again generally fell after L-dopa by an average of 50%. In a short term organ culture system using medullary thyroid carcinoma tissues, calcitonin secretion into the medium was linear with time for 2 h and could be stimulated by dibutyryl cAMP and pentagastrin. L-Dopa, in concentrations from 0.5--3.0 mM, inhibited basal calcitonin secretion (ranging from 25--55%). Addition of the L-dopa decarboxylase inhibitor, alpha-methyldopa, abolished the inhibitory effects of L-dopa. Another L-dopa decarboxylase inhibitor, carbidopa, stimulated calcitonin secretion in vitro; this effect may be independent of the L-dopa decarboxylase-inhibiting properties of this drug since alpha-methyldopa alone did not stimulate calcitonin secretion. It is concluded that the amine precursor L-dopa inhibits calcitonin release in patients with medullary thyroid carcinoma; the in vitro studies suggest that a portion of this effect may involve direct metabolism of L-dopa to dopamine in the tumor tissue itself. The importance of considering the uptake of amine precursors and the subsequent metabolism of these compounds as a modulating site for peptide hormone release from peripheral endocrine tissues is stressed.