[Isolation of the Epstein-Barr virus in suspension cell lines obtained from persons with different forms of hemoblastosis]. Isolation of EBV from patients with different types of haemoblastosis by establishing virus-producing cell lines is described. EBV was isolated from patients with myelogenous leukemia in 71%, Hodgkin's disease--in 22%. EBV was also isolated from 1 patient with lung tumor and leukemogenic reaction. All 8 established cell lines produced EBV in 0.5--3% of cells. The cells of the lines concerned are lymphoblasts and show B-cell characteristics.


[Nucleotide pool of the brains of rats during different stages of dying]. Composition of acid-extractable fraction of nucleotides was studied in brain of dying rats after acute hemorrhage. Within the first 5 min content of ATP, GTP, UTP, GDP, UDP and NAD was distinctly decreased; content of AMP, GMP, IMP, CMP, nucleosides and nitrogen bases was markedly increased. Within the following 30 min of the dying rpocess concentration of CMP and nitrogen bases was increased but content of the other substances studied was decreased as compared with the previous experimental period. Possible effect of the alterations found on reanimation of an organism is discussed.


[Viral oncogens and carcinogenesis].

[Viral oncogens and carcinogenesis]. The modern concepts of tumorigenic virus oncogenes are discussed. The available literature data on the identification of oncogenes of polyoma-, adeno- and oncoviruses and the corresponding protein the products of oncogenes, are reported. Under consideration is the idea of the cellular origin of virus oncogenes. It is suggested that the study of virus oncogenes would offer some new methodical approaches to the identification of cell genes, which impairment results in cell malignification.


[Changes in the quality of hormones in blood plasma and ischemic heart disease risk factors in 40--59-year-old men]. Testosterone, estradiol, hydrocortisone, total cholesterol, triglycerides, cholesterol of alpha-lipoproteins (alpha-CS) were estimated in blood plasma of 124 40--59 years old men examined in the course of an epidemyolodical study (representative selection). Content of insulin was estimated in 112 persons and of growth hormone--in 102 persons. Disbalance of steroid hormones (decrease in alpha-CS) was observed in men with one factor of risk of heart ischemic impairment. Occurrence of two factors of risk of heart ischemic impairment--hypertriglyceridemia and decrease in alpha-CS--was related not only to disbalance of steroid hormones but also to alteration in the ratio of protein hormones--insulin and growth hormone.


[Glycosaminoglycans in the makeup of the lipid-protein complexes of the human aorta]. Lipoprotein complexes were studied in intact parts of human aorta as well as in areas of aorta impaired with atherosclerosis. The arterial walls contained particles, which were similar to pre-beta and beta-lipoproteins as evidenced by immunological properties and by size. At the same time, lipoproteins of the vascular walls differed distinctly from those of blood serum because these former lipoproteins possesed altered electrophoretic mobilities in acetyl cellulose, contained glycosamineglycans, exhibited lower rate of flotation in salt-free solution as compared with the salt-containing solution under conditions of analytical ultracentrifugation. The data obtained suggest that the lipoproteins formed complexes with glycosamineglucans in vascular walls.


[Diagnosis of primary hypercholesteremia--a classification of genetic factors as causes for myocardial infarct]. Cultvated lymphocytes of patients with myocardial infarction with a primary hypercholesterolaemia as well as of unconspicuous control persons take up LDL-cholesterol labelled by 125iodine. Here is evident that lymphocytes of patients with a primary hypercholesterolaemia may incorporate significantly less LDL-cholesterol than healthy control persons. Supplementing the serum lipid determinations and the lipid electrophoresis the described radiochemical investigations at the model of lymphocytes make possible a biochemically exact diagnosis of the primary hypercholesterolaemia and thus an evident genetic analysis of the patients with infarctions.


[Intestinal hemorrhage in Klippel-Trenaunay syndrome]. It is reported on a 21-year-old patient with Klippel-Trenaunay's syndrome and simultaneous intestinal haemorrhage. Anamnesis and histological findings are discussed and the problems of therapy are explained. In the discussion the knowledge of the peripheral angiodysplasias and their differential diagnosis in literature are entered. Comparative considerations concerning the own case are made.


[Electronmicroscopic findings in a malignant hepatoma after oral contraceptives (author's transl)]. In this paper we report the electronmicroscopic findings in a hepatocellular carcinoma of a 24 years old woman after taking oral contraceptives. We saw alterations of the nucleus (finger-like or finger-formed nuclei, nuclear invaginations and intranuclear vesicular membranbodies), changes of the mitochondries (number, polymorphy and osmiophil bodies) as well as changes of lysosomes (myelinshaped residualbodies). The observed and described changes are not specific for malignant tumors of the liver.


Evaluation of continuous labelling data by a practical mathematical method with application to a virus-induced sarcoma of the rat. A simple mathematical function is established which renders the increase of the labelling index during continuous labelling of an exponentially growing tumor. By known values of proliferative fraction, mean cell cycle time and tumor doubling time, the mode and extent of cell loss is determined. Three models for different modes of cell loss are introduced: Model A for cell loss equally affecting proliferative and resting cells; Model B for cell loss predominantly affecting one of the two cell compartments; Model C for cell loss during mitosis. The range of validity of the model function is specified. Experimental data of a Polyoma virus-induced renal sarcoma of the rat provide an example of applicability of the theoretical model.


Multiple functions of thioredoxins.

Multiple functions of thioredoxins. Reduced thioredoxins from microbial and plant cells, both of cytoplasmic or chloroplast origin, are interchangeable in stimulating such diverse enzyme activities as ribonucleoside diphosphate reductase (E. coli), PAPS sulfotransferase (Synechococcus), and fructose-1,6-bis-phosphatase (from spinach) in vitro. It is suggested that reduced thioredoxins are unspecific, multifunctional cellular proteins while in contrast the oxidized froms require specific enzymes for their reduction.


Isolation of single stranded DNA from purified hepatitis A-virus. Hepatitis A-virus was purified from human stools by three purification steps. Virus was identified by radioimmunoassay and purity monitored with immune electron microscopy. Virus particles, serologically and morphologically identical, banded in CsCl in two density ranges at 1.31-1.34 g/cm3 and at 1.41-1.43 g/cm3. Virions of density 1.31-1.34 g/cm3 were shown to contain single-stranded DNA of different size classes. Class I 1.33 kb, class II 4.61 kb in addition a small amount of molecules was detected with lengths up to 15 kb.


[Vaccination and morbidity in Basel school-children (measles, mumps, rubella, chickenpox, cytomegalic inclusion disease)]. Antibodies against diseases of childhood have been determined in fall 1977 taking bloodsamples of 430 15-year old students. Immunity was widespread for measles (96%), chickenpox (93%), mumps (90%) whereas 27% did not have antibodies against rubella. The level of vaccination (measles, mumps, rubella) is increasing fast in younger children. The possibility of being infected by a wild virus will be low in the future for those who have not been vaccinated. Antibodies against Cytomegalovirus were measured in 120 students of normalschool and 107 "gymnasiasts". The proportion between negative and positive was 1.1 for students in normalschool and 2.7 for those attending a higher education.


[Serum lipoproteins in family members of myocardial infarct victims]. Men 40 to 59 years old, first degree relatives of patients with myocardial infarction had a lower HDL-cholesterol concentration than a control group. This is in analogy with the low concentration of HDL-cholesterol observed in the myocardial infarction patients themselves. LDL and VLDL in relatives were not elevated in comparison to the controls. The low HDL-cholesterol could be the expression of a familial defect in cholesterol transport.


[Effect of alloxan diabetes on (Na+ + K+)-activated ATPase in brush border membrane of the mucosal cell of rat small intestine]. In order to elucidate a possible relationship between (Na+ + K+)-activated ATPase and intestinal absorption of actively transported monosaccharides enzyme activity was measured in mucosal cells from alloxan diabetic rats. The general effect of increasing capacity of active, Na+-dependent transport processes in diabetes mellitus is associated with a significantly enhanced (Na+ +K+)-activated ATPase activity in mucosal homogenate from diabetic animals. To study the localization of these effects within the cell we isolated purified brush borders and their substructures. To enable a comparison to be made between preparation procedures of diabetic and control animals the fractions were controlled by electronmicroscopy and by measuring the sucrase activity. In the purified brush border fraction of alloxan treated rats there was no significant increase in (Na+ + K+)-activated ATPase activity. Based on these results we conclude that the (Na+ + K+)-activated ATPase in the basolateral membranes was increased in alloxan diabetes, and it seems very likely that this enzyme is involved in the regulation of Na+-dependent transport processes.


Analysis of function-related interactions of ATP, sodium and potassium ions with Na+- and K+-transporting ATPase studied with a thiol reagent as tool. The paper describes the interaction of ATP, Na+ and K+ with (NaK)-ATPase exploiting the inactivation by reaction with NBD-chloride as an analytical tool for the evaluation of enzyme ligandation with the various effectors. 1. The inactivation of (NaK)-ATPase by reaction with NBD-chloride showing under all conditions studied a pseudo first-order rate rests on the alkylation of thiol groups in or near catalytic centre. ATP bound to catalytic centre prevents from enzyme inactivation by NDD-chloride through protection of these thiol groups from alkylation. Na+ and K+ affect the reactivity of the thiol groups towards NBD-chloride either indirectly via influencing ATP binding or more directly via changing the conformation of catalytic centre. Proceeding from these interrelations, the interaction of the various effectors with the enzyme was analyzed. 2. The K'D-values of various nucleotides determined by our approach correspond to the values obtained by independent methods. As shown for the first time, two catalytic centres per enzyme molecule exist. They exhibit high or low affinity to both ATP and ADP apparently caused by anticooperative interaction of the half-units of the enzyme through intersubunit communication ("half-of-the-sites reactivity"). 3. In the absence of ATP, Na+ or K+ ligandation of (NaK)-ATPase produce opposite effects on the reactivity of the thiol groups of catalytic centres reflecting different changes of their conformation. This corresponds to the well-known antagonistic effect of Na+ and K+ on some partial reactions of (NaK)-ATPase. The Na+ and K+ concentrations required to change thiol reactivity are rather high, i.e. the ionophoric centres for both Na+ and K+ are not readily accessible for cation complexation in the absence of enzyme complexation with ATP. 4. Na+ being without effect on ATP binding to the enzyme also does not influence the inactivating reaction with NBD-chloride while K+ by decreasing ATP binding dramatically decreases the protective effect of ATP. The K+ affinity of the enzyme-ATP complex is by more than two orders of magnitude higher than that of free enzyme. Na+ ligandation of the K+-liganded enzyme-ATP complex reverses the effect of K+ ligandation and produces a protective effect which distinctly surpasses that of the complexation of free enzyme with ATP. Hence, the enzyme molecule carries simultaneously ionophoric centres for both Na+ and K+. 5. The findings that per enzyme molecule ionophoric centres for Na+ and K+, and two catalytic centres with anticooperative interaction coexist corroborate the corresponding basic predictions of the flip-flop concept of (NaK)-ATPase pump mechanism, and explain some peculiar kinetic features of transport and enzyme activities of (NaK)-ATPase.


[Separation of enriched synaptosomes, synaptic vesicles and synaptic plasma membranes]. A rapid and simple method is described for separation of intact synaptosomes, synaptic plasma membranes and vesicles. Two synaptosome fractions were obtained by modified differential centrifugation. The rate zonal zentrifugation in a linear sucrose gradient (very low density) is suitable to obtain fractions highly enriched in synaptic plasma membranes and vesicles. Examination of the prepared fractions was done by enzyme marker activities and electron microscopy


[Concentration and composition of serum lipoproteins in the fetal rat]. 1. Concentration and composition of the "very low density lipoproteins" (VLDL), "low density lipoproteins" (LDL) and "high density lipoproteins" (HDL) and of non-floatable lipids of fetal rat serum (day 22 of pregnancy) were determined by ultracentrifugation, thin-layer chromatographic separation of the floated lipids and quantitation of the lipid and protein moiety. 2. The concentration of VLDL is in the fetal rat by one order of magnitude lower, and that of LDL, 5fold higher than in the adult animal; the concentration of HDL in fetal serum amounts to 60% of the value of adult animals. 3. The composition of LDL and HDL of fetal serum does not differ from that in the serum of adult animals; in contrast, the fetal VLDL have a higher proportion of protein and cholesterol and a lower proportion of triglycerides than the VLDL of adult serum. The electrophoretic mobility of the fetal VLDL is lower than that of adult VLDL.


[Development of serum lipoproteins from fetal to adult rats]. 1. The semi-quantitative development of lipoproteins in the serum (beta-, pre-beta- and alpha-lipoproteins) from fetal (day 18 of intra-uterine development) to adult age was determined by agarose gel electrophoresis and determination of total lipids. 2. A high proportion of beta- and a very low proportion of pre-beta-lipoprotein is typical of the fetal stage. In adult age, however, the beta-lipoproteins are the smallest fraction, and the pre-beta-lipoproteins, the second-strongest fraction. 3. The transition from fetal to adult lipoprotein pattern is more or less abrupt, with the change of the proportion for each of the three lipoprotein fractions occurring at different intervals from birth. Adaptation of the fetal lipoprotein status to that of the adult animal in regard to quantity and mutual relationship of the fractions was observable, at the earliest, 7 weeks after birth.


[Paget's cancer of male breast].

[Paget's cancer of male breast]. The case of a male patient with Paget's cancer of the breast is reported. Radical mastectomy was performed. Two and a half years later the patient was free of symptoms.


Some urgent problems in the treatment of operable breast cancer. The results of 1216 radical mastectomies and 297 conservative surgical interventions performed on 1513 operable mammary carcinoma patients between 1950 and 1962 are analysed on the basis of the 10 years survival rate. It appears that early diagnosis and early operation will give the best result. The bioloical activity of the tumour is the most decisive additional factor.


ACTH and cortisol response to bromocriptine, and results of long-term therapy, in Cushing's disease. Plasma corticotrophin (ACTH) was lowered in 4 out of 5 patients with pituitary dependent Cushing's syndrome (one of whom was studied only after bilateral adrenalectomy) after a single oral dose of 2.5 mg bromocriptine, but plasma cortisols were unaltered in the 3 patients in whom it was measured. Three patients were observed during treatment with bromocriptine for 16 to 87 weeks. One improved symptomatically while maintained on a combination of metyrapone and bromocriptine, but plasma ACTH levels remained high even when the dose of bromocriptine was increased to 20 mg daily. Bromocriptine therapy was discontinued after 16 weeks in the second patient due to the development of mental depression. Her clinical features had not improved during this time. The third patient, who also underwent a course of pituitary irradiation, became, and remains, symptom free, with satisfactory plasma ACTH and cortisol levels for the 87 weeks he has received bromocriptine. The role of bromocriptine in the management of Cushing's disease seems limited despite the fact that plasma ACTH may fall after a test dose of the drug.


Coxsackie B4 viral infection, anti-islet immunity and immunogenetics in insulin-dependent diabetes mellitus. The interrelationship between Coxsackie B4 (CB4) virus antibodies, islet-cell antibodies and HLA B8 was studied in 67 patients with juvenile-onset diabetes mellitus (JOD). In 41 JOD patients (61%) CB4 antibodies (greater than 1:4) were found. A high prevalence (69%) of significant CB4 antibody titers (greater than 1:64) was observed in patients with age at onset between 11 and 20 years compared with those with age at onset below or over that age-range (27% and 33%, respectively). Islet-cell antibodies (ICA) could be detected only in 11 JOD patients, which is probably due to the relatively long mean duration of disease. However, 8 of them presented with significant CB4 antibody production. Although no correlation between CB4 antibodies and the JOD-associated HLA-antigens (B8, Bw15, Cw3 and B7) could be detected in the entire group of JOD patients, a close association between ICA persistence and HLA B8 was found. Furthermore, in the ICA-positive, moderate or high CB4 antibody titers were prevalent. Thus, the present data point to a possible interrelationship between CB4 virus antibodies, persistent ICA production and HLA B8 in a minor proportion of JOD patients. However, it seems premature to assume direct implication of these factors in the pathogenesis of JOD.


Insulinoma with hypoglycemia and normal immunoreactive insulin but with an insulin-like activity restricted to the portal vein. In a 46-year old Caucasian woman, the authors report a B-cell adenoma with plasma immunoreactive insulin (IRI) ranging from 10 to 32 microunits/ml, despite severe spontaneous hypoglycemia. In a peroperative sample withdrawn from the portal vein, normal IRI (40 micromicron/ml) in the presence of high insulin-like activity (290 microunits/ml) was observed by using a biological assay performed on rat epididymal fat tissue. Furthermore, this material did not cross-react with insulin antibodies and was undetectable in systemic venous samples. Although further identification by chromatographic extraction was not performed, the substance secreted by the tumor is probably identical to the non-suppressible insulin-like activity (NSILA) isolated by Froesch and responsible for hypoglycemia in a few cases of extrapancreatic tumors. The absence of this material in systemic samples indicates an immediate removal by a single passage through the liver.


Corona virus induced subacute demyelinating encephalomyelitis in rats: a morphological analysis. Thirty percent of weanling rats infected with JHM murine corona virus developed a subacute demyelinating encephalomyelitis approximately 3 weeks after intracerebral inoculation. Small demyelinating foci were located in the deep cerebral white matter and large, sharply demarcated demyelinating lesions were detectabll preserved in the demyelinating plaques in areas where the lesions extended to the gray matter. Perivascular cuffings, consisting of plasma cells and mononuclear cells, were frequently found. Viral antigen was found mostly in the white matter and in glial cells, leaving neurons unstained. Electron microscopic studies of the early lesions of white matter disclosed two different kinds of cell degeneration which developed prior to the myelin disruption and mononuclear cell infiltration. One was a small pyknotic cell, which is thought to be an oligodendrocyte and the other is a ballooned cell containing abundant microtubules. Virus particles could be demonstrated only in the latter cell type. Discussion about astrocytes as well as oligodendrocytes was made in relation to the initial stage of demyelination caused by virus infection. This animal model may be useful in the analysis of the mechanisms leading to demyelination in subacute or chronic infections.


[The neuropathies of monoclonal gammapathies. Immunofluorescence and immunolabelling in the electron microscopy of immunoglobulins with amyloid structure (author's transl)]. Five cases of peripheral neuropathies occurring with monoclonal gammapathies are studied by means of nerve biopsies. Immunofluorescence and HRP-immunolabelling with the electron microscope are performed. The direct pathogenic role of a subperineurial immunoglobulin deposit is pointed out. The relationship of the heavy chain monoclonal immunoglobulin with amyloid fibrils is discussed.


Herpes simplex virus replication in pheochromocytoma cell line that responds to nerve growth factor. Cultured cells of neural origin (PC-12, pheochromocytoma cell line) respond to nerve growth factor (NGF) by extending neurites. These cells whether treated with NGF or not can be infected with herpes simplex virus and produce progeny virus. Viral antigens are detected on the cell surface and fusion of cells to form polykaryocytes takes place. Nucleocapsids are found within the cell nucleus and enveloped virus in a present both in the cytoplasm and extracellular space. Virus was not observed within the neurites but laterations in the neurite microtubular structure occurred after infection.


Passive transfer of virus induced diabetes mellitus with spleen cells. The study describes passive transfer of diabetes mellitus by transplantation of spleen cells from donor mice infected with a diabetogenic encephalomyocarditis virus. Inbred normal C57 mice were used as both donors and recipients, and other recipients were C57 athymic, nude mice. Transplants were made in two series after 12 and 22 days duration of infection of the donors, respectively. All recipients became diabetic. The possibility of virus transfer with the spleen cell transplant is discussed, and found to be highly improbable. The fact that passive cellular transfer can be effected points to a decisive significance of the thymus dependent lymphocyte system in the pathogenesis of the primary diabetes.


Pulse volume recordings in outer ear canal in pulse synchronous tinnitus. A comparison between ears with Glomus tumour, serous otitis media, and normal ears. With the aid of a volume flowmeter it is possible to record pulse synchromous volumetric changes in the outer ear canal. In 7 ears with glomus tumour in the tympanic cavity and in 5 with serous otitis media, such changes were larger than in 125 persons with a normal middle ear. By changing the ambient pressure in a pressure chamber and instructing the patients not to swallow, the drum can be pushed inward or outward. In all the cases of glomus tumour studied the pulse volumetric change was considerably affected when the drum was pushed inward or outward. In the normal patients the change was no change at all. This means that the pulse volume changes in normals are generated mainly by the vessels in the outer ear canal.


Labyrinthine and somatosensory convergence upon vestibulospinal neurons. In awake cats cells forming the lateral (LVST) and medial (MVST) vestibulospinal tracts were identified by employing antidromic stimulation of the spinal cord. Neuronal responses to bilateral vestibular, forelimb, hindlimb, and neck electrical nerve stimulation were analysed. Extracellular recording in the vestibular nuclei was performed via a glass micropipette saturated with Fast Green, to aid in later histological tract identification. The number of cells projecting to cervical and lumbar regions in the dorsal and ventral division of Deiters' nucleus did not differ significantly. An unexpectedly large number of MVST units was found in the descending nucleus. Some MVST units projected to the lumbar cord but in both the medial and descending nuclei, projections to the cervical cord were in majority. Almost all spinal projecting vestibular neurons received labyrinthine input and more than half received somatosensory input. The units could be separated into several populations on basis of excitatory and inhibitory labyrinthine response latencies indicating multiple pathways. As regards labyrinthine-somatosensory integration the two tracts were found to be quite similar. The extent and complexity of labyrinthine-somatosensory convergence indicate the importance of feed-back mechanisms upon postural controls also at the level of the vestibular nuclei.


Viral culture and electron microscopy of ganglion cells in Meniere's disease and Bell's palsy. Specimens of Scarpa's ganglion during vestibular neurectomy were obtained in 6 cases of Meniere's disease and specimens of geniculate ganglion in 2 cases of total facial nerve decompression and studied by tissue culture methods for detection of possible herpes and cytomegalovirus infection. In electron microscopy inclusions in the form of interwoven yarn-like structures, coarse aggregates of chromatin and light nuclear bodies were found in several vestibular ganglion cells. No typical herpes virus virions could be demonstrated. The culture for viruses all proved finally negative. At present there is no proof that viruses are present in Scarpa's or geniculate ganglions but the possibility remains that the inclusion bodies observed might be viruses inactivated inside the ganglion cells.


Immunocytochemical evidence of SV 40-related T antigen in two human brain tumours of ependymal origin. A series of thirty-time human brain tumours has been screened for the presence or absence of SV 40-related T antigen by the direct and indirect immunoperoxidase techniques. Two tumours (an ependymoma and a choroid plexus papilloma) of ependymal origin revealed markedly positive nuclear staining for T antigen both in in vivo and in vitro. The possible viral etiology of these human brain tumours is discussed in relation to recent human papovavirus isolates.


Inhibition of evoked tongue jerks during the rat's cerebral ventricles perfusion with gamma-aminobutiric acid and beta-phenyl-gamma-aminobutiric acid. The supraorbital branch of the trigeminal nerve of rats in chloralose anaesthesia was stimulated with electrical pulses. The stimulation of the afferent fibres of the trigeminal nerve evoked the retractive jerks of the outstretched tongue. These evoked tongue jerks (ETJ) were recorded on the tape of the line recorder through an isotonic transducer. The caniulae were inserted into the lateral cerebral ventricles by means of streotaxic apparatus and perfusion was performed with inflow of GABA or beta-phenyl-GABA-containing fluid into the right lateral ventricle and outflow through the left lateral ventricle. Perfusion with solution of 0.01 mol/l GABA reduced the amplitude of ETJ 25.9% while perfusion with hypertonic NaCl solution increased it by 19.5%, and perfusion with solution of 0.2 mol/l beta-phenyl-GABA reduced that amplitude by 39.9%.


Brain tumours in the elderly.

Brain tumours in the elderly. The incidence of cerebral tumour in a geriatric department over a seven-year period was 0.9% of all admissions. The distribution of benign, malignant and secondary brain tumours and their histological types were similar to those of younger adults. Apart from small asymptomatic tumours found incidentally at post mortem, most symptomatic tumours were recognized from their characteristic history. In particular, only 0.4% of patients diagnosed as stroke were subsequently found to have a cerebral tumour.


Age changes in collagen characteristics of bone and skin of a short-lived species of reptile. Decalcified bone collagen of older male garden lizards, Calotes versicolor, was less susceptible to digestion by collagenase from Clostridium histolyticum than that from younger individuals. In aged skin the percentage solubility and the soluble/insoluble collagen ratio decreased, with a concomitant rise in insoluble and total collagen. Collagen/unit area increased with advancing age in both dorsal and ventral skin. These results from a non-mammalian poikilothermic vertebrate provide additional evidence in favour of the cross-linkage theory of ageing and suggest a common pattern of collagen ageing in vertebrates.


Osteoporosis: effects of calcium.

Osteoporosis: effects of calcium. Bone loss coincides with the reduced physical activity of both sexes in later years, vigorously aggravated by age-associated hormonal changes in women and chronic inadequate intake of calcium. In the absence of preventive dietary measures, osteoporosis may become a major health problem for America's fastgrowing senior female population. Bone loss and fracture risk may be minimized or reversed by a daily intake of approximately 1 Gm. of calcium derived from the diet or through supplements.


C-type virus-lymphocyte interactions in developing mouse thymus. The appearance of C-type virus particles in thymus cells of Swiss mouse embryos, 11.5 to 15.5 days post-conception age (PCA), was studied with the electron microscope. In thymic rudiments of all specimens examined, virus particles were seen in epithelial cytoplasm, budding from epithelial cell surfaces and in extracellular spaces. Lymphoid cells were first seen in thymic rudiments of 13.5 days PCA, and did not display virus particles at this stage. At 14.5 days PCA, thymic lymphocytes had localized plasmalemmal thickenings of high electron-density which were adjacent to extracellular virus particles. Viruses appeared to be penetrating thymic lymphocytes by viropexis in embryos of 15.5 days PCA. At this stage, many lymphocytes also had cytoplasmic virus-containing vesicles and viral buds at their surfaces. These observations suggest the possibility that, in embryos, C-type viruses are transmitted horizontally from thymic epithelium to early populations of thymic lymphocytes.


Specific subclones derived from a multipotential clone of rat anterior pituitary cells. Several functional subclones of rat anterior pituitary cells were established from our 2A8 clone which apparently contains a heterogenous population of committed and uncommitted cells. On the basis of the hormones secreted into the culture media, as measured by radioimmunoassay, these subclones were divided into four categories, i.e., subclones which secrete (1) ACTH only, (2) prolactin only, (3) prolactin and GH or (4) ACTH, prolactin and GH. None of the subclones produced detectable amounts of thyrotrophic or gonadotrophic hormones. Subclones which secrete a single hormone have shown no change in the type of hormone produced, indicating that these subclones were each derived from a committed cell. The cells of all subclones exhibit a normal diploid karyotype and show good growth characteristics. The cells of the different subclones can be classified by phase contrast microscopy into four categories. However, no clear-cut ultrastructural features have been observed which can be correlated with the different categories of subclones. On the basis of the results a hypothesis is proposed relative to the functional cytodifferentiation of anterior pituitary cells.


Serum and lymph-node collagenase in sarcoidosis. Comparison with angiotensin-converting enzyme. Serum collagenase was significantly elevated in stage II, but not stage I sarcoidosis, in contrast to elevation of serum angiotensin-converting enzyme in stages I and II. Neither serum collagenase nor angiotensin-converting enzyme was elevated in active tuberculosis. Elevated serum collagenase and angiotensin-converting enzyme levels were decreased with steroid therapy. Serum collagenase and angiotensin-converting enzyme were significantly correlated but did not vary identically. Collagenase was not elevated in lymph nodes in sarcoidosis, in contrast to marked elevation of angiotensin-converting enzyme. The results suggest that serum angiotensin-converting enzyme is a more sensitive index of sarcoidosis activity than serum collagenase, which may have an ancillary role in assessment of the disease.


Fibrous histiocytoma of the trachea.

Fibrous histiocytoma of the trachea. The light and electron microscopic features of a fibrous histiocytoma of the trachea that occurred in a 15-year-old Caucasian girl are presented. Emphasis is placed on the aggresive behavior and the importance of early recognition of the lesion in an unusual location.


Detection of early "significant" antibody responses in paired sera by the use of an automated bovine parainfluenza-3 hemagglutination-inhibition system. An automated bovine parainfluenza-3 hemagglutination-inhibition system was used to detect small increases (15--20%) in specific antibody titer between paired bovine sera collected three days apart. Automated and manual parainfluenza-3 hemagglutination-inhibition test procedures were compared as methods for the serologic diagnosis of parainfluenza-3 infection. Fifteen daily serum specimens from each of three animals were analyzed as various possible manual-test and automated-test serum pairs. With the sera from two of the animals, considered to be parainfluenza-3 hemagglutination-inhibition-positive, the manual microtiter test procedure made 18 (69%) false negative diagnoses when using the standard manual-test criteria for "evidence of infection." When a 20% titer increase between three-day serum pairs was considered "serologically significant," the automated system made three (17%) false-negative diagnoses. At a 15% "serologically significant" increase, no false-negative diagnosis was made. The utilization of automated serology for the routine diagnosis of disease should permit treatment and control measures to be initiated early, thereby reducing losses due to infectious disease.


Effect of premenopausal castration and incremental dosages of conjugated equine estrogens on plasma follicle-stimulating hormone, luteinizing hormone, and estradiol. Plasma levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol (E2) were measured serially in 11 premenopausal patients before and after hysterectomy with bilateral salpingo-oophorectomy. One week after operation an incremental dosage regimen of conjugated estrogens (CEE) in tablet form was commenced on a basis of two weeks with therapy (0.3, 0.625, 1.25, and finally 2.5 mg.), with each dose interspersed by two weeks without therapy. FSH, LH and E2 levels were measured at the end of each period with and without therapy. E2 levels fell within 24 hours of operation while FSH and LH levels rose gradually. CEE therapy produced an elevation of E2, but circulating concentrations comparable to the premenopausal values were only maintained during the dosage periods of 0.625 and 1.25 mg. of CEE. In only one instance did CEE succeed in reducing FSH to premenopausal levels, and that was at a dosage of 2.5 mg., in which instance the E2 level was higher than the premenopausal value. LH was never reduced to a premenopausal level. Thus, the data indicate that CEE alone in dosages up to 2.5 mg. per day was unable to reproduce in postmenopausal women the gonadotropin and E2 blood serum levels shown to exist prior to oophorectomy. Usual CEE treatment after menopause, therefore, in itself does not represent physiologic "hormone replacement therapy," if defined as the dosage required to maintain premenopausal circulating concentrations of reproductive hormones.


Neurological disorders and pregnancy.

Neurological disorders and pregnancy. Certain neurological disorders are particularly liable to occur during pregnancy or may be profoundly influenced by it. Moreover, neurological disorders may influence the management of otherwise uncomplicated obstetric cases. Despite the practical importance of this subject, however, little attention has been devoted to it in the literature. Moreover, those papers that have been published are widely disseminated and so are not easily accessible to the many practicing physicians who may be confronted with a clinical problem of this sort. In this communication, a succinct account is provided of the interrelationship between noneclamptic pregnancy and maternal neurological disorders, with emphasis being placed on aspects that are of practical significance in a clinical context. The treatment of neurological disorders that are likely to pose a problem during pregnancy is discussed, and the manner in which these disorders may influence obstetric management is reviewed.


Diagnosis of Sheehan's syndrome using a sequential pituitary stimulation test. Systematic pituitary evaluation was performed in four patients suspected of having Sheehan's syndrome. A sequential pituitary stimulation test, consisting of insulin-induced hypoglycemia followed by stimulation of gonadotropin-(GnRH) and thyroid-releasing hormone (TRH), a metyrapone test, and adrenocorticotropic hormone (ACTH) stimulation test, was performed. All four patients failed to develop a normal increase in serum growth hormone, cortisol, and prolactin (PRL) following insulin-induced hypoglycemia. All patients demonstrated a blunted PRL, follicle-stimulating hormone, and luteinizing hormone response to the combination of GnRH and TRH. Although thyroid stimulating hormone (TSH) response was impaired in all patients, two patients had normal T3 resin uptake and thyroxine, demonstrating minimal TSH reserve maintaining normal baseline free thyroxine index. Metyrapone administration was followed by no increase in 11-deoxycortisol or 17-ketogenic steroids, thereby adding no additional information to the hypoglycemia stimulation. ACTH infusion revealed normal adrenal cortisol response. In conclusion, in patients with suspected postpartum hypopituitarism, a complete pituitary function investigation can be done in a short time by using the described pituitary sequential stimulation test.


Oral administration of theophylline to modify pressor responsiveness to angiotensin II in women with pregnancy-induced hypertension. Normally, women become refractory to the pressor effects of infused angiotensin-II (A-II) early in pregnancy. Gravid women destined to develop pregnancy-induced hypertension (PIH) lose this refractoriness to A-II several weeks prior to the detection of hypertension. Normal pregnant women also lose their A-II refractoriness after treatment with prostaglandin synthetase inhibitors and, in this regard become similar to gravid women who are destined to develop PIH. From this observation, we have concluded that a prostaglandin(s) or a prostaglandin-related substance(s) is likely involved in the mediation of vascular reactivity to A-II during pregnancy. The present study was conducted to ascertain if control of vascular reactivity during pregnancy also involves the cyclic nucleotides. Since theophylline is known to inhibit the action of phosphodiesterase, an action that results in increased cellular levels of cyclic 3',5'-adenosine monophosphate (cAMP), we evaluated the effective pressor dose of A-II before and after the administration of theophylline to women with mild PIH who were beyond the 28th week of gestation. The effective pressor dose of A-II in these women with PIH before theophylline treatment was 7.3 +/- 1.4 ng. times kg.(-1) times min.(-1) (mean and standard error). Following treatment of these women with the equivalent of 500 mg. of theophylline daily for four days, the effective pressor dose of A-II was 16.7 +/- 3.8 ng. times kg.(-1) times min.(-1) (p less than 0.025). These findings are consistent with the view that a prostaglandin(s) synthesized in the arteriole may modulate the vascular refractoriness to A-II by altering the intracellular level of cAMP in vascular tissues.


Fetal lung maturation: human amniotic fluid phosphatidate phosphohydrolase activity through normal gestation and its relation to the lecithin/sphingomyelin ratio. In order to evaluate the relationship between the increase in amniotic fluid phosphatidate phosphohydrolase (PAPase) specific activity and the increase in the lecithin/sphingomyelin (L/S) ratio during normal human pregnancy, PAPase specific activity and the L/S ratio were measured in 171 amniotic fluid samples obtained from 164 women who were at 17 to 42 weeks' gestation. The increase in PAPase specific activity in amniotic fluid is parallel to the increase in the L/S ratio. The correlation between PAPase specific activity and the L/S ratio in amniotic fluid from all gestational ages is highly significant. The relationship of PAPase specific activity in amniotic fluid to PAPase specific activity in gastric fluid was investigated in a study of 97 newborn infants. A highly significant correlation was found between these two values. To ascertain if a relationship exists between the specific activity of PAPase in amniotic fluid and the subsequent development of hyaline membrane disease (HMD), 223 neonates who were delivered within 72 hours of amniotic fluid collection were studied. Only one infant developed HMD out of 170 with amniotic fluid PAPase specific activity equal to or greater than 50 nmoles of orthophosphate released X mg.-1 of protein X hr.-1. On the other hand, the finding of an amniotic fluid PAPase specific activity of less than 50 nmoles was of little value in predicting lung immaturity. We believe that these findings are also supportive of the view that PAPase and surfactant are released from the type II pneumocyte as a closely related structural unit, viz., the lamellar body.


Desoxycorticosterone in normal pregnancy. I. Sequential studies of the secretory patterns of desoxycorticosterone, aldosterone, and cortisol. Plasma concentrations of desoxycorticosterone (DOC) and aldosterone are markedly elevated in pregnancy. Although DOC secretion in nongravid women has been assumed to be dependent mainly on adrenocorticotropic hormone (ACTH), in a previous study of women in the third trimester of pregnancy it was found to be unresponsive to ACTH, dexamethasone, and variations in salt intake. In this study plasma DOC, aldosterone, and cortisol levels, as well as their responses to ACTH stimulation and overnight dexamethasone suppression, were observed sequentially in seven normal women during the course of pregnancy and at three months post partum. Plasma DOC, aldosterone, and cortisol levels rose substantially during gestation, but increments in DOC did not necessarily coincide with those of the other two. Responses of all three corticosteroids to ACTH were enhanced during the first two trimesters compared to the nongravid state; DOC became unresponsive in the third trimester, while aldosterone and cortisol rose to an even greater extent. Elevated maternal DOC was not decreased significantly by dexamethasone at any stage of pregnancy, while plasma cortisol was suppressed. Nonsuppressibility of DOC with dexamethasone and also the lack of correlation of the rise in DOC with the increase in cortisol during the course of pregnancy suggest that increased DOC secretion in pregnancy does not arise from ACTH-dependent pathways of the maternal adrenal. The loss of responsiveness of DOC to ACTH in the third trimester suggests that the maternal adrenals have undergone an alteration in their steroidogenic response to ACTH, but also may indicate that their output of DOC has reached a maximal rate.


Alterations in hepatic fine structure after chronic exposure of rats to dexamethasone. The objective of this study was to determine the effects of chronic dexamethasone (DEX) administration on hepatic ultrastructure and to correlate these changes with plasma lipoprotein levels. Electron microscopic studies were made of hepatocytes from male rats killed 1, 3 and 5 days after DEX (2 mg, twice per day) administration. Three days after treatment plasma lipoprotein levels were highest and hepatocytes contained regions of the cytosome rich in elements of the smooth endoplasmic reticulum (SER). Osmiophilic particles were present in the tubules and vesicles of the SER, in the saccules and vacuoles of the Golgi complex, in secretory vesicles near the cell surface and in the space of Disse. DEX treatments also caused hepatocytes to accumulate tightly packed masses of beta-particles of glycogen in some regions of the cell while other areas displayed dispersed glycogen particles that were associated with the SER. These observations are consistent with the hypothesis that glucocorticoids 1. cause an elevation of plasma lipoprotein levels by increasing hepatic synthesis and secretion of VLDL, which involves the sequential participation of the ER, the Golgi complex and exocytosis of VLDL-containing vacuoles into the space of Disse, and 2. produce a change in the nature of the association of glycogen particles with the SER membranes in response to the physiological state of the animal.


Biochemical and physiologic changes in lungs of rats exposed to a cadmium chloride aerosol. The purpose of this study was to examine the in vivo effects of acute exposure to a cadmium chloride aerosol on the activity of pulmonary enzymes and selected physiologic parameters that are altered by exposure to oxidant agents. Male rats were exposed for 1 hour to 0.5 per cent aerosol of cadmium chloride. At 1,5, and 11 days after exposure to cadmium chloride, exposed rats compared to control rats (data expressed as per cent of control values) had lung-to-body weight ratios of 192, 174, and 140 per cent; lung glucose-6-phosphate dehydrogenase activities of 90, 107, and 135 per cent; lung superoxide dismutase activities of 96, 101, and 132 per cent; tidal volumes of 62, 63, and 89 per cent; respiratory frequencies of 170, 145, and 108 per cent; and lung weight-specific static deflation volumes at 30 cm water of 30, 13, and 31 per cent. A zero-order clearance of cadmium from whole lung was observed, with a half-time of 27.4 days. Light microscopic examination of lung tissue revealed initial pulmonary edema on day 1 that progressed to interstitial pneumonitis on day 5, with some recovery by 11 days after exposure. The cadmium induced biochemical, physiologic, and pathologic alterations were similar to the responses observed in lungs of rats exposed to a wide variety of pulmonary irritants; thus, the changes observed may represent a nonspecific response to tissue injury.


Correlation of clinical and virus-specific immune responses following levamisole therapy of recurrent herpes progenitalis. Patients with herpes progenitalis recurring every 14--28 days were treated with levamisole 150 mg orally twice weekly in an open trial to evaluate the relationship between immunomodulation and clinical response. Eight of 12 patients studied for 4--9 months reported a decrease in the frequency of recurrences. Enhanced virus-specific lymphoproliferative responses were observed in six of eight patients reporting clinical improvement. Herpes-antigen-induced production of leucocyte migration inhibitory factor (LMIF) was similarly enhanced in these individuals. In the four patients reporting no improvement, virus-specific lymphoproliferative and LMIF-generating responses were either depressed or unchanged. No significant alterations in neutralizing antibody titers were observed in any of the patients. Alterations in virus-specific lymphocyte transformation and lymphokine generation observed in vitro thus correlate with changes in clinical course in a manner consistent with the proposed immunomodulatory function of levamisole.


Comparison of the development of resistant strains of Type 1 herpes simplex virus to in vitro antiviral activity of 5-iodo-2'-deoxyuridine or ribavirin. Exposure of HSV/1 to low concentrations of ribavirin during 4--5 passages does not produce ribavirin-resistant virus. IDU resistance was developed by HSV/1 while it was being passed simultaneously. This resistance was seen to develop in both KB and Vero cells. The IDU-resistant virus is also resistant to ribavirin in KB cells.


Effect of ribavirin on Type 2 Herpesvirus hominis (HVH/2) in vitro and in vivo. Ribavirin was found to inhibit five strains of HVH/2 tested in KB cells with the M1C range being 10-100 microgram/ml. In a mouse tail animal model, it effectively reduced HVH/2-induced lesions particularly at the peak lesion periods. Ribavirin was remarkably effective when intravaginal treatment was initiated late in the infection, which suggests that it may have potential for treatment of human cutaneous infections.


Antiviral potential of phosphonoacetic acid. Phosphonoacetate has been found to inhibit specifically the replication of herpes-viruses. A partial inhibition of vaccinia virus represents the only activity outside the herpesvirus class. The drug was found to be a specific inhibitor of the virus-induced DNA polymerases. Normal cellular polymerases were relatively insensitive to phosphonoacetate, resulting in low cellular toxicity. Our working hypothesis is that the drug binds to the enzyme and that initiation of polynucleotide synthesis occurs in the presence of the drug and the required template, substrates, and cations. However, addition of deoxynucleosides to the elongating nascent chain is prevented by the enzyme-bound drug. Kinetic analyses indicated that phosphonoacetate did not interfere with the binding of DNA template to polymerase; and it did not compete with nucleotide substrate binding. The highly specific inhibitory effects of phosphonoacetate allowed for the selection of partially resistant strains of HSV. Resistance of virus to the drug in cell culture was directly correlated with the same relative resistance of the corresponding cell-free DNA polymerases. Phosphonoacetate was also effective therapeutically in herpesvirus skin and ocular infections in animals. Intraperitoneal administration of the drug reduced death and severity of disease in experimental encephalitis in hamsters. High specificity, low toxicity, and reproducible efficacy in lower animals suggested that phosphonoacetate could be a useful new antiviral drug. Sensitivity to phosphonoacetate also is a useful research tool as a genetic marker for herpesviruses.


Inhibition of herpesvirus DNA synthesis by 9-beta-D-arabinofuranosyladenine in cellular and cell-free systems. 9-beta-D-Arabinofuranosyladenine 5'-triphosphate (ara-ATP) is an inhibitor both of DNA polymerase-alpha and -beta from noninfected rabbit kidney cells and of the DNA-dependent DNA polymerase induced by herpes simplex virus Type 1 (strain IES). The studies were performed with partially purified enzymes, and each of the different polymerase preparations contained only one DNA-dependent DNA polymerase species. These enzymes were inhibited in a competitive manner. The HSV-induced DNA-dependent DNA polymerase was 39-fold more sensitive to ara-ATP than was cellular DNA polymerase-beta and 116-fold more sensitive than cellular DNA polymerase-alpha. The affinity of the HSV-induced enzyme for ara-ATP was only slightly influenced by the use of different template/initiators in the enzyme assays. In intact cell systems DNA synthesis was affected by 9-beta-D-arabinofuranosyladenine (ara-A) as indicated by the reduced incorporation of deoxythymidine. In herpesvirus-(strain Lennette)-infected cells, however, ara-A shows no influence on the incorporation on deoxythymidine into cellular DNA, but it substantially reduces the incorporation into viral DNA. Ara-A itself is incorporated into both cellular and herpesviral (strain Lennette, D-316 and IES) DNA during DNA synthesis.


Arabinosyl-N6-hydroxyadenine: a new potent antivirus drug. Abrabinosyl-N6-hydroxyadenine (ara-HA) was tested for its antiherpesvirus and immunosuppressive activities in vivo and in vitro. Mouse strains were used that had been shown earlier to be very susceptible to intraperitoneal (i.p.) or intracerebral (i.c.) inoculation of a virulent strain of herpes simplex virus Type 1 (HSV-1). Susceptible mice were inoculated i.p. or i.c. with a stock pool of HSV-1 and treated with ara-HA (i.p.) beginning at least 24 hr later. The mice were given a 10-day course of drugs and followed for at least 21 days. Similar experiments were carried out with ara-A for comparative purposes. Ara-HA was found to protect mice inoculated with HSV-1 significantly better than ara-A. Lower concentrations of drugs were required and a higher percentage survived. Later challenge of the ara-HA-treated mice with HSV-1 demonstrated that these mice had become immune to HSV-1, indicating that the immune system is not severely affected by this course of ara-HA. A 10-day course of ara-HA, which was found to protect mice from 100 LD50 of HSV-1, reduced the capacity of the mouse lymphocytes to respond to allogeneic cells only slightly. In vitro ara-HA inhibited HSV-1 replication as well as proliferation of lymphocytes exposed to mitogen.


The effect of cyclic nucleotides on virus infection. The yield of influenza virus from infected primary chick kidney cells (PCKC) was enhanced by treatment with dcAMP. In addition, rapid serial passages of the virus in treated chick cells at low multiplicities did also exhibit maintenance of high virus titers. The action of dcAMP upon viral replication could be reproduced in the chick cells by treatment with various agents known to increase intracellular cAMP levels by differing mechanisms. Furthermore, an adrenergic component in the activation of the PCKC cAMP system is indicated by the effects of certain catecholamines on influenza virus yield. Dibutyryl cAMP was shown in the same host to inhibit multiplication of Herpesvirus hominis Type 2, and its effect on other cell-virus systems indicates that its action consists of some host-specific element(s).


The selective inhibition of viral DNA synthesis by chemotherapeutic agents: an indicator of clinical usefulness? A cell culture system has been utilized to measure the effects of drugs on DNA synthesis in uninfected and HSV-(herpes simplex virus)-infected KB cells. DNA from HSV-infected cells was separated into viral and cellular components by isopycnic centrifugation in CsCl gradients. The amount of [3H]thymidine incorporated into acid-insoluble material was measured in the absence and presence of drugs. Dose-response relationships were established by linearly regressing the probit value of the percent inhibition DNA synthesis against the logarithm of drug concentration. Fifty percent inhibitory (I50) concentrations were interpolated from the corresponding regression lines for inhibition of the following: (i) DNA synthesis is uninfected KB cells, (ii) total DNA synthesis in HSV-infected KB cells (iii) cellular DNA synthesis in HSV-infected cells, and (iv) viral DNA synthesis in HSV-infected cells. We have derived an index (SI, selective index) that quantifies the preferential inhibition of viral or uninfected cellular DNA synthesis. This index can be expressed as SI = log10 I50 concentration for DNA synthesis in uninfected cells divided by I50 concentration for viral DNA synthesis in HSV-infected cells. The SI is positive if viral DNA synthesis is inhibited preferentially and negative if uninfected cellular DNA synthesis is more strongly inhibited. A positive SI value of 0.5 was obtained for the clinically useful antiviral drug arabinosyladenine (ara-A) and a value of 0.4 for its metabolite, arabinosylhypoxanthine (ara-H). Although the adenosine deaminase inhibitor coformycin greatly increased the potency of ara-A, the inhibitor did not increase the selectivity of the drug (SI = 0.3). Stallimycin (distimycin A) (SI = 0.3) and phosphonoacetic acid (SI = 0.3) were similarly effective in preferentially inhibiting the synthesis of HSV DNA. In contrast, arabinosylcytosine (ara-C) and ribavirin inhibited DNA synthesis in uninfected cells to a greater degree than viral DNA synthesis (SI = -0.5 and -1.9, respectively). An analysis of the advantages and limitations of this experimental procedure is made and the suggestion is offered that the in vitro determination of a drug's selective index may be a valid predictor of clinical usefulness.


The inhibition of Rauscher leukemia virus and avian myeloblastosis virus DNA polymerases by anthracycline compounds. Studies by other investigators have shown that adriamycin and daunorubicin exhibit antitumor and antiviral activity. A possible antiviral mechanism for the anthracycline compounds is the potent inhibition of viral DNA polymerases. Five anthracycline compounds were tested against purified Rauscher leukemia virus and avian myeloblastosis virus DNA polymerases. All compounds were found to be potent inhibitors of viral DNA polymerase activity. Inhibition was found to be primarily due to the planar ring structure (daunomycinone) common to all of these compounds. The degree of inhibition was dependent on the templates used: activated DNA, synthetic hybrids, poly(rA).dT12-18 and poly(rC).dG12-18, and the synthetic copolymer, poly(DA-dT). Alteration of the group substituent on the planar ring affected the degree of viral DNA polymerase inhibition. The inhibitory effects by anthracycline compounds appear to be relatively specific for viral polymerases.


Effect of a novel adenosine deaminase inhibitor (co-vidarabine, co-V) upon the antiviral activity in vitro and in vivo of vidarabine (Vira-Atm) for DNA virus replication. A new potent inhibitor of adenosine deaminase (co-vidarabine) was used in combination studies with adenine arabinoside (vidarabine, Vira-ATM) to protect this purine nucleoside from enzymatic deamination to the more weakly active metabolite, hypoxanthine arabinoside. Comparing the combination to vidarabine alone, a significant increase (10-fold) of the antiviral activity of the combined drugs was observed against herpes and vaccinia viruses in tissue culture and subcutaneously, against cranial herpesvirus infections in mice. Several other investigators have also recently reported several-fold enhancement of vidarabine activity by newly described deaminase inhibitors. They observed that plaque formation by several large DNA-containing viruses (herpes, vaccinia, varicella zoster) and an RNA-containing oncogenic virus was markedly prevented by the combination compared to vidarabine alone. In animals, enhanced protection (increased survivors) and/or highly significant increase in the life span of dying mice treated with the 2-drug combination, was also observed compared to vidarabine administered singly. These observations in animals clearly indicate that combination studies with vidarabine (Vira-ATM) and co-vidarabine (deaminase inhibitor) deserve serious consideration as future therapy for systemic virus infections in man including herpesvirus encephalitis.


Ovarian neoplasia in ornamental hybrid carp (Nishikigoi) in Japan. Massive abdominal enlargements were observed in 21 adult carp 4--6 years old living in the breeding ponds of carp fisheries in northern Japan. The abdominal enlargement rapidly increased, and the affected fish died within a few months. At necropsy, single or multiple tumors, 1.5--25 cm (average, 10.6 cm) in diameter, were found in the abdominal cavity. Ovarian tissue was identified close to the tumors in 15 cases, and there was no evidence of anatomic continuity between the tumors and other viscera. It seems likely that all of these tumors arose in the ovaries. Histologically, the 21 tumors observed exhibited great variation, but they were composed mainly of various types of cells similar to those of human dysgerminoma, granulosatheca cell tumors, or embryonal carcinomas. Squamous cell nests were found with mesenchymal elements in one tumor. Thus, ovarian tumors in carp may have diverse histogenetic origins and probably arise both from germ cells and from ovarian mesenchyme. Areas of cellular pleomorphism, which suggested a malignant character, were seen in some of the tumors, but no evidence on metastasis was found. Tumor-bearing carp had generally been raised in ponds supplied by wells or underground springs in mountainous areas. Evidence of a possible environmental factors(s) is yet to be demonstrated.


Hepatomas and other neoplasms in the atlantic hagfish (Myxine glutinosa): a histopathologic and chemical study. M. glutinosa is a cyclostome, living in the mud in seawater of high salinity. It probably is a stationary scavenger feeder. About 28,000 hagfish from the Gullmar Fjord were examined during a 5-year period for the occurrence of tumors. Hepatomas were found to be predominant neoplasm, observed at a frequency that decreased from 5.8% in 1972 to 2.9% in 1973 and finally to 0.6% in 1974--76. Islet cell hamartomas and frank neoplasms decreased from 0.5% in 1972 to less than 0.1% in 1973--76. Occasional subcutaneous and mesenterial neoplasms were also observed during 1972--74. In hagfish caught 12 km out in the open sea, the hepatoma incidence decreased from 2.8% in 1972 to 0.9% in 1974. Given this background, it is possible that pollution of the Gullmar Fjord by carcinogenic substances with low biodegradability has occurred until 1972, and this pollution could be of etiologic significance for these hagfish tumors. In fact, the use of PCBs became prohibited by law in Sweden in 1971--72. Severe restrictions were also introduced for the use of chlorinated pesticides, notably DDT, and associated substances (DDD, DDE). Preliminary analyses for the presence of PCBs, DDT (and its metabolites), and aflatoxins (the notorious hepatocarcinogen) were performed by gas chromatography and thin-layer chromatography. Livers (with and without neoplasms) from hagfish caught inside the threshold of the fjord contained about 5 mg/kg of wet weight of PCBs and about 0.1--0.4 mg/kg of dry weight of DDT, DDD, or DDE, whereas those from hagfish caught in the open sea had a much lower PCB concentration (about 0.2 mg/kg of wet weight). No PCBs and no chlorinated pesticides were found in analyses of the mud at the catching site. High PCB concentrations (3 mg/kg of wet weight) were, however, observed in livers from cod living in the  Gullmar Fjord, and it was proposed that bony fish may be the source of hagfish liver PCBs. PCB chromatograms of hagfish livers differed from those of PCB standards and cod liver. This strange pattern, which was not seen in livers from hagfish caught in the open sea, might be explained by an unusual mode of metabolization. The assays for aflatoxins gave completely negative results.


A survey of the hormonal factors that control calcium metabolism. (1) Vitamin D is the precursor of an extremely potent calcium regulating hormone, 1,25(OH)2D3, which is secreted by the kidney. (2) The secretion of 1,25(OH)2D3 by the kidney is influenced by the calcium and phosphorus content of the diet, parathyroid hormone, and 1,25(OH)2D3 itself. (3)Prolactin and growth hormone are importnat regulators of vitamin D metabolism during pregnancy and growth.


[Warts, viruses, cancer (author's transl)]. The malignant transformation of cutaneous lesions in epidermodysplasia verruciformis (E.V.), which lesions are caused by a human papilloma virus (HPV), can be compared to that experimentally induced by the polyoma virus. Thus, HPV would undergo a lytic, transformant and/or abortive cycle in epidermal cells. In this latter case, it would increase cell DNA synthesis and there would be production of cellular antigens coded by the virus. This production of cellular antigens would be the result of the incorporation of part of HPV-DNA into the genome of the cell. The presence of part of HPV-DNA in the cell genome would also increase the susceptibility of infected cells to malignant transformation with other Papova viruses. On practical grounds it is not advisable to use potentially mutagenic treatments such as X or U. V. irradiations in the management of HPV induced lesions in man, especially those of E.V.


[Diarrhea of the newborn animal: nature and mechanism of action of enteropathogenic Escherichia coli in the calf and piglet]. The present knowledge of the enteropathogenic characteristics of Escherichia coli (adhesion and toxinogenesis) is reviewed in the calf, and compared with piglet data. A pathogenic model of E. coli diarrhoea in the calf is proposed, taking into account the electromyographic, bacteriologic and physiologic data.


Results of an epidemiological investigation on viral arteritis in France and some other European and African countries. An original microplate seroneutralization technique was developed in order to study the existence of antibodies against equine viral arteritis. The technique involves a high amount of complement, and has allowed to demonstrate antibodies in 18.5% out of 4,037 horses examined. Titers varied not or little during periods as long as 6 years. Among 3,324 sera samples from French horses, a 15.2% frequency of antibodies was shown. The infection level was not very different between breeds, which is different from results obtained in the U.S.A. with another technique. The infection level increased regularly from 1 to 20 years of age, but, for a constant age, the percentage decreased with the year of birth, between 1950 and 1970. Eight individual infection cases have been identified between 1966 and 1974. It seems that more often the disease occurs without clinical symptoms, or sometimes manifests itself through sporadic abortion. Serological marks of infection have been found at variable levels in all the countries of Europe and Africa investigated. In Africa, a higher level (37.1% among 468 sera) was observed.


Human platelet aggregation and cAMP system--cAMP level, adenyl cyclase, phosphodiesterase. The possibility of a direct and casual relationship between various parameters of the adenosine 3',5'-cyclic monophosphate (cAMP) system, e.g., the level of cAMP, adenyl cyclase activity and phosphodiesterase activity, and platelet aggregation was studied by measuring the effects of various environmental conditions, as well as metabolic inhibitors, aggregating agents and aggregation inhibitors upon these parameters. A competitive binding technique using 3H labeled cAMP was used to determine the level of cAMP in intact platelets, and a high performance liquid chromatographic method was developed to measure the adenyl cyclase and phosphodiesterase activities in the platelet membrane fraction. Although the availability of substrate adenosine triphosphate (ATP) correlated well with the amount of cAMP produced, and in turn the availability of cAMP seemed to have a direct effect upon the reversibility of shape changes induced by the various stimuli, the only effect upon aggregation concerned the extent to which it occurred. No direct correlation of the level of cAMP with either the actual inhibition or activation of the aggregation mechanism was observed.


Phosphonoacetic acid in the treatment of simian varicella. Phosphonoacetic acid inhibited replication of simian varicella virus (Delta herpesvirus) in tissue culture. The drug was tested in patas monkeys 40 h after infection with Delta herpesvirus. A total of 200 mg/kg per day was given intramuscularly, divided into two doses every day for a total of 10 days. The treated monkeys were protected from clinical illness, and Delta herpesvirus was not recovered from their lymphocytes. Complement-fixing and neutralizing antibody titers were significantly lower in phosphonoacetic acid-treated monkeys than in the untreated controls. In animals given the drug alone, there was dermatitis and blackening of the skin and hair, serum glutamic oxalacetic transaminase and serum glutamic pyruvic transaminase enzymes were significantly increased, and liver biopsy revealed diffuse cytoplasmic swelling and granulation of the hepatocytes. The therapeutic range of this drug should be studied carefully before considering its use in severe varicella-zoster infection in humans.


MIF-like activity in non-stimulated and virus infected cell cultures. Macrophage-migration inhibition factor (MIF) is a lymphocyte-derived substance which plays an important role in cell mediated immunity. Soluble factors containing MIF-like activity and produced by non-stimulated and virus-infected non-lymphoid cell cultures have also been reported. In the present study, a MIF-like factor was repeatedly detected in Buffalo green monkey kidney cells infected with mumps and herpes simplex virus type 1 (HSV-1) indicating that this substance is reproducible and can be stimulated by two viruses of widely varying groups. Wistar-38 (WI-38) cell cultures also increased production of this substance in response to mumps but not HSV-1 infection, indicating that the production of this factor is not necessarily induced by all viruses. A factor which stimulated the spread of macrophages was also found to be induced in WI-38 cells by both viruses, suggesting yet another substance produced by non-lymphoid cells in response to viral infection. The ability of non-stimulated WI-38 cells to produce MIF-like activity was also confirmed, and this factor could be further stimulated or opposed by viral infection.


Therapeutic effect of trisodium phosphonoformate on cutaneous herpesvirus infection in guinea pigs. When applied topically, trisodium phosphonoformate (PFA) displayed activity against established cutaneous herpesvirus infections in guinea pigs similar to that exhibited by the closely related phosphonoacetic acid (PAA); however, unlike PAA, PFA was not locally skin irritating. The therapeutic benefits of topical application of PFA were clearly evident when application was delayed for 48 h after virus inoculation, at which time lesions were well developed. The therapeutic effect was dependent on the concentration of PFA and the duration of treatment. PFA exhibited significant activity against established infections when administered intraperitoneally, although it was less effective via this systemic route than when applied topically.


HEp-2 cell- and herpes simplex virus type 1- induced deoxythymidine kinases: inhibition by derivatives of 5-trifluoromethyl-2'-deoxyuridine. 5-Trifluoromethyl-2'-deoxyuridine (F(3)Thd), its free base and nucleotide triphosphate derivative, along with the nucleotide monophosphate and nucleotide triphosphate of deoxythymidine (dThd), were investigated as inhibitors of HEp-2 cell deoxythymidine kinase (dTK) and herpes simplex virus type 1 (HSV-1)-induced dTK. 5-Trifluoromethyluracil did not inhibit cellular or viral dTK. F(3)dThd competitively inhibited phosphorylation of dThd by both the HEp-2 cell- and the HSV-1-induced dTK. The K(mapp) for dThd and the K(Iapp) for the alternate substrate, F(3)dThd, were 3.5 and 22.5 muM for the HEp-2 cell dTK and 63.5 and 71.0 muM for the HSV-1-induced dTK. dThd-5'-PPP at 10 muM inhibited HEp-2 cell- and HSV-1-induced dTK by 94 and 22%, respectively. In comparison, 10 muM F(3)dThD-5'-PPP inhibited HEp-2 cell- and HSV-1-induced dTK 95 and 15%, respectively. These data indicate that F(3)dThd-5'-PPP may mimic dThd-5'-PPP feedback regulation of cellular and viral dTK.


Resistance of spheroplasts and whole cells of Pseudomonas cepacia to polymyxin B. Sucrose-lysozyme spheroplasts were prepared from two strains of Pseudomonas cepacia and tested for susceptibility to polymyxin B and benzalkonium chloride. Spheroplasts were more susceptible than whole cells to benzalkonium chloride but not to polymyxin B. Disruption of the outer membrane layer was not by itself sufficient to render P. cepacia susceptible to polymyxin B.


New medium for rapid screening and enumeration of Clostridium perfringens in foods. A rapid and sensitive procedure for estimating low numbers of Clostridium perfringens has been investigated and compared to methods used currently in the food industry. The new liquid medium, RPM (rapid perfringens medium), was compared with sulfite-polymyxin-sulfadiazine agar and tryptose-sulfite-cycloserine agar in recovery studies with naturally contaminated and with inoculated foods. The medium consists of a mixture of litmus milk and fluid thioglycolate medium fortified with glucose, peptone, gelatin, yeast extract, sodium chloride, and ferrous sulfate. Selectivity is based on an antibiotic system (polymyxin B sulfate and neomycin sulfate) incorporated into the medium, coupled with an incubation temprature of 46 to 48 degrees C for 24 h. Tubes were scored as positive if a stormy fermentation was observed. All tubes demonstrating stormy fermentation were confirmed as containing C. perfringens. Of a total of 774 naturally contaminated food samples, 546 samples (71%) were found to contain C. perfringens with RPM, whereas only 168 (22%) of the samples were positive using sulfite-polymyxin-sulfadiazine agar. C. perfringens was isolated from 71% of 85 other samples using RPM as compared to 14% with tryptose-sulfite-cycloserine agar. Enumeration studies on 14 individual samples using the most probable number technique also demonstrated greater sensitivity with RPM.


Adverse effects of electrical energy applied to the nervous system. Neural prostheses activated by radiofrequency transmission are currently being implanted to treat a variety of clinical problems. It is essential that neither the materials used in these prostheses, particularly the electrodes, nor the stimulus parameters that are employed will cause neural damage. The experiences of investigators engaged in both the experimental laboratory and clinical studies of the effects of electrical stimulation are reported herein.


Effect of thyroid hormone, actinomycin D, cycloheximide and puromycin on TRH-induced secretion of TSH, as studied by pituitary concentration of cyclic AMP and intrathyroidal colloid droplet formation. In an attempt to study the functional relation between pituitary cyclic AMP and TSH secretion in response to thyrotropin releasing hormone (TRH) or thyroid hormone administration, pituitary concentration of cyclic AMP was measured by protein binding assay after in vivo and in vitro administration of test materials (TRH, thyroxine, triiodothyronine, actinomycin D, puromycin and cycloheximide singly or in combination). Small dose of TRH apparently augmented TSH secretion as evidenced by a marked increase of intrathyroidal colloid droplet, but failed to elevate the pituitary concentration of cyclic AMP. Triiodothyronine (T3) and thyroxine (T4) blocked an increase of TSH secretion produced by TRH, but they elevated pituitary concentration of cyclic AMP in vivo and in vitro. Actinomycin D (Act D), puromycin and cycloheximide elevated pituitary cyclic AMP concentration without stimulating TSH secretion. From the data accumulated, it appears that the measurement of total anterior pituitary concentration of cyclic AMP is not useful to evaluate the activity of TSH cells in response to thyroid hormone, TRH and TRH plus thyroid hormone.


The beta-adrenoceptor controlling renin release. The beta-adrenoceptor antagonists, atenolol, metoprolol and propranolol, administered intravenously to anaesthetized rats in doses producing equal beta1-adrenoceptor blocking effects, caused comparable suppression of plasma renin activity (PRA) despite the fact that, at these doses, atenolol and metoprolol exhibited no beta2-adrenoceptor blocking properties. Practolol, an agent specific for beta1-adrenoceptors but possessing intrinsic sympathomimetic activity, caused less marked suppression of PRA. When doses of atenolol, metoprolol, propranolol and butoxamine were selected to achieve equal beta2-blocking effects, PRA was again significantly suppressed by atenolol and metoprolol but not by propranolol or butoxamine. These results do not support the concept that adrenergic release of renin is mediated by beta2-adrenoceptors, but are compatible with the involvement of a beta1-adrenoceptor-mediated mechanism.


[Hormonal regulation of duodeno-pancreatic activity]. After examining the nature and action of the principal gastroduodenal hormones, the most recent findings regarding hormonal regulation mechanisms of duodeno-pancreatic activity are presented, stress being laid on the close interdependence of the various hormones taking part in the complex digestive event. The role of hormone mediators is then examined with special attention to the action of cyclic AMP and prostaglandine.


Cytomegalovirus-induced thrombocytopenia. An unusual case report. A 21-year-old man had severe thrombocytopenia resistant to corticosteroid therapy. Viral studies of cytomegalovirus were 1:1,024. Although the cytomegalovirus titer returned toward normal (1:16 titer) during a period of three months, thrombocytopenia persisted and continued to remain refractory to corticosteroid therapy. Initial bone marrow morphology, platelet size, and platelet survival and sequestration were not typical of idiopathic thrombocytopenic purpura, and it is doubtful that splenectomy would have been helpful. However, when restudied six months later, platelet size was greatly increased, platelet survival was severely shortened (half-time, 2 1/4 hours), and intense splenic trapping was evident. Splenectomy was performed and resulted in normalization of the platelet count. Cytomegalovirus-associated thrombocytopenic purpura is an unusual disease that may take an unpredictable clinical course. Careful study of the mechanism of thrombocytopenia appears worthwhile when early recovery does not occur.


A longitudinal study of the serological and virological status of 18 women infected with cytomegalovirus. 18 women, of known seropositivity, have been followed for between 18 and 66 months (mean 50.2 months) for viral excretion and serological changes. CMV was isolated from 58/146 (39.7 per cent) cultures from various sites, mostly cervix. A total of 129 sera were obtained and each was titrated in the late antigen, early antigen and anti-complement immunofluorescence assays, as well as the complement fixation and microneutralisation tests. From 3 women virus was consistently re-isolated, from 3 others virus was never re-isolated and the remaining 12 women excreted virus intermittently during the period of study. No significant changes in antibody titres could be detected by any of the 5 serological assays in any woman during periods of viral excretion. This suggests that local reactivation of latent CMV infection is not associated with a demonstrable systemic antibody response. Inter-assay correlations were sought for each of the 10 possible combinations of pairs of tests. Significant correlations were found for most pairs of tests, with the notable exception fo those involving the early antigen test. This confirms that antibodies detected in this assay are distinct from those detected by the other 4 serological assays.


Recovery of a Sendai virus variant with temperature-sensitive hemolytic activity from persistently infected cells from mouse brain. A persistently infected cell line designated MB/Senas was established by cultivation of mouse brain cells from four-day-old C3H mice infected intracerebrally at birth with 10(6) PFU of Sendai virus, strain 52. After 5 passages, 0.16 per cent of Sendai52 antiserum (containing two 50 per cent plaque reducing doses/ml of serum) was introduced into the culture medium. The addition of antiserum was accompanied by a rise in cell-associated viral antigen from a level of 5 per cent antigen positive cells to 100 per cent demonstrable by both intracellular and membrane immunofluorescence. A variant of Sendai52 virus, designated Sendaias, was recovered from MB/Senas by inoculation of supernatant medium into chick embryos. Infection of chick embryos at 37 degrees C was abortive. Fifty per cent or less of chick embryos infected at dilutions 10(-1) to 10(-9) yielded detectable virus. Hemagglutination (HA) was weak but could be improved by trypsinization of allantoic fluids. Neuraminidase (NA) activity was barely detectable. Hemolysis (HE) was absent. Propagation of Sendaias virus at 33 degrees C showed no change from weak HA and NA activities but HE activity was now apparent which was temperature sensitive. Mortality of infected chick embryos increased to 100 per cent. HE activity and lethality for chick embryos was thermolabile at 45 degrees C.


Widespread occurrence in Australian marsupials of neutralizing antibodies to a herpesvirus from a parma wallaby. Serum neutralizing antibodies to a parma wallaby herpesvirus (PWHV) have been detected in a wide range of marsupials from different locations across Australia, including several islands. A study of 242 animals (mostly macropods) sampled in the wild showed that 23% had antibodies; a significantly higher frequency (41%) of 116 animals in captivity had antibodies, which were generally at higher levels than those of animals in the wild. Antibodies to PWHV were also detected among parma wallabies from a colony on Kawau Island, New Zealand. The highest antibody levels were found in a group of captive tammar wallabies during recurrent outbreaks of clinical infection. It is suggested that PWHV has evolved along with a marsupial host, and that the high antibody levels among captive animals reflects ease of virus transmission due to crowding, or to conditions of stress leading to expression of latent virus.


Interaction between infectious bursal disease virus and Newcastle disease virus in chickens. The Australian strain of infectious bursal disease virus (IBDV), 002/73, affected the response of chickens to Newcastle disease virus (NDV). The titre of serum antibodies to NDV in chickens infected with IBDV was significantly lower than that of birds infected with NDV alone. It also appeared that IBDV affected NDV excretion from chickens as NDV was more frequently isolated from chickens infected with IBDV, IBDV infection did not alter the pathogenicity of NDV in chickens. This Australian strain of IBDV therefore appeared to be immunodepressive in one-day-old chickens.


A general method for purification of deoxycytidine kinase. Deoxycytidine kinase from a variety of animal tissues binds to Cibacron Blue 3G-A coupled to Sepharose CL-6B. The enzyme can be selectively eluted with ATP to yield single-step purifications of 17-89-fold. The affinity adsorbent is re-usable.


Isolation of human lactate dehydrogenase isoenzyme X by affinity chromatography. Human isoenzyme LDH-X (lactate dehydrogenase isoenzyme X) was isolated from seminal fluid of frozen semen samples by affinity chromatography by using oxamate-Sepharose and AMP-Sepharose. In the presence of 1.6 mM-NAD+, isoenzyme LDH-X does not bind to AMP-Sepharose, whereas the other lactate dehydrogenase isoenzymes do. This is the crucial point in the isolation of isoenzyme LDH-X from the other isoenzymes. The purified human isoenzyme LDH-X had a specific activity of 146 units/mg of protein.


Rapid purification and properties of potassium-activated aldehyde dehydrogenase from Saccharomyces cerevisiae. A method for the purification of yeast K+-activated aldehyde dehydrogenase is presented which can be completed in substantially less time than other published procedures. The enzyme has a different N-terminal amino acid from preparations previously reported, and other small differences in amino acid content. These differences may be the result of differential proteolytic digestion rather than a different protein in vivo. A purification step involves the biospecific adsorption on affinity columns containing immobilized nucleotides in the absence of the substrate aldehyde. Direct binding studies with the coenzyme in the absence of aldehyde reveal 4 NAD sites per tetrameric molecule, each with a dissociation constant of 120 micron. These results conflict with properties of preparations previously reported and may conflict with kinetic models that have aldehyde as the leading substrate. Binding to Blue Dextran affinity columns suggests the presence of a dinucleotide fold in common with other dehydrogenases and kinases.


The mechanism of reaction of fully reduced membrane-bound cytochrome oxidase with oxygen at 176K. 1. The results of non-linear optimization studies on the mechanism of reaction of fully reduced cytochrome oxidase with O2 at 176K are presented. The analysis is carried out on data obtained by means of dual-wavelength multi-channel spectroscopy at three wavelength pairs (604-630, 608-630 and 830-940 nm) and at three O2 concentrations (60, 200 and 1180 micron). The only model that satisfies the triple requirement of a standard deviation within the standard error of the experimental data, good determination of the optimized parameters and a random distribution of residuals is a three-species sequential mechanism. 2. On the basis of the optimized values of the relative absorption coefficients of the intermediates at each wavelength obtained from the present paper together with data from low-temperature trapping, e.p.r. and magnetic-susceptibility studies, the possible valence states of the metal centres in each of the intermediates are discussed.


The mechanism of reaction of ferricyanide-pretreated mixed-valence-state membrane-bound cytochrome oxidase with oxygen at 173 K. 1. The results of non-linear optimization studies on the mechanism of reaction of ferricyanide-pretreated mixed-valence-state cytochrome oxidase with O2 at 173 K are presented. The analysis is carried out on data obtained by means of dual-wavelength multi-channel spectroscopy at four wavelength pairs (444-463 nm, 604-630 nm, 608-630 nm and 830-940 nm) and at two O2 concentrations (360 micron and 520 micron). The only model that satisfies the triple requirement of a standard deviation within the standard error of the experimental data, a random distribution of residuals and good determination of the optimized parameters, is a three-intermediate sequential mechanism. 2. On the basis of the optimized values of the relative absorption coefficients of the intermediates at each wavelength obtained from the present paper together with data from optical wavelength scanning and e.p.r. spectroscopy obtained by low-temperature trapping studies, the possible valence states of the metal centres in each of the intermediates are discussed.


Measurement of adenosine 3':5'-cyclic monophosphate by competitive binding to salt-dissociated protein kinase. An assay for cyclic AMP is described which takes advantage of the high affinity of the dissociated receptor moiety of cyclic AMP-dependent protein kinase I for the nucleotide. The kinase is kept dissociated by salt (800 mM-NaCl/30mM-EDTA). In the presence of a simply prepared heat-stable protein fraction the binding reagent is stable for the time needed to reach equilibrium of binding. A simple procedure [precipitation with poly-(ethylene glycol) followed by DEAE-cellulose chromatography] is described for the separation of protein kinase I from other binding proteins for cyclic AMP in rabbit skeletal muscle. The sensitivity, precision, reproducibility and specificity of the assay compared favourably with those of other cyclic AMP assays. The main advantage of the present assay is its resistance towards non-specific interference from a number of salts, tissue-culture media and substances found in crude tissue extracts. The reliability of cyclic AMP measurement directly in crude tissue extracts was ensured by removal of the assayable cyclic AMP with cyclic nucleotide phosphodiesterase digestion or adsorption with antibody against cyclic AMP, by comparison with measurement in tissue extracts purified by chromatography on QAE-Sephadex or sequentially on Dowex 50, and aluminium oxide as well as by dilution and recovery experiments.


The mode of regulation of pyruvate dehydrogenase of lactating rat mammary gland. Effects of starvation and insulin. 1. The ;initial activity' of the pyruvate dehydrogenase enzyme complex in whole tissue or mitochondrial extracts of lactating rat mammary glands was greatly decreased by 24 or 48h starvation of the rats. Injection of insulin and glucose into starved rats 60min before removal of the glands abolished this difference in ;initial activities'. 2. The ;total activity' of the enzyme complex in such extracts was revealed by incubation in the presence of free Mg(2+) and Ca(2+) ions (more than 10 and 0.1mm respectively) and a crude preparation of pig heart pyruvate dehydrogenase phosphatase. Starvation did not alter this ;total activity'. It is assumed that the decline in ;initial activity' of the enzyme complex derived from the glands of starved animals was due to increased phosphorylation of its alpha-subunit by intrinsic pyruvate dehydrogenase kinase. 3. Starvation led to an increase in intrinsic pyruvate dehydrogenase kinase activity in both whole tissue and mitochondrial extracts. Injection of insulin into starved animals 30min before removal of the lactating mammary glands abolished the increase in pyruvate dehydrogenase kinase activity in whole-tissue extracts. 4. Pyruvate (1mm) prevented ATP-induced inactivation of the enzyme complex in mitochondrial extracts from glands of fed animals. In similar extracts from starved animals pyruvate was ineffective. 5. Starvation led to a decline in activity of pyruvate dehydrogenase phosphatase in mitochondrial extracts, but not in whole-tissue extracts. 6. These changes in activity of the intrinsic kinase and phosphatase of the pyruvate dehydrogenase complex of lactating rat mammary gland are not explicable by current theories of regulation of the complex.


Phosphorylation of glucose in isolated rat hepatocytes. Sigmoidal kinetics explained by the activity of glucokinase alone. The conversion of glucose into glucose 6-phosphate in an extract of isolated rat hepatocytes incubated in the presence of MgATP was studied spectrophotometrically at 340nm and also by a radiochemical procedure based on the release of (3)H from [2-(3)H]glucose. Both methods gave similar results. The glucose-saturation curve was sigmoidal and the shape of this curve was not influenced by the ionic composition of the incubation medium. The activity at 0.5mm-glucose was only 1-2% of V(max.), indicating a virtual absence of low-K(m) hexokinase in the preparation. The radiochemical method was also used for the determination of glucose phosphorylation by intact hepatocytes. The glucose-saturation curve was also markedly sigmoidal, but the s(0.5) (substrate concentration at half-maximal velocity) and the Hill coefficient were larger than in extracts of hepatocytes. These two parameters became smaller when cells were incubated in a medium in which Na(+) ions were replaced by K(+) ions. The increased rate of phosphorylation at low glucose concentration in a K(+) medium was accompanied by an increased rate of metabolite recycling between glucose and glucose 6-phosphate and also by an increased uptake of glucose. In both media phosphorylation of glucose was inhibited co-operatively by N-acetylglucosamine. Calculations indicate that this inhibition would reach 100% at saturation of the inhibitor, although at lower concentrations of N-acetylglucosamine it was smaller than expected from the known K(i) of N-acetylglucosamine for glucokinase. The rate of phosphorylation of glucose was proportional to the amount of glucokinase in hepatocytes from newborn rats and in conditions such as starvation and diabetes in which the total amount of glucokinase in the liver is decreased. In the same conditions, glucose 6-phosphatase activity was either normal or increased. It is concluded that the phosphorylation of glucose in isolated hepatocytes follows sigmoidal kinetics, which can be explained by the activity of glucokinase alone with no participation of low-K(m) hexokinase or of glucose 6-phosphatase.


Calcium ion cycling in rat liver mitochondria. 1. Addition of N-ethylmaleimide to rat liver mitochondria respiring with succinate as substrate decreases both the initial rate of Ca(2+) transport and the ability of mitochondria to retain Ca(2+). As a result, Ca(2+) begins to leave the mitochondria soon after it has entered. Half-maximal effects occur at an N-ethylmaleimide concentration of about 100nmol/mg of protein. 2. The efflux of Ca(2+) induced by N-ethylmaleimide is not prevented by Mg(2+) or by Ruthenium Red at concentrations known to prevent Ca(2+) efflux when exogenous phosphate also is present. Swelling of mitochondria does not accompany N-ethylmaleimide-induced Ca(2+) efflux. 3. Addition of Ca(2+) to rat liver mitochondria in the presence of N-ethylmaleimide produces an immediate decrease in DeltaE (membrane potential), which decreases further to only a slight extent over the next 8min. Concomitant with this is an immediate increase and then levelling off of the -59DeltapH (transmembrane pH gradient). 4. Preincubation of rat liver mitochondria with p-chloromercuribenzenesulphonate, which by contrast with N-ethylmaleimide is unable to penetrate the inner mitochondrial membrane, also prevents Ca(2+) retention. The DeltaE and -59DeltapH respond to Ca(2+) addition in a manner similar to that which occurs when N-ethylmaleimide is present. Subsequent addition of mercaptoethanol produces an immediate increase in both DeltaE and -59DeltapH. At the same time Ca(2+) is rapidly accumulated by the organelles. 5. The above data are interpreted as indicating that under the conditions of Ca(2+) efflux seen here, the mitochondria retain their functional integrity. This contrasts with the uncoupling effect of Ca(2+) seen in the presence of P(i), which generally leads to a loss of mitochondrial integrity. We suggest that a unique mechanism of Ca(2+) cycling is able to take place when mitochondria have been treated with N-ethylmaleimide.


Effect of adrenalectomy on acceleration of gluconeogenesis by calcium ions, adenosine 3':5'-cyclic monophosphate and adrenaline in rat kidney tubules. 1. Gluconeogenesis from pyruvate was measured in renal-cortical-tubules fragments prepared from fed male rats 6-8 days after adrenalectomy or sham adrenalectomy. The response of this process to 3':5'-cyclic AMP and adrenaline was compared in these two states at two Ca2+ concentrations. 2. Adrenalectomy decreased the percentage stimulation of gluconeogenesis by 3':5'-cyclic AMP, but increased percentage stimulation by adrenaline. Cortisol treatment of adrenalectomized rats (50 mg/kg, twice daily for 2 days) did not reverse the change in responsiveness to 3':5'-cyclic AMP and adrenaline. 3. Stimulation of gluconeogenesis by 1 micron-adrenaline was unaffected by 10 micron-propranolol (beta-blocker) in either state. Phentolamine (alpha-blocker; 10 micron) totally blocked stimulation of gluconeogenesis by 1 micron-adrenaline in the sham-operated condition, but was only partially effective in this respect after adrenalectomy.


The involvement of phosphatidate phosphohydrolase and phospholipase A activities in the control of hepatic glycerolipid synthesis. Effects of acute feeding with glucose, fructose, sorbitol, glycerol and ethanol. Rats were fed by stomach tube with a single dose of glucose, sorbitol, fructose, glycerol or ethanol of equivalent energy contents or with 0.15 M-NaCl. They were killed 6 h later and the relative rates of phosphatidate deacylation and dephosphorylation measured in the microsomal and supernatant fractions of the livers. Treatment with sorbitol, fructose, glycerol and ethanol increased phosphohydrolase activities in the microsomal and supernatant fractions. The only significant change in deacylase activity was an increase in the microsomal fraction produced by ethanol. It is proposed that hepatic triacylglycerol synthesis is partly controlled by the balance between phosphatidate phosphohydrolase and phospholipase A-type activities.


Sitosterol in juvenile type II hyperlipoproteinemia. The effect of beta-sitosterol on plasma lipids and lipoproteins was evaluated in a randomized double-blind cross-over trial in 15 children and adolescents with familial hypercholesterolemia over a period of 6 months. Twelve patients completed the study, with good adherence to drug intake. Sitosterol lowered the plasma total choelsterol by 6%, LDL cholesterol by 7% and HDL cholesterol by 15% (P less than 0.05). This insufficient response of total and LDL cholesterol and the marked fall of HDL cholesterol appears to advise against the use of beta-sitosterol granulate in juvenile type II hyperlipoproteinemia.


Hypercholesterolemia in renal allograft recipients. Comparison with non-renal hypercholesterolemia and normal subjects. In order to investigate the degree of similarity between renal transplant and non-renal hypercholesterolemia, serum and lipoprotein lipid compositions were compared in female transplant hypercholesterolemic patients (serum cholesterol greater than 240 mg/100 ml, serum triglyceride less than 150 mg/100 ml) and female non-renal hypercholesterolemic and normal subjects. A number of lipid abnormalities were demonstrated: (1) Serum and LDL cholesterol and phospholipid levels were significantly elevated in both transplant and non-renal hypercholesterolemic patients; (2) Serum triglyceride, VLDL choelsterol, triglyceride and phospholipid, and LDL and HDL triglyceride were significantly increased in transplant hypercholesterolemic patients. Changes of this nature are usually found in hypertriglyceridemia, and were not observed in non-renal hypercholesterolemic subjects. Finally, a number of changes in the ratio of esterified to free cholesterol and in the ratios of other lipoprotein-lipids, most of which did not correspond to any changes found in the common non-renal hyperlipidemias, were also demonstrated.


Relationship between the carbohydrate content of apoproteins of VLDL, IDL, and LDL and the plasma level of these lipoproteins in man. Short dietary effect. A 1,500 Cal test meal was used to study in man the apoprotein B-carbohydrate content and composition of VLDL, IDL and LDL in relation to the variation of plasma lipids and lipoproteins and the VLDL-apoprotein composition. Three essential facts emerge from the study: (1) the mirror image (inverse significant correlation) between VLDL-apo B plasma concentration and N-acetylglucosamine content of apo B; (2) a reduction in total carbohydate concentration of VLDL- and LDL-apo B, 2--4 h after the test meal and a trend to a reduction in the mannose content; (3) a significant correlation between sialic acid and apoprotein B concentration in plasma LDL during fasting. The effect of diet and subsequent fasting on carbohydrates of lipoproteins and the consequence of such changes on lipoprotein uptake are briefly discussed.