Characterization of a cytochrome P-450-dependent steroid hydroxylase system present in Bacillus megaterium. Cell-free extracts from sonically disrupted Bacillus megaterium ATCC 13368 hydroxylated a variety of 3-oxo-delta4-steroids in position 15beta in the presence of NADPH and O2. Ring A-reduced, aromatic and 3beta-hydroxy-delta5-steroids did not serve as substrates for the 15beta-hydroxylase system. Using ion exchange chromatography on DEAE-cellulose and gel filtration on Ultrogel ACA-54 it was possible to resolve the hydroxylase system into three proteins: a strictly NADPH-dependent FMN-containing (megaredoxin reductase), an iron-sulfur protein (megaredoxin), and cytochrome P-450 (P-450meg). The activity of the 15beta-hydroxylase system was fully reconstituted upon combination of these three proteins and addition of NADPH. Megaredoxin had an apparent sulfur to iron ration of 0.98 and showed g-signals at 1.90, 1.93, and 2.06 when analyzed by electron paramagnetic reso0 times and the preparation contained 1 to 2 nmol of cytochrome P-450 per mg of protein. This preparation of cytochrome P-450meg sedimented as a homogeneous zone on sucrose gradients with a sedimentation coefficient of 3.3 S and contained 0.94 nmol of heme per nmol of cytochrome P-450. The oxidized form of cytochrome P-450meg showed absolute absorption maxima at 416, 528, and 565 nm whereas the reduced form showed maxima at 411 and 542 nm. The following scheme is suggested for the electron transport in the 15beta-hydroxylase system in B. megaterium: NADPH leads to megaredoxin reductase leads to megaredoxin leads to cytochrome P-450meg.


Beta-Galactosidase messenger RNA made during recovery from inhibition of protein synthesis is not translated. Bacteria that accumulate RNA in the course of inhibition of protein synthesis are impaired in their ability to synthesize beta-galactosidase during subsequent recovery. By contrast, constitutive enzyme synthesis in recovering cells is normal. Even though no beta-galactosidase is made during recovery from this inhibition, a substantial quantity of beta-galactosidase mRNA (as determined by DNA-RNA hybridization) is made. The beta-galactosidase mRNA made in vivo is functional in vitro. It is capable of directing the in vitro synthesis of a portion of the NH2-terminal beta-galactosidase molecule (in the alpha portion of the molecule). However, this protein is not made in vitro. It is concluded that the beta-galactosidase mRNA that is made during recovery from protein synthesis inhibition, although apparently at least partly normally transcribed in vivo and functional in vitro cannot be translated under these conditions in vivo.


Stoichiometry of carbon monoxide binding by cytochrome c oxidase. The stoichiometry of carbon monoxide binding to beef heart cytochrome c oxidase has been reinvestigated both by titration of the reduced oxidase with CO and by measuring the amount of carboxyhemoglobin that is formed after adding oxyhemoglobin to a solution of the CO-enzyme complex. In the titration experiments the ratio of CO bounds to total heme a present was always less than 0.50 while in the experiments where oxyhemoglobin was added the results were variable and of lower accuracy. These observations do not agree with the recent conclusion of Volpe, J.A., O'Toole, M.C., and Caughey, W.S. (1975) Biochem. Biophys. Res. Commun. 62, 48-53 that CO is bound in a 1:1 ratio with heme a. An explanation for their results is suggested.


The management of soft-tissue sarcomas of the extremities. Of fifty-four patients with a soft-tissue sarcoma of an extremity, having a projected five-year survival rate of 62 per cent, forty-six treated by an  "adequate" surgical procedure (either radical local resection or ablation at an appropriate level, depending on defined circumstances) had a local recurrence rate of 2 per cent. In the other eight patients, whose surgical procedures were not adequate for one reason or another, the local recurrence rate was 100 per cent. The combined recurrence rate after both the adequate and the inadequate procedures was 16.7 per cent. The recurrences were noted prior to thirty months post-operatively and the metastases, prior to sixty months. Histogenesis of the sarcoma, one or more recurrences after previous operations, and treatment by an immediate definitive procedure at the time of biopsy and diagnosis by frozen section had no significant relationship to the rates of local recurrence or metastasis. Adequate radical local resection controlled these sarcomas as well as ablative surgery in terms of local recurrence and metastasis. The significant factors affecting local recurrence that were identified in this study were the location of the sarcoma and the adequacy of the surgical procedure.


Exfoliative cytology interpretation of synovial fluid in joint disease. Criteria for the specific diagnoses of the several arthritides, on the basis of exfoliative cytology examination of fluids aspirated from diseased joints, were derived from examinations of 126 specimens of synovial fluid. They were screened cytologically and wholly on the basis of the cytological findings were classified into seven groups: rheumatoid arthritis, osteoarthritis, traumatic arthritis, gouty arthritis, Reiter's syndrome, pigmented villonodualr synovitis, and septic arthritis. The cytological features of each group appeared relatively constant and reproducible. Subsequent comparison of the clinical and cytological diagnoses showed excellent correlation.


The Budd-Chiari syndrome: angiography and its complications. Four patients with the Budd-Chiari syndrome are presented. Hepatic venography and inferior vena cavography confirmed the diagnosis in these patients. Angiography was used to evaluate the liver and the superior mesenteric and portal venous drainage. Contrast medium complications, ranging from nausea to renal shut-down and death, were encountered in three of these patients, an unusually high number of angiographic complications. The role of angiography in the Budd-Chiari syndrome is assessed.


Membrane behavior of exocytic vesicles. II. Fate of the trichocyst membranes in Paramecium after induced trichocyst discharge. A specific exocytic process, the discharge of spindle trichocyts of Paramecium caudatum, was examined by means of the electron microscope. This exocytosis is induced by an electric shock simultaneously in nearly all of the trichocysts (ca. 6,000-8,000) of a single cell. Single paramecia were subjected to the shock and then fixed at defined times after the shock so that the temporal sequence of the pattern of changes of the trichocyst membranes after exocytosis could be studied. The trichocyst vacuoles fuse with the plasma membrane only for the length of time required for expulsion to take place. After exocytosis, the membrane of the vacuole does not become incorporated into the plasma membrane; rather, the collapsed vacuole is pinched off and breaks up within the cytoplasm. The membrane vesiculates into small units which can no longer be distinguished from vesicles of the same dimensions that exist normally within the cell's cytoplasm. The entire process is completed within 5-10 min. These results differ from the incorporation of mucocyst membranes into the plasma membrane as proposed for Tetrahymena.


Subcellular localization of B apoprotein of plasma lipoproteins in rat liver. Multispecific antigen-binding fragments (Fab) from rabbit antisera against rat very low density lipoproteins (VLDL) and Fab against rat low density lipoproteins that were monospecific for the B apoprotein were conjugated to horseradish peroxidase. Conjugates were incubated with 6-mum frozen sections from fresh and perfusion-fixed livers and with tissue chopper sections (40 mum thick) from perfusion-fixed livers. In the light microscope, specific reaction product was present in all hepatocytes of experimental sections as intense brown to black spots whose locations corresponded to the distribution of the Golgi apparatus: along the bile canaliculi, near the nuclei, and between the nuclei and bile canaliculi. Perfusion fixation with formaldehyde produced satisfactory ultrastructural preservation with retention of lipoprotein antigenic determinants. In the electron microscope, patches of cisternae and ribosomes of the rough endoplasmic reticulum (ER) and particularly its smooth-surfaced ends, vesicles located between the rough ER and the Golgi apparatus, the Golgi apparatus and its secretory vesicles and VLDL particles in the space of Disse all bore reaction product. The tubules and vesicles of typical hepatocyte smooth ER did not contain reaction product, nor did the osmiophilic particles contained therin. The localization obtained in this study together with other evidence suggests a sequence for the biosynthesis of VLDL that differs in some respects from that proposed by others: (a) the triglyceride-rich particle originates in smooth ER where triglycerides are synthesized; (b) at the junction of the smooth and rough ER the particle receives apoproteins synthesized in the rough ER; (c) specialized tubules transport the particle, now a nascent lipoprotein, to the Golgi apparatus where concentration occurs in secretory vesicles; (d) secretory vesicles move to the sinusoidal surface where the particles are secreted into the space of Disse by fusion of the vesicular membrane with the plasma membrane of the hepatocyte.


Characterization of cytoplasmic and nuclear genomes in the colorless alga Polytoma. I. Ultrastructural analysis of organelles. Electron microscope studies have been made on the fine structure of the colorless biflagellate, Polytoma obtusum, with main emphasis on the structural organization of the mitochondria and the leucoplast. Both organelles have been demonstrated to contain DNA aggregates as well as ribosomal particles within their matrix material. Reconstructions from serial showed that (a) the mitochondria were highly convoluted and irregular in shape and size, and (b) the leucoplast was a single cup-shaped entity, with large starch grains, localized at the posterior end, and multiple sites of DNA aggregates. The starch-containing compartments appeared to be interconnected by narrow tubular or sheetlike bridges. Cytoplasmic invaginations into the plastid, often containing mitochondria, were of frequent occurrence, and membranes of mitochondria and the leucoplast appeared to be closely apposed. Membranes elements, both sheetlike and vesicular, were also present in the matrix. The Polytoma leucoplast was, in certain respects, morphologically similar to the plastids of various photosynthetic mutants of Chlamydomonas, most of which show Menedelian segregation. It is suggested that Polytoma arose from a Chlamydomonas-like ancestor, possibly through combined mutational processess of both chloroplast and nuclear genomes. Since Polytoma leucoplasts contain both DNA and ribosomal particles, it is probable that these organelles still possess semiautonomy and limited ability for protein synthesis.


Nucleotidase activity in regenerating liver and in clonal strains of hepatoma and pituitary cells in culture. A nucleotidase of the combined 3'- and 5'-type (nucleotide phosphohydrolase, E.C.3.1.3.31) was present in the cytosol of regenerating rat liver cells, and of rat hepatoma and pituitary cells in culture. The enzyme activity per milligram of cell protein was very similar in regenerating liver and in three of the different cell types. The hepatoma cell strain which showed the slowest growth rate had a three-fold higher basal enzyme activity. After the first days of regenerative growth in rat liver and during early plateau phase of cell growth, there was a 50-120% increase in specific enzyme activity. In the hepatoma cells, the enzyme activities were also compared to the cellular content and synthesis of RNA and DNA. The increase in enzyme activity occurred concomitantly with a reduced incorporation of 3H-thymidine into DNA. The possible physiological role of this nucleotidase in nucleic acid and nucleotide metabolism is discussed.


Induction by short-chain fatty acids of alkaline phosphatase activity in cultured mammalian cells. Short-chain fatty acids, such as propionic, n-butyric,  n-butyric, n-valeric, isovaleric, n-caproic, and n-caprylic acids, induce alkaline phosphatase activity in cultured mammalian cells. Long-chain fatty acids have no similar effects. With B-6 cells (mouse X Chinese hamster cell hybrids), n-butyrate at 2 to 5 mM exhibits the greatest activity. Induction begins exponentially about 24 hours after addition of the fatty acid and continues over 48 hours. Studies on the inducing activity-structure relationship revealed the necessity of a carboxyl and an ethyl or longer alkyl group. n-Butyrate shows a marked synergistic action of induction when added along with other types of inducers: adenosine 3':5'-cyclic monophosphate (cAMP) or 5-bromodeoxyuridine (BrdU). Treatment of other cell lines with either n-buryrate, cAMP, or BrdU revealed a cell-type specific response pattern of alkaline phosphatase. The biological significance of this effect of short-chain fatty acids is discussed.


SV40 transformation of mouse brain cells: critical role of gene A in maintenance of the transformed phenotype. Brain cells derived from the NIH Swiss mouse strain have been established in tissue culture. Astrocytic neuroglial cells, identified by morphology and staining properties, predominate. The brain cell culture was successfully transformed with SV40 wild type virus and with a representative early (A239) and late (C219) mutant. When subjected to growth analysis the A239 transformant displayed selective loss of six characteristics of the transformed phenotype at the restrictive temperature (40.5 degrees C): doubling time, saturation density, ability to grow in low serum, efficiency of growth on plastic and on normal brain cell layers, and cloning in soft agar. Temperature shift experiment demonstrated the reversibility of the differences in saturation density. T-antigen was expressed at both temperatures. Alteration in uptake of 2-deoxyglucose was not a characteristic of the transformed phenotype. The cell lines may be utility in brain culture work and in studies on the mechanism of SV40 transformation.


Separation of vitamin D metabolites and their analogues by high-pressure liquid chromatography. The separation of vitamin D3 metabolites and their analogues by high-pressure liquid chromatography was investigated using a Zorbax SIL column. Each metabolite can be separated effectively with a solvent consisting of 2% methanol-methylene chloride or gradient elution from 0.02% to 6% methanol-methylene chloride. The C-24 epimers of 1alpha, 24-dihydroxy- and 1alpha,24,25-trihydroxy-vitamin D3 are resolved as their free forms, while the epimers of 24-hydroxy- and 24,25-dihydroxy-vitamin D3 can be separated as their trimethylsilyl derivatives.


Study on the efficiency of support-coated open-tubular columns for steroid analysis. Glass support-coated open-tubular columns for the analysis of steroids in biological samples were prepared with varous support materials. The reproducibility of the procedure of preparing the column was investigated and some fundamental chromatographic properties were studied.


Increased plasma protein binding and lower metabolic clearance rate of aldosterone in plasma of low cortisol concentration. During ACTH or cortisol infusion in ten recumbent normal men taking dexamethasone, the metabolic clearance rate of aldosterone increased by 50% as plasma cortisol was raised from low (2 mug/dl) to high concentration (50 mug/dl). Since splanchnic blood flow did not change, a greater efficiency of removal of aldosterone must have occurred, by means of displacement of aldosterone from high-affinity sites on plasma protein. At 37 C, equilibrium dialysis of low-cortisol plasma showed one-third of plasma aldosterone bound to albumin, and 24 to 28% bound to higher-affinity sites on other protein. As plasma cortisol increased, a progressively smaller fraction was tightly bound, approaching zero as transcortin was saturated with cortisol. The addition of large amounts of aldosterone to low-cortisol plasma displaced 14C-cortisol from transcortin binding sites. The results support earlier evidence that a significant fraction of plasma aldosterone is bound to transcortin, from which it is readily displaced by cortisol, resulting in an increased metabolic clearance rate of aldosterone by making a larger fraction available for removal from plasma.


The hormonal activity of the postmenopausal ovary. The origin of plasma sex hormones in postmenopausal women was studied by determining plasma levels under basal conditions, after ACTH stimulation, and after dexamethasone suppression, as well as after hCG stimulation. Values obtained in postmenopausal women were compared with values observed during the follicular phase of the cycle in young women on the one hand, and with values in ovariectomized women of postmenopausal age on the other hand. All sex steroid levels studied with the exception of estrone, were significantly lower in postmenopausal women than in young women during the early follicular phase of the cycle. In ovariectomized women only androgen levels (testosterone, androstenedione, dihydrotestosterone, and to a lesser extent dehydroepiandrosterone,) were lower than in normal postmenopausal women, estrogen, 17 hydroxyprogesterone, and progesterone levels being similar. ACTH increased all plasma steroid levels except estradiol, whereas after dexamethasone, all sex hormone levels were significantly decreased. hCG stimulation finally caused an increase of borderline statistical significance in testosterone, dehydroepiandrosterone, and 17-hydroxyprogesterone levels. We have concluded from this study that the adrenal cortex is almost the exclusive source of plasma estradiol, estrone, progesterone, and 17OH progesterone and the most important source of plasma dehydroepiandrosterone; that the postmenopausal ovary appears to be responsible for about 50% of plasma testosterone and 30% of androstenedione levels; and that hCG stimulation with 5000 IU daily for 3 days, hardly influences steroid secretion by postmenopausal ovaries.


Pituitary-thyroid function in trophoblastic disease. Pituitary-thyroid function was assessed in 12 patients with trophoblastic disease (4 hydatidiform mole, 3 invasive mole, and 5 choriocarcinoma). Thyroid-stimulating activity was detectable, by means of the McKenzie bioassay, in 6 patients (Group 1) but not in the other 6 patients (Group 2). In Group 1 serum thyrotropin (TSH) determined by radioimmunoassay was mostly undetectable and did not respond to the administration of thyrotropin-releasing hormone (TRH) determined by radioimmunoassay was mostly undetectable and did not respond to the administration of thyrotropin-releasing hormone (TRH), while in Group 2 basal TSH was detectable in half of the patients and responded to TRH in all cases. Serum concentrations of total thyroxine (T4) (18.7 +/- 2.0 mug/100 ml, mean +/- SE), free T4 (4.9 +/- 0.04 ng/100 ml), total triiodothyronine (T3) (352 +/- 72 ng/100 ml), and free T3 (0.57 +/- 0.11 ng/100 ml) in Group 1 were statistically greater than those in Group 2 (total T4, 9.2 +/- 1.0 mug/100 ml, free T4, 2.0 +/- 0.2 ng/100 ml; total T3 156 +/- 20 ng/100 ml, and free T3 0.23 +/- 0.03 ng/100 ml). Free T4 and T3 fractions were within normal limits in both groups. After treatment of 5 patients in Group 1, the thyroid stimulating activity determined by bioassay dropped to undetectable levels, the serum concentrations of thyroid hormones decreased to normal limits, and TSH response to TRH became positive. These findings indicate that an abnormal thyroid stimulator, derived from the trophoblastic tissue, stimulated the thyroid hormone secretion from the thyroid gland and in turn suppressed TSH response to TRH in some patients with trophoblastic disease.


Acute administration of 25-hydroxycholecalciferol in man. Serum 25-hydroxyvitamin D responses to oral doses of 25-hydroxycholecalciferol were measured in healthy volunteers. Peak serum 25-hydroxyvitamin D levels occurred 4 to 8 hours after doses of 1.5, 5, and 10 mug per kg of body weight. The mean increments above baseline serum 25-hydroxyvitamin D concentrations at 4 hours were linear over this dose range. Following a single oral dose, serum 25-hydroxyvitamin D levels fell to 33% of their peak values after one week. The serum 25-hydroxyvitamin D responses measured by competitive protein-binding radioassay were similar to the serum 3H responses exhibited by three volunteers who received simultaneous oral doses of 3H 25-hydroxycholecalciferol and 14C-cholecalciferol. The appearance of 14C in plasma was slower and less marked compared with 3H, probably reflecting a more rapid plasma clearance of the 14C-cholecalciferol. The disappearance of 25-hydroxycholecalciferol from the circulation of two volunteers following acute intravenous delivery of 1.0 mg of 25-hydroxycholecalciferol was multiphasic over 10 days. 60% of the dose administered remained in the circulation 24 hours after the dose. When analyzed several days after single 10 mug/kg oral doses of 25-hydroxycholecalciferol to 4 volunteers, the mean rate of decline to total serum 25-hydroxycholecalciferol was slow (t 1/2 = 22 days). When baseline serum 25-hydroxyvitamin D levels were subtracted, however, the apparent serum half-life of administered 25-hydroxycholecalciferol was 12 days, similar to that reported after tracer doses of 3H-25-hydroxycholecalciferol. The rapid and predictable serum 25-hydroxyvitamin D increases following the oral administration of 25-hydroxycholecalciferol suggest its possible therapeutic advantages compared with vitamin D administration.


Modification of adrenal function by the anti-serotonin agent cyproheptadine. The effect of the serotonin antagonist, cyproheptadine (Cypro), on metyrapone-induced stimulation of the pituitary-adrenal axis was evaluated in nine normal subjects. The subjects underwent standard oral metyrapone tests (750 mg every four h, for six doses) while taking no medications and while receiving oral Cypro (4 mg every six h). Cypro administration caused a significant reduction in: 1) baseline 17-hydroxycorticosteroid (17-OH) excretion (-31 +/- 7.2%), 2) the increase above baseline in 24 h 17-OH excretion on the day after metyrapone (-32 +/- 4.9%), 3) serum 11-deoxycortisol concentrations 8 h (-45 +/- 5.5%) and 24 h (-18 +/- 3.6%) after the first dose of metyrapone, and 4) plasma ACTH concentration 8 h (-32 +/- 8.2%) and 24 h (-22 +/- 8.0%) after the first dose of metyrapone. When compared with the control test, Cypro administration did not alter: 1) baseline 17-ketosteroid secretion, 2) the fall in serum cortisol concentration while taking metyrapone, 3) the serum free metyrapone concentration, 4) cortisol metabolism, 5) the adrenal response to ACTH, and 6) assay methods for measurement of serum and urinary corticosteroids. Our data suggest that Cypro can reduce pituitary-adrenal responsiveness by reducing plasma ACTH concentrations. If Cypro acts as a serotonin antagonist, our data lends support to the idea that serotoninergic mechanisms are important in the control of pituitary ACTH secretion in normal human subjects.


The effect of administered ACTH on aldosterone metabolism and secretion. To determine better the overall regulation of aldosterone by ACTH, the effect of administered ACTH on the aldosterone metabolic clearance rate (MCR), the excretion rates of tetrahydroaldosterone (THAldo), and the acid-labile conjugate (ALC) of aldosterone were studied in healthy male volunteers on a sodium intake in excess of 200 mEq/day. In 7 subjects, the aldosterone MCR increased significantly on the third day of ACTH treatment, as did the excretion of THAldo, but there was a decrease in the excretion of the ALC. One subject receiving ACTH for 6 days showed a steady increase in the aldosterone MCR on days 3 and 6 of treatment. In 2 of 3 subjects receiving ACTH treatment for 3-4 days, plasma aldosterone levels and plasma renin activity showed little change, whereas in 1 subject, both plasma aldosterone and plasma renin activity became suppressed. During ACTH administration there was a sustained increase in the excretion of THAldo, a metabolite formed in the liver, (in contrast to a decreasing excretion of the ALC which is made principally in the kidneys) which suggests that the increased aldosterone metabolism in response to ACTH occurred in the liver. Measurements made of the excretion of THAldo show that the ACTH stimulatory effect on aldosterone secretion may not be as transient as previously reported.


Presence of TSH receptor in thyroid neoplasms. The nature of the TSH receptor in adenoma and carcinoma of the thyroid gland was studied using a radioreceptor assay technique. A membrane fraction of tissue homogenate was obtained by discontinuous sucrose gradient ultracentrifugation, and 125I-TSH, labelled by a lactoperoxidase method, was purified with a receptor adsorption method. Both the capacities and the association constants of high affinity receptors (4 x 10(9) M-1) and of low affinity receptors (0.073 x 10(9) M-1) observed in the normal thyroid were almost identical to those of the thyroid of Graves' disease and those of thyroid adenoma. Although the two papillary carcinomas examined were found to have two kinds of TSH receptors, one of the carcinomas showed decreased association constants for both high affinity and low affinity receptors.


Radioreceptor assay for insulin.

Radioreceptor assay for insulin. A sensitive and simple radioreceptor assay (RRA) capable of detecting 10 muU/ml of serum insulin has been developed using the crude membrane fraction of guinea pig kidney and 125I-porcine insulin. Using this assay system, serum insulin during 100 g oral glucose tolerance tests in six males including one obese man was measured. The mean concentration of serum insulin increased from 19 muU/ml during fasting to maximum of 90 muU/ml after 60 min of glucose load and then gradually declined to 38 muU/ml by 180 min. The values obtained by the present RRA agreed well with immunoreactive insulin (IRI).


Receptor-active growth hormone in Laron dwarfism. The hepatic radioreceptor assay for hGH has been applied to the detection of hGH in the sera of patients with high growth hormone dwarfism (Laron dwarfism). Substantial quantities of receptor-active hGH were found in the sera of all 7 patients studied. In one patient, arginine infusion elicited a prompt increase in both immunoactive and receptor-active hGH. These observations suggest that circulating hGH in Laron dwarfism is biologically active and support the concept that the disease may be caused by a generalized defect in hGH receptors.


Progesterone binding by normal and abnormal human endometrium. Cytosols from 75 normal, 36 abnormal, and 5 decidual human endometrial tissue specimens were assayed for the presence of a high affinity, progesterone-specific binding protein. Thirty of the normal and 15 of the abnormal samples were found to contain a binder which would form a high-affinity complex with progesterone but not with cortisol, 17beta-estradiol, testosterone, or 5alpha-androstane-3-,17-dione. Incubation of cytosol with trypsin or incubation for 2 hours at 37 C abolished [3H]progesterone binding by these preparations, indicating the protein nature  and heat-lability of the binder. The average equilibrium constant of dissociation, Kd, of the progesterone-binder complex was 4.0 X 10(-10)M in each phase of the menstrual cycle. The concentration of the binder varied over the cycle, however, with a significant peak at mid-cycle (P = .02). The average saturation values in femtomoles (fmoles)/mg protein ranged from 21 in the early proliferative phase to 64 in the late proliferative samples, dropping to 36 in early secretory and to 3 in the late secretory phase of the cycle. No progesterone-specific binding was detected in decidual samples. Saturable binding was demonstrable in 10 of 22 endometrial hyperplasias, 80-1840 fmoles/mg protein, with high affinity, Kd 3.3 X 10(-10)M. Two other hyperplasia samples bound progesterone, but with lower affinity. Two grade I adenocarcinomas, one grade III adenosquamous carcinoma, and one grade III adenocarcinoma contained the progesterone binder, but in 9 other cancers no detectable binder was present. A benigh adenocanthomyoma was found to contain a progesterone binder (18 fmoles/mg protein with a Kd of 2.5 X 10(-10)M).


Impaired LH release following exogenous estrogen administration in patients with amenorrhea-galactorrhea syndrome. Serum gonadotropin levels were determined in 10 patients with the amenorrhea-galactorrhea syndrome before and following acute iv administration of synthetic LH-releasing hormone (LHRH) or conjugated estrogens, in order to clarify the hypothalamic derangements in the gonadotropin secretion in patients with hyperprolactinemia. The basal prolactin (PRL) levels were elevated in all the patients, and blunted responses to 500 mug of iv synthetic thyrotropin-releasing hormone (TRH) injection were found in 9 out of the 10 patients. The basal levels of LH and FSH were subnormal in 2 and 3 patients, respectively, while those in the remaining patients were normal or slightly elevated. Normal or excessive responses of gonadotropins to 100 mug of iv LHRH were observed in most patients, 9 for LH and 10 for FSH out of 10 patients. In 10 normal cyclic women at the mid-follicular phase (D7-9) and 10 hypothalamic amenorrhea patients without galactorrhea, LH release was found 48 to 72 h after the iv injection of 20 mg conjugated estrogens (Premarin). This LH release following Premarin injection was completely abolished in the patients with amenorrhea-galactorrhea. These data seem to indicate that in patients with hyperprolactinemia, tonic secretion of gonadotropin is maintained fairly well, while of the positive feedback effect of Premarin on the release of LH is impaired. It is suggested that impaired LH release may be partly responsible for anovulation and amenorrhea in patients with hyperprolactinemia.


Impaired release of parathyroid hormone in magnesium deficiency. Parathyroid hormone release and end-organ responsiveness to parathyroid extract (PTE) were evaluated in a 25-year-old woman with magnesium deficiency associated with hypocalcemia and inappropriately low levels of serum  immunoreactive parathyroid hormone (iPTH). End-organ responsiveness to PTE was demonstrated by increases in serum calcium and in urinary phosphorus, cyclic AMP, and hydroxyproline. When the serum calcium was increased from a baseline of 6.9 mg/100 ml to levels of 8.0 mg/100 ml and higher by calcium infusion, the serum iPTH decreased from the low normal range to below the limits of detectability. The intravenous administration of 3 mg/kg of body weight of magnesium led to an abrupt and striking increase in circulating iPTH with a 2-fold increase in one minute, a 6-fold increase in two minutes, and an 8-fold increase in five minutes. The very rapid increase in serum iPTH produced by magnesium infusion in this study suggests an effect of magnesium on hormone secretion rather than an effect on hormone synthesis. The evidence provided by this investigation indicates that the release of parathyroid hormone is impaired in magnesium deficiency and that the level of circulating calcium required for the suppression of parathyroid hormone secretion is lower than that in normal subjects.


A reconsideration of a thyroid-stimulating immunoglobulin as the cause of hyperthyroidism in Graves' disease. Several assays for thyroid-stimulating immunoglobulins in the blood in patients with Graves' disease have been described recently; depending upon the method, different names have been used and distinct entities thus implied. Using an increase in cyclic AMP in the human thyroid slice after 2 h of incubation as an index of thyroid stimulation, we identified thyroid-stimulating activity in all of an unselected series of sera from 11 patients with hyperthyroidism of Graves' disease, but long-acting thyroid stimulator, by mouse bioassay, in only three. The theory is proposed that the thyroid-stimulating immunoglobulin is probably present in all such patients; it may be seen as a polyclonal antibody to a single human antigen that has a variable cross-reaction with a corresponding thyroid antigen in the mouse and in other species.


Urinary excretion of cyclic adenosine 3',5'-monophosphate in children of different ages. The urinary excretion of cyclic AMP was investigated in 97 healthy children, 3 months to 16 years old. When the excretion was expressed as mumol/24 h an increase with age (r = 0.693, P less than 0.001) and an increase with body weight (r = 0.700, P less than 0.001) were found to be quite similar. In relation to surface area, the average excretion for children up to 91/2 years old was 4.45 +/- 1.71 mumol/m2 in constrast with 2.22 +/- 0.66 mumol/m2  in older children (P less than 0.001). The decline appears to be associated with approaching puberty. When cAMP excretion was related to urinary creatinine, an inverse correlation with age was found (r = -0.772, P less than 0.001). In the youngest category, 3 months to 4 years old, the ratio was 9.26 +/- 1.49 mumol/g creatinine vs 4.67 +/- 1.05 mumol/g creatinine in the age group 12 to 16 years old (P less than 0.001), which compares closely with the normal adult average of 4.34 +/- 1.25 mumol/g creatinine found in our previous study. Throughout there was no evidence of sex differentiation.


Viral antibody in the cerebrospinal fluid of patients with acute central nervous system infections. Cerebrospinal fluid (CSF) and sera from 129 patients of a study population of 139 were tested for antibody to herpes simplex, measles,and mumps viruses. Herpes simplex virus antibody was found in three of five patients with laboratory-confirmed herpes simplex infection and in eight patients without serological or virological evidence of current infection with this or other common neurotropic visuses. Eleven of the 139 patients were studied for antibody to lymphocytic choriomeningitis (LCM) virus. Eight of these had laboratory-confirmed LCM infection, and antibody was detected in the CSF of five of them. In one of these five, complement-fixing antibody appeared earlier in the CSF than in the blood. Assay of LCM virus antibody in the CSF may thus indicate infection with LCM virus more rapidly than serological and virological studies. The diagnostic and the possible prognostic significance of herpes simplex visus antibody in CSF remains to be ascertained.


Exposure of thyroid slices to thyroid-stimulating hormone induces refractoriness of the cyclic AMP system to subsequent hormone stimulation. These studies evaluated the influence of an initial exposure of thyroid slices to thyroid-stimulating hormone (TSH) on the subsequent responsiveness to the hormone. Bovine thyroid slices were incubated with or without 50 mU/ml TSH for varying periods and then incubated in hormone-free medium for varying periods. Subsequently, slices were incubated for 20 min with 10 mM theophylline and with or without TSH. Cylic AMP was measured after the third incubation. Phosphodiesterase and adenylate cylase were assayed in homogenates prepared from slices after the second incubation. In some experiments prostaglandin E1, puromycin, thyroxine, and triiodothyronine and propylthiouracil were included in the media. In other experiments, low does of TSH (1 AND 10 mU/ml) were used instead of 50 mU/ml. Slices previously exposed to TSH have decreased responsiveness of the adenylate cyclase-cylic AMP system. Such refractoriness is hormone specific since initial exposure to prostaglandin E1 decreases the subsequent response to this substance but not to TSH. Refractoriness to TSH develops only when the first incubation is at least 30 min. It is not reversed by 5 h of incubation without hormone. Incubation of thyroid slices with puromycin does not eliminate refractoriness. The decreased response to TSH cannot be explained by release of thyroxine, triiodothyronine, or iodide from the slices. Phosphodiesterase activity is not increased during the refractory period. The decreased cyclic AMP response to TSH is associated with diminished response of adenylate cyclase activity to the hormone. Guanosine triphosphate (1 mM) increased adenylate cyclase activity in both control and TSH treated tissue, but the effect was significantly less in the latter. Although with guanosine triphosphate, TSH increased adenylate cyclase activity in TSH treated tissue, the enzyme activity was still less than that present in control tissue incubated with guanosine triphosphate and TSH. NaF caused an equivalent stimulation of adenylate cyclase in both control and TSH treated tissue. These results suggest that the refractoriness represents an alteration in hormone binding or the coupling of the bound hormone to the adenylate cyclase activity rather than any modification of the catalytic site of the enzyme.


Decreased insulin binding to adipocytes and circulating monocytes from obese subjects. Insulin binding to isolated adipocytes from 16 normal and 14 obese patients was studied. The data indicated that, as a group, adipocytes from the obese patients bound significantly less insulin than normal. However, of the 14 obese patients, 5 were not hyperinsulinemic and 4 of these 5 subjects had normal insulin binding. These subjects were also younger, and had the onset of obesity in childhood. When these five patients were separated from the original 14 obese patients, enhanced differences in insulin binding to adipocytes were observed when normals and the remaining 9 obese subjects were compared. Similar findings were obtained with isolated circulating mononuclear cells from these same patients. Presumably the five normoinsulinemic obese patients were not insulin-resistant, and, thus, the data indicate that insulin binding to adipocytes was decreased only in insulin-resistant obese patients. This conclusion was strengthened by finding a highly significant correlation (r=-0.71, p less than 0.001) between insulin binding to adipocytes and fasting plasma insulin level, while a weaker correlation (r=-0.49,p less than 0.01) existed between insulin binding and degree of obesity. Finally, when insulin binding to adipocytes and mononuclear cells from the same individual was compared, a significant positive correlation was found (r=0.53,p less than 0.01). (a) insulin binding to adipocytes and mononuclear cells is decreased in cells from insulin-resistant obese patients; (b) a significant inverse relationship exists between fasting plasma insulin level and insulin binding to adipocytes; and (c) in obesity, events that affect insulin receptors on adipocytes similarly affect insulin receptors on mononuclear cells.


Dihydrotestosterone binding by cultured human fibroblasts. Comparison of cells from control subjects and from patients with hereditary male pseudohermaphroditism due to androgen resistance. Dihydrotestosterone binding was measured in culture fibroblasts from 14 control subjects and from 12 patients with five different types of hereditary male pseudohermaphroditism. Two assays of binding were used--an intact monolayer assay and density gradient centrifugation of cell extracts. In the intact monolayer assay of normal cells the uptake of [3H]dihydrotestosterone consisted of two components. The first was a high affinity component that exhibited saturation at approximately 1 nM dihydrotestosterone. The second was a low affinity component that was not saturable with concentrations of steroid up to 5 nM. Twice the number of high affinity binding sites were present in fibroblasts grown from genital skin (foreskin, labia majora, and scrotum) as from nongenital sites (37 vs. 14 fmol/mg protein). In the density gradient assay in 5-10% sucrose, the major peak of dihydrotestosterone binding was in the 8S region in low molarity buffer and in the 4S region in 0.5 M KCl. High affinity binding was normal in cells from two patients with familial incomplete male pseudohermaphroditism, type 2, an autosomal recessive defect in which dihydrotestosterone formation is deficient, and in cells from a patient with male pseudohermaphroditism due to 17 beta-hydroxysteroid dehydrogenase deficiency, an autosomal recessive defect of testosterone synthesis. High affinity binding was low by both methods in fibroblasts from five patients with complete testicular feminization. Furthermore, binding by both methods was also low in cells from three subjects with familial incomplete male pseudohermaphroditism, type 1, a presumed X-linked recessive disorder of androgen resistance, and in fibroblasts grown from a subject with the incomplete form of testicular feminization. The finding that dihydrotestosterone binding is abnormal in two forms of hereditary androgen resistance in addition to complete testicular feminization suggests either that these disorders are the result of allelic mutations affecting the function of the androgen-binding protein or that normal dihydrotestosterone binding requires the participation of more than one gene product.


The differential impact of work-oriented vs. communication-oriented juvenile correction programs upon recidivism rates in delinquent males. This study examined recidivism rates in work-oriented (N = 30) and communication-oriented (N = 30) juvenile delinquency programs for males. Both groups were matched for age, ethnic origin, educational achievement, and reading scores. Chi-square analyses indicated significant differences in recidivism rates between groups in both residential (chi2 - 6.71, df = 1, p less than .01) and aftercare (chi2 = 3.89, df = 1, p less than .05) programs. In each phase of the treatment program, recidivism was highest in the work-oriented group. The results suggested that (a) facilitation of family interaction and communication is related closely to successful treatment of the delinquent and consequent recidivism; (b) group counseling that provides the youth and parents an opportunity to learn better communication skills appears to improve family cohesion and solidarity; and (c) newer therapeutic approaches in delinquency should concentrate on filial and family-type therapies.


The binding of cyclic AMP to renal brush border membranes. The binding of cyclic AMP to the proximal tubule luminal (brush border) membrane isolated from the rabbit renal cortex was studied. The rate of binding was dependent on temperature; at 37 degrees equilibrium was attained in 45 min, whereas at 0 degrees 120 min was required. The final levels of binding were identical. The binding of 3H-cyclic AMP was reversed by dilution or addition of unlabeled cyclic nucleotide. Debinding was markedly temperature sensitive. Binding was only partially saturable with respect to cyclic AMP concentration, apparently with more than one binding site. The cyclic AMP bound to the membrane was recovered unchanged. When bound to the membrane cyclic AMP was resistant to hydrolysis by endogenous membrane or exogenously added phosphodiesterase. The binding to the membranes was relatively specific for cyclic AMP, although other cyclic purine nucleotides inhibited, cyclic IMP greater than dibutyryl cyclic AMP greater than cyclic GMP. The renal membranes did bind cyclic GMP, but this binding was relatively non-specific. Hormones and drugs, that mediate cyclic AMP generation or renal function, as well as other compounds common to the proximal tubule were without significant effect on cyclic AMP binding. Binding was inhibited by sulfhydryl reacting agents and this inhibition could be blocked and partially reversed by mercaptoethanol.


LH and FSH stimulation of cyclic AMP in specific cell types isolated from the testes. The direct effect of LH and FSH on cyclic AMP levels in specific cell types, isolated from the rat testes, was investigated in vitro. LH significantly stimulated cyclic AMP production in isolated interstitial cells and had only a slight effect on the isolated germ cells. FSH significantly stimulated cyclic AMP production in isolated seminiferous tubules, organ cultures of testes explants, and isolated Sertoli cells, with only a small response elicited in the germ cells. FSH had no effect on the cyclic AMP levels in interstitial cells and either freshly isolated or cultured peritubular cells. These data indicate that the Sertoli cells and interstitial cells are the main cell types in the testes which respond to FSH and LH respectively with increased cyclic AMP production. A possible slight effect of either hormone on the cyclic AMP level in the germ cells has not be ruled out.


Inhibition of cyclic nucleotide phosphodiesterase activity by an endogenous factor. Cyclic nucleotide phosphodiesterase activity of several tissues of rat is inhibited by an endogenous factor isolated from rat adipocytes following exposure of these cells to agents that raise intracellular cyclic AMP levels. The inhibitory action was demonstrated with varying cAMP concentrations from 0.1-400 muM. Enzyme from 10,000 X g supernatant of epididymal adipose tissue was inhibited approximately 2-3 fold more than the plasma membrane of adipocytes by a given concentration of the feedback regulator. Kinetic analysis of cAMP phosphodiesterase of plasma membrane showed that feedback regulator (8.8 U/ml) inhibited the Vmax 48%. The maximum inhibition of phosphodiesterase by feedback regulator (20 U/ml) was about 80%. The apparent Km for cAMP was increased. The ability of phosphodiesterase from several tissues of rat (10,000 X g supernatant) to hydrolyze cAMP and cGMP was tested. Feedback regulator inhibited cGMP hydrolysis in cardiac muscle and 5 other tissues 23-92% more than it inhibited the hydrolysis of cAMP. The physiological significance of this inhibitory effect can begin to be clarified when the feedback regulator is purified to homogeneity and characterized.


The functions of cyclic AMP and calcium as alternative second messengers in parotid gland and pancreas. Biochemical and ultrastructural studies of rat parotid gland slices have led to the identification of alpha- and beta-adrenergic receptors and a cholinergic receptor, all operating within the same secretory cell. While cyclic AMP serves as the second messenger in the beta-adrenergic response of enzyme secretion, Ca++ serves as the second messenger in the alpha-adrenergic and in the cholinergic responses which both lead to K+ release and water secretion. Ca++ also serves as a second messenger for the muscarinic cholinergic receptor in rat pancreas slices in which it causes enzyme secretion. Analysis of this information leads to the conclusion that neither the neurotransmitter, nor the receptor, nor the second messenger are unique for a certain type of response. The latter seems to be dictated by a component of the specific response pathway which is affected by the second or a subsequent messenger. By having different neurotransmitters operate the same response and a single neurotransmitter operate different responses diversity of control is achieved.


The effects of hormones on cyclic adenosine 3':5'-monophosphate accumulation in transitional epithelium of the urinary bladder. Transitional epithelium lining rabbit urinary bladders was isolated and studied in vitro. The homogeneity of the isolated epithelium was demonstrated by light and electron microscopical monitoring as well as cell culture studies. Transitional epithelium responded to epinephrine and prostaglandin E1 (PGE1) in the presence of 2mM 1-methyl, 3-isobutylxanthine (MIX) with increases in intracellular levels of cyclic adenosine 3':5'-monophosphate (cyclic AMP). Corticotropin, aldosterone, insulin, parathyroid hormone and vasopressin were slightly but significantly stimulatory under similar conditions. Glucagon and oxytocin were not stimulatory at the concentrations tested. The effects of epinephrine and PGE1 were potentiated by 2mM MIX 20-fold or greater. The cells were slightly more sensitive to PGE1 then to epinephrine. The prostaglandin produced a noticeable response at about 10nM, while effects of epinephrine were discernible at 0.1muM. Maximal responses to both effectors were seen at about 10muM. The action of 10muM epinephrine, but not 10muM PGE1, was completely abolished by 0.1mM propranolol. Responses to combinations of epinephrine and PGE1 were additive. Cyclic AMP accumulated in the incubation medium of transitional epithelial cells exposed to epinephrine, PGE1, MIX, or combinations of the agonists. The appearance of cyclic AMP in the medium was slow compared to the rate of intracellular accumulation, but reached significant levels following prolonged stimulation.


Femtomole sensitive radioimmunoassay for cyclic AMP and cyclic GMP after 2'0 acetylation by acetic anhydride in aqueous solution. The sensitivity of radioimmunoassays for cyclic AMP and cyclic GMP has been markedly improved to readily detect femtomole (10-15) amounts in tissue extracts by acetylating the cyclic nucleotides at the 2'0 position with acetic anhydride. Acetylation of cyclic nucleotides by acetic anhydride in aqueous solution proceeds more rapidly than the hydrolysis of acetic anhydride to acetic acid thus yielding 100% acetylated cyclic nucleotide. 2'0 substituted cyclic nucleotides have greater affinity for the antibody than the parent cyclic nucleotides because the antibody has been made to a protein conjugate coupled at the 2'0 position. This simple acetylation technique makes it possible to measure cyclic AMP and cyclic GMP in minute quantities of tissue without purification or concentration of the sample.


The effect of Ca++ on cyclic nucleotide phosphodiesterases of superior cervical ganglion. Cyclic nucleotide phosphodiesterase was examined in canine and bovine superior cervical ganglia. Activity in crude supernatant fractions was only slightly stimulated by Ca++ despite the presence of protein activating factor. Three forms of phosphodiesterase were resolved from bovine ganglia supernatant extracts by chromatography on DEAE-cellulose. The first enzyme eluted, (DI), was almost completely specific for cyclic GMP, while the other two (DII and DIII), hydrolyzed both cyclic AMP and cyclic GMP; all were free of heat-stable protein activator. Each enzyme was inhibited by low concentrations of Ca++ in the assay medium. Inhibition by Ca++ was reversed by addition of protein activator, but activity did not increase above the control level. Cyclic AMP hydrolysis by enzyme DII was stimulated by micromolar concentrations of cyclic GMP. This stimulation was reduced by Ca++ unless protein activator was present.


PGE1-mediated cyclic AMP refractoriness: effects of cycloheximide and indomethacin. Human synoviocytes in culture respond to prostaglandin E1 (PGE1) by increasing their intracellular concentration of cyclic AMP. Readdition of PGE1 to cells previously treated with PGE1 elicits no change in the intracellular concentration of cyclic AMP. This refractory state is partially prevented by the inhibitors of protein synthesis, puromycin (PM) and cycloheximide (CH). Indomethacin (IM), which reduces angiotensin tachyphylaxis, does not prevent the occurrence of refractoriness to PGE1 with respect to accumulation of cyclic AMP. This agent does alter the release of cyclic AMP from human synovial cells. We postulate that other factors, independent of new protein synthesis, are necessary for the development of the complete PGE1 refractory state in these cells.


Alterations in the hydrolytic activity, inhibitor sensitivity and molecular size of the rat erythrocyte cyclic AMP phosphodiesterase by calcium and hypertonic sodium chloride. Hypertonic NaCl irreversibly reduced the sensitivity of the rat erythrocyte cyclic AMP phosphodiesterase to inhibition by Ro 20-1724 but not by papavarine. This effect could be prevented by 1 mM EDTA and mimicked by by CaCl2. The reduction in inhibitor sensitivity was associated with an increase in hydrolytic activity and a four-fold reduction in molecular weight as estimated by Sephadex G-200 chromatography. The response to CaCl2, like that to NaCl, was time and concentration dependent and proceeded more slowly in 2 M NaCl. This concentration of NaCl contained sufficient Ca++ as an impurity to account for its effects on inhibitor sensitivity and molecular size. However, the initial and reversible increase in inhibitor sensitivity and hydrolytic activity obtained in the presence of EDTA, probably resulted from the high salt concentration, per se.


Prostaglandin synthetase activity and hormone responsiveness in normal and SV40 transformed WI-38 fibroblasts. Prostaglandin (PG) synthetase activity and selective hormone responsiveness were examined in normal and SV40 transformed WI-38 fibroblasts (VA13-2RA). The transformed VA13-2RA cells have significantly reduced rates of PGE1, PGE2, PGF1alpha and PGF2alpha synthesis as compared to the normal WI-38 fibroblast. The transformed cell in contrast to the normal cell hyperresponds to stimulation by L-epinephrine (10 muM) and PGE1 (8.5 muM) but is unresponsive to bradykinin (BK) as measured by the accumulation of intracellular cyclic AMP. Indomethacin treatment does not significantly alter the PGE1 and L-epinephrine (EPI) responsiveness of the normal WI-38 fibroblast, however it abolishes the BK response in these cells. These results provide further evidence for the dependency of cell activation by bradykinin on the PG synthetase system. No experimental data was found to support the role of PGs as negative regulators of PGE1 and EPI responsiveness in the WI-38 fibroblast. Using the VA13-2RA cells, limited attempts to recover PG synthetase activity comparable to that found in normal WI-38 cells were unsuccessful. The VA13-2RA cell and its normal counterpart represent models for investigating the role of PGs in cell function and the mechanism of BK activation and its effect on cell metabolism.


Cyclic guanosine 3':5'-monophosphate and the regulation of lipolysis in rat fat cells. The addition of the divalent cation ionophore A23187, carbachol, norepinephrine or insulin to rat fat cells elevated cyclic GMP. The increase in cyclic GMP due to these agents was greater at 4 than at 2 minutes after their addition. Cyclic GMP accumulation was also elevated by the addition of 0.1 to 0.5 mM sodium oleate in the presence of 0.1% albumin and by albumin containing added palmitate with an FFA/albumin molar ratio of 6.7. The rise in cyclic GMP due to all agents was markedly reduced in calcium-free buffer. The effects of the various agents on cyclic GMP accumulation in rat fat cells had little correlation with lipolysis. Insulin was an effective anti-lipolytic agent in both the presence and absence of calcium while neither A23187 nor carbachol had any effect on fat cell lipolysis.


Modulation of platelet cyclic nucleotide content by PGE1 and the prostaglandin endoperoxide PGG2. The effects of prostaglandin E1 and prostaglandin G2, the prostaglandin endoperoxide, on platelet cyclic nucleotide concentrations were measured in platelet rich plasma (PRP), and in washed intact platelets. PGE1 was found to be a potent stimulator of platelet cAMP levels in both PRP and washed cells, and to inhibit aggregation in both systems. PGE1 did not change platelet cGMP levels in either PRP or washed cells. PGG2 which is a potent inducer of platelet aggregation, did not affect either the basal cAMP or the basal cGMP concentration. However, PGG2 was found to antagonize the increases in cAMP content in response to PGE1 in both PRP and washed platelets. The addition to our system of a cyclic nucleotide phosphodiesterase inhbitor, theophylline, did not change our findings. It is suggested that PGG2 may induce platelet aggregation by inhibiting PGE1-stimulated cAMP accumulation.


Growth of astrocytoma cells in the presence of prostaglandin E1: effect on the regulation of cyclic AMP metabolism. Human astrocytoma cells (1321N1) in culture respond to pharmacological concentrations of prostaglandins and catecholamines with a marked increase in the accumulation of cyclic AMP. However, growth of 1321N1 cells in the presence of low concentrations (0.003 to 0.1 muM) of prostaglandin E1 (PGE1) results in a marked reduction in the responsiveness of the cells-even to concentrations of PGE1 that normally stimulate maximal accumulation of cyclic AMP. Occasionally, a partial reduction in the responsiveness to catecholamines was observed in cells grown in the presence of PGE1. When it occurred this effect could be correlated with an increase in the cyclic AMP-degradation capacity of the cells. This loss of responsiveness to catecholamines could be completely reversed by 1-methyl-3-isobutylxanthine, a potent inhibitor of phosphodiesterase activity in 1321N1 cells. The consistently observed and more profound desensitization to the effects of PGE1 could  not be correlated with an increase in cyclic AM-degradative capacity. Accordingly, 1-methyl-3-isobutylxanthine was only minimally effective in reversing desensitization to PGE1. It is concluded that the inability of 1321N1 cells grown in the presence of PGE1 to accumulate cyclic AMP upon subsequent challenge with PGE1 is primarily due to a selective desensitization of the PGE1-activated adenylate cyclase.


On the accumulation of adenosine 3':5'-monophosphate in Ehrlich cells and adenylate cyclase desensitization in response to epinephrine. The time course of the action of epinephrine on cAMP levels in isolated mouse Ehrlich ascites cells was studied. Following the addition of hormone (10(-7) to 10(-4) M), cAMP levels reached a maximum within 1 min and declined to about 2 fold basal levels within 10-20 min. The decline phase was not due to the metabolism of epinephrine or accumulation of an inhibitor in the medium. Experiments involving timed additions of theophylline to epinephrine-treated cells suggested that the rate of cAMP synthesis declined progressively with time. Cells treated with epinephrine and then washed free of hormone were shown to be refractory to hormonal stimulation of cAMP levels. Adenylate cyclase prepared from epinephrine-treated cells was shown to exhibit decreased sensitivity to the hormone in in vitro incubations. Resensitization of the enzyme could be achieved by washing pretreated cells free of epinephrine and reincubating them for 1/2 to 2 h. Neither desensitization nor resensitization were prevented by cycloheximide or actinomycin D. Desensitization could not be induced by incubating cels with dibutyryl-cAMP. The desensitization phenomenon is considered to be a mechanism whereby cells protect themselves against the possible harmful effects of high levels of cAMP maintained over a long period of time.


Acetaldehyde: an intermediate in the formation of ethanol from glucose by lactic acid bacteria. Group N streptococci formed acetaldehyde and ethanol from glucose. As the enzymes aldehyde dehydrogenase, phosphotransacetylase and acetate kinase were present this would enable these organisms to reduce acetyl-CoA to acetaldehyde and convert acetyl-CoA to acetyl phosphate and acetate. A pentose phosphate pathway which converted ribose-5-phosphate to glyceraldehyde-3-phosphate was also present. Acetaldehyde could not be formed via the hexose monophosphate shunt or by direct decarboxylation of pyruvate, as the enzymes phosphoketolase and alpha-carboxylase were absent. Phosphoketolase activity was induced in Streptococcus lactis subsp. diacetylactis after growth on D-xylose. Group N streptococci also contained an NAD-dependent alcohol dehydrogenase which reduced acetaldehyde to ethanol while both NAD- and NADP-dependent alcohol dehydrogenase activities were found in Leuconostoc cremoris.


Detection and characterization of differences in plasma  corticoid response to treatments. Ten experiments involving 3013 observations and 103 animals were studied to characterize variation associated with bovine plasma corticoid concentrations (ng/ml). Total corticoids, corticosterone, or cortisol were measured by competitve protein binding assay. Animals were noncystic or cystic, and measurements were made to establish basal concentrations or to estimate response to stimulation by adrenocorticotrophic hormone injection. Data were analyzed by least squares with the nested model: treatment regimen, animal in treatment, sample in animal in treatment, and sampling time to the highest order of regression (up to seventh) which was statistically significant (P less than .05). Estimates of error variances ranged from .31 for basal cortisol in cystic animals to 233.27 for total corticoids in noncystic, stimulated animals. Coefficients of variation among experiments ranged from 33 to 87% with mean 61%. In most analyses, estimates of experimental error were large indicating need for careful attention to experimental design to insure efficient least-cost experimentation. Use of the power function for nested analysis of variance to accomplish this is demonstrated.


Growth, pulmonary, and endocrine function in chronic asthma patients on daily and alternate-day adrenocorticosteroid therapy. Growth, pulmonary, and adrenal-pituitary function were assessed in 20 steroid-dependent asthmatic children and adolescents while they were on daily prednisone therapy. The patients were then switched to equivalent daily methylprednisolone or twice equivalent alternate-day methylprednisolone for a 3-mo period, after which time studies were repeated. The steroid regimens were then crossed so that those patients taking daily methylprednisolone were switched to alternate-day therapy and the alternate-day therapy patients were switched to daily medication for another 3-mo interval, after which studies were repeated again. Abnormal growth, bone maturation, and cataracts were identified. Mean symptom scores at each evaluation were similar. Pulmonary function tests and shunt studies showed little difference between methylprednisolone and prednisone. Endocrine function tests showed a high incidence of disturbed pituitary and adrenal function. A 3-mo period of alternate-day therapy did not alter this.


Peroxidase-conjugate chromatography isolation of conjugates prepared with glutaraldehyde or periodate using polyacrylamide-agarose gel. Fractionation abilities of polyacrylamide-agarose gel (Ultrogel) and dextran gel (Sephadex) column chromatography were compared in isolating horseradish peroxidase conjugates, prepared using two different methods. Utrogel AcA-44 provides an efficient separation of monomer conjugated and nonconjugated immunoglobulins resulting from the two-step glutaraldehyde procedure, Sephadex G-200 does not. Both types of columns eluted the polymer conjugates resulting from the periodate procedure in the void volume; these were hardly isolated from the small amount of monomer conjugate. Unreacted horseradish peroxidase, present in very low quantities after the efficient periodate method and in large amounts after the glutaraldehyde procedure, was separated by both gel types.


Characteristics of a virus causing a pox disease in sheep and goats in Kenya, with observation on the epidemiology and control. The sheep and goat pox viruses isolated in Kenya are not host specific as in the Middle East and India. The virus strains from one species are of a similar pathogenicity for the other, and the same virus appears to occur in the field outbreaks in mixed flocks. A silent infection with no skin lesions occurs in the field outbreaks. The virus grows well in lamb testis cell cultures, it does not haemadsorb nor haemagglutinate and is ether sensitive. Most strains could not readily be adapted to grow on the chorioallantoic membrane of embryonated eggs; one isolation only was made in this way. Staining of the virus inclusions showed that they were of DNA; the virus resembled that of lumpy skin disease in many characteristics. It was possible to cross-protect both species with virus of sheep or goat origin. A vaccine was made from tissue culture virus adsorped onto aluminium hydroxide gel and treated with formalin.


The response to oral poliovaccine in persons aged 16-18 years. Serum neutralizing antibodies to polioviruses were titrated in serum samples from 182 police cadets aged 16-18 years before and, in 168 of the cadets, 6 weeks after vaccination with a single dose of oral polio vaccine (OPV). Faecal excretion of poliovirus was also followed. Vaccination histories were obtained and confirmed whenever possible. Pre-vaccination antibody could not be detected against type 1 in 9-3% cadets, against type 2 in 2-7% and against type 3 in 7-7%. Absence of antibody to at least one virus type was found in 14-3% of the cadets. In 93 cadets in whom vaccination histories could be confirmed 40 had received only inactivated polio vaccine (IPV) previously; of these 23% lacked antibody to at least one virus type, and they had less intestinal immunity to a challenge dose of OPV than those previously given OPV. Only two of the cadets known to have had OPV were non-immune - both had received a single dose following full courses of IPV. However, cadets who had received OPV had their last dose of vaccine more recently (average 4-6 years) than those who had received only IPV (all 12 years or more). The serum antibody response to a single booster dose of OPV, and the faecal excretion of each type of virus after vaccination, showed an inverse relation to the corresponding pre-vaccination antibody concentration. A single dose of OPV did not reliably boost the immunity of those who possessed adequate immunity, and a failure to respond was also observed in a proportion of the cadets with no detectable antibody, mostly in the case of type 3 antibody and particularly if antibody to types 1 or 2 virus was also absent. No evidence was obtained that intestinal immunity could be expected in the absence of detectable circulating antibody. The reasons for the absence of a serological response to OPV in some subjects are discussed and consideration is given to the practical significance of the findings. It is suggested that reinforcement of polio immunity at school-leaving is important, particularly at the present time when many of those aged 16-18 years will have been vaccinated only with IPV. A single dose of OPV is not ideal for this purpose, not only because a small proportion of persons are liable to be left unprotected, but also because failure to produce a reliable boost in persons with adequate immunity at the time of vaccination gives rise to the possibility that they may become susceptible later in adult life.


Mouse or man? Which are pertussis vaccines to protect? Type 1 strains of Bordetella pertussis can infect mouse brain and have been recovered as type 1 organisms after death. When introduced into the naso-pharynx of the marmoset, they immediately acquired agglutinogen 2 or 3, and the resulting type 1,2 or 1,3 infection persisted for many weeks. As in the child, agglutinogens 2 and/or 3 appear to be essential for infection of the marmoset, whereas they are quite unnecessary in mouse brain. A vaccine (extract or whole cell) containing agglutinogen 1 may be sufficient to pass the mouse protection test but it may fail to immunize children. The mouse test is inadequate even for the screening of such extracts.


The importance of agglutinin production in mice in the determination of the definitive serotype of Bordetella pertussis. A schedule for the routine serotyping of strains of Bordetella pertussis based on agglutinin production in mice to the K-antigens has been worked out. Mice have been found as satisfactory as rabbits but far more economical for the production of the very small volumes of serum which are required. Agglutinin production, used in conjunction with direct agglutination, provides definitive information about serotype.


The serotypes of Bordetella pertussis isolated in Great Britain between 1941 and 1968 and a comparison with the serotypes observed in other countries over this period. Classification, by agglutinogens, of 634 isolates of Bordetella pertussis collected from 1971 to 1968 in Great Britain demonstrated that a change from a predominantly 1,2,0,4 serotype (75% of those examined during 1941-4) to a predominantly 1,0,3,0 serotype (73% of those examined during 1966-8) occurred sometime after 1953. Furthermore, evidence from the examination of isolates collected between 1941 and 1953 suggests that the change may have been gradual. Isolates of serotype 1,2,3,4 made up 20-30% of the total of our cross-country selection for the periods 1941-4, 1946-9, 1950-3 and 1966-8, but over shorter periods in individual areas the percentage varied from negligible to as high as half of those isolated. Results from other countries show a similar drift towards a 1,0,3 sertype but more often from a 1,2,3 than from 1,2,0 serotype. The value, in epidemiological studies, of extended information obtained by monospecific typing sera to all six, rather than only two or three agglutinogens, and confirmation of the results by agglutinin production is demonstrated: for instance not all 1,0,3 isolates were identical.


The stability of the serotypes of Bordetella pertussis with particular reference to serotype 1,2,3,4. Strains of Bordetella pertussis in which all the organisms contain agglutinogens 1 and 3 or 1,2 and 4 are easy to identify as serotypes 1,0,3,0 and 1,2,0,4 respectively; and similarly, stable strains of serotype 1,0,3,4 are occasionally found. During repeated subcultures, passage in vivo, and lyophilization and preservation for many years, these serotypes do not change. Mixing 1,0,3,0 and 1,2,0,4 serotypes and culturing them together in vivo and in vitro produces cultures from which organisms of the same two serotypes can be isolated. In contrast, strains which type as 1,2,3,4 are often a heterogeneous group. We have attempted to classify these as "stable", "variable" and "mixed" cultures. Some strains comprise organisms all of which contain the four agglutinogens and are as easy to type as the strains described above. These we have called "stable" 1,2,3,4 strains. Other 1,2,3,4 strains are made up of colonies possessing all four agglutinogens, as shown by agglutinin production, but in amounts varying from day to day so that direct typing is inconsistent. These we have called "variable" 1,2,3,4 strains. The last category, "mixed", is made up of organisms most of which give rise to stable 1,2,3,4 cultures; a few of the component organisms, however, have one or two of the four agglutinogens missing. The importance of the "variable" cultures is emphasized for work on apparent change of serotype, e.g. during infection.


Indirect haemagglutination reaction (LH)-the method of choice for the detection of anamnestic antibodies to varicella - zoster (VZ) virus. The method of indirect haemagglutination is a sensitive quantitative serological reaction for the detection of anamnestic titres of VZ antibodies. The curve obtained by the examination of a representative population sample has shown that the occurrence of VZ antibodies increases with the age so that 60% of children of under-school age and 90% of 15-year-old children have antibodies to varicella-zoster virus. In adult age groups, seropositivity ranges between 90--100%. The incidence and level of antibodies are identical in both sexes and do not decrease in old age groups. The mean titre of indirect haemagglutinating antibodies was 1 : 128.


Localization of antibacterial activity and hydrolytic enzymes in subcellular fractions of rabbit and chicken polymorphonuclear leukocytes. Polymorphonuclear leukocytes of rabbits and chickens after homogenization in 0.34 M saccharose or after multiple freezing and thawing were subjected to differential centrifugation at 150, 800, 10 000 and 50 000 X g. In the fractions obtained in this manner, total bactericidal activity as well as the activity of myeloperoxidase (E.C. 1. 11. 1. 7), catalase (E.C. 1.11.1.6), lysozyme (E.C. 3.2.1.17), cathepsin D (E.C. 3.4.4.23) and E, beta-D-glucuronidase (E.C. 3.2.1.31) and acid phosphatase (E.C. 3.1.3.2) were determined. Antibacterial activity was found in all fractions from rabbit leukocytes, but only in the first fraction from chick leukocytes. The fractions from rabbit leukocytes contained all enzymes under study while in the fractions from chicken leukocytes the presence of myeloperoxidase, catalase or cathepsin E could not be demonstrated. The highest bactericidal activity was found in the second obtained from  the homogenate or rabbit leukocytes. The highest specific activity of myeloperoxidase and homogenate of rabbit leukocytes. The highest specific activity of myeloperoxidase and the lowest activity of cathepsin D were also demonstrated in this fraction. The addition of pepstatin to rabbit leukocytes before their disintegration resulted in the inhibition of the activity of cathepsin D and E and in an increase in the specific activity of myeloperoxidase as well as in total bactericidal activity in the individual fractions. These results testify that microbicidal mechanisms of phagocytes from individual species may differ and when the structure of lysosomes is damaged, the liberated hydrolytic enzymes may gradually inactivate antibacterial substances.


Ultrastructural study of lesions induced in rabbit cornea by Herpes Simplex virus types 1 and 2. Herpes simplex virus types 1 and 2 were used to infect rabbit corneas (without abrasion), and the resulting lesions were compared by light and electron microscopy. The type 1-induced lesion developed more quickly; the infected epithelial cells underwent a progression of defined morphological changes leading to organelle destruction, cell rounding, and death. Virus particles were abundant in the nuclei and cytoplasm of all types of cell in the epithelium, in intercellular spaces, and on the basement membrane. Occasionally, virus was seen to replicate within stromal fibroblasts. In contrast, type 2, although causing similar changes in the epithelial cells, was relatively inefficient in the production of mature virions. Thus type 2 nucleocapsids were synthesized within nuclei in numbers comparable to those of types 1, but intact cytoplasmic virions were uncommon, apparently because of degradation of type 2 virions in cytoplasmic vesicles or tubules. Extracellular type 2 particles were rarely seen. This finding offers an explanation for the relatively slow development of the type 2-induced lesion, but not for its greater severity. All epithelial cells appeared to be capable of supporting replication of either type of virus.


Cytomegalovirus infection in patients receiving immunosuppressive therapy for rheumatologic disorders. Evidence of cytomegalovirus infection was sought in 131 patients attending a rheumatology clinic, 211 blood donors, and 14 patients before and after the initiation of cytotoxic immunosuppressive therapy for a rheumatologic condition. The titer of complement-fixing antibody to cytomegalovirus was significantly related to age and sex, but not to rheumatologic disease. After adjustment for age and sex differences, the proportion of patients treated with corticosteroids who had measurable antibody was lower than that of controls (P less than 0.025). Immunosuppressive therapy with azathioprine or cyclophosphamide did not affect the proportion of patients with antibody, but there was a significantly increased titer of antibody in those who were seropositive (P = 0.04). Cytomegalovirus was isolated from the urine of 20% of patients receiving cytotoxic immunosuppressive drugs, but not from any of the patients receiving corticosteroids or neither form of therapy (P = 0.001). Eight of 14 patients followed prospectively after the initiation of therapy with immunosuppressive drugs became infected with cytomegalovirus as demonstrated by a fourfold or greater rise in complement-fixing titer, viruria, or both. Seven of the eight patients were seropositive before therapy, a finding suggesting that immunosuppression acts largely by reactivating latent infection. It is postulated that immunosuppressive agents alone may account for a large proportion of cytomegalovirus infections seen after allograft transplantation.


Mechanism of injury of virus-infected cells by antiviral antibody and complement: participation  of IgG, F(ab')2, and the alternative complement pathway. Antibody-mediated C-dependent lysis of cell lines infected with herpes simplex type 1 virus, influenza A degrees virus, measles virus, and mumps virus occurred by the alternative C pathway with the participation of IgG antibodies. Lysis occurred only with immune human sera, Mg++ EGTA immune sera, and immune sera depleted of C4 or treated with Fab anti-C4. Lysis did not occur with nonimmune sera, Mg++ EDTA immune sera, and immune sera heated 50 degrees C for 25 min, depleted of factor B or treated with Fab antifactor B. Lysis was restored to heated and factor B immunodepleted immune sera by addition of factor B, but not by addition of an excess of C2. Further studies showed that lysis of HeLa cells infected with measles virus was induced by both immune IgG and F(ab')2 but not Fab' in the presence of a nonantibody-containing human C source. Lysis of measles virus-infected cells was also indpendent of movement of viral antigens on the surface of the infected cells, as inhibition of viral antigen capping by cytochalasin B or sodium azide was not associated with abrogation of immune lysis.


Virus-induced diabetes mellitus. VI. Genetically determined host differences in the replicating of encephalomyocarditis virus in pancreatic beta cells. Beta cells were isolated from strains of mice that were susceptible and resistant to encephalomyocarditis (EMC) viral-induced diabetes mellitus. Beta cells from susceptible mice that were infected in vivo with EMC virus showed higher viral titers, more severe degranulation, and lower concentrations of immunoreactive insulin than beta cells from resistant mice. Immunofluorescence and infectious center assays revealed that pancreas from susceptible mice contained at least 10 times more infected cells than pancreas from resistant mice. Beta cell cultures prepared from susceptible mice and infected in vitro also showed higher viral titers and more severe cytopathologic changes than beta cell cultures from resistant mice. In contrast to beta cell cultures, virus replicated equally well in primary embryo and kidney cell cultures from susceptible and resistant strains of mice. It is concluded that the development of EMC virus-induced diabetes is related to genetically determined host differences in the capacity of the virus to infect beta cells.


The effects of changing the phase and duration of sleep. The relative effects of extended sleep, reduced sleep, and shifts of habitual sleep time on subsequent performance and mood were studied. Ten healthy male university students who regularly sleep 9.5-10.5 hr were the subjects. Measurements were obtained from a 45-min auditory vigilance task, a 5-min experimenter-paced addition task and a mood adjective check list 30 min after awakening, at midday, and in the evening following five electroencephalographically recorded nights of sleep. The experimental treatments compromised at 9.5-10.5 hr habitual sleep condition and four conditions in which the regular sleep period was lengthened, reduced, delayed, and advanced by 3hr. Following each 3-hr altered condition of sleep there was an equivalent decline in vigilance performance and in subjective arousal as measured by the mood scales. Together with other recent evidence, the present results support the hypothesis that acute disruption of the 24-hr sleep-wakefulness cycle produces degradations in human performance largely independent of total sleep time.


Variation in the effects of antidiuretic hormone on the isolated skin of the toad, Scaphiopus couchi. The rate of active sodium transport as measured by short-circuit current across the isolated skin of the toad, Scaphiopus couchi, was elevated following vasopressin (0.2 units/ml) or arginine vasotocin (0.1 units/ml) treatment of skins from active animals at all times of the year tested. Skins from dormant animals showed no such elevation at any time of the year. The rate of active sodium transport was elevated following treatment with dibutyryl cyclic AMP (2.5mM) plus theophylline (10 mM) in all skins tested. The hydraulic conductivity of isolated skins from both active and dormant animals showed no significant change following treatment with vasopressin (0.2 units/ml) or arginine vasotocin (0.1 units/ml except on the first day following emergence from dormancy in the field. A correlation was, therefore, observed between the occurrence of a hydroosmotic response to antidiuretic hormones and the seasonal exposure of S. couchi to standing water. A small but significant elevation of hydraulic conductivity was observed across the skins of dormant toads following treatment with dibutyryl cyclic AMP (2.5 mM) plus theophylline (10 mM) whereas a substantial elevation was observed with the skins of active animals.


Forensic archaeology.

Forensic archaeology. This presentation attempts to demonstrate the value of the use of improved methods in the excavation of buried bodies involved in a criminal investigation. Care in the removal of the skeleton and proper recording will document the interrelationship of all objects found in and around the grave and can, to a certain extent, recreate the event. Carelessness will result in evidence being irretrievably lost. The crime scene investigator's problem, arachaeologically, includes the nature of the remains, dating the time of burial, and techniques of excavation. Archaeological recovery techniques should be adopted in homicide investigation involving buried bodies.


A comparison of molecular weights of foot-and-mouth disease virus RNA fragments determined from lengths and s-rates. A comparison was made of the calculated mol. wt. of RNA fragments from foot-and-mouth disease virus (FMDV) types A12 strain 119, C3 Resende and O1 Brugge. The mol. wt. were calculated by two methods: from the measurements of strand lengths in the electron micrographs and from the observed sedimentation rates (s-rates). RNA extracted from virus by dialysis against water usually had three to four prominent strands of different lengths. Mol. wt. calculated from s-rates (and converted to strand lengths) generally agreed with those measured on electron micrographs. Differences which occurred appeared to be attributable to further breakage during processing for electron microscopy. Major fragment strand lengths range from 0-65 to 2-45 mum. The modal lengths of RNA fragments were preparation-dependent, ranging from 1-25 to 1-95 mum for A12 119, 1-05 to 1-75 mum for C3 Resende, and 1-65 to 2-45  mum for O1 Brugge. There was one fragment length 1-95 mum common to all three types of FMDV RNA and several others which appear in at least two types. Calculations using the molar ratios of nucleotide residues in FMDV RNA, a mol. wt. of FMDV RNA of 2-65 X 10(6) and an internucleotide spacing of 3-17 A indicate that intact FMDV RNA should be 2-62 mum long and therefore would contain approx. 8270 nucleotides. The derived mathematical expression for the relationship between mol. wt. (M) and s-rate (S) giving the best fit for all data was M = 1725 S2-07, a result close to that derived by Spirin (1963) for other single-stranded RNAs.


Deoxyribonucleoside triphosphate pools in herpes simplex type 1 infected cells. Deoxyribonucleoside triphosphate pools were analysed in both exponentially growing and serum starved wild type BHK C13 cells and in a derivative of this cell line which lacks both thymidine kinase and deoxycytidine kinase activities, before and after infection with herpes simplex virus. Serum starved BHK cells had low levels of all four deoxyribonucleoside triphosphates. In exponentially growing cells all pools were expanded, the pool of dCTP being largest and dGTP the smallest. The dATP and dTTP pools were of intermediate sizes. In exponentially growing deoxypyrimidine kinase free cells the pools, with respect to level and distribution, were the same as those observed in wild type cells. After infection with herpes simplex virus there were marked changes in the levels of all deoxyribonucleoside triphosphate pools; the most predominant being a 25- to 50-fold expansion of dTTP pool. The pools of dCTP and dGTP also increased while the pool of dATP was very much reduced. These effects could be observed in both wild type and mutant cells.


Deoxyribonucleoside triphosphate pools in cells infected with deoxypyrimidine kinaseless herpes simplex virus. Deoxyribonucleoside triphosphate pools were analysed after infection of cells with mutant herpes simplex virus which lacks the ability to induce the enzyme deoxypyrimidine kinase. After infection of exponentially growing BHK C13 cells, an increase in all four dNTP pools was observed. However, after infection of cells which themselves cannot incorporate exogenous pyrimidine deoxynucleosides only the purine deoxynucleoside triphosphate pools increased in size. In a system which is non-permissive for virus infection, i.e. resting BHK C13 cells which have been infected with dPyK-, HSV-1, there is an increase in all dNTP pool sizes except for dTTP. A comparison of the changes in dNTP pool sizes after infection with either wild type of dPyK- mutant HSV suggests an important role for dTTP in the control of both the production of the other DNA precursors and of viral DNA synthesis.


The effect of tilorone hydrochloride on the growth of several animal viruses in tissue cultures. Tilorone hydrochloride, at a concentration of 10 mug/ml inhibits the growth of herpes simplex virus type 1 in BS-C-1 cells. The growth of vaccinia virus in BS-C-1 cells is partially inhibited; however, six viruses containing RNA, including four members of the togavirus group grown in chick fibroblasts, are not affected by the drug. The inhibition of the growth of herpes virus by tilorone hydrochloride is greater when the multiplicity of infection is lower than 1 p.f.u/cell and when the drug is added early in the course of infection.


Inhibition of protein synthesis in reticulocyte lysates by poliovirus. Addition of 1 X 10(10) p.f.u. purified poliovirus to 100 mul of a rabbit reticulocyte lysate protein synthesizing system causes a complete inhibition of initiation of protein synthesis. This inhibition is not due to the viral RNA nor to any contaminants of the preparation, but is most likely caused by the viral coat protein.


An improved assay for feline leukaemia virus pseudotypes of murine sarcoma virus. An assay is described for feline leukaemia virus pseudotypes of murine sarcoma virus which increased the virus titre by about 100-fold over conventional assays. The titre is independent of dilution and no secondary focus formation occurs. The assay may be used to study virus neutralization and to detect and type feline leukaemia virus in feline embryo cells by interference.


Mutants of herpes simplex virus types 1 and 2 that are resistant to phosphonoacetic acid induce altered DNA polymerase activities in infected cells. Three mutants of herpes simplex virus (HSV) have been isolated which form plaques in the presence of 100 mug/ml phosphonoacetic acid (PPA). All three mutants (3 from HSV-1 strain 17 syn+, 14 from HSV-1 strain 17 syn, and 19 from HSV-2) induce viral DNA synthesis and viral DNA polymerase activity, and these are much less sensitive to PPA than the wild-type virus. The results support the hypothesis that PPA interacts directly with the viral DNA polymerase protein, at least part of which is virus coded.


Characterization of the minor polypeptides in the foot-and-mouth disease particle. In addition to the four major polypeptides VP1 and VP4, foot-and-mouth disease virus particles contain two minor polypeptides, mol. wt. 40 X 10(3) (P40) and 52 X 10(3) (P52). Extensive purification procedures failed to remove these minor polypeptides from the virus particles. Polypeptide P40 co-electrophoresed in SDS-polyacrylamide gels with VP0, the probable precursor of VP2 and VP4 and was inaccessible to iodination in situ. The second minor polypeptide, P52, co-electrophoresed with the virus infection associated (VIA) antigen found in large amounts in harvests of the virus grown in BHK 21 cells. Polypeptide P52 was shown to be located near the surface of the virus particle by iodination experiments and by its removal on incubating the particles with trypsin or chymotrypsin. Pactamycin mapping showed that this polypeptide was not a precursor of the structural polypeptides. About one copy of P52 and 4 copies of P40 were found in the virus particles sedimenting at 146S. However a larger number of copies was found in those virus particles sedimenting faster than the 146S peak.


Effect of ammonium 5-tungsto-2-antimoniate on encephalomyocarditis and vesicular stomatitis virus infections in mice. Ammonium 5-tungsto-2-antimoniate (HPA 23) protected micr partially or completely against two strains of encephalomyocarditis (EMC) virus and one strain of vesicular stomatitis (VSV) virus. The best protective effect was obtained with EMC strain VR 129 and VSV when a single i.p. injection of HPA 23 was administered shortly before virus inoculation. Mice protected by HPA 23 against EMC strain VR129 had virus titres in the blood and brain similar to those in untreated mice. A synergism between interferon and HPA 23 was observed in mice infected with EMC VR129. Our results demonstrate the in vivo activity of HPA 23 against two lethal viral infections and suggest that, at least in mice infected with EMC, death may not be related solely to virus multiplication.


Herpesvirus proteins: DNA polymerase and pyrimidine deoxynucleoside kinase activities in temperature-sensitive mutants of herpes simplex virus type 2. Eleven temperature-sensitive mutants of herpes simplex virus type 2 strain HG52 were examined for ability to induce DNA polymerase activity in BHK 21/C13 cells. All mutants induced DNA polymerase at a permissive temperature, (31 degrees C) and all DNA-positive mutants at a non-permissive temperature (38 degrees C). Three DNA-negative mutants induced no DNA polymerase (ts 6, ts 9) or very little DNA polymerase (ts 11), at a non-permissive temperature, while ts 1, also DNA negative, induced a little more DNA polymerase than wild-type, often at both temperatures. The DNA polymerase induced by ts 6 at 31 degrees C was temperature-sensitive in vivo, but only slightly so in vitro. These results were confirmed immunologically and suggest that HSV-2 codes for at least part of a DNA polymerase activity, necessary for infection, and that full expression of this enzyme involves at least three viral genes.


Latent herpetic infections following experimental viraemia. The spectrum of tissues harbouring latent herpes simplex virus following intravenous inoculation of mice was defined by in vitro co-cultivation techniques. The virus could be detected in central and peripheral nervous systems (including adrenal medulla), but could not be found in any non-neural tissues. Spinal ganglia were the organs most commonly involved. The relationship of these findings to the natural history of herpetic infections is discussed.


Purification of equine herpesvirus type 1. A method for the purification of enveloped infectious equine herpesvirus type 1 (EHV-1) is presented. Virus from cell culture fluids harvested at 48 h post infection was concentrated by sedimentation and partially purified by differential precipitation with ammonium sulphate. The final steps of purification consisted of two cycles of flotation of virus in pre-formed CsCl density gradients. Yields of infectious virus were about 30% (18 to 44%) of that present in starting material. As judged by electron microscopy, mixed radioisotope labelling, and absence of phosphohydrolase, virus preparations possessed a high degree of purity. Sedimentation of EHV-1 into CsCl density gradients resulted in low recovery of infectious virus. Flotation of virus in CsCl gradients, however, was not deleterious to infectivity of viral preparations.


EEG findings, rapid ACTH test and autonomic nervous symptoms in patients with alopecia areata. Patients of multiple alopecia areata were examined in their EEG findings and rapid ACTH test and described the symptoms concerning the neuroautonomic system. Some abnormal EEG findings were seen in 75 cases out of the examined 105 patients. As to the rapid ACTH test, 10 cases out of 45 subjects were abnormal. A positive correlation was considered between abnormal EEG findings and abnormal value in the rapid ACTH test. It is speculated that the regulation centers in the brainstem and hypothalamus play a great role in pathophysiology of multiple areata.


Trigeminal neuropathy with nasal ulceration: report of two cases and one necropsy. Two cases are reported of progressive trigeminal neutopathy with nasal ulceration. One patient developed signs of spinal cord involvement 15 years after the onset of trigeminal symptoms and died after a total course of 21 years. Necropsy revealed an unusual trigeminospinal system degeneration with deposition of amyloid-like substances in the affected structures. The other patient is alive eight years after the onset of symptoms, the only indication of a lesion outside the trigeminal nerve being a patch of numbness in one leg.


The reinnervated motor unit in man. A single fibre EMG multielectrode investigation. Motor units in patients with motor neurone disease and polyneuropathy were studied with a new multielectrode technique allowing fibre mapping. In these disorders which are characterised by reinnervation the motor unit fibre distribution is changed from a normal scattered pattern to grouping often in clinically uninvolved muscles without definite EMG abnormalities. The changes found with this multielectrode technique in man are in accordance with those found in reinnervated motor units studied histochemically in the experimental animal.


"Satellite potentials" and the duration of motor unit potentials in normal, neuropathic and myopathic muscles. Time-locked activity in the EMG was studied in normal, neuropathic and myopathic muscles, using the averaging technique. Normal and neuropathic muscles seemed in most cases not to differ with regard to the frequency nor the quality of "satellite signals", i.e. small and short potentials joining the main component of the MUP with a constant interval, up to 26 ms in duration. In normal muscles 10% of the MUPs and in neuropathic muscles 12% had a satellite, which, when studied, showed no signs of blocking. However, in 2 patients with an apparent old spinal affection both an increased number and an increased latency of satellite signals were found. In the different myopathies studied 45% of the MUPs recorded consecutively were preceded or followed by an often increased number of tiny satellites. Sometimes the satellites joining the myopathic MUP formed a long (up to 60 ms) "tail" after the triggering component. On the basis of our results we conclude that in myopathies the total duration of MUPs is in certain cases pathologically incrased rather than decreased.


Double-tracer scintigraphy with 67Ga-citrate and 99mTc-sulfur colloid in the diagnosis of hepatic tumors. A method of liver scanning based on a subtraction technique with simultaneous use of two tracers, 67Ga-citrate and 99mTc-sulfur colloid, is described. The subtraction technique isolates radiogallium uptake by the space-occupying hepatic lesions by subtracting interference due to tracer uptake by healthy hepatic tissue. In 82 patients, the method yielded a correct result in 94.7% of the positive scans and in 97.7% of the negatives. Two false positives and one false negative occurred. Very poor results were obtained in the same patients using conventional technetium and gallium scans: only 20.7% of these interpretations were correct. The method proved very helpful in differentiating malignant from benign lesions.


Simple radiometric techniques for rapid detection of herpes simplex virus type 1 in WI-38 cell culture. Two radiometric techniques were developed for detecting the presence of herpes simplex virus type 1 in stationary monolayers of the diploid cell line WI-38. The time of detection was compared to that obtained from visual examination for cytopathic effects in the same cell line. Oxidation of 14C-1-glucose in infected and uninfected cells was determined by 14CO2 production, measured by an ionization chamber, and DNA synthesis was determined by 3H-thymidine incorporation, measured by scintillation counting. Compared to uninfected cells, infected cells showed a 23-26% reduction in 14C-1-glucose oxidation and a 355-498% increase in DNA synthesis 4-6 hr after infection. These changes in cellular metabolism were observed 14 hr before visible signs of cytopathic effects. The increase in DNA synthesis was completely inhibited by viral neutralization with herpes simplex antiserum. Increased DNA synthesis was observed 5 hr after infection with 10,000-3,200,000 TCID50 units of virus. These radiometric methods for the detection of viral effect on cellular metabolism are simple, fast, and objective.


Cyclic AMP content of gingival fluid in women taking oral contraceptives. Gingival fluid samples were collected from 12 healthy adult women who ranged in age from 19 to 34 years. Seven subjects with normal menstrual cycles who received no medications acted as controls while five subjects who had been using oral contraceptives for a minimum of 17 months constituted the experimental group. The women using birth control pills showed a 53% increase in the gingival fluid volume versus the controls. Radioimmunoassay revealed a mean concentration of cyclic adenosine monophosphate (cAMP) in the gingival fluid of 3.8-10(-6)M in the controls and 1.9-10(-6)M in subjects using oral contraceptives, although the total amount of cAMP present in the fluid from both groups was equal. These gingival fluid cAMP levels were a hundredfold higher than those measured in serum and saliva.


Formation of sulfhydryl groups in the walls of Eimeria stiedai and E. tenella oocysts subjected to in vitro excystation. Eimeria stiedai or Eimeria tenella oocysts were incubated in aqueous cysteine hydrochloride (cysHCl) under carbon dioxide (CO2), aqueous cysHCl under air, water under CO2 or water under air, and analyzed for sulfhydryl (-SH) groups. The cysHCl-CO2 treatment produced more -SH groups than the other treatments and was effective in allowing activation of intact and sodium hypochlorite (NaOCl)-treated E. stiedai oocysts as well as NaOCl-treated E. tenella oocysts. The CO2-cysHCl complex may act directly on the oocyst wall, especially in the micropylar region, to unmask lipid-shielded disulfide bridges, which are reduced to -SH groups. The reduction apparently disturbs the protein superstructure of the oocyst wall, promotes opening of the micropyle, and changes the impermeable state of the sporulated oocyst.