Inhibition of Epstein-Barr virus DNA synthesis and late gene expression by phosphonoacetic acid. Growth of lymphoblastoid cells (B95-8, Raji) is not inhibited by the presence of 0.4 mM phosphonoacetic acid. The synthesis of Epstein-Barr virus (EBV) in the producer line B95-8 is completely inhibited, as shown by the total inhibition of viral capsid antigen synthesis. Early viral antigens are made normally in the presence of phosphonoacetic acid, but EBV DNA synthesis is blocked in cells entering the productive cycle. Nonproducer cells in the population replicate the resident EBV DNA by a mechanism that is resistant to phosphonoacetic acid. These results are consistant with the hypotheses that EBV DNA is replicated by two mechanisms, one in the noninduced cell and a different mechanism in the producer cell, and that prior replication of EBV DNA, probably by the second mode, is a prerequisite for late gene expression.


Oligosaccharide moieties of the glycoprotein of vesicular stomatitis virus. Vesicular stomatitis virus contains a single structural glycoprotein whose carbohydrate sequences are probably specified by the host cell. The glycopeptides derived by Pronase digestion of the glycoprotein of vesicular stomatitis virus grown in HeLa cells have an average molecular weight of 1,800. There are multiple oligosaccharide chains on the vesicular stomatitis virus glycoprotein with protein-carbohydrate linkages that are cleaved only by strong alkali under reducing conditions, suggesting that they contain asparagine and N-acetylglucosamine. The oligosaccharide moieties, in addition, appear to be heterogeneous in sequence on the basis of their mobilities during electrophoresis and their sensitivities to cleavage by an endoglycosidase. The carbohydrate-peptide linkage region of the major class of oligosaccharides of the vesicular stomatitis virus glycoprotein has the proposed sequence: (see article).


Autologous immune responses to the major oncornavirus polypeptides in unmanipulated AKR/J mice. The autologous immune response of AKR/J mice to the structural proteins of murine leukemia virus (MuLV) was examined. Immunoglobulins from the renal glomeruli were chemically eluted, separated from antigens, recovered, and tested for immunological reactivity against MuLV structural proteins. Analyzing immune precipitates obtained after mixing radiolabeled Tween-disrupted MuLV preparations with eluates from AKR/J mice on sodium dodecyl sulfategel electrophoresis, we found evidence of antibodies to the major classes of MuLV structural components: gp70, gp45, p30, and one or more proteins in the 10,000- to 15,000-dalton class. Using rate zonal centrifugation we confirmed that the eluates from AKR/J glomeruli contained antibody(s) that bound specifically to p30. These results indicate that AKR/J mice spontaneously mount immune responses against the major oncornavirus polypeptide antigens.


Cellular protein synthesis shutoff by mengovirus: translation of nonviral and viral mRNA's in extracts from uninfected and infected Ehrlich ascites tumor cells. The mechanism whereby picornaviruses inhibit host protein synthesis while their own synthetic processes proceed unabated has remained elusive. One of our approaches to this problem was to study the ability of cell-free extracts derived from uninfected and mengovirus-infected Ehrlich ascites tumor cells to translate viral and nonviral mRNA's under various conditions of incubation. Our results indicate that viral messengers (from mengovirus and encephalomyocarditis virus) and cellular messengers [L cell and Ehrlich ascites tumor poly(A)-containing mRNA's, rabbit globin mRNA, and chicken embryo lens crystallin mRNA] are translated equally well in both extracts. We also examined the simultaneous translation of viral and nonviral mRNA's in extracts from uninfected Ehrlich ascites tumor cells. Our results indicate that under certain conditions mengovirus RNA can suppress completely the translation of globin mRNA. The significance of these results in terms of the shutoff of host protein synthesis is discussed.


Chemical modification of simian virus 40 DNA by reaction with a water-soluble carbodiimide. Superhelical simian virus 40 (SV40) DNA I can be modified with N-cyclohexyl-N'-beta-(4 methylmorpholinium)ethylcarbodiimide (CMC). The reaction produces an increase in the sedimentation velocity of DNA I from 21 to 22.5S and a decrease in its buoyant density in CsCl from 1.694 to 1.688. A comparable shift in buoyant density is observed in a saturated ethidium bromide-cesium chloride gradient where form II, which has been exposed to CMC, shows no shift. The CsCl-buoyant density data allows us to estimate that 108 mol of CMC are bound per mol of SV40 DNA I. In the subsequent paper an alternative procedure has been used to locate CMC sites, and the extent of the regions available to bind CMC have been measured.


Hin D restriction mapping of upaired regions in simian virus 40 superhelical DNA I: considerations regarding structure-function relationships. Superhelical simian virus 40 (SV40) DNA I was reacted with N-cyclohexyl-N'-beta-(4-methylmorpholinium)ethylcarbodiimide (CMC), and the location of CMC sites was mapped using the Hin D restriction endonuclease. The use of 14C-labeled CMC allows a quantitative analysis of the binding to the respective Hin D restriction endonuclease fragments. The percentage of reactivity was 6.54% for fragment A, 3.87% for fragment B, and 2.74% for fragment G. No CMC radioactivity was detected in other fragments. This reactivity is in agreement with the evaluation of binding by buoyant density measurements. The above fragments also contain the sites susceptible to S1 endonuclease action. This adds further support to the view that superhelical DNA can contain regions of localized interrupted secondary structure which may be capable of forming intrastrand hairpin structures if sequence relationships are favorable. The possible structure-function relationships for this model are discussed with the emphasis on transcription.


Structural and growth characteristics of infectious bursal disease virus. The infectious bursal disease virus is not enveloped and has a diameter of 60 nm and a density of about 1.32 g/ml. It contains two pieces of single-stranded RNA with molecular weights close to 2 X 10(6). The capsid is made up of four major polypeptides with molecular weights of 110,000, 50,000, 35,000, and 25,000. The virus replicates in chicken embryo fibroblasts rather than in epitheloid cells. After an eclipse period of 4 h, virus production reaches a maximum about 12 h later. The virus has no structural or biological similarities with defined avian reoviruses, and it cannot be classified in one of the established taxonomic groups.


Blocked 5' termini in brome mosaic virus RNA. All four components of brome mosaic virus RNA have m(7)G(5') ppp (5')Gp as their 5' terminus. The m(7)G can be removed by beta-elimination, resulting in the conversion to pppGp.


Proteins of Epstein-Barr Virus. II. Electrophoretic analysis of the polypeptides of the nucleocapsid and the glucosamine- and polysaccharide-containing components of enveloped virus. Two series of experiments were undertaken to identify the topological location of the structural polypeptides of Epstein-Barr virus. In the first series of experiments, nucleocapsids prepared by detergent treatment of enveloped virus with Nonidet P-40 and sodium deoxycholate were found to be composed of seven polypeptides, VP2, 6, 7.5, 24, 27, 31, ANd 33, which ranged in molecular weight from over 200 X 10(3) to 28 X 10(3). Nine other polypeptides, VP 4, 7, 8, 10, 15, 16, 23, 28, and 29, could be identified in preparations of Epstein-Barr virus nucleocapsids, but the relative amount of this second group of polypeptides was less in preparations of nucleocapsids than in preparations of enveloped virus. The incomplete removal of these polypeptides from enveloped virus by detergent treatment suggests that some of these polypeptides may be components of the envelope or tegument that lie in close proximity to the outer surface of the nucleocapsid In the second series of experiments periodic acid-Schiff-staining and glucosamine-containing components were identified with similar electrophoretic mobility to several of the polypeptides of enveloped virus (VP 5, 8, 9, 11, 12, 13, 14, 15, 16, 17, 28, and 29) that were completely or incompletely removed from purified virus preparations by detergent treatment. The similarity between the polypeptide composition of the nucleocapsids of Epstein-Barr virus and herpes simplex virus was in contrast to the dissimilarity between the nonnucleocapsid polypeptides of Epstein-Barr virus and herpes simplex virus.


Inhibition by zinc of rhinovirus protein cleavage: interaction of zinc with capsid polypeptides. Zinic ions rapidly inhibit virus production in HeLa cells infected with human rhinovirus type 1A and lead to the accumulation of human rhinovirus type 1A precursor polypeptides. The degree to which cleavage of these precursors is inhibited is directly dependent on the quantity of cell-associated zinc. Proteolysis resumes after the removal of zinc-containing medium, and the accumulated viral precursors are cleaved predominantly to stable virus polypeptides. The precursors stabilized at the lowest zinc levels are those that contain capsid protein sequences. Furthermore, added zinc is bound to human rhinovirus type 1A capsids and prevents them from forming crystals. Zinc-resistant mutants display antigenic alterations in coat proteins. These results suggest that zinc complexes with rhinovirus coat proteins and alters them so that they cannot function as substrates for proteases or as reactants in the assembly of the virus particles.


N-Tropic variants obtained after co-infection with N- and B-tropic murine leukemia viruses. Sc-1 cells co-infected with small XC plaque-forming N-tropic and large XC plaque-forming B-tropic murine leukemia viruses produced, in addition to parental types, progeny with the phenotype, large XC plaque morphology, and N-tropism. This phenotype remained stable through end point titration and plaque purification on NIH/3T3 cells and growth on BALB/3T3 cells. These N-tropic viruses (XLP-N virus) grow to unusually high titer and make very large XC plaques.


Cooperative transformation studies with temperature-sensitive mutants of Rous sarcoma virus. Stocks of Rous sarcoma virus Bryan strain were mutagenized using a bromodeoxyuridine treatment immediately after infection. Thirty temperature-sensitive (ts) mutants defective in transformation (td) were isolated by a replica plating technique. Twenty of these mutants were preliminarily characterized and found to be defective in late functions related to transformation. These mutants were used in experiments of cooperative transformation with four Prague strain td ts mutants of different co-transformation group. A small number of Bryan ts mutants were found to cooperate with some of the Prague mutants in transforming chicken embryo cells at the nonpermissive temperature. However, the amount of co-transformation observed was lower than that observed with cooperating Prague ts mutants and no clear-cut pattern of cotransformation was obtained in Prague and Bryan crosses. Indirect evidence indicates that cooperative transformation is the result of recombination events.


Host restriction of Friend Leukemia virus coat protein synthesis. Fv-1 gene-mediated host restriction of Friend leukemia virus replication was investigated in terms of coat protein synthesis. By using the assay of pseudotype formation with vesicular stomatitis virus. it was shown that under restricting growth conditions the availablity of leukemia virus coat protein for pseudotype formation was decreased. These studies appear to eliminate a pure assembly defect as the mechanism of Fv-1 host restriction.


Cell killing by simian virus 40: variation in the pattern of lysosomal enzyme release, cellular enzyme release, and cell death during productive infection of normal and simian virus 40-transformed simian cell lines. Simian virus 40 (SV40) growth on rhesus kidney cells and on the T-22 line of SV40-transformed green monkey kidney (GMK) cells is largely limited by the low plating efficiency of SV40 on these cells. In addition, a fraction of the rhesus kidney and T-22 cells are resistant to infection by SV40. Nevertheless, 72-h viral yields per infected rhesus kidney and T-22 cell are nearly equivalent to that obtained on normal GMK cells and are independent of the multiplicity of infection. Despite the production of high viral yields, infected rhesus kidney and T-22 cells are killed slowly by SV40. Monolayers of these cells are also refractory to plaque formation by SV40. SV40 induces the release of lysosomal N-acetyl-beta-glucosaminidase into the cytoplasmic fractions of rhesus kidney and T-22 cells to an extent equal to that observed during infection of rapidly killed normal GMK cells. In contrast, damage to the plasma membrane, as indicated by the release of the cellular enzymes lactic dehydrogenase and glutamic oxaloacetic transaminase into the overlay media, occurred to a much greater extent in the normal GMK cells than in the rhesus kidney or T-22 cells. Neither a lysosomal hydrolase mechanism nor viral release appear to be responsible for this phenomenon. The different rates and extents of the SV40 cytocidal process on these cells do not result from the differences in the viral plating efficiency on them.


Deoxyribonucleoside triphosphate pools in synchronized human cells infected with herpes simplex virus types 1 and 2. Deoxyribonucleoside triphosphate pools in uninfected and herpes simplex virus type 1 (HSV-1)- and HSV-2-infected KB cells were analyzed to determine whether ribonucleotide reductase functions in vivo in the presence and absence of thymidine (TdR). Previously we showed that HSV-2 replication was inhibited in KB cells blocked in their capacity to synthesize DNA by TdR. HSV-1 replication was not inhibited under these conditions. Both HSV-1 and HSV-2 induced an altered ribonucleotide reductase resistant to dTTP inhibition. Thus, the block to HSV-2 replication apparently was not at the level of reductase. However, the in vitro activity of the enzyme does not necessarily correspond to intracellular conditions. In TdR-blocked HSV-2-infected cells, we found that, while dTTP levels remained high, dCTP concentrations increased. In contrast, KB cells blocked by TdR showed increased dTTP but decreased dCTP levels. We conclude that the HSV-2 enzyme is functional in vivo and that TdR inhibits viral replication by a mechanism other than depletion of dCTP. Infection of KB cells with HSV-1 or HSV-2 altered both dATP and dGTP levels in the presence or absence of TdR. Inhibition of viral replication was not explained by changes in these pools. We suggest that, during infection, HSV-1 induces a virus function(s) not related to reductase which is resistant to TdR, whereas the corresponding HSV-2 function is sensitive. Our evidence shows that the TdR-sensitive function is not in the pathways leading to deoxyribonucleoside triphosphate and may occur at the level of DNA replication.


Early events after infection of Escherichia coli by bacteriophage T5. II. Control of the bacteriophage-induced 5'-nucleotidase activity. The control of activity of the bacteriophage T5-induced 5'-nucleotidase is dependent upon the amount of T5 parental DNA injected into the cell and expressed. When only the first-step transfer DNA is injected and expressed the amount of 5'-nucleotidase activity observed is two to three times the maximum amount observed after normal T5 infection, and inactivation of the enzyme does not occur. Enzyme inactivation occurs only after the remaining DNA is injected, but only limited expression of this DNA is required. The control of the nucleotidase inactivation process is similar to that for the repair of the nicks in parental DNA, and is probably mediated by a class IIa protein.


Antigenic activity of human urothelial tissue. Partially purified extracts from 71 human urothelial tumors and from 75 human urothelial non-tumor tissues were used as competing antigens in competition radioimmunoassay in an effort to detect the presence of 1 of the structural components of type C-ribonucleic and viruses, the p30 core protein. Testing of the tissue extracts was carried out by homologous and heterologous assay systems using 125I-labeled murine (Friend) and feline (Rickard) p30 antigens and anti-feline and anti-murine p30 antisera. The homologous assays were designed to detect the presence of p30 antigens in the human tissue extracts having similar characteristics to the p30 antigens of either the feline or murine type C viruses. The heterologous assay system was designed to detect interspecies antigenic determinants common to murine and feline viruses and also primate viruses. A competing antigen present in 28 per cent of the tissue extracts assayed demonstrated an antigenic protein that competed with the viral p30 protein used in the heterologous radioimmunoassay system and to a lesser extent in the homologous feline radioimmunoassay system. Antigenic competitor proteins were found in tumor and non-tumor tissue. These data suggest that some human urothelial tissues contain at least part of the genome of 1 or more type C viruses. Those tissues that are positive only in the heterologous assay system behave like viral p30 antigens already identified in tissues of several other primates. Those tissues that are positive in the heterologous assay system and the homologous system indicate that the p30 antigenic activity is closely related to that p30 present in feline C-type viruses. Further purification and more detailed characterization of those competing proteins are now under study.


Host range temperature-sensitive mutants of herpes simplex virus type 2. Two small-plaque mutants of herpes simplex virus type 2 (HSV-2) (strain 333), whose growth at 39 C was blocked in certain cell types (cell-dependent temperature sensitivity), were compared compared with parental virus in a number of biological assays. One mutant (no. 69) was found to produce a large number of morphologically normal, but noninfectious, particles; under nonpermissive conditions, these mutant particles were able to interfere with the replication of wild-type HSV-2. The other mutant (no. 74), which is known to belong to a different complementation group, appeared to direct little virus DNA synthesis, even at the permissive temperature. Progeny production and virus DNA synthesis in cells infected by mutant 74 were delayed in comparison with wild-type virus-infected cells. Both mutants were found to be more sensitive to UV irradiation than the parental virus; this was especially marked in the case of mutant 74. Moreover, this mutant was found to have a high transforming efficiency at much lower doses of irradiation than those needed to abolish the cytopathic effect of wildtype HSV-2.


Renal cyst puncture: a non-diagnostic procedure in the infant. A case of metastatic Wilms tumor in a newborn is presented. Cyst puncture was included in the preoperative evaluation and the merits of this procedure in infants are questioned. The differential diagnosis of intrarenal masses in the neonate is discussed.


The prevalence of antibody to infectious bovine rhinotracheitis virus in some game animals of East Africa. Antibody to infectious bovine rhinotracheitis virus was present in the sera of 10 of 25 species of game animals from Kenya, Tanzania and Uganda during 1960-1973. Prevalence of antibody varied considerably between, but not within, species.


An epornitic of duck plague on a Wisconsin game farm. In April, 1973, an acute disease with a high rate of mortality appeared in a flock of 233 ducks and geese at a private game farm. Most of the flock (220) were black ducks (Anas rubripes) and mortality was restricted to them. In May, the remaining live birds were placed in isolation but mortality continued in black ducks and occurred in other species. The overall rate of mortality for black ducks was 93% and the case fatality rate was 97%. No hemorrhaging from either the bill or vent was observed. The most commonly observed gross lesions were extensive fibrino-necrotic plaques covering the mucosal surface of the esophagus, posterior colon and cloaca. Petechial and ecchymotic hemorrhages on visceral organs, particularly the heart, were also common. Virus isolation was attempted from tissues of three black ducks. Duck plague virus was isolated from liver, kidney, spleen and intestine of each. Sixteen black ducks survived the outbreak. Seven of these birds had significant levels of neutralizing antibody to duck plague virus.


MBK neuropathy among spray painters.

MBK neuropathy among spray painters. It has been suggested that the solvent methyl N-butyl ketone (MBK) may cause peripheral neuropathy in humans. An investigation was undertaken after two cases of peripheral neuropathy among spray painters at one work site were reported to the National Institute for Occupational Safety and Health. Twenty-six painters were interviewed and examined. Two were found to have definite peripheral neuropathy and one had a probable case. Although one of these men had been exposed to lead in the past, there are strong reasons to believe that MBK was responsible for his neuropathy. There was nothing to suggest excessive lead absorption in the other two men.


Superior vena caval obstruction due to small-cell anaplastic lung carcinoma. Response to chemotherapy. Among 38 patients with bronchogenic carcinoma, small-cell type, eight patients had superior vena caval obstruction (SVCO) of recent onset. Seven of these patients were initially treated with combination chemotherapy (lomustine, cyclophosphamide, and methotrexate). One patient who had SVCO was treated initially by radiotherapy, with subsequent chemotherapy. In each patient, resolution of the syndrome was prompt, usually was complete by seven days, and was accompanied by objective tumor shrinkage. Symptoms of SVCO were transiently aggravated by the initiation of treatment in only one patient. In one individual treated by chemotherapy alone and in one treated by chemotherapy plus radiotherapy, SVCO recurred after initial control. Chemotherapy is an effective form of management of SVCO caused by small-cell carcinoma of the lung.


Orphan airlift. Enteric pathogens isolated from Vietnamese children immigrating to the United States. Isolation studies for bacterial and parasitic agents were carried out on stool specimens from Vietnamese infants at the time of their mass airlift to the United States. One or more bacterial pathogens were found in 49% of the 367 stool specimens cultured. The isolates included enteropathogenic Escherichia coli (161), Shigella (16), Salmonella (15), but no Salmonella typhi or Vibrio cholerae. Parasites identified in 88 stool specimens included Giardia lamblia (10), Ascaris lumbricoides (7), and Entamoeba histolytica (1). Transmission of agents to volunteers probably occurred, because 48% of 272 adults questioned had diarrhea shortly after caring for the children, and stool cultures from these adults resulted in the isolation of E coli (105), Salmonella (1), and Shigella (3).


The regulation of respiration of guinea pig taenia coli in high-K medium: the role of nicotinamide-adenine dinucleotide, adenosine diphosphate and Ca++. In an attempt to elucidate the regulation mechanism of respiration in the smooth muscle cell, we investigated the roles of nicotinamide-adenine dinucleotide (NAD), adenosine diphosphate (ADP) and Ca++ in the muscle respiration using the tissues and subcellular fractions from guinea pig taenia coli. The tension in the strips of taenia coli increased with a concomitant increase in O2 consumption in high-K medium (40 mM K) containing 2.5 mM Ca. 10(-3) M amytal and 10(-5)M ouabain decreased the high-K induced tension and O2 consumption of the muscle. 10(-4)M 2,4-dinitrophenol (DNP) relieved the decreased respiration induced by ouabain, but not that with amytal. From these data it is suggested that NADH-linked respiration plays an important role in the respiration of the muscle. Ca++ in concentrations ranging from 0.5 to 2.5 mM in the high-K medium resulted in an increase in tension and in O2 concumption progressively. In spectrophotometric observations of subcellular fractions of the taenia coli, ADP increased in absorbance change at 340 m mu. Such occurred in mitochondrial fractions and was initiated by the addition of NADH. Therefore it is deduced that the increase in ADP level of the cytoplasm is primarily due to a contraction triggered by Ca++ thus stimulating respiration. On the other hand, at 0.1 mM of Ca++ concentration, the muscle strip increased O2 consumption without tension development in high-K medium. In the spectrophotometric observations, Ca++ and Sr++ increased the absorbance change in the homogenate and in the mitochondrial fraction. Hence, it seems that one part of the Ca++ entering into the smooth muscle treated with the high-K increased O2 consumption in mitochondia independent of an increase in muscle tension. From these results it is concluded that NADH-linked respiration plays an important role in the smooth muscle respiration in high-K medium and that ADP and Ca++ also play a role in regulating respiration.


["Hypo-alpha-lipoproteinemia" (Tangier-disease) (author's transl)]. Report on a case of Tangier disease in a girl aged 4.11 m. Clinically the tonsils were hypertrophic and of a yellow color. Their stroma contained many cholesterinesters which produced the histologic picture of foam-cell xanthomatosis. The serum contained only traces of alpha-lipoprotein. The level of Cholesterin of the HDL fraction was reduced to one twelvth of the norm. The serum of her father also showed a clearly reduced content of HDL cholesterin. In the patient herself lipolytic enzymes like lecithin-cholesterin-acyltransferase, post-heparin plasma-lipoprotein lipase and hepatic triglycerid-lipase showed normal activity.


[Granulocyte dysfunction. I. Inborn defects (author's transl)]. The insight in the function and dysfunction of granulocytes lately arouses more and more interest. This report summarises our present knowledge. In the first of two chapters the authors review the molecular basis of granulocyte function and the inborn defects of chemotaxis, opsonisation, phagocytosis and intracellular killing of bacteria and fungi.


[Diurnal profiles of plasma aldosterone, cortisol, renin, angiotensinogen and angiotensinases in normal subjects (author's transl)]. Plasma cortisol and renin were estimated in 1 h intervals, plasma aldosterone, angiotensinogen and angiotensinases in 3 h intervals over periods of 24 h in six normal volunteers (age 20-26) under control conditions and subsequently under suppression of ACTH release by dexamethasone. Highest cortisol levels were found around 7 a.m., minimum levels between 9 p.m. and 1 a.m. Dexamethasone reduced cortisol to constantly low concentrations. Aldosterone was highest around 4 a.m. under control conditions and under dexamethasone, and showed lowest concentrations between 4 and 10 p.m. There were no significant differences between mean aldosterone concentrations at corresponding time points of the control and the dexamethasone period. Similar to aldosterone, renen showed peak values around 4 a.m. All mean values at corresponding time points between 7 a.m. and 11 p.m. and the 24 hour mean values of each subject were significantly increased under the influence of dexamethasone. No evidence could be achieved for the existence of circadian rhythms of angiotensinogen and angiotensinases. Dexamethasone did not cause significant changes of these parameters.


[Post-heparin-lipolytic activity in acute ethanol-induced hyperlipoproteinemia (author's transl)]. Post-heparin lipolytic-activity (PHLA) was studied in 10 healthy volunteers ingesting 0.5 g of ethanol/kg body weight initially and 0.1 g/kg body weight and hour over 5 hours. This dose led to enhancement in plasma triglycerides to about 170% of the pre-ethanol values. PHLA was determined before, 15 min, 1 and 5 hours after intake of the initial dose and showed no significant changes. These findings are compared with the results of earlier investigations. It is concluded that acute ethanol induced hyperlipoproteinemia in healthy man seems to be due mainly to enhanced hepatic synthesis of triglycerides and release of very low density lipoproteins and not to decrease catabolism of triglyceride rich lipoproteins.


Defects in granulocyte function in various chromosome abnormalities (Down's-, Edwards'-, Cri-du-chat syndrome). In five infants with autosomal aberrations and diminished resistance to infection (in spite of intact humoral and cellular immune mechanisms) several granulocyte functions (chemotaxis, phagocytosis, intracellular killing and metabolism of killing) were measured. A serum-dependent or a cell-dependent disturbance of phagocytosis of Candida albicans was found in two infants with cat-cry syndrome and one with trisomy 18. In one of these children there was an additional serum dependent defect of the killing of Candida albicans and of Staphylococcus aureus, serum levels of opsonins (IgG, IgM, CH50 and C3) being within normal range. An infant with trisomy 21 showed, in addition to a cellular defect of chemotaxis, a reduced cellular ability of the killing of Staphylococcus aureus and of Escherichia coli in autologous and AB-pool-serum. Phagocytosis of these bacteria remained normal.


The effect of transformation-defective avian oncornavirus mutants on tumor antigen expression. The recent isolation of conditional (temperature sensitive) and nonconditional transformation-defective mutants of avian sarcoma virus strains has facilitated the investigation of the effect of virus transformation on the cell's phenotype, e.g., with respect to morphology, growth pattern, or cell surface antigenicity. Special emphasis was laid on elucidating the correlation between transformed phenotype and tumor antigen expression. All of the tested nontransforming deletion mutants and the majority of the temperature-sensitive mutants were unable to induce tumor antigens in phenotypically untransformed cells. However, 3 temperature-sensitive mutants were found which were able to support the expression of tumor specific surface antigens even at restrictive temperature, when cells otherwise exhibited a normal phenotype. The theoretical and practical implications of this association between normal phenotype and tumor antigen expression are discussed.


Nonrandom distribution of receptors for melanocyte-stimulating hormone on the surface of mouse melanoma cells. An improved bubble method was developed for applying an ultrathin layer of nuclear track emulsion on the surface of cells labeled with I125-MSH. The autoradiographs of I125-MSH binding indicate a nonrandom distribution of receptors on the surface of mouse melanoma cells. It is suggested that MSH receptors are displayed in clusters previous to and independently of their exposure to the hormone.


Chromosomally depleted interspecific hybrid cell clones selected with cytotoxic antisera: utilization in the study of control of murine leukemia virus host-range. A chromosomally stable mouse-Chinese hamster hybrid cell line was subjected to five rounds of selection with cytotoxic antisera raised in rabbits against either the parental mouse 3T3 cells or the parental Chinese hamster Wg-1 cells. Routine karyological analysis of clones isolated at each stage of serum selection revealed that treatment with either serum resulted in a limited loss of chromosomes (compared to the untreated hybrid cell cultured in parallel) and that the pattern of chromosome loss could not be correlated with the particular antiserum used for selection. However, more detailed analysis with the SSC-formamide C-banding technique, which identifies chromosomes containing a mouse centromere region, demonstrated that while large-scale chromosome loss was not achieved as a result of antiserum selection, the limited loss of chromosomes did, in fact, reflect a specific depletion of chromosomes in response to treatment with cytotoxic antiserum. Specific chromosomal elimination was shown to occur as early as the first round of antiserum treatment. Antigenic analysis of the serum-selected clones revealed a quantitative decrease in the expression of the species-specific surface antigens selected against, but no qualitative loss of antigens was detected. The results suggest that treatment with cytotoxic antiserum may select for clones that have lost specific chromosomes bearing genes regulating the expression of species-specific surface antigens, rather than for those demonstrating large-scale depletion of chromosomes bearing the corresponding structural genes. Some of these chromosomally depleted hybrid cell clones have been used (along with pseudotype viruses containing the genome of vesicular stomatitis virus within the envelope of murine leukemia virus, VSV [MuLV]), to study the mechanisms regulating MuLV replication in Chinese hamster cells. The results indicate that the restriction of MuLV replication in Chinese hamster cells operates at two levels: (a) an inability to adsorb to or penetrate Chinese hamster cells; and (b) an additional intracellular block which is dominant in the mouse-Chinese hamster hybrid cell clones examined. This latter block is presently under study.


Structure and function of cholera toxin and hormone receptors. The enterotoxin from Vibrio cholerae is a protein of 100,000 mol wt which stimulates adenylate cyclase activity ubiquitously. The binding of biologically active 125I-labeled choleragen to cell membranes is of extraordinary affinity and specificity. The binding may be restricted to membrane-bound ganglioside GM1. This ganglioside can be inserted into membranes from exogenous sources, and the increased toxin binding in such cells can be reflected by an increased sensitivity to the biological effects of the toxin. Features of the toxin-activated adenylate cyclase, including conversion of the enzyne to a GTP-sensitive state, and the increased sensitivity of activation by hormones, suggest analogies between the basic mechanism of action of choleragen and the events following binding of hormones to their receptors. The action of the toxin is probably not mediated through intermediary cytoplasmic events, suggesting that its effects are entirely due to processes involving the plasma membrane. The kinetics of activation of adenylate cyclase in erythrocytes from various species as well as in rat adipocytes suggest a direct interaction between toxin and the cyclase enzyme which is difficult to reconcile with catalytic mechanisms of adenylate cyclase activation. Direct evidence for this can be obtained from the comigration of toxin radioactivity with adenylate cyclase activity when toxin-activated membranes are dissolved in detergents and chromatographed on gel filtration columns. Agarose derivatives containing the "active" subunit of the toxin can specifically absorb adenylate cyclase activity, and specific antibodies against the choleragen can be used for selective immunoprecipitation of adenylate cyclase activity from detergent-solubilized preparations of activated membranes. It is proposed that toxin action involves the initial formation of an inactive toxin-ganglioside complex which subsequently migrates and is somehow transformed into an active species which involves relocation within the two-dimensional structure of the membrane with direct perturbation of adenylate cyclase molecules (virtually irreversibly). These studies suggest new insights into the normal mechanisms by which hormone receptors modify membrane functions.


Infection in the burned upper extremity. Infection invariably accompanies thermal injury. The degree to which a patient is jeopardized by infection is related to the size and depth of the burn, the density and virulence of the microorganisms colonizing the burn wound, and the competence of his immune defenses. The aim of topical therapy is to limit microbial colonization of the burn wound to levels below those associated with invasive infection of the viable tissue beneath the eschar. The use of effective topical and systemic antimicrobial agents has been associated with the emergence of other bacterial, fungal, and viral infections and a delay in separation of the eschar, presumably caused by the suppression of bacterial débribement of the burn wound. The treatment of fractures in thermally injured patients may require compromise to permit optimal wound care and alertness toward the development of osteomyelitis. Because of the frequency of suppurative thrombophlebitis in burned patients, particular care is needed in the management of intravenous cannulae. The treatment of burns is largely the control of infection. Awareness of the septic complications of thermal injury and constant vigilance against them is critical in successful burn management.


Juvenile nasopharyngeal angiofibroma: radiographic aspects. Because of consistent and unique radiographic features of JNA, it is not necessary, nor is it advisable, to biopsy these tumors to establish a diagnosis. In addition, the carotid arteriograms show the major feeding vessels to the lesion and also completely delineate the periphery of the tumor, thus establishing operability versus non-operability. If a JNA is treated by radiotherapy, the changes effected can be followed by carotid arteriography.


Granular cell myoblastomas.

Granular cell myoblastomas. Granular cell myoblastoma is an uncommon benign tumor. The diagnosis is rarely suspected preoperatively, and the clinician is frequently baffled or frightened by an unfamiliar diagnosis. Twenty-eight patients with the diagnosis of granular cell myoblastoma presented for treatment at Richmond Eye and Ear Hospital and the Medical College of Virginia Hospitals between January 1, 1952, and December 31, 1974. The authors believe that this is one of the largest series of cases reported in the world literature. The tumor presents most often in the fourth decade of life and about equally in the sexes. Of interest is the nearly 5:1 predominance in the Negro race compared to the Caucasian race.


Nasopharyngeal angiofibroma.

Nasopharyngeal angiofibroma. Nasopharyngeal angiofibromas have traditionally been difficult tumors to manage. Their extreme vascularity has led to excessive bleeding during surgical removal, which often obscured the surgical field and contributed to high recurrence rates because of incomplete removal. High recurrence rates have also been due to failure to recognize the full size and extent of these tumors preoperatively by routine physical examination and X-ray techniques. A combined experience of the authors with over 45 cases from the University of Michigan and the University of California, San Francisco, is presented to review the problems in management of these tumors and to present techniques of diagnosis and management which have been used to overcome some of these problems. The most important advances in diagnosis have been the radiographic techniques of laminagraphy and carotid angiography. Previously unrecognized tumor extensions into the areas surrounding the nasopharynx have been quite clearly demonstrated by these techniques. Improved surgical treatment of these tumors by wider surgical exposure and removal, utilizing multiple approaches when necessary, and by better control of bleeding by ligation and embolization of the main arterial supply, has led to fewer recurrences.


Glomus tympanicum--excision by radical mastoidectomy exposure with autograft reconstruction. Wide surgical exposure, total tumor removal and anatomic and functional reconstruction are the goals of tumor management. These goals are met by radical mastoidectomy exposure for the removal of glomus tympanicum tumors and reconstruction by autograft replacement of the posterior osseous canal, and if the tympanic membrane, malleus and incus are removed for tumor exposure, autograft replacement may also be accomplished. The history, physical findings, X-rays and details of surgical management of two patients with glomus tympanicum are reviewed. Wide removal of the posterior osseous canal, just lateral to the VIIth nerve with total replacement of this segment gives excellent exposure of the middle ear space and affords total reconstruction of the posterior osseous canal.


[Value and relevance of metabolic function tests in the diagnosis of primary hyperparathyroidism (author's transl)]. Calcium, phosphate and alcaline phosphatase levels were determined in the serum of 29 patients with suspected primary hyperparathyroidism. Phosphate clearance according to Kyle, 24 hours urine hydroxyproline excretion during collagen free diet, the excretion of cAMP in the 24 h urine during calcium restricted diet were examined with regard to the diagnostic value and relevance as compared to the consumption of laboratory and staff time. The elevation of the serum calcium levels are not specific and only of minor diagnostic value. It has been found that the highest diagnostic value is given by the Kyle-test using 15 mg Ca ions/kg body weight. No false positive results were recorded. The excretion of hydroxyproline and calcium are only of limited value. Serum alcaline phosphatase and cAMP excretion have no diagnostic significance whereas concentration of serum phosphate may have some value.


[Interaction of spin-labeled analogues of vitamin B 6 with the active site of apotransaminase]. Spin-labeled analogues of vitamin B6: 2, 2, 6, 6-tetramethyl-N-oxylpiperydinyl-4-(5' phosphopyridoxyl)-amine (1) and 2, 2, 6, 6-tetramethyl-N-oxyl-piperydinyl-4-(pyridoxal-5')-phosphate (II) are synthesized. There analogues were shown to interact in the equimolar ratio with the active site of cytosol aspartate transaminase. It was proved by CD-titration of apotransaminase with I and II and by competition between the coenzyme and synthesized analogues. The free valency of spin-labeled coenzymes immediately disappears after interaction with the apoenzyme due to iminoxyl group reduction. The binding of I and II with the apoenzyme is accompanied by oxidation of one of the inner cysteine residues. The reactivation of the modified apoenzyme with PLP is not less than 65% of original transaminase activity. The analysis of space-filling atomic models of synthesized compounds allows to conclude that the distance between the centre of pyridine ring of the coenzyme and the modified thiol group is not more than 8 A.


[Study of the nitrogenase from Azotobacter vinelandii by the method of spin labels]. The interaction of nitrogenase with spin labels of four types have been studied. Conclusion about the presence of two SH-groups in the nitrogenase active site (one in Mo-Fe-protein and one in the Fe-protein) have been drawn from the correlation between the degree of inhibition of nitrogenfixing activity by the labels derived from p-Cl-Hg-benzoate and degree of binding of these labels to the nitrogenase molecule. Anaysis of EPR spectra of spin-labeled nitrogenase at 77 degrees K and at room temperature have shown that the labels bind to the free SH-groups and interact with iron containing center (ICC) of nitrogenase through the exchange mechanism. Distance between SH-group and ICC have been found to be 12 A. Spin labels derived from isocyanide have been bound directly to ICC in amount of 6--10 labels per one nitrogenase molecule. Due to the exchange interaction between these labels they give the singlet ESR spectra both at 77 degrees and at room temperature which is characteristic for the closely disposed labels. From this fact a conclusion have been drawn about the cluster structure of ICC. The labels derived from iodoacetamide ana maleimide bind SH- and NH2-groups of nitrogenase molecules. Analysis of temperature dependence of the effective rotational frequency of this labels have revealed a conformational transition in nitrogenase molecule at 19 degrees C, that has made it possible to explain the break in the Arrenius plots of enzyme activity.


[Effect of chain length of dialdehyde oligonucleotides on the rate of their interaction with the three-dimensional matrix of polyacrylamide gel]. The kinetics of the reaction of periodate oxidized oligonucleotides with polyacrylhydrazide gel was studied. The rate of the reaction is proportional to (see article), where M is the molecular weight of the oligonucleotide for the permeable gels. The rate of the reaction with given oligonucleotide decrease as the number of crosslinks in the gel matrix increase; a dramatic decrease of the rate occurs when  oligonucleotides become too large to penetrate into the matrix. The rate of the reactions with short oligonucleotides does not depend upon the viscosity of the medium. ESR method revealed a considerable decrease of the rotational mobility of oligonucleotides captured by the gel matrix. It has been shown  also that an increase of the number of cross-links leads to a decrease of the rotational mobility of the gel chains.


[The regulator effect of fructose-1,6-diphosphate and cyclic adenosine monophosphate on protein and RNA synthesis by isolated mitochondria]. Effects of fructoso-1,6-diphosphate (FDP) and cyclic 3',5'-adenosine monophosphate (cAMP) on various steps of protein biosynthesis in isolated rat liver mitochondria were investigated. It was shown that FDP repressed and cAMP depressed the incorporation of both 14C-amino acid and [3H]uridine into mitochondrial polysomes. Cyclic 2',3'-adenosine monophosphate, a physiologically inactive analog of cAMP, had no depressing effect on the polysomes formation in mitochondria. Effects of FDP and cAMP on the synthesis of mitochondrial RNA at different periods of incubation (5, 10, 30 min) were studied. It was found that FDP repressed the high molecular weight mitochondrial RNA biosynthesis and prevented the mRNA formation. cAMP derepressed the FDP effect. Rifampicin prevented the derepressing action of cAMP. The rate of protein synthesis in the translation system isolated from mitochondria was affected neither by FDP nor by cAMP. Authors concluded that in the mammalian mitochondria the repression of protein synthesis by a glycolytic metabolite (FDP) and its derepression by cAMP represented regulatory mechanism acting at the transcription level like catabolite repression-derepression in microorganisms.


[Evolution of the DNA structure: direction, mechanism, rate]. On the basis of the analysis of frequencies of occurence of pyrimidines of different length, the degree of clustering of DNA of a hundred species belonging to different taxons has been determined. A tendency towards increase in the index of DNA clustering was revealed in the sequence: bacteria, invertebrates, fishes, amphibians, reptiles, birds, mammals. A mechanism is postulated, according to which an increase in the degree of clustering of DNA in the process of progressive evolution of species may be due to accumulation of mutations, Pyr in equilibrium Pur transversions, resulting in an increase in the degree of asymmetry of the complementary chains of DNA. That this mechanism does exist is proved by a positive correlation between the degree of clustering of DNA and the degree of asymmetry of natural DNA chains. The mean frequency of mutation of vertebrates is about 4,6-10(-8) substitutions per nucleotide per year. Evolution of different groups of organisms may be accompanied with an increase in the rate of evolution of DNA structure. With the help of a special computer program, proceeding from the amino acid sequence of cytochromes c in 40 species belonging to different taxons, the degree of clustering of pyrimidines and the degree of asymmetry of complementary chains of DNA cistrons coding for cytochrome c was determined. A general tendency towards an increase in the mean values of the corresponding parametres of structure was found in the following: bacteria, invertebrates, fishes, amphibians, reptiles, birds and mammals. Thus, it was established that "neutral" amino acid substitutions in cytochromes are based on the selection of mutations leading to accumulation of pyrimidines in sense H-chain of DNA, and purines--in the corresponding mRNA. The frequency of mutation in cytochrome c of chordates is about 5,2-10(-8) of amino acid residues per year. It is assumed that the evolution modification of DNA structure may be due to increase in the disturbance stability of translation.


[Reversible bactericidal defect of leukocytes. Pseudomonas meningitis in a child with diabetes mellitus (author's transl)]. A 3-year-old boy with diabetes mellitus contracted meningitis caused by Pseudomonas aeruginosa. The disease lasted for several months in spite of antibiotic treatment. Diabetes mellitus was poorly controlled and hypoglycemia often occured. Immunoglobulin levels were normal. Investigations of leukocyte functions showed (no bactericidal activity), decreased phagocytic activity and very low NBT dye reduction by neutrophils during phagocytosis. Under the administration of polymixin, leukocyte transfusions and stabilization of glycemia the pathological clinical and laboratory findings disappeared within three weeks. Serial investigations of leukocyte function showed a gradual recovery of normal activity.


The syndromes of insulin resistance and acanthosis nigricans. Insulin-receptor disorders in man. In six patients with acanthosis nigricans variable degrees of glucose intolerance, hyperinsulinemia and marked resistance to exogenous insulin were found. Studies of insulin receptors on circulating monocytes suggest that the insulin resistance in these patients was due to a marked decrease in insulin binding to its membrane receptors. When these patients were fasted, there was a fall in plasma insulin but no increase in insulin binding, suggesting that the receptor defect was not secondary to the hyperinsulinemia. The clinical features shared by these cases and several similar ones previously reported may be divided into two unique clinical syndromes: Type A, a syndrome in younger females with signs of virilization or accelerated growth, in whom the receptor defect may be primary, and Type B, a syndrome in older females with signs of an immunologic disease, in whom circulating antibodies to the insulin receptor are found.


Human reovirus-like agent as the major pathogen associated with "winter" gastroenteritis in hospitalized infants and young children. We found a human reovirus-like agent in the stools of 42 per cent of 143 infants and young children hospitalized with acute gastroenteritis between January, 1974, and June, 1975. Half the patients studied by electron microscopy and serologic technics had evidence of infection with the agent. The infection had a seasonal pattern: 59 per cent of those admitted during the cooler months (November to April) shed the agent, with a peak of 78 per cent in December, 1974, and January, 1975, combined. None of the patients admitted during the warmer months (May to October) shed the agent. None of 275 Escherichia coli isolates from 32 patients with diarrhea produced heat-labile enterotoxin, whereas 17 of the 32 had evidence of infection with the reovirus-like agent. In addition, 14 of 40 parents of 37 patients with diarrhea associated with the reovirus-like agent were also infected, but most infectious were inapparent. This agent appears to be the major cause of diarrheal illness in the young during the cooler months.


Possible role for cyclic nucleotides and phosphorylated membrane proteins in postsynaptic actions of neurotransmitters. The postsynaptic actions of some neurotransmitters may be mediated through cyclic nucleotides and cyclic nucleotide-dependent phosphorylation of specific membrane proteins in postsynaptic cells. In addition to providing a molecular basis for the actions of several neurotransmitters and of certain drugs affecting behaviour, the model suggests a mechanism by which neurotransmitter signals may be converted into electrophysiological responses in postsynaptic cells.


Intracellular forms of Epstein-Barr virus DNA in human tumour cells in vivo. Tumour biopsies from Burkitt lymphoma patients, as well as human nasopharyngeal carcinoma cells growing in athymic mice, contain Epstein-Barr virus DNA as covalently closed circular DNA. In addition integrated viral DNA sequences seem to be present.


[Angiographic findings of tentorial branches of the internal carotid artery in gliomas infiltrating the tentorium (author's transl)]. The tentorial branches, originating from the cavernous portion of the internal carotid artery, showed pathological findings in two cases of brain tumors infiltrating the tentorium: a glioblastoma multiforme of the temporooccipital and basal regions and a medulloblastoma diffusely involving the cerebellar hemisphere, vermis brachium pontis and pons. The value and importance of the tentorial branches are emphasized in the diagnosis of glioma infiltrating the tentorium. The percentage of the visualization and the measurements of the visualized segment of these branches were described using selective internal carotid angiograms by the transfemoral catheter technic on 50 presumably normal adults. The percentage of the visualization of the tentorial branches was 24 percents and the average visualized segment measured 15 mm with a range of 5 to 30 mm on conventional angiograms.


[Endocrinological evaluation of sellar and suprasellar tumor cases (the 2nd report);--on postoperative pituitary function (author's transl)]. Fifty-six cases of sellar and suprasellar tumors were examined endocrinologically before and 3 weeks after surgery. 1) Hyporeactive cases of GH & ACTH were more frequently found in pituitary adenoma (100% and 23% respectively than in craniopharyngioma (86% and 14% respectively) before surgery. 2) GH secretion was impaired 3 weeks after surgery in all the cases. 3) ACTH secretion impaired preoperatively in 5 cases of pituitary adenoma improved in 3 cases 3 weeks after surgery. 4) Hyporeactivity of LH, FSH and TSH was found more frequently after surgery than before. 5) Almost all the cases of tuberculum sellae meningioma were endocrinologically normal before surgery. 6) ACTH, LH, FSH and prolactin secretion in cases of tuberculum sellae meningioma was not impaired after surgery, but TSH secretion in these cases became hyporeative in 4 of 5 cases after surgery.


Electrophysiologic changes in uremic neuropathy after successful renal transplantation. The pattern of electrophysiologic abnormalities in uremic neuropathy was studied in 17 patients near the time of successful renal transplantation and for an average of 2 years (4 to 68 months) afterward. Initially, sensory and motor conduction velocities were reduced, distal latencies were lengthened, and evoked action potentials were reduced or dispersed. These abnormalities were most marked in clinically severe neuropathies and were consistent with the combined presence of axonal degeneration and segmental demyelination. Following transplantation, there was a sustained improvement in motor and sensory conduction velocities but less consistent improvement in evoked action potentials. In severe neuropathies, action potentials tended to remain reduced or absent. The results suggest that the main reason for improvement in the neuropathy is segmental remyelination, some fibers remaining degenerate in severe neuropathies. In four patients, late failure occurred in the transplanted kidney, which was accompanied by recurrence of neuropathy. In each patient, a second successful transplant effected improvement for a second time.


Ovarian and adrenal steroid production in the postmenopausal woman. Relative contribution of the ovaries and adrenals to the pool of steroids in postmenopausal women is still the subject of much controversy. Estradiol, delta4-androstenedione, and testosterone were assayed in peripheral blood, adrenal, and ovarian vein blood of 11 postmenopausal patients. Intravenous administration of hCG resulted in increased androgen production by the ovaries but not estrogens while intravenous administration of adrenocorticotrophic hormone did not result in the enhancement of ovarian and adrenal estrogens.


Cellular metabolite distribution and the control of gluconeogenesis in the perfused isolated rat liver. Glucose production was measured in isolated rat livers perfused with 100 ml of blood-free recirculating medium. The gluconeogenic rate using L-alanine as substrate was only 55% of that obtained with L-lactate. The steady-state concentration of gluconeogenic and tricarboxylic acid cycle intermediates were measured in freeze clamped biopsies. Livers perfused with L-lactate displayed higher concentrations of malate, alpha-glycerophosphate and beta-hydroxybutyrate probably as a result of a higher state of reduction of the nicotinamide system. Hexose-phosphate intermediates were also increased when L-lactate was the substrate. Phosphoenolpyruvate and 3-phosphoglycerate were considerably elevated when L-alanine was the glucose precursor. Livers perfused with L-lactate displayed higher cytosolic concentration of all the tricarboxylic acid cycle intermediates except oxaloacetate while glutamate was slightly and aspartate considerably higher when alanine was the substrate. In the mitochondrial compartment the pattern of distribution tended to be the opposite; that is, livers perfused with L-lactate showed lower concentrations of all the intermediates except alpha-ketoglutarate. The mitochondrial: cytosolic metabolite gradients of all the intermediates whose distribution was studied were higher in livers perfused with L-alanine. The relevance of these findings to the observed differences in the gluconeogenic fluxes are discussed.


The relationship between the rate of rise of isometric tension and motor unit recruitment in a human forearm muscle. The recruitment properties of single motor units from the human extensor indicis muscle were investigated during voluntary isometric contractions of different rate of rise but equal amplitude. Both the electrical and the contractile events associated with recruitment were analyzed. The threshold force of recruitment (measured as the total muscle force at firing onset) decreased with increasing rate of rise of isometric tension. This was consistently found for all units. Differences between low and high threshold units indicating a preferential tonic or phasic mode of activation were not observed. The contractile events associated with recruitment were analyzed analoguously to the electrical events. For this purpose, muscle force was measured at the time of the first twitch as it was measured at the time of the first spike. This separate measurement of the electrical and mechanical recruitment of a unit is necessary, because during a change of muscle force, force is different at firing onset and during the subsequent twitch contraction. Muscle tension at the time of the peak of the first twitch contraction was calculated from measurements of the twitch concentration time of the single motor units. In contrast to firing onset, the peak of the first twitch of a motor unit occurs at approximately the same muscle tension no matter how fast the contraction is performed. This is the consequence of the result that the average decrease of the threshold force of recruitment at successively faster contraction has the same value as the corresponding increase of total muscle force during the mean contraction time of the motor units. On the basis of this precise matching between these two changes, the mechanical recruitment of motor units occurs at approximately the same force level irrespective of the rate of rise of tension.


The sensitivity of RNA polymerases I and II from Novikoff hepatoma (N1S1) cells to 3'-deoxyadenosine 5'-triphosphate. The synthesis of ribosomal precursor RNA in Novikoff hepatoma (N1S1) cells is very sensitive to cordycepin (3'-dA). The synthesis of hnRNA, however, is resistant to inhibition concentrations of 3'-dA that completely block the synthesis of 45S ribosomal RNA precursor. We have examined the RNA polymerases present in these cultured cells with regard to their sensitivity to cordycepin 5'-triphosphate (3'-dATP) in an effort to explain the differential inhibition of RNA synthesis observed in vivo. RNA polymerases I and II were characterized on the basis of their chromatographic behavior on DEAE-Sephadex, as well as the response of their enzymatic activities to ionic strength, the divalent metal ions Mn2+ and Mg2+, and the toxin alpha-amanitin. For both enzymes the inhibition of in vitro RNA synthesis by 3'-dATP was competitive for ATP. The km values for ATP and the K1 values for 3'-dATP for the two enzymes were quite similar. RNA polymerase II, the enzyme presumed responsible for hnRNA synthesis, was actually slightly more sensitive to 3'-dATP than RNA polymerase I, the enzyme presumed responsible for ribosomal precursor RNA synthesis. Similar data were obtained when the RNA polymerases were assayed in isolated nuclei. These results indicate that the differential inhibition of RNA synthesis caused by 3'-dA in vivo cannot be simply explained by differential sensitivity of RNA polymerases I and II to 3'-dATP.


A sedimentation study of the interaction of superhelical SV40 DNA with H1 histone. By moving boundary sedimentation it is shown that the interaction of H1 histone with superhelical circular SV40 DNA results in the formation of giant heterogeneous aggregates. The size of these aggregates grows with increasing H1 concentration. s20,w values of some 10 000 S were measured. As compared with open relaxed circular DNA a preferential interaction of superhelical DNA with H1 histone is observed, irrespective of the sign of the superhelical turns which was reversed by the addition to DNA of ethidium bromide. The addition to the H1 complexed aggregates of ethidium bromide effects a progressive breakdown of the aggregates. Furthermore, the superhelicity of DNA is not changed by the addition of small amounts of H1 histone.


[Leucaemoid reaction due to cytomegalovirus infection in a new born infant (author's transl)]. A newborn infant with low birth weight, jaundice, petechiae of the skin and enlargement of spleen and liver is presented. His general condition was severe and progressive aggravation of it was noticed. Bilirubin levels was up to 44 mg/100 ml, most of it indirect and severe anaemia was present. Furthermore leucaemoid reaction of the white cells was developed. Inclusion bodies in the urine and increased title of complement fixation antibodies in the peripheral blood were detected. The diagnosis of cytomegalovirus disease was established. The patient, when he was forty days old, seemed to be fairly well, but mental  retardation was noticed, which persisted at the fifth month of his age.


[Synovial sarcoma of the oral cavity (foramen caecum) Report of a case and review of the literature]. The twelft case of synovial sarcoma of the neck originated in the base of the tongue at the foramen caecum is reported. The patient, a 21 year old man developed a 7 cm. tumor after a 5 month period. A radical neck disection revealed metastases to lymph nodes, to spinal chain and residual tumor infiltrating the parotid gland. The subsequent X-ray studies showed metastases to lung. The patient is well 1 year after surgery. The pathogenesis of synovial sarcomas in regions so distant from normal synovial elements as the neck and other sites is not clear. In the present case and in those reported previously it is probable that the arise from synovial cell lining tendon sheaths or adventitious bursae in the neck.


[Serum glucose-6-phosphatase activity in thyrotoxicosis patients both fasting and following a sugar load]. A study was made of the activity of glucose-6-phosphatase in the blood serum of patients with thyrotoxicosis and in healthy persons and of its change after glucose loading. The activity of the enzyme on fasting stomach proved to be increased in the patients with throtoxicosis. The activity of the enzyme remained unchanged in these persons after glucose loading both during the hyperglycemic and the hypoglycemic phases of the glycemic curve; it remained high till the end of the observation period. In healthy persons, during the hypoglycemic phase of the glycemic curve, the activity of the enzyme was doubled, and at the height of hyperglycemia, after glucose loading- it was no different from the initial value. It is supposed that a high activity of the enzyme in the patients with thyrotoxicosis was associated with reduction in glycogen content in the tissues, along with activation of the glycogenolysis and gluconeongenesis processes.


[The relationship between the level of hypophyseal adrenocorticotropic activity and the character of the tissue distribution of exogenously labeled ACTH]. Experiments were conducted on rats--intact, adrenalectomized and dexamethasone-treated; a study was made of the dynamics of incorporation into the kidneys, liver, adrenal glands, the thyroid gland and elmination from the blood plasma of iodated corticotropine of the hypophysis (Hum-ACTHid-28, Richter). It was revealed that the character of incorporation and elimination of the exogenous labeled ACTH depended on the state of the adrenocorticotropic activity of the hypophysis of the recipient rats. The period of semielimination of the iodated corticotropin of the hypophysis from the blood plasma of intact rats constituted 11.2 minutes. The hormone was not included into the parenchyma of the adrenal cortex. The rate of excretion through the kidney and catabolism in the liver of labeled ACTH increased with elevation of the level of endogenous ACTH in the blood. The period of semiexcretion of ACTH-I131 from the blood plasma of adrenalectomized animals constuted 6.4 min. With decrease of the level of endogenous ACTH in the blood due to the block of the hypophyseal secretion with decamethasone elimination of exogenous labeled hormone from the circulation was markedly retarded T1/2 = 17 min). Under these conditions ACTH-I131 was incorporated into the tissue of the adrenal cortex for 9 min. A conclusions was drawn that the period of semielimination of the exogenous iodated ACTH could be used as a criterion for the assessment of the adrenocorticotropic activity of the hypophysis of the animals-recipients.


[The ultrastructure of pancreatic beta cells in dithizone diabetes and its prevention by sodium diethyldithiocarbamate]. Administration of a nondiabetogenic complex-former-sodium diethyldithiocarbamate (SDEDTC) to rabbits prevented the appearance in beta-cells of the insular apparatus of histological and ultramicroscopic changes with the subsequent administration of a potent diabetogenic agent-ditisone. SDEDTC blocked the insular zinc and prevented ditisone from forming in the islets a complex with this metal producing a destructive action of beta-cells.


[The effect of experimental thyrotoxicosis on the state of the hypothalamo-hypophyseo-adrenal system in rats]. Experiments were conducted on male albino rats (298); a study was made of the effect of experimental thyrotoxicosis of various severity on the state of the hypothalamo-hypophyseal-adrenal system. It was found that the hypothalamo-hypophyseal-adrenal system responded differently to the excess of thyroid hormone, depending on the strength and duration of action. Mild thyrotoxicosis was accompanied by its activation expressed in increased CRF of the hypothalamic extracts, a reduction of the ACTH in the hypophysis and a fall of ascorbic acid content in the adrenal glands. Severe and prolonged thyrotoxicosis was accompanied by a depression of the hypothalamo-hypophysio-adrenal system function. It was concluded that the action of thyroid hormones on the CRF secretion served as the leading mechanism of the mentioned reactions.


Solid tumor models for the assessment of different treatment modalities: systematics of response to radiotherapy and chemotherapy. The change of tumor volumes (efficiency) with local tumor radiation doses from 375R to 3750R and 5-fluorouracil (5-FUra) from 50 to 250 mg/kg was assessed in rats bearing hepatoma 3924A. The data were analyzed utilizing a chi2 technique which fits the logarithmic volume response to polynomials. This provided greater flexibility in selecting different mathematical forms, and allows more accurate description of tumor changes after treatment than the least squares analysis previously used. Quantitative information can be obtained on one tumor following treatment by this method. This information is more analogous to the management of a patient with a solid tumor. The results show a continuous increase in efficiency of radiation throughout the radiation dose range from 375R to 3750R. The efficiency of 5-FUra increased slightly but did not continue to increase with doses of 5-FUra higher than 150 mg/kg. This suggests that factors such as toxicity to the host may prevent further increases of the effectiveness of 5-FUra. The time of minimum tumor volume change after radiation was approximately 6 days and for 5-FUra, 6 days. The time for maximum tumor volume change for 5-FUra was 12 days. There was a slight trend upward for maximum growth for increasing radiation doses from 18 to 22 days. The time of occurrence of both minimum and maximum tumor volume change after treatment showed little relationship to increasing doses of radiation and 5-FUra. Parallel studies have shown that the maximum rate of tumor volume change occurs shortly after the recovery of the host from the effect of 5-FUra. It is feasible, therefore, to use chemotherapy alone or in combination with radiotherapy, and optimize the scheduling of these treatment modalities with recovery of the host from previous therapy.


[The functional activity of rat fetus adrenal cortex in monolayer tissue culture]. A study was made of the functional activity of the adrenal cortex in 15 1/2-, 18 1/2-, and 21 1/2-day rat fetuses and neonates. Adrenal tissue was incubated after its preliminary trypsinization at 37 degrees C for over 2 weeks with and without ACTH addition into the culture medium, in the amount of 0.1 U/ml. The corticosterone level was determined in the culture medium fluorimetrically. The activity of 3 beta-oxysteroid dehydrogenase was determined histochemically. Corticosterone was revealed in the culture medium. The activity of 3 beta-oxysteroid dehydrogenase was found in the cells of the cultivated adrenal glands. Addition of ACTH into the culture medium considerably increased the corticosterone level and elevated the activity of 3 beta-oxysteroid dehydrogenase. It can be considered on the basis of the aforesaid that cells of the primary culture of the adrenal cortex of rat fetuses aged 15 1/2-, 18 1/2-, and 21 1/2-days and of neonates could form, in the process of incubation, corticosterone from the endogenous substrates in the course of 11, 16, 8 and 13 cultivation days, respectively. The presence of ACTH in the culture medium for the adrenal glands of rat fetuses (of all the gestation periods under study) and of neonates intensified the steroidogenesis.


Thyrotropin-ganglioside interactions and their relationship to the structure and function of thyrotropin receptors. Gangliosides inhibit 125I-labeled thyrotropin binding to the thyrotropin receptors on bovine thyroid plasma membranes, on guinea pig retro-orbital tissue plasma membranes, and on human adipocyte membranes. This inhibition by gangliosides is critically altered by the number and location of the sialic acid residues within the ganglioside structure, the efficacy of inhibition having the following order: GD1b greater than GT1 greater than GM1 greater than GM2 = GM3 greater than GD1a. The inhibition results from the interaction of thyrotropin and gangliosides, rather than the interaction of membrane and gangliosides. Fluorescence studies show that the inhibition is associated with a distinct conformational change of the thyrotropin molecule and that the progression from a "noninhibitory conformation" to an "inhibitory conformation" parallels exactly the order of effectiveness in inhibiting 125I-labeled thyrotropin binding. The ganglioside inhibition of 125I-labeled thyrotropin binding appears to be hormonally specific in that it is not affected by albumin, glucagon, insulin, prolactin, follicle-stimulating hormone, growth hormone, or corticotropin. The possibility that a ganglioside or ganglioside-like structure is a component of the thyrotropin receptor is suggested by the finding that gangliosides more complex than N-acetylneuraminylgalactosylglucosylceramide are present in bovine thyroid membranes in much higher quantities than have been previously found in extraneural tissue. The finding that the B component of cholera toxin, which also interacts with gangliosides, has a peptide sequence in common with the beta subunit of thyrotropin, suggests that thyrotropin and cholera toxin may be analogous in their mode of action on the membrane.


Sex differences in binding of human growth hormone to isolated rat hepatocytes. Since liver is a target for growth hormone action, binding of 125I-labeled human growth hormone to enzymatically isolated rat hepatocytes was studied. Specific binding was shown with hepatocytes from both male and female animals. There was a single class of receptors for human growth hormone on cells from males (affinity constant, Ka = 1.16 x 10(9) liters/mole; sites per cell, q = 6200). In males, bovine growth hormone was almost as potent as human growth hormone in displacing bound 125I-labeled human growth hormone, while ovine prolactin was about 1000 times less potent. Cells from female rats bound more 125I-labeled human growth hormone than cells from males. The cells from females contained at least two classes of receptors for human growth hormone. The receptor of highest affinity had the same affinity for human growth hormone as the single receptor found in males (Ka = 0.96 x 10(9) liters/mole). However, there were three to four times as many of these receptors per cell in females (q = 21,000). In females, bovine growth hormone and ovine prolactin were both about 20 times less potent than human growth hormone. Treatment of male rats with estrone produced cells that show the same binding characteristics as females. These results indicate that human growth hormone binds to a somatogenic receptor in hepatocytes from male rats. In females and estrogen-treated males, the receptors that bind human growth hormone recognize lactogenic as well as somatogenic properties. This suggests that the lactogenic and growth-promoting effects of human growth hormone in the rat are mediated by different receptors.


Secretion of macrophage neutral proteinase is enhanced by colchicine. We have studied the effect of colchicine and related compounds on secretion of enzymes by thioglycollated-elicited mouse peritoneal macrophages in culture. Colchicine stimulated secretion of inducible neutral proteinase activities of elastase (EC 3.4.21.11), collagenase (EC 3.4.24.3), gelatinase (pepsin B; EC 3.4.23.2), and azocaseinase 2- to 6-fold for a period of several days, but inhibited the production and release of lysozyme (mucopeptide N-acetylmuramoylhydrolase; EC 3.2.1.17), a noninducible macrophage secretory product. Parallel changes were observed in cell morphology and secretion after treatment with colchicine, Colcemid, and vinblastine, but not with lumicolchicine, and these effects could be gradually reversed by withdrawal of colchicine. Cytochalasin B also stimulated secretion of elastase 2- to 3-fold but did not influence release of lysozyme. These results demonstrate that tubulin-binding drugs may have opposite effects in macrophages than those usually reported for other experimental systems and also provide evidence for the nonparallel discharge of different macrophage secretion products.


Androgen insensitivity in man: evidence for genetic heterogeneity. We have studied ten phenotypically similar patients with complete androgen insensitivity. All of the patients tested had significantly elevated serum luteinizing hormone and plasma androgens within or above the normal adult male range. On the basis of specific dihydrotestosterone binding by skin fibroblasts, we identified two subgroups. Six patients from five different families had undetectable dihydrotestosterone binding, while four patients from two families had normal binding activity. Our results indicate that within the clinical syndrome of androgen insensitivity there are at least two distinct genetic variants. These variants may result from allelic mutations of the same X-linked gene specifying the dihydrotestosterone receptor or, alternatively, from mutations of separate genes both being essential for androgen action in responsive cells.


Assignment of the AK1:Np:ABO linkage group to human chromosome 9. In man-Chinese hamster somatic cell hybrids the segregation patterns of the loci for 25 human enzyme markers and human chromosomes were studied. The results provide evidence for the localization of the gene for adenylate kinase-1 (AK1) on chromosome 9. Since the loci for the ABO blood group (ABO), nail-patella syndrome (Np), and AK1 are known to be linked in man, the ABO:Np:AK1 linkage group may be assigned to chromosome 9.


Genetic variation in serum low density lipoproteins and lipid levels in man. Combined data from 10 different populations indicate that the serum cholesterol and triglyceride levels are higher in Ag(x-) than in Ag(x+) individuals. Although the differences were relatively small, they became more pronounced with increasing age. The Ag(x) antigen, which is associated with the serum low density lipoprotein, is genetically controlled and the present study therefore has revealed a small but significant effect of a single autosomal locus on lipids implicated in the pathogenesis of atherosclerosis. No explanation of the higher cholesterol and triglyceride levels in Ag(x-) than in Ag(x+) persons is apparent.


Production of collagenase and prostaglandins by isolated adherent rheumatoid synovial cells. We have studied cells dispersed with proteolytic enzymes from rheumatoid arthritic synovectomy specimens to determine the cell type(s) responsible for joint destruction. Initially 10-50% of these cells adhered to culture dishes within 24 hr and were of two main types: small, round cells and larger, stellate cells. During 1-4 days of culture, 5-25% had Fc receptors and 25-50% showed brisk phagocytosis. Daily producition, per 10(6) cells of collagenase (EC 3.4.24.3) (after trypsin pretreatment) was up to 70 mug of collagen fibrils lysed per min at 37 degrees (70 units), of prostaglandin (PGE2), up to about 1200 ng, and of lysozyme, up to about 100 mug. Under identical conditions of assay, fibroblasts grown from explants of synovium produced no detectable collagenase or lysozyme, and PGE2 was only 2-4 ng. With the dispersed cell preparations, macrophage markers (Fc receptors and lysozyme) were undetectable after 4 days and PGE2 decreased rapidly after about 7 more days. However, collagenase production continued for 3-25 weeks, and in some cultures, after cell passage. At these later stages, large, slow-growing stellate cells were predominant and could phagocytose carbon particles if incubated for greater than 6-8 hr. Indomethacin (14 muM) inhibited PGE2 but stimulated collagenase production whereas dexamethasone (10 nM) inhibited both. Production of PGE2 and collagenase in large amounts in vitro by these cells suggests that they may be involved in joint destruction in vivo. The precise origin of these synovial cells and the mechanisms responsible for the sustained production of collagenase at a high rate remain unidentified.


Superinfection epithelial nasopharyngeal carcinoma cells with Epstein-Barr virus. Attempts were made to superinfect epithelial explant cell cultures prepared from nasopharyngeal carcinomas with Epstein-Barr virus. Virus-specific markers were observed in such cultures 3 days after superinfection. In addition, expression of Epstein-Barr virus early antigens was observed in epithelial cell explant cultures treated with iododeoxyuridine. The results suggest that epithelial cells of at least certain nasopharyngeal carcinomas possess the receptor for the Epstein-Barr virus and that the latent virus genome can be induced from epithelial cells prepared from such tumors.


Bidirectional regulation of lysosomal enzyme secretion and phagocytosis in human neutrophils by guanosine 3',5'-monophosphate and adenosine 3',5'-monophosphate. The biologic roles of guanosine 3',5'-monophosphate (cyclic GMP) and adenosine 3',5'-monophosphate (cyclic AMP) in the secretion of lysosomal enzymes from, and in phagocytosis by, human neurtrophils were studied. Contact between neurophils and particulate immunologic reactants results in both phagocytosis of the particles and secretion of lysosomal enzymes. These cellular events are accompanied by the accumulation of cyclic GMP and require the presence of extracellular caclium. Acetylcholine, pilocarpine, and cyclic GMP enhance, whereas epinephrine, cyclic AMP, and/or dibutyryl cyclic AMP inhibit, both phagocytosis and lysosomal enzyme secretion. The stimulatory action of cholinergic agents and the inhibitory action of epinephrine are accompanied by the accumulation of cyclic GMP and cyclic AMP, respectively, in human neutrophils. The data suggest that cyclic GMP mediates whereas cyclic AMP inhibits the major functions of human neutrophils. Moreover, by virtue of their effects of cyclic nucleotide accumulation, autonomic neurohormones are capable of modulating human neutrophil function.


Differences in simultaneous cylic adenosine monophosphate concentrations of various rat tissue after heroin administration. Opiate administration has profound effects on neurotransmitter metabolism and neuroendocrine functions. Since cAMP is an intracellular metabolite common to the actions of many of the involved compounds stimulating distinct and specific membrane receptors, cAMP concentrations representative of in vivo conditions in heroin-treated and control animals were determined. Rats were sacrificed by immersion in liquid nitrogen 30 min after heroin injection. Tissue samples, dissected without thawing, were assayed by radioimmunoassay to determine cAMP/mg of protein. Comparisons between treated animals and sham-injected controls revealed significant cAMP concentration increases in hypothalamus, cerebral cortex, cerebellum, adrenal medulla, adrenal cortex, and liver (P less than 0.0005). Cyclic AMP concentrations in heart, skeletal muscle, kidney, and fat were not significantly different from controls. The increased tissue cAMP concentrations associated with the altered physiology of heroin administration apparently represent a pattern of integrated responses to the combination of opiate receptor binding and effects on neurotransmitter metabolism and circulating hormones.


Streptozotocin treatment of streptozotocin-induced islet cell adenomas in rats. Hypoglycemic rats bearing insulin-secreting islet-cell adenomas produced by the combined action of streptozotocin and nicotinamide were treated with streptozotocin. Antitumor response was demonstrated by elevation of blood glucose, reduction in plasma and tumor IRI, and histopathologic changes in the beta-cell neoplasm. The rodent tumor model may serve as a predictive system for selection and investigation of mechanisms of action of future antitumor agents to be used in the treatment of malignant insulinoma in man.


Summer day camp for multihandicapped children. A six-week enriched sensorimotor, communication, and recreational summer day camp for twenty multihandicapped preschool children is described. The program, planned and supervised by therapists and mental  retardation counselors, provided training for students who worked as camp counselors. Each counselor was assigned to work with one child and family and was responsible for assessing, establishing goals, designing appropriate programs, attending the child throughout the day, and relating to the family. The camp provided a trial for use of the Functional Skills Profile (FSP), an assessment battery designed by physical and occupational therapists to measure performance changes in low functioning, developmentally handicapped persons in order to determine the value of therapy programs. The camp is evaluated in terms of improvement in the children, the effectiveness of the student training, and parent satisfaction.


REM sleep predicts subsequent food intake. REM sleep time in a 12 hr period was found to predict accurately food intake in the subsequent 12 hr period in undisturbed cats fed ad lib. In all but one of the cats, the correlation between REM sleep and subsequent food intake was negative. REM sleep was a better predictor of food intake than either waking, slow wave sleep or previous food intake. Cats were then fed only during the 12 hr day period. It was found that REM sleep at night, during which no food was available, no longer predicted food intake.


Enhancement of attention in man with ACTH/MSH 4-10. Normal men were infused for 4 hr with ACTH/MSH 4-10 or a control solution. Behavioral testing after the infusion indicated that subjects who received ACTH/MSH 4-10 were less anxious and had better visual memory than control subjects but the predominant effect of the heptapeptide was to increase visual attention. It was speculated that ACTH/MSH 4-10 may be uniquely coded for attentional functioning.


Induction of neurasthenic musculoskeletal pain syndrome by selective sleep stage deprivation. Two groups of young, healthy, nonathletic volunteers were subjected to selective sleep stage deprivation. Six subjects were deprived of stage 4 sleep and seven subjects of REM sleep. The stage 4 deprived group reported more musculoskeletal symptoms during the deprivation condition than did the REM deprived group. The stage 4 deprived group also showed a significant increase in muscle tenderness between the baseline and deprivation conditions and an altered pattern of overnight change in muscle tenderness in response to deprivation. The REM deprived group did not show either of these changes. These results are discussed in the light of the previously postulated relationship between NREM sleep disturbance and muscoloskeletal pain in patients with so-called "Fibrositis syndrome."


[Malignant synovialoma (author's transl)]. Based upon 351 cases of malignant synovialoma from the literature and 12 own observations clinical and roentgenographic findings, differential diagnosis, therapy, and prognosis are discussed. Because of early recurrencies and metastases early diagnosis is most important for therapeutic results. By means of radical surgery and subsequent intensive irradiation of the previous tumor site and the draining lymphatics a 5-years survival rate of 30-50% can be achieved.


Hepatomography: an experimental technique using an emulsifier. Hepatomography was performed by injecting emulsified Ethiodol into the hepatic artery of 9 beagles. The hepatogram was dense and remained long enough for diagnostic tomography. Contrast material was eliminated in part through the kidneys and disappeared from the liver within 24 hours after injection. The dogs were sacrificed 36 hours to 4 months after contrast injection. Longlasting ill effects were observed in only one dog which died of hepatitis. The death of this prompted the use of a more dilute emulsion which was beeter tolerated by all subsequent animals.


Aspirin as a quantitative acetylating reagent for the fatty acid oxygenase that forms prostaglandins. A selective acetylation of the prostaglandin-forming fatty acid oxygenase (part of the prostaglandin "synthetase" system) occurs with 100 muM concentrations of aspirin (acetylsalicylic acid). The amount of acetylation, measured by counting the [3H]acetyl-protein formed, was proportional to the amount of active, functional oxygenase in a sample. When samples were aged to allow spontaneous inactivation of the oxygenase, the amount of acetylation was proportional to the remaining measurable activity rather than the initial amount of oxygenase protein in the sample. Diethyl dithiocarbamate inhibited the oxygenase activity, but did not interfere with the subsequent acetylation by aspirin. Indomethacin, on the other hand, appeared to inactivate the oxygenase in a manner that interfered only partially with the action of aspirin as an acetylating reagent. The amount of acetylation appeared to be dependent upon the amount of native, undenatured enzyme. The results suggest that the acetylation may be dependent upon an essential functional group or conformation of groups in the catalytic peptide chain(s) that can be destroyed during spontaneous inactivation of the oxygenase, and altered by indomethacin.


Prostaglandin biosynthesis and stimulation of cyclic AMP in primary monolayer cultures of epithelial cells from mouse mammary gland. Cyclic AMP levels in primary monolayer cultures of epithelial cells prepared from mid-pregnant mice are stimulated by prostaglandin E1 and E2. Prostaglandin F1alpha and F2alpha have only a slight effect upon cyclic AMP levels. In the absence of phosphodiesterase inhibitors the rise in cyclic AMP produced by PGE1 is only transient and the levels return to normal within 30 minutes. High concentrations (16 mM) of theophylline are needed to prevent this decline, suggesting that the phosphodiesterase activity of epithelial cells in culture is high. However, theophylline alone produced only a small increase in basal cyclic AMP levels even over a 2-hour period indicating that basal cyclic AMP is turned over more slowly than cyclic AMP produced in response to stimulation with PGE1. Both PGE and PGF synthesis were  monitored using radioimmunoassay procedures previously reported. The observed levels were found to decrease as cell density increased and were sensitive to the addition of agents such as collagen and naproxen.


Heart sarcolemma as a dynamic excitable membrane. Sarcolemma consists of plasma and basement membranes and constitutes the real permeability barrier to the heart cell. It is considered to provide high electrical resistance and capacitance to heart cell and its properties are essentially similar to those of the other excitable membranes. Methods are now available for isolating heart sarcolemma with high specific activities of adenylate cyclase, (Na(+)-K(+) ATPase, Ca(++) ATPase, and Mg(++) APTase. These enzymes are considered to play an important role in heart function by regulating ion movements across sarcolemma as well as by providing signals for various metabolic processes. Sarcolemma possesses different hormone and drug receptors and any alteration in its composition could result in abnormal responses of the myocardium. We believe that heart failure is associated with sarcolemmal defects which can be detected by monitoring the activities of different membrane-bound enzymes and other related processes.


Biochemical properties of cardiac sarcolemma: adenylate cyclase and (Na++K+)-activated ATPase. Morphologically intact plasma membranes from guinea pig ventricles were obtained by exposing isolated cell segments to osmotic shock, followed by extraction of actomyosin in 1 M KC1. These preparations contained approximately 1/6 of the protein and 5-10 percent of the mitochondrial markers present in the original cell preparation. Both adenylate cyclase and (Na++K+)-activated ATPase activities were enriched 3-4 fold. The receptor for epinephrine stimulation of adenylate cyclase was retained. The "basal" ATPase activity of 5-6 mumoles of Pi/mg/hr, measured in 120 mM NaC1 or KC1, was approximately doubled in 100 mM NaC1+20 mM KC1. This increment, the (Na++K+)-activated ATPase, was abolished by 10(-5) M ouabain, the Ki for ouabain being approximately 3x10(-7) M. Adenylate cyclase, which had a basal activity of approximately 0.33 nmole of cyclic AMP produced/min/mg of protein, was significantly stimulated by both l-epinephrine and NaF. Half-maximal stimulation was seen at approximately 5x10(-6) M l-epinephrine. Increasing Ca2+ in the range between 10(-7) and 10(-3) M inhibited basal, l-epinephrine-, and NaF-stimulated adenylate cyclase activities. Basal rates of cyclic AMP production were more sensitive to Ca2+ than was l-epinephrine-stimulated adenylate cyclase activity, so that l-epinephrine stimulation was increased from approximately 60 percent in 0.5 mM ethylene glycol bis (beta-aminoethyl ether) N,N'-tetraacetic acid to approximately 150 percent in 10(-7)M Ca2+ and 400 percent in 10(-5) M Ca2+. The inhibitory effect of Ca2+ on adenylate cyclase activity may represent a negative feedback mechanism by which eelevation of intracellular Ca2+ concentration lowers cellular levels of cyclic AMP and thus reduces Ca2+ influx into the myocardium.


Adenylate cyclase and catecholamine binding in plasma membrane-enriched preparations of cardiac and skeletal muscle. Binding of [3H] epinephrine to plasma membrane-enriched fractions from guinea pig heart and rabbit skeletal muscle was investigated using the micropore filtration technique. [3H] Epinephrine and [3H] norepinephrine were found to be degraded rapidly in aqueous buffer at pH 7.6 and 37 degrees C. Deterioration of the compounds could be prevented by low concentrations of dithiothreitol. Binding of [3H] epinephrine to both membrane preparations was a slow process requiring 60 min to approach equilibrium in the case of cardiac membranes at 37 degrees C, and 20 min for skeletal muscle membranes at 0 degrees C. Binding was antagonized by the unlabeled beta agonists, isopropylnorepinephrine, epinephrine, and nor epinephrine; each was equipotent, in contrast to their different potency is eliciting beta-adrenergic responses and in stimulating adenylate cyclase. A variety of catechol compounds were as effective as antagonists of binding as the catecholamines. Methylation of one ring hydroxyl abolished inhibitory activity. The beta-adrenergic antagonists, propranolol, pronethalol, and dichlorisoproterenol, were not effective in inhibiting binding to either membrane preparation. D-Norepinephrine and L-norepinephrine were equieffective in antagonizing binding of [3H] norepinephrine to skeletal muscle membranes, suggesting that binding was not stereospecific. These and other data led to the conclusion that binding of labeled catecholamine to isolated tissue membranes using the micropore filtration technique probably does not reflect interaction with the specific beta-adrenergic recptor, but more likely reflects a less specific binding of compounds with one or more hydroxyl groups on a ring.


Adenylate cyclase, cyclic nucleotide phosphodiesterase, and norepinephrine binding in rat heart membranes. The method of Kidwai et al. (1971. Biochem. Biophys. Res. Commun. 45:901) offers a rapid and simple technique for the preparation of membrane fractions from rat heart, using sucrose density centrifugation of a 100,000x g pellet. We have investigated the distribution of adenylate cyclase, cyclic AMP phosphodiesterase, and norepinephrine binding activity in these fractions. Specific activity of adenylate cyclase was high in the plasma membrane (PM) and sarcoplasmic reticulum, as well as the nuclear (N) fractions, but 80-90 percent of the total activity was found in the N fraction. Epinephrine stimulation of adenylate cyclase was present in all fractions but did not exceed 25 percent of basal activity. The activity in the mitochondrial fraction was low and insensitive to epinephrine. About 10 percent of phosphodiesterase activity was found in the 100,000 x g pellet. After density gradient centrifugation, 70 percent of this portion was recovered in the N fraction, with the highest specific activity present in the PM fraction. Binding of [3H] norepinephrine was measured by a membrane filtration technique. Binding activity was found in all fractions, and it paralleled roughly the distribution of adenylate cyclase. However, only about 25-30 percent of the binding was blocked by propranolol, except in the PM fraction where binding was not prevented by this drug. Further, a comparison of adenylate cyclase activities with norepinephrine binding yields a turnover number for the enzyme of the order of 10(-2) sec-1, assuming a 1:1 relationship between beta-adrenergic recptor and adenylate cyclase. Since this value seems unrealistic, we suspect that either the binding activity measured is unrelated to the beta-adrenergic receptor or the receptor to cyclase ratio is considerably larger than unity.


Influence of thyroid state on the specific binding of noradrenaline to a cardiac particulate fraction and on catecholamine-sensitive cardiac adenylate cyclase activity. Specific binding of [3H] noradrenaline, possibly to beta-adrenoreceptors, and adrenaline-sensitive adenylate cyclase activity were measured in particulate preparations from the myocardium of hypothyroid, euthyroid, and hyperthyroid rats. Noradrenaline affinity to the binding sites was decreased and the dose-response curve for the stimulation of adenylate cyclase activity by adrenaline was shifted to the right in hypothyroidism. The opposite changes (an increase in noradrenaline affinity and a shift in the adrenaline-adenylate cyclase dose-response curve to the left) were associated with the hyperthyroid state. These findings correlate well with the result of numerous studies according to which thyroid hormone increases and thyroid hormone deprivation decreases the sensitivity of the myocardium to cardiac sympathetic stimulation and circulating catecholamines.