Inhibition of cell-mediated cytotoxicity to chemically induced rat tumours by soluble tumour and embryo cell extracts.

When lymph-node and spleen cells from rats bearing or immunized against aminoazo dye-induced rat hepatomas or 3-methylcholanthrene-induced rat sarcomas were pretreated with 3 M KCl extracts of these tumors, their cytotoxicity for cultured cells from the corresponding tumour was specificially inhibited. In cross-test combinations of target tumour and tumour-bearer effector-cell donor, tumour extracts only inhibited cytotoxic responses when they were derived from the tumour homologous to the target-cell and not when the extract was from the tumour borne by the effector-cell donor. One component of the tumour-bearer cytotoxic response was shown to be directed against tumour-associated embryonic antigens since soluble extracts of embryos or tumour extracts containing embryonic antigens inhibited tumour-bearer cell-mediated cytotoxicity in both specific and cross-test combinations of target tumour and effector cells. These findings are discussed in relation to tumour antigen expression and the immune responses that these tumour-associated antigens may evoke in tumour-bearing rats.


A continuous tumor-cell line from a human lung carcinoma with properties of type II alveolar epithelial cells.

The A549 tumor-cell line, initiated from a human alveolar cell carcinoma, has been continuously propagated in vitro for more than 3 years (more than 1,000 cell generations). These cells have a human karyotype and appear to have been derived from a single parent cell. All A549 cells examined by electron microscopy at both early and late passage levels contain multilamellar cytoplasmic inclusion bodies typical of those found in type II alveolar epithelial cells of the lung. At early and late passage levels, the cells synthesize lecithin with a high percentage of disaturated fatty acids utilizing the cytidine diphosphocholine pathway; such a pattern of phospholipid synthesis is expected for cells believed to be responsible for pulmonary surfactant synthesis. The A549 cell line should permit in vitro analysis of human surfactant synthesis and secretion and possibly provide a source of human surfactant for therapeutic intervention in pulmonary disease states characterized by surfactant deficiency.


Comparative studies of Epstein-Barr virus strains from Ghana and the United States.

A high incidence of oropharyngeal excretion of Epstein-Barr virus (EBV) has been observed in African children with Burkitt's lymphoma (BL) (48%) and matched controls (45%). This compares with an incidence of 77% in American patients with infectious mononucleosis (IM) and 13% in age-matched controls. Cross-neutralization tests between EBV strains derived from BL and IM patients and their sera failed to detect differences in the major neutralizing antigenic components. Cord-blood lymphocytes transformed by American EBV expressed only early viral functions (EBV nuclear and soluble complement-fixing antigens) and produced no detectable transforming activity. By contrast, cord-blood lymphocytes transformed by African EBV strains contained 0.2-0.3% of cells with EBV capsid and early antigen and produced EBV with transforming activity. These cells contained twice as many copies of EBV homologous DNA as the cells transformed by American EBV strains.


Humoral immune responses of cats to mammalian type-C virus p30s.

Natural and experimental cat sera were tested in radioimmune precipitation assays vs purified p30s from FeLV, RD114 and MuLV. Antibodies with specificity for FeLV p30 comparable to hyperimmune sera from heterologous species but of low titer were found in a high percentage of normal cats from households with a high incidence of FeLV and neoplasia. Sera from cats with neoplasms were generally negative. Cats immunized with FeLV gave low-level immune response, also of the same general specificity as heterologous hyperimmune sera. Cat sera do not normally show antibody to RD114 p30 although two immunized weanling cats produced low titered but highly specific p30 antibody. Thus, for both classes of feline type-C virus p30s, there is an evident capability of the cat to mount an immune response to natural or experimental exposure to the respective proteins. The magnitude of the response is between 100 and 1,000 fold below that seen in heterologous species. In contrast, cats immunized with MuLV p30 gave immune responses comparable to those seen in guinea-pigs, rabbits and goats. Several very old cats with carcinoma had antibody which preferentially precipitated MuVL p30. A competition assay using one such serum and labelled MuVL p30 was inhibited by FeVL, RD114, and MuLV p30s. This indicates that the assay is "interspecies" in nature. Among the possible explanations of this reaction category is that it represents antibody to the p30 of an as yet undefined class of feline type-C virus.


Immunity to antigens associated with a cell line derived from nasopharyngeal cancer (NPC) in non-Chinese NPC patients.

Delayed hypersensibility to antigens derived from four lymphoid cell lines was measured in 27 non-Chinese patients with nasopharyngeal cancer (NPC) and 63 non-NPC cancer patients. Of the NPC patients, 17/27 (63%) had a positive skin test response to antigens derived from HKLY-28, a lymphoid cell line which was developed from an NPC biopsy. Only 10/51 (20%) and 1/13 (8%) patients with solid tumors and hematopoietic malignancies, respectively, had positive skin test responses to HKLY-28. Positive skin tests were found less frequently when extracts from cell lines derived from normal individuals or lymphoma patients were utilized, although NPC patients were more reactive to two of the non-NPC derived cell lines than the controls. The NPC patients in this study also had significantly elevated antibody titers to the Epstein-Barr virus (EBV), capsid antigen (VCA) and early antigen (EA). Titers were highest in the patients with more anaplastic nasopharyngeal carcinomas. The skin test and serologic data are consistent with studies in Chinese patients, indicating that NPC in non-Chinese and Chinese patients is biologically similar.


Establishment of EBNA-expressing cell lines by infection of Epstein-Barr virus (EBV)-genome-negative human lymphoma cells with different EBV strains.

Cells of two EBNA (Epstein-Barr virus nuclear antigen)-negative human lymphoma cell lines, BJAB and RAMOS, were infected with two strains of Epstein-Barr virus (EBV). In two different experiments, B95-8 virus-infected BJAB cells revealed a gradually increasing number of EBNA-positive cells. Twenty weeks after infection almost 100% of the cell population expressed this antigen. In contrast, it has not so far been possible to convert RAMOS cells into an EBNA-positive cell line. The initial proportion of 35% EBNA-positive cells declined to about 10% 20 weeks after infection. The development of EBNA-positive multinuclear giant cells was a characteristic feature of infection with B95-8 virus. EA (early antigen) and VCA (virus capsid antigen) appeared in less than 0.1% of the cell population after induction with IUdR only. Infection of BJAB and RAMOS cells with P3HR-1 virus finally resulted in both cases in EBNA-positive lines. In contrast to B95-8 virus, the number of EBNA-positive lines. In contrast to B95-8 virus, the number of EBNA-positive cells remained below 1% during the first 6 to 8 weeks. A sudden increase occurred thereafter, bringing the number of EBNA-expressing cells to almost 100% within the following 4 weeks. During this period, BJAB but not RAMOS cells revealed a small number of EA- as well as VCA-positive cells (less than 0.1%). Thus, reinfection by spontaneously released virus may explain the sudden increase in EBNA-positive BJAB cells. Two distinct patterns of EBNA staining in P3HR-1 virus-infected cells were observed. They may suggest a genetic heterogeneity of this virus preparation.


Attempts to demonstrate virus-specific sequences in human tumors. IV. EB viral DNA in European Burkitt lymphoma and immunoblastic lymphadenopathy with excessive plasmacytosis.

Various biopsies from different European malignant lymphomas, two biopsies from nasopharyngeal carcinomas, and material from non-neoplastic lymph nodes were assayed for the presence of Epstein-Barr virus (EBV) DNA by nucleic acid hybridization. Reassociation kinetics of in vitro-labelled EBV DNA were studied in the presence of tumor DNA. The lymphomas tested included among others follicular lymphomas, germinocytomas, immunoblastic lymphomas and lymphoplasmacytoid immunocytomas. Epstein-Barr viral DNA was demonstrated within the two nasopharyngeal carcinoma biopsies as expected. A histologically typical Burkitt lymphoma as well as an immunoblastic lymphadenopathy with excessive plasmacytosis also contained EBV-DNA. The Burkitt biopsy revealed about 15 EBV genome equivalents per cell. Antibodies against EBV-specific antigens were highly elevated in the serum of this patient. The material of the patient with immunoblastic lymphadenopathy contained 2-3 EBV genome equivalents per cell.


Clonal transformation of human leukocytes by Epstein-Barr virus in soft agar.

The B95-8 strain of Epstein-Barr virus (EBV) induced colony formation of human umbilical cord-blood leukocytes in soft agar medium. One-hit response relationship between the number of colonies and the virus dose was observed with high dilutions of the virus preparation. However, there was a presumed cell-killing effect with low dilutions of virus. The colonies were similarly induced, but with a lower efficiency, in adult peripheral blood leukocyte cultures infected with the virus. The colony-forming activity of EBV was neutralized by anti-EBV-positive but not by negative human sera. The cells in colonies were capable of growing continuously and carried EBV-associated nuclear antigen. Thus, it was evident that the colony formation was caused by clonal transformation by EBV.


Lack of correlation between Marena tumor induction and expression of endogenous avian RNA tumor virus genome.

Infection with Marek's disease virus (MDV) and subsequent tumor development failed to induce the expression of endogenous avian RNA tumor virus genome in Line-15I and Line-7 chickens which lacked such expression at 1 day of age. Titers of avian leukosis virus (ALV) group-specific (gs) antigen detected by the COFAL test and expression of chick helper factor (chf) activity remained relatively constant in birds which expressed these genome functions at hatching time. Endogenous ALV belonging to the E-subgroup was isolated from two of 50 birds; both were controls not exposed to MDV. There was no correlation between expression of ALV genome (gs or chf) at 1 day of age and the rate of tumor development subsequent ot MDV infection. It was concluded that MDV infection can induce tumors without the participation of endogenous ALV genome.


Serum factor levels during the growth of rat hepatoma nodules in the lungs.

The levels of tumour-specific antigen, antibody and specific immune complexes in the sera of rats bearing lung nodules of a chemically induced rat hepatoma, produced by intravenous inoculation of viable tumour cells, have been examined and compared with the levels of serum factors in animals actively immunized by intravenous inoculation of mixed tumour cells and BCG. Animals inoculated with cells alone developed multiple lung nodules which slowly grew, until on day 24 the remaining animals had to be killed due to respiratory distress. In contrast, the animals receiving a mixed inoculum of BCG and tumour cells developed no visible lung nodules and were capable of rejecting a further challenge of tumour cells. Tumour-specific antigen could be detected in the sera of both groups of rats at different times. In actively immunized animals free antigen could be detected from day 3 to day 10 after inoculation of cells BCG whereas in animals receiving cells alone, free antigen could not be detected until day 14 and persisted until the termination of the experiment. Free tumour-specific antibody, however, was found in the sera of rats actively immunized with tumour cells and BCG from day 10 onwards, although in the other group of animals it could not be detected. Conversely, immune complexes of tumour-specific antigen and antibody could be detected from day 10 in rats receiving cells alone and only at day 10 in actively immunized rats. The relevance of these findings in relation to what is known about serum factor levels during tumour growth and regression is discussed.


Fibroblast surface antigen (SF): the external glycoprotein lost in proteolytic stimulation and maligant transfromation.

It was previously shown that the fibroblast surface antigen (SF antigen, SFA) is composed of polypeptides of high molecular weight 210,000 (SF210) and 145,000 (SF145) and that both of these decrease in quantity after transformation of the fibroblasts by Rous sarcoma virus (RSV). The present experiments show that SF210 is a glycoprotein. It is accessible to surface labelling by lactoperoxidase catalyzed iodination. The SF210 molecule is highly susceptible to trypsin on cell surface. Anti-SFA antibodies specifically precipitated the surface labelled polypeptide. The lactoperoxidase iodinated SF210 polypeptide was greatly reduced in cells transformed by RSV. It is concluded from these studies that the large external transformation sensitive (LETS) protein detected by other workers is the same molecule as SF210. Part of the label of surface iodinated fibroblasts did not enter the polyacrylamide gels. This high molecular weight material is also susceptible to trypsin treatment and decreases in quantity after transformation by RSV. The data suggest that it may be antigenically related to SF protein. Treatment of surface of 35S-methionine-labelled cultures with trypsin in concentrations able to initiate proliferation of density-inhibited cells rapidly released SF210 from fibroblast surface. A single high molecular weight polypeptide (mol. wt about 200,000, SF200) was detected in the culture medium. SF210 may thus be a major target molecule of trypsin action. Treatment of cultures with insulin that also stimulated the fibroblasts to initiate proliferation did not result in any detectable alteration in the external glycoprotein SF210. It is concluded that although release of SF210 may be a sufficient trigger to stimulate proliferation in stationary cells, this molecule appears not to be directly involved in initiation of fibroblast proliferation from the G1 (or G0) phase of the cell cycle.


Effect of age on activities of urea cycle enzymes in the bovine.

Eighteen Hereford cows averaging 429.6 +/- 14.5 kg in weight, ranging from 3.33 to 12.12 years of age and consuming 18.2 kg corn silage/animal/day were used to observe the effect of age on activities of the liver enzymes of urea synthesis. Liver samples were taken by aspiration biopsy and homogenized in water for enzyme assay. Plasma urea-nitrogen of all cows averaged 4.20 mg per 100 ml. The enzymes measured were: carbamyl phosphate synthetase (CPS), ornithine transcarbamylase (OTC), argininosuccinate synthetase (AS), argininosuccinase (AL) and arginase (A). Mean enzyme activities of the cows were: CPS 1.57, OTC 44.4, AS 0.61, AL 1.05 and A 203.9 mum product/mg protein/hr. Regression coefficients (b) of enzyme activity (units) on age (years) were 0.021 +/- .018, 0.713 +/- .379, 0.016 +/- .016, 0.032 +/- .014 and 6.73 +/- 5.09 units for the five enzymes, respectively. Age (3.3 to 12.1 years) did not significantly (P less than .05) affect activities of the urea cycle enzymes (per mg protein, per g liver or per W-75 kg/hr) or plasma urea-nitrogen levels in the adult Hereford female at the level of dietary protein fed in this study.


[The angioleiomyoma of the skin].

Twelve cases of angioleiomyoma are reported. These are benign, often painful tumors occurring chiefly on the lower extremities of middle-aged women. Clinical diagnosis is usually possible, and any remaining doubt can easily be resolved by histological examinations.


[The photodynamic effect in Herpes simplex: a review].

Photodynamic action is an extraordinarily potent physicochemical process which is dramatically lethal for herpesvirus and other organisms in vitro. Recent studies in animal models and man indicate that the process is effective against in vivo skin infections as well. Although a great deal is yet to be learned about the application of photodynamic action to clinical disease, current experience indicates that photodynamic action will find a permanent place in the physician's therapeutic armamentarium.


[Diagnostic errors and their reasons in tumors and preblastomatoses of the skin. Results of a statistical evaluation].

Clinical and histological findings and other available data of 6 521 skin excisates were compared with regard to differential diagnosis. It appears from our findings, that "naive" experience must be replaced by secured knowledge concerning frequencies of wrong diagnoses in definite situations, possible causes of errors, age-, sex-, and local distribution of different skin tumours. This knowledge may be a contribution to the improvement of clinical diagnoses.


Morphometric and enzyme histochemical behaviour of the kidney of young rats before and after unilateral nephrectomy.

Four week old male Wistar rats were unilaterally nephrectomized, and one, two, four, fourteen, and twenty-eight days after the operation the remaining kidneys were removed for morphological examination--including measurement of diameters of the tubules--and for histochemical investigation of enzymatic activities in the nephrons. The following enzymes were examined: succinate DH, lactate DH, alpha-glycerophosphate DH, isocitrate DH, glucose-6-phosphate DH, cytochrome oxidase, adenosine triphosphatase, alkaline and acid phosphatases. In comparison with kidneys of control animals of the same age and sex, the hypertrophy of the remaining kidney was found to be, even in these young rats, entirely due to enlargement of preexisting nephrons. The measurements have indicated that the hypertrophy was most distinct between the second and fourth day after uninephrectomy. Four weeks after the operation the diameters of proximal and distal tubules were about 21% and 18% respectively bigger in the uninephrectomized animals than in the corresponding controls. This increase in size was accompanied by some changes in intensity of enzymatic staining reactions; after an initial diminishment, a graduate increase in activity of almost all the observed tubular enzymes was found; this seems to be the expression of the functional compensation by the remaining kidney.


In vitro induction of tyrosinase activity in goldfish (Carassius auratus L.) integument by ACTH and dibutyryl-cAMP.

Tyrosinase was first detected in melanoblasts by the DOPA-oxidase reaction in the presence of catalase in explants of goldfish integument after 12 hr culture with either ACTH (1IU/ml) or DB-cAMP (0.1mM). Melanin did not appear in the new melanocytes until 24 hr. The data indicate that the release of cAMP within the melanoblast in response to ACTH treatment is rapid and the tyrosinase in the melanoblast is released from inhibition and/or activated at least 12 hr prior to melanization of premelanosomes.


The ultrastructural localisation of cadmium.

A modified technique for the ultrastructural localisation of heavy metals is described in this paper. The method involves precipitation of heavy metals as sulphides in the tissue by using (NH4)2 S after brief fixation in glutaraldehyde. The sulphides are, in the presence of a physical developer, then used to catalyse the reduction of silver ions into visible molecular silver. This latter step of physical development has been normally carried out after embedding and sectioning. However, when we followed this method we found that the dark metal sulphide was lost from the tissue during the embedding in epoxy resin. Hence the method was unsuitable for our proposed experiment on the ultrastructural localisation of cadmium. We subsequently modified the technique primarily by treating very thin tissue slices with the developer before dehydration and embedding, thus eliminating any problem from sulphide loss. This modified technique was used to investigate the ultrastructural localisation of cadmium in the kidneys of mice which had been exposed to 50 ppm cadmium in their drinking water for up to eight months. The molecular silver was found to be located mainly in the proximal tubule cells, either as dense clumps in apical vesicles and lysosomes or diffuse grains throughout the cytoplasm of the cells particularly in the basal region. We interpret these results as indicating that cadmium is found in the apical vesicles, lysosomes and cytoplasm of proximal tubule cells.


Pulmonary shunt: a comparison between oxygen and inert gas infusion methods.

Pulmonary shunt measurement was compared using the standard oxygen (Berggren) technique and a new multiple inert gas infusion (Wagner et al.) technique in 11 mongrel dogs with either regional atelectasis or diffuse pulmonary edema. Relative retentions of sulfur hexafluoride, ethane, cyclopropane, halothane, diethyl ether, and acetone were used to calculate intrapulmonary shunt. Relative dilution of oxygen (Berggren method) was used to calculate total right to left shunt. The two methods gave similar results in the range of 20-80%. At low shunts the oxygen method measured higher values. This is because of the greater relative importance of fixed extrapulmonary shunts at this level and, as well, error in the measurement of PO2. Both techniques are suitable for normal clinical use.


Resolution of aflatoxins B1, B2, G1, and G2 by high-pressure liquid chromatography.

Aflatoxins were completely resolved as sharp peaks in the order BU-B2-G1-G2 by high-pressure liquid chromatography on a small particle (10 mum) porous silica gel column in 7-13 min (B1 through G2) by a water-saturated chloroform-cyclohexane-acetonitrile elution solvent (25+7.5+1.0), with detection by ultraviolet absorbance at 360 nm. The relationship between peak height and amount injected was linear over a 5-400 ng range for each aflatoxin. Both retention times and peak heights were highly reproducible, multiple injections of mixed standards giving coefficients of variation of 1.0-1.4% (retention time) and 1.6-2.8% (peak height) for the 4 aflatoxins. Detection was highly sensitive, with mean peak height, mm/ng, of 7.1 (B1), 6.4 (B2), 4.5 (G1), and 4.1(G2), allowing detection of 1-2 ng of each aflatoxin.


Alpha reduced nicotinamide adenine dinucleotide-dependent reductase reactions of rat liver microsomes.

The kinetics of alpha-NADH-dichlorophenolindophenol (DCPIP) and alpha-NADH-cytochrome c reductase reactions of rat liver microsomes showed that the reactio ns proceeded by a ping-pong mechanism, and that the oxidation of alpha-NADH was the rate-determining reaction. The DCPIP-reducing activity with alpha-NADH in the presence of ADP was about 1% of that with beta-NADH. ADP inhibited the alpha-NADH-DCPIP reductase reaction in a competitive manner with respect to alpha-NADH and a value of 1.2 mM  for the inhibition constant was obtained. ADP also inhibited cytochrome b5 reduction with alpha-NADH. More than 90% of cytochrome b5 was reduced under conditions where 90% of the alpha-NADH-DCPIP reductase activity was suppressed with ADP. The reduction of DCPIP with alpha-NADH preceded that of cytochrome b5, but the reductions partly overlapped. From these results, a diversed electron flow from alpha-NADH to cytochrome b5 and electron sharing between cytochrome b5 and DCPIP were indicated. alpha-NAD+ also inhibited the alpha-NADH-DCPIP reductase reaction. Analyses of the inhibition indicated that two types of alpha-NADH-DCPIP reductase reaction existed, one of which was resistant to alpha-NAD+ inhibition. In contrast to the reoxidation of beta-NADH-reduced cytochrome b5, the process was largely monophasic when cytochrome b5 was reduced with alpha-NADH.


Correlation between adenosine 3',5'-cyclic monosphosphate levels, adenylate cyclase activity, and adenosine 3',5'-cyclic monophosphate phosphodiesterase activity in tissue culture cells stimulated by serum.

Changes in the activity of adenylate cyclase [EC 4.6.1.1] of serum-stimulated hamster BHK cells in culture were investigated in relation to changes in the intracellular level of adenosine 3',5'-cyclic monophosphate (cyclic AMP). Addition of calf serum to quiescent cultures decreases the activity of adenylate cyclase, followed by cellular DNA synthesis. Cyclic AMP levels drop in parallel with the decrease in adenylate cyclase activity. Kinetic analysis of cyclic AMP phosphodiesterase [EC 3.1.4.17] revealed that BHK cells contain two forms of the enzyme, one with a Km of 0.77 muM and the other with Km of 17.0 muM. The activity of these enzymes is not affected by the addition of serum to tissue culture cells. The findings indicate that the decrease in adenylate cyclase activity is directly responsible for the decrease in cyclic AMP levels that is observed immediately after serum addition. Low levels of cyclic AMP continue for several hours during serum treatment, followed by a transient increase in cyclic AMP in the early stages of cellular DNA synthesis, at which time the activity of cyclic AMP phosphodiesterase with a low Km shows a slight decrease.


The distribution of pyrophosphatidic acid in nature.

The occurrence of a novel phospholipid, pyrophosphatidic acid, in the lipid extracts of yeasts (23 species), bacteria (E. coli), algae (chlorella), mammalia (human, rabbit, guinea pig, and mouse), insect (cockroach), fish (carp), mollusc (clam), and spermatophyta (spinach) was investigated. Pyrophosphatidic acid was found exclusively in the lipid extracts of several kinds of yeast species, but not in other normal living species (animals, plants, and microorganisms) so far investigated. All of the yeast species containing this lipid belong to the asporogenous yeasts (Cryptococcus neoformans CBS-132, Cryptococcus laurentii Z 6-5, Rhodotorula glutinis H 3-9-1, Rhodotorula rubra AY-2, Kloeckera apiculata KK-3, and Trichosporon cutaneum KC 4-3),  and ballistosporogenous yeast (Sporobolomyces salmonicolor WF 174). In contrast, no detectable amount of pyrophosphatidic acid was found in the cellular lipids of ascosporogenous yeasts.


Organization of lipids in sarcoplasmic reticulum membrane and Ca2+-dependent ATPase activity.

The organization of lipids in sarcoplasmic reticulum membrane was studied with a variety of stearic spin labels and a phosphatidylcholine spin label. The ESR spectra of the spin-labeled membranes consisted of two components, one due to labels in lipid bilayer structure and the other due to more immobilized labels. The relative intensity of the immobilized component increased when the lipid content of the membrane was decreased by treatment with phospholipase A [EC 3.1.1.4] and subsequent washing with bovine serum albumin. Membrane containing 30% of the intact phospholipid, i.e.0.15 mg of phospholipid per mg of protein, showed a spectrum consisting only of the immobilized component (the overall splitting ranged from 58.5 G to 60.5 G). The immobilized component was ascribed to lipids complexed with protein. The fraction of lipids in the two different organizations was determined from the ESR spectrum. The activity of the Ca2+-Mg2+ dependent ATPase [ATP phosphohydrolase, EC 3.6.1.3] was found to increase almost linearly with the lipid bilayer content in the membrane, whereas phosphoenzyme formation was almost independent of the bilayer content. This indicated that the bilayer structure is necessary for the ATPase to attain its full transport activity.


Purification and properties of the intact form of NADH-cytochrome b5 reductase from rabbit liver microsomes.

NADH-cytochrome b5 reductase [EC 1.6.2.2] has been solubilized with Triton X-100 and purified to homogeneity from rabbit liver microsomes. The purified enzyme is essentially free of the detergent and phospholipids and exists in aqueous media as an oligomeric aggregate of about 13 S. Its monomeric molecular weight is about 33,000 and 1 mole of FAD is associated with 1 mole of the monomeric unit. The enzyme catalyzes the reductions by NADH of ferricyanide and 2,6-dichlorophenol indophenol at an activity ratio of 1 : 0.09. Although the intact form of cytochrome b5 is a poorer electron acceptor than its hydrophilic fragment for the purified flavoprotein, electron transfer from the reductase to the intact cytochrome can be markedly stimulated by detergents or phospholipids, which also cause profound enhancement of the NADH-cytochrome c reductase activity reconstituted from the reducatse and cytochrome b5. Upon digestion with trypsin [EC 3.4.21.4], the ability of the reductase to form an active NADH-cytochrome c reductase system with the intact form of cytochrome b5 and Triton X-100 is rapidly lost. This loss of the reconstitution capability can be prevented by preincubation of the reductase with phosphatidylcholine liposomes. Trypsin digestion also results in the cleavage of the reductase molecule to a protein having a molecular weight of about 25,000 and a smaller fragment. The purified flavoprotein can bind to liver microsomes, liver mitochondria, sonicated human erythrocyte ghosts, and phosphatidylcholine liposomes. The reductase solubilized directly from liver microsomes by lysosomal digestion however, is devoid of membrane-binding capacity. It is concluded that the intact form of NADH-cytochrome b5 reductase is an amphipathic protein and its hydrophobic moiety, which is removable by lysosomal digestion, is responsible for the tight binding of the reductase to microsomes and for its normal functioning in the membrane.


Biochemical studies on sulfate-reducing bacteria. XIV. Enzyme levels of adenylylsulfate reductase, inorganic pyrophosphatase, sulfite reductase, hydrogenase, and adenosine triphosphatase in cells grown on sulfate, sulfite, and thiosulfate.

Sulfate-reducing bacteria, Desulfovibrio vulgaris, strain Miyazaki, were grown on either sulfate, sulfite, or thiosulfate as the terminal electron acceptor. Better growth was observed on sulfite and less growth on thiosulfate than on sulfate. Enzyme levels of adenylylsulfate (APS) reductase [EC 1.8.99.2], reductant-activated inorganic pyrophosphatase [EC 3.6.1.1], sulfite reductase [EC 1.8.99.1] (desulfoviridin), hydrogenase [EC 1.12.2.1], and Mg2+-activated ATPase [EC 3.6.1.3] were compared in crude extracts of these cells at various stages of growth. 1) The specific activity of APS reductase in sulfite-grown cells was only one-fourth that in sulfate-grown cells throughout growth. Thiosulfate-grown cells had an activity intermediate between those of sulfate- and sulfite-grown cells. 2) Cells grown on sulfite had lower specific activity of reductant-activated inorganic pyrophosphatase than cells grown on sulfate or thiosulfate. 3) The specific activity of sulfite reductase (desulfoviridin) was highest in sulfite-grown cells. The sulfite medium gave the enzyme in high yield as well as with high specific activity. 4) The specific activities of hydrogenase and Mg2+-ATPase were not significantly altered by electron acceptors in the growth medium.


Effect of glucose and cyclic adenosine 3',5'-monophosphate on the synthesis of succinate dehydrogenase and isocitrate lyase in Escherichia coli.

Repressed respiration of Escherichia coli cells grown in the presence of 2% glucose was derepressed when the cells were incubated in a buffer containing casamino acids. The glucose-repressed cells were deficient in succinate dehydrogenase [EC 1.3.99.1] and isocitrate lyase [EC 4.1.3.1] activities, which increased during the incubation. The increases in respiratory activity and enzyme activity on incubation were repressed by glucose, but except for isocitrate lyase these repressions could be restored by the addition of cyclic adenosine 3',5'-monophosphate. Inhibitors of protein synthesis blocked the increase of enzyme activity on incubation without glucose, or with glucose and the cyclic nucleotide.


The isolation and characterization of glycopeptides and mucopolysaccharides from plasma membranes of an ascites hepatoma, AH 130.

Plasma membranes were isolated from an ascites hepatoma, AH 130, by the fluorescein mercuric acetate (FMA) method. Glycopeptides and mucopolysaccharides were prepared by digesting the membranes with pronase, then by fractionating the digest chromatographically and electrophoretically. Isolated fractions were analyzed for their amino acid and carbohydrate compositions. Results were compared with those for corresponding fractions from AH 66 (J. Biochem. 76, 319-333 (1974)). Mucopolysaccharides and a series of glycopeptides were isolated from the fraction excluded from Sephadex G-50. The mucopolysaccharides were identified as a family of heparan sulfates with different electrophoretic mobilities. The glycopeptides contained serine, threonine, galactose, galactosamine, glucosamine, and sialic acid as the major constituents as aspartic acid and mannose as minor ones. This suggests that most of the carbohydrate moieties are linked to serine or threonine (O-glycosidic), and that some are linked to asparagine (N-glycosidic). No nearly purely O-glycosidic glycopeptides were found in this fraction from AH 130, through they were the major glycopeptides from the AH 66 plasma membranes. In the fraction included in the gel, glycopeptides containing fucose, galactose, mannose, glucosamine, glaactosamine, and sialic acid were found. The presence of galactosamine suggests that some of the glycopeptides are O-glycosidic though most are N-glycosidic. In the corresponding fraction from AH 66, nearly purely N-glycosidic glycopeptides were found.


Action of bacterial collagenase on Ascaris cuticle collagen.

The collagen from the cuticle of Ascaris lumbricoides was digested by Clostridium histolyticum collagenase [EC 3.4.24.3] in the presence and absence of CaCl2. About 1.2 mumoles of amino groups per mg collagen was liberated when the digestion was performed in the presence of 5 mM CaCl2, whereas about 0.5 mumole of amino groups per mg collagen was liberated by digestion in the absence of CaCl2. In contrast, CaCl2 influenced the extent of hydrolysis of rat tail tendon collagen only slightly. The results suggest that CaCl2 is necessary for the hydrolysis of certain regions in the molecule of Ascaris collagen and that such structures may not be present in mammalian collagens.


Studies on a thermophilic RNA polymerase which is active only on poly d(A-T) and poly dAdT.

Two types of RNA polymerases [EC 2.7.7.6], polymerases A and B, exist in thermophilic bacteria, Thermus thermophilus HB8. Polymerase B is apparently like the core enzyme of polymerase A but is active only when an alternating copolymer of deoxyadenylic and deoxythymidylic acids (poly d(A-T)) or a mixture of homopolymers of deoxyadenylic acid and deoxythymidylic acid (poly dAdT) is used as a template. Polymerase B was further characterized to elucidate its relation to polymerase A and to determine why it is inactive on natural DNA's. 1. Polymerase B did not show pyrophosphate exchange activity. Dinucleoside monophosphates did not activate the RNA-synthesizing activity. The results suggested that polymerase B had no initiation and presumably no elongation activities. 2. Polymerase B had about 6 times greater affinity to DNA than polymerase A. The binding of polymerase B to DNA was, however, reversible. The complex of DNA with polymerase A was stable and the polymerase was not removed from the initial complex even when a large amount of DNA was added. 3. E. coli sigma subunit could not stimulate the activity of polymerase B toward DNA's. 4. Polymerase B could utilize poly d(A-T) and poly dAdT as templates, but could not use Bacillus cereus DNA though the structure is reported to be similar to that of poly d(A-T).


Effect of alcohols on polypeptide chain elongation and aminoacyl-tRNA formation.

The effect of alcohols (methanol, ethanol, and propanol) on polypeptide chain elongation was studied. In the E. coli and rat liver cell-free systems, the optimal concentration of Mg2+ decreased with increase of ethanol concentration, although the maximum polyphenylalanine synthesis decreased. Methanol had almost the same effect as ethanol. Propanol decreased the optimal magnesium concentration, but polyphenylalanine synthetic activity was markedly decreased. The shift of optimal Mg2+ concentration by ethanol was also observed in polylysine and polysome-dependent polypeptide syntheses. Even in the presence of spermidine, ethanol caused the shift of optimal Mg2+ concentration. Ribosome-bound Mg2+ was decreased by the addition of ethanol. A study of the effect of alcohols on aminoacyl-tRNA formation with ten amino acids in the absence of added Mg2+ showed that the formation of arginyl-, leucyl-, and valyl-tRNA was stimulated by the alcohols. Valyl-tRNA formation in the presence of alcohols was completely inhibited by EDTA, while that in the presence of Mg2+ was inhibited slightly by EDTA. No PP1-ATP exchange was observed when alcohol was used as the only stimulant of valyl-tRNA formation.


Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 3. Synthesis of deoxyribopolynucleotide segments corresponding to the nucleotide sequence 27-51.

Chemical syntheses of the four deoxyribodecanucleotides, d(T-C-G-A-A-G-T-C-G-A), d(C-G-T-C-A-T-C-G-A-C), d(T-G-A-C-G-G-C-A-G-A), and d(C-T-A-A-A-T-C-T-G-C) are described. These polynucleotides form, respectively, segments 7 to 10 in the plan adopted for the total synthesis of the DNA corresponding to the precursor for the Escherichia coli tyrosine tRNA. The syntheses used the principles of stepwise addition of protected mono- and oligonucleotides to the 3'-hydroxyl end of growing oligonucleotide chains. Detailed schemes used in the present syntheses are shown in Diagrams 1 to 4 in the text. The final products were subjected to extensive chromatography and were characterized as pure by chemical and enzymatic procedures.


Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 10. Enzymatic joining of chemically synthesized segments to form the DNA duplex corresponding to the nucleotide sequence 86-126.

The polynucleotide ligase-catalyzed joining of the eight chemically synthesized deoxypolynucleotides (segments 19 to 26), comprising the nucleotide sequence 86-126 of the DNA corresponding to the Escherichia coli tyrosine tRNA precursor has been investigated. Joining was studied using various combinations of 3, 4, or larger number of segments at a time. The extent of joining was in general low (0 to 40%) for the three-component as well as for the four-component systems. Joining of the five- and six- component systems was more satisfactory with yields from 25 to about 60%. The three duplexes [IVa] to [IVc]were prepared in single step reactions in yields of about 50% and were characterized. Duplex [IVd] could not be prepared in a single step reaction because of the failure of 5'-phosphorylated segment 26 to join to the rest of the duplex. Using a carefully annealed mixture of segments 24, 25, and phosphorylated segment 26, the joining of the latter to segment 24 could be realized in about 25% yield, much activated intermediate being concurrently present.


Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 11. Enzymatic joining to form the total DNA duplex.

The DNA duplex corresponding to the entire length (126 nucleotides) of the precursor for an Escherichia coli tyrosine tRNA has been synthesized. Duplex [I] (Sekiya, T., Besmer, P., Takeya, T., and Khorana, H. G.(1976) J. Biol. Chem. 251, 634-641), corresponding to the nucleotide sequence 1-26, containing single-stranded ends and carrying one appropriately labeled 5'-phosphate group, was joined to duplex [II] (Loewen, P. C., Miller, R. C., Panet, A., Sekiya, T., and Khorana, H. G. (1976) J. Biol. Chem. 251, 642-650) (nucleotide sequence 23-66 or 23-60) was phosphorylated with [gamma-33P]ATP at the 5'-OH ends. Duplex [III] (Panet, A., Kleppe, R., Kleppe, K., and Khorana, H. G. (1976) J. Biol. Chem. 251, 651-657) (nucleotide sequence 57-94 (Fig. 2)) was also phosphorylated at 5'-ends with [gamma-33P]ATP and was joined to duplex [IV] (Caruthers, M. H., Kleppe, R., Kleppe, K., and Khorana, H. G. (1976) J. Biol. Chem. 251, 658-666) (nucleotide sequence 90-126) which carried a 33P-labeled phosphate group on nucleotide 90. The joined product, duplex [III + IV] (nucleotide sequence 57-126) was characterized. The latter duplex was joined to the duplex [I + II] to give the total duplex. The latter contains singlestranded ends (nucleotides 1 to 6 and 121 to 126) which can either be "filled in" to produce the completely base-paired duplex or may be used to add the promoter and terminator regions at the appropriate ends.


Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 12. Synthesis of a DNA duplex corresponding to a sequence of 23 nucleotide units adjoining the C-C-A end.

In continuing the work on the total synthesis of the gene for an Escherichia coli tyrosine suppressor tRNA (accompanying papers) and as a part of a study of the mechanism of transcription of this gene, a 23-nucleotide unit-long DNA corresponding to the previously determined (Loewen, P., Sekiya, T., and Khorana, H. G. (1974) J. Biol. Chem. 249, 217) sequence has been synthesized. The synthesis was carried out by dividing the total duplex into the following five deoxyribooligonucleotide segments, all of which were chemically synthesized: (a) the undecanucleotide, d(A-G-T-G-A-T-G-G-T-G-G); (b)the undecanucleotide, d(T-C-A-C-T-T-T-C-A-A-A); (c) the undecanucleotide, d(G-G-A-C-T-T-T-T-G-A-A); (d) the dodecanucleotide, d(A-G-T-C-C-C-T-G-A-A-C-T); and (e) the heptanucleotide, d(A-G-T-T-C-A-G). All the five synthetic oligonucleotides were characterized by chromatographic and radioactive fingerprinting methods after labeling the 5'-ends with a 32P-phosphate group. Synthesis of the double-stranded DNA duplex was completed by joining 5'-phosphorylated segments 1, 3, and 4 in the presence of segments 2 and 5 using T4-polynucleotide ligase. The DNA duplex was characterized.


Effects of adenosine 3':5'-monophosphate-dependent protein kinase on sarcoplasmic reticulum isolated from cardiac and slow and fast contracting skeletal muscles.

Effects of cyclic adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase were studied in sarcoplasmic reticulum prepared from cardiac and slow and fast (white) skeletal muscle. Cyclic AMP-dependent protein kinase failed to catalyze phosphorylation of fast skeletal muscle microsomes as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cyclic AMP-dependent protein kinase was without effect on calcium uptake by these microsomes. Treatment of cardiac microsomes obtained from dog, cat, rabbit, and guinea pig with cyclic AMP-dependent protein kinase and ATP resulted in phosphorylation of a 22,000-dalton protein component in the amounts of 0.75, 0.25, 0.30, and 0.14 nmol of phosphorus/mg of microsomal protein, respectively. Calcium uptake by cardiac microsomes was stimulated 1.8- to 2.5-fold when microsomes were treated with cyclic AMP-dependent protein kinase. Protein kinases partially purified from bovine heart and rabbit skeletal muscle were both effective in mediating these effects on phosphorylation and calcium transport in dog cardiac sarcoplasmic reticulum. Slow skeletal muscle sarcoplasmic reticulum also contains a protein with a molecular weight of approximately 22,000 that can be phosphorylated by protein kinase. Phosphorylation of this component ranged from 0.005 to 0.016 nmol of phosphorous/mg of microsomal protein in dog biceps femoris. A statistically significant increase in calcium uptake by these membranes was produced by the protein kinase. Increases in protein kinase-catalyzed phosphorylation of a low molecular weight microsomal component and in calcium transport by sarcoplasmic reticulum of cardiac and slow skeletal muscle may be related to the relaxation-promoting effects of epinephrine seen in these types of muscle. Conversely, the absence of a relaxation-promoting effect of epinephrine in fast skeletal muscle may be associated with the lack of effect of cyclic AMP and protein kinase on calcium transport by the sarcoplasmic reticulum of this type of muscle.


Adenine nucleotide changes at initiation of bull sperm motility.

Testicular and cauda epididymal sperm were obtained via catheters previously implanted in the rete testis and proximal vas deferens of bulls and were used to examine the relationships among sperm motility, cyclic adenosine 3':5'-monophosphate (cAMP) level, adenine nucleotide levels, and rates of glucose and oxygen consumption. Testicular, cauda epididymal, and ejaculated sperm contain cAMP-stimulated protein kinase, adenylate cyclase, and nucleotide phosphodiesterase. Treatment of the nonmotile testicular sperm with phosphodiesterase inhibitors resulted in a doubling of cellular cAMP concentration and a 25% increase in their glucose consumption. No change in motility, ATP level, or rate of oxygen consumption was observed. Sperm in neat cauda epididymal semen had flagellating tails but no progressive motility. Dilution of these sperm into glucose-containing buffer resulted in an increase in intracellular cAMP concentration and a decrease in ATP level with concomitant increases in ADP and AMP levels. These biochemical changes occurred within 30 s after dilution and apparently preceded the initiation of progressive motility by most cells. Since sperm in neat cauda epididymal semen became progressively motile when diluted with neat cauda epididymal plasma as well as accessory sex gland fluid or buffer, composition of the fluid surrounding the sperm is not responsible for the initiation of progressive motility upon dilution nor does cauda epididymal plasma contain an inhibitory factor. Perhaps release from contact immobilization provides the stimulation for the initial acquisition of progressive motility by cauda epididymal sperm. We conclude that during epididymal passage sperm develop from a cell physically unresponsive to changes in cAMP concentration to a form which initiates progressive motility upon changes in cAMP concentration.


Alanine and glutamine synthesis and release from skeletal muscle. IV. beta-Adrenergic inhibition of amino acid release.

Alanine and glutamine formation and release were studied using the intact epitrochlaris preparation of rat skeletal muscle. Epinephrine reduced the release of alanine and glutamine in a concentration-dependent manner. Measurable inhibition was observed at 10(-9) M epinephrine, and maximal inhibition was obtained at 10(-5) M. Norepinephrine also reduced alanine and glutamine formation and release but the concentration required for maximal inhibition was approximately 100-fold greater than for epinephrine. Isoproterenol (beta agonist), but not phenylephrine (alpha agonist), reproduced the effects of epinephrine, and propranolol (beta antagonist), but not phentolamine (alpha antagonist), blocked the effect of the catecholamine. N6,O2'-Dibutyryl adenosine 3':5'-monophosphate reproduced the effects of epinephrine and theophylline potentiated the effect of submaximal concentrations of the hormone. Glucagon and prostaglandin E2 had no observable effect on amino acid release. Insulin did not modify the inhibition of alanine and glutamine release produced by epinephrine. Alanine and glutamine formation from added precursor amino acids was unaffected by epinephrine or cyclic adenosine 3':5'-monophosphate. Epinephrine reduced alanine formation in muscles obtained from diabetic rats or animals treated with thyroxine or cortisone. These findings indicate that physiological levels of catecholamines reduce alanine and glutamine formation and release from skeletal muscle. This effect is mediated by a beta-adrenergic receptor and the adenylate cyclase system and can be accounted for by an inhibition of muscle protein degradation.


Turnover of the plasma membrane proteins of hepatoma tissue culture cells.

The turnover of the plasma membrane proteins of hepatoma tissue culture cells was examined by three different methods--loss of polypeptides labeled in situ by lactoperoxidase-catalyzed iodination, loss of membrane polypeptides labeled with amino acid precursors, and loss from the membrane of fucose-labeled polypeptides. In both logarithmically growing and density-inhibited cells the proteins of the membrane are degraded with a half-life of about 100 hours. This is longer than the half-life of total cell protein, 50 to 60 hours, and longer than the doubling time of the cells, about 30 hours. Similar values for the rate of degradation of the membrane proteins were obtained by each of the three techniques. The same fucose-labeled polypeptides are present in the microsomal and the plasma membrane fractions of hepatoma tissue culture cells as analyzed by electrophoresis in dodecyl sulfate-acrylamide gels. But the fucose-labeled polypeptides were lost from the microsomal fraction at a faster rate than from the plasma membrane. Autoradiographic and double labeling techniques using 125I and 131I, or [3H]leucine and [14C]leucine were used to measure the relative rates of degradation of the proteins in the plasma membrane. All of the leucine-labeled polypeptides and the iodinated polypeptides had similar rates of degradation. These results support a model for the biogenesis of the plasma membrane in which the proteins are incorporated and removed in large structural units.


Relationship between microsomal membrane permeability and the inhibition of hepatic glucose-6-phosphatase by pyridoxal phosphate.

Arion et al; (Arion, W. J., Wallin, B. K., Lange A. J., and Ballas, L. M. (1975) Mol. Cell. Biochem. 6, 75-83) propsed a model for glucose-6-phosphatase in which the substrate was transported across the microsomal membrane by a carrier before hydrolysis on the cisternal side. Evidence to support this model has been obtained by studying the inhibition of the enzyme by pyridoxal-P. Pyridoxal-P was a linear noncompetitive inhibitor of glucose-6-phosphatase (EC 3.1.3.9) in freshly isolated ("intact") microsomes from rat liver. Pyridoxol-P was a much less effective inhibitor and no inhibition was observed with pyridoxamine-P. When microsomes were subjected to nitrogen cavitation, treatment with solium deoxycholate, or glutaraldehyde fixation, the Km of glucose-6-phosphatase for glucose-6 P decreased from approximately 6 mM to approximately 2.5 mM; the corresponding change in the Vmax ranged from-10% to +40%. The same procedures decreased the inhibition of glucose-6-phosphatase by pyridoxal-P several-fold. No inhibition by pyridoxal-P was observed in a preparation of glucose-6-phosphatase purified approximately 20 fold (on the basis of Vmax) from micoromes. A nondialyzable inhibitor was apparently formed when intact microsomes were reacted with pyridoxal-P and NaBH4; this inhibition was also reversed by procedures which changed the kinetic properties of glucose-6-phosphatase.


Studies on human serum high density lipoproteins. Self-association of apolipoprotein A-I in aqueous solutions.

Human serum apolipoprotein A-I (apo-A-I), the major protein component of the human serum high density lipoproteins, was studied in aqueous solutions of differing ionic strength and pH by the techniques of sedimentation equilibrium ultracentrifugation and frontal analysis gel chromatography. The ultracentrifugal studies indicate the apo-A-I is a self-associating system that is dependent upon protein concentration, but relatively independent of the nature of the medium. The apparent weight average molecular weights obtained from solutions of initial apo-A-I concentration between 0.2 and 0.9 mg/ml were in the range of 3.0 to 16.7 x 10(4) (monomer molecular weight = 28,014). Of the several models of self-associated examined, that which gave the best theoretical fit was for the monomer-dimertetramer-octamer model. The self-association of apo-A-I in aqueous solutions was further documented by frontal analysis gel chromatography, which not only corroborated the ultracentrifugal results, but also indicated that the multiple species of apo-A-I in solution attain equilibrium rather rapidly. Besides having intrinsic importance, these results indicate that the solution properties of apo-A-I must be established before ligand binding studies are conducted and interpreted.


Testicular protein kinases. Characterization of multiple forms and ontogeny.

Protein phosphokinase activity from the cytosol (105,000 X g soluble fraction) of testes from sexually mature rats has been resolved be DEAE-cellulose chromatography in three forms of protein kinase, cAMP-dependent protein kinases I and II and cAMP-independent protein kinase III. Adenosine 3':5'-monophosphate-binding activity (cAMP-binding activity) was associated with protein kinases I and II but not with protein kinase III. Protein kinases I, II, and III exhibited different pH optima, cyclic nucleotide dependency, and relative substrate specificity. Protein kinases I and II were inhibited by a heat-stable protein inhibitor from rat skeletal muscle, whereas protein kinase III was not inhibited. According to previously established criteria (Traugh, J. A., Ashby, C.D., and Walsh D. A. (1974) Methods Enzymol. 38, 290-299) protein kinases I and II can be classified as cAMP-dependent holoenzymes consisting of regulatory and catalytic subunits. Protein kinase III is a cAMP-independent protein kinase.


ATP-dependent reactions catalyzed by inner membrane vesicles of rat liver mitochondria. Kinetics, substrate specificity, and bicarbonate sensitivity.

Three ATP-dependent reactions catalyzed by the inner membrane of rat liver mitochondria and the ATPase reaction catalyzed by purified mitochondrial ATPase (F1), were studied with respect to kinetic properties, substrates specificity, and sensitivity to bicarbonate. The ATP-dependent transhydrogenase reaction (reduction of NADP+ by NADH) catalyzed by inner membrane vesicles displays typical Michaelis-Menten kinetics in both Tris-Cl and Tris-bicarbonate buffers, with Km (ATP) values of 0.035 mM and 0.054 mM respectively. The Vmax of transhydrogenase activity (25 nmol min-1 mg-1) is the same in Tris-bicarbonate or Tris-Cl buffer. ITP and GTP readily substitute for ATP in the transhydrogenase reaction. The ATP-P1 exchange reaction catalyzed by inner membrane vesicles displays typical Michaelis-Menten kinetics in both Tris-Cl and Tris-bicarbonate buffers with Km (ATP) values of 1.0 mM and 1.4 mM respectively. The Vmax of exchange (200 nmol min-1 mg-1) is the same in either buffer. ITP and GTP do not effectively replace ATP in the exchange reaction.


A major polypeptide component of rat liver mitochondria: carbamyl phosphate synthetase.

One of the major components of rat liver mitochondria detected by gel electrophoresis in sodium dodecyl sulfate is a 165,000 molecular weight polypeptide that makes up 15 to 20% of the total mitochondrial protein. This component appears to be a single molecular species. Evidence is presented here for the identification of this protein with the polypeptide chain of a urea cycle enzyme, carbamoylphosphate synthetase I (EC 2.7.2.5). The 165,000 molecular weight polypeptide was solubilized from mitochondria with Triton X-100 and purified to 90% homogeneity by DEAE-cellulose chromatography. This component co-migrated with carbamyl phosphate synthetase activity when mitochondrial proteins were separated by gel filtration or sucrose gradient centifugation. The identification of the 165,000 molecular weight polypeptide with this activity was also supported by the presence or absence of this protein in a variety of rat tissue mitochondria, in liver and kidney mitochondria from various ureotelic and nonureotelic species, and in fetal rat liver mitochondria.


In vitro bacteriological evaluation of the effectiveness of antimicrobial irrigating solutions.

Nineteen strains of pathogenic aerobic bacteria were exposed for fifteen seconds in vitro to varying concentrations of five antibiotics and of polyvinyl povidone iodine in saline solution. The presence of human plasma in the solution (30 per cent by volume) did not affect bacterial sensitivity to the antibiotics. Most of the bacteria were sensitive to the solutions during that brief exposure. Bacteria which were more sensitive by disc sensitivity tests were more sensitive to the solutions. Polyvinyl povidone iodine sterilized all cultures in concentrations as low as 25 per cent. It is proposed that the use of appropriate antimicrobial solutions as wound irrigants may reduce postoperative infection rates by killing bacteria which contaminate the surface of the wound during operation.


Poland's syndrome.

Forty-three consecutive cases of Poland's syndrome were analyzed and the relevant literature was reviewed. The syndrome is not hereditary and is of unknown origin. It affects males more frequently than females. The clinical features are variable but always include congenital aplasia and syndactyly. The middle phalanges are hypoplastic or absent so that effectively there is only one interphalangeal joint. The syndactyly is usually incomplete and simple. It may involve all fingers and frequently includes the thumb, which then lies in the same plane as the fingers. Poland's syndrome may also include hypoplasia of the nipple and breast, hypoplasia of the upper ribs, herniation of the lung, contracture of the anterior axillary web, and elevated scapula. The arm and more frequently the forearm are hypoplastic. The right side is more often affected than the left. Surgical treatment by separating the syndactyly is recommended. In some cases a digit is removed to produce a three-fingered hand. Surgery is initiated by the age of one year and is completed by the time the child enters school, although periodic revisions may be necessary. Although the hand remains hypoplastic and functional capacity is limited by the inherent skeletal anomalies, surgical treatment improves functional capacity and cosmetic appearance in the majority of patients.


Peripheral neuropathies associated with total hip arthroplasty.

The clinical and electromyographic findings in fourteen patients in whom peripheral nerve damage developed after total hip arthroplasty were reviewed (an incidence of 0.7 per cent in 2,012 procedures). In addition, a prospective study of the clinical, electromyographic, and nerve-conduction findings before and after arthroplasty was performed on twenty-eight patients who had thirty total hip arthroplasties. In the prospective study many patients showed evidence of mild nerve damage, most likely due to operative trauma. Peroneal neuropathies at the knee did not occur in either study. Nerve damage was more frequent in women, but no other predisposing factor could be identified. The prognosis for most patients with severe nerve injuries after total hip arthroplasty is good.


The enhanced production of hyaluronic acid by cultured rat fibroblast cells treated with cyclic AMP and its dibutyryl derivative.

Cells of a newly established rat fibroblast line (SEN) in culture synthesize mucopolysaccharides, which have been identified as hyaluronic acid, chondroitin-4-sulfate and heparan sulfate. Treatment of the cells with adenosine 3':5'-cyclic monophosphate resulted in a marked stimulation of production of hyaluronic acid, but not of the other mucopolysaccharides. Treated cells also showed increased activity of hyaluronic acid synthetase, a reduction in growth rate, and morphological alteration. In addition, 5-bromodeoxyuridine was found to counteract greatly the cyclic AMP effect.


Critical density for relaying in Dictyostelium discoideum and its relation to phosphodiesterase secretion into the extracellular medium.

The critical cell density for relaying in D. discoideum, N*, has been measured as a function of cell density, N, and time after harvesting, t. It has logarithmic dependence on N for 2.5 X 10(4)/cm2 less than N less than 7.5 X 10(5)/cm2 and saturates for N more than 1.0 X 10(6)/cm2. N* is an increasing function of time after harvesting. The phosphodiesterase (PDE) secretion rate on which N* depends is a constant. Expressions were derived which relate N* to PDE secretion and diffusion. They have been fitted to the data from time delay experiments yielding values of the PDE diffusion constant in 2% buffered agar, Dp + (2.25 +/- 0.15) X 10(-9) cm2/s, and the ratio of relaying threshold concentration to signal pulse size, C*/eta = (1.4 +/- 0.05) X 10(5) cm-3. N* has also been measured in the presence of various amounts of added beef heart PDE. The cAMP relaxation rates, I/tauo, due to beef heart PDE were calculated from the N* measurements and found to be proportional to amounts of added PDE for (I/tauo)max less than (10s-1). Finally, two kinds of inhibition have been observed in the PDE secretion. The PDE activity per cell is constant for N less than 8.0 X 10(4)/cm2, and decreases for larger N. It depends only on N for I/tau less than 10 s-1 and is strongly inhibited by extracellular PDE activity above this relaxation rate.


Similarities of the Golgi apparatus membrane and the plasma membrane in rat liver cells.

A Golgi apparatus-rich fraction and a plasma membrane-rich fraction were isolated from a common homogenate of rat liver. Their respective buovant densities, appearances in the electron microscope and 5'-nucleotidase and UDP-galactose ovalbumin galactosyltransferase activities were in accord with published data on separately isolated Golgi apparatus-rich and plasma membrane-rich fractions. Contamination by endoplasmic reticulum and mitochondria was low. Gel electrophoresis of the membrane proteins of the Golgi apparatus-rich and plasma membrane-rich fractions (separately and mixed) showed a close similarity. After Neville's demonstration that electrophoretic patterns of membrane protein subunits from different subcellular fractions are easily distinguishable, the present work demonstrates an unusually close relationship between the Golgi apparatus membrane and the cell membrane. It is possible that membrane similarity may be mediated by the transfer of membrane-bound vesicles from the Golgi apparatus to the cell membrane.


The development of the relaying competence in Dictyostelium discoideum.

The fraction, X2, of a population of D. discoideum cells competent to relay an aggregative signal has been measured as a function of time, t, spent in interphase. X2(t) is less than 0.I up to 5 h, increases slowly to 0.4 by 8 h 20 min, and saturates at I by 10 h. The hypothesis that cellular interactions might enhance X2(t) was tested by mixing populations of cells set into interphase at different times. No interactions were found. External stimulation did not affect X2(t).


Extraction method and thin-layer chromatographic system for the determination of alpha-l-acetylmethadol and metabolites in biological fluids.

An extraction method and thin-layer chromatographic (TLC) system for the determination of alpha-l-acetylmethadol and its known metabolites (methadol, noracetylmethadol, dinoracetylmethadol, normethadol, 6-acetamide-4,4-diphenyl-3-heptanol, and N-methyl-6-acetamido-4,4-diphenyl-3-heptanol) are described. The parent drug and metabolites are extracted from biological fluids with ethyl acetate and separated by TLC using silica gel plates and a developing system of ethyl acetate-methanol-water-ammonia (85:10:1:1). This system may be used to quantitatively determine levels of radiolabeled drug and metabolites by scraping the TLC plates into 3-mm zonal fractions and measuring the amount of radioactivity by scintillation counting. A representative radiochromatogram obtained from an extract of monkey urine is shown.


Partition high-pressure liquid-chromatographic systems for the separation of digitalis glycosides of the cardenolide group.

Several multi-component liquid-systems have been investigated on silica gel SI-60 supports of particle size 10 mum. By using two solvent systems, it was possible to separate 14 digitalis glycosides, ranging from genins of relatively low polarity to the highly polar deacetyl-lanatosides. Solvents with good ultraviolet transparency at 220 nm (lambdamax. for the butenolide ring) were chosen in order to improve the sensitivity of detection. The technique should also permit the determination of by-products and degradation products of these drug substances. Detection limits are as low as 15 ng for a 5 mul injection, and separation times vary between 4 and 20 min. The reproducibility of the retention times and the baseline separations attainable make the systems suitable for quantitative work.


Studies on digitalis. IV. A method for thin-layer chromatographic separation and determination of digitoxin and cardioactive metabolites in human blood and urine.

A thin-layer chromatographic method for the separation of digitoxin and its cardioactive metabolites in one system is described. Pre-coated silica gel plates impregnated with 15% formamide solution in acetone were developed twice in the same direction (running distance 18cm) with ethyl methyl ketone-xylene (50:50) as solvent. The system showed no border-zone effects, and the reproducibility was good. Samples (5 ml) of serum  or urine were extracted with dichloromethane, the extracts were evaporated, the residues were dissolved in 70% ethanol, the ethanol solutions were washed twice with light petroleum and then evaporated, and the residues were dissolved in chloroform-methanol for application to the thin-layer plates. After development, the metabolites were scraped from the plates and analyzed by means of a modified rubidium-86 method. The recovery for the whole procedure was 59%, and the sensitivity of the method permitted the determination of down to 0.5 ng per spot. The method will facilitate the study of digitoxin metabolism in patients undergoing treatment with the drug.


Thin-layer chromatography of homopyrimidazole derivatives.

The chromatographic behaviour of 14 homopyrimidazole derivatives has been studied on thin layers of silica gel, aluminium oxide and ammonia-saturated silica gel. The relationships between structure and chromatographic behaviour, as well as the most suitable systems for separating the individual derivatives, are discussed on the basis of RF values. The delta RMg values, showing the effect on the polarity, are given for certain functional groups.


Fluorescence of tryptophan-containing peptides on paper or silica gel after treatment with formaldehyde, formaldehyde-ozone or formaldehyde-hydrochloric acid.

Sensitive and specific procedures for the chromatographic detection of tryptophan and tryptophan-containing peptides are described. Formaldehyde gas induces strong and characteristic fluorescence from tryptophan and peptides with NH2-terminal tryptophan residues on silica gel. On filter-paper, the detection of small amounts of these compounds requires the additional use of an oxidant, such as ozone. Treatment with formaldehyde-hydrochloric acid was used as a method for inducing fluorescence from tryptophan-containing peptides regardless of the position of the tryptophan residue in the peptide molecule. This reaction is useful for the chromatographic demonstration of small amounts of such peptides on both paper and silica gel. The spectral properties of the fluorophores of such tryptophan-containing peptides are distinctive and serve to distinguish them from all other known biogenic compounds that are capable of giving fluorescence with formaldehyde.


Prolonged corticotropic action of synthetic human ACTH in man.

The corticotropic activities of synthetic human ACTH (revised structure), synthetic porcine ACTH (unrevised structure) and synthetic 1-24ACTH were compared in 8 normal men by measuring the magnitude and duration of the elevation of plasma 11-hydroxy-corticosteroids following the injection of 1 mg of the peptides. The duration of response was significantly longer after human ACTH than following porcine or 1-24ACTH, although the initial responses were identical. This difference was noted after both intramuscular and intravenous administration. The data suggest that the carboxyterminal of the ACTH peptide is of some importance for biological activity and that biological species specificity may exist.


Size heterogeneity of beta-MSH in ectopic ACTH-producing tumors: presence of beta-LPH-like peptide.

Gel chromatographic, immunologic and biologic properties of beta-melanocyte-stimulating hormone (beta-MSH) in tumor tissues obtained from eight patients with the ectopic ACTH syndrome were studied and compared to those of pituitary beta-MSH. Size heterogeneity of immunoreactive beta-MSH was found in all the tumors studied as well as in normal human pituitaries. Both the tumors and pituitaries contained immunoreactive beta-MSH of a larger molecular size than the well-characterized beta-MSH of small molecular size. The large molecular weight beta-MSH also predominated in the plasma. It was found to be bioactive by an in vitro MSH assay, immunologically indistinguishable from human beta-MSH, and chromatographically very similar to beta-lipotropic hormone (beta-LPH). Tryptic  digestion of the large molecular weight beta-MSH under controlled conditions promptly produced bioactive beta-MSH of small molecular size, followed by the appearance of immunologically active but biologically inert fragments. These results suggest that the ectopic ACTH-producing tumor as well as the pituitary elaborate beta-LPH-like peptide which might be the predominant component of immunoreactive beta-MSH in man.


Corticotropic action of an intra-nasally applied synthetic ACTH derivative.

The corticotropic action of a synthetic ACTH derivative, D-Ser1 Lys17,18-1-18ACTH, after intranasal insufflation of 1 mg of the peptide was evaluated in 14 normal volunteers. Plasma cortisol and urinary 17-ketogenic steroids and 17-ketosteroids rose significantly over control values in all subjects (P less than 0.001). Plasma cortisol remained above the normal range for comparable clock times for 18 hr, but fell below 15 mug/100 ml after 12 hr. No evidence for suppression of the hypothalamo-pituitary axis was found the day after ACTH administration, as judged by normal plasma and urinary steroid values and normal diurnal variation. Intranasal administration of long-acting ACTH derivatives may provide a simple, painless and effective way to stimulate endogenous corticosteroid secretion.


The effect of ACTH administration on serum estrogens, LH, and FSH in the aged.

The effect of administration of long acting ACTH on serum levels of estrogens, LH and FSH was studied in aged subjects. Nine women and 5 men between 66 and 90 years of age were examined. One milligram of long-acting synthetic ACTH was given in every 12 h for 2 days. Significant increases in serum estrone and estradiol levels were induced in parallel with serum cortisol at 24 hours and no further change was observed at 48 hours. The estrone/estradiol ratio increased from a control value of 3.0 to 4.0 at 24 hours and to 4.7 at 48 hours (P less than 0.02) compared to control values.


Responsiveness to glucagon in fetal hearts. Species variability and apparent disparities between changes in beating, adenylate cyclase activation, and cyclic AMP concentration.

Previous studies of the ability of the immature heart to respond to glucagon have yielded conflicting results. To test the possibility that the apparent discrepancies might be explained in part by species variability, isolated hearts of fetal mice and rats (13-22 days' gestational age) were studied under identical conditions in vitro. Changes in atrial rate and ventricular contractility were measured in spontaneously beating hearts exposed to glucagon, and activation of adenylate cyclase was assayed in cardiac homogenates. In mice of 16 days' gestational age or less, there was no change in heart rate in response to glucagon; at 17-18 days, minimal responsiveness was present; and after 19 days, 10muM glucagon caused an increase in spontaneous atrial rate of 30 +/- 4% (SEM) (P less than 0.001). Measurement of the extent and speed of volume displacement of the isotonically contracting hearts with a specially constructed capacitance transducer revealed that ventricular inotropic responsiveness also appeared after 17-19 days. Cardiac stores of glycogen were reduced in older hearts exposed to glucagon, but not in those aged less than 16 days. In contrast, glucagon failed to activate adenylate cyclase in homogenates of hearts of fetal mice at any age. Furthermore, glucagon failed to elicit an increase in the concentration of cyclic AMP in spontaneously beating hearts that developed tachycardia. Responses in hearts of fetal rats were distinctly different from those in mouse hearts: at no age was there any change in heart rate, strength of contraction, glycogen content, or adenylate cyclase activation. Thus, there are major species differences in cardiac pharmacological maturation. Although the mouse heart develops the ability to increase its rate and strength of contraction and to undergo glycogenolysis in response to glucagon well before birth, the rat heart does not. In addition, there is an apparent disparity in late fetal mouse hearts between the ability of glucagon to induce functional responses and its ability to stimulate adenylate cyclase and increase cyclic AMP levels. It is impossible, of course, to rule out absolutely the possibility that localized increases in a critical cyclic AMP pool were present but too small to measure in the entire tissue. Nevertheless, the most obvious interpretation of our results is that they are compatible with the hypothesis that glucagon may exert some of its hemodynamic effects independently from the adenylate cyclase-cyclic AMP system in the late-fetal mouse heart.


Renal adenylate cyclase and the interrelationship between parathyroid hormone and vitamin D in the regulation of urinary phosphate and adenosine cyclic 3',5'-monophosphate excretion.

This study examined the role of cyclic AMP in the phosphaturic response to parathyroid hormone in vitamin D-deficient rats. Infusion of purified bovine parathyroid hormone (13.3 mug/h) into control, D-fed, or  D-deficient, thyroparathyroidectomized rats produced a sixfold increase in renal phosphate and cyclic AMP excretion in D-fed rats, but only a two- to threefold increase in both parameters in D-deficient animals. Intravenous injection of parathyroid hormone over the dosage range from 1-50 mug/kg resulted in a dose-dependent increase in phosphate and cyclic AMP excretion with both D-fed and D-deficient thyroparathyroidectomized rats. However, the D-deficient rats responded to these injections of parathyroid hormone with a two- to threefold increase in both renal phosphate and cyclic AMP excretion at the highest dose of 50 mug/kg, whereas the D-fed animals' response was 35-fold and 11-fold over control excretion levels of phosphate and cyclic AMP, respectively. To directly examine the role of the renal cortical adenylate cyclase system in the blunted phosphaturic and urinary cyclic AMP responses to parathyroid hormone in D-deficient rats, we prepared a plasma membrane fraction enriched in this enzyme activity from the renal cortex of D-fed and D-deficient thyroparathyroidectomized rats. The renal cortical adenylate cyclase of D-deficient rats showed significantly (P less than 0.001) less activation by parathyroid hormone over the hormone concentration range from 0.3 to 7.0 mug/ml than was observed with the enzyme prepared from D-fed animals. Basal adenylate cyclase activity and the fluoride-stimulated enzyme activity were not altered by the state of D-deficiency. These experiments demonstrate that the blunted phosphaturic response to parathyroid hormone observed in D-deficient rats is associated with the reduced responsiveness of the renal cortical adenylate cyclase to the hormone. Moreover, the defect in the renal membrane adenylate cyclase system appears to be localized at the level of PTH binding to membrane receptors or, alternatively, at the level of transmission of the hormone-receptor binding signal to the catalytic moiety of this membrane enzyme.


The content and metabolism of cyclic adenosine 3', 5'-monophosphate and cyclic guanosine 3', 5'-monophosphate in adenocarcinoma of the human colon.

Data from cultured cells have suggested that cyclic AMP and cyclic GMP may be important determinants of cell growth and transformation. However, few studies have examined cyclic nucleotide content and metabolism in naturally occurring tumors of man. Accordingly, in the present study we compared cAMP and cGMP levels and metabolism in carcinomas of the human colon to those of the adjacent uninvolved mucosa after therapeutic resection of these tissues. The cAMP content of the tumors, determined in samples frozen 30 min after excision, was significantly lower than that of the adjacent mucosa, when expressed on the basis of tissue wet weight, protein, or DNA content. By contrast, the cGMP content of the tumors was higher than that of the surrounding mucosa if calculated on the basis of tissue wet weight, but this difference did not persist when correction was made for the higher protein or DNA content of the tumors. Incubation of slices of mucosa or tumor with or without theophylline in vitro increased tissue cAMP and cGMP content above levels observed in frozen samples of the same tissue. However, after such incubations cAMP levels in the tumors remained clearly below that of the mucosa, while cGMP content of the two tissues did not differ. The failure of theophylline to abolish differences in cAMP content and the comparable activities of high and low Km cAMP-phosphodiesterase in homogenates of the two tissues suggested that the lower cAMP content of the tumors was a consequence of diminished cAMP synthesis rather than enhanced degradation. This possibility was supported by the reduction in basal and maximal prostaglandin E1 (PGE1)-responsive adenylate cyclase activity found in tumor homogenates relative to those of mucosa, and the lower levels of cAMP in tumor slices after incubation of the tissues with a maximal dose of PGE1 and theophylline. Since NaF-responsive adenylate cyclase activity was not significantly reduced in the tumors, the lower basal and PGE1 activities may not be related to a deficiency of the catalytic unit of the cyclase complex in this tissue. The role of reduced activity of the adenylate cyclase-cAMP system and/or reduced tissue cAMP-to-cGMP ratios in the pathogenesis of colonic carcinoma is uncertain, but these changes might favor unregulated cellular proliferation.


Phosphoinositide metabolism and insulin secretion from isolated rat pancreatic islets.

Since many cell types have been shown to respond to extracellular stimulation with a rapid increase in phosphatidylinositol turnover, the present studies were undertaken to determine whether carbohydrate-stimulated insulin secretion from the isolated rat pancreatic islet is accompanied by detectable alterations in the phosphatidylinositol metabolism of this tissue. We have demonstrated that rat pancreatic islets incubated with tritiated myo-inositol rapidly incorporate radioactivity into islet phosphatidylinositol. Incubation of prelabeled islets with elevated concentrations of carbohydrates which stimulate insulin secretion (D-glucose and D-mannose) results in a decrease in the recovery of lipid-bound radioactivity, whereas incubation with carbohydrates which do not stimulate insulin secretion (D-galactose and myo-inositol) has no effect upon the recovery of lipid-bound radioactivity. Within 2 min of exposure of prelabeled islets to elevated concentrations of D-glucose, a decrease in the recovery of [2-3H]myo-inositol-derived radioactivity in islet phosphatidylinositol can be demonstrated. When islets prelabeled with [2-3H]myo-inositol are perifused with elevated concentrations of D-glucose or D-mannose (but not D-galactose or myoinositol) a rapid and transient increase in the rate of extracellular release of water-soluble radioactivity is observed. Since a significant fraction of the radioactivity released under these conditions is in the form of myo-inositol phosphate, cyclic myo-inositol-1,2-phosphate, and glycerophosphorylmyo-inositol, it is presumably derived from the cleavage of labeled islet phosphatidylinositol. It is speculated that alterations in the metabolism of myo-inositol-containing phospholipids may have a functional role in the process of insulin secretion from the pancreatic beta cell.


Extraosseous calcification. Evidence for abnormal pyrophosphate metabolism in uremia.

The inorganic constituents and crystalline features of extraosseous calcium-phosphate deposits obtained from dialyzed uremic and hypercalcemic patients were studied. Visceral calcification (heart, lung, and kidney) in hypercalcemic patients exhibited either an amorphous or apatitic X-ray diffraction pattern. Uremic visceral calcification consistently gave an amorphous diffraction pattern. Although the calcium content of uremic and hypercalcemic visceral deposits was similar, other inorganic constituents were different. The mean pyrophosphate was 11 +/- 11.8 and magnesium 4.91 +/- 3.86 mg/g in the uremic group as compared to 0.92 +/- 0.24 and 1.36 +/- 1.26 mg/g in the hypercalcemic group (P less than 0.025). After incineration hypercalcemic visceral deposits having an amorphous diffraction pattern were found to generate pyrophosphate supporting the presence of brushite in these deposits. The small amount of pyrophosphate in apatitic deposits from both uremic and hypercalcemic patients actually decreased after incineration and the pyrophosphate content of uremic visceral deposits was unchanged by incineration. It is concluded that in hypercalcemic patients the initial visceral deposit is brushite which is subsequently transformed to apatite. Arterial and tumoral calcium-phosphate deposits in uremic patients were also apatite. Uremic visceral calcium-phosphate deposits are an unique mineral high in magnesium with approximately 30% of the phosphorus present as pyrophosphate. The high pyrophosphate content of these deposits could alter their crystalline structure and prevent the transformation to apatite. The infrared features, high magnesium content of the deposit, and resistance of pyrophosphate in the deposit to hydrolysis by pyrophosphatase suggests that the pyrophosphate may be deposited as the magnesium salt.


Bone pyrophosphate in uremia and its association with extraosseous calcification.

The mean bone pyrophosphate was 0.360 +/- 0.15 mg/g in 8 controls and 1.22 +/- 1.39 mg/g bone in 27 uremic patients (P less than 0.0025). 13 of the 27 uremic patients had bone pyrophosphate levels greater than 2 SD above control values. The ash content of uremic bones with increased pyrophosphate levels (group II) was 56 +/- 9% as compared to 64 +/- 2% in control bones (P less than 0.01) and 60 +/- 7% in uremic bones having normal pyrophosphate levels (P less than 0.1) (group I). The magnesium content of bones in group II was 338 +/- 47 as compared to 211 +/- 13 (P less than 0.0005) in the controls and 294 +/- 73 mmol/kg ash (P less than 0.05) in group I. In group II, but not group I, there was a significant inverse correlation between duration of dialysis and percent bone ash (r = -0.59) (P less than 0.05). A definite relationship existed between elevated bone pyrophosphate levels and soft tissue calcification. In group II the mean pulmonary calcium content was 530 +/- 459 as compared to 32 +/- 26 mmol/kg/ash in group I (P less than 0.0025). All patients with a bone pyrophosphate level greater than 1.4 mg/g bone had extensive pulmonary calcification. It is concluded that the excess bone pyrophosphate present in some uremic patients is either deposited in the apatite crystal in the transphosphorylated form or else as the magnesium salt since the pyrophosphate is resistant to pyrophosphatase and surface adsorption of pyrophosphate is not altered by the increased bone pyrophosphate levels. The excess bone pyrophosphate could disturb bone calcification mechanisms in uremic patients. The association between increased bone pyrophosphate and soft tissue calcification suggests that the disordered pyrophosphate metabolism may be important in the pathogenesis of extraosseous calcification.


Serum lipoproteins and apolipoproteins in rats with streptozotocin-induced diabetes.

The lipoproteins of rats fed a high sucrose diet and made diabetic by administration of 45 mg/kg of streptozotocin were studied. All lipoprotein classes were found to be present in increased concentrations. The apolipoprotein composition of the various lipoprotein fractions was studied by polyacrylamide-gel electrophoresis in the presence of 8 M urea, isoelectric focusing in the presence of 8 M urea, and sodium dodecyl sulfate gel electrophoresis in polyacrylamide gels. In the very low density lipoproteins (VLDL) of diabetic rats, there was a marked alteration in the relative amounts of C proteins by polyacrylamide-gel electrophoresis, and this was found by isoelectric focusing to be primarily a relative increase in C-III-3 apoprotein and a decrease in C-III-O. In addition, in the diabetic rats, the VLDL contained a protein of mol wt 46,000, the A-IV protein, which normally is only present in the high density lipoproteins. In the high density lipoproteins, (HDL) the same alterations in pattern of the C proteins seen in the VLDL were present. Furthermore, the arginine-rich and A-IV protein normally present in HDL could not be detected in the HDL, although the other apolipoproteins are present. Apolipoprotein concentrations were determined by quantitative immunoelectrophoresis. It was found that in the diabetic rats there was an increase in the total amount of apo-B in the plasma, with the increment divided proportionately between the VLDL and the low density lipoprotein (LDL). The total apo-C concentration of plasma increased minimally. The A-IV concentration of plasma increased by 27%; it decreased markedly in the HDL, but appeared in increased amounts in both VLDL and in the d greater than 1.21 fraction. The arginine-rich protein decreased by 63% in the plasma and decreased significantly in the HDL, but increased in VLDL, LDL, and in the d greater than 1.21 fraction. These alterations in apolipoprotein patterns in diabetic animals suggest that the apolipoproteins may play an important role in determining the concentration of various lipoprotein fractions, or may be the result of altered metabolism of the lipoproteins. These lipoproteins with altered apolipoprotein composition may have important biologic differences from normal lipoporteins. Nevertheless, the HDL, despite the fact that it is deficient in some of its major constituents, was unchanged in its cholesterol content.


Altered thyroidal responsivity to thyrotropin induced by circulating thyroid hormones. A "short-loop" regulatory mechanism?

We studied the effect of thyroid hormone administration on responsivity of murine thyroid to exogenous thyrotropin (TSH) in order to explore the possibility that the thyroid gland might be directly inhibited by its own hormones. In the rat both L-thyroxine (T4) and 3,5,3'-L-triiodothyronine (T3) pretreatment inhibited TSH-induced thyroidal ornithine decarboxylase (ODC) activity in vivo in a dose-related manner (half-maximal inhibition, 1.7 mug/rat and 0.6 mug/rat, respectively). Other structurally related compounds exhibited the following inhibitory potencies compared to T4: T3, 283%; triiodothyroacetic acid, 40%; D-T4, 18%; 3,5-L-diiodothyronine, 9%. Monoiodotyrosine, diiodotyrosine, and iodide were not inhibitory. The full inhibitory effect of T4 or T3 was observed when thyroid hormone was administered from 96 to 12 h before TSH and was also seen in hypophysectomized animals. Pretreatment with T4 or T3 in divided doses over 2 1/2 days inhibited TSH-induced increase in [1-14C]glucose oxidation to 14C02 and [3H] leucine incorporation into protein in rat thyroid. In the mouse T4 or T3 pretreatment (0.25-25 mug daily) caused dose-related inhibition of both thyroidal ODC activity and 131I release induced by TSH in vivo. In mice on a low-iodine diet (LID) but not in animals on a regular diet (RD) NaI pretreatment also blunted TSH-induced thyroidal ODC activation and 131I release. When LID or RD mice were pretreated with 12.5-125 mug of T4 or T3 over 2 1/2 days, TSH-induced in vitro stimulation of thyroid cyclic 3',5'-adenosine monophosphate formation was inhibited in a dose-related manner; NaI pretreatment was inhibitory in the LID mouse only. Prior administration of exogenous TSH blunted the activation of thyroid ODC and thyroid hormone release induced by subsequent TSH administration in rat and mouse. These studies indicate altered thyroid responsivity to TSH under the influence of circulating thyroid hormones and suggest the existence of a "short-loop" negative feedback regulating thyroid function.


Ultrasound of the parotid gland.

Five cases of parotid gland masses were studied by ultrasound. Only retention cysts and true cysts of the parotid gland appear sonolucent at low and high gain settings. Benign and malignant tumor present as solid masses except for the Warthin's tumor which is sonolucent at lower gain settings but has a lattice of internal echoes at higher gain settings.


False positive infectious mononucleosis serology in epilepsy.

Positive serum tests for infectious mononucleosis (IM) unaccompanied by the clinical syndrome or blood changes characteristic of the disease were detected in 39/177 (22%) mentally subnormal patients investigated with three different commercially available IM slide tests. Slide positively was still detectable six months later in 27/35 (77%) positive reactors. Positive IM slide tests were significantly associated with anticonvulsant, primarily phenobarbitone therapy and with concomitant elevations of immunoglobulins IgG and IgM. Epstein Barr virus antibodies were not detectable in the sera of 8/38 (21%) positive slide reactors. The implications of these observations are discussed and attention is drawn to the variations in sensitivity, specificity, and comparability of different IM slide tests.


Multifocal malignant histiocytoma.

The pathology of malignant histiocytoma has recently been defined and its malignancy confirmed beyond doubt. A unique example of this tumour is described, a search of the literature having failed to find a previously recorded case with a multifocal origin.


EB virus antibody and infectious mononucleosis in a boarding school for boys.

A study of EB virus antibody was undertaken in a boarding school for boys between 11 and 18 years of age; 35% of boys had EBV antibody when first sampled and a small number acquired antibody each year. Some of these boys had had no recorded illness during the period before the first positive sample and some developed classic infectious mononucleosis which was accompainied by a positive heterophil antibody test and detection of EB virus specific IgM. In a school of 800 boys there were 13 cases of infectious mononucleosis in four years.


An epidemic of diarrhoea in human neonates involving a reovirus-like agent and 'enteropathogenic' serotypes of Escherichia coli.

During December 1974, an epidemic of diarrhoea occurred in the Royal Children's Hospital, Melbourne, in a ward caring for neonates with acute or chronic medical and surgical problems. Electron microscopy of diarrhoeal faeces revealed a reovirus-like particle ('duovirus' or 'rotavirus') known to cause acute enteritis in older children. This virus is considered to have been primarily involved in the aetiology of the epidemic. In addition, three 'enteropathogenic' serotypes of Escherichia coli were isolated from babies during the epidemic, but none produced enterotoxin when tested in ligated ileal loops of rabbits or in monolayers of Y1 adrenal cells. Further epidemics of neonatal diarrhoea must be studied using culture and electron microscopy of faeces to determine the relative importance of this virus and of E. coli in the aetiology of diarrhoea in this age-group.


Urinary cyclic adenosine monophosphate in young adults and elderly subjects.

The 24-hour urinary excretion of cyclic 3',5'-adenosine monophosphate (cAMP) was measured by a protein-binding assay in 55 healthy volunteers (aged 20-35 yr) and in 30 hospitalized elderly subjects (aged 70-93 yr). In the older subjects the mean 24-hour cAMP excretion was significantly lower; the correlation between cAMP excretion and age demonstrated a progressive decrease from the age of 70 to the tenth decade. Many different factors could account for the reduced urinary cAMP excretion in elderly subjects: a decline in the reactivity of the adenyl cyclase-cAMP system related to physiological ageing; reduced physical activity; a reduction in the glomerular filtration rate or decreased production of cAMP by tubular cells in the senile kidney.


Tumor- and drug-induced cutaneous neuro-phospholipidosis.

Drugs and metastatic malignant tumors induce severe alterations in cutaneous axons. The most prominent ultrastructural feature of axonal dystrophy of this type was the presence of multiple lamellated osmiophilic bodies (LOB), similar to those occurring in hereditary disorders with increase of brain gangliosides and lipid storage. Chloroquine, ergotamine, ethaverine and chronic abuse of non-narcotic analgesic drugs seem to be responsible for the drug-induced cutaneous neuro-phospholipidosis, whereas the same condition was found in two patients with metastatic bronchogenic carcinoma and malignant melanoma.


Immunological properties of bovine serum lipoproteins and chemical analysis of their protein moieties.

Four classes of bovine serum lipoproteins were isolated by precipitation with dextran sulfate, ultracentrifugation, and preparative electrophoresis on polyacrylamide gel. Very low density lipoprotein (d less than 1.019 g/ml) was related immunologically to low density lipoprotein-two (d 1.039 to 1.060 g/ml) and high density lipoprotein (d 1.060 to 1.210 g/ml) was related immunologically to low density lipoprotein-one (d 1.019 to 1.039 g/ml), but the two pairs were immunologically distinct. The major N-terminal amino acid of both high density lipoprotein and low density lipoprotein-one was aspartic acid, and that of low density lipoprotein-two was glutamic acid. Very low density lipoprotein had both aspartic acid and glutamic acid as the major N-terminal amino acids. None of the lipoproteins was identical with any other with respect to amino acid composition, but high density lipoprotein and low density lipoprotein-one were similar to each other and different from low density lipoprotein-two. Very low density lipoprotein was similar to both low density lipoprotein-one and low density lipoprotein-two. It is concluded that the proteins of high density lipoprotein and of low density lipoprotein-one are related and are different from that of low density lipoprotein-two. The protein of very low density lipoprotein is related to that of low density lipoprotein-two but may contain polypeptides of high density lipoprotein or low density lipoprotein-one.


Cellular gluconeogenesis by lactating bovine mammary tissue.

The gluconeogenic capacity of mammary tissue of lactating cow was investigated by incubating mammary tissue slices with alanine, glutamate, lactate, pyruvate, or glycerol in conjunction with acetate and glucose (10mM or 1 mM). In no case was any substrate incorporated into glucose per se. In lactose synthesis, glucose was the major source of carbon although glycerol also was incorporated into lactose. Alanine, glutamate, lactate, or pyruvate were not incorporated into lactose at optimum (10 mM) or suboptimum (1 mM) concentrations of glucose. Activity of glucose-6-phosphatase was negligible in mammary tissue, less than 1% of the activity in liver or kidney tissue from the same cows. Pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and fructose-1,6-diphosphatase were in cow mammary tissue, but the activities were lower than in liver. Gluconeogenic substrates were not converted to glucose regardless of whether the incubation contained an optimum (10 mM) or a suboptimum (1 mM) glucose concentration. Consistent with the inability of cow mammary tissue to convert gluconeogenic metabolites to glucose is the virtual absence of glucose-6-phosphatase and the lack of excess gluconeogenic substrates available to the intact mammary gland of lactating cow.


Determination of physical properties of bovine serum lipoproteins by analytical ultracentrifugation.

Pure samples of low density lipoprotein-one (d 1.019 to 1.039 g/ml), low density lipoprotein-two (d 1.039 to 1.060 g/ml), and high density lipoprotein (d 1.060 to 1.210 g/ml) were isolated from bovine serum and their properties studied in the analytical ultracentrifuge. Approach-to-equilibrium experiments indicated that the lipoprotein classes were homogeneous. Molecular weights of the lipoproteins given by this method (low density lipoprotein-one, 1.14 x 10(6); low density lipoprotein-two, 2.37 x 10(6); high density lipoprotein, .576 x 10(6)) agreed well with those obtained by a high-speed-equilibrium method (1.03 x 10(6), 2.15 x 10(6), and .567 x 10(6)). Linear plots of flotation rate (corrected for viscosity) against the density of the medium were obtained in sedimentation velocity experiments and on extrapolation gave values for the hydrated density of 1.032 g/ml for low density lipoprotein-one, 1.044 g/ml for low density lipoprotein-two, and 1.071 g/ml for high density lipoprotein. The density of the material which would have to be added to high density lipoprotein to give it the physical properties of low density lipoprotein one was less than 1 g/ml, which suggested that it was predominantly lipid.


Evaluation of pituitary-adrenal function in patients with chronic bronchial asthma following substitution of steroid treatment with disodium cromoglycate (Lomudal).

In a group of 32 steroid-dependent asthmatic patients an attempt was made to replace steroid treatment with disodium cromoglycate (Lomudal). Withdrawal of steroids was accompanied by a transitory stage of combined corticotropin-Lomudal treatment for 6 to 8 mo. Pituitary-adrenal function was assessed by ACTH and Metopirone test. Before treatment an impairment of pituitary-adrenal function was found in most of our patients, although in 26 patients a normal increment of plasma cortisol was found after ACTH stimulation. At the end of the combined treatment, 17 patients are now on Lomudal with normal pituitary-adrenal function, 9 patients need small quantities of steroids occasionally, and 6 patients are steroid-dependent.