Interference of simultaneous skin tests in delayed hypersensitivity. The interference of two simultaneous skin test reactions of intermediate strength has been studied in the guinea-pig, using four different antigens, i.e. ovalbumin, horse cytochrome c, PPD and oxazolone. Skin test reactions were evaluated at 4, 24 and 48 h by measuring three parameters: increase in skin thickness, diameter of erythema and intensity of erythema. When an Arthus reaction was elicited simultaneously with a delayed hypersensitivity (DH) reaction, no effect on the DH reaction was observed. When two simultaneous DH reactions were elicited with different antigens, the risk of interference appeared to be rather small. When, however, the same antigen was used for both skin tests, suppression of at least one parameter of a DH-reaction was found in almost all experiments. Suppression of one skin test by another one could not be reduced by introducing a large distance between the two skin tests. As complete inhibition of either of the parameters never occurred, multiple skin testing may allow one to obtain a qualitative impression of the state of delayed hypersensitivity; when, however, reliable quantitative data are needed, the performance of more than one skin test at a time should be avoided.


Spontaneous and induced cytotoxic properties of human adherent mononuclear cells: killing of non-sensitized and antibody-coated non-erythroid cells. A method is described which demonstrates that human adherent mononuclear cells have a strong capacity to kill human nucleated target cells. Cytotoxicity was measured by the release of 51chromium from two established cell lines. Freshly prepared monolayers killed both non-sensitized and anti-body coated K562 and CLA-4 target cells. These spontaneous events decreased after short term culture. A second peak of cytotoxic activity was induced by activating the effector cells with endotoxin-treated serum or phytohaemagglutinin. Cytotoxicity was inhibited by silica particles. Studies with alpha-naphthyl esterase showed that 95% of freshly prepared cells and 99% of monolayers cultured for 68 h were monocytes. These studies suggest that adherent monocytes are the predominant cytotoxic effector cell although a contributing effect by the small numbers of contaminating lymphocytes can not be excluded. Evaluation of monocyte cytotoxicity should have a useful role in clinical investigation.


The role of locomotion in lymphocyte migration. In this study, the role of cell locomotion in lymphocyte traffic was investigated following the in vivo fate of 51Cr labelled lymph node cells pretreated in vitro with substances known to modify cell motility. The results obtained show that lymphocyte migration is an energy dependent process, as it could be impaired by sodium azide. That it requires the integrity of microfilaments, as it was modified by Cytochalasin B; and that it is altered by isoproterenol, theophylline, prostaglandin E1 and dibutyryl cyclic-AMP. The relevance of these findings to the process of lymphocyte recirculation is discussed.


Chemical properties of the principle in C. parvum that produces splenomegaly in mice. Suspensions of Wellcome C. parvum strain 6134 produce splenomegaly in mice when injected i.p. in amounts as low as 20 microgram. This lymphoreticular stimulatory activity is extremely sensitive to cell breakage and is abolished by heating for 4 h at 100 degrees. Periodate oxidation of the bacteria destroys their capacity to produce splenomegaly and abrogates the agglutination of intact C. parvum by Con A. Mild HCl hydrolysis also abolished the splenomegaly but phenol:chloroform:ether and chloroform:methanol extractions did not. These results suggest that the relevant stimulatory principle in C. parvum is of carbohydrate nature, and most probably present on the surface of the bacterium.


Lymphocyte surface modulation and glycosphingolipids. Using the fluorescent antibody method, identical localization of ligand and glycosphingolipids was found in human peripheral blood B lymphocytes patched and capped with polyvalent anti-immunoglobulin. cAMP also showed the same pattern of localization. It is suggested that glycosphingolipids are hydrogen bonded to receptor-bearing membrane macromolecules and are borne with them into the ligand-induced patches and caps. Here the asymmetric carbon chains of the glycosphingolipids modify the local lipid environment, leading to activation of the adenyl cyclase in the membrane which produces the cAMP.


Adjuvant for IgE antibody and islet-activating protein in Bordetella pertussis. A new purified protein, with a molecular weight of 77,000, isolated from the supernatant of Bordetella pertussis culture fluid and called islet-activating protein (IAP), had adjuvant activity for IgE antibody in addition to leukocytosis-promoting, histamine-sensitizing and islet-activating activities. None of the three subunits (F1, F2 and F3) was biologically active by itself. All of the four activities were recovered in the complexes of subunits F1.3 and F2.3. These results suggest that the activities are brought about by a common factor inherent in B. pertussis.


Characteristics of a low-molecular-weight factor extracted from mouse tumors that affects in vitro properties of macrophages. A dialysable low-molecular-weight factor capable of affecting in  vitro properties of macrophages was extracted from four different mouse tumors. This factor not only modulates closely related properties of peritoneal macrophages such as spreading and migration but also inhibits lipopolysaccharde-induced tumoricidal activity of these cells. It can be extracted not only from tumor tissues but also from tumor cells grown in vitro. The appearance of this factor is unique to tumors and it is not present in detectable quantities in normal tissues. The factor from one of the tumors, Lewis lung carcinoma, was purified extensively and the partially purified factor retains all the above effects on macrophages. It is not sensitive to pronase or a mixture of bovine spleen phosphodiesterase II, E. coli alkaline phosphatase and pancreatic ribonuclease. The factor is lipid-like in character and it is soluble in both organic solvents and aqueous media. It has ionizable group(s) and is anionic at neutral pH but non-ionic under acidic conditions.


Neutralization of pseudotypes of vesicular stomatitis virus by sera from avian retrovirus-infected hosts. We investigated the capacity of lymphocytes and sera from chickens bearing tumors induced by avian sarcoma viruses (ASV) to interact with phenotypically mixed particles of vesicular stomatitis virus (VSV) and ASV. Immune chicken sera were able to specifically neutralize such VSV pseudotypes. This ability could be absorbed out, however, on purified preparations of avian retroviruses, suggesting that reactivity was primarily against avian retrovirus enveloped components. Supernatant fluids containing phenotypically mixed particles were unable to stimulate division of lymphocytes of tumor-bearing hosts, an ability possessed by culture fluids containing native ASV particles. Polyacrylamide gel analysis was unable to resolve any distinct pseudotype protein, which was not present in either of the parental virus types. Treatment with crude preparations of ultraviolet (UV)-irradiated VSV pseudotype material did not afford immunity against subsequent challenge with live ASV.


Cell surface glycoprotein changes in Epstein-Barr virus-positive and -negative human hematopoietic cell lines. It has previously been shown that differential fucose labelling of many normal and homologous tumor cells, followed by proteolytic release and degradation, yields glycopeptides which upon gel filtration shown an increase in fast-eluting glycopeptides for the tumor cells. This technique has now been applied to cell-surface glycoproteins of different human hematopoietic cell lines. These lines included Epstein-Barr virus (EBV)-carrying lymphoblastoid cell lines of presumed non-neoplastic origin, and malignant EBV-genome-positive Burkitt lymphoma and EBV-negative non-Burkitt lymphoma, leukemia and myeloma lines. As compared with normal peripheral lymphocytes, both the lymphoblastoid type of cell lines and the different types of lines of proven malignant ancestry contained the fast-eluting glycopeptides on their cell surface with very few exceptions. It is therefore concluded that (I) malignant conversion of human lymphoid cell in vivo is commonly, but not obligatorily, associated with a specific change in the composition of the fucosyl glycopeptides, and (2) EBV infection of B lymphocytes does not lead only to the well-documented immortalization in vitro but also, as a rule, to the same type of alteration in fucosyl glycopeptides as was demonstrated for the neoplastic cell lines. It proved possible to distinguish several categories of hematopoietic cell lines due to the effect that pretreatment of the glycopeptides with neuraminidase or mild acid exerted on their subsequent chromatographic behavior.


Establishment and characterization of a human null-cell lymphoblastic lymphoma cell line (K-LL-3). A new cell line (K-LL-3) with exceptional characteristics was derived from a bone-marrow aspirate from a patient with poorly differentiated lymphoblastic lymphoma. The cell line was established and the cells maintained on agar feeders for over 1 year. The morphology and cytochemical staining of the primary tumor and the cell line were remarkably similar. The growth characteristics, chromosome pattern, cell-surface receptors and Epstein-Barr pattern, cell-surface receptors and Epstein-Barr virus studies clearly differentiated this cell line from non-malignant lymphoblastoid cell lines. The assay technique used was able to correlate in vitro colony growth with the patient's clinical course. The cells grew on the agar as colonies rather than as a single-cell suspension, and individual colonies could be aspirated and successfully passaged. The K-LL-3 cells lacked cell-surface markers (cytoplasmic and surface immunoglobulins, Fc receptors, C3 receptors, SRBC) except HL-A and thus were classified as null cells. These cells were EBNA (Epstein-Barr virus-specific nuclear antigen) negative and had a pseudodiploid 46XY karyotype.


Enhancement of 2-acetylaminofluorene liver carcinogenesis in rats fed a choline-devoid diet. The effects of feeding a choline-devoid (CD) or a choline-supplemented (CS) diet on the induction of liver tumors in rats by 2-acetylaminofluorene (AAF) were investigated. Male Sprague-Dawley rats were fed either a CD or a CS diet containing 0.0075% AAF. Three to eight animals were killed 1, 3, 4 and 6 months thereafter. Well to moderately well differentiated hepatocellular carcinomas developed in 50% and 75% of rats fed the CD + AAF diet for 4 and 6 months, respectively. Cholangiocarcinomas developed, after 6 months, in 38% of the animals. No tumor developed in rats fed the CS + AAF diet. The results extend to AAF those previously obtained with DL-ethionine and azaserine showing that feeding a CD diet greatly potentiates the induction in rats of liver tumors by chemical carcinogens.


Reproducibility and relation to specific and non-specific anti-tumor resistance of the tumor "sneaking through" phenomenon. The reproducibility and immunological specificity of the tumor "sneaking through" phenomenon and enhancement of tumor growth were studied in syngeneic and random-bred Syrian hamsters by means of a quantitative modification of the transplantation test. After primary challenge the phenomenon was neither observed in normal animals nor in animals effectively immunized against tumor. However, it was regularly observed in some "immune" animals after secondary challenge. In primary challenge of animals "sneaking through" phenomenon was most often observed in animals pretreated with large doses of heat-inactivated tumor cells. This characteristic could not be transferred with serum of pretreated animals. In contrast to specific tumor immunity, the "sneaking through" pbenomenon appeared to be immunologically non-specific. This was observed in cross-transplantation tests with tumor cells bearing different TSTAs. Thus, TSTA is not an inducer and apparently not a target for a response leading to enhancement of tumor growth in pretreated hamsters. Experiments demonstrating enhanced tumor growth in pretreated animals at the same time demonstrate two other possibly more essential findings: (1) normal animals are naturally resistant to transplantation of 1 to about 1 x 10(3) (or more) tumor cells; and (2) this resistance can be totally abrogated by the pretreatment of normal animals with tumor cell preparations. The preliminary data demonstrate that abrogation of natural anti-tumor resistance in adult hamsters subsequently inoculated with SV40 leads to rapid development of primary tumors in such animals. The development of specific anti-tumor immune response in animals treated with inactivated tumor cell preparations was also studied. Significant non-specific inhibition of sponse in Syrian hamsters treated with inactivated syngeneic tumor cells was observed. The data obtained are considered to demonstrate two anti-tumor defense systems in the animal, i.e., non-specific natural resistance and specific anti-tumor immunity. The first seems to be responsible for elimination of low numbers of tumor cells in the normal organism and also to be esseitial for effective induction and functioning of the specific anti-tumor immunity.


Effect of ACTH on circadian periodicity of nuclear volume and mitotic division in the zona fasciculata externa of the adrenal cortex of mice. In cells of the zona fasciculata externa of the adrenal cortex of mice, the maximal value of nuclear volume is observed in evening and night time, while the mitotic peak occurs in the early part of the day. Ten day subcutaneous injection of 1.5 units of ACTH twice in 24 hr produced nuclear hypertrophy and stimulation of mitotic activity of cells of the zona fasciculata externa. The circadian periodicity of nuclear volume in mice injected with ACTH is disturbed, while the circadian rhythm of mitotic activity is retained.


Reproductive performance of apparently healthy cattle persistently infected with bovine viral diarrhea virus. A Holstein-Friesian bull and three Holstein-Friesian cows were seronegative for bovine viral diarrhea (BVD) virus but were persistently infected with the virus. Virus was isolated from buffy coat cells and nasal and lacrimal secretions during their lifetime, and they remained free of clinical signs of BVD. The three cows were pregnant when purchased, and they gave birth to full-term calves. One calf lived only a few hours, one calf became ill and died within a few days, and one calf became ill and was euthanatized within a few weeks. One cow was then bred and became pregnant but aborted a 7-month fetus. A second cow was bred approximately 5 months after parturition but did not conceive. The third cow was necropsied 6 weeks after calving, because of loss of weight. Although the bull's semen contained BVD virus when seropositive cows were bred, normal calves were born. When seronegative heifers were bred, they became seropositive to BVD virus within two weeks, with higher titers in six weeks. On heifer conceived after one service but aborted a 6-month fetus. Three others continued to have estrous cycles until their titers rose to 1:128, then they conceived and gave birth to normal calves. Another heifer conceived on the first service, had a titer of 1:128 two weeks after breeding, and gave birth to a normal calf.


Enzymes of pyrimidine metabolism in Mycoplasma mycoides subsp. mycoides. The major pathways of ribonucleotide biosynthesis in Mycoplasma mycoides subsp. mycoides have been proposed from studies on its use of radioactive purines and pyrimidines. To interpret more fully the observed pattern of pyrimidine usage, cell extracts of this organism have been assayed for several enzymes associated with the salvage synthesis of pyrimidine nucleotides. M. mycoides possessed uracil phosphoribosyltransferase, uridine phosphorylase, uridine (cytidine) kinase, uridine 5'-monophosphate kinase, and cytidine 5'-triphosphate synthetase. No activity for phosphorolysis of cytidine was detected, and no in vitro conditions were found to give measurable deamination of cytidine. Of the two potential pathways for incorporation of uridine, our data suggest that this precursor would largely undergo initial phosphorolysis to uracil and ribose-1-phosphate. Conversely, cytidine is phosphorylated directly to cytidine 5'-monophosphate in its major utilization, although conversion of cytidine to uracil, uridine, and uridine nucleotide has been observed in vivo, at least when uracil is provided in the growth medium. Measurements of intracellular nucleotide contents and their changes on additions of pyrimidine precursors have allowed suggestions as to the operation of regulatory mechanisms on pyrimidine nucleotide biosynthesis in M. mycoides in vivo. With uracil alone or uracil plus uridine as precursors of pyrimidine ribonucleotides, the regulation of uracil phosphoribosyltransferase and cytidine 5'-triphosphate synthetase is probably most important in determining the rate of pyrimidine nucleotide synthesis. When cytidine supplements uracil in the growth medium, control of cytidine kinase activity would also be important in this regard.


Properties of two cyclic nucleotide-deficient mutants of Neurospora crassa. Studies on the crisp-1 (cr-1), cyclic adenosine 3',5'-monophosphate (cAMP)-deficient mutants of Neurospora crassa were undertaken to characterize the response of these mutants to exogenous cyclic nucleotides and cyclic nucleotide analogs. A growth tube bioassay and a radioimmune assay for cyclic nucleotides yielded the following results. (i) 8-Bromo cAMP and N6-monobutyryl cAMP but not dibutyryl cAMP are efficient cAMP analogs in Neurospora, stimulating mycelial elongation of the cr-1 mutants. Exogenous cyclic guanosine 3'5'-monophosphate (cGMP) also stimulates such mycelial elongation. (ii) Both cAMP levels and cGMP levels found in cr-1 mycelia are lower than those in wild type. However, the levels of both cyclic nucleotides are normal in conidia of cr-1. The data on cr-1 mycelia and those reported earlier in Escherichia coli (M. Shibuya, Y. Takebe, and Y. Kaziro (Cell 12:528-528, 1977) show a previously unexpected relationship between cAMP and cGMP metabolism in microorganisms. The semicolonial morphology of another adenylate cyclase-deficient mutant of Neurospora, frost, was not corrected by exogenous cyclic nucleotides or by phosphodiesterase inhibitors indicating that the frost morphology is probably not caused by low endogenous cAMP levels. The low adenylate cyclase activity and the abnormal morphology of frost may be related separately to the linolenate deficiency reported in the mutant.


Pyridine nucleotide cycle of Salmonella typhimurium: isolation and characterization of pncA, pncB, and pncC mutants and utilization of exogenous nicotinamide adenine dinucleotide. Mutants of Salmonella typhimurium LT-2 deficient in nicotinamidase activity (pncA) or nicotinic acid phosphoribosyltransferase activity (pncB) were isolated as resistant to analogs of nicotinic acid and nicotinamide. Information obtained from interrupted mating experiments placed the pncA gene at 27 units and the pncB gene at 25 units on the S. typhimurium LT-2 linkage map. A major difference in the location of the pncA gene was found between the S. typhimurium and Escherichia coli linkage maps. The pncA gene is located in a region in which there is a major inversion of the gene order in S. typhimurium as compared to that in E. coli. Growth experiments using double mutants blocked in the de novo pathway to nicotinamide adenine dinucleotide (NAD) (nad) and in the pyridine nucleotide cycle (pnc) at either the pncA or pncB locus, or both, have provided evidence for the existence of an alternate recycling pathway in this organism. Mutants lacking this alternate cycle, pncC, have been isolated and mapped via cotransduction at 0 units. Utilization of exogenous NAD was examined through the use of [14C]carbonyl-labeled NAD and [14C]adenine-labeled NAD. The results of these experiments suggest that NAD is degraded to nicotinamide mononucleotide at the cell surface. A portion of this extracellular nicotinamide mononucleotide is then transported across the cell membrane by nicotinamide mononucleotide glycohydrolase and degraded to nicotinamide in the process. The remaining nicotinamide mononucleotide accumulates extracellularly and will support the growth of nadA pncB mutants which cannot utilize the nicotinamide resulting from the major pathway of NAD degradation. A model is presented for the utilization of exogenous NAD by S. typhimurium LT-2.


Catabolite and transient repression in Escherichia coli do not require enzyme I of the phosphotransferase system. Transient and catabolite repression with changes in intracellular concentrations of cyclic adenosine 3',5-monophosphate is produced by glycerol and by glucose-6-phosphate in a strain with a partial deletion of the structural gene for enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system.


Control of cyclic adenosine 3',5'-monophosphate levels by depolarizing agents in fungi. It has been reported that diverse treatments which depolarize the plasma membrane of Neurospora crassa produce rapid increases in cyclic adenosine 3',5'-monophosphate (cyclic AMP) levels. In the current study, membrane active antibiotics, which are known or putative depolarizing agents, were found to produce similar cyclic AMP increases, not only in N. crassa, but also in the distantly related fungi Saccharomyces cerevisiae and Mucor racemosus. Uncouplers of oxidative phosphorylation, which have been found to depolarize Neurospora, also produced cyclic AMP increases in all three fungi. The time course of the cyclic AMP response to these various treatments was similar in all three fungi. The fungal studies and studies on depolarized central nervous tissue suggest that cyclic AMP increases may be produced in response to plasma membrane depolarization in diverse eucaryotic cells. A model is proposed for eucaryotic microorganisms in which membrane depolarization serves as a signal of breakdown of the plasma membrane integrity. The subsequent cyclic AMP increase, in turn, may mediate cellular response to help protect the plasma membrane from chemical and mechanical threats to its integrity.


Metabolism of carbohydrate derivatives by Pseudomonas acidovorans. Wild-type Pseudomonas acidovorans strain A1 was unable to grow on glycerol or glucose as sole source of carbon and energy although it grew well on gluconate. Spontaneous glycerol-positive mutants, which apparently had become permeable to glycerol, were readily isolated, but glucose-positive mutants did not occur. P. acidovorans lacked glucose dehydrogenase and glucokinase, which were sufficient to account for its inability to grow on glucose. Gluconate was degraded exclusively via a noncoordinately induced Entner-Doudoroff pathway. Phosphogluconate dehydrogenase was undetectable. In contrast to P. aeruginosa, P. acidovorans possessed a single glyceraldehyde-phosphate dehydrogenase activity, which was NAD+ specific and constitutive, and an inducible pyruvate kinase. Moreover, growth of glycerol-positive strain K2 on glycerol did not induce any of the enzymes related to metabolism of hexosephosphate derivatives as occurs in fluorescent pseudomonads.


Lipid and cell wall changes in an inositol-requiring mutant of Neurospora crassa. An inositol deficiency in the inositol-requiring (inl) mutant of Neurospora crassa led to changes in the composition of the inositol-containing lipids and the cell wall. On deficient levels of inositol, phosphatidyl inositol decreased by 23-fold, di(inositolphosphoryl) ceramide decreased by 4-fold, and monoinositolphosphoryl ceramide increased slightly. The inositol deficiency also led to an aberrant hyphal morphology and changes in both the amount of cell wall and the amino sugar content of the cell wall. The glucosamine content of the cell wall decreased by 50%, the galactosamine increased by 50%, but no significant changes were found in the content of the cell wall amino sugar precursors, or in the amino acid, glucose, or total hexose content of the cell wall. Inositol-containing compounds were found associated with purified cell wall material. These compounds were bound tightly to the cell wall but could be removed by treatment with alkali, a treatment which disrupts the cell wall integrity. Possible mechanisms of how changes in lipid composition can affect cell wall biosynthesis are discussed.


The formation of enzyme-bound and medium pyrophosphate and the molecular basis of the oxygen exchange reaction of yeast inorganic pyrophosphatase. Yeast inorganic pyrophosphatase, with 10 mM 32Pi and 10 mM Mg2+ present at pH 7.3 TO 7.6, rapidly forms enzyme-bound pyrophosphate equivalent to about 5% of the total catalytic sties on the two enzyme subunits. The enzyme thus appears to bind PPi so as to favor thermodynamically its formation from Pi. The enzyme catalyzes a measurable equilibrium formation of free PPi at a much slower rate. Under similar conditions, the enzyme catalyzes a rapid exchange of oxygen atoms between Pi and water with the relative activation by metals being Mg2+ greater than Zn2+ greater than Co2+ greater than Mn2+. Millisecond mixing and quenching experiments demonstrate that the rate of formation and cleavage of the enzyme-bound PPi is rapid enough to explain most or all of the oxygen exchange reaction.


Purification, properties, and kinetics of D-ribulokinase from Aerobacter aerogenes. The enzyme D-ribulokinase from Aerobacter aerogenes was purified to near homogeneity. The molecular weight, as determined by Sephacryl gel chromatography, is 116,000. The subunit molecular weight, determined by sodium dodecyl sulfate-gel electrophoresis, is 59,000, suggesting that D-ribulokinase is a dimer of identical subunits. Initial rate kinetic studies, involving substrate analogs and products, were carried out. These investigations support a kinetic mechanism of the Random Bi Bi type. Isotope partitioning, utilizing D-[3H]ribulose, indicates that the mechanism is steady state Random Bi Bi.


Amino acid sequence of subunit V of bovine heart cytochrome oxidase, the heme alpha-containing subunit. The complete amino acid sequence of the heme alpha-containing subunit V of bovine heart cytochrome oxidase was determined to be: H2N-Ser-His-Gly-Ser-His-Glu-Thr-Asp-Glu-Glu-Phe-Asp-Ala-Arg-Trp-Val-Thr-Tyr-Phe-Asn-Lys-Pro-Asp-Ile-Asp-Ala-Trp-Glu-Leu-Arg-Lys-Gly-Met-Asn-Thr-Leu-Val-Gly-Tyr-Asp-Leu-Val-Pro-Glu-Pro-Lys-Ile-Ile-Asp-Ala-Ala-Leu-Arg-Ala-Cys-Arg-Arg-Leu-Asn-Asp-Phe-Ala-Ser-Ala-Val-Arg-Ile-Leu-Glu-Val-Val-Lys-Asp-Lys-Ala-Gly-Pro-His-Lys-Glu-Ile-Tyr-Pro-Tyr-Val-Ile-Gln-Glu-Leu-Arg-Pro-Thr-Leu-Asn-Glu-Leu-Gly-Ile-Ser-Thr-Pro-Glu-Glu-Leu-Gly-Leu-Asp-Lys-Val-COOH. The subunit V is a single polypeptide which consists of 109 amino acid residues. The protein contains 48.6% hydrophobic residues and 34.0% hydrophilic residues and it is an acidic protein having a net charge of -3 at neutral pH. The molecular weight of subunit V was calculated to be 12,436 and that for the heme alpha-containing polypeptide was 13,295.


Purification and properties of cyclic AMP-independent glycogen synthase kinase 1 from rabbit skeletal muscle. A cyclic AMP-independent casein (phosvitin) kinase eluted from a phosphocellulose column with 0.35 M KCl also possesses glycogen synthase kinase activity. This kinase, designated synthase kinase 1, is separable from other cyclic AMP-independent protein kinases, which also contain glycogen synthase kinase activity, by chromatography on a phosphocellulose column. This kinase was purified 15,000-fold from the crude extract. Synthase kinase activity co-purifies with casein and phosvitin kinase activities. Heat inactivation of these three kinase activities follow similar kinetics. It is suggested that these three kinase activities reside in a single protein. This kinase has a molecular weight of approximately 34,000 as determined by glycerol density gradient centrifugation and by gel filtration. The Km values for the synthase kinase-catalyzed reaction are 0.12 mg/ml (0.35 micronM) for synthase, 12 micronM for ATP, and 0.15 mM for Mg2+. The phosphorylation of glycogen synthase by the kinase results in the incorporation of 4 mol of phosphate/85,000 subunit; however, only two of the phosphate sites predominantly determine the glucose-6-P dependency of the synthase. Synthase kinase activity is sensitive to inhibition by NaCl or KCl at concentrations encountered during purification. Synthase kinase activity is insensitive to the allosteric effector (glucose-6-P) or substrate (UDP-glucose) of glycogen synthase at concentrations usually found under physiological condition.


Resonance Raman and EPR of nitrosyl human hemoglobin and chains, carp hemoglobin, and model compounds. Implications for the nitrosyl heme coordination state. We report the joint resonance Raman (RR) and electron paramagnetic resonance (epr) study of five- and six-coordinate nitrosyl heme model compounds and of the titled nitrosyl hemoproteins. Both epr and RR spectra fall into two types which, in the models, correspond to five- and six-coordinate nitrosyl hemes. However, neither RR nor epr spectroscopy is highly sensitive to the nature of the bond between a nitrosyl heme and a coordinated nitrogenous base, nor do the results of one technique uniformly correlate with those of the other. It is not possible to use epr spectroscopy as a test for the coordination state of a nitrosyl heme. The position of the highest frequency (depolarized) RR band possibly provides such a test. Any breaking of the very weak bond between nitrosyl heme and proximal histidine in T state human HbNO is more a consequence of tertiary structural features unique to the human alphaNO chains than it is of properties of the T quaternary conformation.


Comparative solvent perturbation of horse heart cytochrome c and Rhodospirillum rubrum cytochrome c2. The extent of exposure of heme to solvent in horse heart cytochrome c and Rhodospirillum rubrum c2 was investigated to determine whether a correlation exists between the properties of these oxidation-reduction proteins and their heme environments. Solvent perturbation absorption difference spectra were measured using ethylene glycol, glycerol, and sucrose at concentrations between 0 and 30%. Cytochrome c appears to exhibit a somewhat greater extent of heme exposure than cytochrome c2 for both the oxidized and reduced states. These results suggest that the lower oxidation-reduction potential of cytochrome c may in part be due to a greater extent of exposure of the heme. The oxidized state of both proteins appears to exhibit a greater exposure than that of the reduced state which is consistent with a more favorable environment for the charge on the ferric heme coordination center.


Primary site and second site revertants of missense mutants of the evolutionarily invariant tryptophan 64 in iso-1-cytochrome c from yeast. The three missense mutants cyc1-132, cyc1-166 and cyc1-189 in the yeast Saccharomyces cerevisiae contain nonfunctional and thermolabile iso-1-cytochromes c and have different replacements of the tryptophan at position 64 which corresponds to the invariant tryptophan residue found in cytochromes c from all eukaryotic species. The cyc1-166 and cyc1-189 mutants contain single replacements of, respectively, serine 64 and cysteine 64, while the cyc1-132 mutant contains a double replacement of glycine 64 and alanine 65 instead of the normal tryptophan 64 and aspartic acid 65. Twenty-three intragenic revertants having at least partially functional iso-1-cytochromes c arose from these three missense mutants by single amino acid replacements of either tryptophan, phenylalanine, tyrosine or leucine at position 64, or by second-site replacements in which the mutant residues at position 64 are retained and the normal serine 45 is replaced by phenylalanine 45. Specific activities of the iso-1-cytochromes c were estimated by growth of strains on lactate medium and are as follows, in terms of the normal, for iso-1-cytochromes c altered specifically in the ways shown: 100% for phenylalanine 64; 25% for tyrosine 64; between 0 and 25% for leucine 64; 100% for phenylalanine 45, cysteine 64; 25% for phenylalanine 45, serine 64; between 0 and 25% for phenylalanine 45, glycine 64, alanine 65; and 0% for serine 64, for cysteine 64, and for glycine 64, alanine 65 iso-1-cytochromes c. The results demonstrate that small residues of glycine, serine, and cysteine at position 64 are incompatible with function; they imply that many of the 10 amino acids accessible by single base-pair substitution but not observed in primary site revertants also are incompatible with function; and they show that large hydrophobic residues of phenylalanine, leucine, and tyrosine at position 64 are capable of restoring at least partial function. The second site revertants indicate that deleterious effects of the three missense mutants can be compensated by the introduction of phenylalanine 45, which may occupy space normally filled by tryptophan 64. Altered shapes of Calpha-band spectra and at least partial instability were characteristics of all iso-1-cytochromes c found lacking tryptophan 64. Apparently, the principal role of the invariant tryptophan is stabilization of the active protein structure, by providing a large hydrophobic group at the proper location.


Heme-associated subunit complex of cytochrome c oxidase identified by a new two-dimensional gel electrophoresis. A Novel two-dimensional system (isoelectric focusing in the first dimension and sodium dodecyl sulfate-urea electrophoresis in the second) was used to determine the pI values of beef heart subunits of cytochrome c oxidase. The results identified a heme-associated complex of Subunits I, II, and IV. Conditions mitigate against migration of free heme.


Reconstitution into liposomes of the glycoprotein of vesicular stomatitis virus by detergent dialysis. The glycoprotein of vesicular stomatitis (VS) virus was selectively liberated from the virion membrane by the dialyzable nonionic detergent, beta-D-octylglucoside. The isolated viral glycoprotein could be rendered virtually free of phospholipid and detergent, under which conditions it formed tail-to-tail glycoprotein micelles in the form of rosettes. When mixtures of viral glycoprotein and egg lecithin were dialyzed free of octylglucoside, glycoprotein vesicles formed spontaneously with spikes protruding in the same external orientation as the VS virion membrane. The glycoprotein vesicles exhibited increased and uniform buoyant density, indicating relative homogeneity in the proportion of glycoprotein and phosphatidylcholine in each glycoprotein liposome. Evidence for similar insertion and orientation of VS viral glycoprotein in both phosphatidylcholine vesicles and virion membrane was substantiated by the finding that proteolytic digestion with thermolysin gave rise to hydrophobic glycoprotein tail fragments in vesicle or virion membranes that migrated identically in polyacrylamide gels.


Inability of parvalbumin to function as a calcium-dependent activator of cyclic nucleotide phosphodiesterase activity. The calcium-sensitive phosphodiesterase-stimulating activity sometimes associated with parvalbumin preparations is due to contaminating (less than 0.1%) amounts of carp muscle CDR (calcium-dependent regulator)-like protein. This protein can be resolved from parvalbumins by Sephadex G-75 chromatography and has many characteristics of the CDR. Parvalbumin itself causes a nonspecific stimulation of phosphodiesterase at all calcium concentrations which, in the presence of CDR, can cause an apparent shift to a lower concentration of the calcium level required for half-maximal stimulation.


Measurement of 14N superhyperfine frequencies in stellacyanin by an electron spin echo method. We have measured the 14N superhyperfine frequencies for weakly coupled nitrogen in stellacyanin and in a model compound Cu(II)-diethylenetriamine-imidazole using a 3-pulse spin echo technique. By making computer simulations of the superhyperfine spectrum, we have been able to show that these frequencies result from the interaction of the remote protonated nitrogen of metal-bound imidazole with Cu(II).


Regulation of phosphatidylcholine metabolism by cyclic AMP in a model alveolar type 2 cell line. The influence of cyclic AMP on the metabolism of phosphatidylcholine, the major component of pulmonary surfactant was examined in a cell line (A549) with type 2 pneumonocyte characteristics. It was found that cyclic AMP increased both the total amount of phosphatidylcholine and disaturated phosphatidylcholine as well as the incorporation of [3H]choline into these fractions. The effect was specific for cyclic AMP since 5'-AMP, adenosine, and cyclic GMP did not alter phosphatidylcholine or disaturated phosphatidylcholine levels. Cyclic AMP had no effect on phosphatidylcholine and disaturated phosphatidylcholine metabolism in another non-type 2 human epithelial cell line (MA-160). Since the ability of various cyclic AMP analogs to increase phosphatidylcholine and disaturated phosphatidylcholine levels was correlated with their ability to activate protein kinase, it seems likely that a protein phosphorylation mechanism is involved in controlling phosphatidylcholine metabolism.


The stereochemistry of NADH utilization by the flavoenzyme monooxygenase orcinol hydroxylase. Ribbons et al. (Ribbons, D.W., Ohta, Y., and Higgins, I.J. (1972) in Molecular Basis of Electron Transport, Miami Winter Symposic Series (Schultz, J., and Cameron, B.F., eds) Vol. 4, pp. 251-274, Academic Press, New York) presented a preliminary report that the flavoenzyme monooxygenase orcinol hydroxylase shows mixed type 4R, 4S stereospecificity with respect to dihydronicotinamide oxidation when resorcinol and m-cresol were used as substrate analogs. With the natural substrate orcinol, 4R chirality was maintained. In kinetic isotope experiments reported here, we demonstrate in fact that orcinol hydroxylase maintains 4R stereospecificity with respect to dihydronicotinamide oxidation with all three substrates, orcinol, resorcinol, and m-cresol. Deuterium and tritium kinetic isotope effects were detected under Vmax conditions with (4R)-[4-2H]-, and (4R)-[4-3H]NADH for all three substrates. No isotope effect was observed with (4S)-[4-2H]NADH and tritium labilization from assays with (4S)-[4-3H]-NADH was negligible in all cases.


Purification of a cyclic nucleotide phosphodiesterase from bovine brain using blue dextran-Sepharose chromatography. The soluble high Km form of cyclic nucleotide phosphodiesterase (EC 3.4.1.17) was purified over 2000-fold from bovine brain homogenates principally using blue dextran-Sepharose chromatography. The purified protein has a specific enzymic activity of 167 units/mg and appears homogeneous when examined by polyacrylamide gel electrophoresis. The enzyme has a molecular weight of 1.26 +/- 0.05 x 10(5) consisting of two apparently identical polypeptide chains. Kinetic measurements indicate that the substrates cyclic GMP and cyclic AMP each have a single Km value, 9 +/- 1 micron and 150 +/- 50 micron, respectively, that the two cyclic nucleotides compete for the same catalytic site, that the blue dye of blue dextran-Sepharose is a competitive inhibitor for the cyclic nucleotides, and that the Vmax with cyclic AMP as substrate is about an order of magnitude larger than that for cyclic GMP. Bovine brain calmodulin stimulates the catalytic rate of the purified enzyme in the presence of Ca2+ by increasing the Vmax associated with each cyclic nucleotide substrate.


Determination of cooperative interaction between clusters in Clostridium pasteurianum 2 (4Fe-4S) ferredoxin. The effect of reducing one 4Fe-4S cluster in Clostridium pasteurianum 2 (4Fe-4S) ferredoxin on the reduction potential of the unreduced cluster has been investigated. While such an effect is suggested by both the x-ray structure of Peptococcus aerogenes 2 (4F-4S) ferredoxin and the polypeptide conformational change on reduction present in clostridial-type 2 (4Fe-4S) ferredoxins, present studies indicate that cluster-cluster cooperative interaction is not strong enough to be of functional importance in these proteins.


Subunit inequivalence in superoxide anion formation during photooxidation of human oxyhemoglobin. Subunit inequivalence in the photooxidation of human oxyhemoglobin was investigated by quantitative analysis of the fraction of alpha and beta chains oxidized after low intensity flash photolysis using white light in quartz cuvettes. This reaction was previously shown to generate methemoglobin and superoxide anion as photoproducts (Demma, L.S., and Salhany, J.M. (1977) J. Biol. Chem. 252, 1226-1230). The present results indicate that superoxide anion photodissociates from the alpha chain about 3 to 4 times more extensively than from beta. This difference was observed in the presence of superoxide dismutase and catalase at both pH 7 and 9, suggesting that photolysis is directly responsible. The reaction of superoxide anion with oxyhemoglobin was also studied with the same analytical methods and no chain differences could be observed. If the electronic structure of the oxyheme complex is viewed as a spin equilibrium between a singlet ground state and a charge transfer configuration, our results may indicate that the uv component of white light perturbs this equilibrium to a greater extent in the oxygenated alpha chain, implying that the separation of energy levels may be smaller in that chain as compared with beta.


Optical detection of a red-shifted Michaelis complex in the reaction of HCN with ferromyeloperoxidase. Evidence for conformational stabilization of high spin iron (II). The reaction of HCN with ferromyeloperoxidase involves the sequential formation of two monocyanide complexes. The first complex, which forms immediately on mixing, is characterized by a red shift in the Soret band of the ferroperoxidase, and a dissociation constant (measured as a Michaelis constant) of 0.67 mM. The second complex arises from the first via a first order process, whose maximal rate is 0.095 s-1 at 25 degrees C, pH 7.0. This more stable complex is characterized by a blue shift in the Soret and alpha bands and by an overall dissociation constant in the region of 4.5 microM. This gives a free energy difference between the two complexes of around 3.0 kcal mol-1 and a difference in optical absorption of 15 nm (Soret). The measured Arrhenius activation energy for the conversion of the high energy, long wavelength complex to the low energy, short wavelength complex is 16.3 kcal mol-1. A larger blue shift is observed on protein denaturation (34 nm), after which the two-step binding reaction is not observed. This, and the magnitude of the activation energy in the spontaneous complex interconversion process, shows that the latter is a conformational process. In addition, it can be concluded that the unknown structural feature of the heme site which is responsible for the anomalous red shift in the optical spectrum of native ferromyeloperoxidase, is also the link between the ligand state of the iron and the protein conformation.


(Na+ + K+)-adenosine triphosphatase of mammalian brain. Catalytic and regulatory K+ sites distinguishable by selectivity for Li+. Li+, K+, and Rb+ are compared as activators of the hydrolysis of p-nitrophenylphosphate by beef brain (Na+ + K+)-ATPase. Previous experiments have established two classes of K+ binding sites that are involved in this reaction: "catalytic sites" have the higher affinity, their occupation is essential for catalytic activity, and they appear to correspond to the extracellular binding sites for active K+ transport; regulatory sites appear to have an allosteric function to "unmask" the catalytic sites. A separate set of Na+-binding regulatory sites bring about a similar unmasking of catalytic sites under phosphorylating conditions. Rb+ can activate p-nitrophenylphosphate hydrolysis both in the presence and absence of Na+ and, thus, can interact effectively with both K+ regulatory and catalytic sites. Li+ does not activate p-nitrophenylphosphate hydrolysis at 25 degrees C in the absence of other monovalent ligands. Li+ does activate when the catalytic sites are exposed by Na+ + ATP. Thus, K+ regulatory and catalytic sites differ in their cation selectivity. At temperatures less than 25 degrees C Li+ is able to activate the phosphatase reaction in the absence of other monovalent ligands: maximum activity occurs at 10-12 degrees C. A plot of the ratio, Li+ activation/K+ activation, as a function of temperature shows that the allosteric transition that unmasks catalytic sites occurs spontaneously with decreasing temperatures.


Arrangement of glucose residues in the lipid-linked oligosaccharide precursor of asparaginyl oligosaccharides. The lipid-linked oligosaccharide synthesized in vitro, in the presence of 1.0 microM UDP-[3H]Glc, GDP-[14C]Man, and UDP-GlcNAc has been isolated and the structure of the oligosaccharide has been analyzed. The oligosaccharide contains 2 N-acetylglucosamine, 9 mannose, and 3 glucose residues. The N-acetylglucosamine residues are located at the reducing terminus. The 3 glucose residues are arranged in a linear order at one of the nonreducing termini in the sequence Glc 1,2--Glc 1,3--Glc--(Man)9 (GlcNAc)2. The structural analysis was made possible largely by the availability of glucosidase preparations of fungal anad microsomal origin which remove glucose residues from the oligosaccharide without releasing mannose residues.


Coordinate overproduction of orotate phosphoribosyltransferase and orotidine-5'-phosphate decarboxylase in hamster cells resistant to pyrazofurin and 6-azauridine. Cells resistant to pyrazofurin and 6-azauridine have been selected from a simian virus 40-transformed Syrian hamster line and from a Chinese hamster lung line. By increasing the concentrations of inhibitors in several steps, mutant cells from both lines have been obtained which resist high concentrations (1 to 5 mM) of the two inhibitors separately or together. Orotidine-5'-phosphate decarboxylase (EC 4.1.1.23), the sixth and last enzyme in UMP biosynthesis, is inhibited by the nucleoside monophosphates derived from pyrazofurin or 6-azauridine. The activity of this enzyme is increased in each resistant cell line tested. Furthermore, there is a parallel increase in each case in the activity of the fifth enzyme of the pathway, orotate phosphoribosyltransferase (EC 2.4.2.10), which is not inhibited by pyrazofurin or 6-azauridine monophosphates, and the amount of increase is up to 67 times the level found in wild type cells. In contrast, the activities of the first three enzymes of UMP biosynthesis remain essentially unchanged in the mutants. Resistant Chinese hamster cells remain sensitive to 5-fluorouridine; this indicates that uridine kinase, the enzyme necessary to convert 6-azauridine to the monophosphate, is still functional.


Kinetic studies of bovine liver fructose-1,6-bisphosphatase. Initial rate kinetic studies with bovine liver fructose-1,6-bisphosphatase were carried out in both directions of the reaction to determine the sequence of product release from the enzyme. Product inhibition by fructose-6-P was found to be S-linear, I-linear noncompetitive relative to fructose-1,6-bisphosphate, whereas inorganic orthophosphate was determined to be linear competitive with respect to the substrate. The kinetics of the reverse reaction were studied by coupling the phosphatase reaction to the aldolase, triosephosphate isomerase, and glycerolphosphate dehydrogenase reactions. The kinetic results were found to be in harmony with the Uni Bi ordered and random sequential mechanisms as well as a Uni Bi ping-pong mechanism. The nomenclature is that of Cleland (Cleland, W.W. (1963) Biochim. Biophys. Acta 67, 104-137). However, nonkinetic considerations, when taken together with the kinetic results, suggest that the steady state ordered Uni Bi mechanism is the most likely possibility. There is evidence that isomerization of the binary complex of enzyme and phosphate occurs in the kinetic mechanism. Although magnesium is required for the reverse reaction, there is no evidence to suggest that the enzyme discriminates between the magnesium-associated or divalent cation-free forms of the substrates.


Oxidation-reduction ratio studies of mitochondria in freeze-trapped samples. NADH and flavoprotein fluorescence signals. The recording of oxidation-reduction-related fluorescence signals of oxidized flavoprotein (Fp) and reduced pyridine nucleotide (PN) from isolated mitochondria at temperatures below -80 degrees C can be accompanished with a high degree of accuracy and a wide dynamic range. The specific low temperature enhancement of the fluorescence signals due to increased quantum yield and to multiple scattering affords increased accuracy and less interference due to screening pigments such as hemoglobin and myoglobin. Since the metabolic processes are arrested and the recording speed can be greatly diminished, the technique can operate with a much smaller concentration of mitochondria than is needed at room temperature, and the method is suitable for localized oxidation-reduction measurements. The Fp and PN signals originate from the mitochondrial matrix space in which they represent the major fluorochromes. Since Fp and PN are near oxidation-reduction equilibrium, the ratio of the two fluorescence intensities, suitably normalized, approximates the oxidation-reduction ratio of oxidized flavoprotein/reduced pyridine nucleotide. Thus, this technique affords a foundation for the resolution of oxidation-reduction states in two and three dimensions.


Purification of murine oncornaviral phosphoproteins using alkyl agarose derivatives.. Methods for the purification of both murine mammary tumor (type B) and murine leukemia (type C) oncornaviral phosphoproteins are described, in which chromatography on alkyl-agarose derivatives is used as the primary fractionation step. Gel filtration or ion exchange chromatography on phosphocellulose was the only subsequent step required for the purification of the type B and type C viral proteins, respectively. The two-step protocols also resulted in the co-purification of a low molecular weight core protein from each virus. Recoveries of the viral proteins purified by this method, based on per cent contribution of individual polypeptides to total virion proteins, were 70% or greater. Radioimmunocompetition analysis of the purified murine mammary tumor virus major core protein as well as analysis of the RNA binding properties of purified low molecular weight type C virus proteins suggests that neither antigenic reactivity nor specific RNA binding characteristics are altered by the purification protocols. The availability of these procedures should aid studies on the possible function and immunochemical properties of the native murine oncornaviral phosphoproteins and may also be extended to the purification of other oncornaviral proteins.


The spatial relationships and structure of the binuclear iron-sulfur clusters in succinate dehydrogenase. Two binuclear iron-sulfur clusters (designated S-1 and S-2) are present in succinate dehydrogenase in approximately equal concentration to that of flavin. The large difference in their midpoint potentials (0 and -400 mV, respectively, in the soluble enzyme) permits the acquisition of individual electron paramagnetic resonance spectra characterized by nearly identical rhombic g tensors (gz = 2.025, gy = 1.93, gx = 1.905). Spin-coupling between the two centers is manifested by broadening and splitting of spectra of reconstitutively active and inactive succinate dehydrogenase, respectively, as the temperature is lowered; relief of power saturation of Center S-1 spectra on reduction of Center S 2; and observation of half-field ("delta ms = 2") signals in the dithionite-reduced enzyme. Saturation behavior of fully reduced dehydrogenase is consistent with the presence of S-1 and S-2 at equivalent concentrations/molecule. Simulation of the spin-coupled spectra, assuming dipolar interaction, provides information on molecular structure. Electron paramagnetic resonance spectra of the enzyme in 80% dimethylsulfoxide are nearly identical to the characteristic binuclear spectra obtained with adrenodoxin. These data provide additional evidence for binuclear structure of both Center S-1 and S-2. The extremely fast relaxation of Center S-2 at low temperatures would imply either an anomalously small value of J or an alternative relaxation mechanism, possibly due to the coupling between S-1 and S-2.


A rapid method for detecting and mapping homology between heterologous DNAs. Evaluation of polyomavirus genomes. A new approach for evaluating homologous sequences among related DNAs is presented. Conventional filter hybridization techniques are employed at 35 degrees C in a range of formamide concentrations in order to perform annealings at effective temperatures as low as Tm -50 degrees C which permits the detection of regions of homology with as much as 33% base mismatch. Under such nonstringent conditions, high levels of specific annealing can be obtained at plateau levels. In combination with the Southern "blotting" technique (1975), this approach can be used to perform biochemical heteroduplex melting experiments. The homology among the genomes of the murine polyoma virus (Py), the simian virus 40 (SV40), and the human papovavirus BK was evaluated using this new methodology.


Carcinogenic effects in the Syrian golden hamster of N-methyl-N-formylhydrazine of the false morel mushroom Gyromitra esculenta. N-Methyl-N-formylhydrazine (MFH) was administered in drinking water as a 0.0078% solution to randomly bred Syrian golden hamsters for life beginning at 6 weeks of age. The treatment gave rise to benign and malignant liver cell tumors, malignant histiocytomas and tumors of the gall bladder and bile ducts. The tumor incidence in these four treated tissues was 43, 34, 11,8%, while in untreated controls it was 0, 0, 0, 0%, respectively. Histopathologically, tumors were classified as benign hepatomas, liver cell carcinomas, malignant histiocytomas, adenomas and adenocarcinomas of the gall bladder, cholangiomas, and cholangiocarcinomas.


Karyotypic variations in human meningioma cell cultures under different in vitro conditions. Examined were how changes of the culture medium, cultivating procedure and cultivating time will influence numerical representation of different karyotypes in a total of 27 cell cultures of meningiomas with two or more cytogenetically distinguishable cell lines. It could be shown that in medium I (80% Mc Coy 5 A, 20% fetal calf serum) more cell lines with a higher degree of hypodipoidy occurred than in medium II (50% TC 199, 40% bovine amniotic fluid, 10% calf serum). The number of cells with normal karyotype was higher in cultures which were grown from trypsinized biopsy material in stationary flasks when compared to particle cultures in roller tubes. Cells with a normal karyotype also increased after 1--6 subcultures. By demonstration of SV 40 tumor antigen it could be shown in two cultures that these normal mitoses derived from tumoral and not from stromal tissue.


Distribution of T- and B-cells and of immunoglobulin-producing cells in tumor tissue of patients with nasopharyngeal carcinoma. Various subtypes of 17 European nasopharyngeal carcinomas are examined by immunocytological and immunohistological methods. The T- and B-Cells in the inflammatory infiltrate are determined quantitatively and correlated with the amount and type of intracellular immunoglobulin. This data is compared with peripheral T-Cell values and the serum anti Epstein-Barr virus (EBV) titers, and finally with the tumor type and stage. Three of the 17 patients had marked local immunoglobulin producing cells, 8 only moderate, and 6 had nearly none. The intracellular immunoglobulin was primarily IgG/kappa, and no significant increase of IgA producing cells was observed as suggested by others. No clearcut correlation was found between local T- and B-Cell infiltrates and the tumor type, stage of disease, or anti EBV titers in the patients' serum. The possible implication of this data is discussed.


Karyology and tumorigenicity of a simian virus 40-transformed Chinese hamster cell clone. A pseudodiploid clone of chinese hamster cells tranformed in vitro with Simian virus 40 (SV40) was isolated from soft agar and injected into nude mice through three successive passages with a short in vitro cultivation between each animal inoculation. the original clone and the three subsequent tumor populations were characterized in regard to SV40 T antigen staining, modal chromosome number, and Giemsa-banded karyotype. All tumor cell lines maintained the pseudodiploid mode, as well as the positive sv40 t antigen staining. Nonrandom chromosomal changes included loss of one of the X chromosomes, additions of abnormal variants of chromosomes No. 1 and No. 2, the appearance of unidentified marker chromosomes, and the loss of autosomes No. 5, No. 6, and No. 11. The deletion of one of the X chromosomes occurred with about the same frequency in all cell lines. Additions of abnormal chromosomes No. 1 and No. 2 tended to recur more often in the tumor cell lines than in the original clone. the appearance of marker chromosomes, as well as the loss of autosomes No. 5, No. 6, and No. 11 demonstrated a correlation with tumorigenicity. Yet, the three successive passages of the cells through the animal did not select for a tumor population with a single, homogeneous karyotype.


Biochemical genetic analysis of pyrimidine biosynthesis in mammalian cells. II. Isolation and characterization of a mutant of Chinese hamster ovary cells with defective dihydroorotate dehydrogenase (E.C. 1.3.3.1) activity. A mutant (A204) of Chinese hamster ovary cells (CHO-K1), which is deficient in dihydroorotate (DHO) dehydrogenase (E.C. 1.3,3.1) activity, has been isolated by a replica plating procedure. The mutant does not show a requirement for exogenously added pyrimidines. Examination of intact cells shows that the mutant accumulates a large amount of carbamyl aspartate and is markedly but not totally deficient in biosynthesis of orotate from earlier precursors of pyrimidine biosynthesis, including aspartate and dihydroorotic acid, when compared to wild-type cells. Analysis of cell-free extracts of mutant and wild-type cells shows that the mutant is deficient in DHO dehydrogenase activity, possessing ca. 5% of the wild-type activity. this evidence leads to the conclusion that this mutant, A204, is in fact partially deficient in DHO dehydrogenase, and that in these cells it is this enzyme which carries out the fourth step of de novo pyrimidine biosynthesis.


Cholera toxin stimulates division of 3T3 cells. Cholera toxin was used in an attempt to inhibit epidermal growth factor stimulated 3T3 cell division. Instead, cholera toxin alone at low concentrations (10(-10) M), was able to stimulate cell division and could augment EGF stimulated cell division. The mitogenic effect of cholera toxin can occur despite a dramatic increase in the intracellular levels of cAMP in 3T3 cells. Cholera toxin stimulated mitogenesis could not be mimicked by choleragenoid, the binding but inactive subunit of cholera toxin, or by other agents which elevate cAMP levels in 3T3 cells.


Effects of angiotensin II and angiotensin II antagonist saralasin on cell growth and renin in 3T3 and SV3T3 cells. Components of the renin-angiotensin system were studied in established cell culture lines of 3T3 and SV3T3 mouse fibroblasts. The renin content in 3T3 cells was significantly higher than in virus-transformed SV3T3 cells. With time after infection, renin decreased in Simian virus 40 transformed cells, while it increased steadily in mock-infected 3T3 cells. In contrast to renin, angiotensinase activity was higher in SV3T3 cells. Angiotensin II stimulated cell proliferation in 3T3 mouse fibroblasts and decreased their renin content in a dose-related manner. In contrast, saralasin, an angiotensin receptor antagonist, inhibited cell growth in 3T3 and SV3T3 cells and caused an increase of cellular renin concentration. The angiotensin fragments angiotensin (2-8) heptapeptide and angiotensin (4-8) pentapeptide had no effect on cell growth. A significant negative correlation was found between cell proliferation and renin levels in 3T3 and SV3T3 cells irrespective of the treatment. Our results indicate (1) that angiotensin II may be involved in cell growth regulation, (2) that a negative feedback exist between angiotensin II added and intracellular renin content, and (3) that virus infection causes a decrease in intracellular renin synthesis, while non-specific angiotensinase activity is increased under this condition.


Evidence suggesting that a cyclic AMP-dependent protein kinase is a positive regulator of proliferation in Cloudman S91 melanoma cells. Wild-type Cloudman S91 melanoma cells have a retarded rate of division when agents which raise cyclic AMP levels such as melanotropin, protaglandin E1, or cholera toxin are supplemented to the culture medium. A mutant cell line was isolated which had the opposite response, i.e., the mutant grew very slowly unless agents which raised cyclic AMP levels were present (Pawelek et al., '75a). In this report evidence is presented indicating that the molecular basis for the mutant phenotype resides in the major cyclic AMP-dependent protein kinase found in the cells. The mutant kinase had increased thermolability and an elevated activation constant for cyclic AMP over the corresponding wild-type kinase. It is proposed that the elevated requirement for cyclic AMP for the proliferation of cAdep cells is related to the elevated activation constant of the kinase, suggesting that the kinase is a positive regulator of proliferation in Cloudman S91 cells.


Postmenopausal virilization due to ovarian stromal hyperthecosis. A 53-year-old woman presented with hirsutism of long duration and virilization of recent onset. She was gravida 12 para 9. The 17-ketosteroids were normal, gonadotropins were in the post-menopausal range, and the serum testosterone was in the male range. After bilateral oophorectomy, the testosterone became normal. Ovarian pathology revealed stromal hyperthecosis.


Cyproheptadine treatment of Nelson's syndrome: restoration of plasma ACTH circadian periodicity and reversal of response to TRF. Administration of cyproheptadine for four months to a patient with Nelson's Syndrome was associated with evidence of skin lightening, decreased plasma ACTH concentrations, return of a normal circadian periodicity of plasma ACTH concentrations, and lack of responsiveness of plasma ACTH concentrations to TRF administration, in contrast to the response seen in the untreated state.


Production of diglyceride from phosphatidylinositol in activated human platelets. Human platelets generate diglyceride within 5 s of exposure to thrombin. Production of diglyceride is transient. 15 s after the addition of thrombin, the levels of diglyceride have increased up to 30-fold, but decrease thereafter. Prior incubation of platelets with 2 mM dibutyryl cyclic AMP prevents both the generation of diglyceride and the secretion of serotonin. Acetylsalicylic acid (100 microgram/ml), which completely inhibits prostaglandin endoperoxide synthesis, does not block diglyceride production and serotonin secretion induced by thrombin. Based on studies examining the incorporation of [3H]arachidonic acid into diglyceride of prelabeled platelets exposed to thrombin, it is concluded that neither phosphatidic acid nor triglyceride is the source of the diglyceride. Phosphatidylinositol appears to be the most likely source, both because its loss of radiolabel is sizable and rapid enough to account for the appearance of radiolabel in diglyceride, and because a phosphatidylinositol-specific phosphodiesterase, described in this report, exists in platelets. The phosphatidylinositol-phosphodiesterase, which produces diglyceride and inositol phosphate, requires Ca+2 and shows optimal activity at pH 7. The enzyme does not act upon phosphatidylcholine, phosphatidylethanolamine, or phosphatidylserine.


Fluoride-mediated activation of the respiratory burst in human neutrophils. A reversible process. Fluoride ion (F-) is an effective activator of the respiratory burst in neutrophils, as indicated by its ability to induce O2- production by these cells. Other halide ions did not activate the burst, Cl-, in particular appeared to antagonize the effect of F- on O2- production. F- stimulated O2- production showed a requirement for Ca++, but was independent of other exogenous cations. Neither phagocytosis nor degranulation were necessary for respiratory burst activation by F-. The effect of F- on the respiratory burst was reversible. Washing the cells after treatment with F-, while they were still producing large amounts of O2-, returned them to the resting state. They could then be stimulated again to produce O2- in amounts equivalent to those originally produced. Our experiments indicated that restimulation did not represent the activation of a population of cells that had not been activated during the initial exposure to F-, nor did it represent serial activation of different subpopulation of the O2- forming enzyme molecules present in a given cell. Rather, our data suggest that the entire population of O2- forming enzyme molecules was activated in a reversible fashion by F-.


Ricinoleic acid stimulation of active anion secretion in colonic mucosa of the rat. Perfusion of the colon with ricinoleic acid produces fluid and electrolyte accumulation. The mechanism of these changes in water and electrolyte movement is uknown. These studies were designed to determine whether ricinoleic acid effects active ion transport across isolated rat colonic mucosa. 0.5 mM Na ricinoleate produced significant increases in potential difference (3.8 +/- 0.5 mV) and short-circuit current (Isc) (99.2 +/- 10.1 muA/cm2). The increases in Isc produced by Na ricinoleate were inhibited by both removal of bicarbonate and chloride and by the presence of theophylline. The hydroxy fatty acid also resulted in a significant decrease in net Na absorption from 4.7 +/- 0.8 to 0.1 +/- 0.7 mueq/h cm2 and reversed net Cl transport from absorption (+ 4.5 +/- 0.9) to secretion (-2.2 +/- mueq/h cm2). In parallel studies 0.5 mM Na ricinoleate increased mucosal cyclic AMP content by 58%. The concentrations of Na ricinoleate required to produce detectable and maximal increases in both Isc and cyclic AMP were the same. These results provide evidence in support of the concept that hydroxy fatty acid-induced fluid and electrolyte accumulation is driven by an active ion secretory process.


Glucocorticoid receptors and actions in subpopulations of cultured rat bone cells. Mechanism of dexamethasone potentiation of parathyroid hormone-stimulated cyclic AMP production. We have previously shown that bone cells possess glucocorticoid receptors and that, in addition to being inhibitory to cell growth, glucocorticoid treatment potentiates the ability of parathyroid hormone (PTH) to stimulate cyclic AMP (cAMP) formation. This study extends those observations to specific subpopulations of bone cells and explores the mechanism of the cAMP augmentation. Subpopulations of cultured bone cells derived from 20-d-old fetal rat calvaria were enriched for "osteoblast-like" (OB) and "osteoclast-like" (OC) cells by sequential collagenase digestion. OC cells released during the first 30 min of collagenase digestion were characterized by low alkaline phosphatase activity, a cAMP response to salmon calcitonin (CT), but only a small cAMP response to bovine PTH. In contrast, OB cells released between 30 and 120 min of collagenase digestion, possessed high alkaline phosphatase activity, responded with a large cAMP rise to PTH, but exhibited no response to CT. Glucocorticoid receptors, with similar properties, were demonstrated in both populations (K(d) congruent with 5 nM, N(maximum) congruent with 400 fmol/mg cytosol protein). Dexamethasone equivalently inhibited cell growth and alkaline phosphatase activity in both populations. Dexamethasone potentiation of cAMP generation occurred after PTH but not CT stimulation. A greater enhancement of cAMP generation observed in OB cells appears to result from two glucocorticoid actions: (a) stimulation of adenylate cyclase and (b) inhibition of phosphodiesterase. Only the latter mechanism was found in OC cells. Dexamethasone-treated cells showed an increase in both sensitivity and maximal response of cAMP to PTH. The possible relationship of these actions to the mechanism of glucocorticoid-induced osteopenia is discussed.


In vitro suppression of serum elastase-inhibitory capacity by reactive oxygen species generated by phagocytosing polymorphonuclear leukocytes. Human polymorphonuclear leukocytes (PMN) phagocytosing opsonized antigen-antibody complexes, produce dialyzable species of activated oxygen which are capable of partially suppressing the elastase-inhibiting capacity (EIC) of whole human serum or purified human alpha1-proteinase inhibitor. Serum EIC was partially protially protected by superoxide dismutase, catalase, or mannitol, suggesting that hydroxyl radical, formed by interaction of superoxide radical and hydrogen peroxide, might be responsible for this effect. NaN3 also partly protected EIC, implicating myeloperoxidase-mediated reactions as well. An artificial superoxide rradical-generating system, involving xanthine and xanthine-oxidase, could be substituted for phagocytosing PMN with resultant EIC suppression. These results are consistent with previous demonstrations of the release of potent oxidants by stimulated PMN, as well as earlier studies from our laboratory showing sensitivity of alpha1-proteinase inhibitor to inactivation by oxidants. Oxidative inactivation of proteinase inhibitors in the microenvironment of PMN accumulating at sites of inflammation may allow proteases released from these cells to more readily damage adjacent connective tissue structures.


Portal vein invasion demonstrated by ultrasound. Two patients evaluated by ultrasound for abdominal masses were found to have thrombus within their central portal veins. Their cases are presented with angiographic correlation. Both are proven cases of hepatocellular carcinoma. The importance of evaluation of the portal vein in patients with liver neoplasms is discussed.


Comparison of enzyme-linked immunosorbent assay (ELISA) technique and complement-fixation test for estimation of cytomegalovirus IgG antibody. The ELISA technique has been found to be reliable for the detection and titration of cytomegalovirus-specific IgG antibody in serum. It is about six times more sensitive than the CF test although some discrepancies were found between the antibody titres determined by the two methods.


Multiple malignant Spiegler tumors with brachydactyly and racket-nails. Light and electron microscopic study. Multiple malignant Spiegler tumors were observed on the scalp of a 67-year-old female patient with brachydactyly and racket-nails affecting all fingers of both hands. The occurrence of similar clinical features in the family of the patient is suggestive of a hereditary disorder. The light and electron microscopical findings are described.


Potentiation of guanosine 3':5'-monophosphate accumulation in C-6 glial tumor cells. C-6 glial tumor cells treated with norepinephrine and sodium azide accumulated cyclic GMP to concentrations approximately 10-fold greater than the sum of the separate responses. Isoproterenol, but not phenylephrine, was an effective substitute for norepinephrine, and the response was blocked by propranolol and sotalol. Nitroprusside, but neither cyanide nor isobutyl-methylxanthine, replaced azide. The potentiation was not affected by removal of CA2+ OR Na+ from the extracellular medium and was not blocked by cocaine. The potentiative accumulation of cyclic GMP in C-6 cells differs from the recently described stimulation by catecholamines of soluble guanylate cyclase of renal cortex. The potentiative phenomenon is compared with the few known instances in which cyclic AMP augments cyclic GMP formation and may be associated with synergistic modifications of cellular functions.


Effect of sodium fluoride on cyclic AMP production in rat hepatocytes. The effect of NaF on cAMP production was studied in hepatocytes isolated from fed and fasted rats. A four-six fold increase in hepatocyte cAMP production was observed in the presence of 10-20 mM NaF in cells isolated from either fed or fasted rats. The maximal stimulation of cAMP production was observed after a 10 min incubation in the presence of 1 mM theophylline. However, as little as 0.05-0.15 mM NaF induced a significant increase in cAMP production. It was also found that NaF would alter the production of glucose in isolated rat hepatocytes. When hepatocytes from fed rats were incubated with 0.05-5 mM NaF there was an increase in amount of glucose released from endogenous sources. Also NaF resulted in a decrease in lactate and pyruvate production. Similarly NaF stimulated glucose production in hepatocytes from fasted rats. The maximal stimulation was observed with about 0.15-0.25 mM NaF. At NaF concentrations greater than 1.5 mM a decrease in glucose production was observed. It is concluded that NaF increases the level of cAMP and alters glucose metabolism in intact hepatocytes.


Acute suppression by PGF2 alpha on LH, epinephrine and fluoride stimulation of adenylate cyclase in rat luteal tissue. Injection of PGF2 alpha (250 microgram/rat) 15 min prior to isolation of corpora lutea (CL) from PMSG treated immature rats significantly reduced the LH stimulation of adenylate cyclase in CL membranes. The LH stimulation did not return to normal even 24 h after PGF2 alpha injection. A transient decrease in epinephrine and fluoride stimulation of AC was also observed, the response returning to normal 6-12 h after PGF2 alpha treatment. In vitro incubation of whole isolated CL with 0.005 micrometer or higher concentrations of PGF2 alpha markedly reduced the LH and fluoride stimulation of AC in the CL membranes. Exposure of CL to PGF2 alpha for 15 min in vitro reduced the LH and fluoride response. The results are discussed in relation to the suppressive action of PGF2 alpha on LH receptors in CL, and a mechanism is proposed to explain the discrepancy in time relation between our results and the LH receptor studies. The proposed mechanism might also explain the transient decrease in epinephrine and fluoride stimulation of AC.


A simple direct assay of 3',5'-cyclic nucleotide phosphodiesterase activity based on the use of polyacrylamide-bononate affinity gel chromatography. A rapid, simple, and direct assay for 3',5'-cyclic nucleotide phospho-diesterase activity is based on the effective separation of cyclic AMP, cyclic GMP or cyclic CMP from their corresponding 5'-nucleotides and nucleosides by chromatography on a polyacrylamide-boronate gel. The affinity of the boronate residue for cis-diols results in the retention of 5'nucleotides and nucleosides while 3',5'-cyclic nucleotides are not retained. The coelution of all 5'-nucleotides and nucleosides allows for the accurate assessment of phosphodiesterase activity in preparations contaminated by other purine metabolizing enzymes such as 5'-nucleotidases and nucleotide and nucleoside deaminases. Phosphodiesterase activity assayed by this means yields linear reaction kinetics with respect to time and amount of enzyme protein. Low blank values obtained allow for detection of as little as 2-3% conversion of substrate to product.


Compatibility of various materials with oral tissues. I: The components in composite restorations. The basic ingredients in composite restorative materials include: monomers, polymerization stabilizers, color stabilizers, polymerization initiators, polymerization accelerators, inorganic reinforcing fillers, and coupling agents. Typical members of each category are discussed.


Effects of the aflatoxins on ATPase activities in mouse and rat liver. The effects of the aflatoxins on ATPase activities in mouse and rat tissues were investigated in vitro. The hepatic oligomycin-sensitive (O.S.) Mg++ ATPase was inhibited significantly. The order of inhibition was G1 greater than B1 greater than G2 greater than B2. Mouse O.S. Mg++ ATPase was more sensitive than the corresponding rat enzyme. The oligomycin-insensitive (O.I.) Mg++ ATPase activities in rat and mouse liver were not altered. Although aflatoxins G1 and B1 were more potent inhibitors of hepatic O.S. Mg++ ATPase, no concentration-response was observed, whereas aflatoxins G2 and B2 inhibited enzyme activity in a concentration-dependent manner. Spectral analysis of aflatoxin G1 solutions suggested that solubility was not related to the observed effects. In addition, the effects of aflatoxin B1 and G1 on mouse brain microsomal Na+-K+ ATPase were examined. Although aflatoxin B1 was more potent that G1, both mycotoxins significantly inhibited enzyme activity in a concentration-dependent fashion.


Multiangle light scattering flow photometry of cultured human fibroblasts: comparison of normal cells with a mutant line containing cytoplasmic inclusions. Multi-angle light scattering flow photometry was used to study the light scattering properties of normal cultured fibroblasts and a mutant fibroblast line containing cytoplasmic lysosomal inclusions. The effect of glutaraldehyde fixation on the light scattering properties of the cells was also examined and correlated with their ultrastructure. Normal fibroblasts showed uniform organelle distribution with few vacuoles or dense bodies in the cytoplasm while the mutant line showed abnormal cytoplasmic inclusions of varying morphology, density and lucency. As predicted by light scattering theory, the mutant cells containing the cytoplasmic inclusions scattered more light at large angles (greater than theta = 1.85 degrees) than did the normal cells. Glutaraldehyde fixation decreased light scattering at small angles (less than theta = 1.85 degrees), increased light scattering at larger angles (greater than theta = 1.85 degrees) in both normal and mutant cells and enhanced resolution of the light scattering signatures. The mutant line scattered 2-3 times more light at a wide angle (greater than theta = 12.74 degrees) than did the normal cells. These data suggest that abnormal lysosomal storage inclusion bodies in the cytoplasm of the cells can be detected by differential light scattering methods.


Autofluorescence of viable cultured mammalian cells. The autofluorescence other than intrinsic protein emission of viable cultured mammalian cells has been investigated. The fluorescence was found to originate in discrete cytoplasmic vesicle-like regions and to be absent from the nucleus. Excitation and emission spectra of viable cells revealed at least two distinct fluorescent species. Comparison of cell spectra with spectra of known cellular metabolites suggested that most, if not all, of the fluorescence arises from intracellular nicotinamide adenine dinucleotide (NADH) and riboflavin and flavin coenzymes. Various changes in culture conditions did not affect the observed autofluorescence intensity. A multiparameter flow system (MACCS) was used to compare the fluorescence intensities of numerous cultured mammalian cells.


Differences in flow cytometry and 3H-thymidine analyses of perturbed human lymphocytes. A calf thymocyte crude aqueous extract was tested for DNA synthesis inhibitory activity using phytohemagglutinin-stimulated human peripheral blood lymphocytes. Inhibition of DNA synthesis was assayed using tritiated thymidine and flow cytometry. Although the calf thymocyte crude extract inhibited tritiated thymidine incorporation by over 50%, only very slight changes in the flow cytometric analysis were observed. When dibutyryl-cyclic adenosine monophosphate was used as an inhibitor, a correlation in terms of the inhibition of tritiated thymidine to the inhibition by flow cytometry was observed.


Subacute infection with temperature-sensitive vesicular stomatitis virus mutant G41 in the central nervous system of mice. I. Clinical and virologic studies. Inoculation of three- to four-week-old BALB/c mice with temperature-sensitive (ts) vesicular stomatitis virus mutant G41 produced a subacute neurological disease, initially characterized by development of lethargy, hunched posture, and ruffled fur within five to seven days after infection. More than 90% of infected mice developed these clinical signs. In approximately 60% of infected mice, the initial neurological signs proceeded to striking hind-limb paralysis and weight loss. These signs usually appeared by seven to nine days after infection and lasted for 21-28 days. Only 16% of the mice died as a result of infection; death usually occurred eight to 12 days after infection. Most of the infected mice recovered from the acute phase of disease and appeared normal by four weeks after infection. However, hind-limb paralysis persisted in 4% of the mice for as long as the mice were observed, i.e., 42 days. The mutant ts-G41 was recovered from the brains and spinal cords of infected mice for the first seven days after infection. Peak titers of virus were modest, 10(4)-10(5) pfu/ml in brain tissue and 10(3)-10(4) pfu/ml in spinal cord tissue. Virus isolated after in vivo infection was temperature-sensitive and thus not revertant wild-type virus. Although virus was recoverable by homogenization for only the first seven days of infection, use of cocultivation techniques permitted the detection of ts-G41 in brains and spinal cords of infected animals for as long as 21 days after infection. Virus recovered by cocultivation was also temperature-sensitive.


Subacute infection with temperature-sensitive vesicular stomatitis virus mutant G41 in the central nervous system of mice. II. Immunofluorescent, morphologic, and immunologic studies. Inoculation of three- to four-week-old BALB/c mice with temperature-sensitive (ts) vesicular stomatitis virus (VSV) mutant G41 produced a subacute neurological disease mainly localized in the spinal cord. Meningitis and diffuse microglial infiltration of the anterior horns of the spinal cord were seen starting six days after infection when neuronal degenerative changes could be seen. Infection of neurons was demonstrated by immunofluorescence microscopy five days after infection. Two to three weeks after infection, loosening of the neuropil was evident due to neuronal dropout, and the mononuclear infiltration had become perivascularly distributed and had changed in character because of a striking increase in plasma cells. These cells together with Russell bodies became the main inflammatory cellular component about four to five weeks after viral inoculation. Starting eight days after infection, several foci of primary demyelination could be found in the anterior columns of the spinal cord. Immunological responses appeared within four days after infection when both neutralizing antibody and stimulation of specific spleen lymphocytes could be demonstrated. Serum antibody responses peaked at 21-28 days but remained elevated for up to 153 days. Stimulation of spleen lymphocyte cells peaked at 10-21 days and also remained elevated for as long as 116 days. The presence of both inflammatory changes and immunological responses to VSV mutant ts-G41 for prolonged periods is characteristic of persistent viral infections. Infection of BALB/c mice with ts-G41 thus represents the first in vivo example of persistent viral infection utilizing ts mutants of VSV.


Serologic studies of transmission of hepatitis A in humans. In 1968, studies of infectious hepatitis in volunteers were reported. Immunologic procedures for serologic study of the hepatitis A virus were not available at that time, and only the clinical and biochemical parameters of the disease were reported. Serial serum specimens from the participants in the study were retained; these specimens had been taken before inoculation and up to more than 100 days after inoculation. When a radioimmune assay for antibody to hepatitis A virus was developed, the series of sera was analyzed retrospectively. Forty-four male volunteers were involved in a series of three studies. Twenty (46%) of the volunteers were found to be initially immune to hepatitis A virus. Eighteen susceptible volunteers (with no preexisting antibody) were challenged with infectious virus. Eight of these volunteers developed clinical hepatitis and seroconverted; one seroconverted without evidence of clinical disease; and nine neither seroconverted nor had evidence of clinical disease. The radioimmune assay provided a method for diagnosis of immune status and of the acute disease caused by hepatitis A virus.


Isolation of a cytotoxin-enterotoxin from Entamoeba histolytica. A heat-labile material with cytotoxic and enterotoxic activities was isolated from axenically cultivated Entamoeba histolytica. The cytotoxin-enterotoxin was partially purified from the particulate-free supernatant of sonicated trophozoites by ammonium sulfate precipitation and gel filtration. Cytotoxic activity, assayed on monolayers of HeLa or BHK-21 cells, was restricted to proteins that eluted in the molecular weight range of 25,000--35,000 daltons. Cytotoxicity was demonstrated at protein concentrations as low as 2--4 microgram/ml, was heat-labile (75 C, 30 min), and was inhibited by specific immune IgG and by an undetermined factor in nonimmune serum. Enterotoxic activity of the partially purified toxin was demonstrated by induction of fluid secretion in ligated rabbit ileal loops. The cytotoxin-enterotoxin of E. histolytica may play an important role in the production of diarrhea and mucosal injury in amoebic colitis.


Chronically persistent infection with human cytomegalovirus in human lymphoblasts. Cells from a line of human lymphocytes originating from a leukemic patient were persistently infected with human cytomegalovirus. Assays of infectious centers and fluorescent antigen staining indicated that 1%--10% of the cells were infected. It appears that persistent infection is due to an equilibrium between the release of virus by infected host cells and the growth of uninfected cells rather than to defective virus or temperature-sensitive mutants.


Travelers' diarrhea among U.S. Army troops in South Korea. A prospective study of diarrhea was conducted among 98 U.S. Army soldiers during their first six weeks in South Korea. Diarrhea developed in 54 (55%) of 98 soldiers and had a mean duration of five days. Infections with Salmonella, Shigella, Vibrio, enterotoxigenic Escherichia coli, enteroviruses, and intestinal parasites were uncommon. Four (8%) of 50 soldiers with documented diarrhea, two (6%) or 32 with a history of diarrhea, and one (3%) of 29 who denied gastrointestinal symptoms had serologic evidence of a recent rotavirus infection. The etiology of diarrhea among U.S. soldiers who had recently arrived in South Korea differed from the etiology among travelers in warmer climates, where enterotoxigenic strains of E. coli were responsible for the majority of cases. Further efforts are needed to define other enteric pathogens in the etiology of diarrhea among new arrivals in different parts of the world.


Seroimmunity to polioviruses in U.S. Army recruits. Titers of neutralizing antibody to poliovirus types 1, 2, and 3 were determined for serum specimens obtained from 268 U.S. Army recruits. Among those tested, 20.9% lacked neutralizing antibody to one or more types of poliovirus, and 1.1% lacked antibody to all three types. An analysis of demographic data showed that age of less than 18 years, schooling for less than 10 years, and residence in the northeastern United States were associated with higher percentages of recruits lacking neutralizing antibodies to polioviruses in serum.


Complement-enhanced neutralizing antibody response to varicella-zoster virus. Humoral immunity following infection with varicella-zoster virus (VZV) was evaluated by a complement-enhanced neutralization test. The specificity of the test was examined and its sensitivity compared with that of the assay which measures antibody to VZV-induced membrane antigen (FAMA). Generally, the titer of neurtalizing antibody was two- to fourfold higher than the FAMA titer. The absence of neutralizing activity at a serum dilution of 1:4 indicated susceptibility of the donor to VZV infection and correlated with an absence of FAMA (titer, less than 1:2). A survey of susceptible leukemic children exposed to chicken pox revealed that several recipients of zoster immune globulin had a subclinical infection, as manifested by seroconversion and persistence of neutralizing antibody to VZV. Results of these studies indicate that the complement-enhanced neutralization test is a sensitive and specific assay for determination of humoral immune status with regard to VZV in healthy and immunosuppressed individuals.


Relapse of pneumonia caused by cytomegalovirus in two recipients of renal transplants. Two cases of relapse of pneumonia caused by cytomegalovirus (CMV) in recipients of renal transplants were studied. Between episodes of pneumonia, both patients recovered completely as judged by X-ray clearing and by resolution of fever and respiratory symptoms. CMV was isolated during each episode of pneumonia from lung tissue, pleural fluid, or buffy coat cultures. Serologic studies revealed a rise in titer of antibody, confirming recent infection with CMV during the first epidose of pneumonia in each patient. Intranuclear inclusions consistent with CMV infection were detected in each of the three episodes where lung tissue was obtained. Special stains and cultures for Pneumocystis carinii, fungi, Nocardia, and mycobacteria yielded negative results. The renal allograft continued to function in both patients after they recovered from the first bout of pneumonia, and the relapse was temporally related to reinstitution of aggressive immunosuppressive therapy. The relapse of pneumonia emphasizes the need for caution when immunosuppressive therapy is reinstituted after recovery from serious CMV infection.


Presence of two distinct types of rotavirus in infants and young children hospitalized with acute gastroenteritis in Mexico City, 1977. Between December 1976 and January 1978, infection with rotavirus was detected by electron microscopy in 61 (25%) of 242 infants and young children hospitalized with acute gastroenteritis at two hospitals in Mexico City. This type of infection was more frequent in autumn than in winter. The presence of virus could also be detected by the electrophoretic pattern of its segmented, double-standed ribonucleic acid (RNA) in the stool samples taken from 52 (85%) of the 61 patients who were shown by electron microscopic examination to excrete rotavirus. Two distinct patterns, previously called 2s and 21, were observed, which were distinguishable by the relative migration of the second, 10th, and 11th viral RNA segments in gel electrophoresis; the 21 pattern was observed much more frequently than with the 2s pattern.


Evaluation of indirect hemagglutination and its inhibition in the differentiation between antibodies to herpes simplex virus types 1 and 2 for seroepidemiologic studies: use of a II/I index threshold of 85 and an assay of type-specific antibodies. Two parameters, a II/I index threshold value of 85 and an assay of type-specific antibodies to herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), were assessed, for seroepidemiologic purposes, for their efficacy in the establishment of past infections with HSV-1 and HSV-2 as indicated by the results of tests for indirect hemagglutination and its inhibition. Neither criterion alone established past infection with either HSV-1 or HSV-2. However, by combination of the two parameters, antibodies to HSV-1 and HSV-2 could be differentiated for seroepidemiologic studies.


Primary infection with Epstein-Barr virus in infants in the United States: clinical and serologic observations. Previous studies in Ghana had shown that primary infections with Epstein-Barr virus in infants under the age of two years remain silent and evoke antibody responses different from those seen in infectious mononucleosis. In order to determine whether or not these observations were limited to Africa, 80 American infants presenting with minor infectious complaints were studied serologically; 14 (17.5%) showed evidence of recent or current primary infections with Epstein-Barr virus. The clinical features of these 14 infants were similar to those of the other 66 and did not suggest a diagnosis of infectious mononucleosis, nor were there histories of a recent infectious mononucleosis-like illness. Ten (72%) had antibodies to the early antigen complex induced by Epstein-Barr virus; however, these antibodies were directed, as in the Ghanaian infants, against the restricted rather than the diffuse components, in contrast to the pattern generally observed in infectious mononucleosis. Possible reasons for the differences between the responses of infants and those of older individuals to primary infection with Epstein-Barr virus and to the early antigen complex are discussed.


Role of Norwalk virus in outbreaks of nonbacterial gastroenteritis. Twenty-five separate outbreaks of nonbacterial gastrointestinal illnesses were studied serologically for evidence of infection with the Norwalk virus and the rotaviruses that affect humans. Eight of 25 outbreaks appeared to be related to the Norwalk virus. In one of the 25 outbreaks, there was evidence of rotavirus infection. These observations suggest that the Norwalk virus or serologically related agents play an important role in epidemic nonbacterial gastroenteritis in adults and older children.


Ornidazole and anaerobic bacteria: in vitro sensitivity and effects on wound infections after appendectomy. The sensitivities of 68 clinical isolates of Bacteroides fragilis, 18 of Clostridium perfringens, and 11 of other Clostridium species were tested against ornidazole alone and in combination with ampicillin and gentamicin. A concentration of 3.1 microgram of ornidazole/ml inhibited 98% of the strains of B. fragilis, with greater sensitivity when ampicillin and gentamicin were also present. A concentration of 6.2 microgram of ornidazole/ml inhibited 16 of 18 strains of C. perfringens and all 11 strains of other Clostridium species. Concentrations in serum and tissue were determined after intravenous infusion of 500 mg of ornidazole 15 min prior to appendectomy. During the operation the concentration in serum was 7.90 +/- 0.57 microgram/ml, and in appendix tissue, 5.26 +/- 0.60 microgram/g. In the series of 200 patients undergoing appendectomy, six patients treated with ornidazole and 12 patients treated with placebo developed a wound infection. In patients with perforated appendix, the rate of wound infection was 7.1% in those given ornidazole and 63.6% in those given placebo (P = 0.004). Not a single B. fragilis was isolated from appendix swabs or wound exudates after prophylaxis with ornidazole.


Clinical significance of serum angiotensin-converting enzyme levels in sarcoidosis. Determinations of SACE activity were performed in 80 patients with sarcoidosis, 55 normal controls, and 29 patients with asthma, by the spectrophotometric method of Cushman and Cheung. SACE levels were significantly higher in both untreated and steroid-treated patients with sarcoidosis than in normal controls: 46.2 +/- 20.6 (S.D.), nm/min/ml, 38.1 +/- 23.1, and 26.8 +/- 1.8, respectively. There were no significant differences between steroid-treated and untreated patient groups. However, an inverse correlation was observed between SACE levels and steroid dose, suggesting the possibility of a dose dependency for steroid-induced depression of SACE. Elevated SACE levels (2 S.D. above mean controls) were present in 67% of untreated sarcoidosis patients. The prevalence of elevated SACE levels was not significantly higher when the patient population was examined with respect to duration of disease, radiological stage, and the presence of abnormality in pulmonary function tests. When patients were divided according to the frequency of clinical criteria of disease activity, the presence of two or more criteria was associated with elevated SACE levels in 88% of patients. But SACE levels were elevated in 32% of patients judged to have dormant disease by clinical criteria. SACE levels had an 81% accuracy in prediction of disease activity and a 79% accruacy for prediction of inactivity. It was concluded that SACE elevations have definite diagnostic value in sarcoidosis and are helpful in establishing the presence of disease activity but are not sufficient to fully separate active from inactive disease groups.


The ultrastructure of cholesterol granuloma of the middle ear: an electron microscope study. The ultrastructure of giant cells in cholesterol granuloma is described, with particular reference to their role in the absorption of cholesterol needles. Cholesterol is a highly resistant substance in its crystalline form and it is confirmed that the giant cells are endowed with endoplasmic structures and organelles and are partly crowded with lysosomes, as evidence of the active role of the giant cells in the absorption and removal of cholesterol formed in the granuloma from blood, mucus or necrotic material.