Genetic transmission of mammary tumour virus by GR mice. By immunodiffusion assay (ID-test) milk samples of mice of several strains and of F1-hybrids of the GR strain were tested for the presence of mammary tumour virus (MTV) antigens. The results clearly demonstrated that the presence of viral antigens in the milk of the first lactation period is restricted to mice harbouring endogenous MTV-GR. Viral antigens were detectable in about 50% of the milk samples collected during the first (occasionally the second) lactation periods of mice of the segregating backcross I (Bc I) populations: DBAfX(DBAfXGR), AKRX(AKRXGR), BALB/cX(BALB/cXGR) and C57BLX(C57BLXGR), indicating that one dominant gene is responsible for the presence of viral antigens in the milk of GR mice. The proposed gene symbol is Mtv-2. Milk samples from female mice of three different Bc II populations were tested for the occurrence of viral antigens. In the first Bc II: [BALB/cX(BALB/cXGR)]XBALB/c 33 out of 51 mice, descending from ID-positive mothers, had ID-positive milk and only one out of 71 mice, which were the progeny of ID-NEGATIVE Bc I mothers, was ID-positive. These results may be influenced by the MTV transmitted extrachromosomally via the milk of the mother. The two other Bx II populations were derived from Bc I fathers, either [BALB/cX(BALB/cXGR)] or [(BALB/cXGR)XBALB/c] f and BALB/c females. The results obtained with these Bc II populations suggested that 6 Bc I fathers were heterozygous for Mtv-2. Since the segregation ratio (60:29) in the Bc II population (progeny of these 6 Bc I male) deviates significantly from the expected 1:1 ratio, one may assume extrachromosomal transmission of MTV via the seminal fluid of the father to the progeny. A close correlation was found between the presence of MTV antigens in the milk and the occurrence of both early mammary tumours after hormone treatment and spontaneous mammary tumours before the age of 13 months. These results suggest that the early appearance of mammary tumours in the GR strain and the early expression of MTV antigens in the milk appear to be controlled by the same genetic factors.


Transformation of rabbit kidney cells by BKV(MM) human papovavirus. Primary rabbit kidney cells were transformed by BKV(MM), a papovavirus isolated from the urine of a child with the Wiskott-Aldrich syndrome. The transformed cells contained BK T-antigen, but no antigen that reacted with SV40 U-antiserum. The transformed cells failed to produce tumors in nude mice, and BKV (MM) was not rescued from transformed cells by cell fusion or chemical induction methods. The transformed cells supported the growth of rabbit kidney vacuolating virus (RKV), and could be used to quantitate RKV by plaque formation under an agar overlay.


Isolation from a transmissible lymphoid tumour (TLT) lymphoblastoid cell line of a herpesvirus similar to Marek's disease virus. A chicken lymphoblastoid cell line (TLT)-6855 originally established from an avian oncornavirus-induced lymphoma (Siegfried and Olson, 1972) was studied for the presence and expression of Marek's disease virus (MDV) genome. By nucleic acid hybridization a significant amount of MDV DNA was found in this cell line. This virus DNA, however, was not expressed in either virus-specific intracellular or membrane antigens or the MD-associated tumour-specific surface antigen (MATSA). Moreover, MDV-specific antigens could not be activated in this cell line by treatment with 5-IdUrd. In several experiments, when chickens were inoculated with the cell line a herpesvirus was repeatedly isolated from the kidneys. This herpesvirus was antigenically similar to MDV but was low in oncogenicity for chickens.


Epstein-Barr virus antibodies in tonsillar carcinoma patients;. Sera from 18 tonsillar carcinoma patients and from 18 matched control subjects were examined for the presence of antibodies to viral capsid antigen (VCA), early antigen (EA) and nuclear antigen (EBNA) of EB virus. Antibodies to all three antigens were found more frequently and in significantly higher titres in the tonsillar carcinoma patients than in control subjects.


Column separation of viral capsid antigen (VCA)-positive cells from VCA-negative cells in an Epstein-Barr virus (EBV)-producing lymphoid line,. Virus producing, VCA (viral capsid antigen)-positive cells could be selectively removed from Epstein-Barr virus (EBV)-carrying, virus-producer P3HR-1 cultures by two different methods of column passage. In the first, the virus-producing cells were covered with human EBV antibody and subsequently passaged through anti-human-Ig columns. In the second methods, untreated P3HR-1 cells were allowed to pass through columns of EBV receptor-positive cell lines or, as controls, EBV receptor-positive cell lines or, as controls, EBV receptor-negative cells. The majority of the VCA-positive cells were selectively removed by both techniques. The second method involves the attachment of EBV-producer cells, known to accumulate viral envelope material in their plasma membrane, to EBV receptors. In view of recent evidence indicating an association between EBV and complement receptors, human and mouse complement were tested for their ability to block this attachment. Fresh mouse and human complement regularly exerted blocking activity, whereas heat-inactivated human serum did not block. Heat-inactivated mouse serum did block occasionally, but the effect was more irregular than with fresh mouse serum. Trypsin treatment of the EBV-receptor colum abolished its ability to retain VCA-positive cells.


The presence of Herpesvirus Saimiri genomes in virus-transformed cells. Herpesvirus saimiri (H. salmiri) -transformed cells contained both types of viral DNA, unique L-DNA and highly repetetive H-DNA. DNA from spleen and lymph-node autopsies of two tumor-bearing marmoset monkeys contained 0.14-0.75% viral L-DNA -AND 0.115-1.08% H-DNA. This amount of H-DNA would be equivalent to the presence of 14-130 M-genomes per diploid tumor tissue cell. Six virus-transformed lymphoid cell lines, two of them virus-producing, contained 0.69-2.27% H-DNA and more than 0.72-1.95% L-DNA. These concentrations of H-DNA sequences correspond to 83-274 M-genome copies per lymphoid tissue culture cell. The majority of viral genomes in transformed non-producer lymphoid cell lines appeared to be defective, since part of the L-sequences present in virions were found to be deleted in the genome copies of transformed cells. There was a relative excess of repetitive H-sequences in all transformed cells in regard to the ratio of H-DNA/L-DNA in M-genomes of H. saimiri virions.


FeLV epidemiology in Los Angeles cats: Appraisal of detection methods. We investigated the reliability of the fixed cell indirect fluorescent antibody (IFA) peripheral blood smear test as an index of systemic infection with FeLV. Positive results with this test were found to correlate well with detectable FeLV p30 antigen in bone-marrow smears by IFA, in serum and tissue by competition immunoassays, and with type-C particles in bone marrow or spleen by electron microscopy. Most cats with lymphoma, anemia or infectious peritonitis were positive for FeLV and showed a poor or absent antibody response to FeLV p30, gp70, and FOCMA antigens. Most older cats with lymphoma, carcinoma, or sarcoma were negative for FeLV expression and also lacked these FeLV-related antibodies. Detectable immunologic response to FeLV p30 and gp70 proteins and a high-titered FOCMA antibody response were generally restricted to certain healthy cats exposed to FeLV. Antibody to endogenous RD-114 viral p30 and gp70 was not detected in any of a large number of feline sera tested. The prevalence of FeLV-related diseases and immunologic responses to FeLV in healthy cats was directly correlated with the degree of FeLV exposure. By using the IFA blood smear and FOCMA antibody tests one can monitor the horizontal spread of FeLV in multi-cat household and accurately predict the FeLV disease susceptibilty or resistance of individual cats in each environments.


Symmetrical features in polypeptide hormone-receptor interactions. Symmetrical features were observed in the amino acid sequences of some biologically active peptides. It is suggested that this approximate symmetry is reflected in the conformations of the peptides at their respective biological receptors, and has arisen by natural selection as both peptides and receptors evolved to optimise their mutual fit. It follows that the binding site for each peptide at its receptor would share the same symmetry element. This would arise if the peptide binds to two symmetrically related similar or identical submits in the receptor.


Synthesis and biological activities of two ACTH-analogues containing L-norarginine in position 8. Two new ACTH-analogues, an octadecapeptide amide and a tetracosapeptide containing the lower homologue of arginine (norarginine) in position 8, have been synthesized by the generally accepted method. Special attention was paid to the synthesis of the required tetrapeptide representing the 7-10 sequence, which was obtained either by direct introduction of L-nitronorarginine or by amidination of the gamma-amino function in a protected peptide containing alpha, gamma-diaminobutyric acid. Biological activity determination showed that the shortening e arginine side chain in position 8 results in the formation of active ACTH-analogues.


Free radicals in pyrimidines:single crystals of dihydro-6-methyl uracil irradiated and observed at 77 K. Electron-spin-resonance measurements have been made on single crystals of dihydro-6-methyl uracil, which were irradiated by 4-0 MeV electrons at 77 K. At low temperature, four radicals have been identified. Two, the C5 and C6 hydrogen abstraction radicals, were also studied in a previous work. On exposure of U.V., the C6 abstraction radical was found to convert into the C5 abstraction radical. Annealing at room temperature reversed the process. The other radicals identified are that formed after H-atom addition to O4, and that formed after H-atom abstractions from N1, C5, and C6. The latter was not present in deuterated crystals. Both radicals were found to be unstable at intermediate temperatures. Thermoluminescence glow-curves were found to correlate to the formation and the decay of these radicals. Semi-empirical INDO MO-calculations have been performed for identification purposes.


Cytologic diagnosis of adenoviral epidemic keratoconjunctivitis by direct immunofluorescence. A direct immunofluorescence technique for the diagnosis of acute adenoviral keratoconjunctivitis and pharyngoconjunctivitis was found to be a reliable, sensitive, and specific technique for the detection of soluble adenoviral antigens in epithelial cells on conjunctival scrapings of patients with epidemic keratoconjunctivitis. Of 25 patients with clinical findings consistent with epidemic keratoconjunctivitis or pharyngoconjunctival fever, all had positive diagnostic scrapings by direct immunofluorescence.


Interaction of adrenergic agents with alpha-melanocyte-stimulating hormone and infrared irradiation of the iris in the rabbit eye. Breakdown of the blood aqueous barrier in the rabbit eye induces a protein leakage into the aqueous humor, seen as a flare in the anterior chamber. A barrier damage was induced by topical prostaglandin E2(PGE2), infrared irradiation of the iris, or alpha-melanocyte-stimulating hormone (alpha-MSH) given subcutaneously. The aqueous flare was measured quantitatively by means of a photoelectric instrument. The interference of adrenergic antagonists and agonists on the breakdown of the barrier was tested. The alpha-adrenergic antagonist phentolamine and the beta-adrenergic antagonist propranolol, given intravenously, had no effect on exogenously administered PGE2, but both antagonists reduced the flare response to infrared irradiation which is supposed to exert its effect via endogenous prostaglandin release. The alpha-MSH response was unaffected by phentolamine, whereas propranolol abolished the flare response to alpha-MSH totally. The PGE1 response was unaffected both by the alpha-adrenergic agonist noradrenaline and the beta-adrenergic agonist terbutalin sulfate, administered topically. Noradrenaline, however, inhibited the flare response to infrared irradiation and facilitated the flare response to alpha-MSH. Terbutalin sulfate worked synergistically with both infrared irradiation and alpha-MSH. It is assumed that alpha-MSH exerts its effect on the barrier via enhanced beta-adrenergic activity, whereas the effects caused by infrared irradiation seem conditioned by intact alpha- as well as beta-adrnergic receptor sites.


Retinal neoplasia and dysplasia. I. Induction by feline leukemia virus. Feline leukemia virus, a naturally occurring C-type virus (oncorna virus), was injected systemically and intraocularly into fetal and newborn kittens. A light microscopy study of the resultant ocular tumor changes was carried out. One animal developed an intraocular tumor of apparent retinal origin. Eight animals showed intraocular neoplastic cell infiltration. The most common ocular change observed was retinal dysplasia. Sequential histologic examination of enucleated eyes showed diffuse intraocular inflammation after viral inoculation. In the developing retinas progressive disorganization and necrosis were documented, with subsequent reorganization into cell clumps and dysplastic rosette structures. The retinal pigment epithelium showed proliferation and intraretinal migration. The mature retina exhibited full-thickness folds and tubes associated with infoldings of the retinal pigment epithelium.


The value of enzyme histochemical techniques in classifying fibre types of human skeletal muscle. 1. Adult skeletal muscles with no apparent disease of the neuromuscular system. Fibre-type classification of human skeletal muscle into type I and type II fibres is mostly based on their slight or strong staining with the myosin adenosine triphosphatase reaction. In order to evaluate the reliability of this screening technique a combined histochemical and biochemical study was performed on normal and diseased skeletal muscle of human subjects. In the present investigation activities of enzymes which play a role in the aerobic and anaerobic pathways and which can characterize fibre type, were examined in muscle specimens, with no apparent disease of the neuromuscular system. Special attention is given to the maximal activities of phosphofructokinase and fructose-1,6-diphosphatase, the rate limiting enzymes for the regulation of the glycolysis and glyconeogenesis, respectively. A most important feature of the biochemical findings is the constancy of the activity ratios of the examined enzymes. From these results and from the histochemical results it can be concluded that in apparently normal adult human skeletal muscle the ATP-ase technique for type I and type II typing is reliable. For fibres with an intermediate intensity of staining with the myosin ATPase technique of typing it is also necessary to apply other enzyme histochemical techniques.


Composition and enzyme activities of Spiroplasma citri membranes. Spiroplasma citri was cultured in three different media that supplied cholesterol and fatty acids from: (i) horse serum, (ii) pleuropneumonia-like organism (PPLO) serum fraction, or (iii) bovine serum albumin-fatty acid-cholesterol. The ability of PPLO serum fraction to support growth varied by lot number. Neither PPLO serum fraction nor the bovine serum albumin medium supported growth as well as the horse serum medium. Analysis of cholesterol, lipid phosphorus, and membrane protein showed the horse serum- and PPLO-grown cells to be indistinguishable, but the bovine serum albumin-grown cells were deficient in lipid phosphorus. The three cultures did not show markedly different fatty acid compositions, but, in all cases, the cultures preferentially incorporated palmitic acid and discriminated against linoleic acid. Cultures grown for different times from logarithmic growth through a degenerative phase showed relatively constant ratios of cholesterol/protein and lipid phosphorus/protein. Fatty acid composition was also relatively constant at the different stages. Adenosine triphosphatase and p-nitrophenyl phosphatase were mainly associated with the membrane, whereas reduced nicotinamide adenine dinucleotide oxidase was either readily removed or not associated with the membrane. The reduced nicotinamide adenine dinucleotide oxidase was inactivated at temperatures above 35 degrees C.


Inhibition of aminoacyl-transfer ribonucleic acid synthetases and the regulation of amino acid biosynthetic enzymes in Neurospora crassa. Growth conditions that result in the accumulation of the tryptophan intermediate indoleglycerol phosphate or of the histidine intermediate imidazoleglycerol phosphate cause mycelia of Neurospora crassa to exhibit an immediate and sustained increase in the differential rate at which the biosynthetic enzymes of the tryptophan, histidine, and arginine pathways are synthesized. These accumulated intermediates are shown to be inhibitors of the activity of aminoacyltransfer ribonucleic acid (tRNA) synthetases, as judged by an in vitro esterification assay. The tryptophan intermediate is shown to inhibit the charging of tryptophan, and the histidine intermediate is shown to inhibit charging of histidine. The inhibitions noted are consistent with the finding that the level of charged tRNATrp is decreased significantly in cells that have accumulated indoleglycerol phosphate and that of tRNAHis is decreased significantly in cells that have accumulated imidazoleglycerol phosphate. These results are interpreted as support for the involvement of aminoacyl-tRNA species in mediating cross-pathway regulation of the tryptophan, histidine, and arginine biosynthetic pathways as proposed in Lester's polyrepressor hypothesis (G. Lester, 1971). the correlations noted lead to the conclusion that Neurospora utilizes regulatory mechanisms that have the ability to react to changes in the level of charging of tRNA species.


Isolation and characterization of Saccharomyces cerevisiae mutants defective in glycerol catabolism. Mutants of the yeast Saccharomyces cerevisiae that are defective in the catabolism of glycerol were isolated, and two types of mutants were obtained. One type was deficient in glycerol kinase activity, whereas the other type was deficient in sn-glycerol 3-phosphate dehydrogenase activity. Genetic analysis indicated that each mutant strain owed its phenotype to a single nuclear mutation, and that the two mutations were complementary. The mutations were not linked to each other or to any of 10 loci tested. In addition, neither mutation was centromere linked. Possible mechanisms for the regulation of these enzymes were tested by growing the parental strain in the presence of various carbon sources.


Purification and some properties of phospholipase C (alpha-toxin) of Clostridium perfringens. 1. Phospholipase C[EC 3.1.4.3] was purified from the culture filtrate of Clostridium perfringens by successive chromatographies on CM-Sephadex, DEAE-Sephadex, and Sephadex G-100. During the purification it was noted that, beside the monomer form of the enzyme which was purified, a part of the enzyme existed in active polymerized forms. 2. The purified preparation gave a single band on polyacrylamide gel electrophoresis and gave a single precipitin line in immunodiffusion with the National Standard gas gangrene (C. perfringens) antitoxin, indicating the homogeneity of the preparation. 3. The specific lecithin-hydrolyzing activity of the purified preparation was comparable to that of a preparation obtained by affinity chromatography, which had the highest specific activity previously reported. 4. The molecular weight of the purified enzyme was estimated to be 43,000 by SDS-polyacryl-amide gel electrophoresis, although the same preparation gave a molecular weight of 31,000 as determined by gel filtration on Sephadex G-150. From this and the above finding that a part of the enzyme exists in active polymerized forms, the discrepancy among reported values for the molecular weight of C. perfringens phospholipase C can be accounted for. 5. For maximum hydrolytic activity toward lecithin, the enzyme required sodium deoxycholate (SDC) and Ca2+ ions. In the presence of 6 mM Ca2+, the optimal molar ratio of SDC to lecithin for maximal hydrolytic activity was about 0.5 for dipalmitoyl lecithin and about 1.0 for egg lecithin. The effects of various divalent cations on the enzymatic hydrolysis were also investigated. 6. The effects of sodium deoxycholate and Ca2+ ions on the enzymatic hydrolysis are discussed, based on their possible roles in mixed micelle formation.


Dihydropteridine reductase from bovine liver. Purification, crystallization, and isolation of a binary complex with NADH. Dihydropteridine reductase [EC 1.6.99.7] was purified from bovine liver in 50% yield and crystallized. The physicochemical properties of the purified enzyme were quite similar to those of sheep liver dihydropteridine reductase. During the course of purification, however, the enzyme was found to be separated into 2 major peaks together with minor peaks by column chromatography on CM-Sephadex, and one of the major peaks was identified as a binary complex of the enzyme with NADH. The reductase-NADH complex was also prepared in vitro and crystallized. Upon addition of quinonoid-dihydropterin to the complex, NADH was oxidized and released from the enzyme. The amount of bound NADH was calculated to be 2 moles per mole of the reductase. The occurrence of the reductase-NADH was calculated to be 2 moles per mole of the reductase. The occurrence of the reductase-NADH complex in bovine liver extract as a predominant form was in accord with the pyridine nucleotide specificity for NADH as a coenzyme. The results further support the view that NADH is the natural coenzyme of this reductase.


A complex of collagenase with low molecular weight inhibitors in the culture medium of embryonic chick skin explants. Collagenase inhibitors with molecular weights of about 6,000 and 12,000 were isolated from latent chick skin collagenase treated with 3 M NaI and from the culture medium of embryonic skin explants. It is suggested that these inhibitors, which are possibly derived from connective tissue macromolecule metabolites, are candidates for regulating factors of collagenase activity in vivo.


Some enzymatic properties of peptidyl dipeptide hydrolase (angiotensin I-converting enzyme). Peptidyldipeptide hydrolase [angiotensin I-converting enzyme, EC 3.4.15.1] was inhibited by inorganic and organic phosphorus compounds tested, except for beta-glycerophosphate, 5'-AMP, and 5'-ADP, at the reagent concentrations used. Orthophosphate and pyrophosphate nonspecifically inhibited the enzyme activity. The enzyme was also inhibited specifically by carboxylates. The degree of inhibition by aliphatic monocarboxylates increased in proportion to their chain length up to C14. Aromatic and omega-phenylalkylcarboxylates also inhibited the enzyme activity. The enzyme was noncompetitively inhibited by acetate, 3-phenylpropionate and laurate. The Ki's for acetate, 3-phenylpropionate, and laurate were 60, 3.3, and 2.5 mM, respectively.


Multiple molecular forms of phosphoprotein phosphatase. Separation of four forms of the rabbit skeletal muscle enzyme. Phosphoprotein phosphatase [phosphoprotein phosphohydrolase EC 3.1.3.16] in the soluble fraction of rabbit skeletal muscle, when assayed with phosphorylase a[EC 2.4.1.1] from rabbit skeletal muscle and phosphohistone as substrates, was resolved into three active fractions (Fractions I, II, and III in order of elution) by DEAE-cellulose column chromatography. Sucrose density gradient centrifugation showed that these fractions were composed of subfractions of different molecular size (I: 7.3S and 4S; II: 8S and 4S; III; 6.7S). Components with larger molecular size in the major fractions, II and III, were dissociated to a molecular size similar to that of the smallest component on freezing in the presence of mercaptoethanol. These results indicate that phosphoprotein phosphatase from skeletal muscle occurs in multiple forms very similar to those of the liver enzyme reported previously (Kobayashi, Kato and Sato (1975) Biochim. Biophys. Acta. 373, 343-355).


Two types of sarcoplasmic reticulum-orthophosphate interactions observed with dye probe. The interaction of Pi with sarcoplasmic reticulum (SR) isolated from rabbit skeletal muscle was studied using bromocresol purple (BCP) as a probe and a dual-wavelength spectrophotometer. Two kinds of absorption-intensity changes controlled by a low concentration of Ca2+ (greater than 10(-6)M) were observed after addition of Pi; an increase phase (in the presence of Ca2+), and a decrease phase (in the presence of EGTA). The increase phase was rapid, Ca2+-dependent, Mg2+-enhanced (depressed by high Mg2+ concentration) and not inhibited by PCMB and was suggested to reflect the formation of an SR-Pi complex. The decrease phase was slower than the increase phase, and was strongly inhibited by the low concentration of Ca2+. It required Mg2+, and was completely inhibited by p-chloromercuribenzoate or deoxycholate. It was suggested to reflect the formation of SR-Pi (phosphorylated protein). ATP inhibited this phase by converting it completely to an SR-MgATP phase. PPi was effective for inducing the decrease phase but PPPi was not. From measurements of these phases, the association constants of the SR-Pi complex and SR-Pi at pH 8.8 in the reaction scheme, SR + Pi in equilibrium SR-Pi in equilibrium SR-Pi, were calculated as 5.4 X 10 M-1 and 1.8 X 10(3) M-1, respectively. From the completely different responses of SR-Pi and SR-Pi observed with BCP a marked difference in the conformations of these enzyme states was suggested.


Relationship between tubulin SH groups and bound guanine nucleotides. Guanine nucleotides bound to both the non-exchangeable sites (N sites) and exchangeable sites (E sites) of tubulin were completely released after 7 moles of SH groups per tubulin subunit (55,000 molecular weight) had reacted with PCMPS. The blockage of 2 moles of SH groups in the glycerol-reassembly buffer or 1 mole of SH groups in glycerol-free reassembly buffer resulted in complete loss of tubulin polymerizability. However, under both sets of experimental conditions, the amount of guanine nucleotides released from the E sites was less than 8% and the loss of total guanine nucleotides was only 5%. Addition of GSH did not induce reassociation of released guanine nucleotides, although it restored tubulin polymerizability. These results indicate that the loss of tubulin polymerizability on blockage of the SH groups was not due to dissociation of bound guanine nucleotides and that the binding sites of the nucleotides were independent of the SH groups in tubulin required for polymerization. Furthermore, blockage of SH groups did not change the ratio of GTP to GDP bound to tubulin.


The structure and function of ribonuclease T1. XXII. Tryptic cleavages of the single lysyl and arginyl bonds in ribonuclease T1. 1. When ribonuclease T1 [EC 3.1.4.8] was treated with trypsin [EC 3.4.21.4] at pH 7.5 and 37 degrees, activity was lost fairly slowly. At higher temperatures, however, the rate of inactivation was markedly accelerated. The half life of the activity was about 2.5 h at 50 degrees and 1 h at 60 degrees. 3'-GMP and guanosine protected the enzyme significantly from tryptic inactivation. 2. Upon tryptic digestion at 50 degrees, the Lys-Tyr (41-42) and Arg-Val (77-78) bonds were cleaved fairly specifically, yielding two peptide fragments. One was a 36 residue peptide comprizing residues 42 to 77. The other was a 68 residue peptide composed of two peptide chains cross-linked by a disulfide bond between half-cystines -6 and -103, comprizing residues 1 to 41 and 78 to 104. 3. When the trinitrophenylated enzyme, in which the alpha-amino group of alanine-1 and the episolone-amino group of lysine 41 were selectively modified, was treated with trypsin at 37 degrees, the activity was lost fairly rapidly with a half life of about 4 h. In this case, tryptic hydrolysis occurred fairly selectively at the single Arg-Val bond. Thus the enzyme could be inactivated by cleavage of a single peptide bond in the molecule, an indication of the importance of the peptide region involving the single arginine residue at position 77 in the activity of ribonuclease T1.


Selective solubilization of apolipoproteins from hen's egg yolk very low density lipoprotein with guanidine hydrochloride and urea. The selective solubilization of apo-very low density lipoprotein (apo VLDL) of hen's egg yolk was achieved from intact VLDL with guanidine hydrochloride (GuHCl) or urea. The amount of extracted apoVLDL increased with increase of the reagent concentration. GuHCl was more effective than urea and more than 60% of apoVLDL was solubilized with 6 M GuHCl. Previously we reported the presence of five major apoVLDL components, GPI, ApoA, GPII, ApoB, and ApoC in order of size, and found that GPI and GPII were periodic acid-Schiff staining positive, while ApoA, ApoB, and ApoC were negative. With GuHCl or urea, GPI and GPII were easily solubilized, while ApoA and ApoB could not be extracted. The solubilized apoVLDL was rich in carbohydrates, especially sialic acid, compared with the residual apoVLDL. However, only slight differences in amino acid compositon were found between the soluble and the residual apoVLDL. After the partial removal of apoVLDL with GuHCl or urea, VLDL retained its particulate nature, and no destruction of the lipid core was observed. These results were interpreted as indicating that the release of apoVLDL with GuHCl or urea occurred from the surface of the VLDL particle and that the selectively solubilized apoVLDL fractions, such as GPI and GPII, were weakly bound to lipids on the surface of VLDL, while ApoA and ApoB were tightly associated with the VLDL particle.


Induction of ornithine decarboxylase in cultured mouse L cells. II. Effects of additions of amino acids and serum. Induction of ornithine decarboxylase [EC 4.1.1.17] (ODC) in mouse L cells by components of the culture medium was investigated. It was found that further addition of amino acid  mixture, but not of calf serum, to confluent cells of 5 day culture induced ODC and that this induction was accelerated by actinactinomycin D. In salt solution, addition of either amino acids or serum alone did not cause full induction, but addition of both together did. This induction, in contrast, was inhibited by actinomycin D. Induction by insulin, but not by cyclic AMP, was enhanced by a higher concentration of amino acids. These results can be explained by supposing that in non-growing cells there is stable RNA which is involved in ODC induction, possibly mRNA of ODC, and that the observed induction is caused by inhibition of enzyme degradation and accelerated translation, while in growing cells this RNA is unstable and ODC induction is controlled at the level of transcription.


Properties of immunoreactive glucagon fractions of canine stomach and pancreas. The present study was designed to identify the physicochemical, immunologic, and biologic properties of the immunoreactive glucagon (IRG) moieties of canine gastric fundus and to compare them with those of the canine pancreas. Acid-alcohol extracts of the gastric fundus and pancreas of dogs were subjected to Bio-Gel P-10 chromatography, The elution profiles of extracts of both organs revealed IRG peaks in the Mr = 2,000 3,500, and 9,000 zones; in the gastric extracts, a void volume peak was also present. On the basis of Sephadex G-150 rechromatography and sucrose density gradient ultracentrifugation the latter IRG was estimated to have a Mr = 65,000. Incubation of fundic IRG65,000 in 8 M urea failed to alter its elution position. Its pI was 6.4, while fundic IRG3,500 had a pI of 6.15 and pancreatic glucagon 6.25. Fundic IRG9,000 had a pI of 4.5 and pancreatic IRG9,000 4.65. Dilution curves of these three fundic and two pancreatic IRGs were parallel to crystalline beef-pork glucagon. The glycogenolytic activity of fundic IRG3,500 and IRG65,000, measured in the isolated rat liver system, was not different from that of immunoequivalent amounts of dog pancreatic glucagon or crystalline beef-pork glucagon. Both fundic and pancreatic IRG9,000 were devoid of glycogenolytic activity and lacker adenylate cyclase stimulating activity and 125I-glucagon displacing activity when tested on partially purified rat liver membranes. Fundic IRG65,000, however, stimulated adenylate cyclase and displaced 125I-glucagon to the same degree as immunoequivalent amounts of pancreatic glucagon. Fundic IRG3,500 was more active than pancreatic glucagon in stimulating adenylate cyclase activity. This was not clearly attributable to differences in binding to liver cell membranes.


Mechanism of regulation of adenylate cyclase activity in human polymorphonuclear leukocytes by calcium, guanosyl nucleotides, and positive effectors. This study presents the results of a kinetic investigation of adenylate cyclase in human polymorphonuclear leukocytes. In the presence of a saturating concentration of substrate (1 mM), the basal activity was increased severalfold by increasing Mg2+ from 1 to 25 mM. A Hill coefficient of 1.9 was obtained for Mg2+ or ATP. The data suggest cooperative interactions between the substrate binding sites in the neutrophil adenylate cyclase complex. It has been observed that guanyl-5'-yl imidodiphosphate (Gpp(NH)p) (S0.5 = 10 MUM) significantly increased and Ca2+ (S0.5 = 0.5 MM) significantly decreased only the Vmax without affecting the Hill coefficient or S0.5 for ATP. The Hill coefficients for Ca2+ or Gpp(NH)p were 0.9 and 0.8, respectively. The Hill coefficient for Ca2+ was not changed by the increased Gpp(NH)p concentrations. It appears that neutrophil adenylate cyclase has distinct binding sites for Gpp(NH)p and Ca2+, one for each compond. The binding of ligands is not changed by the other effectors and the action is directed only toward the Vmax of the enzyme. The stimulatory action of positive effectors (prostaglandin E1, isoproterenol, histamine) was enhanced by Gpp(NH)p and depressed by Ca2+. No preferential stimulation by Gpp(NH)p nor inhibition by Ca2+ of the action of the positive effectors has been found. The data suggests that only one type of catalytic subunit responds to the action of several positive effectors. Extracellular Gpp(NH)p or Ca2+ do not affect the cyclic adenosine 3':5'-monophosphate (cAMP) level in whole neutrophils and the effect of positive effectors on cAMP production is also not significantly changed by 5 mM Ca2+ or 0.1 mM Gpp(NH)p. Ionophore A23187 in the presence of 5 mM Ca2+ enhances Ca2+ entry into cells and decreases the basal cAMP formation. It appears that Gpp(NH)p or Ca2+ act only at the intracellular site of the adenylate cyclase complex.


Incorporation of dietary cis and trans isomers of octadecenoate in lipid classes of liver and hepatoma. Groups of rats bearing Morris minimal deviation hepatoma 7288CTC were fed a fat-free diet supplemented with either 0.5% safflower oil (diet A), 15% safflower oil or free acids (diets Band C), or 15% safflower oil or free safflower fatty acids (diet D) for 4 weeks. A group of normal rats was also fed diet D. Triglycerides, cholesteryl esters, phosphatidylcholines, and phosphatidylethanolamines isolated from livers and hepatomas of animals on each diet were analyzed quantitatively for positional isomers in the cis- and trans-octadecenoate fractions. When sufficient samples could be obtained, the cis- and trans-hexadecenoate fractions were also analyzed. Plasma from normal rats on diet D was analyzed in the same manner. The octadecenoate fractions of all hepatoma and liver lipid classes from animals fed diets A, B, and C were greater than 95% the cis isomers. Trans isomers accounted for approximately 15, 30, 50, and 70% of the octadecenoate fractions isolated from liver triglycerides, cholesteryl esters, phosphatidylcholines, and phosphatidylethanolamines, respectively, of animals fed diet D. In contrast, all hepatoma lipid classes from animals on diet D contained the same approximate percentage of trans isomers (15 to 20%). Oleic and vaccenic acids were the major positional cis-octadecenoate isomers of all liver and hepatoma lipid classes from animals fed diets A, B, and C. The ratios of oleic to vaccenic, unaffected by diets A, B, and C, differed for each lipid class in liver, but the ratios were similar for the two hepatoma neutral lipid classes and for the two phospholipid classes. The cis-octadecenoate fractions from all liver and hepatoma lipid classes of animals fed diet D consisted predominantly of the delta9, delta11, and delta12 isomers. The cis delta10 isomer, which was a major isomer of the diet, was almost excluded from liver, hepatoma, and plasma lipids. The positional isomers of the trans-octadecenoate fractions from liver and hepatoma triglycerides and cholesteryl esters exhibited the same approximate distribution as the trans fatty acids of diet D. In contrast, the 10-trans-octadecenoate, like 10-cis-octadecenoate, was almost excluded from the phospholipids of liver and plasma. Unlike liver, the hepatoma phospholipids contained 10-trans-octadecenoate at approximately half the percentage of neutral lipids. Because diet D contained no hexadecenoic fatty acids, the occurrence of trans-hexadecenoate isomers in liver and plasma lipids indicated a chain shortening process. Predominance of the 8-trans-hexadecenoate isomer indicated a preference of the 10-trans-octadecenoate isomer for chain shortening.


Magnetic resonance studies of the manganese guanosine di- and triphosphate complexes with elongation factor Tu. Analysis of titration data of EF-Tu-GDP with Mn(II) where free and bound Mn(II) were determined by proton relaxation rate of water (PRR) yields one tight Mn(II) binding site and a value of 2 muM for the dissociation constant of Mn(II) from the EF-Tu-MnGDP complex, K'A. The dissociation constant of manganese nucleotide from the ternary EF-Tu-MnGDP complex, K2, 0.2 muM, was derived from the known value of Ks, the dissociation constant for the binary EF-Tu-GDP complex, and the titration data of the ternary complex with excess GDP as titrant. The apparent number, n, of rapidly exchanging water ligands coordinated to bound Mn(II) in the ternary complex EF-Tu-MnGDP is estimated from the frequency dependence of the PRR of the complex to be approximately 1. The value of n and the values of PRR enhancements, epsilont = 4.3 for EF-Tu-MnGDP at 21 degrees, 24.3 MHZ and epsilont = 4.1 for the ternary GTP complex, are unusually low for protein-Mn-nucleotide complexes. The antibiotic X5108 which induces GTPase activity in EF-Tu-MgGTP was shown to bind stoichiometrically to EF-Tu-MnGDP and thereby change the PRR enhancement of the complex from 4.3 to 7.4. The characteristic broad lines in the EPR spectra of Mn(II) nucleotides are strikingly narrowed upon binding of Mn(II) nucleotides to EF-Tu. The long electron spin relaxation times inferred from the EPR spectra indicate a limited access of solvent water to the first coordination sphere of Mn(II) in its EF-Tu-nucleotide complexes. The frequency dependence of the PRR indicates that the electron spin relaxation time, T1e, is the dominant process modulating the Mn(II)-H2O interaction of the EF-Tu-MnGDP complex and consequently determines the correlation time. The value of T1e, estimated from the PRR experiments to be 2.5 ns at 21 degrees, is consistent with the lower limit of T1e obtained from the line widths of the EPR spectrum of the complex. Upon binding of a stoichiometric quantity of the antibiotic X5108, the EPR spectrum of EF-Tu-MnGDP is severely broadened indicating greater access of solvent water to the manganese coordination sphere, i.e. an opening of the nucleotide binding site as already suggested by the increased PRR enhancement.


Proteolytic activation of rat liver adenylate cyclase by a contaminant of crude collagenase from Clostridium histolyticum. Treatment of rat liver plasma membranes with various commercial preparations of crude collagenase from Clostridium histolyticum at concentrations as low as 1 mug/ml, resulted in activation of the adenylate cyclase system. Maximal activation occurred at 50 to 100 mug/ml of collagenase, and promoted a 2- to 3-fold increase in the basal activity as well as in the activities stimulated by catecholamines, glucagon, fluoride, or GTP. This was due to an increase in the maximal velocity of the cyclizing reaction without any increase in the affinity of the enzyme for its substrate. Treatment of plasma membranes with crude collagenase did not induce gross structural modifications as judged by electron microscopic examination. 5'-Nucleotidase activity was slightly inhibited and ATPase activity remained unaffected. The stimulatory substance was nondialyzable, thermolabile, and inhibited by both EDTA and -SH reagents, thus appearing to be a protein. The following observations suggest the effects observed were due to other protease(s) present in crude collagenase: (a) only crude collagenase was active on liver adenylate cyclase: treatment with purified collagenase from C. histolyticum or from Achromobacter iophagus gave no stimulation; (b) the stimulatory activity was irreversible since washing of the membranes after treatment was without effect; (c) crude collagenase contained no lecithinase or sphingomyelinase activity under our conditions of adenylate cyclase assay; (d) after chromatography on Sephadex G-100, the activator appeared as a peak in the 30,000-dalton region and was clearly separated from the collagenase and clostripain peaks, but coincident with elastolytic and caseinolytic activities; (e) the effect of crude collagenase could be prevented by addition of elastin in vitro and was mimicked by purified elastase from hog pancreas. It remains to be seen whether the effects observed result from an increase in the catalytic constant of adenylate cyclase, or an unmasking of new catalytic sites.


Biosynthesis of ferritin in rat hepatoma cells and rat livers. I. Synthesis and assembly of protein subunits of ferritin. Cell fractions were prepared from ACI rat livers and from rat hepatoma cell clone M-5123-C1. Radioimmunoassays of ferritin and of its protein subunits in various cell fractions after biosynthetic labeling with [14C]leucine were done by means of ferritin-specific and subunit-specific rabbit antibody. In both ACI rat livers and M-5123-C1 hepatoma cells free polyribosomes synthesized approximately 81% of the protein subunits of ferritin, and membrane-bound polyribosomes synthesized the rest. In both polyribosomal fractions, [14C]leucine-labeled subunits were detected earlier than [14C]leucine-labeled ferritin and apoferritin (5 min as against 30 min after initiation of a pulse). Time sequence studies of the shifts of biosynthetically labeled subunits and ferritin through different cell compartments provided evidence for vectorial transport of subunits and of ferritin, the direction of transport being from the two polyribosomal systems to the smooth membrane compartment and to the cytosol.


Biosynthesis of ferritin in rat hepatoma cells and rat livers. II. Binding of iron by ferritin protein. Ferritin and its protein subunits in rat hepatoma cell clone M-5123-C1 were biosynthetically labeled with [14C]leucine and 59Fe. Radioimmunoassays of ferritin/apoferritin and of protein subunits in the free polyribosome, membrane-bound polyribosome, smooth membrane, and cytosol fractions were done with ferritin-specific and subunit-specific rabbit IgG antibodies at various time intervals after pulsing. Much more 59Fe was bound by ferritin/apoferritin than by subunits in all of the cell fractions. Binding of iron to subunits may have been a random process. When hepatoma cells were simultaneously pulse-labeled with 59Fe and [14C]leucine, uptake of much of the 59Fe by ferritin occurred relatively early, in comparison to incorporation of [14C]leucine, in all of the cell fractions examined. Thus, 59Fe was readily incorporated into pre-existing ferritin. We conclude that most, if not nearly all, of the iron is incorporated after assembly of protein subunits.


Effect of variations in lipopolysaccharide on the fluidity of the outer membrane of Escherichia coli. The lipid hydrocarbon chains in the outer membrane of gram-negative bacteria appear from previous experiments to be less mobile than in the cytoplasmic membrane. To determine whether lipopolysaccharide, a unique outer membrane component, is a cause of this restricted mobility, outer membranes differing in the amount of lipopolysaccharide, and the length of the polysaccharide side chain, were prepared from Escherichia coli J5. Cytoplasmic membranes were prepared for comparison. The probes, 5- and 12-doxylstearate, were introduced into these membranes, electron spin resonance spectra were analyzed, and the order parameter (S) and empirical motion parameter (tau0) were calculated. Outer membrane preparations containing long chain lipopolysaccharide were much less fluid by these criteria than were preparations containing short chain lipopolysaccharide. Removing about 40% of the lipopolysaccharide from the former preparations greatly increased their fluidity. The lipid in the cytoplasmic membrane preparations was more fluid than in the outer membrane and cytoplasmic membranes were similar to each other regardless of the composition of the outer membrane. These results indicate that lipopolysaccharide, and especially the polysaccharide portion, directly or indirectly causes the restricted mobility of the lipid hydrocarbon chains observed in the outer membrane.


Inhibition and inactivation of bovine mammary and liver UDP-galactose-4-epimerases. Bovine liver and mammary UDP-galactose-4-epimerases were investigated with respect to various inhibitors and inactivators. Uridine nucleotides and NADH are potent inhibitors with Ki values in the low micromolar range. The NAD+/NADH ratio may be an important physiological control mechanism for it affects markedly the activity of the enzyme with 50% inhibition occurring at a ratio of 20:1. In the presence of uridine nucleotides binding of NADH to the epimerases is enhanced. Consequently, the effect of changes in the NAD+/NADH ratio in vivo would not be immediately apparent as uridine nucleotides would slow down the displacement of NADH by NAD+. Neither uridine nor galactose 1-phosphate inhibits the purified enzymes as previously reported with the impure liver enzyme. Uridine nucleotides provide almost total protection against the apparent first order inactivation of the epimerases by trypsin and allow determination of dissociation constants. NAD+ partially protects against trypsin inactivation. Inactivation with various sulfhydryl reagents is complex and the results indicate that at least three sulfhydryl groups may be modified before total inactivation occurs. Partial inactivation occurs upon modification of the epimerases with 2-hydroxy-5-nitrogenzyl bromide. Some protection against this modification is provided by the combination of NAD+ and UDP.


Isolation, chemical characterization, and biophysical properties of three different abnormal lipoproteins: LP-X1, LP-X2, and LP-X3. Three different but related abnormal lipoprotein species, LP-X1, LP-X2, and LP-X3, have been isolated from cholestatic plasma by ethanol precipitation and zonal ultracentrifugation. All three populations are rich in phospholipids (64.9 to 67.5%) and cholesterol (23.0 to 26.8%) but poor in cholesteryl esters (0.4 to 1.9%), triglycerides (1.8 to 3.2%), and protein (3.2 to 6.7%) with differences in chemical composition which result in buoyant densities (1.038, 1.049, and 1.058, respectively) to allow their separation. LP-X1, LP-X2, and LP-X3 exhibited apparent flotation rates of 17.3, 9.7, and 3.2 Svedbergs and Stokes radii of 339, 343, and 294 A, respectively. As determined from circular dichroic measurements, the protein constituents of all three particles possessed a high degree of alpha helical structure (41 to 65%). Each LP-X particle exhibited abnormally low fluidity as evaluated by electron paramagnetic resonance. All of the particles contained human serum albumin and the C-proteins as major protein constituents, but only LP-X2 and LP-X3 contained apolipoprotein A-I and apolipoprotein E.


Interaction of human and bovine A-1 apolipoproteins with L-alpha-dimyristoyl phosphadicylcholine and L-alpha-myristoyl lysophosphatidylcholine. The major protein components from human and bovine high density serum lipoproteins (apo-A-I proteins) were investigated in their interactions with L-alpha-myristoyl lysophosphatidylcholine and L-alpha-dimyristoyl phosphatidylcholine. Complex formation was followed by 25 degrees by observing changes in fluorescence polarization, rotational relaxation times (ph), and CD spectra of the proteins, covalently labeled with fluorescent dimethylaminonaphthalene sulfonyl groups. Monomeric human apo-A-I and initially oligomeric bovine apo-A-I interact with similar efficiency with the same amphiphiles. During binding of L-alpha-myristoyl lysophosphatidylcholine and L-alpha-dimyristoyl phosphatidylcholine, the structure of both proteins changes drastically, exhibiting about 35% increases in secondary structure with the phospholipid and about 20% increases with the lysophospholipid. Simultaneously, regions of the proteins adjacent to the fluorescent probes become more mobile. With the bovine protein, binding of both amphiphiles results in changes in the oligomeric structure: dissociation with L-alpha-myristoyl lysophosphatidylcholine and dissociation or rearrangement with L-alpha-dimyristoyl phosphatidylcholine. The complexes formed in the presence of excess L-alpha-myristoyl lysophosphatidylcholine are flexible structures with considerable rotational freedom. The largest rotational unit in these complexes has ph = 60 ns for both proteins; by comparison, the monometic human apo-A-I has a ph = 73.5 ns. Interactions of both proteins with the lysophospholipid take place well above its critical micelle concentration, but probably involve binding of monomeric lipid. With L-alpha-dimyristoyl phosphatidylcholine the complexes are different from those formed with the lysophospholipid. They have limiting ph values of 250 +/- 50 and 280 +/- 60 ns with the human and bovine proteins, respectively. These ph values are consistent with particles of the general size of human high density lipoprotein rather than of liposomes and indicate the formation of distinct, relatively small structures upon interaction of the self-associated lipid with the apo-A-I-proteins.


Physical properties of isolated complexes of human and bovine A-I apolipoproteins with L-alpha-dimyristoyl phosphatidylcholine. Human or bovine A-I apolipoproteins in solution form complexes with sonicated L-alpha-dimirystoyl phosphatidylcholine at 23 and 37 degrees, but not at 8 degrees, suggesting a strong dependence of the interaction on the physical state of the lipid (phase transition temperature 23 degrees). Complexes were isolated by gel filtration on a Sepharose 4B column and were subsequently analyzed for protein and lipid content, molecular weight, and physical state of the lipid portion. The average stoichiometry of all complexes, regardless of the initial concentrations or ratios of protein and lipid, was constant: 90 +/- 20 mol of phospholipid/mol of protein monomer, suggesting a highly cooperative interaction. Sedimentation equilibrium experiments indicated homogeneous macromolecular preparations and gave molecular weights around 235,000 (+/- 15%) for the complexes, with the human and bovine apo-A-I proteins contributing 77,000 (+/- 10%), i.e. about three protein subunits per complex. The lipid portion of the complexes retained some characteristics of a bilayer: it had a broad phase transition with a midpoint at 25.5 degrees as reported by the fluorescence polarization of the lipophilic probe diphenylhexatriene. Above the phase transition temperature the mobility of the phospholipids in the complexes with both apo-A-I proteins was considerably decreased relative to the pure L-alpha-dimyristoyl phosphatidylcholine dispersion; below the phase transition temperature the opposite was true, i.e. the protein fluidized the lipids. The results indicate that apol-A-I proteins interact stoichiometrically with L-alpha-dimyristoyl phosphatidylcholine vesicles above the gel to liquid-crystalline transition temperature of the lipid, promoting the destruction of vesicles and the formation of well defined particles of the general size of high density serum lipoproteins.


Direct assignment of the cysteinyl, the slowly exchangeable, and the aromatic ring 1H nuclear magnetic resonances in clostridial-type ferredoxins. We have directly assigned the 1H NMR corresponding to the cysteinyl protons, the slowly exchangeable protons, and the aromatic ring protons in the 1H NMR spectrum of Clostridium acidi-urici ferredoxin by isotopic labeling and 13C NMR decoupling techniques. We also show that the resonance pattern in the 8- to 20-ppm (from 2,2-dimethyl-2-sialapentanesulfonic acid) region of the 1H NMR spectra of oxidized Clostridium acidi-urici, Clostridium pasteurianum, Clostridium perfringens, and Peptococcus aerogenes ferredoxins are very similar, and we assign the resonances in this region by analogy with the spectrum of C. acidi-urici ferredoxin. The 1H NMR spectra of the beta protons of the cysteinyl residues of these ferredoxins differ, however, from the 1H NMR spectra of equivalent beta protons of the methylene carbon atoms bonded via a sulfur atom to [4Fe-4S] clusters in synthetic inorganic analogues. In the spectra of the synthetic compounds, the beta protons appear as a single resonance shifted 10 ppm from its unbonded reference position. In the spectra of oxidized clostridial ferredoxins, the cysteinyl beta protons appear as a series of at least eight resolved resonances with shifts that range from 6 to 14 ppm, relative to the free amino acid resonance position. This difference in the spectra of the protein and the synthetic compounds probably results from the fact that the equivalent beta protons of the synthetic compounds are not constrained and are free to rotate and thus assume the same average orientation with respect to the [4Fe-4S] cluster. The shift pattern in the 9- to 14-ppm region is identical in three different clostridial ferredoxins. This suggests that the molecular environments of the corresponding cysteinyl residues are identical. Significant differences in the resonance positions occur, however, in the 14- to 18-ppm region, suggesting that the physical environments of these cysteinyl residues differ. This may reflect differences in the orientation of the corresponding cysteinyl residues relative to the [4Fe-4S] clusters or differences in charge density at the cysteinyl beta protons or both. The slowly exchangeable protons were identified by comparing the 1H NMR spectra of ferredoxins reconstituted in H2O and 2H2O. The remaining resonances in the 8- to 20-ppm region were assigned to each of the 2 tyrosyl residues in C. acidi-urici ferredoxin. This was done by comparing the 1H NMR spectra of C. acidi-urici [(3',5'-2H2)Tyr]ferredoxin and C. acidi-urici [PHE2]ferredoxin with that of C. acidi-urici native ferredoxin.


Initiation of synthesis of messenger RNA of deoxynucleotide kinase by oligoribonucleotides. The effects of nucleoside triphosphates and oligoribonucleotides on the initiation of synthesis of messenger RNA of the T4 phage-specific enzyme, deoxynucleotide kinase, have been studied. The procedure involved incubation of T4 DNA, purified RNA polymerase from Escherichia coli, and selected nucleotide compounds during a brief period to permit initiation of RNA synthesis. Further initiation was arrested by the addition of ribampicin, and completion of the transcription of the newly initiated RNA was permitted to take place in the presence of the full complement of nucleoside triphosphates. After translation of the messenger RNA into phage-specific enzymes, the measured activities of the latter whe first incubation period. The effectiveness of individual nucleoside triphosphates, when present singly or in combination during the initiation period, was compared to that when all four nucleoside triphosphates were available. ATP alone was extremely effective as an initiator of the synthesis of the messenger RNA for deoxynucleotide kinase. The addition of UTP to ATP not only enhanced the magnitude of initiation but also affected the kinetics of ATP interaction with T4 DNA and RNA polymerase during the initiation period. Several oligoribonucleotides including a series ApA to ApApApA, UpU to UpUpUpU, and the heteropolymers, Ap1pU and ApApApU, were tested as initiators of kinase mRNA synthesis. A sequence of nucleotides in the promoter region of T4 DNA for the deoxynucleotide kinase gene has been proposed as a result of these experiments.


Glycogen synthase kinases. Distribution in mammalian tissues of forms that are independent of cyclic AMP. Extracts of rat tissues contain kinases which catalyze the conversion of glycogen synthease from the glucose 6-phosphate-independent (I) form to the glucose 6-phosphatate-dependent (D) form. These kinases were stimulated by adenosine 3':5' monophosphate (cyclic AMP). The glycogen synthase kinase activity ratio (activity in the absence of cyclic AMP divided by activity in the presence of cyclic AMP) varied from 0.28 to 0.97. The activity ratio for histone kinase in the same extracts ranged from 0.11 to 0.29. The levels of glycogen synthase kinase varied by a factor of 80 in the following rat tissues (given in order of decreasing enzyme activity): kidney, liver, stomach mucosa, lung, brain, heart, skeletal muscle, and adipose tissue. In the same tissues the levels of histone kinase varied by only a factor of 6 and did not correlate with the levels of glycogen synthase kinase. A modification of the method of Walsh et al. ((1971) J. Biol. Chem. 246, 1977-1985) was developed for purification of the heat-stable inhibitor of cyclic AMP-dependent protein kinases (inhibitor). The modified procedure resulted in good yields of highly purified inhibitor and was much simpler than the previously described procedure. This inhibitor completely inhibited cyclic AMP-dependent histone kinase activity of the extracts but much of the glycogen synthase kinase activity was not inhibited. The portion of glycogen synthase kinase that was insensitive to the inhibitor was: stomach mucosa, 95%; brain, 90%; liver, 82%; kidney, 81%; lung, 68%; adipose tissue, 65%; skeletal muscle, 63%; and heart, 54%. This histone kinase activity in the extracts and hte ratio of glycogen synthase kinase to histone kinase activity of purified catalytic subunit of the cyclic AMP-dependent protein kinase was used to calculate for each extract the glycogen synthase kinase activity contributed by the cyclic AMP-dependent protein kinase. Based on these calculations, the portion of the glycogen synthase kinase which was due to kinases independent of cyclic AMP was: kidney, 97%; liver, 91%; lung, 89%; brain, 87%, heart, 85%; stomach mucosa, 84%; adipose tissue, 38%; and skeletal muscle, 33%. A significant portion of the glycogen synthase kinase activity, but virtually none of the cyclic AMP-dependent histone kinase activity, of these extracts could be adsorbed to phosphocellulose columns. Liver extracts contained, in addition, a form of glycogen synthase kinase which was not adsorbed to phosphocellulose and which could be separated from the cyclic AMP-dependent protein kinase by additional chromatography. These studies demonstrate that kinases independent of cyclic AMP account for most of the glycogen synthase kinase activity of many tissues. The widespread distribution and high concentrations of these enzymes suggest that they are of physiological importance.


Biological cross-reactivity of rat testis phosphodiesterase activator protein and rabbit skeletal muscle troponin-C. Phosphodiesterase activator protein and troponin-C have been purified from rat testis and rabbit skeletal muscle, respectively. The two proteins appear to be structurally distinct since the activator protein migrates faster than troponin-C on sodium dodecyl sulfate-polyacrylamide gels. Each of the calcium-binding proteins will, however, substitute for the other in their respective biological systems. Testis activator protein forms a complex with rabbit muscle troponin subunits TnI and TnT soluble in low salt. This hybrid complex (AIT) can regulate rabbit skeletal muscle actomyosin ATPase activity. AIT regulation, although influenced by free Aa2+ levels, is distinct from that of native troponin. Likewise, muscle troponin-C can substitute for activator protein in the stimulation of cyclic nucleotide phosphodiesterase. Troponin-C will fully stimulate phosphodiesterase although its affinity is 600-fold lower than that of activator protein. Ca2+ regulation studies demonstrate that both proteins require micormolar levels of free Ca2+ to induce phosphodiesterase activation. Activator protein requires 1.2 x 10(6) M and troponin-C, 1.9 X 10(6) M free Ca2+ for half-maximal stimulation of phosphodiesterase. The biological cross-reactivity of these proteins supports the sequence homology recently reported by Watterson et al. (Watterson, D.M., Harrelson, W.G., Keller, P.M., Sharief, F., and Vanaman, T.C. (1976) J.Biol. Chem. 251, 4501-4513). In addition, this preliminary study suggests that this nonmuscle troponin-C-like protein potentially may function in other Ca2+-regulated cellular events in addition to its moculation of cyclic nucleotide levels.


Preferential usage of glycyl-tRNA isoaccepting species in collagen synthesis. Chick embryo tRNA charged with [3H]glycine was incubated in an in vitro protein-synthesizing system using polysomes isolated from either chick embryo liver or calvaria. Using collagenase digestion to measure the fraction of protein synthesized which was collagenous, the results indicate that in the calvaria system approximately 65% of the incorporated [3H]glycine was in collagen. The incorporation of [3H]glycine into protein from individual isoaccepting species was determined by chromatography on a reversed phase system of the charged tRNA before and after incubation in the polysome systems. In the calvaria system, a single tRNAGly species cognate to GGU and GGC and which is found in unusually large amounts in collagen-synthesizing tissues was used preferentially in collagen-synthesizing tissues was used preferentially in collagen synthesis. In the liver system, the rate of incorporation was similar to the calvaria, but no collagen synthesis was detected and only a relatively small preferential usage of any of the four major isoaccepting species was observed. These results support the notion that the complement of tRNA found in a cell may be adapted to the synthesis of a particular protein. It is also possible that under certain circumstances, collagen synthesis may be controlled in vivo at the translational level by the concentration of particular tRNA species.


Resection-arthrodesis for malignant and potentially malignant lesions about the knee using an intramedullary rod and local bone grafts. Twenty patients with malignant or potentially malignant tumors (osteogenic sarcoma, giant-cell tumor, synovial-cell sarcoma, chondrosarcoma, and chondroblastoma) located in the proximal end of the tibia or distal end of the femur were treated by local resection and arthrodesis employing an intramedullary rod and autogenous segmental cortical grafts obtained from the same extremity. Use of a customized bent, fluted rod in the most recent cases provided more stable fixation. There was only one local recurrence. In properly selected patients, the method provided a stable extremity that permitted resumption of a vigorous life-style within approximately one year.


Experimental sensory reinnervation of the median nerve by nerve transfer in monkeys. Anastomosis of the superficial radial nerve, the dorsal cutaneous branch of the ulnar nerve, or both to the distal cut end of a widely resected median nerve in monkeys was followed by successful sensory reinnervation of the thumb, index finger, and long finger within thiry-five to forty weeks. Success was ascertained by the presence of an intact anastomosis as observed grossly without any evidence of spontaneous regeneration of the median nerve. Reinnervation was confirmed by histological and histochemical reactions observed in the Meissner's corpuscles in the skin innervated by the median nerve. The demonstration of nerve fiber and the presence of normal specific and non-specific cholinesterase reactions exhibited by the Meissner's corpuscles in the cholinesterase preparations were considered the histological criteria for successful reinnervation. These histological and histochemical observations may explain the reported functional sensory recovery in clinical cases when similar nerve transfers were done.


A radioimmunoassay for 3,3',5'-L-triiodothyronine (reverse T3): assessment of thyroid gland content and serum measurements in conditions of normal and altered thyroidal economy and following administration of thyrotropin releasing hormone (TRH) and thyrotropin (TSH). The present report describes the development of a radioimmunoassay for 3,3',5'-L-triiodothyronine (reverse T3) which is performed on unextracted serum. Utilizing this radioimmunoassay, 21 normal subjects had a mean (+/-SD) serum reverse T3 level of 60 +/- 12 ng/100 ml, 17 of 19 hyperthyroid patients had elevated serum reverse T3 levels, and 10 of 11 hypothyroid subjects had decreased serum reverse T3 concentrations. Thyroidal secretion of reverse T3 was assessed by measurements in samples obtained from the internal carotid artery and jugular vein of sheep following the administration of thyrotropin releasing hormone (TRH) or bovine thyrotropin (TSH). Reverse T3 levels were increased 45-60 min after TRH administration, but TSH administration produced inconsistent alterations in reverse T3, although 18 of 27 samples obtained after TSH injection were higher than their average respective baseline concentration and the mean peak reverse T3 level was 14% higher than baseline. Following TRH administration to 10 normal human subjects, mean serum reverse T3 levels significantly increased from 53.6 ng/100 ml to 56.3ng/100 ml (P less than .05). The thyroid gland content of reverse T3 in human autopsy material was 6.5 +/- 1.5 microng/g tissue. Both pregnancy and estrogen administration were associated with increases in serum reverse T3 concentrations presumably because of their ability to augment thyroxine binding globulin synthesis.


Circadian rhythms in the urinary excretion of cyclic 3',5'-adenosine monophosphate (cyclic AMP) and cyclic 3',5'-guanosine monophosphate (cyclic GMP) in human subjects. Significant circadian rhythns in urinary excretion of cyclic AMP, cyclic GMP, creatinine, 17-hydroxycorticosteroids and inorganic phosphorus were demonstrated in three normal subjects (two males and one post-menopausal female) who collected serial 4-h specimens for periods of 33, 22, and 14 days, respectively. Peak excretion of the above substances occurred, respectively, at approximately 1500-1700, 2300-0200, 1700-1800, 1100-1300, and 1800-2200 h. The estimated amplitudes of the rhythms expressed as a percentage of the mean 4-h excretion rate were 12-13%, 9-13%, 9-15%, 30-78% and 22-26%, respectively. With the possible exception of cyclic GMP, all of the observed rhythms appeared to be sinusoidal, with normal periods of 24 h. The rhythms in both cyclic AMP and creatinine excretion, but not in the remaining substances may be explained largely in terms of the known rhythm in glomerular filtration rate. The factors responsible for the rhythm in cyclic GMP excretion are unknown.


Demonstration of dual rhinovirus infection in humans by isolation of different serotypes in human heteroploid (HeLa) and human diploid fibroblast cell cultures. The ability to isolate rhinoviruses in human heteroploid cell cultures was investigated by inoculating HeLa cells (HeLa M) with specimens previously shown to be positive in human diploid cell cultures. The 135 positive specimens selected were representative of 22 different rhinovirus types, and 4 to 9 specimens were available for each serotype. Specimens were inoculated into human diploid fetal tonsil fibroblasts (FT), HeLa cells with 30 mM Mg2+, and HeLa cells without increased Mg2+. One hundred twelve rhinovirus strains (83%) were reisolated in FT cells, whereas 76 rhinovirus strains (56%) were recovered in HeLa cells with 30 mM Mg2+. All strains recovered in FT were the same serotype as that originally recovered in diploid cells, but five of the HeLa cell isolates (3.7% of total specimens) were different serotypes, indicating dual rhinovirus infections. Four rhinovirus serotypes, (3, 42, 48, and 70) were recovered in HeLa but not in diploid cells; these serotypes were rare in our previous studies. Isolation of rhinovirus in FT cells was usually accomplished at first passage, whereas rhinovirus cytopathic effects in HeLa cells were not observed at first passage, but required one, two, or (rarely) three blind passages. Only 28 rhinoviruses (21%) were recovered in HeLa cells without increased Mg2+; however, three serotypes, types 16, 36, and 58, were recovered as effectively in HeLa cells, with or without added Mg2+, as they were in FT cells. In general, rhinoviruses were less efficiently recovered in HeLa cells; however, certain serotypes may be detected better by HeLa cells.


Isolation of simian virus 40 from a newborn child. Rubella virus and simina virus 40 (SV40) were isolated from a newborn child suffering from neurological and anatomical anomalies. The SV40 isolate was very similar to SV40 strain 777 by electron microscopic, biological, and immunological criteria.


Inhibition of thyroid-stimulating hormone stimulation of protein kinase, glucose oxidation, and phospholipid synthesis in thyroid slices previously exposed to the hormone. Prior exposure of thyroid slices to thyrotropin (TSH) induced refractoriness to subsequent stimulation of the cyclic AMP system by the hormone. Although the inhibition is incomplete, we examined whether the reduction in cyclic AMP was sufficient to alter other metabolic effects of TSH. Bovine or dog thyroid slices were incubated with or without 5-100 mU/ml TSH for 1-2h, washed, and then incubated without hormone for 1-2h. Half of the slices not exposed to TSH initially were then incubated with buffer and half were exposed to 5-100 mU/ml TSH. Slices initially incubated with TSH were also incubated with or without TSH in the third incubation. During the refractory period, TSH activation of protein kinase was inhibited even though the hormone still caused some increase in cyclic AMP concentrations. However, protein kinase activity was fully responsive to dibutyryl cyclic AMP when slices were incubated with it during the third incubation. Stimulation of glucose oxidation by TSH was significantly decreased in thyroid slices previously incubated with the hormone. During refractoriness, stimulation of glucose oxidation caused by prostaglandin E1 and dibutyryl cyclic AMP was also significantly diminished but that due to acetylcholine was not. Thus even though dibutyryl cyclic AMP could fully activate protein kinase activity during refractoriness, its effect on glucose oxidation was still inhibited, suggesting that the metabolic block responsible for this refractoriness was distal to activation of protein kinase. Stimulation of 32Pi incorporation into phospholipid by TSH and acetylcholine was also inhibited during refractoriness. Despite reduction of the stimulatory effect of TSH, binding of 125ITSH was not modified by prior incubation of thyroid slices with TSH. These results indicate that changes in the TSH  receptor are not responsible for the development of refractoriness and other metabolic sites besides activation of adenylate cyclase appear to be involved.


Metabolism of vasoactive peptides by human endothelial cells in culture. Angiotensin I converting enzyme (kininase II) and angiotensinase. Cultured endothelial cells provide a model for the study of interactions of vasoactive peptides with endothelium. Endothelial cell cultured from veins of human umbilical cords contain both angiotensin I converting enzyme (kininase II) and angiotensinase activities. Intact monolayers of cells can both activate angiotensin I and inactivate bradykinin when the peptides are added to culture flasks in protein-free medium. Intact suspended cells or lysed cells convert angiotensin I to angiotensin II, inactivate bradykinin, and hydrolyze hippuryldiglycine to hippuric acid and diglycine. These actions are inhibited by SQ 20881, the specific inhibitor of converting enzyme. The kininase activity of endothelial cells was partially inhibited by antibody to human lung converting enzyme. Endothelial cells also inactivate longer analogs of bradykinin, such as kallidin, methionyl-lysyl bradykinin, and bradykinin coupled covalently to 500,000 mol wt dextran. The endothelial cells retained converting enzyme activity through four successive subcultures, indicating that the enzyme is synthesized by the cells surface, and it is apparently a marker for endothelial cells, since cultured human fibroblasts, smooth muscle cells, and baby hamster kidney cells do not have it. Endothelial cells also contain an aminopheptidase which hydrolyzes both angiotensin II and the synthetic substrate, alpha-L-aspartyl beta-naphthylamide. The angiotensinase activity increased when the cells were lysed, which suggests that the enzyme is localized within the cells, Hydrolysis of both alpha-L-aspartyl beta-naphthylamide and angiotensin II was inhibited by omicron-phenanthroline, indicating that the enzyme is an A-tipe anigotensinase.


Alpha-1-antitrypsin bodies in the liver. The cytoplasmic bodies in hepatocytes thought to indicate possession of the Z allele for alpha 1-antitrypsin deficiency were found in necropsy in 10 of 64 adults with cirrhosis, four of nine with hepatic fibrosis, and four of 15 with hepatocellular carcinoma. They were also found in six of 76 adults with severe panacinar emphysema, and in four of a control series of 110 adults with neither emphysema nor liver disease. The association of the bodies with each of the three liver diseases was statistically significant, but the association of the bodies with emphysema was not. It is considered probable that heterozygous (PiMZ) alpha 1-antitrypsin deficiency is associated with an increased incidence of cirrhosis, hepatic fibrosis, and hepatocellular carcinoma.


25-Hydroxyvitamin D levels in patients treated with high-dosage ergo- and cholecalciferol. 25-Hydroxyvitamin D (25-OHD) levels were measured in 39 patients with metabolic bone disease or hypoparathyroidism who had been treated with a constant high dose of vitamin D2 or D3 for at least 12 weeks. Plasma 25-OHD levels rose with increasing dosage, the relationship between dose and plasma level being approximately linear whether or not the dose was expressed on a weight-corrected basis. A therapeutic range of 25-OHD to be expected when patients with these conditions are treated with vitamin D has been established. There may be certain exceptions in which plasma 25-OHD levels within the range are associated with either an inadequate response to treatment or, conversely, the hypercalcaemia of vitamin D toxicity. There was no correlation between plasma calcium level and 25-OHD concentration in the group of patients studied. There was also no difference between the dose/25-OHD relationship of patients treated with vitamin D2 and that of patients receiving vitamin D3. Ten patients were started on treatment with large doses of vitamin D during the period of the study. The rate of rise of plasma 25-OHD was followed during treatment. The incremental rise in 25-OHD was calculated at the end of the first week of treatment in terms of dose per unit body weight. The rate of rise of plasma 25-OHD level was highly correlated with the dose used. Plasma 25-OHD levels after one weeks' treatment were only 15-20% of the expected steady-state level on the same dosage. The importance of a high priming dose when a rapid response is needed is thus emphasised.


Morphology and the natural history of cribriform adenocarcinoma (adenoid cystic carcinoma). Forty-three examples of cribriform adenocarcinoma (adenoid cystic carcinoma) of mixed glandular origin are reported. Structural studies emphasise the classical cribriform pattern which indicates the capacity of this neoplasm to behave as both an epithelial and a connective tissue type tumour. In terms of survival, it does not appear to be an malignant as other forms of carcinoma arising in similar anatomical locations. The five-year crude survival rate (56%) compares favourably with that of other carcinomas of the palate and paranasal sinuses. A recurrence rate of the order of 20% may persist up to 10 years after primary treatment, and while this is not inimical to longer survival there is clearly a high morbidity.


The mobile receptor hypothesis in hormone action: a general model accounting for desensitization. The characteristics of the hormone interaction with their cell receptors and of the related adenylate cyclase activation are complex. Among the theoretical models which have been proposed to account for this mechanism, the mobile or floating receptor model has been discussed (3-5). The purpose of this work is to deduce several particular models from the general mobile receptor model on the basis of thermodynamic considerations. The analysis of some of these models shows that the mobile receptor hypothesis contains in itself the little understood kinetic phenomenon of hormone desensitization.


The involvement of prostaglandin endoperoxide formation in the elevation of cyclic GMP levels during platelet aggregation. Arachidonic acid- or collagen-induced aggregation was accompanied by a progressive elevation in the level of cyclic GMP in washed human platelets with no significant alteration in the concentration of cyclic AMP. The extent of the increase in cyclic GMP was proportional to the concentration of arachidonic acid added. Enhanced accumulation of cyclic GMP produced by arachidonic or collagen was prevented by prior exposure of platelets to aspirin or indomethacin. Prostaglandin endoperoxide G2 caused platelet aggregation and an increase in cyclic GMP concentration; neither event was blocked by prostaglandin synthesis inhibitors. These results indicate that the generation of prostaglandin endoperoxides is a step in the sequence of events in platelet aggregation leading to the enhanced accumulation of cyclic GMP.


Decrease in calcium transport associated with phosphoprotein phosphatase-catalyzed dephosphorylation of cardiac sarcoplasmic reticulum. Phosphoprotein phosphatase activity is found in preparations of sarcoplasmic reticulum isolated from canine heart when assayed with either phosphate or phosphorylated sarcoplasmic reticulum as substrate. Phosphoprotein phosphatase-catalyzed dephosphorylation of the 22,000 dalton phosphoprotein of cardiac sarcoplasmic reticulum is stimulated markedly by MnCl2 (5 mM) and to a lesser extent by MgCl2 (5 mM); inorganic phosphate (50 mM) and NaF (25 mM) are inhibitory. Dephosphorylation of this 22,000 dalton phosphoprotein is correlated with a decreased initial rate of calcium transport. The close structural and functional relationship of phosphoprotein phosphatase to the cardiac sarcoplasmic reticulum suggests a possible role of this enzyme in reversing the relaxation-promoting effects of catecholamines on the intact heart.


Milk fever in dairy cows. VIII. Effect of injected vitamin D3 and calcium and phosphorus intake on incidence. In a field trial, effects of prepartal intermuscular injection of 10 million units of vitamin D3 on incidence of milk fever were examined both in relation to intake of calcium and phosphorous during the dry period and previous history of milk fever. Based upon intake of calcium and phosphorus cooperating herds were grouped as feeding: 1) greater than .53% of the total ration dry matter as calcium and greater than .28% as phosphorus: 2) less than .47% as calcium and greater than .28% as phosphorus; 3) greater than .47% to less than .53% as calcium and greater than .22% to less than .28% as phosphorus. Injections of vitamin D3 given approximately 1 wk prepartum reduced incidence of milk fever in cows with previous history of milk fever in all three groups but had no effect in cows with no previous milk fever. Incidence of milk fever was lower in group 3 than for cows of groups 1 and 2 with previous milk fever and than for cows of group 1 with no previous history. The results indicate that careful control of calcium and phosphorus intake during the dry period at .5% calcium and .25% phosphorus of the dry matter of the total ration will limit milk fever incidence to about 10%. Injections of vitamin D3 as described will reduce inicidence of milk fever further in cows with previous milk fever but not in cows with no previous milk fever.


The persistence of poliovirus of poliovirus in activated sludge treatment. A model activated sludge treatment plant was used which was functionally very similar to a full scale plant. It was inoculated with poliovirus either continuously or with a single dose and the amounts of virus in the influent, mixed liquor and effluent were monitored regularly. The distribution of the virus in the liquid and solid phases of the mixed liquor was very unequal with about 85% of the virus associated with the suspended solids fraction. Only small amounts of virus were recovered from the effluent and after inoculation was stopped virus rapidly became indetectable. The efficiency of the plant with regard to removal of virus was closely related to its capacity to remove suspended solids and the adsorption of virus to solids and its inactivation is briefly discussed.


Experimental transplacental transmission of porcine cytomegalovirus. Six serologically negative sows were infected by intranasal instillation of porcine cytomegalovirus (PCMV) between 31 and 85 days of pregnacy. Four sows showed an afebrile anorexia and lethargy 14-25 days after infection and all 6 developed significant increases in indirect immunofluorescent (IIF) antibody titres within 35 days. Virus was recovered from nasal and/or cervical swabs from 2 sows during life and from lung macrophage cultures after death. At term the sows were killed and their fetuses harvested by caesarean section. The number of mummified and stillborn fetuses increased from 4/63 in 6 previous litters to 18/60 in the 6 present litters. Nine of 43 fetuses born alive were reared in isolators for up to 6 weeks but the majority were killed for examination on the day of birth. Virus was isolated from 16 piglets from 4 of the 6 litters examined; it was isolated most frequently from lungs and liver but also from spleen, kidney, brain and nasal mucosa. Unsuckled day-old pigs had insignificant IIF titres, irrespective of whether they were excreting virus or not. The 5 congenital excretors which were reared all died within 7 days but no death occurred among their 4 litter-mates. Post-natal infection of 2 of these piglets reared in contact with congenitally infected pigs was suggested by the recovery of virus from nasal swabs 17 and 27 days after birth and the subsequent rise in IIF titre to 1/256 by day 42.


Biologic and chemical characterization of HLA antigens in human serum. HLA antigens of both the A and B loci were shown to be associated with the high density lipoprotein fraction of serum prepared by ultracentrifugal flotation. HLA-A9 antigens were purified 100-fold with essentially complete recovery by a simple procedure of high density lipoprotein preparation involving precipitation with polyanions and ultracentrifugal flotation. The purified lipid-associated antigen was immunogenic since it elicited the formation of cytotoxic xenoantibodies in rabbits. Serum HLA-A9 antigens were found by immunoprecipitation and gel electrophoresis to consist of a 45,000 m.w. heavy chain associated with beta2-microglobulin. The size of the HLA-lipid complex (less than 190,000 m.w.) and of the HLA-deoxycholate complex (less than 102,000 m.w.) suggests that HLA antigens are shed into plasma as a complex of a single HLA molecule and a single beta2-microglobulin chain, associated with boundary lipid.


Cell-mediated immune responses in patients with recurrent Herpes Simplex infections. II. Infection-associated deficiency of lymphokine production in patients with recurrent herpes labialis or herpes progenitalis. Herpes simplex virus antigen-induced lymphocyte proliferation and production of leukocyte migration inhibitory factor (LMIF) and lymphocyte-derived interferon were studied in normal individuals and patients with recurrent Herpes labialis and Herpes progenitalis. Virus-specific lymphoproliferative responses were regularly detected in patients with recurrent infection irrespective of the clinical stage of infection. In contrast, transient deficiencies in herpes-specific lymphoid production of both LMIF and interferon were regularly documented at the time of and immediately before herpes simplex-induced vesicular eruptions. During the convalescence, pronounced production of these mediators in response to antigenic stimulation with inactivated virus antigen preparations were regularly detected. The biology of these fluctuations in lymphokine production is evaluated and discussed.


Human monocyte-macrophage-mediated antibody-dependent cytotoxicity to herpes simplex virus-infected cells. Human peripheral blood mononuclear cells which mediate antibody-dependent cellular cytotoxicity (ADCC) against herpes simplex virus- (HSV) infected target cells consist of both adherent (MA) and nonadherent (MNA) effector cell populations. These two cell populations can be distinguished by their different phagocytic properties and morphologic appearance, their requirement for antibody in the ADCC reaction, and the rapidity with which they lyse target cells in the presence of immune serum. The MA cells are predominantly phagocytic and have the morphologic characteristics of monocyte-macrophages, whereas the MNA cells are nonphagocytic and appear to be small to medium-sized lymphocytes. Optimal expression of ADCC by MA cells requires higher concentrations of immune serum than does MNA cell-mediated ADCC. MA-mediated cell killing is first detectable by 8 hr and reaches completion after 24 hr of incubation. In contrast, MNA-mediated ADCC produces target cell damage by 2 hr and reaches completion at 8 hr of incubation. Unlike MNA effector cells, the MA effector cells are profoundly inhibited after preincubation with either latex or silica particles. The HSV immune status of the donor had no effect on the ability of either cell population to mediate ADCC. These data demonstrate the participation of both nonadherent mononuclear cells, presumably K cells, and monocyte-macrophages, in ADCC directed against HSV-infected target cells.


Identification of the Schwann cell as a peripheral nervous system cell possessing a differentiation antigen expressed by a human lung tumor. Recent studies of the plasma membrane antigens of a human lung tumor (oat cell carcinoma) indicated that the tumor expressed at least two normal differentiation antigens undetectable in normal respiratory epithelium. One antigen was characteristic of certain endodermally derived epithelial cells of the digestive system; the other antigen was characteristic of certain neural crest-derived cells in the peripheral nervous system. The present studies were undertaken to identify the reactive cell type in the peripheral nervous system. Since similar cells in the rat peripheral nervous system expressed a cross-reactive form of this antigen, and since pure cultures of different rat nerve cell type were available, the following approach was possible. Cultures of pure neurons, pure Schwann cells, pure fibroblasts, neurons and Schwann cells, and neurons, Schwann cells, and fibroblasts were assayed for this antigen with rabbit anti-oat cell carcinoma plasma membrane antiserum absorbed with normal lung and liver. The indirect immunofluorescence method on both whole, viable cell and fixed cell substrates was used. Only Schwann cells expressed the antigen; Schwann cells in the presence of neurons expressed the antigen much more strongly than did pure Schwann cells. It was concluded that the oat cell carcinoma of the lung expressed a differentiation antigen present on Schwann cells.


Stimulation of human peripheral blood lymphocytes by periodate, galactose oxidase, soybean agglutinin, and peanut agglutinin: differential effects of adherent cells. Blastogenic responses of normal human peripheral lymphocytes to three distinct groups of mitogens were studied: Group I--phytohemagglutinin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM); Group II--soybean agglutinin (SBA) and peanut agglutinin (PNA); and Group III--galactose oxidase (GO) and sodium periodate (IO4-). SBA was mitogenic for human cells, and this effect was enhanced by treating the cells with neuraminidase (NA). PNA was mitogenic only after cells had been treated with NA. GO was effective before and activity was increased after lymphocytes were treated with NA. Responses to Group II and III mitogens were more variable than were those to Group I mitogens. Studies with purified T and B cells indicated that SBA and PNA were T cell mitogens, whereas IO4- and GO failed to stimulate either T or B cells. Adding macrophages back to this system indicated that they were both T cell mitogens with strict macrophage requirements. T cell responses to SBA and PNA were enhanced over responses to unfractionated cells to a degree that could not be explained simply by enrichment of the cultures with T cells. Removal of adherent cells from unfractionated cell suspensions again revealed a marked enhancement of responses to SBA and PNA, a consistent decrease in responses to IO4-, and a variable decrease in responses to GO. Similar results were found with 14C-leucine and 3H-uridine incorporation,  as well as 3H-thymidine for the assessment of bastogenic response. Mechanisms responsible for these differential effects of macrophage depletion on lymphocyte responses to different groups of mitogens are yet to be determined. Either different mitogens require different lymphocyte to macrophage ratios for optimal stimulation, or some mitogens (i.e., SBA and PNA) form inhibitory complexees in the lymphocyte-macrophage mixture. In any case, variability in response to mitogenic agents in normal as well as pathologic states may be dependent on adherent cell populations, rather than on the lymphocytes themselves.


Cell-mediated immune responses in recurrent herpesvirus infections. I. Lymphocyte proliferation assay. Studies in immunosuppressed and immunodeficient patients indicate that the cell-mediated immune response appears to be responsible for controlling reactivated herpesvirus infections. In this study, the various parameters of a herpesvirus (types 1 and 2) antigen specific lymphocyte proliferation assay were optimized and used to evaluate individuals with clinical, recurrent HSV-1 and HSV-2 infections. Normal individuals with neutralizing antibody to HSV-1 or HSV-2 responded to virus antigen in culture as well as individuals with recurrent disease. Normal individuals without neutralizing antibody responded with a significantly lower response. Specificity of the lymphocyte proliferation assay was observed most strikingly in normal individuals with a rare HSV-1 infection during the vesicular eruption. Specificity was also observed by determining the ratio of the response to HSV-1 as compared to the response to HSV-2. Evaluated in this manner, individuals with recurrent HSV-1 infections had significantly higher ratios than individuals with HSV-2 infections and vice versa. Data from individuals with recurrent disease was compared to that of normal individuals to determine whether the former demonstrated a specific alteration in this response. Individuals with recurrent disease were found to have higher neutralizing antibody titers than normals. The neutralizing antibody titers in normal individuals correlated well with the lymphocyte proliferation assay results, whereas a similar evaluation in individuals with recurrent disease gave a negative correlation. The ratio of HSV-1 response/HSV-2 response also demonstrated a suppressed response in recurrent infections to the homologous virus during active disease, which disappeared when the individual was convalescent. These studies indicate that individuals with recurrent HSV infections have virus antigen specific alterations of their cell-mediated immune response, which can be associated with their disease.


Specificities of killing by cytotoxic lymphocytes generated in vivo and in vitro to syngeneic SV40 transformed cells. Cell-mediated immunity to SV40-transformed C3H and C3H-SW cell lines was measured by using both 51Cr and 125IUdR release assays. Killing by cytotoxic cells generated on in vitro sensitization of immune spleen cells with syngeneic SV40 cells by either assay is specific for syngeneic SV40 transformants. Cytolysis mediated by in vitro sensitized cells is ablated by treatment of the effector cells with anti-theta serum and complement. Intraperitoneal immunization with syngeneic SV40 cells yields two distinct killer-cell populations in the peritoneal exudate when assayed by 125IUdR release. The first, nylon wool nonadherent and sensitive to anti-theta and complement, is indistinguishable from the killers generated in vitro. The second population, present in larger numbers and more efficient on a per-cell basis in killing of SV40 targets than the first, is nylon adherent and is not removed by treatment with anti-theta and complement. This second population will kill any SV40 transformed target, whether syngeneic or allogeneic. The possible roles of T cell and non-T cell effectors in rejection of syngeneic SV40 tumors are discussed.


Characterization of the blastogenic and cytotoxic responses of normal mice to ecotropic C-type viral gp71. Spleen cells from normal (B6C3)F1 mice demonstrated natural cytotoxic reactivity mediated by a "null" cell population which, in part, had immunologic specificity for the major envelope glycoprotein (gp71) of the endogenous ecotropic murine leukemia virus. In contrast, the cytotoxic reactivity reflected in spleen cells of NIH Swiss nude mice apparently had no immunologic specificity. A significant level of blastogenic response could be generated in vitro by using normal (B6C3)F1 and AKR spleen cells in the presence of gp71. This reactivity was highly type specific. Moreover, using normal spleen cells from (B6C3)F1, both secondary blastogenic responses and cytotoxic T cell responses could be induced in vitro with purified soluble viral gp71. These findings extend further our previous studies on the existence of a natural immune response in normal mice to their endogenous MuLV by providing in vitro evidence for the expression of cell-mediated response in addition to the humoral response and that at least two effector cell types are operable in the cell-mediated phase.


Influence of H-2 complex on susceptibility to infection by murine leukemia virus. Titers of infectious ecotropic MuLV in mouse spleen were examined after deliberate infection. In congenic mice differing only in H-2 haplotype, a gene (or genes) within the H-2 complex determined either a high virus titer (H-2k, H-2d, H-2a) or a low titer (H-2b, H-2q). Susceptibility to high virus titers was inherited as a dominant trait. Kinetic studies revealed similar initial patterns of infection in both groups, with a fall in titer in the "resistant" strain occurring from week 6 through 10 after infection. Anti-VEA antibody titers differed significantly between the groups, but no mechanistic role for antibody in eliminating virus was apparent. Genes outside the H-2 complex were shown to influence MuLV titers after infection as well.


Stimulation of mouse migration inhibitory factor (MIF) production form MSV-immune lymphocytes by soluble tumor-associated antigen: requirement for histocompatible macrophages. Spleen cells from C57BL/6 mice immunized with murine sarcoma virus (MSV) are capable of producing migration inhibition factor (MIF) in response to stimulation with a specific tumor-associated antigen prepared by solubilization with 3 M KCL. We have previously demonstrated that this response is T cell-dependent. Further investigations into the effector cells involved in the production of MIF have revealed that spleen cells from mice immunized with MSV cannot produce MIF when stimulated with tumor extract if the population has been previously depleted of macrophages. However, the response can be restored by adding nonimmune syngeneic macrophages but not by allogeneic macrophages. The inability of allogeneic macrophages to provide this function was not due to their increased suppressor activity since in mixing experiments they did not interfere with the ability of immune spleen cells to produce MIF. Furthermore, they were not defective since they could supply this "cooperative function" to appropriate F1 mice. The results indicate that macrophages are required for stimulation of MIF by soluble tumor antigens and that for efficient interaction the macrophages and lymphocytes must share some genetic similarities.


A comparative study of detection methods for Aleutian disease viral antibody. Four methods of detecting and quantitating mink antibody against Aleutian disease (AD) virus were compared. Counterelectrophoresis, modified, counterelectrophoresis, immunofluorescence, and complement fixation were performed blindly on 274 serum samples. All four methods were reliably specific for AD antibody. Immunofluorescence was less reproducible than the other systems. Immunofluorescence complement fixation were 4- to 8-fold more sensitive than regular or modified counterelectrophoresis, but were limited by background staining and anti-complementary activity, respectively, when used to detect small amounts of antibody in undiluted sera.