Angiotensin I-converting enzyme from guinea pig lung and serum. A comparison of some kinetic and inhibition properties. The angiotensin I-coverting enzyme (peptidyldipeptide hydrolase, EC 3.4.15.1) was isolated from both guinea pig lung and serum; Km and V values were determined using both angiotensin I and hippurylhistidylleucine as substrates. Km values for the lung enzyme were 3.1 mM for hippurylhistidylleucine hippurylhistidylleucine and 0.076 mM for angiotensin I. Inhibition studies were performed and I50 values were obtained with the following inhibitors: angiotensin II (lung, 1.9 - 10(-5) M; serum, 1.7 - 10(-5) M), bradykinin (lung, 2.6 - 10(-6) M; serum, 2.1 - 10(-6) M), and pyrrolidone-Lys-Trp-Ala-Pro (lung, 7.9 - 10(-8) M; serum, 5.6 - 10(-8) M). Both enzymes were glycoproteins and were inhibited by concanavalin A. A maximum inhibition of 35% initial enzymatic activity was observed for both enzymes at a concanavalin A concentration of 4 - 10(-4) M suggesting that the sugar moieties of each enzyme are similar. Both enzymes required NaCl for activity and were inhibited by EDTA. A comparison of kinetic and inhibition properties indicates that both enzymes are quite similar.


Extraction of collagenase from the involuting rat uterus. Collagenase (EC 3.4.24.3) activity can be measured directly in homogenates of the involuting rat uterus. Latent forms of collagenase are activated by a brief exposure to trypsin; trypsin activity is then blocked with soybean trypsin inhibitor. Homogenizing conditions have been developed that permit 90-95% recovery of the total active and latent collagenase activity in a 6000 X g pellet, where it is presumably bound to its collagen substrate. This insoluble activity can then be extracted by heating to 60 degrees C for 4 min in 0.04 M Tris - HCl buffer, pH 7.5, containing 0.1 M CaCl2. Methods are presented for the estimation of the recovery of collagenase in the extracts; this approximates 65-70% of the total. Small amounts of activity can also be extracted from rat liver and kidney. This extraction procedure should be of use in purifying collagenase without culturing the enzyme-producing tissue and in the direct assay of tissue collagenase activity. The activity extracted from rat uterus has been proven to be collagenase by its characteristic pattern of collagen breakdown products on disc electrophoresis and by the split of tropocollagen at interband 41 as shown by electron microscopy of reconstituted fragments. The activity is inhibited by EDTA, and this inhibition is not reversed by calcium or zinc ions.


Specific modification of the GTP binding sites of rat 5'-adenylic acid aminohydrolase by periodate-oxidized GTP. 1. Rat skeletal muscle AMP deaminase (AMP aminohydrolase, EC3.5.4.6) can be inactivated by incubation with the periodate-oxidized analogue of the enzyme inhibitor GTP. 2. Nucleoside triphosphates and KCl at high concentrations protect against inactivation, while ADP has no effect. 3. The inactivation can be reversed by the addition of GTP and amino acids and made irreversible by reduction with NaBH4. This indicates that, in the binding of the oxidized GTP to the enzyme, a Schiff base is formed between the aldehyde groups of the inhibitor and amino groups of the enzyme. 4. The kinetic properties of the reduced (oxidized GTP)-AMP deaminase derivative indicate that the loss of activity results from an increase in Km while no appreciable change in V is observed; consequently, the enzyme shows positive homotropic cooperativity even in the presence of optimal KCl concentration. 5. Since the treated enzyme shows kinetic properties similar to those of the native enzyme in the presence of GTP, and since the loss of sensitivity to GTP is directly proportional to the degree of inactivation, it is concluded that the oxidized GTP specifically modifies the binding sites for GTP. 6. Binding of the radioactive oxidized GTP shows that two binding sites for this reagent exist in the AMP deaminase molecule.


A study of phosphoglycerate kinase in human erythrocytes. I. Enzyme isolation, purification and assay. The enzyme ATP-3-phospho-D-glycerate-1-phosphotransferase (EC 2.7.2.3) (phosphoglycerate kinase) has been isolated from human red cells in crystalline form by a modification of the method of Yoshida and Watanabe (1972) J. Biol. Chem. 247, 440-445). The crystalline enzyme was further purified by electrofocusing using carrier ampholytes (pH 7-9). The isoelectric point of phosphoglycerate kinase was estimated to be 8.75. The specific activity of purified phosphoglycerate kinase from electrofocusing was 2200 units per mg of protein at pH 8.3 (37 degrees C). Enzyme activity was assayed in the forward direction leading from 1,3-diphosphoglycerate to a 3-phosphoglycerate using a fluorimetric procedure for NAD-coupled enzymes for the measurement of the reaction rate at very low substrate concentrations. The auxiliary indicator enzymes were added in excess to yield true initial velocity kinetics, i.e. with no time lag upon addition of substrate (1,3-diphosphoglycerate). This was established theoretically using a mathematical model and confirmed experimentally. Further phosphoglycerate kinase was shown to be the rate-limiting step when the assay conditions were varied.


The refolding of lactate dehydrogenase subunits and their assembly to the functional tetramer. The renaturation process of different lactate dehydrogenase isozymes (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) from their unfolded subunits was investigated using a number of techniques. (a) kinetics of activity regain, (b) the kinetics of fluorescence change of fluoresecence change of the protein tryptophans, (c) kinetics of regain of the fluorescence properties of a covalently attached fluorescence probe (fluorescein) and (d) the kinetics of assembly, by following the intermediate oligomeric species appearing in the assembly pathway from monomers to tetramers. The results indicate that the unfolded polypeptide is converted to the active oligomeric species by the following scheme: Denatured subunit I leads to partially refolded subunit II leads to folded subunit III leads to dimer IV leads to tetramer. Step I and step II are first-order where step II is rate limiting. The ligands NAD+ and NADH accelerate step II, thus converting step I to the rate-limiting process. The fact that partially folded lactate dehydrogenase subunits are capable of co-enzyme binding may indicate the possible role of these ligands in the assembly of lactate dehydrogenase in vivo. Steps III and IV were found to be fast. The intermediate formation of an enzyme dimer which then dimerizes to the tetrameric species is found to be the major assembly pathway. Only a small portion of the lactate dehydrogenase tetramer is formed through the intermediate formation of a trimer intermediate.


Effect of periodate oxidation on the structure and properties of glucose oxidase. In order to elucidate the molecular structure of glucose oxidase (beta-D-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4) and the roles of its carbohydrate moiety, chemical, physiochemical and immunological experiments were performed with enzyme samples before and after periodate oxidation. Hydrodynamic parameters indicated that the native enzyme was a globular protein with values of 1.21 for the frictional ratio and 43 A for the Stokes radius. The enzyme contained about 12% carbohydrate by weight, of which the main component was mannose. The periodate treatment decreased the carbohydrate content to about 40% of its original value. Slight modifications were detected in the absorbance spectrum and the content of arginyl residue. However, no significant alteration was brought about by this treatment in the catalytic parameters, immunological reactivities of the gross structure, not in the secondary and quaternary structures of the protein moity. Thermal denaturation temperature (about 72.5 degrees C) and the enthalpy of denaturation (about 450 kcal/mol) were common to the native and the periodate-oxodozed enzymes. The native was found to be quite resistant to sodium dodecyl sulfate and fairly stable to urea and heating. The periodate-oxidized enzyme was also stable to heat treatment, but it showed a diminished stability when denaturing agents were present. Kinetic analyses of the thermal inactivation processes showed that the entropy of activation was greatly decreased by the denaturing agents, especially in the case of the periodate-oxidized enzyme. It is concluded that the carbohydrate moiety of the enzyme plays a role in increasing the stability of the protein moiety, but does not directly participate in the catalytic activity, the immunological reactivity, or in maintaining the conformation of the enzyme protein.


Lysine- and lysine-plus-threonine-inhibitable aspartokinases in Bacillus brevis. Two aspartokinase (ATP:L-aspartate 4-phosphotrasferase, EC 2.7.2.4) enzyme activities have been identified and partially purified from Bacillus brevis. Aspartokinase I is subject to both inhibition and repression by lysine, and has a molecular weight in the region of 110 000. Aspartokinase II is a lysine-stabilised enzyme, inhibited multivalently by lysine plus theonine and has a molecular weight in the region of 95 000. This attern of aspartokinase activity has not been described previously and is unusual in that one end product (lysine) regulates two isoenzymes catalysing the first reaction of a branced biosynthetic pathway. In the absence of lysine, aspartokinase II changes to a more unstable non-inhibitable enzyme. Both enzymes are stabilised by sulphydryl reducing agents and have similar affinities for ATP, aspartate and lysine. However, there is no evidence for a view that they are products of a common gene. Problem concerned with the regulation of aspartokinase activities in Bacillus species are discussed.


Substrate specificity of vetebrate collagenase. Substrate specificity of purified tadpole collagenase (EC 3.4.24.3) has been studied using eleven synthetic peptides. A pentapeptide, t-butyloxycarbonylprolylalanylglycylisoleucylalanine amide, was susceptible to the action of the enzyme and an octapeptide, acetylprolylglutaminylglycylisoleucylalanylglycylglutaminylarginine ethyl ester, was proposed to be the best substrate for vertebrate collagenase among the peptides tested.


Evidence for the selective release of lysosomal proteinases in fasted rabbits. The enzyme responsible for the conversion of "neutral" to "alkaline" fructose 1,6-bisphosphatase (EC 3.1.3.11) by removal of a 7000 dalton peptide (converting enzyme, Proteinase I) has been shown to be localized in rat liverlysosomes. Lysosomes also contain a specific proteinase (Proteinase II) that catalyzes the release of a small peptide from the NH2-terminus of the native subunits. In fasted rabbits Proteinase II is released into the cytoplasm, together with Cathepsin A, but Proteinase I remains associated with the lysosomal fraction. Increased osmotic fragility of liver lysosomes in fasted rabbits has also been observed, but this increased fragility does not result in the release of Proteinase I. The appearance of Proteinase II in the cytoplasm may be due either to its selective release from the lysosomes, without release of Proteinase I, or its localization in a different lysosomal fraction. Changes in lysosomal structure induced by fasting may play a dual role in : 1) the mobilization of amino acids for gluconeogenesis and 2) the modulation of activity of gluconeogenic enzymes.


[Network of interactions between unlinked genes: synergistic and antagonistic regulation of iso-1-cytochrome c, iso-2-cytochrome c and cytochrome b2 synthesis]. Five chromosomal genes, CYPI to CYP5 involved in the regulation of the synthesis of iso-1-cytochrome c, iso-2-cytochrome c and cytochrome b2 are described. The function of these genes was studied either by varying the proportion of the mutated and wild type alleles in the cell vy varing the growth conditions, or else by transforming the mutants into sigma-cytoplasmic petites. We have shown a network of genetic interactions which regulate the synthesis of three structurally different proteins : iso-1-cytochrome c, iso-2-cytochrome c and cytochrome b2, by two unlinked genes : CYC1 and CYP1, one of which (CYC1) is the structural gene by iso-1-cytochrome c. Within this network the interactions are proportional to the gene dosage and are either antagonistic or synergistic depending on the allele combination and the protein studied. The mutated alleles cyp1 stimulate the synthesis of iso-2-cytochrome c, inhibit the synthesis of iso-1-cytochrome c, while the cytochrome b2 synthesis is also inhibited but by a combination of cyp1 mutated alleles CYC1 wild type allele. Other loci, CYP2, CYP3, CYP4 and CYP5 were also studied in various allelic combinations. They show some interactions between them or with CYC1 locus but these interactions are different and less pronounced than those involving loci CYP1 and CYC1.


Studies of the regulation and reaction mechanism of the carbamyl phosphate synthetase and aspartate transcarbamylase of bakers' yeast. Kinetic studies of the carbamyl phosphate synthetase activity (CPSase) of bakers' yeast revealed an absolute requirement for K+ ions ; KM values for two of the substrates, glutamine and bicarbonate, were found to be 5 X 10(-4) M and 3 X 10(-3) M respectively. CPSase activity of the purified enzyme aggregate (M.W. 800,000) was extremely sensitive to UTP with a Ki of 2.4 X 10(-4) M. The purine nucleotide intermediate, XMP, was a strong activator of CPSase, acting at a site different from the regulatory site at which UTP binds ; XMP activation diminished at high concentrations of the substrate Mg-ATP. Studies of the reaction mechanism of CPSase revealed that it involved the sequential addition of the substrates bicarbonate and Mg-ATP, liberation of ADP, addition of glutamine, binding of ATP and then release of ADP and the product carbamyl phosphate. Studies of the reaction mechanism of the aspartate transcarbamylase (ATCase) of the aggregate yielded data which were not compatible with any of the usual models ; whichever reaction mechanism is ultivately found to fit the data, it will probably prove applicable both to the ATCase of the aggregate and to the disaggregated ATCase subunit (MW 138,000).


Further characterization of yeast RNA polymerases. Effect of subunits removal. Two forms of yeast RNA polymerase A are resolved by phosphocellulose chromatography. One of these, called RNA polymerase A, is lacking two polypeptide chains of 48,000 and 37,000 daltons. The properties of the two enzymes are compared in the present paper. RNA polymerase A transcribes d(A-T)n with a similar efficiency as the complete enzyme, but it is comparatively much less active with native DNA. The two enzymes can also be differentiated on the basis of their ionic strength and divalent cation requirements. RNA polymerase A has a particularly low activity at high salt and low Mg2+ concentrations. Thermal inactivation curves of the two enzymes are different when residual activity is assayed with native DNA. In contrast with d(A-T)n as template the apparent inactivation curves of the two enzymes are identical. The data suggest that the two dissociable polypeptide chains play an important role in transcription. The template specificity of yeast RNA polymerase B was further investigated using SV40 DNA-FI as template. RNA polymerase B is able to retain [3H]SV40 DNA-FI on nitrocellulose filters but the enzyme-DNA complex is very unstable. The observation that RNA polymerase B can transcribe to some extent a supercoiled DNA but not a linear double stranded template supports the hypothesis that the enzyme needs some unpaired DNA structure to initiate transcription.


[Purification and mechanism of action of a plant galactokinase]. The previously described galactokinase from Fenugreek seeds, has been purified by affinity chromatography on a column of galactosamine-CH Sepharose. This material ensures a more specific fixation than does ATP-Sepharose. A 400 fold purification was achieved in a single step, with a 80 per cent yield. Km's for galactose and for Mg/ATP2- complex were respectively 0.54 x 10-3 M and 5, 10-3 M. Galactose-1-phosphate is a competitive inhibitor of galactose while the inhibition for Mg-ATP2- is not a competitive one. The Mg-ADP complex is a non-competitive inhibitor of both galactose and Mg-ATP2-. Moreover, the Km of the enzyme for M-ATP2- complex is modified when 2-deoxy- and 6-deoxy-galactose are used instead of galactose. These results are consistent with an ordered sequential mechanism for this galactokinase: galactose binds to the enzyme before Mg-ATP2-, and galactose-1-phosphate is the last reaction product liberated. The affinity of the kinase for 6-deoxygalactose is lower than for 2-deoxygalactose. This observation reveals the importance of the hydroxyl in C6 position for the binding on the enzyme.


Isolation and properties of a cyclic guanosine-monophosphate sensitive intracellular ribonuclease from Bacillus subtilis. A ribonuclease was isolated and completely purified from sporulating cells of Bacillus subtilis. This RNase has a M.W. of about 150,000 daltons. It hydrolyzes single stranded RNA and single stranded synthetic polynucleotides yielding nucleoside 5'-monophosphates. The enzyme is an exonuclease which degrades polynucleotides from the 3'-end in the direction of the 5'-terminal. The RNase activity is strikingly inhibited by cGMP and to a lesser extent by cAMP. This inhibition (Ki = 0.1 mM) is of a non competitive nature. It appeared that in addition to the inhibition site, the enzyme contains a high affinity binding site for the two cyclic mononucleotides (K (cAMP) = 8.3 x 10-8; K (cGMP) = 2.5 x 10-7). The RNase activity is also strongly inhibited by spermidine. This inhibition appeared to be due to the polyamine binding with the RNA, thus lowering the affinity of the substrate for the active site of the enzyme. This RNase may play a role in vivo in selective degradation of newly synthesized mRNA during sporulation.


Alteration of poly (ADP-Rib) synthesis during progesterone- caused gene expression in oviducts of quails. The biological model of the selective induction of RNA synthesis in oviducts of estrogen stimulated immature quails by progesterone has been used to clarify whether poly (AD-Rib) is involved in DNA transcription. The chromatin-bound as well as the soluble poly (ADP-Rib) polymerase has been isolated from oviducts and the optimal reaction conditions have been determined. The activities, as measured by the incorporation rates of NAD+ into poly (ADP-Rib), of both, chromatin-bound "endogenous" polymerase (in the absense of "exogenous" DNA and histones) and soluble enzyme (native DNA-lysine-rich histone ratio: 4.3) from progesterone treated quail oviducts, have been determined to be only 30 per cent and 46 per cent respectively, as compared with the activities of the enzymes from the controls. This decrease in incorporation rates is apparently not due to an increased poly (ADP-Rib) degrading enzyme activity. Poly (ADP-Rib) synthesis in vivo was determined by incorporation studies with the precursor (14C) ribose. 12 h after intraperitoneal administration, 0.014 per cent of the total radioactivity was recovered in the oviduct histone fraction, 0.011 per cent in the oviduct nonhistone fraction and 0.009 per cent in the oviduct "HCIextract" containing the histone subfractions f1, f2 and f3. Among these histone subfractions f1 is ADP-ribosylated to the largest extent. ADP-ribosylation of f1 is less extensive in progesterone-stimulated oviducts (65 per cent) than in the controls (100 per cent). The present results suggest that in course of the selective, progesterone-induced DNA transcription the poly (ADP-Rib synthesis might drop.


Antibodies to herpes simplex virus types 1 and 2: a comparative study using indirect haemagglutination in carcinoma of the cervix and in other malignant conditions. The authors used a technique of indirect haemagglutination to compare levels of antibody to Herpesvirus simplex types 1 and 2 in groups of patients with carcinoma of the cervix, together with other malignant conditions and in comparison to controls. A significant difference in the mean titre of antibody to type 2 virus is revealed between the carcinoma group and the B controls. The significance of the antibody assays is assessed by a study of the specificity of the antibodies found and by an assay of antibody which cross reacts with Herpesvirus simplex type 1.


Cytopathogenic XC assay for bone marrow from mice infected with murine leukemia viruses. Virus production "in vitro" by bone marrow cells of murine leukemia virus-infected BALB/c mice was determined with a modified reverse XC cell assay. Bone marrow of mice infected by either Rauscher leukemia virus or Soulé myeloid leukemia virus was cocultivated with XC cells and quantification was done by counting syncytia. During the development of both erythroblastosis and myeloid leukemia, the number of syncytia induced by cocultivation increased. High numbers of syncytia were especially obtained with myeloid leukemia cells; the phenomenon was evident as early as one month following inoculation with virus and before the onset of conspicuous clinical signs of disease.


[Blood platelet cyclic AMP and prostaglandin E1 before and during post-operative venous thrombosis]. The intracellular level of cyclic AMP and PGE1 in platelets of 19 patients before and on the third day after surgery was measured. Among these 19 patients, 11 developed venous thrombosis in the legs early after surgery. We found no correlation between the occurrence of thrombosis and the initial level of cyclic AMP of PGE1. Nevertheless, after surgery the mean values of cyclic AMP or PGE1 in both the groups with or without thrombosis were significantly decreased. For PGE1 levels statistical analysis of variance showed a significant difference between the individual variations, pointing out an "individual factor".


Interactions between diverse proteinoids and microspheres in simulation of primordial evolution. Experiments demonstrating an incorporation of different enzymelike activities into a single preparation of proteinoid microspheres provide a conceptual basis for the primitive lengthening of protometabolic pathways. An enhancement of one enzymelike activity by another proteinoid in the same microsphere has been found. This effect, plus the pathway-lengthening propensity of combinations of microspheres, indicates selective advantages contributing to adaptive protoselection. Data reported in this paper also bring into purview the concept of internally controlled variation. Inferences are derived for the origin of protosexuality in protocells. When allowance is made for a closer relationship to the environment than that needed in contemporary selection, the fundamental mechanistic requirements of protoevolution are regarded as met by the proteinoid microsphere.


Phenotypic expression of galactokinase deficiency in heterozygous and homozygous subjects: in vivo and in vitro studies. Cultured fibroblasts derived from a patient homozygous for galactokinase deficiency, his parents, and controls had similar rates of growth in culture media where the only hexose was glucose. However, in media where the only hexose was galactose there was almost no growth of homozygous mutant cells or of maternal heterozygous cells and slight growth of paternal heterozygous cells. Growth of control cells was initially slow, but after a lag period (which coincided with increasing galactokinase activity) growth reached approximately the same levels as in glucose medium. In all cell lines there was a direct relation between the degree of enhancement of galactokinase activity and the ability of cells to adapt to growth in media where the only hexose was galactose. Erythrocyte galactokinase activities in a series of 24 children children with congenital cataracts aged 2-16 years were similar to those in 26 controls. One child in each of the cataract and control groups had 40-50% of mean control activity and was considered to be a potential heterozygote. Galactokinase deficiency (homozygous and heterozygous) is considered to be an uncommon cause of childhood cataracts. Nevertheless, it is an important cause since early dietary treatment can prevent or reverse lens opacities. The heterozygous state may be expressed phenotypically in the patient by the appearance of cataracts and in cultured cells by their defective growth in media where galactose is the only hexose.


Immediate and long-range effects of maternal viral infection in pregnancy. A brief description of the pathogenesis and immediate and long-range outcome following certain maternal viral infections in pregnancy has been presented. As evidence has accumulated from the follow-up of infants with specific congenital infections, it is clear that the consequences can be disastrous but not just for the obviously affected neonate - the normal-appearing infant at birth may have problems which appear much later. So-called "silent" congenital infections of the central nervous system may be a more frequent cause of mental retardation and specific learning disorders than we realize.


Hallux syndactyly--ulnar polydactyly--abnormal ear lobes: a new syndrome. A new syndrome is described. Its distinguishing features are hallux syndactyly, ulnar polydactyly and abnormal earlobes with other roentgenographic abnormalities along the medial border of the foot. It is inherited as an autosomal dominant.


[Role of alimentary hyperglycemia in the pathogenesis of several disorders of lipid metabolism]. Alimentary hyperglycemia in rabbits was found to be accompanied by increase in cholesterol level, as well as in the amount of triglycerides in the blood sera. Progressive increase in the serum concentration of pre-beta lipoproteids (serving as endogenous tryglyceride carrier) has also been demonstrated. A short-term feeding with cholesterol against the background of hyperglycemia in rabbits appeared to result in early marked atherosclerotic changes, as compared to those in control group of normoglycemic animals.


[Elaboration of chloramphenicol acetyltransferase by cells of E. coli K-12 under conditions altering the intracellular concentration of cyclic adenosine-3',5'-monophosphate]. The influence of cAMP, ACTH and glucose on the stimulation of chloramphenicol acetyl transferase synthesis in the cells of E. coli CSH-2/R222 and E. coli WZ-78/R222. (cya855) was examined. Glucose proved to decrease the enzyme synthesis in E. coli CSH-2/R222, causing catabolite repression; 5 mM of cAMP and 1000 mug/ml ACTH overcame the latter. The enzyme synthesis in E. coli WZ-78/R222 was insensitive to the catabolite repression; as to ACTH - it failed to cause stimulation ofchloramphenicol acetyl transferase synthesis in E. coli WZ-78/R222.


[Effect of cyclic adenosine-3', 5'-monophosphate on cells of experimental lympholeukemia L-5178]. A study was made of the effect of cyclic adenosine-3',5'-monophosphate (cAMP) dibutyril-cAMP and theophylline (phosphoesterase inhibitor - an enzyme transforming adenosine-3'-5'-monophosphate into adenosine-5'-monophosphate) on the intensity of proliferation (by the increase in the content of nucleic acids in the culture), DNA synthesis (by the H3-thymidine incorporation) and on the transplantation properties (the capacity to repopulation in the animal organism) of leukemic cells of the L-5178 strain. It was found that cAMP in a concentration of 0.8 mM considerably inhibited the H3-thymidine incorporation, retarded the proliferation and decreased the transplantation capacity of leukemic cells. Theophylline and dibutyril-cAMP had a comparatively low inhibitory capacity on the DNA synthesis, proliferative activity and the transplantation properties of the cells.


[Detection and study of a protein factor mediating stimulation of disulfide reductase activity by cyclic 3', 5'-AMP and other effectors]. DSR stimulation in the supernatant fraction under the effect of 3', 5'-AMP 10(-7) M, ATP 5.10(-5) M, Mg2+5.10(-5) M, EDTA 5.10(-4) M and protamine 5 mg/ml was mediated through a factor which was readily sorbed by BaSO4, Al (OH)3 and activated carbon, and was easily eluated with a 10-fold increase of the buffer molarity. Barium eluates of the liver and of the brain restored the effect of 3', 5'-AMP eliminated by BaSO4, crosswise and equally. Apparently, the sorbed factor was a protein since it was not dialyzed, very thermolabile and readily inactivated by a low trypsin concentrations. The factor restored the DSR activation eliminated or decreased by the protein proteinkinase inhibitor (PPKI). A linear relationship between active quantities of the PPKI and the protein factor were in favour of the fact that the protein factor was affiliated or identical to proteinkinase. A possible biological significance of the DSR activation and the hypothesis on the existence of a special protamine-sensitive proteinkinase is discussed.


[Enzyme activity of the bile and liver after disruption of its innervation and bile loss]. Continuous loss of bile in rats with a bile reservoir applied to the common bile duct caused an increase in specific activity of malic dehydrogenase, lactic dehydrogenase, glutamic dehydrogenase, glucose-6-phosphoric dehydrogenase, alkaline and acid phosphatase, urokinase and histidinase in the liver homogenates by the 7th day; the specific activity decreased by the 10th day. Disruption of innervation of the liver caused a sharp decrease of the ATP content and the abovementioned specifc activity in this organ. In continuous loss of bile there were revealed oscillations in the activity of the above-mentioned enzymes and sorbitol dehydrogenase in bile from the 1st to the 10th day of the experiment. Marked changes in the oscillations in the dysinnervated liver were in favour of the fact that those oscillations coursed under the control of the nervous system.


[Effect of early thymectomy on the growth of transplanted mammary gland tumors developing in mouse line C3Hf]. Mammary tumours which developed in factorless C3Hf mice when transplanted to MTV-S+ and MTV-S- recipients were found to grow at the same rate' early thymectomy failed to influence their growth. The observed accelerated growth of mammary tumour transplants from C3H/He mice to MTV-S+ recipients, as compared to the MTV-S- ones, and inhibition of the tumour growth after early thymectomy seemed to be connected with the peculiarities of the immunological reaction to the virus-induced antigens.


[Catecholamine concentration in the hypothalamus following alteration of pituitary adrenocorticotropic function]. Norepinephrine, epinephrine and serotonin levels were studied in male rabbit hypothalamus and midbrain under the influence of different factors potentiating the hypophysial adrenocorticotropic function. Intensification of this adrenohypophysial function was accompanied by a certain decrease in the hypothalamic and midbrain norepinephrine level and by the appearance of epinephrine normally absent here. The role of serotonin in the regulation of the investigated pituitary function was not revealed since potentiation of this function was observed both with the lowered and with the elevated concentrations of this monoamine in the hypothalamus.


[Changes in leukocyte enzyme activity during experimental amyloidogenesis and in patients with amyloidosis]. The activity of hydrolytic and redox myeloperoxidase enzymes was determined in the neutrophils and lymphocytes of the peripheral blood of patients with secondary amyloidosis and also of the animals with amyloidogenesis and amyloidosis (caseine model). During the amyloidogenesis the activity of hyocrolytic enzymes and of myeloperoxidase in the neutrophils was found to decrease; this was particularly marked at the stage of the initial amyloid deposition. Changes in the enzyme activity in the animals against the background of already developed amyloidosis coincided with such in the blood cells of patients with secondary amyloidosis. The results obtained are discussed from the aspects of resorption theory.


[Effect of microvessel injury on the frequency of tumor cells' lodging in them]. The action of local laser-induced microvascular injury on the frequency of tumour-cell lodgement and on the ability of these cells to migrate out of the microvessel lumen was studied. Intravital TV-microscope with a videotape recorder and an ultra-voilet laser was used. Ascites Saidel hepatoma cells were injected into the blood stream of rats. Lodgement of tumour cells in the mesenteric vessels was observed but rarely (11%). The lodged cells adhered to the microvascular wall and gradually migrated from the lumen. The injury of the wall with laser beam results in the formation of microthrombi and in increase in the frequency of lodgement of the tumour cells in these vessels: 82% against 11%, but migration of these cells decreased. The data obtained supported the significance of the microthrombus formation in the metastatic process. However, this role is not one-sided since on the background of increased frequency of adhesion of the tumour cells there was noted a decrease in their motility in the course of several hours after the lodgement in the microvessels.


Treatment of Nelson's syndrome by pituitary implantation of yttrium-90 or gold-198. Eight patients with Nelson's syndrome were treated with a pituitary implant of yttrium-90 or gold-198 four to 16 years after adrenal surgery. All had considerable pigmentation. One already had cranial nerve abnormalities and visual field defects and had had both a craniotomy and deep x-ray treatment. Radiographs showed that the pituitary fossa was abnormal in seven patients. A biopsy performed in six cases showed mucoid (or basophil) adenoma in all. In the four specimens examined ACTH was identified by electron microscopy or immunofluorescence, or both. Patients were followed up after pituitary implantation for three months to 12 years. All showed decreased pigmentation, and six became normal. Four patients regained normal ACTH levels and the other two studied had decreased levels. In no case did new cranial nerve disease or further sellar expansion develop since operation, and two patients showed remodelling of the sella. Complications were temporary leakage of cerebrospinal fluid and diabetes insipidus in one patient and gonadotrophin deficiency in another.


The uptake of L-glutamate by the retina. The accumulation of L[14C]glutamate by the isolated rat retina has been studied. When retinae were incubated at 37 degrees C in a medium containing L-[14C]glutamate, tissue/medium ratios of about 40:1 were achieved after 60 min. The labelled L-glutamate was rapidly metabolised and after 10 min about 50% of the radioactivity in the tissue amino acids was present as glutamine, aspartate, and 4-aminobutyrate (GABA). The process responsible for L-glutamate uptake showed many of the properties of an active uptake system: it was temperature sensitive, sodium dependent, inhibited by metabolic inhibitors and showed saturation kinetics. The saturable uptake process could be resolved into two components; a 'high' affinity process (apparent Km = 21 muM, Vmax = 35 nmoles/min/g tissue) and a 'low' affinity process (Km = 630 muM, Vmax = 881 nmoles/min/g tissue). The 'high' affinity and 'low' affinity uptake processes for L-glutamate appeared to have identical properties in the retina. The uptake of L-glutamate was not specific and was inhibited by other acidic amino acids including D-glutamate but not by neutral or basic amino acids. The retinal uptake of L-glutamate is not likely to be due to a homoexchange phenomenon because the retina was capable of achieving a large net uptake of glutamate and the efflux of L-[14C]glutamate from the tissue was not increased by the addition of non-radioactive L-glutamate to the incubation medium. Autoradiographic studies indicated that the sites for glutamate uptake are largely in the neuroglial Muller cells.


Cytochemical localization of glucose-6-phosphatase activity in the central nervous system of the rat. The histochemical detection of glucose-6-phosphatase (G-6-Pase) in neurons of the CNS has been confirmed at the level of electron microscope. Both glucose-6-phosphate (G-6-P) and alpha-glycerophosphate (alpha-gP) can be used as substrates to localize the reaction product of this enzyme, which we have found in all cell types of the cerebral cortex, cerebellum and brain stem. The reaction was most prominent in large neurons, such as the Purkinje cells of the cerebellum and the pyramidal cells of the cerebral cortex. This is due to their extensive content of rough and smooth endoplasmic reticulum, the ultrastructural sites of G-6-Pase activity. It was possible to measure quantitatively the hydrolysis of G-6-P and alpha-gP in brain homogenates and also in microsomal fractions, the biochemical correlate of the cytochemically demonstrable activity. These results call for a reappraisal of the previous biochemical evidence, which negates the existence of brain G-6-Pase, and consequently a reassessment of current concepts pertaining to the metabolic regulation of brain glucose.


Effects of morphine upon the lamina V type cells activities in the dorsal horn of the decerebrate cat. The effects of morphine (2 mg/kg i.v.) upon the transmission of nociceptive messages at the  spinal level have investigated in decerebrate cats by studying its effects on the activities of lamina V dorsal horn interneurons. In contrast to previous results obtained on the spinal cat, morphine had little or no effects on lamina V type cells in the decerebrate preparation. The mean values for spontaneous activity and responses to natural noxious stimulation were practically identical before and after morphine administration. Moreover, no significant depressive effect was found on responses induced by supramaximal transcutaneous stimulation. However, for this type of activity a depressive effect was revealed, if only the late component of units which presented bimodal responses were considered. We were unable to demonstrate after morphine administration an increase of the  descending inhibitory effects induced on lamina V cells by stimulation of the central inferior  nucleus of the raphe. Additional experiments using reversible spinalization (by cooling the cord at the thoracic level) suggest that the lack of effect of morphine on decerebrate animals could be explained by the fact that in this preparation, descending inhibitory influences are strongly exacerbated and thus may mask the depressive effects of this drug. These results indicate that the direct electrophysiological evidence of an increase of the descending control systems after morphine administration must be performed in the  intact preparation in order to avoid the effects ot their exacerbation in the  decerebrate state.


Brain energy metabolism of the conscious rat exposed to various physiological and pathological situations. Using a time-sharing fluorometer-reflectometer, pyridine nucleotide (NADH) and flavoprotein (Fp) fluorescence, as well as reflected light at the excitation wavelength, were measured and correlated with the electrical activity of an awake cerebral cortex. Exposure of the rat to a nitrogen atmosphere (anoxia) led to an increase in signals representing the reduction of pyridine nucleotides and flavins, with very similar kinetics. Inducement of partial ischemia by bilateral carotid artery ligation led to an increase in NADH, accompanied by a very small effect on the electrical activity (ECoG). In most animals, 2-3h after ligation, the ECoG  became flat or depressed. Exposure of this ischemic cerebral cortex to KC1 solution caused depression of the electrical activity without metabolic response probably due to the limitation of oxygen supply. The metabolic state of an awake cerebral cortex was identified by exposing the brain to various levels of  oxygen, epileptoform activity, spreading depression, hyperbaric pressure of oxygen and an uncoupler. From our results we conclude that the awake cerebral cortex is close to the resting state, state 4, rather than to the active state, state 3.


Regulation of rat adrenal dopamine beta-hydroxylase. II. Receptor interaction in the regulation of enzyme synthesis and degradation. Rat adrenal gland dopamine beta-hydroxylase is under neuronal regulation from the splanchnic nerve and hormonal control via adrenal cortical glucocorticoids. The regulatory systems act in different ways; neuronal stimuli induce dopamine beta-hydroxylase synthesis while hormonal stimulation inhibits enzyme degradation. Despite these mechanistic differences, both systems require a normally innervated cholinergic receptor to exert their effect. The enzyme response to either neural stimulation or ACTH administration is blocked by splanchnic denervation. Glucocorticoid stimulation of dopamine beta-hydroxylase, however, can occur after adrenal denervation, suggesting that ACTH acts on a receptor which requires splanchnic innervation, but glucocorticoids act distal to the receptor. Similar results were obtained when the effect of these manipulations were studied on phenylethanolamine N-methyltransferase, another enzyme in the catecholamine biosynthetic pathway. A model attempting to unify these and earlier findings is presented, in which the splanchnic nerve is involved in regulating both adrenal cortical glucocorticoidogenesis (by allowing ACTH to act on glucocorticoid synthesis) and adrenal medullary catecholamine biosynthesis (by induction of enzyme synthesis.).


CYCLIC AMP/*SECRETl cyclic AMP into push-pull perfusates in freely moving rats. Cyclic AMP was found in perfusates of push-pull cannula's, chronically placed in the lateral ventricle of freely moving rats. After addition of norepinephrine, dopamine or adenosine to the perfusates the output of cyclic AMP was enhanced, whilst serotonin and histamine were found to be ineffective. The effects of noradrenaline and dopamine on cyclic AMP were found to be mediated by different receptors: haloperidol antagonized only the dopamine response. The effect of norepinephrine was mimicked by isoprenaline, a beta-adrenergic agonist, but was not blocked by propranolol. L-DOPA, given intraperitoneally, increased cyclic AMP levels in the perfusate. We conclude that the release of cyclic AMP into the extracellular medium is a normally occurring phenomenon in brain tissue.


Factors affecting uptake and retention of technetium-99m-diphosphonate and 99m-pertechnetate in osseous, connective and soft tissues. The bone scanning complex, 99mTc-Sn-EHDP, consisting of the nuclide technetium-99m, stannous ion and ethane-1-hydroxy-1,1-diphosphonate, administered intravenously is retained in soft tissues in proportion to increasing calcium content of the tissues. Within bone tissue, the retention is proportional to vascularity and to surface area of calcium phosphate in bones and not necessarily to calcium and phosphate concentration. The nuclidic agent 99mTcO4-BUT NOT THE 99MTc-diphosphonate is selectively taken up by the thyroid and this uptake can be blocked by administering sodium perchlorate. Among the connective tissues studied, the tracheal cartilage seems to have the greatest potential to calcify with increasing age of the animal and man. Soft tissue does not retain the bone scanning complex 99mTc-Sn-EHDP but does retain 99mTcO4-.


Effect of estrogen on the collagenolytic activity of rat bone. Rats were injected with 17 beta-estradiol benzoate intramuscularly and the collagenolytic activity of the rat bones was assayed by measuring the degradation of purified reconstituted 3H-proline- and 3H-hydroxyproline-labeled collagen. A diminution of the collagenolytic activity which could be recovered from the culture medium of estrogen-treated rat bone was observed. However, the addition of estrogen to the enzyme using the same assay revealed no diminution of collagenolytic activity. These results suggest that the diminution of collagenolytic activity in the culture medium of estrogen-treated rat bone is due to an alteration in the production or activation of collagenolytic activity.


Status of tRNA charging, trinucleotide acceptor sequence and tRNA nucleotidyltransferase activity in the human placenta. Samples of tRNA isolated from the cell sap of full-term human placenta were found to have a low capacity for accepting amino acids in the presence of partially purified synthetase preparations made from placental or rat liver cell sap. Gel electrophoresis of placental tRNA showed that part of this could be accounted for by gross degradation. The proportion of chargeable tRNA carrying amino acids was estimated by periodate oxidation followed by stripping and then charging with labeled amino acids. Only 50% of chargeable placental tRNA was in the charged state when isolated, whereas 87% of freshly isolated rat liver tRNA was found to be charged with amino acids. A fraction from placental cell sap was shown to have tRNA nucleotidyltransferase activity. When placental tRNA was incubated with this fraction and [3H]ATP or [3H]CTP, ATP was incorporated into about 12% of the tRNA molecules and CTP into 5-7%. When rat liver tRNA was used in place of placental tRNA, [3H]ATP was incorporated into less than 5% of the tRNA molecules. By using snake-venom diesterase over short periods of incubation, it was confirmed that the ATP had been incorporated terminally as AMP into the placental tRNA. These observations show that, in contrast to rat liver tRNA, tRNA prepared from human placenta is poorly charged with amino acids, many of the molecules lack the acceptor trinucleotide and there is extensive degradation beyond this stage.


The synthesis of apoproteins of very low density lipoproteins isolated from the Golgi apparatus of rat liver. The incorporation of [3H]leucine in vivo into very low density lipoproteins (VLDL) from the rat hepatic Golgi apparatus and serum was studied. A Golgi-rich fraction isolated on a discontinuous sucrose gradient between 0.5 and 1.1 M was found to contain VLDL having common antigenic determinants with serum VLDL. The incorporation of the [3H]leucine into the Golgi VLDL and serum VLDL suggested a precursor-product relationship. Analysis of the apoproteins of the Golgi VLDL by polacrylamide gel electrophoresis revealed protein bands with similar mobility to those of serum VLDL, except that the former contained virtually no rapidly migrating peptides with the mobility of serum apo-C-II and apo-C-III. The pattern of incorporation of the [3H]leucine into the apoproteins was similar in VLDL from Golgi apparatus and serum, except for the absence of radioactivity in the area of the gel of Golgi apo-VLDL corresponding to apo-C-II and apo-C-III. The radioactive amino acid was incorporated predominantly into the Golgi apo-VLDL bands with similar mobility to apo-B and an apoprotein or group of apoproteins containing the arginine-rich peptide of serum VLDL. In vitro incubation of the Golgi VLDL with [3H]leucine-labeled HDL resulted in the acquisition of a number of proteins, including the rapidly migrating proteins. Administration of colchicine prior to the injection of [3H]leucine resulted in the appearance of gel bands and radioactivity in the apo-C-II and apo-C-III areas of Golgi apo-VLDL, suggesting that these can be acquired if secretion of VLDL is slowed or inhibited. The hepatic Golgi apparatus was then divided into fractions of predominantly forming face (GF3) or secretory granules (GF1). After polyacrylamide gel electrophoresis of the apo-VLDL from GF, no visible bands or incorporation of [3H]leucine was found in the region of apo-C-II or apo-C-III. However VLDL from GF1, showed visible and radioactive bands in the apo-C-II and apo-C-III area although they represented a much smaller proportion of the total apoprotein than was found in the corresponding serum apo-VLDL. In the isolated perfused liver the percentage incorporation of [3H]leucine into the rapidly migrating apoproteins of Golgi VLDL was considerably less than that found in the corresponding apoproteins of perfusate VLDL, where circulating C lipoproteins are virtually absent. The data indicate that nascent VLDL begins to acquire the C-II and C-III apoproteins during its passage through the Golgi apparatus but that the main acquisition occurs during or after secretion into the space of Disse.


The induced biosynthesis of 7-dehydrocholesterols in yeast: potential sources of new provitamin D3 analogs. The effect of low concentrations of a specifically designed sterol-24-transmethylase inhibitor, 25-aza-24, 25-dihydrozymosterol (10) on sterol production in Saccharomyces cerevisiae was examined. The synthesis of cholesta-5,7,22,24-tetraen-3beta-ol (4), its 7,22,24 analog (15) and the 7,24 analog (5) coupled with the availability of zymosterol (6) and cholesta-5,7,24-3beta-ol (3) derivatives facilitated a search for these sterols in cultures treated with this inhibitor. When S. cerevisiae was grown in the presence of 1.3 and 5 muM 10, it produced no ergosterol but accumulated zymosterol (6), cholesta-5,7,22,24-tetraen-3beta-ol (4) and related C27 sterols (3 and 5). These results indicate blockage of the side chain methylation that normally occurs during the biosynthesis of ergosterol in yeast by compound 10 is efficient. The cholesta-5,7,22,24-tetraen-3beta-ol is a close structural analog of provitamin D3 (7-dehydrocholesterol). The inhibited yeast thus provides a source of a potentially new provitamin D3 substitute.


Versatility of oxazolidine spin labels--a model study with acyl-alpha-chymotrypsin. The p-nitrophenyl ester of N-oxyl-4',4'-dimethyl-3-oxazolidinebutyric acid was synthesized. The resonance spectrum of the acyl-alpha-chymotrypsin intermediate of this substrate was found to have more motional freedom at the enzyme active site as compared to the acyl-enzyme prepared from the p-nitrophenyl esters of 1-oxyl-2,2,5,5-tetramethyl-3-carboxypyrrolidine and 1-oxyl-2,2,6,6-tetramethyl-4-carboxy-1,2,5,6-tetrahydropyridine. The flexibility and the versatility of Keana's oxazolidine spin labels as covalent conformational probes is discussed.


Diphosphoinositide metabolism in bovine adrenal medulla. (1) A phosphatidylinositol kinase (EC 2.7.1.67) of a chromaffin vesicle membrane preparation isolated from bovine adrenal medulla was characterized. Its activity towards endogenous and exogenous phosphatidylinositol was very similar to the kinase activity of the microsomal fraction prepared from the same tissue. (2) Phosphomonoesterase (EC 3.1.3.36) and diesterase activity hydrolysing membrane bound phosphatidylinositol 4-phosphate was located mainly in the microsomal fraction. No hydrolytic activity was present in the vesicle membrane. (3) Phosphorylation of chromaffin vesicle membrane phosphatidylinositol did not increase calcium-binding by the membranes.


Cyclic nucleotides in spinal cells.

Cyclic nucleotides in spinal cells. The most striking effects of intracellular injections of adenosine 3'5'-cyclic monophosphate (cAMP) into spinal mononeurons in cats are a speeding-up of the action potential, both its rising and falling phase, and a potentiation of the after-hyperpolarization; the latter porbably indicates a marked enhancement of Ca2+ influx. In this respect, cAMP and guanosine 3'5'-cyclic monophosphate (cGMP) have similar actions, though cAMP appears to be more potent. It is suggested that through this mechanism, cyclic nucleotides may play an important role in synaptic facilitation. Changes in resting membrane potential and resistance are less conspicuous or predictable. By contrast, both agents, when injected into unresponsive cells, presumed to be neuroglia, regularly cause a drop in membrane resistance; this is associated with hyperpolarization and therefore likely to reflect an increase in membrane K+ conductance.


Response of serum cholesterol and triglycerides to hormone treatment and the relation of pretreatment values to mortality in patients with prostatic cancer. Cholesterol and triglycerides were measured in plasma samples from patient with cancer of the prostate before and after 3 months treatment with either Premarin, Provera, Provera and diethylstilbestrol, or diethylstilbestrol alone. Cholesterol was also measured before and after one of three doses of diethylstilbestrol or placebo. Pretreatment cholesterol levels at 196 +/- 1.3 mg per 100 ml (X +/- SE, N = 1093) were significantly lower than these reported for similar age group noncancer controls. Significant increases occurred with some of the estrogen treatments. Pretreatment cholesterol levels showed a significant negative correlation with age in Stage III and IV patients of both studies and a positive correlation with hemoglobin in Stage III patients of both studies. Pretreatment triglyceride levels at 120 +/- 1.9 mg per 100 ml (X +/- SE, N = 1089) were similar to levels reported for noncancer controls of similar age. Estrogen treatment produced a significant increase in triglyceride levels. Serum triglycerides were significantly correlated with hemoglobin, weight, and cholesterol and  negatively correlated with age, Analysis of covariance for both cholesterol and triglycerides showed highly significant treatment effects, but no stage effects and no stage-treatment interactions. It showed that the pretreatment value is of extreme importance for predicting or explaining the 3-month value. Death rates were calculated by level of pretreatment cholesterol or pretreatment triglycerides for all Stage II and IV patients, all treatments combined, and for Study 2 and Study 3 separately. No consistent trends were evident for cholesterol. Spearman correlation coefficients between category of initial triglyceride value and rank of death rate were computed to test for a quadratic effect. When the absolute values of the initial triglyceride values minus the overall mean were correlated with the death rate, a significant negative correlation was found for all causes of death and for deaths due to cardiovascular disease and prostatic cancer. These results indicate that the death rate is highest near the overal mean for initial triglyceride values and decreases as the initial values deviate above or below the mean. Initial triglyceride levels appear to have potential as indicators of risk of death in patients with prostatic cancer. The percentage of patients dead at 1 year by initial triglyceride levels, measured only in Study 3, revealed a pattern similar to that observed for the death rate, that is, the highest percentages were associated with values near the overall mean.


Response of plasma fibrinogen and plasminogen to hormone treatment and the relation of pretreatment values to mortality in patients with prostatic cancer. Fibrinogen and plasminogen were measured in plasma samples from prostatic cancer patients before and after 3 months of treatment with either Premarin, Provera, Provera and diethylstilbestrol, one of three doses of diethylstilbestrol, or placebo. Plasminogen levels generally were increased significantly with the estrogens but were unchanged following placebo or Provera treatment. Pretreatment plasminogen levels in Study 3 were significantly lower (p less than .001) than in Study 2. Plasminogen pretreatment levels were significantly correlated with age, hemoglobin, body weight, and blood pressure. Fibrinogen pretreatment levels were significantly elevated above normal. They were not significantly with age, hemoglobin, body weight, or blood pressure. Fibrinogen levels generally were significantly decreased by the estrogens. Comparisons of means of pretreatment fibrinogen and plasminogen levels from patients dying during the first year of the study with the mean pretreatment levels of the patient group alive after 1 year on study yielded no significant differences. Death rates were calculated by pretreatment plasminogen or fibrinogen for all treatments of all Stage III and Stage IV patients combined for Study 2 and Study 3 separately. Such rates were calculated for all causes combined and for deaths from prostatic cancer or cardiovascular disease separately. The levels of plasminogen were significnatly negatively correlated with death rate from all causes combined and with cardiovascular disease considered separately, but not with death from prostatic cancer. The levels of fibrinogen were signigicantly positively correlated with death rates from all cuses and nearly significantly with prostatic cancer, but not cardiovascular disease. Elvated pretreatment fibrinogen levels were associated with an increased proportion of deaths at 1 year from all causes and from cancer of the prostate.


Early assessment of normal tissue tolerance of fast neutron beam radiation therapy. Between September 10, 1973 and May 20, 1975, 94 selected patients were treated with fast neutron beams generated by bombarding an "intermediate" beryllium target with 21 MeV deuterons. The clinical material included: 58 patients with head and neck cancers; 27 patients with cerebral glioblastoma multiforme; and 9 patients with a variety of other neoplasms. Of the 67 patients treated only with neutron beams, 31 received two increments per week and 36 received three increments per week. The calculated doses in most patients were 1800 rad n gamma (neutrons + gamma components) delivered in 6 weeks. Seventeen patients were treated with two increments of neutron beams and three increments of 60Co photons weekly to calculated total doses of 780-1050 rad n gamma + 3360-3780 rad 60Co gamma delivered over 50-54 days. Ten patients were treated with neutron bean "boosts" of 300-900 rad n gamma in three to seven increments over 8-23 days following conventional radiation therapy. Treatment has been well tolerated, with only 9 of 94 patients not completing the anticipated course. In three of these, treatment was dixcontinued because of intercurrent problems. Except for those patients with glioblastoma multiforme, treatment complications have not been dose-related. In four patients, complications probably were related to persistent cancer. Neutron beam irradiaiton may have been a factor in the neurologic deterioration of five patients with glioblastoma multiforme who did not complete treatment.


Humoral immunity to herpes simplex viral-induced antigens in smokers. Antibodies to herpes simplex viral-induced antigens (HSVIA) were assayed by an indirect immunofluorescent technique in 93 regular cigarette smokers, 75 of whom also imbibed alcoholic beverages. Controls were 94 nonsmoking, nondrinking volunteers matched with the smoker for age and sex. IgA antibodies to HSVIA were detected six times more frequently in the sera of smokers than in nonsmoking controls, p less than .0005. IgG and IgM anti-HSVIA were detected with comparable frequencies in both groups, but the antibody titers were significantly higher in the smoking group than in controls, p less than .05. IgA anti-HSVIA was detected more frequently in smokers with 10 or more pack-years of cigarette exposure than in smokers with 10 or more pack-years of smoking (p less than .05) or in matched nonsmokers (p less than .02). IgA antibody titers to HSVIA were significantly higher in cigarette smokers who drank alcoholic beverages than in smokers who did not drink, p less than .025. This study demonstrates that cigarette smoking and the use of alcoholic beverages are associated with heightened humoral immunity to HSV-induced antigens in a population at high risk for development of squamous cell carcinomas of the head and neck region.


Trophoblastic pseudotumor of the uterus: an exaggerated form of "syncytial endometritis" simulating a malignant tumor. Twelve cases of a hitherto unrecognized pseudotumorous trophoblastic invasion of the myometrium are analyzed. Human chorionic gonadotropin (HCG) was identified by an immunoenzyme technique in the cytoplasm of the invasive cells. The lesion may be localized and only superficially invasive or deeply invasive and have a gross appearance suggesting a neoplasm in the excised uterus. In either case, this process has been confused with various types of malignant tumors, most often choriocarcinoma, from which it may be distinguished by an absence of the characteristic dimorphic population of cytotrophoblast and cyncytiotrophoblast. Most of the patients, who ranged in age from 19 to 41 years, presented with amenorrhea uterine enlargement and were thought to be pregnant, although only four of them had positive pregnancy tests. The most serious complication was uterine perforation, which occurred spontaneously in one patient and during curettage in five others. One patient died as a result, but the other 11 are alive without evidence of disase 6 months to 12 years after the diagnosis. Of the 11 surviving patients, four received no treatment other than curettage. Because this trophoblastic lesion is likely benign and simulates a malignant tumor on pathologic examination, the term "trophoblastic pseudotumor" has been chosen to designate it.


Direct immunoperoxidase staining for Regan isoenzyme of alkaline phosphatase in human tumor tissues. Immunoperoxidase staining for Regan isoenzyme of alkaline phosphatase was performed on cryostat sections of five human tumor tisssues. With a direct immunoperoxidase staining for the localization of Regan isoenzyme at the light and electron microscope levels, sections previously fixed with 0.05 M phosphate-buffered 4% paraformaldehyde were reacted with rabbit antisera to human placenta alkaline phosphatase conjugated to horseradish peroxidase. Comparison of conventional histochemistry and immunohistochemistry for Regan isoenzyme indicated that strong specific immunoperoxidase staining appeared on the cell membrane surface, and a diffuse one, in the cytoplasm of lung and colon cancer tissue cells showing L-phenylalanine-sensitive alkaline phosphatase. No immunoperoxidase reaction was obtained in tumor cells showing sensitivity to L-homoarginine or lacking aklaline phosphatase activity.


Primary mucinous adenocarcinoma of thyroid gland. A mucin-producing carcinoma in the thyroid gland found in a 44-year-old man was first thought to be a metastatic carcinoma, possibly from salivary gland. However, follow-up examinations for 8 years have not demonstrated another neoplasm, and it seems reasonable to conclude that this lesion was a rare primary mucinous adenocarcinoma of the thyroid gland.


Risk and prognostic factors in trophoblastic neoplasia. Three hundred and seventeen patients with gestational trophoblastic tumors were investigated and treated between 1957-1973. The risk of trophoblastic tumor was influenced by the outcome of the antecedent pregnancy (hydatidiform mole, non-mole abortion, term delivery) and the ABO blood groups of the mating couple; it was also influenced by the patient's age. The response to treatment with chemotherapy and , where appropriate, with surgery and radiotherapy, was influenced prfoundly by several factors. These included 1) the outcome of the antecedent pregnancy, 2) the total body burden of tumor at the time treatment stated as reflected by the urinary output of human chorionic gonadotrophin (CG), 3) the interval between the antecedent pregnancy and the start of chemotherapy, 4) the ABO groups of the mating couple, 5) the extent of mononuclear cell infiltration in the tumor, 6) the immunological status of the patient at the start of treatment, 7) the size of tumor masses, 8) the site of metastases and particularly the presence of intracranial metastases, and possibly by 9) the age and 10) the parity of the patient. A detailed study of the HLA antigens of the patient, her husband, and antecedent child has shown no positive effect on risk or prognosis. These data provide a basis for a scoring system that allows the prognosis to be defined at the time of diagnosis and facilitates tisk of drug resistance. Applied retrospectively to the cases from which the scoring system was generated, prognostic groups with survival rates ranging from 0-100% can be defined. Unfavorable prognostic factors combine so as to increase the probability of drug resistance.


Management of small cell carcinoma of the lung: therapy, staging, and biochemical markers. A chemotherapeutic regimen consisting of BCNU, cyclophosphamide, vincristine, and procarbazine was evaluated in 43 patients with small cell carcinoma of the lung. The majority of patients received radiation therapy of the primary tumor, but chemotherapy alone was utilized in a group of patients with widely disseminated disease. In addition to thorough staging with radioisotope scans and bone marrow biopsies, a study of calcitonin and histaminase as biochemical markers was performed. The BCVP chemotherapy resulted in a complete and partial response rate of 53% when given alone or in conjunction with radiotherapy. The survival data are preliminary, but the complete responders do have a statistically significant better survival (mean of + -95 days) than the partial responders and nonresponders. Hypercalcitonemia was not detected in our patients, but elevated histaminase activity was found in eight of 24 patients with small cell carcinoma and in only one of 19 patients with squamous and large cell carcinoma.


Recurrent cystosarcoma phyllodes: a clinicopathologic study of 32 cases. A retrospective study was made of recurrent mammary cystosarcoma phyllodes seen at Memorial Sloan-Kettering Cancer Center from 1932 to 1972. Clinicopathologic findings, clinical course, treatment, and survival are described. It is apparent that cystosarcomas may recur and several years may elapse between reucrrences. We found that 18% of the benign and 8% of the malignant cystosarcomas recurred and malignant "transformation" occurred in two of the 28 recurrent benign cystosarcomas.


Pretranslational control of tryptophan oxygenase levels in Morris hepatoma and host liver. Tryptophan oxygenase is present and hormonally inducible in host livers but is absent in transplanted Morris hepatomas examined under basal conditions as well as in hormonally induced animals. Studies were performed to determine whether the absence of tryptophan oxygenase in hepatomas is mediated by an alteration in the translational efficiency or the level of the messenger RNA (mRNA) for tryptophan oxygenase. The tissue level of the specific mRNA coding for tryptophan oxygenase was quantitated in an mRNA-dependent Krebs ascites cell-free protein-synthesizing system. The enzyme levels and mRNA activities in host livers and hepatomas from control rats and rats given injections of an inducing dose of hydrocortisone were compared; they indicate that the induction of tryptophan oxygenase in host livers by hormones is accompanied by a proportional increase in the level of its mRNA, whereas in the transplanted hepatomas the tryptophan oxygenase catalytic activity and the mRNA coding for this enzyme were undetectable in both control and glucocorticoid-induced animals. No functional mRNA for tryptophan oxygenase could be detected in the total polyadenylate-containing mRNA isolated from the Morris hepatoma cells. The hepatomas contained normal levels of cytoplasmic glucocorticoid receptor that could bind glucocorticoid, undergo "activation," and translocate to both normal and neoplastic nuclei. Thus, deletion of tryptophan oxygenase in hepatomas is a consequence of the absence of the gene product, i.e., the tryptophan oxygenase mRNA, which codes for its synthesis; this is not due to detectable alterations in the ability of the glucocorticoid receptor to bind the steroid hormone, or of the hormone-receptor complex to undergo activation, or of the activated steroid-receptor complex to bind to nuclei derived from the hepatoma or normal liver.


Levels of C-type viral p30 antigens in lymphoma-resistant mice. Until relatively recently, interest has largely centered upon the causal role of oncogenic viruses especially with respect to the development of murine lymphomas. Host factors have recently come to the fore and are considered to be effective here, where we note that, in spite of relatively high levels of C-type viral antigen in the AKR X CBA F1 mouse, this hybrid remains relatively lymphoma resistant. Evidence points to an overriding host factor in this situation that is dominant with respect to tumor resistance and furthermore independent of the viral load at least as judged by levels of p30 viral antigen, an assumption confirmed by xc plaque assay.


Differences in transcription between transformed mouse cells in culture and the tumors that they produce in syngeneic mice. RNA-DNA saturation hybridization experiments were used to determine the degree to which cellular DNA sequences are transcribed in cultured mouse cells transformed by polyoma virus and in tumors that develop after injection of these cells into syngeneic mice. A general, overall reduction in the proportion of the genome transcribed was found in tumors compared to the proportion in cultured transformed cells. When explants of the tumor cells were grown in culture, the level of transcription of both repetitive and nonrepetitive DNA returned to that observed in the original transformed cells.


Immunization against feline oncarnavirus disease using a killed tumor cell vaccine. Specific pathogen-free cats were immunized with an inactivated feline oncornavirus tumor cell vaccine. Immunized cats produced high antibody titers to the feline oncornavirus-associated cell membrane antigen and were protected from oncogenic feline sarcoma virus challenge. However, immunization did not produce virus-neutralizing antibody nor did it prevent viremia.


Binding of metabolically activated benzo(a)pyrene to nuclear macromolecules. The binding of metabolically activated [3H]benzo(a)pyrene ([3H]BP) to the DNA, RNA, histones, and nonhistones of isolated rat liver and lung nuclei was studied. Conditions for optimal binding to the nuclear components were determined. Upon incubation with isolated liver nuclei and reduced nicotinamide adenine dinucleotide phosphate, [3H]BP was able to bind to nuclear components. The binding appeared to be covalent in nature. Treatment of the rats with 3-methylcholanthrene induced the nuclear aryl hydrocarbon hydroxylase (AHH) activity and also increased the level of carcinogen binding. The addition of rat liver microsomes to the incubation systems greatly enhanced the level of [3H]BP binding to the macromolecules in the nuclei from both the control and 3-methylcholanthrene-treated rats, and the maximal levels of binding obtained with these two types of nuclei were similar. The binding was inhibited by 7,8-benzoflavone or glutathione. Lung nuclei from control rats had very low AHH activity and did not exhibit appreciable carcinogen binding, whereas those from 3-methylcholanthrene-pretreated animals had slightly higher AHH activity and caused low levels of binding. The binding of [3H]BP to lung nuclei was greatly enhanced by liver microsomes but only slightly by lung microsomes, which had rather low AHH activity. Several lines of evidence indicate that, in the control experiments (no reduced nicotinamide adenine dinucleotide phosphate added), the radioactivity associated with the macromolecule fractions is probably a background value rather than due to the binding caused by a specific interaction between benzo(a)pyrene and cytochrome P-450. The present study clearly demonstrates that a carcinogen activated at the microsomes can enter into the nucleus and react with its macromolecules; the carcinogen can also be activated by the monoxygenase system of the nuclear envelope. It appears that both the endoplasmic reticulum and the nuclear envelope are potentially important sites of carcinogen activation.


Inhibition of antibody-complement-mediated killing of tumor cells by hormones. Line 1, a chemically induced guinea pig hepatoma, is susceptible to killing by anti-Forssman immunoglobulin M antibody and guinea pig complement. When these tumor cells are pretreated at 37 degrees with 10(-4) to 10(-11) M concentrations of the polypeptide hormone insulin, with the catecholamine L-epinephrine-HCl, or with the glucocorticoid steroids hydrocortisone sodium succinate or prednisolone sodium succinate, the cells show a marked reduction in their suseptibility to killing by antibody and guinea pig complement; pretreatment at 0 degrees is ineffective. Similar results were obtained with another antigenically distinct guinea pig hepatoma (line 10) when tested with anti-Forssman immuno-globulin M or specific antitumor antibodies and human complement. The ability of the hormones to render the cells resistant is dependent on time, temperature, and hormone concentration. The effect of hormone treatment is maximal between 30 and 60 min and is reversible within 4 hr even in the continued presence of hormone. Treatment of line 1 cells with up to 10,000-fold greater concentrations of the less biologically active or inactive analogs, DL-epinephrine, beta-estradiol, testosterone, or proinsulin has no effect on the susceptibility of the cells to killing by antibody and guinea pig complement. The effect of hormone treatment is not due to a direct inactivation of bound or fluid-phase complement components by the hormones or to a decrease in the ability of the cells to bind complement-fixing antibody.


Inhibition of de novo pyrimidine nucleotide and DNA synthesis and growth of cultured Novikoff rat hepatoma cells and other cell lines by pyrazofurin (NSC 143095). Pyrazofurin (PYF), a C-riboside, inhibited the replication of cultured Novikoff rat hepatoma cells, HeLa cells, and mouse L-cells at concentrations as low as 0.1 to 10 muM, but Novikoff cells were more sensitive than the cells of the other two cell lines. Inhibition of cell replication was completely prevented by the presence of 0.1 to 1 mM uridine in the medium, and partly by the presence of other pyrimidine, but not purine nucleosides. A 2- to 4-hr treatment of the cells with 10 muM PYF resulted in a 2-fold increase in the rate of incorporation of uridine into the acid-soluble pool and nucleic acids, while the rate of incorporation of adenosine into RNA was reduced about 85%. The incorporation of adenosine and deoxyuridine into DNA were reduced about 85 and 50%, respectively. The results are consistent with the view that PYF inhibits the de novo synthesis of pyrimidine nucleosides. The inhibition of cell replication seems to be due mainly to an inhibition of DNA rather than RNA synthesis, resulting from a rapid depletion of the pyrimidine deoxynucleotide pool, since addition of thymidine and deoxycytidine reversed the inhibition of DNA synthesis and cell replication by PYF. PYF must enter the cells to exert its toxicity since the toxicity of PYF was reduced 70 to 80% by the presence of 8 muM Persantin, a potent inhibitor of the facilitated and simple diffusion of various substrates, in the medium. If PYF is incorporated via normal nucleoside salvage pathways, its affinity for the nucleoside transport system(s) and kinases, must be low since, even at a concentration of 1 mM, it had only a slight effect on the initial rates of incorporation of various nucleosides into the nucleotide pool.


Nitrosourea combinations in lung cancer. BCNU, CCNU, and methyl-CCNU have undergone extensive trial in multiple drug combinations for bronchogenic carcinoma. The addition of a nitrosourea appears to be an improvement over cyclophosphamide used alone in oat cell carcinoma and over the two drug combination of cyclophosphamide and methotrexate in both adenocarcinoma of the lung and oat cell disease. Encouraging response rates have been seen in squamous lung cancer with multiple-drug combinations of a nitrosourea, an alkylating agent, vincristine, and bleomycin with or without adriamycin. The nitrosoureas have been easily incorporated, at reduced doses, into multiple-drug regimens with cumulative myelosuppression seen only when the interval between nitrosourea doses is less than 6 weeks. Conclusions about the ultimate role of these compounds in lunb cancer treatment must await (a) comparative trials of combinations with and without a nitrosourea, and (b) further exploration of new approaches to increase their therapeutic index.


The incidence of premature contractions in coronary patients during the sleep-awake cycle. Mean heart rates and their variability indices during REM sleep and the frequency of cardiac arrhythmias during the sleep-awake cycle were determined in a group of coronary patients with disturbances of cardiac rhythm. In some patients there was a temporal relationship of atrial and ventricular premature contractions with slow-wave sleep, REM sleep or wakefulness. However, when they were considered as a group, no significant correlation could be observed. Furthermore, the sinus arrhythmia characteristic of REM sleep was not found to be exaggerated in those patients.


Insulin: fundamental mechanism of action and the heart. Insulin accelerates the entry of glucose and amino acids into muscle cells by acting upon the 'carrier-facilitated' transport mechanism. For glucose this process is passive and leads to equilibration of intracellular and extracellular concentrations. In heart muscle, glucose transport is a rate-limiting step for glucose uptake. During hypoxia and ischemia the heart turns to anaerobic glycolysis for energy production and therefore, maximal glucose transport becomes important. Insulin is necessary to insure proper protein synthesis, probably at the level of membrane-bound polyribosomes. However, during myocardial hypoxia, insulin alone cannot restore the associated depression in protein synthesis. Although insulin hyperpolarizes the cell, a change in the ratio of intracellular to extracellular activities of potassium is not its primary mode of action. An insulin-induced configurational change in the plasma membrane could simultaneously account for the effects of insulin on sodium and potassium permeability and the action on facilitated transport. Intracellular levels of cyclic adenylate may be reduced by insulin in adipose tissue because of inhibition of adenyl cyclase or stimulation of phosphodiesterase. However, at this time there is little evidence that insulin alters cyclic AMP levels in the heart. Insulin secretion is depressed in patients with heart disease in proportion to the reduction of cardiac index sustained. Since the ischemic heart is dependent upon glucose as the major fuel, insulin lack may deprive the heart of adequate substrate.


Differential beta adrenergic sensitivity of atrial and ventricular tissue assessed by chronotropic, inotropic, and cyclic AMP responses to isoprenaline and dobutamine. Cat atrial tissue was more sensitive to isoprenaline (USP:isoproterenol) and dobutamine than ventricular tissue. Chronotropic response of atrial strips occurred at lower agonist concentrations than inotropic responses of papillary muscle. Cyclic AMP responses in atrial slices also occurred at lower agonist concentrations and were of much greater magnitude than the cyclic AMP responses in ventricular slices. However, the disporportionately greater sensitivity of atrial tissue was more marked with isoprenaline than with dobutamine.


On the fine structure of the small, heavily pigmented non-pyramidal cells in lamina II and upper lamina III of the human isocortex. With the aid of a newly developed technique for the successive examination of both the Golgi and pigment picture of individual neurons (Braak, 1974a) Braak (1974b) demonstrated that within lamina II and upper lamina III of the human isocortex, heavily pigmented non-pyramidal cells are distributed irregularly and sparsely. The lipofuscin pigment granules serve as excellent internal markers to identify these non-pyramidal cells in ultrathin sections. This favourable circumstance facilitates the study of these interneurons in the electron microscope. The heavily pigmented non-pyramidal cells are small, spherical to avoid with diameters of about 12-15 mum. One pole of the cell comprising a large cytoplasmic area gives rise to a few dendrites, while the other pole is occupied by the nucleus and in some cases is in close apposition to another nerve cell body. The nucleus is deeply invaginated by the large cytoplasmic area and occasionally displays nuclear inclusions. Among the usual organelles distributed within the large cytoplasmic area the mitochondria with a moderately electron dense matrix are abundant and the coarse lipofuscin pigment granules are the most striking elements. The latter contain densely packed filamentous or tubular material and a single vacuole. The perikaryon rarely receives more than 3 type I and type tii synapses per section per cell, whereas the dendrites receive numerous synapses of both type I and type II. Within the apposition zone to another nerve cell body (which in no case is a heavily pigmented non-pyramidal cell) puncta adhaerentia occur and also contacts in which the cleft of 8 nm is intersected by a dense stratum. Some of the ultrastructural findings are summarized in the schematic drawing of Figure 15.


Cell differentiation of the fetal rat anterior pituitary in vitro. Adenohypophysial primordia of rat embryos at 13 to 15 days gestation were cultured in Parker 199 synthetic medium for 2 to 11 days. At the end of the culture period their fine structure and the presence of immunoreactive trophic hormones using the peroxidase-labeled antibody technique were investigated. The degree of differentiation in the glands depends largely on the age of the embryos furnishing the explants. Cultured pituitaries explanted on the 13th day of gestation contain only ACTH-positive cells and about 15% of the cells are granular. The granules are 50-100 nm in diameter in some cells, while in other cells they range from 50 to 200 nm. In cultivated adenohypophysial primordia of embryos on the 15th day of intrauterine life ACTH, prolactin, LH and TSH cells are evident, but only the same two kinds of granular cells can be observed with the electron microscope. The extent of cytodifferentiation in the glands explanted on the 14th day of gestation is intermediate between the two other groups. The data suggest that the fetal rat pituitary has the capacity of self-differentiation but to a lesser extent than that of the in situ hypophysis.


Multinucleate cells in thymus of C3H mice. Multinucleate epithelial cells occur in the thymus of C3H mice. They are poorly differentiated and scarce, but are more numerous in the medulla than in the cortex. Their increase in number with age is particularly significant between the first and the third months especially for cells with a large number of nuclei, and may be related to thymic involution. Viral particles of type C, similar to those described in murine leukemias, are found in mono- and multinucleate medullary epithelial cells.


The influence of social rank on adenohypophysial cell activity in Salmo irideus. With special reference to basophil ACTH-cells of the proximal pars distalis. In nine cell types of the adenohypophysis in untreated adult rainbow trout, histologically different activity phases, seasonal changes in activity, and the relation between certain cell types and the interrenal gland, thyroid or gonads were investigated by light and, occasionally, by electron microscopy. Special attention was given to the effect of social rank on the synthetic activity in adenohypophysial cells of trout kept in small groups in which a social hierarchy with one (light) dominant and several (dark) submissives is established. Cell types in the rostral pars distalis were azocarminophil (I) or amphiphil (II). Proximal pars distalis cell types were slightly basophil (IV), orangeophil (V), strongly basophil (VI) or chromophobe (VII). In the pars intermedia, cell types were amphiphil (VIII) or very slightly basophil (IX). Type III was a non-secretory supporting (?) cell. Histologically different activity phases abounded in type IV cells, which mainly occurred in the proximal pars distalis but were also found dispersed in the rostral pars distalis, the pars intermedia and the neurohypophysis. Influences of social rank were pronounced in type IV cells. Phases with a high synthetic activity were exclusively found in submissive animals, phases with a low synthetic activity occurred in dominants. As a positive relation existed between type IV cell activity and the social rank dependent activity of the interrenal gland, it was suggested that type IV cells produce ACTH. In (dominant) male trout treated with DOCG or ACTH, colloid-containing type IV cell phases, reflecting accumulation of the secretory product, were found. This supported the earlier suggestion that ACTH in the trout is produced in the basophil type IV cells and not, as reported in the literature, in cells comparable to type II.


Temperature-sensitive mutants of Balb/3T3 cells: description of a mutant affected in cellular and polyoma virus DNA synthesis. A temperature-sensitive Dna- mutant (ts-2) of the mouse cell Balb/3T3 is characterized. Studies with synchronized cells indicate that the defect is in DNA synthesis itself, rather than in progress toward its initiation. ts-2 supports polyoma DNA synthesis after infection at 33degreesC but not at 38degreesC. Viral DNA synthesis begun at 33degreesC is inhibited upon shift to 38degreesC. A procedure is proposed by which viral DNA synthesis can be used to distinguish different classes of cell Dna- mutants.


A quantitative study of ultramicroinjection of macromolecules into animal cells. Improvements in the technique of ultramicroinjection of macromolecules into animal cells are described. The method is based on the Sendai virus-induced fusion of animal cells with erythrocyte ghosts containing trapped macromolecules. Fusion of hepatoma tissue culture (HTC) cells with ghosts prepared by hemolysis of erythrocytes in the presence of cytochrome C is much more efficient than fusion with ghosts prepared in the presence of bovine serum albumin (BSA) as in previous investigations. La+++ is more fficient in promoting fusion and less toxic to cells than Mn++, which was used previously. Thus in all subsequent experiments, erythrocytes were hemolyzed in the presence of cytochrome C plus other macromolecules to be trapped, and the resultant ghosts fused in the presence of La+++. The percentage of HTC cells which fused with ghosts reached 80% in many experiments. Ghosts containing 125I-BSA were used to measure the number of BSA molecules injected into HTC cells. About 10(6) BSA molecules were injected per fused cell. The overall efficiency of injection was low (about 0.02% of the starting material).


Preferential synthesis of viral late RNA by nuclei isolated from SV40 lytically infected cells. Nuclei from SV40-infected monkey cells were isolated late in lytic infection and their cell-free transcriptional activity was characterized. 3H-RNA synthesized in vitro was hybridized to excess quantities of separated SV40 DNA strands which were each covalently bound to Sepharose. It was found that 3-5% of the newly synthesized RNA is virus-specific and that the plus-strand DNA, coding for late RNA sequences, is transcribed at a rate about 15 times higher than that of the minus-strand DNA, which codes for early RNA sequences. This indicates that transcriptional control has a major role in determining the relative abundancy of early and late RNA classes in lytically infected cells.


Mapping of poly(A) sequences in the electron microscope reveals unusual structure of type C oncornavirus RNA molecules. We have synthesized a convenient electron microscope label for mapping poly(A) sequences. Short lengths of poly(dT) are polymerized onto nicked circular SV40 DNA with the enzyme terminal deoxynucleotidyl transferase. An RNA or DNA molecule of interest is treated with glyoxal, hybridized briefly with the poly(dT) circles, and spread for microscopy; poly(A) stretches are clearly marked because they are attached to the poly(dT) on the easily recognized SV40 duplex circles. The RNAs of several type C oncornaviruses were examined by this method. The endogenous feline virus(RD-114), the endogenous baboon virus (BKD), and the woolly monkey sarcoma virus (WoMV) all contain a dimer of RNA subunits held together in a central secondary structure feature we call the dimer linkage structure. Both ends distal to the dimer linkage structure hybridize to the SV40-poly(dT). Assuming both poly(A)s are on the 3' ends of the subunits and that both subunits are identical, the two identical subunits are held together by interactions between sequences close to the 5' ends.


RD-114, baboon, and woolly monkey viral RNA's compared in size and structure. The molecular weights, subunit compositions, and secondary structure patterns of the RNAs from an endogenous baboon virus and from a woolly monkey sarcoma virus were examined and compared to the properties of the RNA of RD-114, an endogenous feline virus. The high molecular weight RNA extracted from each of these three viruses has a sedimentation coefficient of 52S, and a molecular length, measured by electron microscopy, of 16-20 kb (kb=kilobase, 1000 nucleotides). Each such RNA is a dimer, containing two monomer subunits of 8-10 kb in length (molecular weight 3 X 10(6) daltons). The two monomer subunits are joined at their non-poly(A) ends in a structure called the dimer linkage structure. The appearance of this structure is somewhat different for the different viruses. The dimer linkage dissociates at temperature estimated to be 87 degrees C in aqueous 0.1M Na+ for RD-114 and baboon viral RNAs, but at the lower temperature of 66 degrees C for woolly monkey RNA. All three viral RNAs have two large loops of similar size and position symmetrically placed on either side of the dimer linkage structure. Since the baboon virus is partially related to RD-114, and the woolly monkey virus is unrelated to either of the other two, the dimer linkage and symmetrical loops are surprisingly similar and may well be common features of type C virus RNAs.


Cloned mouse mammary cell lines requiring androgens for growth in culture. Clonally derived cell lines have been established from the mouse mammary tumor Shionogi carcinoma 115. Physiological concentrations of androgens markedly increase the growth rate and saturation density of these clones. The cells fail to grow in 1% fetal bovine serum-supplemented medium, but addition of 10-50 nM androstanolone or testosterone results in resumption of exponential growth. Some other androgens, but not estrogens or progesterone, stimulate growth of these cells. Steroid receptors can be isolated from the cells. In addition to their effect on growth, androgens also cause a marked alteration in the cell and colony morphology of the clones.


High frequency variation in mammary tumor virus expression in cell culture. Clonal derivatives of C3HMT murine mammary cell lines in culture demonstrate conversion of mammary tumor virus (MMTV) expression at a rate of appriximately 6 per 100 clones. This alteration is largely unidirectional from a relatively high level (MMTV(H)) to a 10 fold lower level (MMTV(L)). This high rate of MMTV(L) variant conversion is in apparent contrast to the presumably mutational rate (approximately 3 per million cells) that governs development of resistance to 6-thioguanine (TG) in the same mammary cells. In somatic cell hybrids between different MMTV TGr clones and mouse or hamster TK- cells, expression of constitutive levels of MMTV and responsiveness to dexamethasone induction is dominant. Thus MMTV expression is regulated by at least two levels of positive control, constitutive expression and glucocorticoid stimulation, but the former is subject to a high rate of variant formation.


The chick chorioallantoic membrane as a model system for the study of tissue invasion by viral transformed cells. The chick chorioallantoic membrane (CAM) was used as an assay system to investigate the the invasive properties of viral transformed NIH/3Y3 cells. Scanning electron microscopy demonstrated that single Kirsten sarcoma virus (KiSV)-transformed cells passed between the epithelial cells of the  CAM ectoderm within 6 hr of application, while viable NIH/3T3 cells did not penetrate the ectoderm within 24 hr. The transformed cells entered the mesoderm of the CAM and formed tumors of proliferating cells. The application of 5 X 10(5) KiSV-transformed cells resulted in the formation of donor cells resulted in the formation of the donor cell tumors within 5 days in 43% of the membranes. No tumors were formed when as many as 5 X 10(6) NIH/3T3 cells were applied to the membrane. NIH/3T3 cells transformed by the Abelson leukemia virus or the Moloney sarcoma virus also ivaded the CAM and formed tumors of proliferating cells within the mesoderm, while cells infected with the Moloney leukemia virus did not. NIH/3T3 cells inoculated onto the CAM 8 days after infection and transformation with KiSV formed tumors with a frequency similar to that of KiSV transformed cells that have been passaged in culture for many generations. Cells that formed invasive tumors within the mesoderm also attracted loops of host blood vessels.


The selection of virus-resistant Chinese hamster ovary cells. We have selected Chinese hamster ovary (CHO) cells resistant to infection by encephalomycarditis (EMC) virus. Thus far, we have obtained five lines resistant to EMC, all of which manifest different phenotypes. Three of the five are not persistently infected with virus, while two lines produce infectious virus and grow in its presence. The nonpersistently infected lines exhibit different resistance profiles to the other viruses we have tested, and they are stable in nonselective growth conditions. Their resistance appears to be due to a genetic alteration in the cell.


Identification of regions of the SV40 genome which contain preferred SV40 T antigen-binding sites. SV40 T antigen binds to SV40 DNA. Using a series of purified SV40 DNA restriction fragments, we have obtained evidence indicating that the antigen preferentially binds to three specific regions. These binding regions are contained within Endo R-Hin d(II + III) A, B, and C.


Isolation and characterization of Sendai virus DI-RNAs. When passaged at high multiplicity, four strains of Sendai virus all showed evidence that they contained defective interfering (DI) particles. RNA isolated from nucleocapsids of cells infected with the high multiplicity passage stocks was found to consist of only minor amounts of nondefective genome length RNA and major amounts of smaller RNAs, the DI-RNAs. These DI-RNAs were found to have unusual and variable sedimentation properties in sucrose gradients, but were found to represent unique segments of the viral genome by length measurements in the electron microscope and by hybridization. A striking feature of the DI-RNAs is their ability to form circular structures, indicating that the ends of the DI-RNA are complementary. The implications of this finding in terms of the mechanism of genome replication is discussed.


A significant lag in the induction of ovalbumin messenger RNA by steroid hormones: a receptor translocation hypothesis. Although ovalbumin and conalbumin mRNA accumulate in the same tubular gland cells of the chick oviduct in response to estrogen or progesterone treatment, the kinetics of induction are markedly different. Conalbumin mRNA begins to accumulate within 30 min after estrogen administration, whereas there is a lag of approximately 3 hr before ovalbumin mRNA begins to accumulate, as measured by three independent assays. The kinetics of estrogen-receptor binding to chromatin indicate that these sites are saturated within 15 min of estrogen administration to the chicks, demonstrating that the lag is not due to slow uptake of the steroid. Suboptimal doses of estrogen produce the same lag, but the resultant rate of ovalbumin mRNA accumulation is lower than with an optimal dose. Partial induction of ovalbumin mRNA by a low dose of estrogen does not shorten the lag with an optimal dose. With progesteone, there is a lag of about 2 hr before either ovalbumin or conalbumin mRNA begins to accumulate. Treatment of chicks with hydroxyurea shortens the lag for ovalbumin induction with either hormone. Inhibition of protein synthesis with emetine does not prevent the accumulation of either ovalbumin or conalbumin mRNA. With cycloheximide, however, ovalbumin mRNA accumulation can be prevented. The existence of a lag suggests that there are intermediate steps between the binding of steroid receptors to chromatin and the induction of ovalbumin mRNA. There are basically two models to explain these delays in response: one involving the accumulation of an essential intermediate, and the other involving a rate-limiting translocation of steroid receptors from initial nonproductive chromatin-binding sites to productive sites. Several aspects of the kinetics of ovalbumin mRNA induction are more consistent with the latter model.


[Histiocytic medullary reticulosis and herpetic necrotizing inclusion hepatitis]. A male aged 47 suffered from the syndrome of histiocytic medullary reticulosis terminating lethally within one year. Because of the finding of a necrotizing inclusion hepatitis of the herpetic type at autopsy, intranuclear inclusions were retrospectively searched for in the tumour tissue. Inclusions were found, on the one hand, in a bioptic sample obained from a lymph node at the beginning of the disease and, on the other hand, in lympatic tissue and bone marrow obtained at autopsy. The relation of histiocytic medullary reticulosis to the herpetic infection has been discussed.


[Malignant nesidioma with 2 types of secretory granules in tumor cells]. A case has been described of a male aged 38 with a malignant nesidioma. The clinical examination disclosed marked hepatomegaly, variable hypertension associated with cool sweating, agitation and transient hyperglycaemia. The primary tumour as well as the metastases in the liver weighing over 13 kg were histologically of trabecular structure and stained positively with the methods of Bodian and grimelius. The impregnation for D-cells according to Rohoznická and Vortel was positive in isolated cells only. Ultrastructurally, the tumor cells could be divided in two types. The more numerous Type I cells contained rather small secretory granules morphologically resembling A granules of islet cells, and staining positively with impregnation methods for A-cells on the ultrastructural level. Type II cells contained, in addition to the formerly described ones, another type of granules appearing as large, slightly osmiophilic ones characteristic of the D-cells of Langerhan's islets and positive with the impregnation methods for D-cells. The type II tumour cells must be therefore regarded as a mixed cell type containing two types of secretory granules.