Stabilizing effect of magnesium chloride and sucrose on Sabin live polio vaccine.

A trivalent vaccine was stabilized with (a) 70% sucrose, (b) MgCl2 1M, and (c) 35% sucrose plus MgCl2 1/2M. A portion of each batch was kept at +4 degrees C and 22-25 degrees C. No change in titre for all 3 preparations was recorded after 9 weeks storage at +4 degrees C. While the potency of vaccines containing MgCl2 alone or mixed with sucrose and kept at +25-28 degrees C for 5 weeks was not altered, the vaccine containing only sucrose was less stable, and a drop of titre was noticed after 2 weeks of storage at +22-25 degrees C. Monovalent polio vaccines were also stabilized as above and kept at +4 degrees C or at -20 degrees C. It was found that regardless of the type of stabilizer used, 82 to 97 per cent of potency was retained after 9 months of storage at +4 degrees C or at -20 degrees C.


Smallpox eradication: progress and problems.

In 1958, the Eleventh World Health Assembly, on the proposal of the USSR, approved a resolution for world-wide smallpox eradication. In that year alone the disease occurred in 59 countries, and in addition many other areas experienced imported cases. When the intensified eradication programme began in 1967, there were 33 countries with endemic smallpox and two more countries subsequently became endemic. These 33 countries had a population of over 1,200,000,000 and were located in four of the six WHO regions. The most important endemic area was in Asia, but the disease was also endemic in South America and in Africa, south of the Sahara. The intensified campaign quickly narrowed the endemic area. In April 1971, the last case was reported from Brazil, the stronghold of the disease in the Western Hemisphere. By 1973, with the exception of those in the Horn of Africa, smallpox transmission was interrupted in all African countries. The last case in Asia occurred in Bangladesh where variola major made its last stand. The last known case of smallpox in the world was reported from Somalia on 27 October 1977. If no more cases are discovered it will be possible, in two years from that date, to certify that smallpox has been eradicated from all areas of the world, and Member States of the World Health Organization will be able to celebrate an unprecedented victory for preventive medicine. The complete eradication of smallpox is not only the liberation of the world from one of its most dangerous diseases, but also provides an example of what can be achieved when countries throughout the world join together with a common aim.


[A broad program of vaccination in the Ivory Coast].

This paper is a description of a vaccination programme against the most frequent communicable diseases among children: measles, tuberculosis, diphtheria, tetanus, whooping-cough and poliomyelitis. The programme is to be carried out in three different climate zones (town, forest and savanna) and over a period of six years. The project is supported by US-AID and UNICEF.


Monolayer culture of animal cells with the gyrogen equipped with tubes.

The gyrogen with tubes has been the object of repeated trials carried out over a period of some years. The aim of these trials was essentially to multiply BHK cells in monolayer and to reproduce the FMD virus in one operation. At the laboratory stage, the gyrogen consisted of 3 rolling flasks; at the pilot stage, it equalled 100 rolling flasks; and at the industrial stage which will be tested in the coming months, it should equal 500 flasks. The culture of cells on a glass support was not in itself new; the difficulty appeared when replicating the virus in order to obtain the high parameters necessary for the production of vaccine. Everything was done so that finally cell culture and viral multiplication could be carried out in situ in the same apparatus.


Cell culture on beads used for the industrial production of foot-and-mouth disease virus.

The microcarrier culture technique has been applied to a pig kidney cell line. Microcarriers consisted of DEAE Sephadex A 50 beads washed and containing carboxymethyl cellulose. Sifted beads gave better results than unclassified material. Cells for inoculum were prepared in Roux flasks. The two types of fermentors which were used (operating capacity 100 l and 150 l) gave similar results. The growth of the cells can easily be followed by microscopic observation and cell count. The yields of cells per volume of spent medium were three times higher on microcarrier than in Roux bottles; the average doubling time was not modified. Foot-and-mouth disease virus can be produced with cells grown on beads. Vaccines prepared with these viruses induced good protection for pigs.


Multiplication of normal and virus-infected diploid cells cultivated on microcarriers.

In prelminary studies, growth of diploid cells (fetal calf buccal cells and chick embryo fibroblasts on dextran microcarriers was studied and compared to growth in monolayer. Growth coefficients obtained were always greater (by 2 fold) when microcarriers were used. Moreover, this technique had the advantage of reducing the number of manipulations and hence the risk of contamination. In two series of experiments, buccal epithelial cells were infected with vescicular stomatitis virus. Good virus multiplication was obtained.


Alterations of membrane glycoproteins in malignant cells.

Comparisons have been made of the carbohydrates bound to membrane proteins of control and malignant cells in culture. It has been found that most individual glycoproteins of control and malignant cells differ in their carbohydrate components. Differences between control and malignant cells have been foundin the glycoproteins of the various membrane systems (surface, nucleus, mitochondria, endoplasmic reticulum). It is suggested that the extensive changes in bound carbohydrates of the malignant cell permit the cell to live and divide but isolate the cell functionally. The extensive structural changes may also be responsible for the cascading of involvement of various processes within the malignant cell.


A human neoplastic hematopoietic cell line producing a fibroblast type of interferon.

1301 is a subline of the EBV-genome negative T leukemia line CCRF-CEM. IF was induced by Sendai virus. By use of neutralizing antisera and by studies of some physical properties it could be shown that IF was of the fibroblast type. This was confirmed by its antiviral effect on heterologous cell lines.


The use of frozen BHK21 C13 cells to control the biological parameters for cell and foot-and-mouth disease virus growth.

In order to control the four primary variables (cells, serum, medium and physical conditions) in a cell or virus growth system it is important to have a reliable and constant positive control with which to compare any variable of the system components. The use of aliquots of a frozen cell population (stored at -136 degrees C) for this purpose is the subject of this paper. Using such cells, tests have been established for the control of sera, media, cell susceptibility and the quality of serum treated DEAE Sephadex A50 beads.


Large-scale concentration and purification of virus suspension from microcarrier culture for the preparation of inactivated virus vaccines.

Because of the presence of bovine serum proteins in poliovirus and rabiesvirus suspensions harvested from microcarrier cultures, a concentration and purification process has been developed in order to remove these proteins. The process comprises clarification by filtration, concentration by ultrafiltration, gel filtration on Sepharose 6B and column chromatography on DEAE-Sephadex A50. The serum protein content of the virus suspensions if reduced to a serum dilution of less than 1: 10(6) at a virus antigen recovery rate of 70 to 80%.


Some aspects of FMDV-production in growing cells and a closed system for concentration of FMDV by polyethylene glycol.

Different commercially available peptone preparations, all derived by enzymatic digestion of meat, were tested for their ability to replace the individual amino acids and polyethylene glycol (PEG)-treated serum in BHK suspended cell culture growth medium. Cell growth was not impaired if the individual amino acids were replaced by 3 g/l of peptone in combination with 3 g/l LAH and 5% PEG-treated bovine serum. The concentration of the latter could be reduced to 1% by gradually lowering the concentration in successive cell passages. No significant differences in growth stimulating properties were observed between the peptone preparations. The virus produced was concentrated and partly purified by precipitation with PEG, collection of the precipitate by filter-aid filtration and direct elution from filter cake in the filter by pumping one tenth the original volume of buffer through the filter. The whole procedure was performed in a simple closed system, offering maximum guarantee of bacteriological sterility and preventing virus aerosol formation in the surroundings.


Purification of poliovirus obtained from human diploid cells by means of DEAE-Sepharose.

Mahoney, MEF and Saukett poliovirus strains were grown on human diploid cells (MRC 5) and concentrated on Amicon filter. Concentrated viral suspensions containing 3H-uridine labelled virus were mixed with 14C-amino acid labelled cell extracts containing calf serum and passed through a DEAE-Sepharose column. Fractions eluted at various ionic strengths were analyzed for infectivity, radio-activity and serum content by counter immunoelectrophoresis in the presence of a rabbit anti-calf serum. A peak containing 40--60% of the input infectivity was easily obtained by elution with 0.01 M phosphate buffer at pH 7. The virus peak did not contain 14C or calf serum and its purity was controlled by PAGE-SDS electrophoresis and electron microscopy. This technique may be useful in the large-scale purification of viruses used in the preparation of inactivated vaccines.


Construction of hybrid viruses in vitro and their possible use for the production of specific viral or cellular proteins.

The use of plasmid and bacteriophage vectors for cloning of DNA segments from procaryotes and eucaryotes is described. A similar approach is being developed with DNA viruses like simian virus 40 or adenovirus 2. Such hybrid viruses may permit the amplification of specific genes and the overproduction of the corresponding proteins.


[The surgical treatment of Klippel-Trenaunay syndrome (author's transl)].

Three cases of congenital angiodysplasies of the extremities, classified as Klippel-Trenaunay syndrome, underwent to surgical treatment. These cases, clinically similar, have shown a quite different pathogenesis, according to which the surgical treatment has been modified. Once again we recommend a complete diagnostic study every time one find varicosities of the extremities which are atipical for time of appearance, territories involved or for aspect and thus be congenital varicosities with complex pathogenesis.


Virus particles in normal prostate tissues of mice from ten strains, with special reference to type-B virus particles.

Prostate tissues of a total of 61 normal mice from 10 different strains, including high (C3H/Dm, RIII/Dm, and A/Dm) and low (BALB/c/Dm, C3Hf/Bi/Dm, and C3Hf/He/TEX) mammary cancer, and high (AKR/Dm) and low (CBA/J/Cr, SJL/J/Cr, and C57/BL/6/TEX) leukemia strains were examined by electron microscopy for the presence of virus particles. These studies demonstrated that type-B virus particles were present in normal prostate tissues of some old mice from all the three high (C3H/Dm, RIII/Dm, and A/Dm) and one low (BALB/c/Dm) mammary cancer strains. They further demonstrated that varying number of type-C virus particles were present in prostate tissues of some young and old mice, including those from all the 10 strains, especially in a larger number in older mice, and that intracisternal type-A virus particles were observed in all the mice examined. Immunological characterization of type-B virus particles by fixed immunofluorescence tests and immunoelectron microscopy revealed that type-B virus particles in normal prostate tissues of old C3H/Dm and A/Dm mice are morphologically and immunologically similar to the mouse mammary tumor virus. Thus, prostate tissues of mice can be a potential source of horizontally transmitted mammary tumor virus in mice of at least some high mammary cancer strains.


Fusion of XC cells by murine type-C viruses: cinematographic observations.

Continuous observations on the processes of multikaryon formation by purified murine type-C virus, produced by a line of murine myeloma cell cultures and Rauscher leukemia virus, were carried out by cinematography. The process first began by fusion of two mononucleated cells and additional cells continued to introduce their cytoplasm and nucleus one after another to give rise to multinucleated giant cells. These findings strongly suggested that the formation of multikaryons is due to a cellular fusion process and not to endomitosis.


Establishment of cell lines from the wild rodent Millardia meltada and tests for endogenous virus.

Four cell lines were established from the wild rodent, Millardia meltada; an untransformed cell line (MM-D), a non-producer (NP) cell line transformed with murine sarcoma virus (MM-CL4), and SV40-transformed cell lines (MM-8 MM-663). MM-D is epithelioid and contact-inhibited, whereas MM-CL4, MM-8, and MM-663 are fibroblastic and grow piling up. No endogenous virus was induced in Millardia cell lines by a long-term culture with various inducers. Millardia cell DNA has no nucleotide sequence homology with cDNA of either murine leukemia virus or rat endogenous virus.


Immunofluorescence studies for hepatitis A virus and hepatitis B surface and core antigen in liver biopsies from patients with acute viral hepatitis.

Immunofluorescence studies for hepatitis A virus and hepatitis B surface and core antigen were performed on liver biopsies from 48 patients with acute viral hepatitis. Hepatitis A virus was detected in 11 out of 17 patients with type A hepatitis and was not found in patients with type B or non-A non-B hepatitis. Hepatitis B surface and core antigens were detected in 2, hepatitis B core antigen alone in 1, and hepatitis B surface antigen alone in 4 out of 24 patients with type B hepatitis. Neither hepatitis B surface nor core antigen were found in patients with type A or non-A non-B hepatitis. One patient with both type A and B hepatitis were all negative for hepatitis A virus, and hepatitis B surface and core antigens. Immunofluorescence examination for hepatitis A virus in human liver biopsies appears to be a sensitive and specific technique and might be valuable in the search for possible chronic carriers of hepatitis A virus.


Phosphomonoesterases in the male genital system of some mammals.

The various male genital organs of the experimental animals used in this investigation (rat, guinea pig, rabbit, cat and dog) showed a widely different localizations of alkaline and acid phosphatases. Alkaline phosphatase was presented as secretory, stromal, nuclear and vascular, while with acid phosphatase, distinction was made only between secretory and nuclear phosphatases. Although the morphological distributions of both enzymes were sometimes overlapping, they were not identical.


Plasma prednisolone levels and adrenocortical responsiveness after administration of prednisolone-21-phosphate as a retention enema.

Plasma prednisolone levels have been measured by radioimmunoassay after oral and rectal administration to healthy volunteers and to patients with idiopathic proctocolitis. The amount of prednisolone absorbed from a 20 mg retention enema given to patients with proctocolitis was about 44% of that absorbed from the same dose orally administered. Adrenocortical response to synthetic ACTH in patients receiving prolonged rectal therapy was either normal, or only slightly impaired, and this may be related to the pattern of steroid absorption rather than to the total amount absorbed.


Effect of ouabain on Na,K-ATPase and electrolyte transport in the dog ileum in vivo.

It is currently believed that the rate and direction of sodium transport in the small intestine may be regulated by the activity of Na,K-ATPase in the basolateral cell membrane. We tested this hypothesis by selectively infusing ouabain, a known inhibitor of Na,K-ATPase, into the mesenteric artery supplying a perfused loop of ileum in 18 dogs. Before ouabain infusion there were significant correlations between the activity of Na,K-ATPase and net and lumen to plasma fluxes of sodium and chloride. After ouabain, there was no significant change in sodium and chloride transport, unidirectional fluxes or transmucosal potential difference, despite a 50% reduction in Na,K-ATPase activity. Furthermore, there was no significant correlation between Na,K-ATPase activity and sodium or chloride transport after ouabain. The only statistically significant effect of ouabain infusion was a reduction in the rate of bicarbonate secretion. Thus, the results of our experiments suggest that mucosal Na,K-ATPase is not a rate-limiting step in the absorption of sodium and chloride in the dog ileum, though it may be an important facilitative factor.


Turcot's syndrome and its mode of inheritance.

Two sisters with Turcot's syndrome, in which malignant cerebral neoplasms are associated with colonic polyposis, are presented. Cases reported in the literature, including some familial cases, have also been analysed. In familial cases, sex was unrelated to the occurrence of this disease and it was found only among siblings of the same parents and not in other members of the family. There was consanguinity in the parents of the patients in two out of three families. We therefore concluded that the mode of inheritance in this condition is autosomal recessive and that it is genetically distinct from the ordinary form of familial polyposis coli. Support is lent to the absence of an association between the two disorders by a difference in the number, size, and distribution of the colonic polyps found in Turcot's syndrome as compared with familial polyposis coli.


Cyclic AMP and cyclic GMP levels in human colonic mucosa before and during chenodeoxycholic acid therapy.

Previous experimental studies suggest that bile salt-induced colonic fluid secretion is mediated by adenosine 3':5'-phosphate (cyclic AMP). Two biopsy specimens of colonic mucosa were obtained endoscopically before and after different periods of therapy (five, 10, or 15 days), from each of 21 patients receiving chenodeoxycholic acid. A rise of cyclic AMP intracellular levels was found, but only after five and 10 days of treatment was the increase statistically significant when compared with basal levels. Similar changes were observed for guanosine 3':5'-phosphate (cyclic GMP), but percentage increases were higher than for cyclic AMP. Initial diarrhoea disappeared spontaneously, and at 15 days the levels of both cyclic nucleotides were not significantly different from basal levels. Our findings suggest that colonic adaptation to increase in luminal bile salt levels is related to changes in intracellular levels of cyclic nucleotides and support the hypothesis that not only cyclic AMP, but also cyclic GMP may play an important role in producing bile salt-induced diarrhoea in man.


Relaxin and collagen metabolism.

The influence of the peptide hormone relaxin on collagen metabolism was studied in the symphysis pubis of the mouse. In the tissue the content of water and of acid soluble collagen in relation to total collagen is increased by hormonal treatment. Total collagen calculated in relation to the dry weight is decreased. Collagenase which was also detected in the symphyses of the controls is slightly enhanced. In serum collagen peptidase and collagen peptidase inhibitor as well as cyclic AMP exhibit distinctly increased levels. The effects can be suppressed by administration of relaxin-specific antisera. The data make clear that in the symphysis relaxin activates the collagenolytic system.


Surface localization of apolipoprotein AII in lipoprotein-complexes.

Lipoprotein particles reconstituted from the apolipoprotein AII (apo AII) component of human serum high density lipoprotein, phosphatidylcholine and lysophosphatidylcholine were covalently linked to the imidoester groups of a polystyrene residue. Apo AII was proteolytically digested with thermolysin after delipidation. The covalently bound peptides remaining at the resin were cleaved and separated by combined two-dimensional electrophoresis/chromatography. The peptides were isolated, hydrolyzed and their amino acid composition determined. They were assigned to the apo AII sequence. Since the imidoester groups on the surface of the resin carrier cannot react with buried lysine residues, this method gives strong chemical evidence for the spreading of the apo AII polypeptide chain over the surface of the lipoprotein particle, as far as the sequence carrying lysine residues between residue 22 and 55 of each symmetrical half is concerned.


Conformational analysis of serum apolipoprotein AII in lipoprotein complexes with bifunctional crosslinking reagents.

Apolipoprotein AII isolated from human serum high density lipoproteins was recombined with phosphatidylcholine to yield homogeneous particles of 80-120 A diameter. The radioactive bifunctional crosslinkers dimethyl [1,1'-14C]suberimidate and dimethyl 4,4'-dithiobis([1-14C]butyrimidate) were reacted with these particles. The kinetics of the reactions and the localisation of the crosslinked lysines of the polypeptide chains were determined. Thermolysin hydrolysis followed by two-dimensional separation of the peptides and isolation of the mono- and bifunctionally modified peptides allowed the assignment of the crosslinked peptides of the apoprotein AII seqeunce. The crosslinking pattern indicates a close-neighbour relationship (13-15 A) of the peptide chains between amino acid residues 3, 23, 46 and 55 of the symmetrical halves of the apo AII molecule. A reconstruction of the secondary structure of Apo AII in the lipoprotein complex on the basis of theoretical calculations is given and correlated with the chemical data.


Hepatoblastoma with adenomatoid renal epithelium.

A case is described in which a hepatoblastoma is associated with a renal malformation where adenomatoid epithelium lines Bowman's capsule and the adjacent part of the proximal tubule. The significance of this is discussed in relation to the association of developmental malformations with neoplasia.


Deinstitutionalization in the absence of consensus.

The process of deinstitutionalization began almost unnoticed in 1955 as state hospital populations started to decline, and it proceeded without adequate planning and without development of a social consensus. The inevitable result was strong criticism, severe personal dislocations, and, with rare exceptions, programmatic chaos. The authors trace and describe the reasons for the growing polarization about deinstitutionalization among such groups as mental health professionals, public officials, families, advocacy groups, citizens, and unions. They also note that between 1950 and 1970 the total institutionalized population in the U.S. was not reduced but simply shifted. Deinstitutionalization should focus not on the location of care but on the broader problem of improving the lot of persons with chronic illness, regardless of its cause or time of onset, the authors suggest. They outline the basic elements of a service and financing system to meet both the daily-living and the specifically medical needs of the chronically ill.


Does insulin dependent diabetes mellitus have a viral etiology?

Clinical, epidemiological, and experimental observations suggest that common viruses may play an etiologic role in at least some cases of insulin dependent diabetes mellitus. The evidence is summarized and critically assessed in this presentation.


Malignant soft tissue tumors (malignant fibrous histiocytoma, pleomorphic liposarcoma, and pleomorphic rhabdomyosarcoma): an electron microscopic study.

A comparative ultrastructural analysis of malignant soft tissue tumors (malignant fibrous histiocytoma, pleomorphic liposarcoma, and pleomorphic rhabdomyosarcoma) revealed similar ultrastructural features in this group of tumors. However, by electron microscopy these tumors can be differentiated on the basis of cytoplasmic and extracytoplasmic features (myosin filaments, lipid droplets, and perinuclear intermediate filaments, for example). This is even true of less well differentiated tumors and tumor cells. These findings support and amplify the concept of a common histogenesis for tumors of mesenchymal origin. Paradoxical features observed by light microscopy warrant further study by electron microscopy if the correct diagnosis is to be made in atypical cases, such as apparent malignant fibrous histiocytoma with cross striations.


Does the Z gene variant of alpha-1-antitrypsin predispose to hepatic carcinoma?

In 10 of 56 patients with primary liver carcinoma the nontumorous hepatocytes contained diastase resistant, periodic acid-Schiff positive and alpha-1-antitrypsin positive (immunoperoxidase technique) globules. This is a frequency of 18 per cent among patients with liver carcinoma against 6 per cent in an unselected autopsy series. The tumors were of the hepatocellular or mixed type in nine of the 10 patients, and the frequency among such patients was 23 per cent. We consider that these globules indicate a carrier of the protease inhibitor allele of the Z gene variant (single or double).


Budd-Chiari syndrome caused by undifferentiated small round cell tumor of the liver in an adult.

An undifferentiated small round cell tumor of the liver in a 60 year old black male is described. Although the light microscopic findings in this tumor were strongly suggestive of embryonal rhabdomyosarcoma, the ultrastructural study disclosed no myofibrils or other specific characteristics. Thus, we classify this tumor as an embryonic tumor or primitive mesenchymal tumor of the liver.


Triplex gene dosage effect of TPI and G3PD in a human lymphoblastoid cell line with partial trisomy 12p13 and 18p.

The EBV-induced lymphoblastoid line established from a patient carrying a duplication of the distal part of chromosome 12 short arm (12p13) retained the original partial trisomy and displayed the same triplex gene dosage effect for TPI and G3PD as found in the patient's RBC and WBC.


Poliovirus-induced suppression of lymphocyte stimulation: a macrophage-mediated effect.

Poliovirus was shown to suppress the in vitro response of human mononuclear blood cells stimulated with phytohaemagglutinin (PHA), pokeweed mitogen (PWM), tuberculin purified protein derivative (PPD) or allogeneic cells. The suppression required infectious virus and the presence of macrophages. Experiments with combined cultures of human lymphocytes stimulated with PHA, PWM, PPD or allogeneic cells, and different numbers of autologous macrophages indicated that both lymphocyte stimulation and the inhibitory effect of poliovirus increased with increasing ratio of macrophages to lymphocytes. The response of human lymphocytes to PHA also was enhanced when autologous macrophages were replaced with murine macrophages. Mouse hepatitis virus inhibited this enhancing effect whereas poliovirus failed to do so. The findings confirm our previous suggestion that poliovirus inhibits stimulation of lymphocytes by suppressing the enhancing effect of macrophages.


The in vitro effects of propranolol and atenolol on neutrophil motility and post-phagocytic metabolic activity.

Propranolol at concentrations of 1 x 10(-6) to 1 x 10(-4) M consistently increased neutrophil motility as measured in Boyden chambers. The effects were not due solely to stimulation of random migration and chemokinesis but also of directional motility. Propranolol, over a similar concentration range, caused inhibition of post-phagocytic cell metabolic activity (hexose monophosphate shunt, nitro-blue tetrazolium reduction and protein iodination) without any detectable effect on the ingestion rate of Candida albicans. Atenolol had no effect on any of these neutrophil functions. Both drugs were without effect on glycolysis and intracellular cyclic AMP levels. Propranolol however, at concentrations which stimulated cell motility, caused increased intracellular cyclic GMP levels. It is suggested that propranolol may stimulate neutrophil motility by promoting increased intracellular cyclic GMP levels or by decreasing neutrophil superoxide production.


Induction of suppressor cells by sodium periodate: the opposite expression of periodate-induced lymphocyte activation in spleen cells of normal and nude mice.

Normal mouse spleen cells treated with sodium periodate for 10 min at 4 degrees are stimulated to undergo blastogenesis and to incorporate thymidine. In contrast, periodate treatment does not stimulate DNA synthesis in nude mice spleen cells. The effect of such treatment on the antibody response in vitro induced by sheep red blood cells (SRBC) and by trinitrophenyl polyacrylamide (TNP-PAA), a T-independent antigen has been evaluated. Periodic-induced proliferation is accompanied by a marked inhibition of the immune response to both of these antigens in normal mouse spleen cells. Mild oxidation of nude cells with periodate markedly enhances their anti-TNP response elicited by TNP-PAA. The data suggest that periodate-induced immune suppression is associated with T lymphocytes. Furthermore, periodate-treated cells are capable of suppressing the in vitro antibody response of untreated normal cells.


Evidence for the role of superoxide radicals in neutrophil-mediated cytotoxicity.

Human peripheral neutrophils became cytotoxic to chicken red blood cells (CRBC) in the presence of lectins as assessed by release of 51chromium from labelled target cells. Phytohaemagglutinin (PHA) and concanavalin A (Con A), which caused time-dependent and dose-dependent cytotoxicity over a concentration range of 25--400 microgram/ml, also caused significant generation of superoxide radicals as measured by ferricytochrome C reduction. Pokeweed mitogen, which does not induce cytotoxicity over the same concentration range, was unable to promote superoxide generation by neutrophils. PHA-induced generation of superoxide paralleled and appeared to precede PHA-dependent cytotoxicity. Superoxide dismutase (SOD), which enzymatically destroys superoxide, caused moderate inhibition of PHA-dependent cytotoxicity over the concentration range of 100--500 microgram/ml whereas catalytically inactive enzyme had no effect. Incubation under oxygen-depleted conditions caused a marked decrease in both PHA-induced superoxide generation and cytotoxicity relative to that obtained with neutrophils incubated aerobically. These findings suggest a central role for superoxide radicals in causing target cell damage in this model of neutrophil-mediated cytotoxicity.


Studies on the mechanism of macrophage aggregation induced by lymphokines.

Macrophage aggregation (measured by a light-scattering technique) induced by unpurified lymphokine preparations has been investigated. This has been shown to be Ca2+ or Mg2+ dependent and to be inhibited by a number of agents known to affect cellular physiology and biochemistry. The divalent cation ionophore A23187 causes a macrophage aggregation superficially similar to that induced by lymphokines, although differences were found. It is suggested that a biochemical response by the macrophage is required for aggregation induced by lymphokines, and thus measurements of macrophage aggregation allow a study of the early events in the response of macrophages to lymphokines.


Molecular approach to the epidemiology of swine vesicular disease: correlation of variation in the virus structural polypeptides with serological properties.

Variation has been observed in the structural polypeptides of swine vesicular disease viruses isolated from the United Kingdom and Hong Kong. Despite the limited number of isolates examined, several distinct polypeptide patterns were obtained when the virus structural proteins were examined by polyacrylamide gel electrophoresis. Isolates from outbreaks in the United Kingdom which were known to be connected gave the same polypeptide pattern, whereas viruses with different polypeptide patterns could not be traced to a common source. The different polypeptide patterns were obtained consistently and were not altered by passage of the virus in tissue culture. In general, isolates with identical polypeptide patterns could not be distinguished by neutralization or antibody blocking tests or by competition radioimmunoassays. However, isolates with different polypeptide patterns could be differentiated by antibody blocking tests or radioimmunoassay. The correlation between the serological tests and the polyacrylamide gel electrophoresis analyses illustrates the value of analyzing structural polypeptides in the epidemiological study of swine vesicular disease.


Leukocyte cytotoxicity in a persistent virus infection: presence of direct cytotoxicity but absence of antibody-dependent cellular cytotoxicity in horses infected with equine infectious anemia virus.

Antibody-dependent cellular cytotoxicity and direct cytotoxicity assays were performed with equine infectious anemia virus-infected target cells, equine leukocytes, and equine anti-equine infectious anemia virus antibody to determine whether these mechanisms play a role in controlling viral replication in equine infectious anemia. Direct cytotoxicity was observed by using peripheral blood mononuclear cells from 7 of 10 infected horses. Antibody-dependent cellular cytotoxicity was not observed. The antibody-dependent cellular cytotoxicity reaction in horses was then studied by using sheep erythrocytes and trinitrophenylated sheep erythrocytes as target cells. Lysis of these target cells was mediated by neutrophils, monocytes, and lymphocytes. The reaction was activated by antibody of the immunoglobulin G class but not by immunoglobulin G(T). Furthermore, immunoglobulin G(T) efficiently inhibited immunoglobulin G in this function.


Effect of viruses on luminol-dependent chemiluminescence of human neutrophils.

The effects of Newcastle disease, herpes simplex, vaccinia, encephalomyocarditis, vesicular stomatitis and reoviruses on in vitro function of neutrophils were studied in Ficoll-Hypaque-separated polymorphonuclear leukocytes (PMN) employing the technique of luminol-dependent chemiluminescence. Newcastle disease, herpes simplex vaccinia, and reoviruses depressed chemiluminescence by 98, 65, 46, and 29%, respectively, while encephalomyocarditis and vesicular stomatitis viruses had no inhibitory effect. None of the viruses affected phagocytosis or PMN viability. These observations suggest significant alteration of neutrophil function by interaction with several viruses in in vitro settings. It is suggested that similar changes in PMN function may occur during in vivo viral infection.


Interaction between 6/94 virus, a parainfluenza type 1 strain, and mouse macrophages.

The 6/94 virus, a type 1 parainfluenza virus recovered from multiple sclerosis brain cells after lysolecithin-induced fusion of these cells with African green monkey kidney cells (CV-1), has been found to grow in splenic and peritoneal macrophages obtained from outbred and different strains of inbred mice. Macrophages from C57BL animals were least susceptible to infection, a resistance apparently partially age related. The virus also has been found to replicate in IC21 cells, a line of simian virus 40 virus-transformed mouse macrophages. Viral growth was detected by development of hemadsorption in infected cells, followed by the appearance of infectious virus. The growth of 6/94 virus had different kinetics in mouse macrophages, in the standard continuous cell lines L, 3T3, and CV-1, and in primary mouse kidney and mouse embryo cells. The virus produced in macrophages could be passed in series to other macrophage cultures. In addition, once infected, the cultures continued to produce virus, and permanently infected cell lines were thus obtained. Macrophages from immunized mice with high titers of humoral neutralizing antibodies were found variably able to support virus growth.


Interaction between 6/94 virus, a parainfluenza type 1 strain, and unstimulated mouse lymphocytes.

6/94 virus was found to grow in unstimulated splenic mouse lymphocytes depleted of monocyte macrophages. Both egg-grown virus and virus produced in mouse macrophage cultures induced the development of hemadsorption and the appearance of new infectious virus in the lymphocyte cultures. An antigenic relative. Sendai virus, on the contrary, was quickly inactivated in these lymphocyte cultures, in agreement with previous reports in the literature. The T lymphocyte population seemed to be the fraction principally involved in the replication of 6/94 virus. The immunization of mice with 6/94 virus and the appearance of high levels of humoral neutralizing antibodies did not inhibit completely the susceptibility of their lymphocytes to the infectious agent.


Interaction between 6/94 virus, a parainfluenza type 1 strain, and human leukocytes.

6/94 virus, a parainfluenza type 1 virus recovered by lysolecithin fusion of multiple sclerosis brain cell cultures with CV-1 cells, replicated in monocyte macrophages and lymphocytes from normal human donors and from a patient with multiple sclerosis. In macrophage cultures, hemadsorption-positive cells and high levels of infectious virus became apparent within 24 to 48 h after infection, persisted for 6 days, and then began to decrease. Phytohemagglutinin-stimulated macrophages yielded similar titers of virus, but the levels were maintained for a longer period of time. Macrophage-produced virus appeared to be infectious for other macrophages in the same culture. Both unstimulated and phytohemagglutinin-stimulated lymphocytes also supported virus replication. Significantly higher titers were produced in the stimulated cultures, T cell-enriched populations producing more virus than unseparated populations whether stimulated or unstimulated. The presence or absence of antibodies to the virus in the donors did not appear to influence the levels of virus obtained in any of the leukocyte cultures. However, an increase in blastic forms after 6/94 virus infection was noted in lymphocytes from donors with antibodies as revealed morphologically and by increased incorporations of tritiated thymidine. Furthermore, 6/94 virus-infected lymphocytes, unlike Sendai virus-infected lymphocytes, were able to respond well to mitogenic stimulation by phytohemagglutinin.


Immunity to foot-and-mouth disease virus in guinea pigs: clinical and immune responses.

Clinical and immune responses were determined for guinea pigs infected with different doses of foot-and-mouth disease virus (FMDV) type A12, strain 119, administered by different routes. Vesicles developed on the tongue or heel pad 1 day after these areas were intradermally inoculated with FMDV. However, vesicles did not develop on the feet and tongue until 3 to 4 days after the intradermal inoculation of FMDV in the flank skin or after intracardiac or subcutaneous inoculation. Infected guinea pigs developed neutralizing antibody, immediate skin reactivity of the Arthus type (4 h), and delayed skin reactivity. In addition to a delayed skin response, the presence of a cell-mediated immune response to FMDV was shown by the specific production of macrophage migration inhibition factor by peritoneal exudate cells in response to FMDV. Kinetic studies showed that neutralizing antibodies were detected at 3 days postinfection, and Arthus and delayed skin reactivity were detected at 4 days postinfection. Some guinea pigs developed either mild or subclinical infections. Regardless of the dose of infectious virus, the route of inoculation, the severity of disease, or the time of clinical onset of disease, infected guinea pigs developed similar immune responses.


Characteristics of infection of B and T lymphocytes from mice after inoculation with cytomegalovirus.

Viremia was produced by inoculating intravenously BALB/c mice with murine cytomegalovirus. Virus was detected in plasma and granulocytes only during the first 8 days after infection. Lymphocyte-associated viremia, detectable by cocultivation on syngeneic or allogeneic fibroblasts, persisted for at least 4 weeks. Eight to 10 days after infection, sonicated lymphocytes had no demonstrable free virus. When whole lymphocytes with no demonstrable free virus were enclosed in a Millipore chamber and placed on a fibroblastic feeder layer, T cells produced free virus but B cells did not. Compared to normal calf serum, specific hyperimmune serum reduced B cell-associated infectious centers by 74% and T cell-associated infectious centers by only 38%. Normal mouse sera reduced by 36% and 30% infectious center production by B cells and T cells, respectively. Lymphocytes enriched with Fc receptor-positive cells produced significantly more infectious centers than receptor-negative cells.


Shigella infection of henle intestinal epithelial cells: role of the host cell.

The process of Henle 407 embryonic intestinal epithelial cell infection by Shigella flexneri 2a M42-43 was studied in an in vitro model system. The role of the Henle cell was assessed. It was established that entry of S. flexneri into cells was suppressed by reagents which inhibit uptake of particles by phagocytic cells. The compounds tested included cytochalasin B, dibutyryl-cyclic adenosine monophosphate, choleragen (Vibrio cholera enterotoxin), iodoacetate, and dinitrophenol. Cytochalasin B inhibited infection at concentrations of 1.0 mug/ml or greater. Dibutyryl-cyclic adenosine monophosphate at concentrations of 1 mM and choleragen at 0.1 mug/ml caused significant suppression of infection. Iodoacetate or dinitrophenol, at 0.1 mM concentrations, inhibited internalization of virulent shigellae, and a combination of these compounds inhibited infection at 0.01 mM concentrations. Preincubation of Henle cell monolayers with the combination of iodoacetate and dinitrophenol (0.05 mM) also inhibited infection markedly. The data suggest that infection of epithelial cells by S. flexneri in vitro is accomplished by an endocytic process induced by virulent bacteria. The process appears to be similar to uptake of particles by phagocytic cells. Ultrastructural analysis by transmission electron microscopy provided corroborative evidence of phagocytosis of shigellae by Henle cells in that intracellular bacteria were often observed within membrane-limiting vacuoles resembling phagosomes.


A rat model for study of bilirubin conjugation by a cultured cell/ artificial capillary liver assist device.

The Gunn rat, congenitally lacking the conjugating enzyme glucuronyl transferase, provides an animal model for the study of bilirubin conjugation by an extracorporeal liver assist device (LAD) composed of cultured cells and artificial capillaries. Rat hepatoma cells of the H4-II-E cell line able to form conjugated bilirubin glucuronide from bilirubin were grown to tissue density by circumfusion culture of the LAD before symbiotic hemoperfusion experiments lasting up to 24 hours. Rat bile was analyzed by thin layer chromatography for bilirubin glucuronide which was formed by the device and then excreted by the rat.


Simultaneous presence of EBNA-positive and colony-forming cells in peripheral blood of patients with infectious mononucleosis.

EBNA-positive lymphoblast cells were detected in 0.1 to 0.9% of the T-cell-depleted lymphocytes obtained from peripheral blood samples of five patients with infectious mononucleosis (IM). The same blood specimens from four of the five patients contained cells that formed EBNA-positive colonies in soft agar containing EBV antibodies. The ratio of the colony formers to EBNA-positive cells was higher in blood samples taken early in the disease than in those obtained in later stages of the disease. The present results strongly suggest that EBV-transformed cells are present in the peripheral circulation of IM patients and that such cells can directly give rise to immortalized cell lines in vitro.


Relationship between the Epstein-Barr virus genome and nasopharyngeal carcinoma in Caucasian patients.

In order to explore whether undifferentiated nasopharyngeal carcinoma (NPC) shows a regular association with Epstein-Barr virus (EBV), regardless of the geographical and ethnic origin of the patient, a correlated histopathological and nucleic acid hybridization study was performed on biopsies from Caucasian patients with nasopharyngeal carcinomas and from various controls. Among 12 undifferentiated NPCs, 11 were positive for EBV-DNA, with multiple copies of the viral genome per cell. Serological tests showed elevated anti-VCA and anti-EA(DA) titers. Six NPCs with various degrees of squamous differentiation, four malignant lymphomas of the nasopharynx and seven carcinomas located outside the nasopharynx were EBV-DNA negative. These findings further stress the uniqueness and regularity of the association between EBV-DNA and undifferentiated NPC. Clearly, the association extends over geographical barriers and holds true not only in the previously studied, moderate-incidence African ethnic group, but also in the low-incidence Western patients.


Macrophage-dependent, NK-cell-independent "natural" surveillance of tumors in syngeneic mice.

The present study, which was designed to further characterize the "natural" T-independent rejection of syngenetic tumours (Greenberg and Greene, 1976), has revealed the following points: (1) no detectable DBA/2 NK cell activity was demonstrated against the syngeneic tumour lines studied, and these tumours were indensitive to NK cells from high-activity strains; (2) in addition the tumour frequencies in old and young mice receiving small tumour inocula were identical, in contrast with the reported decline in NK cell activity with age, suggesting that the surveillance of small inocula of these tumours was NK-cell-independent; (3) injection of silica intraperitoneally enhanced the frequency of tumours in normal and immunodeficient AT x BM mice, suggesting that the rejection mechanism was macrophage-dependent; (4) the effects of silica injection were maximal if administered 3 days prior to tumour injection, indicating that the period of time in which the rejection mechanism must act was very limited; (5) silica markedly decreased the survival of AKR mice dying of spontaneous tumours, providing evidence that the effect of this agent was not limited to model systems but would influence the appearance of spontaneous tumours; (6) reticuloendothelial stimulants such as mycobacterium butyricum and proteose peptone decreased the tumour frequency of small tumour inocula, indicating that the effector mechanism can be stimulated; and (7) soluble tumour antigen enhanced the tumour frequency in normal and immunodeficient mice, suggesting that the specific receptor molecule of the surveillance mechanism was not thymus-dependent.


Effect of interferon on chronic infection of human cells by xenotropic type-C viruses.

Different preparations of human interferon inhibit virus production in human cells chronically infected by a variety of type-C xenotropic viruses. Some of these viruses have been incriminated in the development of leukemia in primates. The characteristics of blocking of viral multiplication are similar to those described for the effect of mouse interferon on ecotropic viruses. The amount of free virus in culture supernatants is strongly decreased while intracellular protein p30 stays unchanged or is slightly increased. On the other hand, the inhibitory effect is reversible. The withdrawal of interferon results in a rapid increase in virus production as detectable in supernatant fluids. In the light of these results it is suggested that human interferon might be useful in the treatment of some blood malignancies suspected of being related to infection with xenotropic type-C viruses.


Enhancement by asbestos of oncogenesis by Moloney murine sarcoma virus in CBA mice.

Five micrograms of finely ground crocidolite asbestos (UICC standard sample) were injected intraperitoneally into 3-week-old CBA mice, together with 10(5) FFU of Moloney murine sarcoma virus. Altogether 44 out of 61 mice (72.1%) so treated developed palpable intraperitoneal tumours, and half of these died of such tumours within 100 days. The same amount of quartz and carbon similarly administered gave lower tumour incidences, namely, 19.4% (3.2% fatal) and 11.9% (1.5% fatal) respectively. Only 1 out of 59 mice inoculated with the virus alone developed a palpable intraperitoneal tumour, and this regressed spontaneously within 10 days of its first appearance. No tumours were encountered in mice treated with either asbestos, quartz or carbon alone. All the neoplasms had the appearance, under the light microscope, of anaplastic sarcomas. Most of them were confined to the serosal surface of the abdominal cavity, although invasion of underlying tissues was observed in some of the animals that died. Electron microscopical examination of tumours revealed the presence of C-type particles budding off from cellular surface. Some neoplastic cells showed characteristic features of mesothelial lining cells. The role of milky spots (taches laiteuses) in oncogenesis by asbestos and virus, especially in the induction of mesothelioma, is discussed.


Therapy of adrenocortical hydroxylase deficiencies in acne vulgaris.

Twenty women with acne vulgaris who presented to a gynecologist's office with the complaints of hirsutism, infertility, menorrhagia, or oligomenorrhea were subjected to a 24-hour cosyntropin (ACTH) infusion to detect partial 11- or 21-adrenocortical hydroxylase deficiencies. All patients demonstrated enzymatic defects and were placed on prednisone therapy. The six patients with moderate acne either showed improvement of their acne or conceived. Of the 14 patients with mild acne, seven showed improvement of their acne or conceived; whereas, the remainder showed no improvement. Patients with acne and gynecologic signs and symptoms should be evaluated for the presence of 11- or 21-adrenocortical hydroxylase deficiencies.


Granular cell tumor of the tongue.

The granular cell tumor has been and still is a debatable lesion in several aspects. The etiology and pathogenesis are still unknown. Granular cell tumors may occur everywhere in the body, but the oral cavity is a favorite location. The majority of the oral lesions are found in the dorsum and the borders of the anterior two-thirds of the tongue. Seven patients with a granular cell tumor of the tongue are described. The clinical and microscopic findings have been discussed. In all cases an excisional biopsy had been aimed at. In spite of incomplete excision in six out of the seven cases, local recurrences were noticed in only one patient. In one patient "metastatic" occurrence of granular cell tumors was noticed.


Follow-up study of 14 oral granular cell tumors.

The granular cell tumor is an uncommon benign lesion with a predilection for the oral cavity. Fourteen tumors from 13 patients have been reviewed with a follow-up period ranging from 7 months to 21 years with an average of 7.1 years. Women were affected twice as often as men, and the age of the patients varied from 14 to 75 years. Eleven of the lesions were located on the tongue, the remaining on the lower lip, sublingual papilla, and the buccal mucosa. Multiple lesions were found in one patient.The tumors varied in size from 3 to 25 mm in diameter and were all firm to hard in consistency. All the lesions were treated by local excision. Pseudoepitheliomatous hyperplasia of the overlying epithelium was seen in four patients. In spite of a possible inadequate removal of 10 of the lesions where granular cells extended to the margin of excision, no recurrence was seen. A simple but deep local excision is the recommended treatment.


Two qualitatively different effects of hyperthermia on acid phosphatase staining in mouse spleen, dependent on the severity of the treatment.

Heating the lower body of the mouse for up to 1.5 hours at temperatures above 41.0 degrees C causes an increase in splenic lysosomal acid phosphatase activity. For mouse temperatures up to 42.3 degrees C the change is probably due to enzyme activation, which reaches a maximum 1.5 hours after heating and then decays in a way which may be related to the transient ability of moderate hyperthermia to potentiate X-ray damage. At temperatures above about 42.5 degrees C hyperthermia results in a qualitatively different lysomal response, probably due to an increased lysosomal membrane permeability. The change is observed immediately after heating and remains high for at least 4 hours. The resultant release of hydrolases into the cytoplasm may be involved in the irreversible cell damage caused by severe hyperthermia.


Suppression of induced beta-galactosidase synthesis by cysteamine and its reversion by gamma-irradiation in the presence of ascorbate.

The induced synthesis of beta-galactosidase in E. coli was found to be inhibited by cysteamine. This inhibitory effect of the SH compound was antagonized by the addition of ascorbate followed by gamma-irradiation with relatively low doses. The cAMP level which, it has been suggested, plays a role in the radioprotective action of cysteamine, is stabilized by ascorbate against changes induced by irradiation.


Enhancement of radiation killing of cultured mammalian cells by cordycepin.

Asynchronous populations of rat hepatoma cells (H4) in log-phase growth survived a 3-hour exposure to cordycepin (3'-deoxyadenosine), and RNA antimetabolite, in a simple exponential fashion with a 'DO' of 43.8 microM/l. When cordycepin-treated cells were exposed to X-irradiation, the resultant survival levels were much lower than one would expect were the agents simply additive. Patterns of X-ray survival of cells treated with cordycepin were dependent on drug concentration, the predominant effect being to decrease the DO of the X-ray survival curve. The increased sensitivity of cells exposed to cordycepin to subsequent X-ray treatment persists for longer than 4 hours after drug administration. Although immediate cordycepin post-treatment of X-irradiated cells is less effective than pre-treatment, the interaction is still significant. Cordycepin treatment did not appear to reduce split-dose recovery or to inhibit the rejoining of single-strand breaks as measured by DNA sedimentation in alkaline-sucrose gradients.


Hydrated electron-initiated main-chain scission in peptides: an e.s.r. and spin-trapping study.

The reactions of hydrated electrons (eaq-) with 19 tripeptides were investigated. Hydrated electrons were produced by gamma-radiolysis of aqueous peptide solutions containing sufficient sodium formate to remove hydroxyl radicals and hydrogen atoms. t-Butanol was also used to scavenge hydroxyl radicals. The short-lived radicals formed by the reactions of eaq- with the peptides were spin-trapped with t-nitrosobutane to form stable nitroxide radicals and identified by e.s.r. spectroscopy. The tripeptides studied contained two glycine residues. Following the addition of eaq- to tripeptides, C-N bond scission was observed at three sites. Cleavage occurred between the nitrogen of the ammonium group and the alpha-carbon and between the nitrogen of the peptide linkage and the adjoining alpha-carbons. The radicals corresponding to each of these three types of scission were identified. From a comparison of the radical yields of the reaction of eaq- with ala, (ala)2, and poly-DL-ananine with an average degree of polymerization of 1800, it was shown that eaq-, can react with many carbonyl groups of poly-DL-alanine, leading to main-chain scission. Analogous reactions of eaq- with proteins and enzymes may be expected to lead to loss of biological activity.


"Triple infections" (fungal, bacterial and viral) in immunosuppressed renal transplant recipients.

In a period of 5 years, 8 out of 77 renal transplant patients showed simultaneous fungal, bacterial and viral infections. Candida albicans was found in all cases. The most severe bacterial complications were infections with Klebsiella, Pseudomonas and Staphylococcus aureus. Cytomegalovirus, persistent HBsAg positive hepatitis, herpes zoster, and herpes simplex infections were also found. Seven patients died of bacterial superinfection and miliary tuberculosis. The data presented show that "triple infections" are associated with high mortality and that miliary tuberculosis occurred frequently in immunosuppressed renal transplant recipients.


Models of electrolyte absorption and secretion by gastrointestinal epithelia.

An attempt has been made to account for the electrolyte transport properties of several gastrointestinal epithelia in terms of relatively simple cell models. In doing so, it becomes apparent that the complex patterns of electrolyte absorption and secretion by tissues such as in vitro rabbit ileum may actually represent a combination or superposition of several basic transport processes that can be more readily identified in other epithelia. Thus, attempts to explain the effects of agents such as cyclic AMP in terms of a single mechanism of action may prove to be unproductive for such tissues. Clearly, our approach has involved certain speculations and, undoubtedly, oversimplifications. Only those processes which appear to be responsible for sodium and chloride transport have been dealt with in any detail; much remains to be learned about the processes responsible for the absorptive and secretory movements of other ions, principally HCO3. However, this analysis may be useful in bringing a degree of uniformity to a complex area and, hopefully, has identified certain important gaps in our understanding of electrolyte transport at the cellular level which require further investigation.


[Bronchiolo-alveolar cell carcinoma].

15 cases of bronchiolo-alveolar cell carcinoma were observed at the Dept. of Surgery of the University Hospital of Zurich from 1961 to 1976. The mean age of these patients was 56 years. The sex distribution was 13 males and 2 females. Five cases were discovered accidentally; the symptoms of the remaining 10 were uncharacteristic. Cytology, bronchoscopy and mediastinoscopy were negative at early stages. Diagnosis was made by histologic examination of the specimens only. Needle biopsy, performed routinely over recent years, proved helpful in 4 cases. In 12 out of 14 patients, lobectomy was considered 'radical'; nevertheless, only 2 survived more than 5 years. The disseminated form of the bronchiolo-alveolar cell carcinoma is characterized by a particularly poor prognosis. According to our experience and to the literature, only the solitary, nodular type shows a more favourable outlook.


Use of protein A-coated colloidal gold particles for immunoelectronmicroscopic localization of ACTH on ultrathin sections.

ACTH was localized in dissociated porcine adenohypophysial cells using a novel indirect EM immunocytochemical technique. Incubation of ultrathin resin sections in anti-ACTH was followed by incubation with protein A-coated colloidal gold particles. Protein A binds specifically to the Fc part of the IgG molecule, and thus the ACTH-containing secretory granules became labelled with electron-dense gold particles. With this method, the dissociated porcine ACTH cells was identified as containing numerous round or ovoid 170--300 nm secretory granules.


Ultrahistochemical investigations of dog lung surfactant with ruthenium red and iodoplatinate reactions.

Dog lungs have been fixed by immersion and submitted to two histochemical procedures. An iodoplatinate reaction technique to demontrate choline phospholipids stains cell membranes, inclusion bodies of type II alveolar epithelial cells and tubular myelin figures of pulmonary surfactant, the latter as electron-dense lines measuring 5 nm. The ruthenium red procedure gives rise to an intense contrast of the free surface of alveolar epithelium. The 5 nm-lines of the pulmonary surfactant are seen as electron-lucent lines, but bordered by electron-dense rims. Though both techniques have limitations in their interpretation, which are discussed in this paper, they demonstrate the tubular myelin figures to be a highly organized mixture of phospholipids and glycoproteins.


Experimental canine anaphylaxis: cyclic nucleotides, histamine, and lung function.

We studied the effects of Ascaris suum antigen (iv) in 32 natively allergic, anesthetized dogs. After thoracotomy, bilateral cervical vagotomy, and propranolol, samples of peripheral lung frozen in situ were obtained for measurement of cyclic nucleotides and histamine. Following Ascaris, lung histamine decreased 20.4 +/- 3.7% (mean +/- SE), cAMP increased 391 +/- 122%, and cGMP increased 110 +/- 20% with increased plasma histamine and physiological changes of anaphylaxis. No significant changes occurred in 10 control dogs. Release of histamine, reflecting immunological degranulation of mast cells, correlated closely with the physiological effects of anaphylaxis. beta-Adrenergic stimulation with isoproterenol prevented these physiological effects of anaphylaxis in three dogs studied. Furthermore, the level of cAMP induced in lung tissue by beta-adrenergic stimulation in these dogs correlated with the degree of inhibition of immunologically induced histamine release. These results illustrate the suitability of this experimental preparation to study the biochemical and physiological mechanisms of anaphylaxis in vivo.


Factors affecting the physical performance of the rat in hyperbaric environments.

Experiments were designed to partition the effect of inspired oxygen tension (PIO2), gas density (rho), and ambient pressure (P) at rest and during exercise in rats. At rest oxygen uptake (VO2) was reduced by 19.5% (P less than 0.01) in 3 ATA air and by 32.1% (P less than 0.01) in 10 ATA air from 1 ATA air control of 29.3 +/- 1.21 (SE) ml/min.kg; there was no difference in VO2 between 1, 3, and 10 ATA He mixtures containing 20% O2. During exercise, VO2 was depressed by 36.2% (P less than 0.01) in 3 ATA air from 1 ATA air value of 55.3 +/- 3.23 ml/min.kg; VO2 was unaffected in 3 ATA He containing 20% O2 as compared to that of 46.5 +/- 2.43 ml/min.kg at 1 ATA He-O2 ambient. These VO2 values were compared to that obtained from normoxic ambients for separation of effects of elevated PIO2, P, and rho on VO2 at rest and during exercise. The effect of PIO2 was deduced by comparing VO2 in normoxic and hyperoxic He and N2 at a given P and rho. The density effect was obtained by comparing the VO2 in various densities at a given P and PIO2. The P effect was obtained by comparing the VO2 in various ambient pressures at a given rho and PIO2. Summation of the effects of P, rho, and PIO2 sufficiently approximates the changes in VO2 at rest. The same procedure underestimates the actually measured VO2 in all N2 ambients.


Maturation of ventilation and ventilatory pattern in normal sleeping infants.

Noninvasive studies of ventilation and ventilatory pattern were performed serially in 15 normal infants in the first 4 mo of life during REM and quiet sleep with the barometric method. We measured tidal volume (VT), total respiratory cycle time (Ttot), inspiratory time (Ti), expiratory time (TE), mean inspiratory flow (VT/TI), and respiratory "duty cycle" (TI/Ttot). Vt, Ttot, TI, TE, VT/TI, and VT/Ttot but not TI/Ttot increased with age. In all age groups, Ttot, TI, and TE but not VT/TI were greater in quiet than in REM sleep. In the first 2 mo of life, VT was greater in quiet than in REM sleep; in the older infants, VT/Ttot was smaller in quiet than in REM sleep. TI/Ttot was not dependent on sleep state. Thus, because VT/Ttot = VT/TI X TI/Ttot, the increase in VT/Ttot with age results from an increase in mean inspiratory flow rather than from changes in respiratory "duty cycle". Further, the "on-switching" as well as the "off-switching" of inspiratory activity depends on sleep state.


Oxygen extraction by canine hindlimb during hypoxic hypoxia.

Regional and total blood flow and O2 uptake (VO2) were measured in 10 anesthetized paralyzed dogs in which venous outflow from the left hindlimb (less paw) was directed through the femoral vein. After being ventilated on room air during a 40-min control period, they were ventilated on 12.0% O2 for 40 min, 9.2% O2 for 40 min, and again on room air for 40 min. A second group of 10 was treated the same after they were given 1.0 mg/kg propranolol to block beta-adrenergic receptors. Neither limb blood flow nor cardiac output changed significantly during moderate hypoxia in either group; both increased in severe hypoxia but only in the no-beta-block group. Limb VO2 was not decreased significantly in either group during moderate hypoxia but total VO2 was decreased in the beta-block group. Total VO2 was decreased in both groups during severe hypoxia but limb VO2 was maintained in the beta-block group. beta-Block prevented the fall in total and limb peripheral resistance seen in severe hypoxia but did not alter the consistently more efficient utilization of total O2 delivery shown by the limb in comparison to the whole body by higher O2 extraction ratios and lower venous O2 pressure. beta-Vasodilator receptors evidently played an active part in the vasodilatation seen during severe hypoxia.


Reduced lung volume during behavioral active sleep in the newborn.

In a previous study of newborn infants we observed overall rib cage collapse during active sleep and postulated that the lungs also could be deflated, leading to reduced oxygen stores and circumstances favoring the rapid development of hypoxemia during apnea. In this study, thoracic gas volume (TGV) has been measured directly by occlusion plethysmography in six normal babies during behavioral quiet and active sleep and related to the different movements of the rib cage and abdomen-diaphragm that occur during each sleep state. TGV was significantly reduced in each baby during active sleep and was associated with rib cage deflation and increased abdomen-diaphragm excursions. The average reduction of TGV was 31% when compared with the volume in quiet sleep and did not depend on the order in which the sleep states were tested. The reduced lung volume in active sleep could have implications for the regulation of breathing in that state. A reduction of lung oxygen stores in active sleep suggests an age-related vulnerability of the young infant to hypoxemia.


Inert gas transport in the microcirculation: risk of isobaric supersaturation.

This paper is concerned with the theretical background and implications of isobaric supersaturation and bubble formation in the microcirculation following an abrupt shift from one inspired inert gas to another. The use of more than one inert gas, simultaneously or sequentially, has become common in diving and presents risks as well as potential benefits. A review of microcirculatory model useds, theoretical approaches to decompression, and order of magnitude calculations indicates that present empiricisms are inadequate for predicting such supersaturation phenomena. This is true whether based on the familiar assumption of perfusion-limited behavior or its diffusion-limited counterpart. The "chromatographic"  model used here, which considers both perfusion and axial diffusion in tissue cylinders, shows that these combined effects can produce unexpectedly high local supersaturation. The implications include new possibilities for the experimental evaluation of gas transport models as well as practical risks of inert gas shifts in diving and certain diagnostic procedures.


Solubility and partition coefficients for gases in rabbit brain and blood.

Solubility and partition coefficients for argon, methane, neon, nitrogen, and sulfur hexafluoride in normal saline, rabbit blood, and exsanguinated rabbit brain were determined in a two-step process that involved equilibration of the various aliquots, and subsequent analysis by gas chromatography. Brain-blood partition coefficient was specifically higher than one for argon, neon, and sulfur hexafluoride, and questionably significant (P less than 0.025) for nitrogen.


Solubility of inert gases in homogenates of canine lung tissue.

The solubility of sulfur hexafluoride (SF6), ethane, cyclopropane, halothane, diethyl ether, and acetone in homogenates of dog lung tissue were measured and compared with values obtained in dog blood. The measurements were made to provide data for a method for determining distribution of ventilation, blood flow, and tissue volume (Physiologist 20: 95, 1977) and for reasons discussed, the blood was not washed from the tissue prior to homogenization. All gases except SF6 were significantly more soluble in blood than  lung tissue, whereas SF6 was 3.7 times more soluble in tissue than blood. It was further found that SF6 is 5 times more soluble, and ethane is twice as soluble in tissue obtained from lungs containing blood than in tissue obtained from rinsed lungs, suggesting that measurements of parenchymal solubility made on tissue from sinsed lungs may be considerably in error for some lipid-soluble gases.


Arousal, ventilatory, and airway responses to bronchopulmonary stimulation in sleeping dogs.

We studied waking, ventilatory, and reflex tracheal smooth muscle (TSM) responses to tracheobronchial irritation or lung inflation in three sleeping dogs. The dogs breathed through a cuffed endotracheal tube, and airflow was measured with a pneumotachograph. TSM tone was monitored directly by measuring pressure in the water-filled cuff of the endotracheal tube. Mild degrees of tracheobronchia irritation, produced by squirting 0.1--1.0 ml of water into the lower trachea, or by having the dogs inhale one breath of acetic acid vapor (concentration 17 ppm), generally caused arousal from slow-wave sleep (SWS), but not from rapid-eye-movement (REM) sleep. During wakefulness the irritant stimuli caused coughing and TSM constriction; during SWS or REM sleep, these responses occurred only if the stimulus first produced arousal. In contrast, stimulation of pulmonary stretch receptors by lung inflation did not cause arousal, but readily produced apnea and TSM relaxation during SWS. The results indicate that cough and airway smooth muscle constriction in response to bronchopulmonary irritant stimuli do not occur in the absence of arousal, and that arousal responses to such stimuli are depressed in REM sleep.


Exhaustive exercise, endurance training, and acid hydrolase activity in skeletal muscle.

The activity of eight acid hydrolases and two energy metabolism enzymes were assayed from homogenates of predominantly red (proximal heads of m. vastus lateralis, m. vastus medialis, and m. vastus intermedius) and predominantly white (distal head of m. vastus lateralis) skeletal muscle of mice belonging to one of the following groups: 1) sedentary controls, never trained or exhausted; 2) exhausted controls, exhausted once by running on a treadmill 5, 10, or 20 days before killing; 3) trained mice, exercising until killed; 4) exhausted trained mice, exercising until exhausted 5, 10 or 20 days before killing, not exercising during that period; and 5) detrained mice, terminating training 5, 10, or 20 days before killing. In untrained but not in trained animals, exhaustive exercise caused, 5 days afterward, fiber necrosis and a marked increase in the activities of beta-glucuronidase, beta-N-acetylglucosaminidase, arylsulphatase, ribonuclease, deoxyribonuclease, cathepsin D, and cathepsin C, especially in red muscle fibers. Training increased the activities of citrate synthase, beta-glucuronidase, and cathepsin D in both muscle types and those of beta-N-acetylglucosaminidase, arylsulphatase, and cathepsin C in red muscle. Effects of detraining were minor. Exhaustive exercise causes lethal and evidently also sublethal fiber injuries manifesting themselves as an activation of the lysosomal system of muscle fibers 5 days later. Training affects cellular homeostasis by causing an apparent resistance to the damaging effects of exhaustive exercise. Moderately increased hydrolase activities may reflect increased turnover in endurance-trained muscles.


Effects of high oxygen exposure on bioenergetics in isolated type II pneumocytes.

O2-mediated alterations in cell energy metabolism may play a role in structural and functional abnormalities described in type II pneumocytes (T-II-P) following in vivo hyperoxia. Bioenergetic alterations produced by hyperoxia (95% O2) were therefore examined in a culture-maintained cell line derived from T-II-P. Exposure of cell monolayers to 95% O2 for 96 h results in a significant decrease in O2 consumption (from 0.52 +/- 0.07 to 0.30 +/- 0.08, P less than 0.01), suggesting impaired mitochondrial energy provision. In addition, there are increased rates of aerobic lactate production (from 2.89 +/- 0.52 to 3.84 +/-0.80, P less than 0.05) with loss of Pasteur effect, indicating a shift to glycolytic metabolism at relatively high PO2's. These metabolic changes are not accompanied by altered activities of critical mitochondrial (cytochrome oxidase) or glycolytic (pyruvate kinase, phosphofructokinase) enzymes. Altered cell bioenergetics following hyperoxia may this represent an important secondary mechanism leading to functional abnormalities in T-II-P.


Diaphragmatic muscle tone.

It is generally believed that there is a scarcity of muscle spindles in the diaphragm and that there is no tonic activity at end expiration. This conclusion is based mainly on animal studies and the difficulty in differentiating tonic electromyogram activity from noise. We have, however, found a number of muscle spindles in the newborn human diagphragm, concentrated in the region of the central tendon. We also tried to detect tonic activity by decreasing it (by rapid-eye movement (REM) sleep or anesthesia) or increasing it (with abdominal loading). During REM sleep in five infants and five adults, using subcostal electrodes were observed a marked fall in tonic activity (P less than 0.001) compared to non-REM or quiet sleep. We also observed a reduction in diaphragmatic tonic activity with halothane anesthesia (P less than 0.001). With esophageal electrodes in adult subjects, there was a rise in tonic diaphragmatic activity proportional to the amount of abdomina load (P less than 0.001). We conclude that there are muscle spindles in the human diaphragm and that there is tonic activity at end expiration.


Dibutyryl cyclic GMP and hyperventilation promote rat lung phospholipid release.

Ventilation of rats at high inspiratory pressures raises lung tissue content of guanosine 3',5'-cyclic monophosphate (cGMP). Hyperventilation in rabbits augments release of phospholipid into lavage fluid. Can cGMP, in the absence of hyperventilation, increase lung phospholipid release? Sprague-Dawley rats are injected with [14C]palmitate, and after 1.5 h are anesthetized and ventilated for 20 min. Three groups are ventilated at peak inspiratory pressures (PIP) of 10 cmH2O, while saline, dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP), or dibutyryl cGMP (DBcGMP) is infused into the pulmonary artery. In a fourth group, saline is infused into the pulmonary artery, but ventilation is performed with PIP of 25 cmH2O. Lung tissue and lavage fluid are then analyzed for phospholipid (PL) content and for incorporation of [14C]palmitate into lavage and tissue PL fractions. Ventilation at increased pressure and infusion of DBcGMP are associated with increases in release of labeled PL into lavage fraction. The findings suggest that the increase in lavage PL release associated with hyperventilation is, at least in part, mediated by cGMP.


Pulmonary interdependence of gas transport.

We have examined the interaction of convection and gas-phase diffusion among parallel pathways of the human lung by solving the differential equation for gas transport in a solid geometric model. Two trumpet-shaped units with a threefold differences in volume flow were joined at a branch point that could be varied in position along the airway tree. Because diffusion dominates gas transport in peripheral airways, or when time for diffusion is large, alveolar concentrations are more homogeneous than predicted from volume flows when the branch point is peripheral to respiratory bronchioles, or when total flow rate is small. When the branch point is in the larger airways, subtending large units of lung, diffusion is less important, so that alveolar concentration of each unit depends almost completely on its volume flow. These simulations provide a possible explanation for experimental findings of partial separation of inert gases of differing diffusivities and of improved O2 exchange when dense gases are breathed.


The ultrastructure of the epithelium of the ducts of the Harderian and lacrimal glands of the turkey, fowl and duck.

The ultrastructure of the epithelium in the Harderian and lacrimal gland ducts of the turkey, fowl and duck has been investigated. The findings establish that the ducts are fundamentally similar in morphology, although species differences occur. The duct epithelia are composed of cuboidal, columnar or pseudostratisfied columnar cells with many secretory vesicles as well as goblet cells. PAS-positive fibrillary rods and crystalline inclusions occupied the cisternae of the rough endoplasmic reticulum of the epithelial cells of turkeys. The crystals, which were thought to evolve from the rods, had a mean periodicity of 7.0 nm. In chicks between 1 and 3 days of age, but not in poults and ducklings, the duct epithelia displayed large intracytoplasmic deposits of glycogen which disappeared by the seventh day.


Morphological and cytochemical study of a hypothalamochiasmatic perivascular neuronal system.

The topographical localization and some cytochemical characteristics of groups of perivascular neurons in the hypothalamus and in the optic chiasma have been studied. In the anterior hypothalamic area there are neuronal groups located around the blood vessels which penetrate the optic chiasma still associated with neurons. These perivascular neurons show some characteristics similar to those observed in neurosecretory hypothalamic neurons, but differ from them in that they show a greater intensity of thiaminopyrophosphatase and nucleoside-diphosphataseactivities. It is suggested that these neuronal groups may constitute a special hypothalamochiasmatic-perviscular-neurosecretory system (H. Ch. P. N. S.).


Transport of proteins across the mitochondrial outer membrane. A precursor form of the cytoplasmically made intermembrane enzyme cytochrome c peroxidase.

Cytochrome c peroxidase, a cytoplasmically made enzyme located between the inner and outer membrane of yeast mitochondria, is synthesized as larger precursor in a reticulocyte cell-free lysate as well as in pulsed yeast spheroplasts. When the pulsed spheroplasts are chased, the precursor is converted to the mature apoprotein. When the in vitro synthesized precursor is incubated with isolated yeast mitochondria in the absence of protein synthesis, it is cleaved to the mature form; the mature form co-sediments with the mitochondria and is resistant to externally added proteases. These results, in conjunction with those reported earlier (Maccecchini, M.-L., Rudin, Y., Blobel, G., and Schatz, G. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 343-347) suggest that the mechanism of protein transport into the mitochondrial intermembrane space is quite similar to that of protein transport into the matrix or the inner membrane.


Stereochemistry of hydrolysis by snake venom phosphodiesterase.

[18O]Adenosine 5'-O-phosphorothioate-O-p-nitrophenyl ester was prepared by saponification of the bis (-O,O-p-nitrophenyl ester) with K18OH. Only the diastereoisomer with the Rp configuration si a substrate for snake venom phosphodiesterase. The asymmetrically labeled [18O]adenosine 5'-O-phosphorothioate formed in this reaction was converted enzymatically to [18O]adenosine 5'-(1-thiodiphosphate) with the Sp configuration. The position of the 18O label, either bridging [1,2-mu-18O] or nonbridging [1-18O] was then determined. The results show that the reaction catalyzed by snake venom phosphodiesterase takes place with retention of configuration at phosphorus. This indicates that the hydrolysis proceeds via a covalent nucleotide enzyme intermediate.


Hyperfine structure resolved by 2 to 4 GHz EPR of cytochrome c oxidase.

Using low frequency 2 to 4 GHz EPR at 10 K, we have resolved previously unseen hyperfine structure associated with the EPR-detectable copper signal of cytochrome c oxidase. The observed hyperfine structure appears consistent with hyperfine coupling to copper; although to account for all of the observed structure, an additional magnetic interaction is required as well. This work points out the utility of the 2 to 4 GHz EPR technique for resolving electronic hyperfine structural information from copper and possibly other paramagnetic sites in biomolecules when random variation in electronic g values is a cause of EPR line-broadening.


Affinity labeling of catalytic subunit of bovine heart muscle cyclic AMP-dependent protein kinase by 5'-p-fluorosulfonylbenzoyladenosine.

Incubation of 5'-p-fluorosulfonylbenzoyladenosine with the catalytic subunit of bovine cardiac muscle cyclic AMP-dependent protein kinase led to the formation of an inactive enzyme irreversibly modified with approximately one mol of reagent per mol of subunit. The inactivation reaction followed pseudofirst order kinetics. The rate of inactivation at various reagent concentrations exhibited saturation kinetics implying that the reagent reversibly binds to the enzyme prior to inactivation. The addition of MgATP, MgADP, or MgAMP-PNP to the reaction mixture fully protected the enzyme from inactivation by 5'-p-fluorosulfonylbenzoyladenosine. The reagent was demonstrated to be a competitive inhibitor of MgATP with a Ki of 0.235 mM. Metal-free nucleotides were without effect upon the reaction rate while metal ions alone accelerated the inactivation rate up to 7-fold. The inclusion of casein or synthetic peptide substrate in the incubation mixture did not affect the reaction kinetics. Reaction of 5'-p-fluorosulfonylbenzoyladenosine with the kinase subunit exhibits all of the characteristics of affinity labeling of the MgATP-binding site.


Calcium dependence of hormone-stimulated cAMP accumulation in intact glial tumor cells.

The Ca2+ content of glial tumor (C6) cells was reduced approximately 5-fold by repeated treatment with media containing ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA) without loss of cellular viability. The ability of the cells to accumulate cAMP in response to beta-adrenergic agonists was reduced 60 to 70% following Ca2+ depletion. Ca2+ did not affect the apparent KACT for norepinephrine, nor did it change the concentration of propranolol required to produce 50% inhibition of the maximal norepinephrine response. Phentolamine did not alter the Ca2+ dependence of the response. The binding of dihydroalprenolol by intact C6 cells was not influenced by Ca2+. Furthermore, pretreatment with norepinephrine did not affect the Ca2+ dependence of cAMP accumulation. The effects of Ca2+, therefore, appeared to be exerted on components of the adenylate cyclase system other than the catecholamine receptor. Micromolar free Ca2+ concentration in the extracellular medium were sufficient to restore a maximal norepinephrine response to Ca2+-depeleted cells. The effect of Ca2+ on cAMP accumulation in response to hormone was immediate and was rapidly reversible upon the addition of EGTA in excess of the cation. Cells in media containing Ca2+ exhibited a characteristic biphasic time course of cAMP accumulation; with Ca2+-depleted cells cAMP was accumulated more slowly and the subsequent decline in cAMP content was also reduced. Verapamil, an inhibitor of plasmalemmal Ca2+ influx, decreased the Ca2+-dependent component of the cAMP accumulation when added prior to the cation. The effect of Ca2+ on cAMP accumulation was reduced more extensively by pretreatment of cells at 45 degrees C under Ca2+-depleted (80% loss) than under Ca2+-restored (30% loss) conditions. Trifluoperazine at micromolar concentrations decreased the Ca2+-dependent increment in accumulation of cAMP in Ca2+-restored cells. This inhibition was not overcome by increasing concentrations of norepinephrine or of extracellular Ca2+.


Observations on the mechanism of the reversible epimerization of GDP-D-mannose to GDP-L-galactose by an enzyme from Chlorella pyrenoidosa.

An enzyme fraction from the green alga Chlorella pyrenoidosa that catalyzes the reversible epimerization of guanosine 5'-diphosphate D-mannose to guanosine 5'-diphosphate L-galactose brings about the incorporation of tritium from  tritium-labeled water into the hexosyl moieties of those sugar nucleotides. The hexoses were degraded by periodate oxidation whereby the tritium was found to be equally distributed between carbon atoms 3 and 5. That observation was taken to imply that the epimerizations proceed via ene-diol intermediates.


Biosynthesis of the Ca2+- and Mg2+-dependent adenosine triphosphatase of sarcoplasmic reticulum in cell cultures of embryonic chick heart.

The biosynthesis of the Ca2+- and Mg2+-dependent adenosine triphosphatase of sarcoplasmic reticulum was studied in cell cultures of embryonic chick heart. Rates of synthesis were estimated from the incorporation of tritium-labeled leucine into the ATPase. Newly synthesized ATPase was isolated from cells by immunoprecipitation. Radioactive leucine incorporation into the ATPase was determined by gel electrophoresis of the immunoprecipitates and counting of gel slices containing the ATPase band. Accumulation of the ATPase was estimated from the concentration of Ca2+ and Mg2+-dependent, hydroxylamine-sensitive phosphoprotein in the whole cell membrane fraction of cultured cells. Embryonic heart cells cultured in a medium which permitted cell proliferation showed approximately linearly increasing rates of ATPase synthesis and accumulation/culture plate as the cells proliferated. When cells were cultured in a serum-free medium, cell proliferation was inhibited and there was no sustained increase in the rate of ATPase synthesis or accumulation. Inclusion of isoproterenol or dibutyryl cyclic AMP at concentrations of 10 microM up to 1 mM in serum-free culture medium failed to stimulate significantly ATPase synthesis.


Cytochrome c oxidase from bakers' yeast. Photolabeling of subunits exposed to the lipid bilayer.

Yeast mitochondria and purified yeast cytochrome c oxidase incorporated into micelles of the nonionic detergent Tween 80 were equilibrated with the hydrophobic aryl azides 5-[125I]iodonaphthyl-1-azide or S-(4-azido-2-nitrophenyl)-[35S]thiophenol. The azides were then converted to highly reactive nitrenes by flash photolysis or by illumination for 2 min and the derivatized cytochrome c oxidase subunits were identified by gel electrophoresis and radioactivity measurements. 5-[125I]Iodonaphthyl-1-azide labeled mainly the three mitochondrially made Subunits I to III and the cytoplasmically made Subunit VII. Subunits IV to VI or cytochrome c bound to the purified enzyme were labeled 9- to 90-fold less. Essentially the same result was obtained with S-(4-azido-2-nitrophenyl)-[35S]thiophenol except that Subunit V was labeled as well. In contrast, all seven subunits as well as cytochrome c were heavily labeled when the enzyme was dissociated with dodecyl sulfate prior to photolabeling with either of the two probes. These data indicate that all subunits of yeast cytochrome c oxidase except Subunits IV and VI are at least partly embedded in the lipid bilayer of the mitochondrial inner membrane.


Membrane-dependent guanine nucleotide binding and GTPase activities of soluble protein from bovine rod cell outer segments.

Soluble proteins can be extracted by osmotic shock of purified rod (photoreceptor cell) outer segments that have intact plasma membranes. The soluble proteins include a component that contains tightly bound GDP-Exchange of this GDP with exogenous nucleotide is catalyzed by (and requires) the membranes from the outer segments. ATP does not participate in these reactions. Approximately one-half of the binding sites in the soluble component require GTP as the source of exogenous nucleotide; the remainder accept GTP or GDP with equal facility. When exogenous GTP is the source of bound nucleotide, it is found in the complex in the form of GDP. Exchange of bound nucleotide with GTP is stoichiometrically related to GTPase activity; this activity is highly dependent upon the presence of both membranes and soluble protein. The soluble nucleotide binding protein was purified by making use of the fact that it binds tightly to the membranes (under conditions of moderate ionic strength) in the absence of GTP and can be eluted by solutions containing low concentrations of GTP (but not GDP or ATP, nor can it be eluted by GTP-free solutions of low ionic strength). The purified protein contains two polypeptide chains of molecular weights 41,000 and 37,000; these are the major species that can be extracted from the outer segments by osmotic shock, and they constitute approximately 7% of the total protein of the isolated organelle.


Synthesis and secretion of corticotropins, melanotropins, and endorphins by rat intermediate pituitary cells.

The synthesis and secretion of various intermediate pituitary proteins was studied by using dispersed intermediate pituitary cell suspensions. Control studies indicated that the isolated cells were obtained in good yield and that after more than 24 h in culture the isolated cells continued to synthesize a collection of proteins similar to those found in freshly extracted intermediate pituitary tissue. Rat intermediate pituitary cells synthesized a molecule (Mr = 30,000; called 30K) that contained antigenic determinants for beta-endorphin, gamma-lipotropin, corticotropin (ACTH), and 16K fragment (the NH2-terminal region of mouse tumor cell pro-ACTH/endorphin). This 30K molecule, two high molecular weight forms of ACTH(13K and 20K), and 16K fragment were all shown to be glycoproteins. Continuous labeling and pulse-chase incubations were used to define the intracellular biosynthetic processing of the 30K molecule. After a 15-min pulse incubation the 30K molecule was the only labeled protein containing antigenic determinants for beta-endorphin, gamma-lipotropin, ACTH, or 16K fragment. A beta-lipotropin-like molecule served as a biosynthetic intermediate in the production of proteins similar to beta-endorphin and gamma-lipotropin. Methionine-enkephalin and alpha-endorphin were not major products in the intermediate lobe cells. Molecules similar to alpha-melanocyte-stimulating hormone and corticotropin-like intermediate lobe peptide (ACTH(18-39)) were also derived from the same 30K molecule; 20K ACTH served as a biosynthetic intermediate in this conversion. In rat intermediate pituitary cells ACTH(1-39) was not a major final product of the intracellular biosynthetic processing of the 30K molecule. The 30K molecule also served as a precursor to a protein similar to mouse tumor cell 16K fragment and related smaller proteins. With rat intermediate pituitary cells, pulse-chase experiments utilizing [35S]methionine demonstrated almost quantitative conversion of the 30K precursor into labeled proteins similar to beta-endorphin and alpha-melanocyte-stimulating hormone. In the absence of added secretagogues, small amounts of all of the smaller proteins derived from the 30K precursor were secreted coordinately into the culture medium.


Modulation of protein synthesis by nerve growth factor.

The effects of nerve growth factor (NGF) and two analogs of cAMP have been studied in the PC12 line of rat pheochromocytoma cells. We have used two-dimensional gel electrophoresis to detect more than 800 proteins from control and NGF-treated cells. Of these proteins, none were qualitatively repressed in response to NGF, and no new proteins appeared after NGF treatment. Visual inspection of the gels showed that approximately 5% of the proteins were detectably increased or decreased in rate of synthesis by NGF, and each of these changes was mimicked by both cAMP analogs. The two-dimensional gel data were further analyzed by a computerized scanning system. This analysis has revealed many significant changes that are smaller than those detected by eye. Approximately 25 to 30% of the proteins analyzed were found to be altered in rate of synthesis by 30% or more. Statistical analysis has shown that the response to NGF and the response to dibutyryl cAMP are highly correlated, even down to changes as small as 30%. No proteins were found to be significantly altered by both dibutyryl cAMP and 8-bromo cAMP, but not by NGF. These results show that NGF causes only quantitative modulations of protein synthesis in PC12 cells, and these data strongly suggest that the response of PC12 cells to NGF is mediated by cAMP.


Crystallographic symmetry and coenzyme binding properties of D-glyceraldehyde-3-phosphate dehydrogenase from the tail muscle of Palinurus vulgaris.

Crystals of apo- and holo-D-glyceraldehyde-3-phosphate dehydrogenase from the tail muscle of the Mediterranean lobster Palinurus vulgaris, previously found to be suitable for single crystal microspectrophotometric studies of catalytic activity in the crystalline state, have been examined by x-ray crystallography. The two forms are isomorphous, space group C 2 with cell dimensions a=128.4 A, b=99.9 A, c=80.3 A,beta=113.4 degrees. These data are consistent with a molecular weight of 73,000 in the crystallographic asymmetric unit, indicating that the tetrameric molecule possesses an exact 2-fold axis both in the presence and in the absence of NAD+. Analysis of the intensity distribution of conventional x-ray precession photographs shows that two further noncrystallographic diads are present and that the molecule has the 2 pseudo 22 symmetry found in other D-glyceraldehyde-3-phosphate dehydrogenases. Binding of NAD+ to apoenzyme in solution, at 25 degrees C, is anticooperative and it can be satisfactorily described by assuming two classes of coenzyme binding sites.


The purification and properties of a double-stranded DNA-binding protein encoded by the gene D5 of bacteriophage T5.

We have purified a DNA-binding protein from bacteriophage T5-infected cells. The protein is the product of the T5 gene D5 and is produced in quantities ultimately exceeding 2% of the total cell protein. The protein has no tendency to aggregate and exists in solution as a monomer of about 29,000 daltons. Although the protein binds with a high affinity to both single-stranded (fd, T7) and duplex DNA (T5, T7), there is a preferred binding to duplex DNA. The binding of the gene D5 protein to duplex DNA is cooperative with an estimated dissociation constant of 6.27 x 10(-10) M and leads to the production of a more condensed form of the DNA. In contrast, binding of the gene D5 protein to single-stranded DNA is noncooperative with an estimated dissociation constant of only 1.85 x 10(-8) M and produces a sedimentation shift in the DNA proportional to the added mass of the protein. The protein product of the gene D5 of bacteriophage T5 is known to be required for both T5 DNA replication and for the inhibition of early phage gene expression. We also show here that the protein is required to initiate transcription of the late region of the T5 genome.


Kinetic studies on the effect of the heme iron(III) on the protein folding of ferricytochrome c.

The three-dimensional conformation of ferricytochrome c results from specific folding of the polypeptide chain around the covalently bound heme so that His-18 and Met-80 are axially coordinated to the Fe(III). The Fe(III)-free, porphyrin protein has an intrinsic viscosity, sedimentation coefficient, and circular dichroism indicative of a compact, globular protein conformation comparable to the holoprotein. Both the porphyrin protein and ferricytochrome c are reversibly denatured by guanidinium chloride. Refolding of the porphyrin protein occurs in essentially a single, exceptionally rapid kinetic phase (tau = 14 ms, 0.75 M guanidinium chloride, pH 6.5, 25 degrees C); whereas refolding of ferricytochrome c occurs in two slower kinetic phases (TAU 1 = 0.10 S, TAU 2 = 20 S) UNDER COMPARABLE CONDITIONS. The presence of Fe(III) in the metalloporphyrin of ferricytochrome c thus has a major effect on the protein folding kinetics. The slow kinetic phase is evidently due to this effect of Fe(III) and not to the slow cis-trans isomerism of the peptide bond of proline residues as has been suggested.


A poly(dT)-stimulated ATPase activity associated with simian virus 40 large T antigen.

Highly purified SV40 large T antigen exhibits an ATPase activity which can be stimulated approximately 7-fold by the DNA homopolymer poly(dT). The poly(dT)-stimulated enzyme can hydrolyze various ribonucleotide and deoxyribonucleotide triphosphates, with ATP and dATP serving as the best substrates. Purified large T antigen hydrolyzes ATP to ADP and Pi, with a maximum specific activity of 13.5 mumol of inorganic phosphate released per h per mg of protein. Of the various natural and synthetic polynucleotides tested, poly(dT) was by far the best activator. Long chain poly(dT) molecules are much more effective activators than are short chain length oligo(dT) molecules. The highly purified large T antigen contains no detectable protein kinase activity.