Hepatitis B antigen and auto-antibodies in chronic liver diseases in Hong Kong.

The frequency of occurrence of hepatitis B antigen (HBAg) and certain tissue autoantibodies [antinuclear antibody (ANA), smooth muscle antibody (SMA) and mitochondrial antibody (MIA)] were studied with the microtiter complement fixation and immunofluorescence techniques respectively in a group of patients suffering from chronic liver diseases.  These were chronic hepatitis (30), cirrhosis of the liver (66) and hepatocellular carcinoma, mostly with underlying cirrhosis (100).  A group of closely matched hospital in-patients served as controls.  HBAg was found in high frequency in the patients with liver disease (60% in chronic hepatitis, 36.4% in cirrhosis and 49% in hepatocellular carcinoma) whereas tissue auto-antibodies were found in lower frequencies (16.7%, 10.6% and 13% in the three groups respectively).  However, in both the frequency was significantly higher than that in the controls (9.2% for HBAg and 0.8% for auto-antibodies).  There was a negative correlation between HBAg and tissue auto-antibodies in the group of patients with liver disease when taken as a whole (x2=14.3, P less than 0.001).  These results suggest a possible aetiological role played by hepatitis virus B in hepatocellular carcinoma through chronic hepatitis and cirrhosis in Hong Kong while the mutual exclusion between HBAg and auto-antibodies supports the hypothesis of heterogeneity in the aetiology of chronic liver diseases.  The patients with auto-antibodies may belong to the auto-immune category but no definate conclusion can be reached until the role played by hepatitis virus A in chronic liver diseases is clarified when more reliable techniques for its identification are available.


Inappropiate ADH secretion associated with massive vincristine overdosage.

A patient with acute lymphocytic leukemia is described who developed meningeal leukemia 14 months after the initial diagnosis was made.  As part of his antileukemic therapy, at that time, he received prednisone and vincristine, given prophylactically to maintain a bone marrow remission.  He inadvertently received 15 mg of vincristine instead of 1.5 mg.  Following this overdosage he developed pancytopaenia, mild neurotoxicity and subsequently a grand mal seizure associated with the delayed onset of hyponatremia.  This was presumed to be due to the inappropriate secretion of antidiuretic hormone (ADH) secondary to vincristine toxicity.  This responded to fluid restriction and anti-epileptiform therapy.


Ectopic adrenocorticotrophic hormone production and neoplasm.

Three patients presented with severe hypokalaemic, hypochloraemic alkalosis, muscle weakness and later cutaneoud pigmentation.  There were few signs of Cushing's syndrome.  Two had bronchial and one pancreatic neoplasm.  Plasma cortisol and urinary steroid metabolites were extremely elevated and synacthen stimulation showed significant adrenal capacity even with adrenal metastases.  Aminoglutethamide reduced plasma cortisol in two patients.


Inheritance of a lethal immunodeficiency disease of Arabian foals.

A fatal syndrome of certain Arabian foals which begins at about 25 days of age (range 14 to 46 days) and which runs a course of about 23 days (range 13 to 42 days) is described. The syndrome, which affected 17 foals on a single farm is further characterised by pneumonia, in some instances by dermatitis (dermatophilosis) and other infections, together with a progressive decline in health till death at about 49 days of age (range 34 to 77 days), despite intensive therapy. Four of the foals, on histopathological evidence, had adenviral pneumonia, in 2 foals there was histopathological evidence of an immunodeficiency disease and an adenovirus was isolated from 1 foal. It is argued, however, that the 17 foals were affected by a single, specific syndrome (an immunodeficiency disease) and that this disease is inherited as a simple, recessive, autosomal gene.


Bluetongue in sheep and goats.

A description is given of the symptomatology, pathology and pathogenesis of bluetongue in sheep and goats. The economic significance of the disease in South Africa is discussed.


Bluetongue: the disease in cattle.

Most researchers in South Africa found that although BT virus could be isolated from apparently healthy cattle and from inoculated cattle the virus did not produce overt clinical disease in cattle. However, when epizootics were reported outside Africa, clinical signs were observed in cattle in Israel, Palestine, Syria, Portugal, and Spain. Most natural BT infections in cattle in the United States do not result in overt clinical signs. However, in certain infected herds, approximately 5% of the cattle show from mild to severe disease. Except for severe cases, spontaneous recovery is usual. The clinical diagnosis of BT in cattle is difficult and requires laboratory assistance. Culicoides variipennis can serve as a vector of BT virus from cattle to cattle, cattle to sheep, sheep to cattle, and sheep to sheep. In utero transmission occurs in cattle and can result in abortion, hydraencephaly, congenital deformity, and birth of viraemic calves which may or may not develop BT antibody. Calves inoculated in utero or those born to infected dams may have a persistent viraemia with or without BT antibody. tone such animal has been held in insect-secure quarters and has continued to harbour virus for 3 years. Bluetongue virus was isolated from the semen of experimentally infected bulls. Calves inoculated with BT virus and also given an immuno-suppressant developed marked clinical disease in 8 to 12 days. Bluetongue virus is very closely associated with the erythrocytes of infected cattle, sheep, and goats. Cattle are considered important and relatively long-term virus reservoirs. In attempts to determine the maximum period of viraemia in cattle it is necessary to inoculate washed erythrocytes, rather than whole blood, and to use susceptible sheep as the assay system rather than embryonated chicken eggs.


Bluetongue-like disease of deer.

A comparison has been made of the disease produced in white-tailed deer by the viruses of epizootic haemorrhagic disease and bluetongue. The similar nature of these diseases in deer and of some of the viral properties has been described. Although these two viruses are considered to be distinct, it is possible by employing an unnatural procedure to produce antibody which will demonstrate a minor antigenic component common to both viruses. Biological features that differ between the two viruses have also been noted.


Ibaraki disease and its relationship to bluetongue.

Ibaraki disease, an epizootic disease of cattle in Japan resembling bluetongue, is characterized by fever and lesions affecting the mucous membranes, the skin, the musculature and vascular system. Degeneration of striated muscular tissue is observed in the oesophagus, larynx, pharynx, tongue and the skeletal muscles. Oedema and haemorrhage are marked in the mouth, lips, abomasum, around the coronets, etc., and are occasionally followed by degeneration of the epithelium leading to erosions or ulcerations. Severe lesions affecting the oesophageal and laryngopharyngeal musculature cause difficulty in swallowing which in turn produces dehydration and emaciation, and occasionally the aspiration pneumonia, which constitute the major causes of death of affected animals. These clinical and pathological findings indicate the similarity of the disease to bluetongue in sheep and cattle. Ibaraki disease was first recognised in Japan in 1959 and 1960. Seasonally its occurrence is limited to late summer and autumn, and geographically to the central and western parts of Japan, roughly south of 37 degrees north latitude. It is absent from the higher altitudes. The seasonal and geographical incidence suggests the possibility of an arthropod vector; but direct evidence for such a vector is still lacking. Serological data suggest the presence of Ibaraki virus on Bali Island in Indonesia and in Taiwan. The disease can be transmitted serially in calves by the intravenous inoculation of blood obtained at the height of a febrile reaction. Ibaraki virus can be isolated in bovine cell cultures from both natural and experimentally produced cases of the disease. The virus multiplies and induces cytopathic effects in primary cultures of bovine, sheep and hamster lung origin, and L cells; but it does not grow in primary cultures of horse and swine kidney nor in HeLa cell cultures. The virus is readily passaged serially in 4 to 5-day-old eggs by yolk-sac inoculation and incubation at 33.5 degrees C. It multiplies in the brains of mice of any age after incracerebral inoculation but younger mice give a better viral growth and develop encephalitis. No evidence has been obtained that rabbits and guinea pigs are susceptible to Ibaraki virus...


Serological methods in the diagnosis of bluetongue.

The viral antibodies in the serum of cattle, sheep and deer are not detectable in the ordinary direct CF test. We have shown that the so-called "non complement-fixing" viral antibodies in the serum of these animal species can be demonstrated by the MDCF test. The MDCF test, as well as the micro AGP test, are group reactive, with all BT isolates studied. However, it is possible with these tests to differentiate antigenically BT virus from EHD virus. The FAT was also useful in differentiating BT virus grown in TC cells from the EHD virus. In the PRN test, all the BT virus isolates studied cross reacted and although quantitative differences were frequently observed, no obvious antigenic classification was possible with the antiserums used in this work. Reactions between the BT viral isolates and the EHD virus were all within the limits of what is presently considered to be non-specific inhibition.


The comparative sensitivity of sheep and chicken embryos to bluetongue virus and observations on viraemia in experimentally infected sheep.

The virus titre in sheep blood samples received from BT-suspected cases in the field was assayed in sheep and in chicken embryos. These infected blood samples represented 3 different BT virus types: 4, 10 and 16. Three identical experiments were performed, one with each of the 3 different virus types. Ten-fold dilutions of the infected blood samples were prepared and 1 ml of each blood dilution was inoculated IV into series of 10 to 12-month old susceptible sheep; at the same time 0.1 ml of each dilution was inoculated IV into series of 10 to 13-day-old chicken embryos. The virus titre was found to be similar when assayed in the two host systems. There was no correlation between the amount of virus inoculated and the severity of symptoms in the inoculated sheep. The virus content in daily blood samples collected from the experimental sheep was assayed by IV inoculation of CE. Virus was isolated from all the reacting sheep and was detected sometimes as early as 1 day PI and as late as 30 days PI. A high titre of log10 4.0 to 7.0 per 1 ml of blood was recorded during several consecutive days before and after the onset of clinical signs. There seemed to be an inverse ratio between the amount of virus inoculated and the number of days the virus persisted in the bloodstream. The neutralisation index in day 22 serum samples was 3.5 to 4.5. Virus was isolated from some of the reacting sheep on the day that these antibody levels were recorded. Since the comparative simultaneous titrations of BT virus in sheep and in CE yielded similar results, the IV inoculation of CE is advocated as the routine method to be employed for laboratory diagnosis of this disease.


The epizootiology of bluetongue: the African situation.

Bluetongue virus is transmitted biologically by various species of Culicoides, notably C. pallidipennis and C. variipennis. Factors such as rainfall, temperature and relative altitude, which influence the breeding of the insect vectors also govern the incidence and distribution of the disease. The host range of bluetongue virus includes sheep, cattle, goats and various antelopes. Many other, as yet unidentified hosts could perhaps harbour the virus and influence the epizootiology of the disease. The close relationship between C. pallidipennis and cattle is indicated and the efficient mechanism for virus maintenance which this relationship constitutes is emphasised. It is further postulated that sheep are not essential for the continued survival of bluetongue virus, but merely function as accidental or indicator hosts.


Epizootiology of bluetongue: the situation in the United States of America.

Bluetongue was first reported in the United States in 1948 in sheep in Texas. The virus has now been isolated from sheep in 19 States. When the disease first occurs in a flock, the morbidity may reach 50 to 75% and mortality 20 to 50%. In subsequent years, the morbidity may be only 1 to 2% with very few deaths. Difference in breed susceptibility has not been observed. Natural bluetongue infection has not been observed in Angora or dairy goats. Bluetongue virus was first isolated from cattle, in Oregon, in 1959. The virus has now been isolated from cattle in 13 States. In cattle, the disease is usually inapparent but can cause mild to severe clinical disease and neonatal losses. Natural clinical bluetongue has also been reported in bighorn sheep, exotic ruminants in a zoo, mule deer, and white-tailed deer. Serological evidence of exposure to the virus has also been found in other species of ruminants in the wild. Inoculation of virulent bluetongue virus, vaccine virus, or natural disease can cause congenital deformities and neonatal losses in calves, lambs, and white-tailed deer fawns. Culicoides is considered the important insect vector of bluetongue. The virus has also been isolated from sheep keds and cattle lice. U.S. field strains of the virus fit into four serologic groups. No cross reactions were found between bluetongue and epizootic haemorrhagic disease of deer viruses. Cattle are considered significant virus reservoirs. It is necessary to use washed erythrocytes, rather than whole blood, and to inoculate susceptible sheep, rather than embryonated chicken eggs, to detect longer-term viraemia in cattle.


The control of bluetongue in an enzootic situation.

On account of the wide host range of bluetongue virus and its biological transmission by insects, control of the disease in an enzootic situation is based primarily on the active immunisation of susceptible animals as well as on the prevention of contact between the insect vectors and the susceptible hosts. In spite of their unquestionable value, the egg attenuated vaccines which are currently employed for prophylactic immunisation, have certain shortcomings. The existence of 16 known serotypes of bluetongue virus makes it difficult to achieve a very wide spectrum of immunity in sheep vaccinated once or twice only. The problems which are experienced with the immunisation of lambs born in spring are indicated. The present vaccine can also present problems when used in breeding animals. Furthermore, the costs involved in the annual vaccination of large numbers of animals are considerable. The need for a vaccine for cattle is indicated. Work is also being conducted at present on the development of an inactivated vaccine for use in sheep. The use of novel virological techniques may aid in the future development of absolutely safe and highly efficient vaccines against bluetongue.


Bluetongue certification -- Australian policy.

Australian quarantine policies with respect to BT are based on regarding this disease as one of high risk and major potential economic importance to our ruminant population. There are deficiencies in our knowledge of world distribution, epidemiology and pathogenesis. There may be unknown vectors and unsuspected animal reservoir hosts. The international distribution of BT could be extending through the movements of insects or cattle. If introduced into Australia, the cattle and sheep populations would probably be at continuous risk as eradication would be difficult or impossible. Costs to the sheep industry at least could be high and productivity gravely affected. To reduce the probability of introduction, imports of ruminants and semen have, for the last 16 years, been restricted to a very small number of countries presumed free from BT. Future policies will almost certainly be based on these same considerations taking into account scientific advances in diagnosis and virus detection. The establishment of an off-shore high security quarantine station will facilitate imports of ruminants even from known BT-infected countries.


Control of bluetongue in an epizootic situation: Australian plans.

Epizootics of BT outside Africa have occurred in the Middle East, Indian subcontinent, Spain and Portugal, and the United States of America. The disease has only been eradicated from a major region once, this being Spain and Portugal in 1956-60 when it was achieved by quarantine, compulsory vaccination and slaughter of some infected animals. Because of the serious economic effect that BT would have on the Australian sheep industry the policy in this country is to attempt eradication, if at all feasible. The prospects of eradication will be greatly enhanced if the disease can be diagnosed quickly, when the outbreak is still localised. The laboratory diagnosis of BT involves inoculation of blood samples, collected from febrile animals on the suspect property, into groups of susceptible sheep in insect-proof quarters. The diagnosis would be confirmed at the reference laboratory in South Africa by serological and other identity tests. The diagnosis of an outbreak that originated in the northern cattle areas might be very difficult, because of the probable mild clinical nature of the disease in cattle. It is suggested that sentinel sheep flocks be maintained in these areas in strategic places near likely points of entry. Control measures, based on current knowledge of the epizootiology and pathogenesis of bluetongue; are discussed. The disease is transmitted by Culicoides that ingest infected blood from viraemic ruminants. Control is based on reducing the number of viraemic ruminants in the infected area, reducing the population density of Culicoides, and reducing the availability of susceptible ruminants. It is vital to prevent the movement of potentially infected ruminants to new localities. Contingent plans for the eradication of an epizootic of BT in Australia have been prepared. These call for the creation of two control zones. In the inner infected area, which extends for about five miles around known infected farms, there would be an intensive disinsection programme with aerial and ground spraying. Ruminants in the infected area would probably be slaughtered. The quarantine zone would extend for another 50 miles and in this area there would be prohibition of the movement of ruminants from farms. Regular clinical inspections and serological surveys would be carried out to detect secondary outbreaks. If control measures failed, it might be necessary to mount urgently a massive vaccination campaign to prevent disastrous losses to the Australian sheep industry. The difficulties in obtaining sufficient quantities of monovalent vaccine from overseas at short notice are discussed. Plans have been made to produce BT vaccine in Australia, in a specially designed high security laboratory, should the need arise.


Eradication of subgroups A and B lymphoid leukosis virus from commercial poultry breeding flocks.

A specific-pathogen-free pedigreed breeding population of White Leghorns was determined to be free of lymphoid leukosis subgroups (LL-A and LL-B) on the basis of the standard resistance-inducing factor (RIF) test of sera of individual birds. The fourth pedigreed generation (G-4) was the first to test free of viruses of both sub-groups. In three subsequent pedigreed generations (G-5 through G-7) no viremic birds were found. The RIF test of sera was also used to detect carriers infected with LL-A and LL-B in sample populations of commercial broiler, brown-egg layer, and other White Leghorn breeding lines. When only known nonviremic dams were used for reproduction, in only one instance were viremic birds detected in samples of their progeny in succeeding generations. Some implications for a virus-free breeding program are discussed.


Morphological changes of chicken tracheas and tracheal organ cultures infected with avian infectious bronchitis virus studied in scanning electron microscope.

Chicken tracheas infected in vitro in organ culture and in vivo with avian infectious bronchitis virus (IBV) were studied by scanning electron microscopy. In in vitro infection in organ culture the columnar epithelial cells lining the tracheal ring lost their cilia and rounded up in 48 hr. There were numerous elevations and holes on the infected cell surface. Subsequently the cells sloughed off. In in vivo infection the tracheal epithelial cells lost their cilia in 4 days. These cells without cilia appeared bulging, and the surfaces were covered with microvillus-like structures. Subsequently a pseudomembrane was present in most areas. In 10 days postinfection the cilia started regenerating, and in 12 days postinfection there was complete regeneration of cells and cilia, giving a normal appearance to the trachea.


Rous sarcoma regression in chickens resistant or susceptible to Marek's disease.

In 4 experiments, strains of chickens relatively susceptible (S-strain, P-line) or resistant (N-line, PDRC) to Marek's disease (MD) were challenged with Rous sarcoma virus (RSV) at 6-8 weeks old. RSV tumors occurred in 94-100% of the birds in each strain, but the number with tumors that regressed during the 40-46-day experiment varied from 7% (S-strain) to 91% (PDRC). The N-line and P-line birds, derived from a random-bred flock with selection only for susceptibility or resistance to MD, regressed their tumors at about equal rates (respectively 59 and 65%). Thus, although the genetic strains differed in ability to regress RSV tumors, the difference was not necessarily related to genetic resistance or susceptibility to MD. Other birds in 2 of the experiments, infected 2-4 weeks earlier with JM or GA isolants of MD virus, had significantly (P less than 0.01) lower incidence of RSV tumor regression than did controls provided that they developed gross lesions of MD.


Growth of Mycoplasma synoviae in a medium supplemented with nicotinamide instead of B-nicotinamide adenine dinucleotide.

The cultivation of Mycoplasma synoviae (MS) on growth medium requires supplementation with B-nicotinamide adenine dinucleotide (NAD), an expensive and relatively unstable compound. Strains of MS were adapted (selected) to grow in a medium supplemented instead with nicotinamide, a stable and inexpensive substance that can be heat-sterilized in the medium without impairing its growth-promoting properties. MS plate agglutinating antigens grown in nicotinamide-enriched media were comparable in yield and serological reaction to antigens grown with NAD supplementation.


Avian encephalomyelitis virus in chicken pancreatic cell cultures.

Monolayer cell cultures consisting of epithelioid cells were made from pancreatic tissue of 10-to-13-day-old chicks. The maximum virus titer of the cell-culture fluid was obtained 8 days after inoculation with an embryo-adapted avian encephalomyelitis virus (AEV). Virus titers also increased in cell cultures inoculated with a chick-pancreas-passed AEV or a field isolant. Cell cultures inoculated with 3 strains of AEV maintained virus titers of 10(2.9)-10(3.7) 50% embryo-infective doses/ml for 15-20 days. In other cell cultures from pancreatic tissues of chicks preinfected orally with the chick-pancreas-passed AEV or the field isolant, the virus titers decreased for several days after cultivation and thereafter increased and persisted until at least the 25th or 30th day. Neither a cytopathic effect nor any inclusion body was observed in the cell cultures infected with AEV. No AEV-antigen-positive cell was detected by direct fluorescent-antibody technique.


Transformation of cultured rat liver cells by murine sarcoma viruses.

Cultured liver cells (TRL-2 cells), originating from a BD-6 rat, an inbred black rat strain, and possessing several marker enzymes, and characteristic ultrastructures of hepatic cells, were infected with murine sarcoma viruses (MSV: Kirsten and Gazdar strains). On day 5-6 after virus inoculation, the monolayered epithelial cells focally began to pile up forming small polypoid projections, and on day 7 round-shaped cells were released from the polypoid foci. These cells first grew in a chain or as an islet of cells, but at confluent stage, they grew in suspension with some epithelioid cells attaching to the bottom of the flasks. Virus titer and focus count gave a one-hit dose response. However, 100 to 1,000 times higher titer of MSV was necessary to transform TRL-2 cells than to transform syngeneic fibroblasts, which suggested a higher repression of MSV in the epithelial cells. Those transformed TRL-2 cells showed only murine group-specific virual antigens. One of the minimal MSV-producing, MSV-transformed TRL-2 lines was subcutaneously transplanted into weanling BD-6 rats, producing progressively growing tumors with possible histology of hepatocellular carcinoma.


Serotyping of avian infectious bronchitis viruses by the virus-neutralization test.

The antigenic relationships of 12 strains of infectious bronchitis virus (IBV) were evaluated by a virus-neutralization procedure similar to that used in typing human rhinoviruses. Such a procedure consists of reciprocal neutralization tests performed by reacting 32-320 EID50 or plaque-forming units of virus with 20 antibody units of antiserum. Eight serologic groups were identified by chicken embryo assay, and 4 by plaque-reduction (90%). In general, serologic groupings were not distinct but reflected numerous intergroup relationships. The contrasting results exhibited by indicator systems are viewed as differences in the accuracy of the methods employed. It is suggested that before an IBV classification scheme can be proposed, agreement must be reached on the most suitable indicator system, techniques must be standardized, and reference viruses and antisera distributed to several institutions for comparison testing. In addition, cross-protection studies are needed to determine the importance of IBV serotypes and/or variants on vaccine efficacy.


T-lymphocyte cell lines derived from patients with acute lymphoblastic leukemia.

Several lymphoid cell lines with thymus-dependent lymphocyte (T-cell) characteristics have been established from 2 patients with acute lymphoblastic leukemia (ALL). These cell lines are considered to be leukemic T-cells on the basis of their abnormal chromosome constitution, growth in heterologous animals, ability to form rosettes with sheep red blood cells and the absence of immunoglobulin production, and destruction by antithymus cell sera. Sera from patients with leukemia did not react with these cultured cells but there was a strong reaction with infectious mononucleosis sera despite the fact that the cultured leukemia T-cells had no detectable Epstein-Barr virus (EBV) nor its genome.


Biochemical and immunologic characterization of an oncornavirus glycoprotein.

Studies were carried out on an oncogenic C-type virus, Scripps leukemia virus (SLV), produced by a lymphoblastoid cell line (SCRF 60A) from a New Zealand Black (NZB) mouse. A 70,000 dalton glycoprotein on the virion surface constitutes about 10% of the virion amino acids and about 50% of the glucosamine. The protein can be isolated from surface iodinated SCRF 60A cells as well as from a line of nonproducer cells. This protein reacts with sera which neutralize Moloney, Kirsten, Rauscher, AKR, and, of course, Scripps viruses. These data suggest that this is the principal antigen detected by immunofluorescence on the surface of cells infected with murine leukemia virus (MuLV) and that this antigen may be involved in virus neutralization.


Spontaneous Herpesvirus saimiri lymphoma in owl monkeys.

Three examples of spontaneous malignant lymphoma were observed in owl monkeys 23, 81, and 183 days after arrival in our laboratories. The pathological features of the disease were analogous to experimentally induced Herpesvirus saimiri lymphoma. H. saimiri was recovered from 2 animals (it was not attempted from one case) and one isolate was shown to reproduce characteristic H. saimiri malignant lymphoma. Each monkey originated in Peru in contrast to our usual source of owl monkeys which originate in Barranquilla, Colombia. Samples collected from owl monkeys in Peru did not reveal antibodies to H. saimiri nor were virus isolated from cocultured leukocytes. Squirrel monkeys in the same geographical location all carried H. saimiri. The observations indicate that H. saimiri lymphoma can occur as a spontaneous disease and that the virus can cross the same taxonomic lines in nature as in the laboratory.


Experiments to vaccinate marmoset monkeys against malignant lymphoma.

Humoral antibodies can be induced in cotton topped (CT) marmosets with killed Herpesvirus saimiri (HVS) vaccines. The serum antibodies delayed, but did not prevent, malignant lymphoma in actively and passively immunized monkeys after inoculation with 10(3.8)TCID50 of cell-free HVS. Experiments are in progress to determine whether the vaccines are able to protect against smaller challenge dosages of HVS. The immunized monkeys were not resistant against transplantation of tumor cells. Infection with Epstein-Barr virus (EBV) and Marek's disease virus (MDV) did not protect against the tumors caused by superinfection with HVS. Attenuation of HVS was not achieved by passaging the HVS genome through different monkey species.


Comparisons of surface markers on Herpesvirus-associated lymphoid cells of nonhuman primates and established human lymphoid cell lines.

Herpesvirus saimiri (HVS)-owl monkey lymphoid cells were found to have high levels of surface receptor for sheep erythrocytes and erythrocytes of 3 other species. These HVS-lymphoid cells lacked a receptor for modified complement. Lymphoid cells of one HVS-owl monkey line showed evidence for the presence of surface immunofluorescent staining with anti-kappa chain serum. Cells of an established HVS-marmoset lymphoid line had similar surface markers. Of 4 established human lymphoid cell lines, all lacked a receptor for sheep erythrocytes, 3 showed evidence for the presence of receptor for modified complement, and 3 showed immunofluorescent evidence for the presence on the surface of both heavy and light chain immunoglobulin. Preliminary data on an established Epstein-Barr virus (EBV)-owl monkey lymphoid cell line indicated a lack of receptor for sheep erythrocytes, presence of receptor for modified complement, and surface immunofluorescent staining with both anti-heavy and anti-light chain sera.


EB virus: malignant lymphoma in cottontop marmosets following inoculation and recovery of the virus from cells of an experimental tumor maintained in organ culture.

The oncogenic potential of Epstein-Barr virus (EBV) was investigated in cottontop marmosets. Neoplasia resembling human malignant lymphomas, reticulum cell sarcoma type, occurred following inoculation of materials containing EBV. One of 4 monkeys that received autologous cells transformed in vitro by EBV developed lymphoma in mesenteric lymph nodes seven and one-half months after inoculation. Three of 4 marmosets inoculated with cell-free EBV developed lymphoma. The latent period for given with EBV accelerated the course of disease. Nevertheless malignant lymphoma occurred in an animal given only cell-free virus. Six of 8 marmosets inoculated with EBV demonstrated antibodies to the virus. Four marmosets not exposed to the virus, of which 2 received immunosuppressive drugs, have not developed tumors, nor EBV antibodies. EBV antigen detectable by immunofluorescences has been found in 2% of cells shed from one tumor maintained in organ culture. These results imply that EBV is capable of inducing malignant lymphoma in at least one primate species. Additional experimental evidence is required, however, before its oncogenic capacity in this host can be accepted without reservation.


A comparative study on the presence of C-type viral particles in placentas from primates and other animals.

C-type particles are found in human, baboon, marmoset, rhesus, patas, and cynomolgus monkeys. A cebus monkey and two chimpanzee placentas have failed thus far to show the presence of these particles. Of the nonprimate tissues examined one mouse and one guinea pig placenta were positive whereas a rabbit placenta was negative. Variations in number of particles present in tissues as well as number of animals of a given species containing particles are noted. The isolation of foamyviruses from placenta cultures makes critical evaluation and interpretation of findings imperative. Several considerations suggest that the presence of foamyviruses is coincidental.


Human amnion and lung tissue culture systems for possible detection and study of human RNA tumor viruses.

A human lung and an amnion cell line were identified as highly susceptible to transformation by the rhabdomyosarcoma-114 (RD-114) virus pseudotype of murine sarcoma virus (MSV). MSV transformation on these two cell lines demonstrated a) "one-hit" kinetics with an MSV stock which contained a 100-fold excess of MSV over its detectable associate RD-114 helper virus and b) only a slight increase (2X) in focus-forming titers by the addition of optimal concentrations of RD-114 helper virus. These findings indicated that the primary MSV interaction with those amnion and lung cells was that of non-productive transformation; and this was confirmed by the isolation of sarcoma-positive leukemia (helper) virus-negative (S+Lminus) cells from cloned terminal foci of MSV transformed human amnion and lung cells. These MSV-susceptible human cell lines are the first human cells identified as capable of demonstrating the defective nature of MSV. Human candidate oncornaviruses have not, however, been detected to date with the use of normal lung and amnion cells and their S+Lminus derivatives as indicator systems. These cell lines were useful for the isolation and identification of a new RD-114-like virus from a cat cell line.


Complement-fixation-inhibition as a test for antibodies in cats and humans to C-type RNA tumor virus antigen.

The complement-fixation-inhibition (CFI) test was evaluated as a means of detecting humoral antibodies in cat sera and in human sera to mammalian C-type RNA virus interspecies antigen(s). CFI antibody titers of greater than or equal 1:2 were detected in sera from all tumor bearing (23) and normal cats (23), however, sera from most germ free cats were negative. When the same cat sera were tested for blocking antibody by the paired radioiodine labeled antibody technique the correlation between the radioimmune assay and CFI tests was 85%. Sera from 378 cancer patients and 193 normal people were tested for antibodies to the mammalian oncornavirus interspecies-specific antigen in the CFI test. This test used a rabbit antiserum prepared toward a purified feline leukemia virus (FeLV) interspecies antigen. Disrupted Rauscher murine leukemia virus (RLV) was used as source of interspecies antigen in the CFI test. A significantly (P=0.01) higher number of reactions occurred with sera from patients with lymphosarcoma (70.4%), osteosarcoma (41.0%), reticulum cell sarcoma (56.7%), and rhabdomyosarcoma (31.8%) as opposed to sera from normal individuals (6.2%). Of 51 sera from patients with acute lymphocytic leukemia 23.5% (P=0.05) were reactive. Of the sera from 88 breast cancer patients 22.7% reacted, as opposed to 7.8% of 116 normal females and 13.9% of 43 patients with benign breast disease. CFI antibody titers were shown to be dependent on RLV antigen concentration. Absorption with human A and B red blood cell (RBC) and Forssman antigen did not reduce the CFI titers in human sera whereas absorption with RLV reduced them significantly. By indirect radioimmunoelectrophoresis the antibody in selected human sera was shown to be an IgG.


Immune response of healthy and leukemic cats to the feline oncornavirus-associated cell membrane antigen.

Feline leukemia is an infectious disease caused by a horizontally transmitted virus. Infection of animals or cultured cells with feline oncornaviruses results in the expression of a specific cell membrane antigen, feline oncornavirus-associated cell membrane antigen (FOCMA). The humoral antibody response to FOCMA is directly correlated with tumor progression. The measurement of this antibody is a useful tool for determining virus exposure. Using this procedure it was determined that cats living in leukemia "cluster" households as well as cats used as contact controls in virus injection experiments have a risk of infection of 90% or higher.


Quantitative and qualitative differences in DNA complementary to avian myeloblastosis virus between normal and leukemic chicken cells.

Hybridization of avian myeloblastosis virus (AMV) RNA with DNA immobilized on filters or in liquid with a vast DNA excess was used to measure the viral specific DNA sequences in chicken cells. Newly synthesized viral DNA (v-DNA) appears within an hour after infection of chicken embryo fibroblasts (CEF) with avian oncornaviruses. A fraction of newly synthesized v-DNA becomes integrated into the cellular genome and the remainder gradually disappears. A covalent linkage between v-DNA and cellular DNA was demonstrated to exist in CEF and in leukemic myeloblasts by alkaline sucrose velocity sedimentation. Hybridization of AMV RNA in DNA excess has revealed that there are 2 clases of viral specific sequences within normal as well as in leukemic cells. The 2 types of sequences differ in their rate of hybridization. The amount of both types of DNA sequences is about 2 times higher in leukemic cells than in normal cells. Both the fast- and slowly reacting sequences in leukemic cells exhibit a higher Tm (2 degrees C) than the respective DNA sequences in normal cells. Furthermore, when nucleotide sequences in AMV RNA complementary to normal DNA are removed first by exhaustive hybridization with normal DNA, the residual RNA only hybridizes with leukemic DNA but not with normal DNA. These results suggest that leukemic cells contain viral specific DNA sequences which are absent in normal cells. Endogenous v-DNA has been shown to be integrated in cellular DNA region(s) with a reiteration frequency of approximately 1,200 copies per cell and each integration unit appears to have a size approximately equivalent to the 35S RNA subunit of the viral genome. Viral sequences acquired after infection appear to be integrated in the unique region of cell DNA, or in tandem with the endogenous viral sequences.


Control of reversion of Moloney virus sarcoma virus transformed cells.

Mouse cells transformed by the Moloney murine sarcoma virus (MSV) native to the species can give rise to revertants which are supersusceptible to a second cycle of transformation. The MSV retransformed cells can give rise to several complex functional states even after several cycles of cloning: a) Formation of sarcoma positive, leukemia negative (S+Lminus) type cells of which some sublines may be inducible for MSV by halogenated pyrimidines; b) detection of initially SminusLminus cells which spontaneously become transformed and S+Lminus; c) the derivation of flat murine leukemia virus positive (MuLV+) cells which have an atypical MuLV and may become MSV+ as well as MuLV+; d) the release of free MSV from some cell clones with an apparent absence of MuLV. A general feature of all the above variants is a failure of detection of spontaneous reversion occurring after the second cycle of transformation. The nature of MuLV spontaneously released is that of a poorly replicating MuLV which exhibits cross-interference with other MuLV pseudotypes of MSV and the envelope which is most similar to that of Moloney leukemia virus (MLV). The examination for spontaneous reversion in human S+Lminus cells transformed by the same type of genome capable of good frequency of reversion in mouse cells, did not yield human revertant cells.


Detection of Epstein-Barr virus DNA in human tumors.

The presence of Epstein-Barr virus (EBV) DNA in biopsies of human tumors was tested by complementary RNA (cRNA) hybridization on membranes and DNA-DNA reassociation kinetics. DNA-DNA reassociation kinetics can detect as few as 1 EBV genome in every 50 cells, and the cRNA method can detect EBV genomes if 2 or more are present in a cell. Twenty-three out of 24 African Burkitt's lymphoma biopsies, 18 out of 23 African nasopharyngeal carcinomas (NPC), and 8 out of 24 other African tumors were positive for EBV DNA. Three cases of American Burkitt's lymphoma were tested by the cRNA method, and EBV DNA was not detected. Three out of 25 American tumors other than American Burkitt's lymphoma contained 0.1 to 0.3 genomes per cell of EBV DNA.


In vivo interactions between murine leukemia and sarcoma viruses.

Experiments have been performed with the aim of elucidating the nature and the extent of the in vivo interactions between murine leukemia viruses (MuLVs) and murine sarcoma virus (MSV). BALB/c and CBA mice, injected neonatally with Graffi or passage A Gross viruses (MuLV-Gi, MuLV-G), have been inoculated as young adults with murine sarcoma virus, Moloney strain (MSV-M). A higher percentage of nonregressing sarcomas appeared in these animals, sometimes accompanied simultaneously by leukemia. The immune reactivity of mice receiving MuLV-Gi at birth was found to be significantly depressed when evaluated by the hemolytic palque-forming cell (PFC) technique. However, in mice infected with MuLV-Gi and MSV-M the number of PFC ranged within the control values or slightly increased. The potentiation of MSV-M oncogenicity following infection with MuLV was studied in a more natural situation. Adult AKR mice, known to release endogenous MuLV continuously, were injected with MSV-M. The incidence of induced sarcomas was similar to that observed in control BALB/c mice inoculated with MSV-M. Moreover, tumors developed with a very long latent period. On the other hand, the great majority of tumors showed no regression and ultimately killed the host. Additional experiments, making use of immunologic manipulation of the host and Fl hybrids, suggest that the relative resistance to MSV-M oncogenesis in AKR mice is influenced by genetic and immunologic factors. MSV recovered from MSV-M-induced tumors in AKR and C58 mice was typed by highly specific mouse antisera. The results clearly showed that formation of a new MSV pseudotype occurred in vivo, the endogenous Gross virus acting as helper.


Hexose transport in sarcoma virus transformed cells.

Avian and mammalian fibroblast cultures transformed by type C sarcoma viruses show a dramatic enhancement of the rate of hexose transport at the beginning of transformation which is quantitatively and qualitatively different from that seen by variation in culture conditions of nontransformed control cells. The identification of this change as being a transport alteration independent of total glucose metabolism has been shown by use of nonmetabolizable analogues, 2-deoxyglucose, 3-O-methylglucose, and L-glucose. Increased transport rates were not dependent on levels of hexokinase activity. Transport studies of 3-O-methylglucose confirmed these conclusions and further revealed an additional altered nature of hexose transport after transformation by sarcoma virus. 3-O-methylglucose was not only transported more rapidly in the transformed cells than in the parental nontransformed cells, but the sugar "infiltrated" into the transformed cells despite the inhibitory effect of cytochalasin B. This was not seen with control cells. The sarcoma cells were also able to transport L-glucose in contrast to lack of uptake by nontransformed cells. Under conditions in which cell toxicity was not a factor, 2-deoxyglucose and several other sugars present in culture media inhibited transformation by sarcoma viruses. These same sugars reduced the incidence of sarcomas produced by virus in vivo when administered daily to test animals. The transport changes also correlate well with the transformed state as found by other laboratories using temperature-sensitive mutants and revertant cell lines. Collectively these data suggest that manipulation of transport systems may prove useful for control of certain malignancies.


Biochemical properties of wild mouse oncornaviruses with lymphomagenic and neurotropic activities.

The close immunologic relation between the group-specific and polymerase proteins of the wild mouse derived and the established strains of mouse type C oncornaviruses, and the lack of any unusual structural polypeptide or RNA components in the wild mouse virions, eliminate the possibility of detectable contamination by a class of virus different from the mouse oncornavirus class in the wild mouse virus preparations. Liquid hybridization studies with a wild mouse viral 70S 3H-RNA and cellular DNA under conditions of DNA excess, suggest that a significant fraction but not all of the virus-specific nucleotide sequences is present in all normal and tumored wild mouse tissues tested. These virus related sequences may possibly be attributed to a hypothetical endogenous inherited type C virus genome(s) carried by all wild mice or to an infection by one or more but not all of the different exogenous strains of wild mouse type C viruses which could possibly be present in the virus preparation used. The findings are consistent with wild mouse derived type C viruses being either entirely exogenous or a mixture of endogenous and exogenous viruses. This interpretation is also consistent with the earlier observation that certain wild mouse type C viruses are exogenously transmitted, transplacentally, and via milk. The possible relation of the virus heterogeneity or the distinct characteristics of the virus surface molecules to the diverse pathogenicity of the wild mouse oncornaviruses is discussed.


Immunological control of virus-induced tumors in primates.

Cells infected by oncogenic viruses may transform, may develop a latent carrier state, or may be destroyed but understanding of the control of the results of infection is incomplete. Even if cells transform, ultimate development of a tumor may be immunologically controlled. For example, cells of some marmoset species transform after infection with RNA tumor viruses, and animals react to the transformed cells with cell-mediated and humoral immune responses. Both virus specific and cross-reacting cell membrane antigens have been demonstrated. Immune deficiency accelerates tumor growth or causes recurrence of a regressing tumor. In contrast certain simian herpesvirus (Herpesvirus saimiri, HVS and Herpesvirus ateles, HVA), which cause no or minor disease in their natural hosts, induce lymphomas or lymphoblastic leukemias in other primate species. The immune response of the natural host species to HVS is greater than that of animals developing malignancies after experimental infection. HVS and HVA share many properties with Epstein-Barr virus (EBV) of man, including antigens appearing early and late during infection and their related antibody responses but no evidence exists that they induce malignancies in their natural hosts. However, if induction is as infrequent as that with EBV and Burkitt's lymphoma (BL), we have not observed sufficient numbers of squirrel or spider monkeys to have seen a BL-like tumor. Interference with the immune systems of animals carrying HVS or HVA may induce tumor development, and clarify our understanding of the relationships between EBV and BL.


Horizontal transmission of feline leukemia virus in cats.

Traditionally, cancer has not been considered an infectious disease although some multiple cases of leukemia in man and cattle have been reported. The discovery that feline lymphosarcoma was associated with an RNA virus (feline leukemia virus(FeLV)) meant that infectious transmission of the disease was a possibility. The critical question was whether the predominant method of transmission from one animal to another was 'vertical' (via the gametes) or 'horizontal' (via contagion or infection). A number of epidemiological studies have shown that the chances of healthy cats contracting lymphosarcoma are greatly increased when a cat with the disease lives in close proximity. It does not matter whether the healthy cats are related to the sick animal or not. It has also been established that viremic normal cats have an approximately 900 times greater chance of developing leukemia than cats whose FeLV status is unknown. Infectious FeLV is present in the excretions and blood of viremic animals. In the natural environment, feline lymphosarcoma occurs in clusters. The results in pet cats have been supported by experiments with cat colonies under controlled conditions and prove that horizontal transmission of FeLV occurs. This does not mean that epigenetic (infection in utero or via the milk) or vertical transmission cannot also occur. It should be possible to break the cycle of horizontal transmission of the virus by vaccination and thus control FeLV-related diseases.


Cytocidal action of anticancer antigens: evaluation of the sensitivity of cultured animal and human cancer cells.

The cultured cell lines Yoshida ascites sarcoma, L-1210 mouse leukemia, OAT cell line derived from human lung cancer of the oat cell type, and P3HR-1 cell line derived from Burkitt's lymphoma have been used for the cell-killing kinetics study of anticancer agents and evaluation of the sensitivity of cells using the soft agar cloning assay method. It has been found that there are 2 types of actions: 1) Type I (cytocidal and concentration-dependent action). The dose survival curves of Type I agents fit the equation log S=log nminuskD (S, surviving fraction; D, concentration of agents; n and k are constant). The sensitivity of cells can be expressed by mean lethal dose 90% (MLD90=1/k). Four cell lines were compared on this basis, and some problems concerning the chemotherapy of human cancer are discussed. Alkylating agents and anticancer antibiotics belonged to this group.2) Type II (cytostatic and time-dependent action). The dose survival curves fit the Gompertz equation S=exp[(minusbeta/alpha)(1minus e-aD)] (beta, population reduction parameter; alpha, constant). The exposure survival curve is negative exponential, indicating that exposure time rather than concentration is the key fo effective cell killing of Type II agents. Difficulties in expression of sensitivity to Type II agents are discussed. Antimetabolites, Vinca alkaloids, and L-asparaginase belonged to this group. The cell-killing kinetics of anticancer agents and comparison of the sensitivity of cells may provide some indications not only of optimal dosage schedules but also of a new approach in screening systems for truly effective agents for human cancer.


Nonhuman primate models for lymphoma, leukemia, and other neoplasms.

Herpesvirus saimiri (HVS) is an oncogenic virus for a variety of nonhuman primates. HVS does not produce overt disease upon inoculation in the natural host (squirrel monkey) but consistently induces neoplasms including lymphomas and lymphocytic leukemias in 4 other species of monkeys. Various drugs inhibit replication of HVS in vitro including cytosine arabinoside and adenine arabinoside. In addition, the lymphoma and leukemia induced in owl monkeys responds to vincristine and prednisolone, cyclophosphamide, cytosine arabinoside, and human interferon. Of the various chemical carcinogens studied, the antitumor agent procarbazine induces neoplasms in a variety of species including monkeys. Thus far this compound has induced acute myelogenous leukemia (AML), lymphoma, and hemangiosarcomas in macaques. We have induced primary liver tumors in macaques with several nitrosamines and aflatoxin B1 and these tumors produce alpha-fetoprotein (AFP) which can be assayed for both diagnosis and therapy. Thus far, therapy of hepatocellular carcinoma has been most successful with surgical resection; and the tumor mass and serum AFP have been less responsive to single agent chemotherapy. These nonhuman primate models are useful for an understanding of the cause, diagnosis, prevention, and treatment of the human disease.


Biohazards and simian viruses.

Nonhuman primates are extensively used in laboratories as experimental animals. It is necessary, however, to realize that their employment may be dangerous to man and other species of primates, if recognition of their flora and fauna, especially viral, is not considered and if appropriate controls are not followed. Several outbreaks have occurred, resulting in high mortality and morbidity of man and simian. A number of recommendations are provided which, if followed, will minimize the waste of time, money, and life.


In vitro transformation of cells from human neoplasms.

The possible involvement of RNA tumor virus genomes in human cell transformation was investigated. Forty-nine cell cultures from neoplastic, normal, or embryo tissues were examined for transformation, following inoculation of murine leukemia virus (MuLV), feline leukemia virus (FeLV) grown in human cells, or bone marrow aspirates from leukemia patients. Five cultures exhibited transformation (1 after inoculation of MuLV grown in human cells; 4 after inoculation of human leukemic bone marrow), and 4 were established as cell lines. They were derived from giant cell tumor and fibrosarcomas. The established transformed cells formed colonies in soft agar, grew progressively in immunosuppressed mice, and carried antigens common to FeLV and MuLV. Although virus particles were not seen in these cultures, 68S RNA was detected in their media. Medium from nontransformed parent cultures also contained 68S RNA but in amounts about 15 times less than in transformed cultures. Transformed human cells passaged in mice produced both type C virus particles and 68S RNA. Antigens common to MuLV and FeLV were found in these particles. However, the results of biological and serological studies indicate their difference from conventional MuLV and FeLV. The relationship of this virus and 68S RNA found in transformed cultures remains to be determined.


[Liver damage caused by laxatives. A contribution to the hepatotoxicity of 4,4'-(2-quinolylmethylene)-diphenol-hydrochloride].

A female patient developed a recurrent hepatitis-like liver damage after ingestion of a laxative containing 4,4'-(2-quinolyl-methylene)-diphenol-hydrochloride. After cessation of the drug the clinical picture improved. The hyperbilirubinemia decreased and the definitely elevated GOT, GPT, alkaline phosphatase and gamma-GTP became normal. Histologically hepato-cellular damage was seen with intra-hepatic cholestasis. As a cause for these symptoms, resembling those after taking oxyphenisatin-containing preparations, immunological procedures were thought responsible, such as "unpredictable hepatic drug reactions".


Metabolism of D- and L-norgestrel in humans.

Levels of DL-norgestrel, D-norgestrel, L-norgestrel and norethisterone were measured in blood by radioimmunoassay after oral administration of the progestin to 3 healthy male subjects and the half-lives calculated. Half-lives for D-norgestrel were much lower than those of L-norgestrel and similar to those of norethisterone. Measurement of norgestrel in blood can therefore be misleading unless the biologically active D-isomer is estimated. Since there was no difference in half-lives between norgestrel and norethisterone the greater potency of the former is probably due to it more strongly binding to target organ receptors.


Methanol extraction residue of BCG in the treatment of transplanted rat tumours.

Subcutaneous growth of immunogenic chemically induced rat sarcomata and a hepatoma was restricted when cells were injected into syngeneic animals in admixture with MER. Rats rejecting mixed inocula were immune to further challenge with the same tumour. Growth of a chemically induced mammary carcinoma which lacks detectable immunogenicity was suppressed when low cell inocula were injected in admixture with MER or intact BCG organisms, although animals were not immune to re-challenge. These studies indicate that clinically MER may be a suitable alternative to BCG for contact suppression of tumour growth or incorporation into tumour cell:adjuvant vaccines for active immunotherapy.


An electron microscopic study of tumour cell adhesiveness induced by aggregation promoting factor from rat ascites hepatoma cells.

A substance capable of inducing tumour cell aggregation, which is supposed to be a glycoprotein showing noncytotoxicity, was separated from rat ascites hepatoma cells and partially purified by chromatography. Adhesiveness of rat ascites hepatoma cells induced by this substance was characterized by gradual development of known binding structures during a period of 24 h after contact with the substance; simple apposition and intermediate junctions developed in the early stage, and desmosomes and focal tight junctions in the later stage. It was assumed that the substance might be involved in the development of such binding structures as a triggering mechanism of tumour cell adhesiveness.


Inappropriate production of collagen and prolyl hydroxylase by human breast cancer cells in vivo.

Thirty-two scirrhous cancers of breast have been examined to determine the origin of the collagen stroma in these tumours. Employing two immunohistochemical techniques it has been shown that the malignant epithelial cells in 30 of these tumours contain not only collagen but also prolyl hydroxylase, a key enzyme in collagen biosynthesis. Neither this enzyme nor collagen was detectable in the spindle cells in the stroma of these tumours. Neither the epithelium in normal breast, that in fibrocystic disease and in fibroadenomata, nor the malignant epithelium in two medullary cancers of breast contained either collagen or prolyl hydroxylase. These results strongly suggest that the malignant epithelium of scirrhous breast cancers produces its own collagen stroma and that the scirrhous reaction in these tumours is not a host response to tumour invasion. The production of collagen and prolyl hydroxylase by breast cancer cells (of the scirrhous type) therefore represents another example of inappropriate protein production by a human tumour.


Electron spin resonance study of changes during the development of a mouse myeloid leukaemia. I. Paramagnetic metal ions.

The blood, spleen and liver of mice were examined by means of electron spin resonance (e.s.r.), throughout the course of myeloid leukaemia induced by intravenous injection of leukaemic spleen cells. In blood, marked increases in the concentrations of iron transferrin and ceruloplasmin occurred within the first 3 days after injection. In the spleen, changes in the concentrations of paramagnetic copper and iron complexes were detectable by about the 5th day, before any measurable splenic enlargement, whilst in the liver changes were detectable by about the 8th day. The changes occurring in blood, spleen and liver during the development of leukaemia appear to be related and they are discussed in terms of iron transport.


Loss of anti-tumour immunogenicity of a somatic cell hybrid line with increasing subculture.

Good immunoprotection was afforded by A9/SEWA somatic hybrid cells in the C3H mouse/C3H Py tumour system, confirming results previously obtained in the A.SW mouse/SEWA tumour system. However, in this study the immunogenicity decreased with increasing serial subculture of the hybrid line and concomitant chromosome loss.


Serum antibody responses to an ascitic variant of rat hepatoma D23.

Antibody was detected by membrane immunofluorescence tests in sera of rats bearing an ascitic variant of a transplanted hepatoma, and in concentrated cell-free ascitic fluid. Ascites hepatoma cells were also shown to have immunoglobulin, possibly tumour specific antibody, bound to their surface. The kinetics of antibody responses to ascites hepatoma and hepatoma cells from solid tumours were compared: both tumour types gave positive reactions by the third day after implantation; antibody was present throughout subsequent tumour growth with the ascites whereas antibody was not detected after tumour became palpable in rats injected with hepatoma cells from solid tumour. Antibody responses to ascites tumour were investigated in rats bearing solid hepatoma tumour. Subcutaneous hepatoma did not influence the antibody response to ascites, but rats bearing intraperitoneal tumours showed a diminished serum antibody response to ascitic hepatoma.


Effects of 5-fluorouracil on the cell kinetic and growth parameters of hepatoma 3924A.

The effect of 5-fluorouracil (5-FU) on the growth and cellular proliferation of hepatoma 3924A was studied using the following parameters as indices of tumour response: (1) volume measurements, (2) cell kinetic analysis including estimates of both growth and cell loss fractions, (3) changes in tumour histology and (4) tumour DNA content and DNA synthesis. Of a series of single intraperitoneally injected doses (25-300 mg/kg body weight), 150 mg/kg interrupted tumour growth most effectively with minimal toxicity within 168 h, and after 10 days treated tumour volumes were only 42% of untreated tumour size. Doses of 25 mg/kg failed to change the rate of growth while 300 mg/kg exceeded the LD50. Alterations of both tumour cell proliferation and histology developed well in advance of changes observed in growth. A dose of 150 mg/kg body weight blocked the transition of cells from G1 through S for a 24 h interval when cell kinetics were measured by 3H-TdR autoradiography. However, 3H-UdR incorporation into DNA following 5-FU suggested that cellular recovery from the drug was delayed for an additional 24 h. Concurrently, significant losses of tumour tissue and tumour DNA occurred during the first 48 h with an expected increase in both necrotic and connective tissue. During the subsequent 120 h both tumour and necrotic tissue had returned to non-treated levels, while kinetic analysis revealed (a) a slight reduction in the cell cycle time and growth fraction and (b) an increased cell loss factor. The observations from this tumour model system suggest that before using tumour volume or weight as an index of therapeutic response, the relationship between the kinetics of tumour cellularity and tumour volume must be defined.


A new method for monitoring subclinical virus infections of the central nervous system in mice.

The course of a subclinical virus infection of the central nervous system (CNS) of mice has been followed by measuring spontaneous motor activity using Animex equipment. A significant drop in activity occurred on the 5th and 6th day after inoculation with the A774 (avirulent) strain of Semliki Forest virus (SFV) before histological changes were apparent but at the same time as maximum brain virus titre. The relationship of the fall in activity to changes in the pattern of weight gain, food and water intake was also investigated.


Morphological and virological investigations of cell strains cultured from the brain in Jakob-Creutzfeldt disease and subacute sclerosing panencephalitis.

Cell strains were established in culture from fragments of the brain from 2 cases each of Jakob-Creutzfeldt (JC) disease and subacute sclerosing panencephalitis (SSPE). After about 12 weeks strains from the former spontaneously formed persistent heaped up nodules of cells which appeared to produce reticulin-like fibrils as well as confluent sheets of rounded and spindle, fibroblast-like cells. Similar sheets of cells were obtained from the cases of SSPE but the only nodules formed were smaller and ephemeral. Attempts to detect virus in all 4 strains were made by inoculation of supernatant fluids into cultures of other laboratory cells, haemadsorption, co-cultivation, electron microscopy and immunofluorescence, and testing for interferon production. No evidence was found by any of these methods of the persistent presence of virus in the strains. Immunofluorescence revealed a probable anti-glial cell IgM autoantibody in one case of JC disease. Morphologically some cells resembled astrocytes and others fibroblasts. Those from JC disease contained more vacuoles and redundant membranes than did those from the cases of SSPE, features that are particularly striking in brain cells in human and animal cases of the spongiform encephalopathies.


Hypersensitive and insensitive extremities. The dermatological aspects.

The importance of physically intact hands and feet is taken for granted and consequently not appreciated until they are functionally impaired by vascular or neural injury of disease. The pathogenesis and clinical manifestations of cutaneous lesions complicating hypersensitive and insensitive limbs are described. The current therapeutic approaches and measures for these conditions are presented. Protection and prophylaxis trauma for neurologically impaired parts are emphasized.


Intraendothelial tubular aggregates in experimental wounds.

Intraendothelial tubular aggregates were found in small dermal blood vessels in three healing superficial wounds in patients receiving systemic corticosteroids. Similar inclusions were also demonstrated in two wounds in healthy subjects, but were not found in normal (unwounded) skin of either healthy subjects or steroid-treated patients. Tubular aggregates of similar structure have previously been described as occurring in 'connective tissue' or 'auto-immune' diseases, including lupus erythematosus and dermatomyositis, both in the skin and in internal organs. Although it has been suggested that such aggregates are of viral origin, their morphogenesis and significance remain undertermined. Our findings do not support a viral cause, but suggest that these structures are probably a product of regenerating endothelium.


Generalized bullous and haemorrhagic lichen sclerosus et atrophicus. Marked improvement with ACTH.

An unusual case of lichen sclerosus et atrophicus is described, in which lesions were bullous and haemorrhagic and extended over almost the entire surface of the skin. A marked improvement followed treatment with ACTH.


Radiography in functional lacrimal testing.

A functional study of the lacrimal drainage apparatus was made in 25 patients. Angiographin was instilled into the conjunctival sac and serial radiographs were taken. The films were interpreted in conjunction with the intubation macro-dacryocystograms. Although this is a useful procedure, the use of angiographin compares unfavourably with ultra-fluid Lipiodol in a similar procedure.


Homonymous hemianopia in multiple sclerosis. With report of bilateral case.

A patient with multiple sclerosis and bilateral retrochiasmal visual field defects is reported. Homonymous field defects are rare in multiple sclerosis despite the frequency of pathological involvement of the retrochiasmal visual pathways. A higher incidence might be found with a higher index of suspicion and careful visual field testing with qualitative confrontation technique. Other reasons for the infrequent clinical detection of retrochiasmal lesions are considered. Such lesions may exist without demonstrable defect. This may be explained by anatomical factors, for example, fibre arrangement, or physiological factors, such as, geniculate or retrogeniculate integration. Lesions producing demonstrable defects may be asymptomatic because they: affect only the peripheral field, are small scotomas that do not impair visual acuity, affect only one eye, or occur late in the course of disease when masked by optic nerve involvement.


Collagenase and other proteinases in the cornea of the retinol-deficient rat.

1. Enzymes that may contribute to liquefaction of the cornea in retinol-deficient animals and in man have been studied using rat cornea. The established technique of culturing tissue fragments and determining the activity of collagenase (EC 3-4-24-3) and other enzymes in the medium after different periods of culture was used. 2. A collagenolytic system was detected in the media from cultures of rat corneas. This system probably consists of at least two enzymes, a collagenase and a neutral proteinase. 3. Both proteolytic and specific collagenolytic activity were greater in media from retinol-deficient rat corneas. The hydroxyproline level increased in parallel with the increase in enzyme activity. 4. In the final stages of retinol deficiency the cornea is invaded by granulocytes and other cells of the blood and we suggest that destruction of cornea collagen may be due largely to the activity of the enzymes from these cells.


Conformational energy refinement of horse-heart ferricytochrome c.

The reported X-ray structure of horse-heart ferricytochrome c has been refined by conformational energy calculations, using a three-stage computational procedure. In stage I, the atomic positions are adjusted to conform to idealized bond lengths and bond angles characteristic of small amino acid derivatives, while yet remaining as close as possible to the X-ray coordinates. In stage II, atomic overlaps are eliminated by adjusting the backbone and side-chain dihedral angles to minimize the nonbonded energy, hydrogen-bonded energy, and rotational energy contributions. In the final stage of refinement, the electrostatic energy and a more accurate hydrogen-bonded energy treatment are considered, in addition to the energy contributions of stage II. A "fitting potential" of gradually decreasing strength is imposed in both stages II and III, in order to keep the computed structure as similar to the x-ray structure as is consistent with a low-energy conformation. The final computed structure of cytochrome c exhibits a very low conformational energy (-504 kcal/mol) and also closely resembles the X-ray structure (RMS deviation = 0.77 A for all atoms). However, a special treatment was required in order to alter the location of the phenyl ring of phenylalanine-82. In contrast to the originally published X-ray structure, which shows the phenyl ring pointing away from the heme, the phenyl ring in the computed structure is tucked into the heme crevice, in a position similar to that observed in the reduced form of tuna cytochrome c, in the oxidized form of Rhodospirillum rubrum cytochrome c2, and also in the recently determined structure of oxidized tuna cytochrome c.


The influence of amino acid substitutions on the conformational energy of cytochrome c.

Conformational energies have been evaluated for each of the staggered side-chain conformations associated with the 261 amino acid substitutions known to occur among 60 eucaryotic species. At least 86% of these substitutions can be sterically accommodated (one at a time) within the structure of horse-heart cytochrome c resulting from conformational energy refinement. Simultaneous incorporation of all pertinent amino acid substitutions found in eight representative species into the refined horse-heart structure is also shown to be sterically possible, with few exceptions. In two cases (Pekin duck cytochrome with 10 substitutions and Samia cynthia cytochrome with 24 substitutions), all substitutions could be readily incorporated, and the total energies associated with their computed structures differed by less than 10 kcal/mol from that of horse-heart cytochrome c. In the cytochromes from rattlesnake (22 substitutions), tuna (18 substitutions), and Neurospora crassa (36 substitutions), tyrosine could not be substituted for phenylalanine at position 46, within the constraints of the calculations. However, when all of the remaining substitutions were incorporated into these three cytochromes, their computed conformational energies differed by less than 30 kcal/mol from that of horse-heart cytochrome c. Between two and four amino acid substitutions cause high energies in the cytochromes from human, baker's yeast, and cotton seed, but all of the remaining substitutions are consistent with a low energy conformation. These results suggest that the structures of homologous proteins may be even more similar than has previously been recognized. Substitutions of all possible amino acid types at the invariant positions (where all eucaryotic cytochromes c bear the same amino acid) have revealed some cases where different amino acids can be accommodated, thus demonstrating that the biological constraints on amino acid substitutions are often different from the purely steric constraints investigated in this work.


Cobalt-cytochrome c. I. Preparation, properties, and enzymic activity.

An improved procedure for the preparation of cobalt-cytochrome c has been developed. Various factors influencing the cobalt insertion process are discussed. The optical spectra of cobalt-cytochrome c suggest a six-coordinated species. The spectral shifts occurring with oxidation-reduction are compared with those observed for deoxy-cobaltohemoglobin and ferrocytochrome c and attributed to the effect of d(z2) electron on stereoelectronic interactions between the axial ligands and the porphyrin pi systems. Cobalt-cytochrome c has Em,7 = -140 +/- 20 mV as compared to an Em,7 of +250mV for ferrocytochrome c. An explanation for this negative Em,7 is offered. Cobaltocytochrome c is oxidized by cytochrome oxidase at about 45% of the rate for native cytochrome c. On the other hand cobalticytochrome c was not reduced by microsomal NADH or NADPH cytochrome c reductase nor by mitochondrial NADH or succinate cytochrome c reductase. It appears that the integrity of the reductase binding site is destroyed and the oxidase binding site has been modified by cobalt substitution.


Controlled digestion with trypsin as a structural probe for the N-terminal peptide of soluble and membranous cytochrome c.

When purified bovine cytochrome c1 is digested with trypsin under controlled conditions, the heme polypeptide is preferentially converted from a species of molecular weight 30,600 to a heme polypeptide of molecular weight 29,000. The trypsin sensitive peptide bond is located in the N-terminal region of the cytochrome. Both the reduced and oxidized cytochrome are susceptible to hydrolysis by trypsin at the same locus, but the reduced cytochrome is cleaved at an initial rate approximately twofold greater than the oxidized cytochrome. Membranous cytochrome c1, as occurring in cytochrome b-c1 complex or succinate-cytochrome c reductase complex, is not susceptible to trypsin proteolysis under similar conditions, nor after more extensive treatment of the membranes with trypsin, in spite of the fact that cytochrome c1 presumably comes into contact with cytochrome c at the membrane surface during electron transport. These findings are consistent with a model for the structure of cytochrome c1 in situ in which the cytochrome is an integral membrane protein, located primarily in the membrane continuum, while still having the heme-containing portion of the protein available at the membrane surface for electron transfer to cytochrome c.


The specificity of induced conformational changes. The case of yeast glyceraldehyde-3-phosphate dehydrogenase.

The specificity of induced conformational changes and of the probes used to detect them has been investigated in yeast glyceraldehyde-3-phosphate dehydrogenase. Cyanylation of the active-site SH groups in two of the four identical subunits of glyceraldehyde-3-phosphate dehydrogenase has no effect on reactivity of the unmodified SH groups toward the cyanylating reagent (2-nitro-5-thiocyanogenzoic acid, NTCB) but results in total loss of catalytic activity. Cyanylation of the dicarboxamidomethylated enzyme was four orders of magnitude slower than with the unmodified enzyme in contrast to cyanylation of the dicyanylated enzyme. Cyanylation by NTCB as well as alkylation by iodoacetate and acylation with beta-(2-furyl)acryloyl phosphate are enhanced in the presence of NAD+ while alkylation by iodoacetamide is inhibited by NAD+. In the absence of NAD+, hydrolysis of the acylated enzyme is faster than phosphorolysis while the reverse is true in the presence of NAD+. NAD+ accelerates hydrolysis of the 3-phosphoglyceroylated enzyme about 60-fold but decreases the rate of hydrolysis of the furylacryloylated enzyme by a factor of 17. Other examples of the specificity of the induced conformational changes and the probes are described. The conformational changes induced by NAD+ make the protein specifically reactive toward its physiological substrates and less reactive toward extraneous competing compounds.


Thermotropic lipid clustering in tetrahymena membranes.

The effect of temperature on the core structure of endoplasmic reticulum membranes has been visualized directly in cells of the poikilothermic eukaryote Tetrahymena pyriformis by freeze-etch electron microscopy. Moreover, the effect of temperature on the smooth microsomal membrane vesicles isolated from these cells, as well as on the extracted membrane lipids, has been examined by fluorescence probing, electron spin resonance, proton nuclear magnetic resonance, and calorimetry. Freeze-etch electron microscopy of T. pyriformis cells, equilibrated at different temperatures between 28 and 5 degrees, reveals the emergence of smooth areas on the fracture faces of endoplasmic reticulum membranes at temperatures below similar to 17 degrees. In this temperature range, we also find discontinuities in the glucose 6-phosphatase activity, in the fluorescence intensity of 8-anilino-1-naphthalensulfonate, in the partition of 4-doxyldecane, and in the separation of the outer hyperfine extrema of 5-doxylstearic acid in the microsomal membranes. These membranes apparently contain at least two lipid environments of different fluidity as indicated by the 12-doxylstearic acid spin-label. Proton nuclear magnetic resonance of the extracted membrane lipids indicates an abrupt change of the fatty acid chain mobilities at temperatures below similar to 17 degrees. This, however, is not due to a true thermal liquid crystalline in equilibrium crystalline phase transition. Calorimetric measurements also support this conclusion. The thermotropic alterations observed within the membranes are interpreted to be due primarily to a clustering of "rigid" liquid crystalline lipid environments which exclude membrane-intercalating proteins.


A spin-label study on fusion of red blood cells induced by hemagglutinating virus of Japan.

Fusion of red blood cells (RBC) induced by hemagglutinating virus of Japan (HVJ) has been studied using a phosphatidylcholine spin label. The spin label was readily incorporated and diffused into the lipid bilayer portion of the viral envelope. The exchange broadening in the electron spin resonance (ESR) spectrum of densely labeled virus disappeared rapidly when the virus was mixed with RBC at 37 degrees. The spectrum gradually approached that of the host cell spin labeled with the phosphatidylcholine label. The results directly indicate transfer and intermixing of phospholipid molecules between the viral envelope and RBC membrane. The transfer reaction was strongly dependent on temperature. No transfer was observed at lower temperatures where the virus adsorbed to the cell and caused aggregation but no hemolysis and fusion. The transfer rate remained negligibly small until 19 degrees and increased rapidly between 25 and 30 degrees. The virus-induced hemolysis showed similar temperature dependence. The transfer rate was greatly reduced under inhibitory conditions of fusion: glutaraldehyde treatment of RBC, trypsin treatment of HVJ, or the presence of concanavalin A. Only slight transfer was observed from fusion-inactive influenza virus to RBC. The transfer was greatly enhanced by the help of HVJ. The close parallelism suggests that the transfer and intermixing are necessary steps to the cell fusion. The transfer rate was dependent on fluidity of the host cell membrane and independent of the viral dose. The virus-induced transfer of phospholipid molecules between RBC's was also detected by the spin label. Its temperature dependence was quite similar to that for the virus-to-cell transfer. The intercellular transfer was nearly proportional to the viral dose.


High molecular weight forms of adrenocorticotropic hormone in the mouse pituitary and in a mouse pituitary tumor cell line.

Denaturing solvents have been used to determine the molecular weight of the adrenocorticotropic hormone (ACTH) activity in mouse pituitary, in an ACTH secreting mouse pituitary tumor cell line (AtT-20/D-16v), and in the tissue culture medium from the pituitary tumor cells. ACTH activity was quantitated by radioimmunoassay and by bioassay. It is possible to utilize guanidine hydrochloride or sodium dodecyl sulfate in characterizing the multiple forms of ACTH because treatment of porcine ACTH (the 39 amino acid polypeptide form of ACTH, alpha(1-39)), pituitary extracts, tumor cell extracts, and tumor cell tissue culture medium with these denaturants does not diminish the immunological ACTH activity. Based on gel filtration in the presence of guanidine hydrocholoride, extracts of the pituitary tumor cells and the mouse pituitary contain three distinct molecular weight classes of ACTH activity. The major form of ACTH has a molecular weight similar to alpha(1-39) (molecular weight 4000-5500), but there are significant amounts of two higher molecular weight forms of ACTH: molecular weight 6500-9000 and molecular weight 20,000-30,000. The 6500-9000 molecular weight form of ACTH is the major form of ACTH in the tissue culture medium; there is no peak of alpha(1-39) size ACTH in the medium. In the radioimmunoasay all three forms of ACTH generate competitive binding curves parallel to that of porcine alpha(1-39); in the bioassay (stimulation of steroidogenesis in a mouse adrenal tumor cell line) the dose response curve for each of the molecular forms of ACTH is parallel to that for porcine alpha(1-39).


Thyrotropin-releasing hormone regulates the number of its own receptors in the GH3 strain of pituitary cells in culture.

Thyrotropin-releasing hormone (TRH), a hypothalamic tripeptide, binds rapidly and reversibly to specific membrane receptors on GH3 cells, a clonal strain of rat pituitary cells grown in culture. GH3 cells were incubated for 1-72 hr with unlabeled TRH, washed, and then incubated for 1 hr with [3H]TRH. Under these conditions 80% of any bound, unlabeled TRH exchanges with [3H]TRH in the medium, and the amount of radioactivity bound to the cells gives a measure of the number of TRH receptors. In GH3 cells, the number of available TRH receptors decreased from 92% of control after 1 hr to 35% after 48 or 72 hr of incubation with unlabeled TRH. Binding of [3H]TRH to both intact control and TRH-treated cells was half-maximal at 8 nM [3H]TRH, but the maximum amount of [3H]TRH bound was decreased by 75% in cells previously incubated for 48 hr with unlabeled TRH. Equilibrium binding studies were performed using membrane fractions prepared from control cells and cells previously exposed to TRH for various periods. The dissociation constant of the TRH-receptor complex was the same in all cases, but the maximum amount of TRH bound decreased progressively in membrane fractions from cells incubated with TRH for 1-51 hr. TRH receptors were not found in cytoplasmic fractions of control or TRH-treated cells. The loss of TRH receptors was reversible within 4 days. In the continued presence of the tripeptide the number of receptors remained low for 12 days. After incubation for 2 days with different concentrations of TRH, the number of receptors was decreased to 33% of control at 100-300 nM TRH, and half of this decrease occurred at about 1 nM TRH; half-maximal biological responses occur at 2 nM TRH. The biologically active Ntau-methylhistidyl derivative of TRH also effected a loss of receptors, while three inactive analogs of TRH did not cause reductions in the number of TRH receptors. In cultures incubated for 40 hr with cycloheximide, protein synthesis was inhibited by 85%, but the number of TRH receptors was 76% of control suggesting that the receptor has a long half-life. When GH3 cells were incubated with cycloheximide plus TRH, the number of TRH receptors decreased by only 23% as compared to a decrease of 73% in cells incubated with TRH alone, suggesting that receptor loss is partially dependent on active protein synthesis. We conclude that in GH3 cells TRH regulates the number of its own receptors.


Photoaffinity labeling of adenosine 3',5'-cyclic monophosphate binding sites of human red cell membranes.

To identify and investigate the cAMP binding sites of human red cell membranes a photoaffinity analog of cAMP, 8-azidoadenosine 3',5'-cyclic monophosphate (8-N3cAMP), has been synthesized. This analog activates cAMP-dependent protein kinase(s) in the red cell membrane. It exhibits tight, but reversible binding to the membranes which is competitive with cAMP. Photolysis of [32P]-8-N3cAMP with red cell membranes results in covalent incorporation of radioactive label onto two specific membrane proteins. This incorporation requires activating light and is reduced to background levels with addition of low levels of cAMP. Prephotolysis of 8-N3cAMP completely abolished its ability to photolabel membrane proteins. Both the reversible and photocatalyzed binding of 8-N3cAMP show saturation kinetics. The molecular weights of the two primarily labeled proteins are approximately 49,000 and 55,000. The differential effects of cAMP, ATP, and adenosine on the photocatalyzed incorporation of [32P]-8-N3cAMP onto these two proteins suggest that they have biochemically different properties. The potential usefulness of this compound for investigating various molecular aspects of cAMP action is discussed.


Photoaffinity labeling of a protein kinase from bovine brain with 8-azidoadenosine 3',5'-monophosphate.

8-Azidoadenosine 3',5'-monophosphate (8-N3-cAMP) containing 32P has been used as a photoaffinity label specific for the adenosine 3',5'-monophosphate (cAMP) binding site(s) present in a partially purified preparation of soluble protein kinase from bovine brain. 8-N3-cAMP and cAMP were found to compete for the same binding site(s) in this preparation, as determined by a standard filter assay. When this protein preparation was equilibrated with [32P]-8-N3-cAMP, and then irradiated at 253.7 nm, the incorporation of radioactivity was predominantly into a protein with an apparent molecular weight of 49,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. This labeled protein comigrated in the gel with the only protein which is endogenously phosphorylated by [gamma-32P]ATP, a protein which has been shown to be the regulatory subunit of the protein kinase (H. Maeno, P. L. Reyes, T. Ueda, S. A. Rudolph, and P. Greengard (1974), Arch. Biochem. Biophys. 164, 551). The incorporation of [32P]-8-N3-cAMP into this protein was half-maximal at a concentration of 7 x 10(-8) M. In accordance with a proposed mechanism involving the formation of a highly reactive nitrene intermediate upon irradiation of the azide, the incorporation of radioactivity into protein was maximal within 10 min of irradiation, and was almost eliminated by preirradiation of the photolabile ligand. Moreover, this incorporation was virtually abolished by a 50-fold excess of cAMP, but not by AMP, ADP, ATP, or adenosine. We suggest that 8-N3-cAMP may prove to be a useful molecular probe of the cAMP-binding site in receptor proteins and report its use in conjunction with sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a highly sensitive and selective radiochemical marker for cAMP-binding proteins.


DNA binding by cyclic adenosine 3',5'-monophosphate dependent protein kinase from calf thymus nuclei.

Cyclic adenosine 3',5'-monophosphate (cAMP) dependent protein kinase and proteins specifically binding cAMP have been extracted from calf thymus nuclei and analyzed for their abilities to bind to DNA. Approximately 70% of the cAMP-binding activity in the nucleus can be ascribed to a nuclear acidic protein with physical and biochemical characteristics of the regulatory (R) subunit of cAMP-dependent protein kinase. Several peaks of protein kinase activity and of cAMP-binding activity are resolved by affinity chromatography of nuclear acidic proteins on calf thymus DNA covalently linked to aminoethyl Sephrarose 4B. When an extensively purified protein kinase is subjected to chromatography on the DNA column in the presence of 10(-7) M cAMP, the R subunit of the kinase is eluted from the column at 0.05 M NaCl while the catalytic (C) subunit of the enzyme is eluted at 0.1-0.2 M NaCl. When chromatographed in the presence of histones, the R subunit is retained on the column and is eluted at 0.6-0.9 M NaCl. In the presence of cAMP, association of the C subunit with DNA is enhanced, as determined by sucrose density gradient centrifugation of DNA-protein kinase complexes. cAMP increases the capacity of the calf thymus cAMP-dependent protein kinase preparation to bind labeled calf thymus DNA, as determined by a technique employing filter retention of DNA-protein complexes. This protein kinase preparation binds calf thymus DNA in preference to salmon DNA, Escherichia coli DNA, or yeast RNA. Binding of protein kinases to DNA may be part of a mechanism for localizing cyclic nucleotide stimulated protein phosphorylation at specific sites in the chromatin.


Optical and magnetic resonance studies of formate binding to horse liver catalase and sperm whale myoglobin.

The binding of formate ion, a substrate for the peroxidatic reaction of catalase, has been investigated by magnetic resonance techniques. Comparative studies of formate binding to ferric myoglobin have also been performed. The nuclear magnetic relaxation (NMR) rate of formate and water protons is enhanced by the presence of ferric horse liver catalase. The enhancement is not changed significantly by the addition of cyanide, indicating that water and formate are still bound in the presence of cyanide. Formate proton to heme iron distances determined by magnetic resonance techniques indicate that formate does not directly bind to the heme iron of catalase or myoglobin but to the globin, and NMR relaxation occurs as a result of outersphere mechanisms. Evidence that water forms an innersphere complex with the iron atom of the catalase heme is presented. In similar experiments with ferric myoglobin, the addition of cyanide caused a large decrease in the enhancement of the proton relaxation rate of both formate and water, indicating the displacement of water and formate from the heme and the vicinity of the heme, respectively. Broad, high-spin, ferric ion electron paramagnetic resonance absorptions of catalase and myoglobin at room temperature obtained in the presence and absence of formate show that formate does not alter appreciably the heme environment of catalase or myoglobin or the spin state of the heme iron. Studies on the binding of formate to catalase as monitored by changes in the heme absorption spectrum in the visible region show one-to-one stoichiometry with heme concentration. However, the small changes observed in the visible region of the optical spectrum on addition of formate ion are attributed to a secondary effect of formate on the heme environment, rather than direct binding of formate to the heme moiety.


A heat stable paraoxonase (O,O-diethyl O-p-nitrophenyl phosphate O-p-nitrophenyl hydrolase) from Russell's viper venom.

Fractionation of Russell's viper venom revealed separate phosphohydrolase activities directed against p-nitrophenyl phosphate, bis(p-nitrophenyl) phosphate, p-nitrophenylthymidylic acid, and O,O-diethyl p-nitrophenyl phosphate (paraoxon). On gel fractionation, the first two activities are eluted ahead of the latter. They could be resolved further by phosphocellulose cation exchange chromatography. The hydrolytic activities directed against p-nitrophenylthymidylic acid hydrolyzing component is heat labile, while the paraoxon hydrolyzing component manifests an unusually high degree of heat stability. Gel filtration yields 9600 for the molecular weight of the "paraoxonase". This enzyme, as all known enzymes of this type, requires the presence of a divalent cation. Maximum activity is obtained in the presence of Ca2+. In the presence of Sr2+ the reaction rate is 50% of that of Ca2+; other divalent cations show lower activities. The presence of the enzyme is species specific. Of four species tested, only Russell's viper venom showed significant paraoxonase activity. Enzyme activity is intact following incubation with iodoacetate of p-chloromercuribenzoate. Activity is partially preserved even in the presence of 8 M urea.


Functional consequences of modifying highly reactive arginyl residues of fructose 1,6-bisphosphatase. Loss of monovalent cation activation.

Modification of pig kidney fructose 1,6-bisphosphatase with 2,3-butanedione (in the presence of AMP) results in the loss of activation of the enzyme by monovalent cations. Under these conditions about 8 arginyl residues per mole of enzyme were modified. No other residues were modified. No loss of monovalent cation activation occurs when modification with 2,3-butanedione is carried out in the presence of AMP plus the substrate fructose 1,6-bisphosphate and 3.2 less arginyl residues were modified. Since fructose 1,6-bisphosphatase contains 4 subunits, it is suggested that one arginyl residue per subunit plays an essential role in monovalent cation activation of the enzyme. Studies on sulfhydryl group reactivity toward 5,5'-dithiobis(2-nitrobenzoic acid) explain the protection exerted by fructose 1,6-bisphosphate against the loss of monovalent cation activation in terms of an enzyme conformational change induced by substrate, which makes unreactive the essential arginyl residue. The results of the present paper, as well as previous evidence, are discussed in terms of the mechanism of monovalent cation activation of fructose 1,6-biphosphatase.


Characterization of Novikoff hepatoma mRNA methylation and heterogeneity in the methylated 5' terminus.

KOH digestion of methyl-labeled poly(A)+ mRNA purified by (dT)-cellulose chromatography produced mononucleotide and multiple peaks of a large oligonucleotide (-6 to -8 charge) when separated on the basis of charge by Pellionex-WAX high-speed liquid chromatography in 7 M urea. Heat denaturation of the RNA before application to (dT)-cellulose was required to release contaminants (mostly 18S rRNA) that persisted even after repeated binding to (dT)-cellulose at room temperature. Analysis of the purified poly(A)+ mRNA by enzyme digestion, acid hydrolysis, and a variety of chromatographic techniques has shown that the monucleotide (53%) is due entirely to N6-methyladenosine. The large oligonucleotides (47%) were found to contain 7-methylguanosine and the 2'-0-methyl derivatives of all four nucleosides. No radioactivity was found associated with the poly(A) segment. Periodate oxidation of the mRNA followed by beta elimination released only labeled 7-methylguanine consistent with a blocked 5' terminus containing an unusual 5'-5' bond. Alkaline phosphatase treatment of intact mRNA had no effect on the migration of the KOH produced oligonucleotides on Pellionex-WAX. When RNA from which 7-methylguanine was removed by beta elimination was used for the phosphatase treatment, distinct dinucleotides (NmpNp) and trinucleotides (NmpNmpNp) occurred after KOH hydrolysis and Pellionex-WAX chromatography. Thus Novikoff hepatoma poly(A)+ mRNA molecules can contain either one or two 2'-0-methylnucleotides linked by a 5'-5' bond to a terminal 7-methylguanosine and the 2'-0-methylation can occur with any of the four nucleotides. The 5' terminus may be represented by m7G5'ppp5' (Nmp)lor2Np, a general structure proposed earlier as a possible 5' terminus for all eucaryotic mRNA molecules (Rottman, F., Shatkin, A., and Perry, R. (1974), Cell 3, 197). The composition analyses indicate that there are 3.0 N6-methyladenosine residues, 1.0 7-methylguanosine residue, and 1.7 2'-0-methylnucleoside residues per average mRNA molecule.


Nuclear ribonucleoprotein complexes containing U1 and U2 RNA.

Nuclear ribonucleoprotein (RNP) complexes that contain the U1 and U2 RNA of chromatin of Novikoff hepatoma cells were extracted with 0.01 M Tris-HCl (pH 8.0) after the nuclei were initially washed with 0.075 M NaCl and 0.025 M EDTA (pH 8.0). These RNP complexes were purified by chromatography on Sepharose 6B columns and centrifugation on sucrose density gradients. The identity of the U1 and U2 RNA in these particles was established by their electrophoretic mobility in polyacrylamide gels and their T1 RNase fingerprints which were identical with those of authentic U1 and U2 RNA (R. Reddy et al. (1974), J. Biol. Chem.249, 6486-6494; H. Shibata et al. (1974), Mol. Cell. Biochem. 4, 3-19). The nuclear riboncleoproteins had a buoyant density of 1.47 g/ml in CsCl gradients. Two-dimensional polyacrylamide gel electrophoresis of their proteins showed these RNP complexes contain 10 polypeptide spots, of which two are phosphorylated in vivo.