[Formation of nitrosyl complexes of nonheme iron (2.03 complexes) in animal tissues in vivo]. Formation or dinitrozyl non-haem iron complexes (2.03 complexes) in white rats' tissues in vivo has been studied. The formation of these complexes has been examined in the liver kidneys and small intestines when injecting p/o NaNO2 to the rats. Most of the complexes have been found in the liver. They have not been detected in the heart, spleen, muscle, cerebrum and marrow in vivo. 3-4-fold content of 2.03 complexes has been determined while introducing iron and NaNO2 in running water. This effect has not been found when introducing iron and NaNO2 simultaneously. The mechanism of 2.03 complex formation in the animal tissues in vivo has been proposed. According to this mechanism these complexes are formed in the tissues when Fe-NO from nitrozyl non-haem from complexes originating in blood passes into SH-group of protein in tissues. The formation of 2.03 complexes in vivo has been found in blood uniform elements.


[Free radical centers in the chromatophores and preparations of Rhodospirillum rubrum reaction centers]. The light-induced free radical ESR signals in chromatophores and reaction center preparation of R. rubrum were studied. It has been shown that the properties of the signals in both systems were strictly identical. At the microwave powers higher than 10 mW the ESR signal at g approximatley 2.00 is saturated and its asymmetry arises. Changes of the ESR signal shape may be explained by the superposition of P870+ signal with that from the ubisemiquinone. Addition of ferricyanide to chromatophores induces in the dark a new ESR signal characterized by a linewidth of 4.5 Oe which presumably arises from the antenna bacteriochlorophyll aggregates, because this signal is absent in the reaction center preparations.


Perinatal development of glucoeneogenic enzymes in rabbit liver. Activities of the 4 hepatic gluconeogenic enzymes: glucose-6-phosphatase, fructose-1,6-diphosphatase, pyruvate carboxylase, particulate and cytosolic phosphoenolpyruvate carboxykinase (PEPCK) have been measured in fetal rabbits (22, 25, 28, 30 and 31 days of gestation) and in fasted or suckling newborns (1 and 2 days after birth). Between days 25 and 31 of gestation, fructose 1,6-diphosphatase and particulate PEPCK activities represent 50% of adult (pregnant female) activities, while pyruvate carboxylase is present at adult values during the same period. Glucose-6-phosphatase is low and cytosolic PEPCK absent in fetal liver until 30 days of gestation and increase significantly during the day preceding birth. Al the enzymes show a further increase after birth independently of the nutritional status of the animals (starved or suckling).


The effect of fluphenazine upon social and vocational functioning in remitted schizophrenics. Thirty-six remitted schizophrenics who participated in an outpatient study of fluphenazine decanoate or oral fluphenazine vs placebo given for a year were examined for the effect of drug treatment upon social and vocational functioning. Only the period prior to any clinical relapse was evaluated. We found no difference between those on drug or placebo, and conclude that antipsychotic drugs, at least in the context of an aftercare clinic offering a rich spectrum of nonpharmacological services, and when combined with antiparkinson medication, do not interfere with social and vocational functioning.


Antibacterial effect of some combinations of pesticides of pure cultures of microorganisms. The investigations conducted showed that the antibacterial effect of dinitro-o-cresol, cuprozane, and trichlorfon is due to their chemical structure and to the species of microorganisms on which they act. Dinitro-o-cresol and Cuprozane possess selective toxicity with respect to B. anthracis and Rh. gracilis and cuprozane with respect to Ch. perfringens. The method of evaluating the effect of combinations of chemical compounds consisting of two or more components which have been developed made it possible to detect the antibacterial effect of mixtures consisting of the indicated pesticides. It was established that the combinations studied possess synergic and additive effects, which also depend on the chemical structure of the pesticides and the species of microorganisms.


In vitro transforming activity of Epstein-Barr virus (EBV). II. Differences between M81 and B95-8 EBV strains. Lymphocytes from 38 human cord blood and 9 adult circulating blood were aliquoted and infected in parallel either with the B95-8 EBV strain (produced by cotton-top marmoset lymphocytes transformed by EBV originating from an infectious mononucleosis line) or with the M81 EB virus (produced by callithrix jacchus marmoset lymphocytes transformed by EBV originating from a nasopharyngeal carcinoma (NPC) derived line). Significant differences were observed in the lymphoblastoid lines obtained and involved cell morphology, cell growth and synthesis of viral antigen. Cord blood lymphocytes infected with M81 virus resulted in lymphoblastoid lines where EA and VCA synthesis and production of virions took place, whereas this was not observed in B95-8 induced lines.


Chemotaxis of human leucocytes: II. Effects of lectins, colchicine, cytochalasin B, cyclic nucleotides and immuno-stimulatory products. Drugs or products acting as chemo-modulators were assayed for their effect on the chemotactic activity of human normal neutrophils by a method of direct microscopic observation. Lectins (PHA-P and Con A) have an inhibitory effect at the level of 10 micrograms/ml. Colchicine and Cytochalasin B have also an inhibitory effect at the level of 0.25.10(-6)M/ml and 0.20 micrograms/ml. For the cyclic nucleotides: dibutyryl cAMP and products such as norepinephrine, aminophylline that raise the cellular level of cAMP have an inhibitory effect, and whereas cGMP and carbamylcholine have a stimulatory effect Imidazole and levamisole, have some enhancement of chemotaxis but it is not significant. Lectins by binding to cell surface receptors which assume the recognition of a chemotactic gradient in the environment, colchicine and cytochalasin B by interfering with the cytoskeletal elements (microtubules and microfilaments) cyclic nucleotides involved in the metabolic activity during the cell motility. The data show that the integrity of one of these three sequences is necessary for the normal activity of chemotasxis. Animal and human models (i.e. mutant mouse bg/bg and Chediak-Higashi syndrome) give some clue of their interrelation.


Virus induced diabetes and the immune system. II -- Evidence for an immune pathogenesis of the acute phase of diabetes. The infection of normal DBA2 and BALB/C nu/ + mice with 2 X 10(2,6) TCID50 of the M variant of the EMC virus induced pathological mean glucose values of 426 +/- 99 mg/100 ml in DBA2 mice and 292 +/- 130 mg/100 ml in BALB/C nu/ + mice on day five following infection. As diabetic animals died afterwards, mean glucose values decreased in the surviving animals on day seven and fourteen. The infected immunodeficient BALB/C nu/nu mice with thymus aplasia did not show abnormal mean glucose values or higher standard deviation of the means (114 +/- 37 mg/100 ml) when compared to uninfected controls (117 +/- 23 mg/100 ml). This demonstrates that a complete thymus-dependent immune system seems to be necessary for the development of the acute stage of virus-induced diabetes in the mouse.


Hepatitis A infection and primary hepatocellular carcinoma. The prevalence of antibody to the hepatitis A antigen (anti-HAV) has been studied in Senegal, among patients suffering from primary hepatocellular carcinoma (PHC) and among healthy blood donors. Anti-HAV was found in 62.5% of 64 cases of PHC as compared to 64% of 50 blood donors. Anti-HAV was as prevalent among PHC patients who evidenced chronic hepatitis B infection as among those without markers of chronic hepatitis B infection. These data suggest that hepatitis A infection is not associated with PHC in Senegal.


Abnormal red cell membrane phosphatase activity in an unidentified congenital hemolytic anemia. The erythrocyte membrane contains a neutral phosphatase, which was studied with p-nitrophenyl-phosphate as the substrate. The enzyme was investigated in four members of the same family, suffering from a congenital spherocytic hemolytic anemia. Although the condition was transmitted as a dominant trait, it was not identified to the common hereditary spherocytosis. The phosphatase, instead of having a Michaelis-Menten kinetics, displayed a dramatically biphasic kinetics: substrate excess generated partial inhibition of the enzyme. We consider that such an abnormality, which was never encountered before, is a distinctive feature of a given type of congenital hemolytic anemia.


Electron spin resonance (esr) investigations on blood of patients with leukemia. ESR investigations of lyophilized blood of patients with acute lymphatic leukemia exhibit an increase in spin concentration and an additional peak not present in control samples. This peak disappears almost completely concomittantly with a reduction in spin concentration after treating the patients with prednisolone, vincristine, and doxorubicin or after addition of 5mM of CuCl2 to blood samples. The results show that the ESR spectra of lyophilized blood samples can be utilized to follow the effect of therapy. Moreover, they suggest that the leukemic blood possesses a high concentration of antioxidants.


Lithium side effects in a routine lithium clinic. Approximately two thirds of a group of bipolar patients attending the routine lithium clinic suffered sid effects. These patients tended to have a lower plasma and erythrocyte lithium concentration and a lower erythrocyte/plasma lithium ratio than those who had no side effects. Explanations of this unexpected finding are discussed.


The antidromic compound action potential of the hypothalamo-neurohypophysial tract, a tool for assessing posterior pituitary activity in vivo. A sensitive technique was developed for measuring continuously the summated neural activity of the rat hypothalamo-neurohypophysial tract. Large compound potentials of 4-9 mV peak amplitude were recorded from the fibre tract in response to electrical stimulation of the neurohypophysis. It was estimated that all fibres in the tract were electrically activated and contributed to the compound potential. It was shown that a decrease of peak amplitude could be used as a measure of changes in endogenous neural activity. The decrease in peak amplitude during an increase of endogenous neural activity was due to fatigue and temporal spread of the compound potential, and also to collision of antidromic and orthodromic action potentials. Decrease of the compound potentials was related to the intensity of vaginal distension, haemorrhage and plasma hyperosmolality, and it correlated with neurohypophysial hormone release. This method may usefully complement hormone assays and single cell analysis.


Behavioral and biochemical analysis of GABA-mediated inhibition in the early chick embryo. Exogenous gamma-aminobutyric acid (GABA) decreased spontaneous motility in 4-, 6-, 7-, 9-, and 13-day chick embryos; the younger embryos were more sensitive. Neither the positional isomers of GABA, alpha-aminobutyric acid (AABA) and beta-aminobutyric acid, nor the principle GABA catabolite, succinic acid, decreased motility in 4-day embryos. Several semi-rigid GABA analogues decreased motility in 4-day embryos with a potency that paralleled their effectiveness in displacing [3H]GABA in ligand-binding studies. The effects of AABA and GABA on hind-limb motility were quantitatively similar in thoracic spinal and sham-operated 7-day embryos. Bicuculline and picrotoxin elicited absolute motility increases at 6, 7 and 9 days of incubation. Picrotoxin and two bicyclophosphate GABA antagnoists elicited relative motility increases while bicuculline elicited an absolute motility increase at 4 days. The two bicyclophosphates increased motility with a potency that paralleled their electrophysiological effectiveness. L-Glutamic acid decarboxylase (GAD) activity was detected in the embryonic lumbar spinal cord at all ages examined (3--7 days) using a new radiometric cation-exchange method. Gamma-Aminobutyric acid transaminase (GABA-T) activity was detected in the lumbar spinal cord at the earliest age examined (day 5). Both GAD and GABA-T activity were detected at earlier ages than previously reported. GABA receptors, and the enzymes necessary for the synthesis and degradation of GABA, all appear to be present at (or before) the onset of spontaneous motility. GABA-mediated transmission appears to be present at 6 days and perhaps as early as 4 days.


Evaluation of receptor function in ACTH-responsive and ACTH-insensitive adrenal tumor cells. Two variant cell lines (Y6 and OS3), derived from the ACTH-sensitive mouse adrenocortical tumor clone Y1, are defective in the ACTH-sensitive adenylate cyclase system. This study further characterizes the nature of the defects in Y6 and OS3 cells using ACTH1-10, ACTH4-10, and cholera toxin. In Y1 cells, ACTH1-39, ACTH1-10, and ACTH4-10 stimulated steroidogenesis to the same maximum level with Kd' values of 5 x 10(-11) M, 5 x 10(-7) M and 10(-4) m respectively. ACTH1-10 (0.4 mM) and ACTH4-10 (3.2 mM) increased the accumulation of adenosine 3',5'-monophosphate (cAMP) in Y1 cells two- to three-fold. Cholera toxin increased steroidogenesis and cAMP accumulation in Y1 cells with Kd' values of 0.4 ng/mL and 9 ng/mL respectively. Y6 and OS3 cells responded to added cholera toxin with increased cAMP accumulation and increased steroidogenesis but did not respond to ACTH1-39, ACTH1-10, or ACTH4-10 at concentrations effective in Y1 cells. These data are interpreted to suggest that Y6 and OS3 cells are defective in a process or component that links the principal binding regions of the ACTH receptor to the catalytic subunit of the adenylate cyclase system. Attempts to were made to assess the interactions of ACTH with the principal binding regions of the ACTH receptor by analysis of binding of radioactive, iodinated ACTH1-24. ACTH binding, however, showed low affinity, high capacity, and no target-tissue specificity, and was considered not to be useful in evaluating the integrity of the ACTH receptor.


Low density lipoprotein binding, internalization, and degradation in human adipose cells. Human adipose tissue derives its cholesterol primarily from circulating lipoproteins. To study fat cell-lipoprotein interactions, low density lipoprotein (LDL) uptake and metabolism were examined using isolated human adipocytes. The 125I-labelled LDL (d = 1.025-1.045) was bound and incorporated by human fat cells in a dose-dependent manner with an apparent Km of 6.9 + 0.9 microgram LDL protein/mL and a Vmax of 15-80 microgram LDL protein/mg lipid per 2 h. In time-course studies, LDL uptake was characterized by rapid initial binding followed by a linear accumulation for at least 4 h. The 125I-labelled LDL degradation products (trichloroacetic acid soluble iodopeptides) accumulated in the incubation medium in a progressive manner with time. Azide and F- inhibited LDL internalization and degradation, suggesting that these processes are energy dependent. Binding and cellular internalization of 125I-labelled LDL lacked lipoprotein class specificity in that excess (25-fold) unlabelled very low density lipoprotein (VLDL) (d less than 1.006) and high density lipoprotein (HDL) (d = 1.075-1.21) inhibited binding and internalization of 125I-labelled LDL. On an equivalent protein basis HDL was the most potent. The 125I-labelled LDL binding to an adipocyte plasma membrane preparation was a saturable process and almost completely abolished by a three- to four-fold greater concentration of HDL. The binding, internalization, and degradation of LDL by human adipocytes resembled that reported by other mesenchymal cells and could account for a significant proportion of in vivo LDL catabolism. It is further suggested that adipose tissue is an important site of LDL and HDL interactions.


An approach to measuring the in vivo transformation of glycerol to a very low density lipoprotein triglyceride. This paper describes a new method which permits measurement of the steady-state rate of transformation of serum glycerol to a very low density lipoprotein (VLDL) triglyceride in vivo in dogs. Although the turnover of glycerol and the turnover of VLDL triglyceride glycerol have both been previously measured, the rate of transformation of the former into the latter has not. While there is considerable dog-to-dog variation in the absolute turnover and transformation rates, the relationship between the various rates is quite constant. Thus, 13% of the serum glycerol which normal fasting dogs utilize is converted to VLDL triglyceride. The remaining 87% is converted to other products. Also, 28% of VLDL triglyceride glycerol in these dogs is derived from serum glycerol. The balance, 72%, is derived from other sources. The procedure described here can be used to quantitate the contribution of glycerol to VLDL in a number of conditions in which glycerol and (or) VLDL triglyceride metabolism is altered, thereby providing another way to gain insight into the metabolism of VLDL. Even more generally, the principles developed here can be applied to estimate the transformation of other precursors to other products in vivo.


Multiple effects of serum low-density lipoprotein on cultured human fibroblasts. The effects of serum low-density lipoproteins (LDL) were studied in cultures of human skin fibroblasts grown in medium supplemented with human serum deficient in lipoproteins and in platelet factor. The LDL led to a temporary increase in the rate of cell replication, to increases in the cell content of protein and cholesterol, to an increase in average cell size, and to an increased secretion of glycosaminoglycans. The increases in cholesterol and protein were proportional to the increase in cell size, suggesting that the additional protein and cholesterol were of a structural, rather than a storage, nature. The increase in cell protein during the first few days of exposure to LDL was due to a decrease in the rate of protein degradation. Ultrafiltration of the serum to remove substances of molecular weight less than 30,000 did not reduce the basal rate of cell proliferation but did prevent the stimulation of proliferation by LDL; it did not alter the effect of LDL on cell protein and cholesterol, indicating that the latter responses are independent of the mitogenic action. The response of cells from diabetic donors did not differ from that of normal cells.


Presence of herpes simplex virus type 2 DNA in hamster cells oncogenically transformed by ultraviolet-inactivated virus. Herpes simplex virus type 2 (HSV-2) DNA has been detected by molecular hybridization in hamster fibroblast cells oncogenically transformed by ultraviolet-irradiated virus. At early passages after cloning in soft agar, about 40% of the HSV-2 genome was present in all the transformed cell lines at one to six copies per cell. In cell lines derived from tumors induced by these cells, the same percentage of the HSV-2 genome was also found with more uniform number of copies (between two and three). Thus the presence of viral DNA seems to be necessary for the maintenance of the transformed state in these cell lines.


Studies of two temperature-sensitive mutants of Mengo virus. Studies of the synthesis of viral ribonucleates and polypeptides in cells infected with two RNA- ts mutants of Mengo virus (ts 135 and ts 520) have shown that when ts 135 infected cells are shifted from the permissive (33 degrees C) to the nonpermissive (39 degrees C) temperature: (i) the synthesis of all three species of viral RNA (single stranded, replicative form, and replicative intermediate) is inhibited to about the same extent, and (ii) the posttranslational cleavage of structural polypeptide precursors A and B is partially blocked. Investigations of the in vivo and in vitro stability of the viral RNA replicase suggest that the RNA- phentotype reflects a temperature-sensitive defect in the enzyme. The second defect does not appear to result from the inhibition of viral RNA synthesis at 39 degrees C, since normal cleavage of polypeptides A and B occurs in wt Mengo-infected cells in which viral RNA synthesis is blocked by cordycepin, and at the nonpermissive temperature in ts 520 infected cells. Considered in toto, the evidence suggests that ts 135 is a double mutant. Subviral (53S) particles have been shown to accumulate in ts 520 (but not ts 135) infected cells when cultures are shifted from 33 to 39 degrees C. This observation provides supporting evidence for the proposal that this recently discovered particle is an intermediate in the assembly pathway of Mengo virions.


Myoinositol biosynthesis by Sertoli cells, and levels of myoinositol biosynthetic enzymes in testis and epididymis. Levels of glucose-6-phosphate cyclase (myoinsitol-1-phosphate synthase, EC 5.5.1.4) and myoinositol-1-phosphate phosphatase (myoinositol-1-phosphatase, EC 3.1.3.25) were determined in extracts of testes from10-, 20-, and 30-day-old rats, and in extracts of Sertoli cells, germinal cells, and epididymides. The specific activity of the cyclase was approximately 1/10th that of the phosphatase in all extracts found to contain either enzyme. Among cells in the testis examined, Sertoli cells had highest levels of enzymes required for inositol biosynthesis from glucose, while spermatocytes and round spermatids did not have detectable activity. Spermatozoa from the epididymis also had no detectable cyclase or phosphatase activity. In contrast, extracts of washed epididymides contained exceedingly high specific activities of these enzymes. Primary cultures of Sertoli cells, maintained in a chemically defined medium without added inositol, released inositol into the medium during three successive 24-h periods. The amounts released were greater in cells stimulated by dibutyryl cyclic AMP. Results were interpreted to indicate that inositol in the fluid of seminiferous tubules most probably originates from Sertoli cells, which synthesize inositol from glucose. Additional inositol in the fluid of epididymal tubules could readily be provided by metabolism of glucose by epididymal epithelial cells


Photooxidation of NADH by 2,3-butanedione: a potential source of error in studies on active site arginyl residues. Incubation of NADH or NADPH with 2,3-butanedione in aqueous solution results in photooxidation of the reduced pyridine nucleotides under conditions of ordinary laboratory lighting. Maximum rates of photooxidation are obtained at pH 7 and with light at a wavelength of 410 nm. This reaction could lead to artifactual results in experiments on the role of arginyl groups in enzymes in which a reduced pyridine nucleotide is used to protect the active site residues from modification by 2,3-butanedione.


Restoration of virulence of axenically cultivated Entamoeba histolytica by cholesterol. The lost pathogenicity of two strains of Entamoeba histolytica, one isolated in 1924 and the other in 1967, grown in axenic culture for the past 5 and 6 years respectively, was restored by supplementing the culture medium with cholesterol through a number of transfers. The number of passages in the cholesterol-supplemented medium, necessary to restore a certain degree of pathogenicity of the two strains in hamsters, was proportional to the total time of in vitro cultivation of the strain, and not just the time of cultivation under axenic conditions. Pathogenicity, once restored, persisted for a long time after cholesterol treatment was stopped.


Infection of inbred strains of mice with Sendai virus. A comparative study of the consequences of Parainfluenza type 1 (Sendai) virus infection in inbred (C57B1/6J, C57Br, CBA, DBA) strains and a randomly bred (Swiss white) strain of mice showed significant mortality in the inbred strains but not in the randomly bred ones. This difference may be partly related to the high levels of virus growth obtained in the lungs of the inbred strains contrasted to little or no virus growth in the lungs of the Swiss white mice. A similar difference between these mice was found in the incidence of virus involvement of a variety of mouse tissues. These differences in mortality and in vivo growth of virus were not clearly mimicked in antiviral-antibody production in these different mouse populations.


Second-step concentration of viruses in drinking and surface waters using polyethylene glycol hydroextraction. In our laboratory, virus adsorbed to talc--Celite layers is eluted with 100 mL of 10% fetal calf serum (FCS) in normal saline (pH 9.0). A further 10-fold reduction in the volume of the eluate was necessary before its inoculation into cell cultures. A 100-mL volume of an experimentally contaminated sample was placed in a dialysis sac and hydroextracted overnight (4 degrees C) with polyethylene glycol (PEG) 6000. The viscous material remaining in the sac was resuspended in 10 mL of Earle's balanced salt solution. After membrane filtration (0.2 micron), the concentrate was plaque assayed in BS-C-1 cells. Using this technique, recoveries of five laboratory-adapted enteric viruses (polio 1, echo 6, coxsackie B5, coxackie A9, and reo 3) and four freshly isolated enteric virus strains (polio 1, echo 1, coxsackie B3, and reo) ranged from 87 to 97%. In comparative tests, PEG hydroextraction was simpler and superior to organic flocculation.


[Induction and repression of the collagenase synthesis in Acinetobacter sp]. The synthesis of collagenase in Acinetobacter sp. was found to be inducible by denatured collagen and by its high molecular weight fragments. The presence in the inducer of part of the tertiary structure appear to be indispensable. On the other hand, an addition of Casamino acids, meat protein hydrolysate, or a mixture of amino acids with a similar composition to gelatin does not stimulate collagenase synthesis. Enzyme production was severely repressed in the early phase of growth by glucose, arabinose, and ribose, single amino acids, proline, hydroxyproline, alanine, glutamic acid or casein acid hydrolysate. A mechanism of repression similar to catabolite repression was involved in the phenomenon caused by carbohydrates. However, the fact that cyclic adenosine 3'5-monophosphate did not overcome the repression caused by amino acids or Casamino acids, in contrast to classical catabolite repression, suggests that these two forms of repression may be distinct.


High yields of coatless spores of Clostridium perfringens strain 8--6 in a defined medium. Coatless spores of mutant strain 8--6 of Clostridium perfringens were formed reproducibly at levels exceeding 10(7)/mL in a defined medium. Confirming previous observations, the spores are quite stable in water and resistant to heat, octanol, and ethanol. They also resist 5% phenol. Electron microscopy confirms, with further evidence, the probable absence of any structure exterior to the cortex. The levels of sporulation attained are sufficient to permit recovery and purification for future studies on physicochemical and germination properties. The defined medium makes possible studies on spore coat (and exterotoxin) biosynthesis and coat assembly.


The superiority of combination chemotherapy over single agent chemotherapy in small cell lung carcinoma. From June 1974 to October 1976, 288 patients with small cell undifferentiated lung carcinoma were entered into a randomized, controlled study comparing the two noncycle-active induction regimens of cyclophosphamide vs. the combination of cyclophosphamide, doxorubicin and imidazole carboximide (DTIC). Patients were stratified by extent of disease, previous radiotherapy and performance status. Responding patients and those who did not progress were then randomized to receive their initial regimen alone, or their initial regimen with added cycle-active therapy (vincristine, hydroxyurea and methotrexate). While only 4/34 (12%) evaluable patients treated with cyclophosphamide achieved a response (greater than 50% regression), a final total of 119/217 (57%) evaluable patients on the three drugs have responded (p = 0.005). The survival curve for all the combination-treated patients was significantly better than for those treated with cyclophosphamide alone (p = 0.012). There was no demonstrable statistical superiority in length of remission or survival for patients on the combination who received in addition cycle-active consolidation therapy. In the combination chemotherapy group, survival duration was longer for patients with limited disease than extensive disease (p = 0.035). There was a strong correlation between quality of remission produced by the combination and survival.


Radical radiation therapy of advanced lung cancer: evaluation of prognostic factors and results of continuous and split course treatment. One hundred patients with inoperable (80) or unresectable (20) bronchogenic carcinoma without evidence of spread beyond the thorax and supraclavicular nodes were planned for radical radiotherapy. Seventy-six patients received continuous irradiation (6000 rads in 30 treatments in 6 weeks, TDF 99) and 24 received split course therapy (2 courses of 2500 rads in 10 treatments with a 3 week break between courses, TDF 88). Forty-three patients had squamous cell carcinoma or adenocarcinoma and were considered to have favorable prognostic factors, while 57 patients had unfavorable prognostic factors: undifferentiated large cell or small cell carcinoma, supraclavicular metastases, SVC obstruction, superior sulcus tumors, or bone erosion in continuity with the tumor. Ninety-two patients completed the planned course of treatment. In patients completing treatment, local control of cancer within the irradiated volume was achieved in 58.5% of continuously irradiated patients and 45.4% of patients receiving split course therapy. Median survival was 1.2 months in patients not completing treatment and 12 months for the patients who completed treatment; 19% of the total group survived 3 years. Median and 3 year survivals of 14 months and 20.4% and of 9 months and 11% were observed for patients treated continuously and by the split course techniques, respectively. Corresponding survival figures for patients with favorable and unfavorable prognostic signs were 21 months and 26%, and 4 months and 11%, respectively. Implications of these data for treatment planning and patient selection for radical radiotherapy in bronchogenic carcinoma are discussed.


Delayed radiation necrosis of brainstem related to fast neutron beam irradiation: case report and literature review. A 42-year-old man developed necrosis of the brainstem 10 months following fast neutron irradiation of a recurrent adenocystic carcinoma of the right submandibular salivary gland. The neoplasm had been diagnosed 15 years earlier. Neck dissection and several local excisions failed to control local extension, so that 7000 rad 4.8 MeV x-rays were administered. The right mandible and part of the tongue and palate were resected for recurrence and chemotherapy and transfer factor were given. Because of cranial neuropathy and erosion of the skull base, fast neutron radiation (2.080 rads) was administered five and one-half years after the x-ray therapy, but to a nonoverlapping field. Ten months later, rapidly progressive symptoms and signs of multiple cranial nerve palsies on the right side and left hemihypesthesia and hemiplegia appeared. Clinical manifestations of brainstem necrosis lasted 4 months. Postmortem examination demonstrated radionecrosis of pons and upper medulla, predominantly on the right side. This case illustrates the efficacy of neutron radiation in eradicating local carcinoma and also the serious complications of this therapeutic modality.


Elevated prolactin levels in bronchogenic carcinoma. The frequency and significance of hyperprolactinemia was studied in 21 consecutive, untreated male patients with bronchogenic carcinoma. Seven patients (33%) were found to have elevated serum prolactin (hPRL) levels. No correlation was demonstrated between increased hPRL levels, tissue histology, or tumor burden. L-dopa suppression and/or TRH stimulation tests were obtained in three untreated patients with hyperprolactinemia. The results of these tests were considered normal suggesting hypothalamic control.


The value of carcinoembryonic antigen in patients with carcinoma of the lung. Carcinoembryonic antigen levels in 682 lung cancer patients have been studied in order to assess their value in the screening of high-risk populations, monitoring total surgical ablation and projecting the effectiveness of therapy. The initial values are shown to be related to the histology of the tumor and to the extent of the disease. All histologic types of lung cancer produce elevated CEA but adenocarcinoma characteristically produces higher values than small, large or squamous cell carcinomas. CEA has its most precise value in distinguishing at an early date cured patients subsequent to surgical resection from those patients who will eventually fail because of recurrent disease.


Histopathology of nasopharyngeal carcinoma: correlations with epidemiology, survival rates and other biological characteristics. A total of 363 cases of nasopharyngeal carcinoma (NPC) in Singapore were classified into squamous cell carcinoma (SCC; 73 cases), non-keratinizing carcinoma (NKC; 178 cases) and undifferentiated carcinoma (UC; 172 cases). Possible biological differences between these histologic types and between tumors with and without lymphocytic infiltration were investigated by correlations with survival rates and with selected epidemiologic, immunovirologic, and immunogenetic data on the disease. The 5-year survival rates following radiotherapy were 25.3% for all cases and 58.8% for tumors restricted to the nasopharynx. The 5-year survival rate for SCC was poorer than for the combined NKC and UC groups (p less than 0.05). The 3-year survival rate was better for tumors with lymphocytic infiltration (p less than 0.05), but there were no differences in the 5-year survivals. The survival rates were better in females (p less than 0.01) and in the younger age groups (p less than 0.01). There were no significant correlations between histopathology of NPC and the distributions of cases by age, sex, HLA antigen profiles, or cell-mediated immune status. Squamous cell carcinoma was associated with lower levels of antibodies to the Epstein-Barr nuclear antigen (p less than 0.05), but there were no differences with respect to antibodies against other EBV related antigens. These findings support the view that SCC, NKC, and UC of the nasopharynx, as defined in the WHO classification, are variants of a fairly homogeneous group of neoplasms in the Singapore population.


Relationship between survival and histologic type in small cell anaplastic carcinoma of the lung. Survival and histologic subtype were compared in 61 patients with small cell anaplastic lung cancer. Patients with lymphocyte-like (oat cell) and fusiform histologies treated with chemotherapy had longer median survivals than the polygonal and other varieties on the same treatment. Likewise, when detectable disease was confined to the chest and supraclavicular nodes, the patients with lymphocyte-like and fusiform types lived longer. The improved survival in the lymphocyte-like and fusiform categories accounted for most of our improved overall median survival rates with the COPP regimen in small cell lung cancer. Survival in the polygonal and other types was not appreciably different from that seen in non-small cell lung cancer (squamous and adenocarcinoma). It may be possible to refine treatment plans on the basis of cell type so as to further increase survival in small cell anaplastic lung carcinoma.


Chemotherapy of nonirradiated malignant gliomas. Phase II: study of the combination of methyl-CCNU, vincristine, and procarbazine. Twenty-eight adult patients with nonirradiated malignant gliomas of the brain were administered a combination of methyl-CCNU (130 mg/m2, p.o., day 1), vincristine (2 mg/m2, i.v., day 1) and procarbazine (100 mg/m2, p.o., days 2 to 15) (MVP), scheduled to be given at successive 6 week intervals. Nineteen (67.9%) were not responsive to MVP and 9 (32.1%) were. Of 16 who had previous partial resection of their tumors, 8 (50%) responded to MVP and 8 (50%) did not. Of 12 who had previous biopsy, only 1 (8.3%) responded. Overall 1-year survival rate for the 28 patients was 28.6%. Major side effects of MVP were leukopenia, thrombocytopenia, pulmonary emboli, and thrombophlebitis, detected mainly during the first 20 to 24 weeks of treatment.


Absence of carcinoembryonic antigen-like material in mesothelioma: an immunohistochemical differentiation from other lung cancers. This study is to examine the potential usefulness of immunohistochemical staining for carcinoembryonic antigen (CEA)-like material in the differential diagnosis of mesotheliomas (12 cases) from other lung cancers (14 cases) that had been previously diagnosed by transmission and scanning electron microscopy and conventional light microscopy. Indirect immunofluorescent staining for CEA was carried out on formalin-fixed paraffin-embedded sections, and the slides were examined under code. All 9 cases of diffuse mesothelioma were negative, and all 12 cases of adenocarcinoma and bronchioloalveolar carcinoma were positive for CEA-like material. Three localized mesotheliomas and a carcinoid tumor were also negative. A squamous cell carcinoma was positive. A positive immunohistochemical result for CEA-like material in lung cancers will raise the possibility of its being of bronchial epithelial origin.


Sclerosing hemangioma of the lung: an ultrastructural study. A well-circumscribed nodular mass was excised from the lingula of the left lung of a 40-year-old-female. The lesion was discovered on routine chest x-ray examination and corresponded to a typical "sclerosing hemangioma" described by Liebow and Hubbell. Electron microscopic study revealed unequivocal epithelial cells in both the irregular spaces and the solid areas of the tumor; some of these cells were identical to granular pneumonocytes. The true vascular component was sparsely distributed. We concluded that "sclerosing hemangioma" was a misnomer because we were dealing with an essentially epithelial lesion.


Cell kinetic analyses of human malignant brain tumors (gliomas). Administration of 3H-thymidine, either with or without mitostatic agents, to 24 patients with various gliomas revealed 1) in viable areas a labeling index (LI) for glioblastomas equal to 5-15%, 2) an LI of 1% or less in well-differentiated gliomas, and 3) the LI of anaplastic astrocytomas was intermediate and reflected the extent of anaplasia. Growth fraction was 0.14-0.44 in malignant gliomas (average 0.31 +/- 0.10), and cell cycle time obtained by means of stathmokinetic analysis was 75.6 +/- 45.7 hr. With few exceptions, the patients whose tumors showed an LI of 5% or more died within 1 year after the onset of disease, and no patients whose tumors at biopsy had an LI of over 5% survived more than 6 months. In contrast, there was a far better prognosis for patients whose tumors demonstrated an LI of less than 1%.


Virus-associated hemophagocytic syndrome: a benign histiocytic proliferation distinct from malignant histiocytosis. Nineteen patients whose bone marrow smears showed histiocytic hyperplasia with prominent hemophagocytosis were found to have a clinicopathologic syndrome associated with active viral infection. High fever, constitutional symptoms, liver function, and coagulation abnormalities and peripheral blood cytopenias were characteristic findings. Hepatosplenomegaly, lymphadenopathy, bilateral pulmonary infiltrates, and skin rash were often present. Fourteen of the patients were immunosuppressed. Active infection by herpes group viruses was documented in 14 patients and by adenovirus in 1. The bone marrow of most patients also showed decreased granulopoiesis and erythropoiesis with normal to increased numbers of megakaryocytes. Treatment generally consisted of supportive therapy and withdrawal of immunosuppressive drugs. Thirteen patients recovered. Lymph node biopsy and autopsy material showed generalized histiocytic hyperplasia with hemophagocytosis. The relationship of this disorder to familial hemophagocytic reticulosis, familial erythrophagocytic lymphohistiocytosis, histiocytic medullary reticulosis, and malignant histiocytosis is discussed. Immunosuppressive and cytotoxic therapy may be contraindicated in the treatment of this virus-associated syndrome.


Carcinogenic effect of N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine, a postulated proximate pancreatic carcinogen in Syrian hamsters. N-Nitroso(2-hydroxypropyl)(2-oxopropyl)amine (HPOP) proved to be a potent carcinogen in Syrian golden hamsters. The compound is an in vivo metabolite of N-nitrosobis(2-hydroxypropyl)amine, N-nitrosobis(2-oxopropyl)amine (BOP), and N-nitroso-2,6-dimethylmorpholine and a postulated proximate pancreatic carcinogen in hamsters. As with BOP, HPOP induced a higher incidence of pancreatic ductular adenocarcinomas than did N-nitrosobis(2-hydroxypropyl)amine and N-nitroso-2,6-dimethylmorpholine, and these neoplasms showed a great tendency for invasion and metastasis. Also, HPOP induced tumors of the forestomach, liver, gallbladder, kidneys, and vagina (as did BOP). However, HPOP [unlike BOP, but like N-nitrosobis(2-hydroxypropyl)amine and N-nitroso-2,6-dimethylmorpholine] led to tumor development in the nasal cavity, larynx, trachea, intestine, Harderian gland, lips, and flank organ. The possible mechanisms of HPOP carcinogenicity are discussed.


Comparison of virus-positive and virus-negative cases of feline leukemia and lymphoma. One hundred eighty-four cases of feline leukemia and lymphoma diagnosed in Boston from 1972 through 1976 were investigated. Fifty-eight % of these cases were lymphoma, and 42% were leukemia. Sixty-seven % of the cats had positive fluorescent antibody tests for circulating feline leukemia virus. The rest (33%) were virus negative. Clinically and epidemiologically, the virus-positive and virus-negative cases were remarkably similar except for their age at diagnosis. Virus-negative cats tended to be older (mean age at diagnosis, 4.9 years) as compared to virus-positive cats (3.5 years). For 22 cases of leukemia and lymphoma diagnosed after the age of 8 years, 15 were virus negative. The minimum mean induction period (time from first positive virus test to diagnosis of cancer) for 19 cats that were virus positive and healthy at their first test was 16.7 months (range, 2 to 41 months).


Content of gonadotropins in cultured human malignant cells and effects of sodium butyrate treatment on gonadotropin secretion by HeLa cells. Twenty-one of 82 human cell lines examined for production of human chorionic gonadotropin and its subunits (HCG-alpha and HCG-beta) produced either one or both subunits at some phase in their growth. Of these, 14 produced an excess amount of free alpha subunit, and seven produced HCG-beta or complete HCG without evidence for free alpha subunit synthesis. Five of the HCG-producing cell lines also contained or secreted the beta subunit of human luteinizing hormone. CBT cells derived from a glioblastoma multiforme and JAR choriocarcinoma cells secreted significant amounts of the beta subunit of human luteinizing hormone, while three other cell lines (breast carcinoma MCF-7, HeLa S3, and melanoma A375) produced small amounts of the beta subunit of human luteinizing hormone but did not appear to secrete it. Two cell lines (the melanoma line A375 and the SV40-transformed line SV80) appeared to contain small amounts of human follicle-stimulating hormone. Sodium butyrate caused a 40-fold induction in the secretion of both HCG-alpha and HCG-beta by HeLa S3 cells, but the total amount of HCG-alpha secretion induced was 800-fold greater than that of HCG-beta. Induction was blocked by actinomycin D (1 microgram/ml) and cycloheximide (5 microgram/ml) but was not affected by 1-beta-D-arabinofuranosylcytosine at a concentration (5 microgram/ml) that blocked DNA synthesis 99%. These results indicate that a number of malignant human cell lines produce the subunits of both placental and pituitary gonadotropins and that there is frequently an excess secretion of the free alpha subunit common to these hormones.


An infectivity assay for bovine leukemia virus using the immunoperoxidase technique. This report describes the development of a 7-day infectivity assay for bovine leukemia virus (BLV) which is based on the induction of the major internal virion antigen (a protein with a molecular weight of 25,000) in susceptible indicator monolayer cell cultures. In this assay, the antigen is detected in the indicator cells by the immunoperoxidase antibody technique using a specific rabbit antiserum to a BLV protein with a molecular weight of 25,000. The immunoperoxidase infectivity assay is specific, quantitative, reproducible, and more sensitive than the previously described syncytia induction assay. The immunoperoxidase infectivity assay can be applied to the detection of BLV-infected cells and provides a reliable method for the direct diagnosis of BLV infection in cattle.


Lymphocyte transformation for the detection of herpesvirus-induced tumor-associated antigens. Cell-mediated immunity to fibroblasts transformed by herpes simplex virus type 2 was investigated with a lymphocyte assay system. The assay system was first standardized with phytohemagglutinin, a nonspecific stimulator of blastogenesis. Hamster splenic and blood lymphocytes reacted to phytohemagglutinin with a dose-response curve similar to that reported for other rodent species. Splenic lymphocytes from hamsters bearing isografts, induced by herpes simplex virus type 2, were transformed by cell-free virus-induced tumor antigens. The reactions with cell-free tumor antigens were dose-dependent and paralleled the findings with phytohemagglutinin. The initial transformation response of immune lymphocytes to homologous tumor antigens occurred after 72 hr incubation with antigen. Immune splenic lymphocytes from hamsters were also significantly stimulated with antigens obtained from cells productively infected with herpes simplex virus type 2. Immune lymphocytes were not stimulated with heterologous antigens from simian virus 40-transformed mouse or hamster cells. Likewise, lymphocytes from hamsters sensitized to cells transformed by simian virus 40 reacted with both simian virus 40-transformed mouse and hamster cells but did not react with cells transformed by a heterologous virus. The results suggest that under defined conditions a lymphocyte transformation assay may be useful for the specific detection of common viral-induced antigens on tumor cells.


Enhanced transformation of xeroderma pigmentosum variant cells by ultraviolet light-irradiated simian virus 40. The role of DNA repair in transformation was investigated by infecting repair-deficient xeroderma pigmentosum (XP) variant cells, XP variant heterozygous cells, and normal human fibroblasts with simian virus 40 which had been irradiated by ultraviolet light. The transformation frequencies obtained were compared to those observed for unirradiated virus. While normal and heterozygous cells showed no differences between transformation frequencies using either irradiated or untreated virus, two XP variant cell lines were transformed 2- to 7-fold more readily with irradiated virus than with unirradiated virus. XP variant cells were also found to produce lower than normal quantities of virus following infection with either damages or undamaged virus, suggesting that increased viral production was not contributing to the increased transformation seen for these cells. Finally, the proportion of cells which repair ultraviolet light-irradiated simian virus 40 was found to be similar for wild-type and XP variant cells, suggesting that enhanced transformation in the mutant cells was not associated with a reduction in the numbers of cells which repair damaged virus. Several possible mechanisms to account for the increased transformation of XP variant cells by ultraviolet light-irradiated simian virus 40 are proposed.


Ornithine decarboxylase activity, cell proliferation, and tumor promotion in mouse epidermis in vivo. The effect of different phorbol esters and of mechanical treatment on the activity of ornithine decarboxylase in mouse epidermis in vivo was investigated. The strong promoter 12-O-tetradecanoylphorbol-13-acetate as well as the weak promoters phorbol dibenzoate and the 12-O-tetradecanoylphorbol-13-acetate analog 12-O-tetradeca-2-cis, 4-trans-6,8-tetraenoylphorobol-13-acetate strongly increased the activity of the enzyme and the intraepidermal level of putrescine, with a maximum at 5 hr after application, when applied in doses which evoke comparable proliferative and irritant responses in skin. The hyperplasiogenic but nonirritant and almost nonpromoting 4-O-methyl ether of 12-O-tetradecanoylphorbol-13-acetate did not show such effects. Mechanical removal of the uppermost horny layer led to a considerable increase of ornithine decarboxylase activity after 4 to 8 hr, while skin massage showed only a minute effect under conditions in which both treatments exhibit about the same mitogenic efficiency. Neither manipulation promotes tumor development. After skin massage, the induction of ornithine decarboxylase was influenced neither by treatments which alter the cyclic adenosine 3',5'-monophosphate level in epidermis (inhibition of phosphodiesterase, beta-adrenergic stimulation, and injection of dibutyryl cyclic adenosine 3',5'-monophosphate) nor by injection of epidermal G1 chalone. The results indicate that no clear-cut correlation exists between epithelial cell proliferation, development of hyperplasia, and tumor promotion on the one hand and an activation of epidermal ornithine decarboxylase on the other.


The potential histogenic relationship of the peripheral nerve to synovioma. The belief that synoviomas differentiate from the synovial membrane has been widely accepted. Absolute proof is lacking, mainly because precancerous synovial atypia has not been documented in human tissue. During the last 50 years, pain has emerged as one of the significant features of synovioma, occurring very early in the course of the disease in some patients. Gross relationship to nerve, cumulative histological data, and theoretical relationships with three other cancers of neural origin suggest the nerve sheath as an alternate parent tissue for synovioma. Three cases illustrating nerve involvement are included.


Incorporation of HLA antigens into the envelope of RNA tumor viruses grown in human cells. Virion antigens of feline leukemia virus are associated with HLA antigens in the membrane of human cells infected with the virus. Feline leukemia virus produced by these cells incorporates host membrane antigens into the virion envelope, making the virus susceptible to lysis by antisera directed to HLA or to uninfected cells plus complement.


Defective reactivation of ultraviolet light-irradiated herpesvirus by a Bloom's syndrome fibroblast strain. We have used the technique of host cell reactivation of UV-irradiated herpes simplex virus type 1 as a measure of the repair capacity of three Bloom's syndrome skin fibroblast strains. At low multiplicity of infection (less than 6 x 10(-4) plaque-forming unit/cell), reactivation of the virus by the Bloom's syndrome strains was indistinguishable from that by normal strains. Reactivation at higher multiplicities was measured using an infectious centers assay. At 3 plaque-forming units/cell, survival of UV-irradiated herpes simplex virus was higher in all cell strains as a result of the multiplicity reactivation effect. This effect was, however, much smaller in one Bloom's syndrome strain, GM1492, than in either the normal strains or the other Bloom's syndrome fibroblasts. The defect in GM1492 was manifest only at relatively high multiplicates of infection. Thus, at 0.01 plaque-forming unit/cell, the GM1492 strain appeared normal, using the infectious centers assay. Clonal survival of the UV-irradiated GM1492 fibroblasts was also normal. Caffeine at 4 mM had little effect on either virus or cell survival following UV irradiation. The results indicate that the Bloom's syndrome strain GM1492 may be deficient in one of the cellular functions responsible for the multiplicity reactivation effect. These effects include complementation and recombinational events. Alternatively, the GM1492 strain may have a defective UV repair system which becomes saturated at high levels of damage.


Temperature-dependent alteration in immunogenicity of tumor-associated transplantation antigen monitored via paraformaldehyde fixation. Paraformaldehyde-fixed mKSA tumor cells of BALB/c mice were shown to retain tumor-associated transplantation antigen (TATA) activity to a degree comparable to that of X-ray-inactivated tumor cells under the optimal conditions of 1% paraformaldehyde for 30 min at 37 degrees. Unexpectedly, the TATA activity of cells fixed below 10 degrees was greatly reduced. This temperature effect was reversible. TATA activity was restored if the cells were returned to 37 degrees before fixation. Fixation at all temperatures for longer than 2 hr or at paraformaldehyde concentrations greater than 1% also caused a decrease in immunogenicity. Spleen cells from mice immunized with tumor cells fixed at 37 degrees were able to more effectively neutralize tumor growth in the Winn assay compared with those from mice immunized with cells fixed at 0 degrees. Immunization with paraformaldehyde-fixed tumor cells was completely specific. Mice immunized with an antigenically unrelated tumor were not rendered immune to tumor challenge. Fixed tumor cells could be stored for at least 1 month without loss of TATA activity.


A thymus cell marker in murine leukemia virus-induced lymphomas of rats. A specific marker for an immature population of thymus cells in the rat was shown by the rosette formation between thymus cells and guinea pig erythrocytes. This method was used to classify murine leukemia virus-induced rat lymphomas. Eight of nine Gross virus-induced rat lymphoma lines, which originated in the thymus, formed rosettes; whereas Friend, Rauscher, or Moloney virus-induced rat lymphoma lines, which originated in either the thymus, spleen, or mesenteric lymph nodes, did not form rosettes. The percentage of the total cells which formed rosettes in the Gross lymphoma lines decreased with in vivo passages. If the tumor cells were exposed to trypsin treatment, then the tumor cells would form rosettes. Lymphoma lines which lacked rosette-forming cells did not show rosette formation after trypsin treatment. An immunofluorescence test showed that none of the lymphoma lines induced by Gross, Friend, Rauscher, or Moloney viruses carried the surface immunoglobulin characteristic of B-cells. These results suggest that Gross lymphomas may be derived from the thymic cortex and that Friend, Rauscher, or Moloney lymphomas may be derived from either mature thymus cells (non-rosette-forming cells) or from a subpopulation of the B-cell series which does not have the surface immunoglobulin G receptor.


Influence of adrenal corticosteroids on the susceptibility of cats to feline leukemia virus infection. The natural resistance of adult specific-pathogen-free cats to feline leukemia virus (FeLV) was abrogated by treatment with various doses of a synthetic corticosteroid, methylprednisolone acetate (MPA), prior to either oral-nasal or i.p. inoculation of FeLV. Persistent viremia was induced in 82% (18 of 22) of MPA-treated cats versus 11% (1 of 9) of age-matched control cats. MPA-treated FeLV-inoculated cats developed prolonged lymphopenia (2 to 8 weeks postinoculation) and a delayed antibody response to the feline oncornavirus-associated cell membrane antigen. The distribution of FeLV group specific antigen in tissues of MPA-treated, FeLV-inoculated cats suggested that corticosteroids enhanced susceptibility to FeLV by impairing early viral containment in the reticuloendothelial and lymphoid tissues.


Interaction of thoracic irradiation and chemotherapy on local control and survival in small cell carcinoma of the lung. The records of all patients with small cell carcinoma of the lung seen in the Division of Therapeutic Radiology at The Medical College of Wisconsin from 1971 through 1977 were reviewed. Of 69 patients (34 with limited disease and 35 with extensive disease), 35 received irradiation only, nine received local plus total-body irradiation, and 25 received irradiation plus chemotherapy. In spite of the prompt response of the tumor, 16 of 35 patients who were only irradiated failed locally. Two patients who received local plus total-body irradiation failed in the chest. Only four of 25 irradiation plus chemotherapy patients failed locally in spite of much lower doses of thoracic irradiation. Limited-disease patients who had local control by irradiation had a slight increase (29 vs 22 weeks) in median survival. Patients receiving irradiation plus chemotherapy (three with limited and 22 with extensive disease) survived for a median of 56 weeks if the local tumor was controlled and for a median of 28 weeks if there was local failure. Regardless of therapy, survival beyond the median is greatly influenced by local tumor control.


Phase II evaluation of vindesine in patients with non-small cell carcinoma of the lung. Fifty-two patients with non-small cell carcinoma of the lung were treated iv with doses of vindesine at 3--4 mg/m2/week iv. Partial responses occurred in all histologic types in ten of 46 adequately treated patients, for an overall response rate of 22%. Patients not previously treated with chemotherapy had a higher response rate than those who had received prior chemotherapy (33% versus 12%). Reversible peripheral neuropathy occurred in all patients, and was generally of a mild to moderate degree. Mild leukopenia was seen frequently, with a median wbc nadir of 2900/mm3. As reported with the older vinca alkaloids, platelet-sparing with occasional episodes of thrombocytosis occurred with vindesine. It is concluded that vindesine is an active agent in non-small cell carcinoma of the lung and that further studies in previously untreated patients are indicated.


Phase II evaluation of thioTEPA for treatment of central nervous system tumors. Nineteen patients with primary malignant gliomas were treated with thioTEPA. It was found to be ineffective in 13 evaluable patients.


Liproprotein inhibitor of bone marrow cells in tumor-bearing rats. The role of a plasma inhibitor of erythropoiesis is evaluated in rats with Walker-256 carcinoma (W-256). Plasma from tumor-bearing rats was treated by gel filtration chromatography (Sephadex G-150) and fractions were combined into four pools on the basis of mol. wt. Inhibitory activity was assayed by adding an aliquot of the plasma fractions to normal rat marrow cells which were cultured for 24 hr with and without erythropoietin. 59Fe-heme synthesis, [3H]thymidine DNA synthesis, and 14C-leucine protein synthesis were studied. The results indicated that cultures containing the high mol. wt. pool (greater than 400,000 daltons) had significantly decreased heme, DNA and protein synthesis. This inhibitor also diminished the response to erythropoietin in polycythemic mice. The lower mol. wt. pool stimulated heme synthesis in vitro. To identify the inhibitor further, plasma lipoprotein classes were isolated by density gradient ultracentrifugation. The very low density lipoprotein (VLDL) and chylomicron fractions markedly inhibited DNA, protein and heme synthesis. Low density and high density lipoprotein fractions were inactive. A lipoprotein inhibitor of erythropoiesis was also identified in cancerous ascitic fluid, and to a lesser degree, in normal rat plasma. We suggest that this VLDL inhibitor of marrow erythropoiesis is a contributing factor in the anaemia of cancer.


The mechanism of sensory transduction in the sensilla of the trochanteral hair plate of the cockroach, Periplaneta americana. The trochanteral hair plate of the cockroach leg contains approximately 60 hair sensilla that are deflected by a joint membrane during flexion of the leg. Previous work has shown that the organ is a mechanoreceptor which limits leg flexion during walking by reflex connections to flexor and extensor motoneurons. Functional analysis of the largest sensilla has shown that their behaviour may be well approximated by a velocity detector followed by a unidirectional rectifier. We report here the results of an examination of the largest sensilla by scanning and transmission electron microscopy in an attempt to correlate the structure with the known functional elements. Each hair is innervated by a single sensory dendrite which is surrounded by an electron dense dendritic sheath. The dendrite terminates below the hair shaft in a tubular body containing a parallel array of microtubules embedded in an electron dense matrix, while the dendritic sheath extends beyond the tubular body to form the walls of the ecdysial canal. At the proximal end of the tubular body the dendritic sheath and sensory dendrite are anchored to the cuticular socket by a fibrous dome which seems to form a flucrum around which the tubular body can be deflected by movements of the hair. We suggest that the basis for the detection of velocity shealth and the tubular body. The structure is also discussed with relation to the mechanism of sensory transduction and the possible causes of the unidirectional sensitivity.


Infundibular neurons of the human hypothalamus simultaneously reactive with antisera against endorphins, ACTH, MSH and beta-LPH. In man, discrete neurons of the infundibular (arcuate) nucleus contain compounds that can be stained with anti-endorphin (alpha and beta), anti-ACTH, anti-MSH (alpha and beta) and anti-beta-LPH immune sera (I.S.). In the fetus, certain neurons stain with anti-beta-endorphin or anti((17--39)ACTH starting from the 11th week of fetal life. At the ultrastructural level, these neurons contain elementary granules that are immunoreactive with anti-beta-endorphin. In the adult, neurons immunoreactive with anti-beta-endorphin are found in the infundibular nucleus. Their axonal fibers terminate around blood vessels in the neurovascular zone and in the pituitary stalk, or establish contacts with non-immunoreactive perikarya of the infundibular nucleus. These neurons can be stained with anti(17--39)ACTH and anti-beta-endorphin I.S. The most reactive are also stained moderately with anti-alpha-MSH, anti-beta-MSH, anti-beta-LPH, anti-alpha-endorphin, or anti(1--24)ACTH I.S. These results indicate that, in man, compound(s) identical with or immunologically related to endorphins, beta-LPH, ACTH and MSH are secreted by certain hypothalamic neurons. These agents probably originate from a common precursor molecula similar to the so-called pro-opiocortin.


Electron microscopic study of the giant cells in the olfactory bulb of labyrinth fish (Belontiidae, perciformes). The giant cells in the olfactory bulb of labyrinth fish, most likely belonging to the nervus terminalis, show ultrastructural features of active synthesis of secretory material. Dense core vesicles (70--100 nm in diameter), found in the perikaryon as well as in the axon, are the possible storage sites of the secretory substance. Its chemical nature is unknown. In some of these vesicles acid phosphatase is demonstrable. Large membrane-bound bodies (up to 2000 nm in diameter), either containing an electron dense matrix or debris of cytoplasmic organelles, are also acid phosphatase-positive, suggesting their lysosomal nature. Some other ultrastructural characteristics of these cells are also described.


The distribution of endogenous chicken retrovirus sequences in the DNA of galliform birds does not coincide with avian phylogenetic relationships. The chicken is a domesticated form of Red Jungle-fowl (Gallus gallus), which belongs to the Pheasant family (Phasianidae) within the order Galliformes. Domestic chickens carry the genome of the endogenous retrovirus RAV-O as DNA sequences integrated into host chromosomes transmitted through the germ line. We have examined the presence and distribution of RAV-O-related sequences in the DNA of Red Junglefowl and other closely related species of Junglefowl, as well as more distantly related Pheasants and Quail. DNA sequences homologous to RAV-O were analyzed by molecular hybridization in liquid and after electrophoresis of restriction endonuclease fragments. The presence of RAV-O-related sequences in avian DNA does not correlate with phylogenetic relationships. Under stringent conditions of hybridization in liquid, DNA sequences homologous to RAV-O cDNA were detected at high levels (greater than 80% homology( only in the genomes of the domestic chicken and its phylogenetic ancestor, the Red Junglefowl (Gallus gallus). The DNA of two other species of Gallus (G. sonnerati, Sonnerat's Junglefowl and G. varius, Green Junglefowl), of Ring-necked Pheasant and of Japanese Quail contained sequences with less than 10% homology to RAV-O cDNA. Under conditions permitting mismatching, however, Ring-necked Pheasant DNA hybridized up to 50% of the RAV-O cDNA, and Quail DNA 24%, whereas the extent of hybridization to Sonnerat's and Green Junglefowl DNA was not markedly increased. Analysis of restriction enzyme digests revealed several distinct fragments of DNA hybridizing to chick retrovirus cDNA in both Red Junglefowl and domestic chicken, and multiple fragments in DNA from two species of Phasianus. No fragments with sequences related to chicken retroviruses were found, however, in digests of DNA prepared from Sonnerat's, Ceylonese and Green Junglefowl, from two other Pheasant genera (Chrysolophus and Lophura), or from one Quail genus (Coturnix). Thus the DNA of three Junglefowl species closely related to Gallus gallus lacked RAV-O sequences while the DNA of more distantly related Phasianus species showed significant homology. These results show that RAV-O-related sequences have not diverged together with the normal host genes during the evolution of the Phasianidae. Although RAV-O sequences are endogenous in all domestic chickens and Red Junglefowl studied thus far, it appears that the RAV-O genome has been introduced relatively recently into the germ line of Gallus gallus, following speciation but before domestication, and independently of the related sequences found in members of the genus Phasianus.


The roles of the simian virus 40 tumor antigens in transformation of Chinese hamster lung cells. Simian virus 40 mutants and deletions between 0.54 and 0.59 map units direct the synthesis of defective 20K t antigens (Crawford et al., 1978). These deletion mutants transformed actively growing CHL cells nearly as efficently as did wild-type virus, in either the focus formation assay or the growth in soft agar assay. In contrast, when CHL cells were in a resting state during infection, the transformation frequency of the mutants relative to wild-type dropped approximately 50 fold. The presence of the phorbol ester, TPA, diminished this difference. CHL cell lines transformed by the deletion mutants and selected by the focus assay grew almost as efficiently in soft agar as lines transformed by wild-type SV40. Both produced tumors in nude mice. The function of the 20K t antigen is discussed.


Loss of integrated viral DNA sequences in polyomatransformed cells is associated with an active viral A function. Rat cells transformed by polyoma virus contain, in addition to integrated viral DNA, a small number of nonintegrated viral DNA molecules. The free viral DNA originates from the integrated form through a spontaneous induction of viral DNA replication in a minority of the cell population. Its presence is under the control of the viral A locus. To determine whether the induction of free viral DNA replication was accompanied by a loss of integrated viral DNA molecules in a phenomenon similar to the "curing" of lysogenic bacteria, we selected for revertants arising in the transformed rat populations and determined whether these cells had lost integrated viral genomes. We further investigated whether the viral A function was necessary for "curing" by determining the frequency of cured cells in populations of rat cells transformed by the ts-a mutant of polyoma virus following propagation at the permissive or nonpermissive temperature. A large proportion of the revertants isolated were negative or weakly positive when assayed by immunofluorescence for polyoma T antigen and were unable to produce infectious virus upon fusion with permissive mouse cells. The T antigen-negative, virus rescue-negative clones can be retransformed by superinfection and appear to have lost a considerable proportion of integrated viral DNA sequences. Restriction enzyme analysis of the integrated viral DNA sequences shows that the parental transformed lines contain tandem repeats of integrated viral molecules, and that this tandem arrangement is generally lost in the cured derivatives. While cells transformed by wild-type virus undergo "curing" with about the same frequency at 33 degrees or 39 degrees C, cells transformed by the ts-a mutant contain a much higher frequency of cured cells after propagation at 33 degrees than at 39 degrees C. Our results indicate that in polyoma-transformed rat cells, loss of integrated viral DNA can occur at a rather high rate, producing (at least in some cases) cells which have reverted partially or completely to a normal phenotype. Loss of integrated viral DNA is never total and appears to involve an excision event. The polyoma A function (large T antigen) is necessary for such excision to occur. In the absence of a functional A gene product, the association of the viral DNA with the host DNA appears to be very stable.


The structure of the termini of the DNA of Epstein-Barr virus. We have studied the DNA of Epstein-Barr virus (EBV) isolated from the B95-8 strain of that virus (Miller and Lipman, 1973). When EBV DNA is partially digested with lambda-exonuclease and allowed to reanneal, up to 50% of the full-length molecules circularize. The arrangements of nucleotide sequences containing the terminal repeats identified in this circularization experiment have been determined. Those fragments of viral DNA generated by digestion with restriction endonucleases which are terminal and contain the terminal repeats have been identified by their sensitivity to digestion of full-length DNA by lambda-exonuclease and by virtue of their being partially homologous to one another. The population of DNA molecules in the B95-8 strain of EBV was found to be nonuniform. The nonuniformity results from different molecules having different numbers of a 0.37 megadalton terminal repeat at each end. About 70% of molecules have four terminal repeats at one end, while four equal classes, each comprising approximately 25% of the population, have one, two, three or four repeats at the other end. The arrangements of nucleotide sequences identified as being terminal in virion DNA were studied in the intracellular circular viral DNA of cells transformed by a single particle on EBV. All fragments produced by digestion with endonucleases and scored as being terminal in virion DNA were absent from intracellular circular DNA. An additional fragment was identified in the digests of intracellular DNA of each transformed clone. The molecular weights of the new fragments equal the sum of the molecular weights of two terminal fragments which are joined upon intracellular circularization of viral DNA.


Structure of the 3' hairpin termini of four rodent parvovirus genomes: nucleotide sequence homology at origins of DNA replication. The nucleotide sequences of the 3' termini of the DNA from four autonomous rodent parvoviruses have been determined. The terminus of each genome exists as a Y-shaped hairpin structure involving 115 or 116 nucleotides. The sequence of this region of DNA is highly conserved and shows no evidence of internal sequence heterogeneity, a characteristic which is observed in the terminal nucleotide sequence of the helper-dependent, adeno-associated viruses (Berns et al., 1978a). The implications of these results with respect to the models of parvovirus DNA replication are discussed.


Sequence of inverted terminal repetitions from different adenoviruses: demonstration of conserved sequences and homology between SA7 termini and SV40 DNA. We have established the nucleotide sequence for the inverted terminal repetition of human adenovirus type 3, a subgroup B adenovirus. The repetition, which is 136 bp long, shows a high degree of homology with the known sequence for the inverted repetition of adenovirus type 5 (Steenbergh et al., 1977) a subgroup C adenovirus. Partial sequence information convering 120 bp of the inverted terminal repetitions of human serotype 12, a subgroup A member, and of simian adenovirus type 7 has also been obtained. A comparison of the established sequences shows that the terminal repetitions, in particular the first 50 bp from the ends, contain sequences that have been well conserved in adenovirus evolution. For instance, only six mismatched base pairs were detected among the first 50 bp in the repetitions of simian adenovirus type 7 and human adenovirus type 5, although the homology between simian adenovirus 7 and human subgroup C adenoviruses was estimated to be only 30%. A 14 bp sequence located 9-22 nucleotides from the ends is present in DNAs from all the human serotypes examined as well as in simian adenovirus 7 DNA. Furthermore, the simian adenovirus 7 repetition contains a 21 bp sequence which is present in SV40 DNA, close to the origin of DNA replication.


SV40 recombinants carrying rabbit beta-globin gene coding sequences. We have constructed and propagated two SV40 recombinants in which different portions of the viral late gene region are replaced by a rabbit beta-globin coding sequence derived from a cloned cDNA (Maniatis et al., 1976). One of these recombinants, which retains the promoter, leader and intervening sequences for a viral late mRNA, directs the synthesis of a stable SV40-globin hybrid transcript. Using a sensitive radioimmunoassay, we have shown that this globin RNA is translated in infected monkey cells. A second recombinant, which retains the late region promoter but lacks the RNA splicing region, produces neither a stable globin transcript nor any detectable beta-globin. These experiments indicate that some sequence within a 500 bp SV40 segment, presumably the splicing region, is required for the accumulation of stable mRNA. They also demonstrate how hybrid transducing viruses can be used both to characterize viral regulatory sequences and to produce new gene products in cultured cells.


SV40 recombinants carrying a functional RNA splice junction and polyadenylation site from the chromosomal mouse beta maj globin gene. We have introduced a fragment of the chromosomal mouse beta major globin gene into SV40 and used the resultant hybrid virus to infect cultured monkey kidney cells. The mouse DNA fragment, which contains an intervening sequence and a poly(A) addition site, has been inserted in both possible orientations relative to the SV40 late region promoter. While the fragment is transcribed regardless of orientation, the RNA splice signal and poly(A) addition site are utilized only when the fragment is inserted in the "sense" orientation. Thus genomic mouse signals for both splicing and polyadenylation are recognized across species boundaries. Furthermore, since only an 18 bp segment was included on the 5' border of the intervening sequence, we can define a maximum length for this splice signal.


Effect of organotin compounds and hexachlorophene on brain adenosine cyclic 3',5'-monophosphate metabolism. The effect of triethyltin (TET), triphenyltin (TPT), hexachlorophene (HCP) and cuprizone on adenosine cyclic 3',5'-monophosphate (cyclic AMP) production in rat brain was examined both in vitro and in vivo. TET and TPT inhibited basal adenylate cyclase activity of brain homogenate at a concentration as low as 1 microM in vitro but these compounds had no effect on norepinephrine (NE) and dopamine(DA)-stimluated enzyme activity. HCP and cuprizone failed to inhibit adenylate cyclase activity. In vivo TET given intravenously at a dose rate of 10 mg/kg decreased the cyclic AMP content of cerebrum, but not of medulla. TPT and HCP give intravenously and intraperitoneally respectively failed to decrease the cyclic AMP content of the cerebrum. In the case of TET the reduction in cyclic AMP content of the cerebrum was prevented by maintaining the rats normothermic after treatment. On the basis of these results the inhibition of adenylate cyclase produced by TET in brain homogenates in vitro would not appear to be involved in the development of nervous changes associated with acute TET toxicity, or in the production of progressive brain oedema caused by TET, HCP and cuprizone.


Field and experimental studies of poxvirus infections in rodents. Data are presented from serological and virological investigations of natural and experimental infections, in rodents, with different poxviruses. The demonstration of poxvirus antibodies (virus neutralizing antibodies, antihaemagglutinins, and precipitins) in the kidneys and/or lungs of rodents from Europe and Africa, and the isolation of poxviruses from them, all indicate that rodents may be poxvirus carriers. Isolates from rodents differed in their properties. Some, from Turkmenistan rodents or from white rats caught near Moscow, appeared to be very close to cowpox virus, while others (from Zaire rodents) were identical to variola-like (whitepox) viruses found earlier in monkeys in the same region. The results suggest that rodents that carry the virus closely similar to cowpox virus might be a source of infection for other animal species.


[Vasoactive intestinal peptide (VIP): specific receptors and adenylate cyclase activation in a human prolactin-secreting pituitary tumor]. Receptors for the Vasoactive Intestinal Peptide (VIP) were characterized in particles enriched in plasma membranes obtained from a human prolactin-secreting pituiatry tumor. Native VIP inhibited competitively the binding of 125I-VIP to the particles and stimulated cyclic AMP production; both these effects were observed at concentrations of VIP as low as 10(-11)-10(-10) M, which are compatible with VIP concentrations in the hypothalamopituitary portal blood.


[Cooperative non-specific binding of the cyclic adenosine 3'--5'-monophosphate receptor protein (CRP) from Escherichia coli to double-stranded thymus and lambda pgal DNA]. Either free or combined with cAMP, CRP binds cooperatively to double-stranded thymus and lambda pgal DNA. The affinity of CRP for both DNAs in these non-specific interactions is increased by cAMP without noticeable change in the degree of cooperativity. Values of the intrinsic association constant, cooperativity parameter, and site size of DNA were determined from ultracentrifugal investigations under near-physiological ionic conditions.


[Effects of EDTA on (Na+-K+) ATPase bound to human erythrocyte membranes]. Membranes preincubation with high EDTA concentration induced an alteration on (Na"-K+) ATPase kinetic behaviour. This effect can be related to a change in calcium content tightly bound to the membranes.


Suppression of renin secretion in the rat kidney by a nonvascular alpha-adrenergic mechanism. We studied the effect of alpha-adrenergic stimulation, using phenylephrine, on basal and isoproterenol-provoked renin secretion in the isolated perfused rat kidney. Infusion of phenylephrine increased renal perfusion pressure and prevented the response in renin secretion to isoproterenol. No suppression of basal secretion was observed. Renal vasoconstriction was abolished, and the response in renin secretion to isoproterenol was restored by alpha-adrenoceptor blockade with phenoxybenzamine. In contrast, when renal vasoconstriction was prevented by dihydralazine, suppression of renin release by phenylephrine still occurred. These observations support an inhibitory effect of a nonvascular alpha-adrenergic mechanism on renin release. We suggest that the alpha receptor mediating this effect is related directly to the juxtaglomerular cell.


Effect of acetylcholine on the norepinephrine-induced positive chronotropy and increase in cyclic nucleotides of isolated rabbit sinoatrial node. Transmural stimulation of, or application of nicotine to, the isolated rabbit sinoatrial (SA) node resulted in initial negative and late positive chronotropy. Simultaneous application of acetylcholine and norepinephrine produced a similar biphasic chronotropic effect. These procedures produced an initial increase in cyclic guanosine 3':5'-monophosphate (cyclic GMP) and a delayed elevation in cyclic adenosine 3':5'-monophosphate (cyclic AMP). The initial and late effects on rate and nucleotide levels were inhibited by pretreatment with atropine and propranolol, respectively. Pretreatment with atropine shortened the time of maximum increase in cyclic AMP level and heart rate from 3 to 1 minute after the simultaneous application of acetylcholine and norepinephrine and enhanced the positive chronotropic effect. Physostigmine prolonged the duration of the increase in cyclic GMP and negative chronotropic effect after the simultaneous application. These results suggest that when acetylocholine and norepinephrine are present simultaneously in the SA node region, the former interacts predominantly with muscarinic receptors and stimulates the cyclic GMP system, which in effect delays the cyclic AMP elevation and reduces the positive chronotropic effect of norepinephrine. However, these effects of acetylcholine cannot be explained solely on the basis of changes in the cyclic GMP level, because sodium nitroprusside produced a marked elevation of the cyclic GMP levels without decreasing the heart rate and did not affect the norepinephrine-induced increase in pacemaker rate and cyclic AMP. Sodium nitroprusside may affect cyclic GMP pools other than those susceptible to acetylcholine. These cyclic GMP pools may not exert chronotropic effects.


Effects of the oral converting enzyme inhibitor, SQ 14225, in a model of low cardiac output in dogs. Dogs with thoracic caval constriction retain sodium and develop ascites and edema. The role of the renin-angiotensin-aldosterone system in this model of low output failure was evaluated before, during, and after administration of the new orally active converting enzyme inhibitor, 2-D-methyl-3-mercaptopropanoyl-L-proline (SQ 14225). The acute response to the initial oral dose of SQ 14225 (10 mg/kg) consisted of a striking fall in plasma aldosterone concentration (PAC) from 22.7 and 29.9 ng% to 10.7, 11.9, and 11.0 ng% (P less than 0.05) after 67.5, 112.5, and 157.5 minutes; sodium excretion increased from 1.9 and 1.9 mu Eq/min to 19.9, 22.4, and 17.8 mu Eq/min. Arterial pressure and filtration fraction decreased (P less than 0.05), and plasma renin activity (PRA) increased (P less than 0.05) after the initial dose of SQ 14225; clearance of paraaminohippuric acid (PAH) and creatinine did not change significantly. The daily responses for 3-4 days to SQ 14225 (35 mg/kg per day, given as doses of 10, 10, and 15 mg/kg) were a decrease in PAC from 50 +/- 15 and 32 +/- 10 ng% to 10 +/- 4 ng% on the 4th day, a value not statistically different from normal (P greater than 0.05), and an increase in sodium excretion from 2.9 to 2.0 mEq/day to 5.7, 10.0, 32.4, and 32.9 mEq/day on a sodium intake of 35 mEq/day (P less than 0.05 for the last 2 days). Arterial pressure and creatinine clearance decreased (P less than 0.05). PRA increased transiently on day 1 of SQ 14225 and then returned toward control levels, and clearance of PAH was unchanged. These data demonstrate an important role for aldosterone and the renin-angiotensin system in the retention of sodium and in ascites formation in dogs with thoracic caval constriction.


Decreased cardiac beta-adrenergic receptors in deoxycorticosterone-salt and renal hypertensive rats. The development of experimental deoxycorticosterone-salt (DOCA-salt) and renal artery clip hypertension in rats is associated with alterations in the sensitivity of the myocardium to adrenergic stimulation. We studied beta-adrenergic receptors and isoproterenol-stimulated adenylate cyclase in myocardial membranes from hypertensive rats to determine whether this altered sensitivity is associated with any change in beta-adrenergic receptors. The specific binding of the beta-adrenergic antagonist, 125I-iodohydroxybenzylpindolol, was used to measure numbers and affinities of receptors in myocardial membrane preparations. Cardiac membranes from both DOCA-salt and renal hypertensive rats showed significantly fewer beta-receptors than did membranes from control, normotensive rats. Receptor affinity remained unchanged. This decrease was from 110 +/- 19 to 49 +/- 5 fmol/mg protein for DOCA-salt hypertension and from 110 +/- 18 to 75 +/- 16 fmol/mg protein for renal artery clip hypertension. Isoproterenol-stimulated adenylate cyclase activity also was lower in membranes from hypertensive rats, whereas basal and fluoride-stimulated activities were unchanged.


Serum cortisol and 11 deoxycortisol by liquid chromatography: clinical studies and comparison with radioimmunoassay. We describe a liquid-chromatographic procedure for separating and measuring cortisol and 11-deoxycortisol in serum. We quantitated these steroids in patients who were undergoing various tests of pituitary and (or) adrenal function and compared the results with those obtained by two radioimmunoassays done in two different laboratories. Results of 48 tests done in 37 functionally normal humans are presented. Cortisol values for sera collected in the morning as determined by liquid chromatography were (mean +/- SD) 134 +/- 54 micrograms/L. Serum cortisol concentrations increased from 136 +/- 65 to 321 +/- 80 micrograms/L 60 min after injecting synthetic corticotropin and increased from 107 +/- 46 to 242 +/- 31 micrograms/L after insulin-induced hypoglycemia. Serum cortisol decreased from 142 +/- 49 to 26 +/- 20 micrograms/L after oral administration of metyrapone, while 11-deoxycortisol increased from less than 10 to 210 +/- 53 micrograms/L. Serum cortisol measured less than 10 micrograms/L the morning after oral ingestion of dexamethasone. Results of the dynamic tests of adrenal function correlated well with previously reported studies. However, the cortisol values obtained by our technique were generally lower than those obtained by radioimmunoassay, possibly owing to lack of specificity of the latter methods used here for comparison. In contrast, values for 11-deoxycortisol were the same by both methods. The present studies confirm the usefulness of liquid chromatography for measuring these two steroids in serum during tests of pituitary and adrenal function. Future refinements of the technique should continue to increase its clinical applications.


Identification of alpha1-lipoproteins in crossed immunoelectrophoresis. Evans Blue dye binds selectively, but with different avidities, to five major antigens in human serum. The anodic mobility of the antigen-dye complexes is greater than that of the antigens alone in crossed immunoelectrophoresis, which is of practical value for identification. We used this characteristic to show that in some human sera there is a population of alpha1-lipoprotein molecules that migrates electrophoretically in the beta-lipoprotein region, where in conventional zone electrophoresis it could be mistaken for beta-lipoprotein. We also demonstrate that horses, unlike rabbits, rarely make precipitins to human serum alpha1-lipoprotein. Equine antiserum to human serum therefore generally is useless for detecting this important serum antigen in immunoelectrophoresis.


Comparison of two micromethods for determination of lipoprotein cholesterol in plasma. We compared the results obtained by a micromethod for the determination of plasma lipoprotein cholesterol, in which electrophoresis is used to separate the lipoprotein fractions (beta-, pre-beta-, and alpha-lipoproteins), with those determinations with ultracentrifugation (low-density, very-low-density, and high-density lipoproteins). Precision of determination (coefficient of variation, CV, %) was the same for beta- and low-density lipoproteins (1.6%), and for pre-beta- and very-low-density lipoproteins (3.7%); however, determination of alpha-lipoprotein cholesterol was more precise (1.4%) than that of high-density lipoprotein cholesterol (3.1%). Analytical recovery of lipoprotein cholesterol was the same for both methods (98--100%) and the results were closely correlated (r = 0.943). The procedure has been used to determine the cholesterol content of plasma lipoprotein fractions of apparently healthy adults (both sexes). Lipoprotein cholesterol concentrations in our population sample compare well with those reported for other groups of similar age, in particular Stanford long-distance runners.


1,25-Dihydroxycholecalciferol in human serum and its relationship with other metabolites of vitamin D-3. A competitive protein binding assay for 1,25-dihydroxycholecalciferol has been developed using the hormone's nuclear receptor protein from chick intestinal mucosa. This nuclear receptor protein can be stored at -70 degrees C for several months and bound and free hormone can be separated easily with dextran coated charcoal. Results obtained using this assay agree well with those reported by other groups of workers. Serum levels of other vitamin D-3 metabolites, namely 25-hydroxycholecalciferol and 24,25-dihydroxycholecalciferol have also been measured and are shown in relation to 1,25-(OH)2D3 levels.


Enzymes of collagen biosynthesis in skin and serum in dermatological diseases. I. Enzymes of the skin. Immunoreactive prolyl hydroxylase protein, prolyl hydroxylase activity and galactosylhydroxylysyl glucosyltransferase activity were measured in skin biopsies from 54 patients with various dermatological disorders and compared with results from 15 control samples; The mean prolyl hydroxylase activity was significantly elevated in case of keloids (P less than 0.001) and lichen ruber planus (P less than 0.02) and the mean galactosylhydroxylysyl glucosyltransferase activity in keloids (P less than 0.005), lichen ruber planus (P less than 0.02) and psoriasis (P less than 0.05), and high activities of these two enzymes were also occasionally found in other skin diseases and active systemic connective tissue diseases. These two enzyme activities correlated highly significantly (P less than 0.001) with each other in the total disease material. Immunoreactive prolyl hydroxylase protein was significantly elevated in psoriasis (P less than 0.02) lichen ruber planus (P less than 0.005), but not in keloids, where very high activities of the corresponding enzyme were found. Active prolyl hydroxylase constituted about 1.3% of total immunoreactive prolyl hydroxylase protein in normal skin, but as much as 10% in cases of keloids.


Enzymes of collagen biosynthesis in skin and serum and dermatological diseases. II. Serum enzymes. Serum immunoreactive prolyl hydroxylase protein and galactosylhydroxylysyl glucosyltransferase activity were measured in 54 patients wtih various dermatological diseases and compared with corresponding values in 32 control subjects. These serum enzymes were at the control level in the great majority of the patients, and no correlation was found between serum and skin enzymes, except for one weak correlation in lichen ruber planus. Some patients with psoriasis, lichen ruber planus, keloids, erythema nodosum or chronic discoid lupus erythematosus, however, did have at least one of these enzymes elevated in the serum, and a significant correlation (P less than 0.01) between the two enzymes was found in the total disease material. Thus it does seem that diseases limited only to the skin can sometimes raise these serum enzyme levels. The mean levels of serum immunoreactive prolyl hydroxylase and galactosylhydroxylysyl glucosyltransferase activity were significantly elevated (P less than 0.001) in active systemic connective tissue diseases such as systemic lupus erythematosus, scleroderma or dermatomyositis compared with the controls, and 4 out of 7 values for immunoreactive prolyl hydroxylase and 3 out of 7 for galactosylhydroxylysyl glucosyltransferase activity were above the 95% confidence limit of the controls. Since the levels of the skin enzymes were not elevated in most of these patients, however, the main sources for the elevated serum enzymes were probably tissues other than the skin.


Apoenzyme of aspartate aminotransferase isozymes in serum and its diagnostic usefullness for hepatic diseases. Aspartate aminotransferase in the sera of normal subjects and of patients with hepatic diseases has been immunologically separated into two isoenzymes, cytosolic aspartate aminotransferase and mitochondrial aspartate aminotransferase. The activity of the isoenzymes was measured in three different buffer solutions with or without pyridoxal 5'-phosphate. To attain maximal activation, the apoenzyme of mitochondrial fraction must be preincubated with pyridoxal 5'-phosphate longer than that of the cytosolic fraction in either of the three reaction mixtures. In most sera the activity of both isoenzymes increased substantially in the presence of pyridoxal 5'-phosphate regardless of the type of buffer solutions. Both the apoenzymatic activity and the ratio of apo- to holo-enzymatic activity of each of the isoenzymes varied among samples from the patients with hepatic diseases. However, significantly high ratios of apo- to holo-enzymatic activity of both isoenzymes were observed in the patients with hepatoma in contrast with those with other hepatic diseases. These findings suggest that the simultaneous measurement of both apo- and holo-enzyme activities of aspartate aminotransferase isoenzymes may be useful in the clinical assessment of hepatic diseases.


Kinetics of plasma corticosteroids during metyrapone infusion in man. The effect of metyrapone on plasma kinetics of cortisol (F), 11-deoxycortisol (S), corticosterone (B), 11-deoxycorticosterone (DOC), aldosterone (Aldo) and 17 hydroxyprogesterone (17 OHP) was studied in ten normal prepubertal children intravenously infused with the drug (1 gm/1.73 m2 body surface area in 4 h). Similar falls in F, B and Aldo were observed during the first hour expressing similar sensitivity of the 11 hydroxylases to the drug. The effect of the compensatory increase of endogenous ACTH could be detected after 1 hour by the rise in 17 OHP. However the concentrations of the 11-hydroxy compounds, F, B and Aldo, remained low throughout the infusion period. From the second hour onwards, facilitation of the synthesis of DOC was noticed. After the end of infusion, the levels of the precursors decreased and F and B were restored to initial values. Aldo remained low probably due to the inhibitory effect of the elevated DOC on the renin-angiotensin system.


Plasma cyclic-AMP response to parathyroid hormone in Turner's syndrome and Albright's hereditary osteodystrophy. Purified bovine parathyroid hormone (BPTH) given by injection to five patients with Turner's syndrome, and seven healthy volunteers produced a significant rise in plasma cyclic AMP reacing a maximum within 10 min. In a pseudohypoparathyroid patient there was no increase. Urinary excretion of cyclic AMP exceeded the normal in three out of four patients with Turner's syndrome. Thus, if there is a relationship between Turner's syndrome and Albright's osteodystrophy it is with the incomplete form known as pseudo-pseudohypoparathyroidism.


The short metyrapone test: comparison of the plasma ACTH response to metyrapone and insulin-induced hypoglycaemia. Plasma ACTH levels in response to metyrapone and insulin hypoglycaemia were compared in subjects with normal pituitary-adrenal function. After a single dose of 2 g of metyrapone given with a snack at midnight, the ACTH level was 468 ng/l +/- 66 )SEM) at 07.30 h the next morning (mean increment approximately nine fold over normal morning values). After insulin-hypoglycaemia the peak ACTH level was 369 ng/l +/- 31 (SEM). Peak ACTH levels greater than 200 ng/l were achieved in twenty of twenty-one (95%) subjects after metyrapone and twenty of twenty-four (83%) after insulin. No major side effects were noted after metyrapone. It is concluded that the short single-dose metyrapone test produces at least as strong and consistent a stimulus to ACTH release as the standard insulin-hypoglycaemia test in normal subjects. A direct assay of ACTH avoids misinterpretations which are inherent in a judgement based on compound S increase only. The short test has significant practical advantages over the classical metyrapone test, and provides a convenient and sensitive method of assessing the negative feedback ACTH control mechanism. It may be particularly useful in detecting minor degrees of pituitary suppression. The value of this test in clinical practice for the investigation of patients with hypothalamic-pituitary diseases in comparison to the classical tests of ACTH stimulation has yet to be demonstrated.


Salivary tumours--experience over thirty years. A clinicopathological survey of tumours of salivary glands seen in a special surgical clinic at the Christie Hospital and Holt Radium Institute is reported. Nine hundred and seventy-seven benign and malignant tumours have been seen within the past 30 years. The results of treatment are included with particular attention to those of the parotid glands which present the greatest therapeutic problem. The approach to surgical treatment of pleomorphic adenomas is described in detail and guidelines offered as to the procedure that should be used based on full exploration and assessment of each tumour. The need for adjuvant irradiation depends on the standard of surgery that is done. The relationship between carcinomas and benign adenomas is discussed and the need for surgery in all cases is stressed in order to make a definite histological diagnosis in view of the wide range of pathological abnormality encountered.