A new method for receptor autoradiography: [3H]opioid receptors in rat brain. Opioid receptors can be labeled with [3H]ligands in lightly fixed tissue sections mounted on microscope slides. The preparation of these sections does not seem to alter any of the known characteristics of opioid receptors. The light microscopic autoradiographic distribution of these binding sites can be observed if one attaches emulsion-coated coverslips to these slides to obtain autoradiograms. The distribution of [3H]diprenorphine binding sites determined by this in vitro method is identical to the distribution found in earlier studies utilizing in vivo labeling of opioid receptors. In addition, [3H]opioid peptide binding sites and [3H]dihydromorphine binding sites may be similar, perhaps identical, to those for [3H]diprenorphine, an opiate antagonist. This method has several important advantages over earlier methods for determining the autoradiographic localization of receptors. It is possible, by washing, to reduce nonspecific binding to low levels. Since receptor labeling is performed with single tissue sections mounted on slides, one can examine different receptors in different but adjacent sections. One can use ligands that are not entirely suitable for in vivo labeling (For example, one can use [3H]peptides which do not normally cross the blood-brain barrier). One can perform autoradiographic studies after labeling tissues under a wide variety of conditions (For example, one can examine the effects of various ions and nucleotides on ligand binding distributions). This technique seems to be free of various artifacts, such as edge artifacts, which were common by techniques used in our laboratory earlier. Since one does not have to load an entire animal with radioactive ligand as one must with in vivo labeling, this approach is economically advantageous. One can use postmortem tissues, including that from humans to study receptor distribution with a high anatomical resolution.


[Mechanism of lymphocyte activation by periodic acids. Blastogenesis and induction of immunomodulator populations]. Mild periodate treatment is mitogenic for T lymphocytes. With murine spleen cells periodate oxidation is effective between C8 and C9 on sialyl acid residues. With human blood lymphocytes this oxidation occurs between C7 and C8 of these residues. In vitro immune response is inhibited by periodate treatment. Activation of an immunosuppressive T lymphocyte population is obtained. Similar results are performed with human blood lymphocytes.


[Chromatin proteins and genetic control]. Gene expression is in great part under the control of proteins associated with DNA: histones which can be phosphorylated and acetylated, non-histone proteins which include enzymes, hormone receptors and other regulatory proteins. Phosphorylation seems to play an important role in chromatin regulation.


[Regulation of glycogen metabolism in the liver and hepatic glycogenosis due to phosphorylase system deficiency]. Glycogen synthesis and breakdown in the liver are tightly controlled through different mechanisms. The purpose of this review is to describe some properties of the enzymes involved in the glycogen metabolism and the sequence of events by which glucose, allosteric effectors and hormones control this metabolism in the liver. Clinical, genetic and biological aspects of the phosphorylase and the phosphorylase kinase deficiencies are examined. The enzymatic analysis of the haemolysates from the patients allows discrimination of these two types of glycogenosis.


Analysis of paramagnetic centers in X-ray-irradiated enamel, bone, and carbonate-containing hydroxyapatite by electron spin resonance spectroscopy. Carbonate-containing hydroxyapatite, enamel, and bone were irradiated by an X-ray and investigated between 77 degrees and 350 degrees K by means of electron spin resonance (ESR) spectroscopy. The ESR spectrum of enamel irradiated at 77 degrees K in vacuum and observed at the same temperature was almost the same as that of the carbonate-containing hydroxyapatite. The temperature dependence of signal intensities confirms a spin-energy exchange between the mineral and organic constituents in bone, but in enamel no or very little spin-energy exchange between the mineral and organic constituents. Considerable similarity among the ESR spectra of enamel, bone, and carbonate-containing apatite was obtained after X-ray irradiation in air at 300 degrees K with both an X-band and a Q-band ESR spectrometer. The Q-band spectrum can be interpreted in terms of two paramagnetic species. One is identified as a CO3(3-) anion radical which has an axial symmetry with g factors of 2.0029 and 1.9972. The other species is found to be centered at g = 2.0019.


The calciuria of increased fixed acid production in humans: evidence against a role for parathyroid hormone and 1,25(OH)2-vitamin D. We measured mineral and acid balances, serum iPTH, urinary cAMP/creatinine, and plasma concentrations of 25OHD and 1,25(OH)2D in 7 healthy adults during control conditions and during increased fixed acid production achieved either by the administration of NH4Cl (N = 3) or by increased dietary protein intake (N = 4). When acid production was increased, the subjects were in positive acid balance and negative Ca balance because of increased urinary Ca excretion. Serum iPTH fell slightly but urinary cAMP and the plasma levels of vitamin D metabolites did not change. W conclude that the accelerated skeletal and urinary losses of Ca that occur when fixed acid production is increased are not contributed to nor compensated for by the parathyroid-vitamin D endocrine systems.


Pleomorphic C-type virus particles in bone tissue of strain 101 mice and (C3H X 101)F1 hybrids. The proximal tibial metaphysis of apparently healthy strain 101 mice, 3-4 weeks old, and (C3H X 101)F1 hybrids, 3-48 weeks old, was studied by electron microscopy. Budding, immature, and mature (C-type virus particles were found within trabecular bone tissue of 3 of 8 strain 101 and 4 of 12 (C3H X 101)F1 mice. The particles were most common in lacunae of aging osteocytes and were only occasionally associated with osteoblasts. Although the morphology of budding and immature particles appeared to be identical with that of typical C-type viruses, most of the mature forms of particles showed atypical structure and size. The electron-dense core was very large and not clearly defined, measuring approximately 70-130 nm in diameter. This diffuse core sometimes completely filled the space within the envelope of the particles. The diameters of the pleomorphic mature C-type particles ranged from approximately 90 to 150 nm. The possible association between the production of pleomorphic C-type virus particles by bone cells and spontaneous osteomagenesis in 101 and (C3H X 101)F1 mice is discussed.


Cortisol enhancement of PTH-stimulated cyclic AMP accumulation in cultured fetal rat long bone rudiments. Cortisol in concentrations from 10 nM to 10 microM produced a dose-related inhibition of basal and PTH1-stimulated 45Ca and [3H]-hydroxyproline release from cultured fetal rat forelimb rudiments. PTH-stimulated cyclic AMP generation however was not diminished by cortisol; in contrast, at a concentration of 1 microM cortisol produced a 57% increase in PTH-stimulated bone cyclic AMP content. The stimulatory effect of cortisol on cyclic AMP content appeared to be the result of reduced phosphodiesterase activity, since this effect was not seen in the presence of 10 mM theophylline. It is concluded that cortisol inhibition of PTH-induced resorption in long bones is not accompanied by reduced cyclic AMP generation.


Long-term culture of cells from bone affected by Paget's disease. Cells obtained from surgical bone specimens of eight patients with Paget's disease of bone were maintained in culture for up to 8 months and seven passages. The doubling time during the period of maximal cell growth ranged from 4 to 12 days. Evidence consistent with the hypothesis that many of the cells were bone cells included the following: (a) histochemical techniques demonstrated staining of some cells for alkaline phosphatase or acid phosphatase and succinic dehydrogenase; (b) parathyroid extract stimulated increased uptake of 3H-thymidine and 3H-uridine; (c) parathyroid extract suppressed and salmon calcitonin stimulated uptake of 3H-proline; and (d) crystalline calcium deposits were found within cells and extracellularly. Ultrastructural analysis revealed that three of the eight cultures contained cells whose nuclei had inclusions which were almost identical to those found in the osteoclast nuclei of all patients with Paget's disease. The maintenance of cells derived from pagetic bone in long-term culture should aid in testing the hypothesis that Paget's disease represents a slow virus infection of bone.


Study of an asymmetric ESR signal in x-irradiated human tooth enamel. An asymmetric ESR signal and an outer doublet signal centered at g = 2.00 produced in human tooth enamel by X-irradiation have been studied over a temperature range from liquid helium temperature to 380 degrees K. The line shape of the asymmetric signal for an enamel crystallite is Lorentzian at room temperature. The temperature dependence of the integrated intensity of the asymmetric signal suggests that the signal follows a singlet-triplet model with the exchange interaction of 0.022 eV. Below about 60 degrees K the asymmetric signal becomes broad as the temperature is decreased. The ENDOR line of the asymmetric signal shows that there exists interaction between the centers and protons. The temperature dependence of the integrated intensity of the double signal is analogous to that of the asymmetric signal.


From beta-lipotropin to beta-endorphin and 'pro-opio-melanocortin'. Studies on the biosynthesis of beta-LPH on the one hand, and of ACTH on the other, have produced a new concept, that of a single precursor form which contains three active molecules. Thus, it is proper to name such a precursor 'pro-opio-melanocortin.' The concept that beta-LPH was a precursor molecule was first put forward in 1967 and was based on both structural forms and biological activities. The discovery that morphine-like substances are part of the C-terminal fragment of beta-LPH brought an additional important biological side product. That, together with the recent demonstration of ACTH as part of a still larger precursor, constitutes an exciting model for the study of peptide hormone biosynthesis. We have shown unambiguously that beta-endorphin is the result of a maturation process from the large precursor, while beta-LPH is an important and transient intermediary. Since it is also present in the brain, our recent results using pars intermedia cells can be applied to study the fabrication and degradation of these molecules in the brain. We expect to see it established that all other neuropeptides are also biosynthesized as larger precursor molecules whose structure at the site of cleavage could well be constituted of two basic amino acids like in the pro-opio-melanocortin.


Enzymatic modifications of bovine adrenocortical angiotensin II receptors. Several classes of proteolytic enzymes were used to gain an insight into the biochemical composition of the antiotensin II (ATII) receptor prepared from bovine adrenal cortices. Exposure of the receptor fractions to trypsin reduced their capacity to bind [3H]ATII. Phospholipases A2 and C similarly inhibited the [3H]ATII binding process, while phospholipase D had no effect. Binding was stimulated following addition of phosphatidylcholine but inhibited by lysophosphatidylcholine. Neuraminidase had no influence on [3H]ATII affinity for binding, while beta-galactosidase reduced binding of the radioligand. Concanavalin A did not displace [3H]ATII bound to receptor fractions. Very little aminopeptidase activity was detected in the receptor fraction, relative to the homogenate. The data suggest that the ATII recognition sites contain protein moieties, while phospholipids may play an essential role in ATII binding. Galactose units may form a part of the ATII receptor not directly associated with the binding site. The peptidase studies indicate that ATII probably cannot be hydrolyzed to its des-Asp1 metabolite at or near the site of binding.


Evidence for two adenine derivative receptors in rat ileum which are not involved in the nonadrenergic, noncholinergic response. The receptors mediating inhibition of the rat ileum by adenosine and adenine nucleotides were studied. ATP and ADP were more potent than AMP or adeonsine. Theophylline antagonized the effects of adenosine and AMP but not those of ATP or ADP. Preparations desensitized to ATP or ADP were still inhibited by adenosine and vice versa. The nonadrenergic, noncholinergic inhibition produced by field stimulation or nicotine was not attenuated by the presence of theophylline or desensitization to ATP. These data indicate that more than one adenine derivative receptor is present in rat ileum and that ATP and adenosine are unlikely candidates for the unknown transmitter.


An opiate receptor on frog sciatic nerve axons. The effect of meperidine (3 X 10(4) M) on the action potential of frog sciatic nerve was examined by means of the double sucrose gap technique. Meperidine decreased the amplitude, maximum rate of depolarization, and maximum rate of repolarization of the action potential but had no effect on the resting potential. This depression in amplitude and maximum rate of rise was partially blocked by naloxone (1 X 10(-8) M) while the maximum rate of depolarization was further depressed. The data suggest that the effect of meperidine is due to two mechanisms, a nonspecific local anaesthetic like effect and an opiate receptor mediated effect.


Mg2+--Ca2+--Na+ interactions in uterine smooth muscle: studies with cAMP. The influence of variation in the extracellular concentrations of Na+, Mg2+, and Ca2+ in the depolarizing medium on isoproterenol-induced increases in cAMP levels and relaxation was studied in rat uterus. Isoproterenol (10(-8) M) failed to increase cAMP levels in the high-K+ medium containing no Na+. When 80 mM Na+ was present in the medium, isoproterenol caused increases in cAMP levels similar to those observed in nondepolarized uterus. A similar effect of 2.5 mM Mg2+ was observed on the cAMP response. These effects of Na+ and Mg2+ were antagonized by increasing the extracellular concentration of Ca2+. The simultaneous presence of 80 mM Na+ and 2.5 mM Mg2+ did not produce an additive effect on the cAMP responses.


Interferon, antibody responses and protection induced by an intranasal infectious bovine rhinotracheitis vaccine. The interferon and antibody response induced by an intranasal infectious bovine rhinotracheitis vaccine was followed in 22 calves over a nine month period and the ability of these vaccinated calves to withstand challenge with virulent infectious bovine rhinotracheitis virus was assessed. Interferon was detected two to three days post-vaccination and disappeared by the tenth day. Nasal and serum antibodies appeared by day 7 and persisted for nine months. The calves challenged three days postvaccination came down with disease typical of infectious bovine rhinotracheitis, whereas calves challenged three weeks, three months or nine months postvaccination resisted infection.


Neurologic involvement in glucagonoma syndrome: response to combination chemotherapy with 5-fluorouracil and streptozotocin. A 34-year-old man presented with classic glucagonoma syndrome manifested by weight loss, dermatitis, stomatitis, anemia, and mild diabetes mellitus. The diagnosis of glucagonoma was made by light and electron microscopic demonstration of a metastatic alpha cell carcinoma in a liver biopsy specimen. Plasma glucagon concentration was abnormally high. The patient also had symptoms and signs of involvement of the central nervous system. Radionuclide and CAT scans of the brain, negative CSF cytology and myelography excluded the possibility of metastases or other space-occupying lesions. Glucagon was demonstrated in the CSF. We postulate that the neurologic symptoms were due to direct or indirect effect of this hormone on the brain. Following therapy with streptozotocin and 5-fluorouracil, the patient had a subjective and objective clinical and hormonal remission of his disease including amelioration of his neurological impairment.


Peripheral neuropathy associated with cis-dichlorodiammineplatinum (II) treatment. Peripheral neuropathy developed in two cases of recurrent bladder carcinoma treated with Cis-dichlorodiammineplatinum (II) (DDP) at a dose of 75 mg/m2 IV every 3 weeks. The neuropathy appeared in both patients while they were achieving a subjective and objective response to DDP. The neuropathy was reversible in one patient. In the other patient, a disabling sensory neuropathy progressed despite cessation of therapy. The temporal relationship between the neurological symptoms and the administration of DDP implicate this drug as the possible causative agent in the peripheral neuropathy.


The relation between Epstein-Barr virus antibodies and clinical symptomatology and immunodeficiency in patients with Hodgkin's disease. Anti-Epstein-Barr virus (EBV) titers were measured in the sera of 37 patients with Hodgkin's disease and in 40 normal controls. The patients were grouped according to histologic type, clinical symptomatology (relapse or remission), and their immune state (immunodeficient or non-immunodeficient). Anti-Epstein-Barr nuclear antigens (EBNA) and antiviral capside antigens (VCA) titers were higher in patients with Hodgkin's disease than in the controls. Anti-EBNA titers were significantly higher in patients with lymphocyte predominance, and anti-VCA titers were significantly higher in patients with mixed cellularity. Patients in clinical relapse had higher anti-EBV antibody titers than patients in remission or those in the control group. Immunodeficent pateints had significantly higher anti-VCA titers than either the non-immuno-deficient or the control cases. We believe high anti-EBV titers are related to immunodeficiencies. The relationship between Hodgkin's disease and EBV is discussed.


Angiomatoid malignant fibrous histiocytoma: a distinct fibrohistiocytic tumor of children and young adults simulating a vascular neoplasm. This article describes 41 examples of an unusual fibrohistiocytic sarcoma which occurred primarily in the extremities of young individuals between the ages of 5 and 25 years (median 13 years). It manifested as a nodular subcutaneous growth that seldom caused tenderness or pain, and clinically was often mistaken for a hematoma or a hemangioma. Grossly, the tumor presented as a circumscribed, multinodular or multicystic, hemorrhagic mass that ranged in size from 0.7 to 10 cm (median 2.5 cm). On microscopic examination, it consisted principally of 1) solid arrays or nests of fibroblast- and histiocyte-like cells, not infrequently containing varying amounts of intracellular hemosiderin or lipid, 2) focal areas of hemorrhage or hemorrhagic cyst-like spaces, sometimes occupying the major portion of the tumor, and 3) aggregates of chronic inflammatory cells, chiefly lymphocytes and plasmacytes, a feature that caused confusion with a lymph node metastasis in several cases. Follow-up information, available in 24 patients, revealed a variable clinical course. Twenty-one patients were alive, 11 with recurrence (including one with 9 recurrences in a 21-year period) one with recurrence and metastasis and one with metastasis. Three patients had died of metastasis 1, 3, and 13 years respectively, after the initial surgical therapy. The exact histogenesis is still obscure. Most likely it is a tumor of fibroblast- and histiocyte-like cells, akin to malignant fibrous histiocytoma, but different in its age incidence, microscopic appearance and behavior.


Mixed malignant mesenchymal tumor of the cerebellar vermis. In an example of mixed malignant mesenchymal tumor of the cerebellar vermis, striking osteoblastic differentiation was present along with extensive intradural spinal invasion. The CNS sarcomas and their variants are discussed as well as the extraskeletal osteosarcomas. Comparison is made to the teratoid tumors and other mixed and unusual posterior fossa tumor elements, and the capability of midline posterior fossa tissues for multipotential differentiation is considered.


Ultrastructural comparison of hepatoblastoma and hepatocellular carcinoma. The ultrastructural characteristics of fetal liver, two hepatoblastomas and two hepatocellular carcinomas were compared. Tumor cells of hepatoblastoma disclosed monotonous nuclei, poorly-developed cytoplasmic membrane system, abundant free ribosomes and prominent glycogen granules. Thos of hepatocellular carcinoma revealed comparatively pleomorphic nuclei, well-developed cytoplasmic membrane system, a few free ribosomes and numerous glycogen granules. Fetal liver showed monotonous nuclei, well-developed RER abundant free ribosomes and prominent glycogen granules. Young mesenchymal cells with well-developed RER and continuous basal lamina surrounding the epithelial cells were detected in both cases of hepatoblastoma but not in those of hepatocellular carcinoma. Tumor cells of hepatoblastoma in a case showed intramitochondrial crystalloids and thick bundles of fibrils in the cytoplasm.


Ovarian low-grade stromal sarcoma with thecomatous features: a critical reappraisal of the so-called "malignant thecoma". A case of low-grade ovarian stromal sarcoma in a postmenopausal woman is described. Although pelvic recurrences of the tumor followed 5 and 7 years after the original surgery, the patient has remained well and without evidence of tumor 3 years since the last operation. Histopathologic, electronmicroscopic, and hormonal studies are described. There was evidence of estrogenic stimulation by the theca elements of the tumor in this patient. Cases previously reported in the world literature as malignant thecoma were analyzed, and most of them were considered inadequately documented; indeed most of them were probably either sarcomatoid granulosa cell tumors, stromal sarcomas, or fibrosarcomas. If a thecoma ever becomes malignant, the tumor cells dedifferentiate so that they cannot be recognized any longer as theca cells; instead, they proliferate as a stromal sarcoma or fibrosarcoma. It is proposed, therefore, that the term "malignant thecoma" not be used. On the other hand, very rare malignant ovarian stromal tumors do exist, consisting of undifferentiated stromal cells, fibroblasts, and theca cells, which can show evidence of hormonal activity.


Acquired myeloperoxidase deficiency and recurrent infections in a patient with acute myelomonocytic leukemia. Acquired myeloperoxidase deficiency has been reported in several hematological malignancies. The clinical course of a patient with acute myelomonocytic leukemia is described which was characterized by staphylococcal infections prior to therapy and again during a period of relapse. Neutropenia was not a feature of these two periods but in vitro studies revealed decreased bacterial killing capacity and decreased neutrophil myeloperoxidase activity. Infectious complications were not observed during drug-induced remission when bacterial killing capacity and myeloperoxidase activity were improved toward normal. These observations suggest that the myeloperoxidase deficient neutrophils were derived from leukemic progenitors.


Benign mixed tumor of the trachea.

Benign mixed tumor of the trachea. A case of mixed tumor, salivary gland type, removed by segmental resection of the trachea is reported. Only thirteen acceptable and five probable tracheal mixed tumors have been found in the world literature. The behavior of this variety of tumor in the trachea appears to be similar to its counterpart in other sites and distinctly different from the more frequently encountered epithelial tumors: squamous cell carcinoma and adenoid cystic carcinoma.


Urinary hexosaminidase in patients with lung carcinoma. The specific activity of urinary hexosaminidase was determined in 58 patients with various histological types of lung carcinoma. Compared to an apparently healthy control population, 32/35 patients with widely disseminated disease showed elevated values and 18/23 patients with disease confined to the chest had normal hexosaminidase values. Detailed studies of 18 patients indicated that declining hexosaminidase values were associated with effective therapy, rising values generally accompanied progressive disease.


Evidence for clustering of hepatitis B virus infection in families of patients with primary hepatocellular carcinoma. Family member of 13 patients with hepatitis B surface antigen (HBsAg) positive primary hepatocellular carcinoma (PHC) were tested for the presence of hepatitis B virus-associated antigens and antibodies. Of the 122 members examined, circulating HGsAg was detected in 47 (39%), antibody to HBsAg (anti-HBs) was found in 37 (30%), and antibody to hepatitis B core antigen (anti-HBc) alone was present in 13 (11%). The relatives with the highest frequency of HBsAg positivity were the offspring of the propositus, followed by the nieces and nephews and the grandchildren. Anti-HBs and anti-HBc were detected most often in the spouses and non-blood relatives. Evidence for past and present hepatitis B virus (HBV) infection was more frequently found in the Asian family members when compared to the non-Asians. The e antigen (HBeAg) was present in 38% of the HBsAg positive individuals, including four with PHC; antibody to HBcAg (anti-HBe) was rarely detected. These results indicate that clustering of HBV infection was commonly present in family members of patients with PHC. The HBsAg positive individuals may be major contributors to the endemic pool of the virus, and may themselves be potential cases of chronic active type B hepatitis, cirrhosis, and PHC.


Neurological complications of malignant germ cell tumors of testis: biology of brain metastases (I). Central nervous system metastases are a common complication of disseminated germ cell tumors of the testis. They occurred in 16% of 242 patients treated and in 25% of the patients who died in our VAB chemotherapy series. Pulmonary metastases preceded or coincided with the development of brain metastases. The frequency of brain metastases differed with the histology of the primary tumor. They occurred in 13% of pure embryonal carcinomas, 18% of mixed tumors containing embryonal or choriocarcinoma elements, and 83% of pure choriocarcinomas. Embryonal carcinoma and choriocarcinoma were the principle histologies found in brain metastases. Characteristically, pure choriocarcinoma deposits in the brain were multiple (8/9) and cerebellar involvement was common (5/9). Pure embryonal carcinoma CNS metastases were typically single (6/8) or very few and cerebellar involvement was not observed. The interval from the diagnosis of malignancy to the diagnosis of brain metastases was longer for embryonal carcinoma than for pure choriocarcinoma (23 mos. vs. 6.5 mos.). Survival following the diagnosis of brain metastases was poor. There was a tendency toward longer survival for histologically pure embryonal carcinoma deposits in the brain than for the pure choriocarcinomas (6.5 mos. vs. 1 mo.).


Breast cancer in a man 30 years after radiation for metastatic osteogenic sarcoma. A man developed breast cancer 30 years after chest radiation for metastatic osteogenic sarcoma. At least eight previous cases of radiation-associated breast cancers have been reported in males, but this man represents the first to develop breast cancer following a childhood malignancy.


Carcinogenic chemicals enhance mouse leukemia virus infection in contact-inhibited culture: a new simple method of screening carcinogens. Carcinogenic chemicals enhanced infection in contact-inhibited cells with mouse leukemia virus. A correlation was found between the enhancing effects and the carcinogenicities of the 40 chemicals tested. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate did not enhance viral infection.


Adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate phosphodiesterase activities in 1,2-dimethylhydrazine induced colon adenocarcinoma. The intracellular concentrations of adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) in 1,2-dimethylhydrazine (DMH) induced rat colon tumors were previously reported to be one-half and twice, respectively, that measured in normal rat colon tissue. The results of this present study indicate that the total amount of cyclic nucleotide hydrolysis by the phosphodiesterase enzymes (PDE) could account for the lower cAMP and elevated cGMP noted in cancer tissue. PDE activities in homogenates prepared from normal and neoplastic tissue showed general similarities in both their hydrogen and metal ion dependencies with optimum degradation of nucleotides occurring at pH 7.2-7.8 in the presence of either Mg2+ or Mn2+ ions. The enzymes from both tissue types were inhibited to a similar extent by papaverine and theophylline. Distribution studies indicated PDE activities were similar throughout the entire length of the normal colon; consequently, the anomalous activities measured in the tumors were attributable to the specific cell population and not to the particular site at which malignancy arose. Thus, the findings of this study suggest that one cause for lowered cAMP and elevated cGMP levels in DMH induced colon adenocarcinomas was the amount of cyclic nucleotide hydrolysis by PDE in the malignant tissue.


Glycolytic enzyme activities in malignant cells grown in vitro and in vivo. Cell proximity has a profound effect on the glycolytic enzyme levels observed in mammalian cells grown in vitro. Hexokinase activity generally increased with the increasing cell density of both untransformed and virus-transformed cultures. In contrast, phosphofructokinase and pyruvate kinase activities were generally elevated only in crowded cultures of transformed cells. A spontaneous experimental mouse tumor cell line (EMT6) exhibited cell crowding-dependent elevations in glycolytic enzyme levels when cultured in vitro. The glycolytic enzyme levels of experimental mouse tumors grown in vivo, on the other hand, were constant and normally less than those observed in cultured EMT6 cells.


Inhibitors for the mutagenicities of colon carcinogens, 1,2-dimethylhydrazine and azoxymethane, in the host-mediated assay. Inhibitory effects of several chemicals on the mutagenicities of 1,2-dimethylhydrazine (DMH) and azoxymethane (AOM) for Salmonella typhimurium G46 in the host-mediated assay were investigated. They were carbon disulfide (CS2), tetraethylthiuram disulfide (disulfiram, DSF), sodium diethyldithiocarbamate (SDDC), ethylene-bis(dithiocarbamato) manganese (Maneb), pyrazole (PZ), aminoacetonitrile hydrogen sulfate (AAN), and sodium selenite (SE). All the compounds, except for SE, inhibited the mutagenicities of DMH and AOM.


Mutagenicity of chamuvaritin: a benzyldihydrochalcone isolated from a medicinal plant. The mutagenic effects of chamuvaritin, dihydrobenzylchalcone isolated from Uvaria chamae, were investigated using Salmonella typhimurium tester strains TA92, TA94--98, TA100--1535, TA1537 and TA1538. The phytochemical was mutagenic in tester strains TA98 and TA100 and required activation by the hepatic S-9 microsomal enzyme preparation.


Fine structure of the rabbit adrenal cortex and the effects of short-term ACTH administration. The fine structure of the rabbit adrenal cortex was investigated. The parenchymal cells display the ultrastructural features of steroid-producing cells, and also contain numerous electron-dense bodies frequently located near intercellular canaliculi, when open into the subendothelial space. Short-term ACTH-administration induced a noticeable decrease in the volume of the lipid compartment in the cells of all three cortical zones and a significant increase in the volume of dense bodies in the cells of zona fasciculata and zona reticularis. The hypothesis that these dense bodies are secretory granules is discussed in the light of biochemical evidence showing that ACTH increases the concentration of both corticosterone and cortisol in the decapsulated-enucleated adrenal homogenate and does not affect the activity of two lysosome-marker enzymes (i.e., acid phosphatase and beta-glucuronidase).


Nuclear inclusions in the nerve cells of the sphenopalatine ganglion of the dog. Large numbers of nuclear inclusions have been found in the nerve cells of the sphenopalatine ganglia of six healthy adult dogs. Their morphological characteristics are similar to those previously described elsewhere. The presence of simple and granular bodies in normal cells seems to support the hypothesis that these nuclear structures might be considered as normal nuclear organelles related to cellular metabolic activity.


Localization of the ASV src gene product to the plasma membrane of transformed cells by electron microscopic immunocytochemistry. The cellular location of the src gene product (p60src) of the Schmidt-Ruppin strain of avian sarcoma virus has been determined by electron microscopic immunocytochemistry in Schmidt-Ruppin ASV-transformed NRK cells, and the amount of the protein in different regions of the cell has been quantified. The protein is concentrated on the inner surface of the plasma membrane, particularly under ruffles, and it is highly concentrated on the inner surface of the membrane near junctions connecting adjacent cells. Small amounts of p60src were detected in the cytoplasm and in the perinuclear Golgi region of the cell. No significant localization was detected in control NRK cells or in NRK cells transformed by the Kirsten strain of murine sarcoma virus. The presence of p60src on the inner surface of the plasma membrane indicates that the changes in cell growth, cell shape and cell membrane structure noted in ASV-transformed cells are due to an initial action of p60src at the cell membrane.


Evidence for various degrees of motional freedom of the "boundary" lipid in cytochrome oxidase. The cytochrome oxidase-lipid complex from beef heart mitochondria after various degrees of lipid extraction has been studied by electron spin resonance spectroscopy using spin labelled fatty acids and phospholipids. With cytochrome oxidase at the lowest lipid content (below 0.2 mg/mg of protein) i.e. at the level sometimes referred to as the "boundary" lipid, with spin labelled fatty acids an immobilized spectrum is observed. However, when spin labelled phospholipids are used under the same conditions, a mobile component is also observed. A quantitative estimation of the spectral components by computer analysis has been performed. The difference in behaviour of the spin labelled fatty acids and phospholipids suggest that the part of the residual lipid of the complex, which in some conditions is apparently immobilised, may exhibit in other conditons a considerably high degree of mobility.


Mechanism of action of collagenase on the blood-brain barrier permeability. Increase of endothelial cell pinocytotic activity as shown with horse-radish peroxidase as a tracer. The ultrastructural mechanism of the protease induced blood-brain barrier permeability-increase was studied with horse-radish peroxidase as a tracer. After intravenous injection of collagenase or pronase, a significantly increased number of pinocytotic vesicles was found in brain capillary endothelial cells. alpha-Chymotrypsine did not exert such an action.


A transient rise in cyclic AMP levels following chemotactic stimulation of neutrophil granulocytes. A short transient rise of cyclic AMP is observed within 1 minute after primary stimulation of neutrophils with chemotactic serum peptides containing classical anaphylatoxin (CAT). A second administration of these peptides after two minutes failed to produce a second peak of cAMP. Human serum albumin (HSA) which has chemokinetic but no chemotactic activities did not change cAMP levels. There was no significant change in cGMP levels within 1 minute following stimulation of rabbit neutrophils with chemotactic peptides or HSA.


Creatine kinase measurement in the 1977 CAP Enzymology Survey: anomalies explained. In the recently published results of the 1977 College of American Pathologists Enzymology Survey, Some anomalous results were obtained in measuring the creatine kinase (EC 2.7.3.2) activity of the samples. The cause of these anomalies was discovered to be an arginine kinase (EC 2.7.3.3) contaminant in one of the enzyme pools used to make the samples. The contaminant shows cross reactivity with creatine phosphate and is activated by dithioerythritol but not the other commonly used thiol activators.


Separation and partial characterization of new apoproteins from human plasma high density lipoproteins. In four patients receiving parenteral fluids, human plasma high density lipoproteins were obtained by sequential ultracentrifugation and delipidated. Apoproteins were resolved by gel filtration on Sephadex G-200, DEAE-cellulose chromatography, and polyacrylamide gel electrophoresis. Sephadex-Fraction V was unusually large (11--33% compared to 3--7% in normal subjects) and was found to contain seven new apoprotein components of an apparent molecular weight (by SDS gel electrophoresis) between 8000 and 11 000. Amino acid analysis showed that all these peptides had a high glycine and arginine content and a very low content of threonine and valine. Isoelectric focusing gave isoelectric points ranging from 5.00 to 8.00. In two patients injected with L-[1-17C]-leucine, incorporation of radioactivity into these peptides gave specific activities of the same order of magnitude as apo C-II and apo C-III. IT may be postulated that these peptides increased significantly when proteins are deficient in the diet or have low levels in plasma. The structural and functional significance of these peptides remains to be determined.


Effects of thyroid hormones on liver binding sites for human growth hormone, as studied in the rat. Several weeks after thyroidectomy (T), female rats stopped growing, and their pituitary GH content had decreased to less than 2--3% of the values found for age-matched controls (C). The liver membranes of such animals were explored with human GH (hGH). It was found that in the severely hypothyroid T rat, the number, but not the affinity, of the lactogenic binding sites was markedly reduced. Treatment of these rats for 3 weeks with 1.75 micrograms or T4 or 0.5 micrograms T3/100 g body weight/day restored growth, increased pituitary GH content and restored the number of liver lactogenic binding sites were practically to normal. As regards the lactogenic binding sites, similar results were obtained when the severely hypothyroid rats were treated with a much lower T4 dose (0.2 microgram/100 g/day): this dose was clearly growth promoting, and restored to normal both the low circulating GH levels and the pituitary PRL content of the severely hypothyroid rat. The changes in plasma PRL were not clear. The lactogenic binding sites on liver membranes from rats which were both thyroidectomized and hypophysectomized were decreased in number. Treatment with 0.5 microgram T3/100 g/day for 30 days (but not for 12 days) resulted in an increase in the number of lactogenic binding sites, though it did not affect growth or the undetectable plasma GH levels. The effect on the lactogenic binding sites was less marked than in T rats with an intact pituitary. It would appear that minute amounts of thyroid hormones are needed for maintenance of liver lactogenic binding sites; it is possible that this not only occurs through mechanism(s) which involve the pituitary, but also through others which do not. The possible role of these receptors in growth processes is not yet clearly understood.


Changes of urinary cyclic AMP excretion and plasma parathyroid hormone levels before and after parathyroidectomy in patients with primary hyperparathyroidism. Urinary cyclic AMP excretion and plasma parathyroid hormone(PTH) levels were examined in three patients with primary hyperparathyroidism before and after parathyroidectomy. Plasma PTH and urinary cyclic AMP in the individual patients decreased in parallel following parathyroidectomy. During surgery there was a statistically significant correlation between PTH levels and cyclic AMP excretion in individual patients. These findings support the claim that the rate of urinary cyclic AMP excretion reflects endogenous PTH activity in patients with primary hyperparathyroidism.


Evaluation of resection of pituitary microadenoma for the treatment of Cushing's disease in patients with radiologically normal sella turcica. Six patients with Cushing's disease and a radiologically normal sella turcica have been treated by transsphenoidal hypophysectomy. Microadenomas were found and removed in five. One patient was in complete remission by 2 months and three by 6 months post-operatively. Two of the remaining patients (one with and the other without a surgically demonstrable tumour) were not cured after 8 and 15 months follow-up, respectively. Our data suggest that pituitary tumours are present in the majority of patients with Cushing's disease. The possibilities of incomplete adenomectomy or of a tumour buried within the gland should be considered in patients in whom pituitary surgery does not induce remission. In one patient, subsequently proven to have a pituitary adenoma, the disease remitted pre-operatively with cyproheptadine, suggesting hypothalamic regulation of the tumour and/or direct drug action at the pituitary level.


Studies on guinea-pig macrophage migration inhibitory factor (MIF). I. Glycoprotein nature and net charge. Guinea-pig macrophage migration inhibitory factor (MIF), obtained by the stimulation of sensitized lymph node cells with tuberculin PPD, was characterized as a glycoprotein by the following criteria: (a) its activity is destroyed by 0.02 M sodium periodate; (b) when MIF-containing culture fluids are subjected to precipitation by perchloric acid (final concentration 1 M), the inhibitory activity is recovered in the supernatant; and (c) MIF binds to Sepharose-linked concanavalin A and can be eluted with methyl-alpha-D-glucopyranoside. When MIF-containing culture supernatants are fractionated by isoelectrofocussing, migration inhibitory activity is recovered in a fraction with an isoelectric point of 4.4--4.6.


Glomus tumor treated by prostaglandin inhibition. Report of a case. Glomus tumor is a very painful pericytal lesion of the arteriovenous anastomotic complex that controls circulation in a limb. Glomus tumor usually involves a digit. Prostaglandin inhibition may control the glomus tumor pain, but surgical removal is the cure. When the condition is discovered early, or when there is no gross evidence of tumor, thermography and localized anesthetic blocks are invaluable in arriving at the proper diagnosis.


Ultrastructure of cartilage in heritable disorders of connective tissue. Ultrastruct of cartilage were examined in Marfan syndrome, Menkes kinky hair syndrome, achondroplasia, asphyxiating thoracic dysplasia, mild diastrophic dysplasia and mucopolysaccharidoses I and III. Ruthenium red staining revealed decrease of proteoglycans in cases with Marfan syndrome and kinky hair syndrome, and increase in cases with osteochondrodysplasia and mucopolysaccharidosis III. This morphologic tendency coincided with the result obtained by biochemical analysis of glycosaminoglycan contents in cartilage matrix from cases with Marfan syndrome (decreased content) and asphyxiating thoracic dysplasia (increased content). It was postulated that proteoglycan content in cartilage matrix might be related to excessive or reduced skeletal growth in Marfan syndrome or osteochondrodysplasia.


Mucosal enzymes in human inflammatory bowel disease with reference to neutrophil granulocytes as mediators of tissue injury. 1. Biopsies of rectal mucosa were obtained for histology and enzyme analysis from 32 patients with inflammatory and functional bowel disorders, and the biopsies were classified morphologically as active colitis, quiescent colitis or normal. 2. Supernatant fractions of biopsy homogenates were assayed for their content of the proteolytic enzymes alpha-chymotrypsin, elastase and cathepsin D, and of protein, unsaturated vitamin B12-binding capacity, lysozyme, myeloperoxidase and N-acetyl-beta-glucosaminidase. 3. Mean unsaturated vitamin B12-binding capacity was significantly raised above normal in the active colitic mucosa, and mean lysozyme activity was raised above normal in both active and quiescent mucosae. 4. In active colitic mucosa there was no rise above normal in mean activities of any of the proteolytic enzymes, though a significant fall below normal occurred in mean N-acetyl-beta-glucosaminidase activity in the active colitic group.


Selective ion monitoring in clinical chemistry. The principles of selective ion monitoring are described. Choice of instrumentations, derivatives, and internal standards is discussed. The most important factors influencing sensitivity, specificity, and precision are summarized. Applications of selective ion monitoring for quantitative assay of steroids, fat soluble vitamins, triglycerides, prostaglandins, biogenic amines, amino acids, carbohydrates, and several other organic compounds of clinical interest are critically reviewed. It is concluded that isotope dilution selective ion monitoring is one of the most sensitive and accurate techniques presently available for quantitation of a large number of endogenous compounds of clinical interest. In view of the high accuracy, the technique is useful not only for the clinical chemists dealing with determination of specific compounds which are difficult to analyze by other methods, but also for those dealing with quality control of routine analyses of simple organic compounds.


Changes in optic glass-fibers due to X-ray irradiation. The purpose of this study was to clarify the relationship between x-ray irradiation and changes in the functional properties of flexible fiberscopes. Scanning electron microscopy, electron spin resonance, and thermoluminescent dosimetry techniques were used to determine the nature of the changes and their possible reversibility. Decreases in light transmission were observed at exposures above 5 roentgens (R), color changes were observed above 25 R, and electron spin resonance absorption changes occurred above 100 R. These changes were proportional to the x-ray doses. The x-ray exposure rates of the segmental and the subsegmental bronchi are about 20 percent that of the patient's thoracic skin surface. The above-mentioned studies showed that, for optimal results, a fiberscope should not be exposed to more than 15 minutes of fluoroscopy by a TV apparatus per week.


Free radicals in cancer.

Free radicals in cancer. It has been hypothesized that free radicals play a significant role in cancer. A historical review indicates a series of rises and falls in the acceptance of this hypothesis and it remains controversial. The strongest evidence for a critical role of free radicals in cancer is based on electron spin resonance (e.s.r.) data from lyophilized (freeze-dried) tumours. Recent results indicate that such data are artifactual in the sense that the observed signals are not directly related to free radicals existing before lyophilization. These data also indicate, however, that some of the observed changes are reproducible and may be indirectly linked to biophysical or biochemical changes that occur in tumour cells. A possible key to such a link is via antioxidants, especially ascorbic acid. It is now feasible to do experiments to definitively determine: (1) the generality of the effect of lyophilization on e.s.r. spectra of tumours; (2) the molecular nature of the free radicals observed in lyophilized tumours and normal tissues; (3) the effect of redox reactions and substances on the observed e.s.r. spectra; and (4) the occurrence of free radical changes during carcinogenesis.


A disruption of pachytene DNA metabolism in male mice with chromosomally-derived sterility. DNA metabolism was analyzed in spermatocytes of mice that were sterile either because of X-autosome or autosome-autosome translocations, or because of trisomy. In the strains analyzed, spermatogenic development is arrested by metaphase I or soon thereafter. In all such strains a disruption of the normal pattern of pachytene DNA metabolism occurred. Prepachytene metabolism appeared normal. Disruption was manifest in both the level of endogenously generated nicks during pachytene and in the distribution of nicks among the different DNA sequence classes. Nicking was more intense in the steriles and tended to be randomized in distribution. Satellite DNA underwent pachytene nick-repair in the steriles but not in fertile controls. The repair capacity of spermatocytes from steriles was equal to that of the fertiles; the higher frequency of nicks in the steriles was due to a persistence of nicking activity.


A rationale for opiate withdrawal symptomatology. The discovery of opioid peptide transmitters and the delineation of their interactions with the noradrenergic locus ceruleus (LC) has led to our proposing that opiate effects on mood and physiological responses result from the opiate-induced inhibition of the LC. Withdrawal of opiates removes this "tonic" inhibition of the LC and could readily result in a piperoxane-like release from inhibition. The hypothesis that noradrenergic hyperactivity underlies the physiologic and affective changes seen in opiate withdrawal and spontaneous panic seen in man can be readily tested in man by evaluating the efficacy of drugs which inhibit the LC and block piperoxane- and LC-elicited increases in specific behaviors for the treatment of opiate withdrawal and naturally occurring panic-anxiety states in man.


Rapid adaptation to neuronal membrane effects of ethanol and low temperature: some speculations on mechanism. There is increasing evidence that ethanol exerts its primary effect at neuronal membranes by influencing specific lipid--protein or lipid--lipid interactions that control the state of organization of a specific membrane component; for example, a specific lipid--protein complex that controls a particular physiological property. This implies that tolerance to ethanol is the result of a change in the composition and/or state of organization of this critical membrane component. This altered state confers ethanol resistance. It may or may not have an effect on function in the absence of ethanol. One basis for these speculations comes from experiments using a sensitive and specific neurophysiological assay -- the rate of decay of posttetanic potentiation (PTP) at an identified synapse in an isolated, perfused Aplysia ganglion. We review evidence that PTP decay rate is strikingly accelerated by ethanol (a membrane-fluidizing agent) and strikingly decelerated below a transition temperature, presumably reflecting a transition in the structure of a membrane component. The ethanol and low-temperature effects are antagonistic. The system develops adaptation (tolerance) to either ethanol or low temperature within hours of its exposure. Tolerance persists for at least 12 hours, the longest interval tested thus far. In the absence of ethanol and at normal temperature the system behaves normally, that is it shows no "physical dependence". The system also has the remarkable property that when it becomes tolerant to either of these treatments, it shows tolerance to the other treatment that normally has the opposite effect. Therefore, the adaptation to either treatment cannot be a simple change in membrane composition governing overall membrane fluidity. A hypothesis which could explain the bidirectional cross-tolerance is considered in which adaptation to both treatments involves a shift from a homogeneous to a more heterogeneous composition of the critical membrane component, for example increasing heterogeneity in boundary lipid surrounding a critical membrane protein. It is becoming increasingly clear that ethanol exerts its primary effect by altering cell membrane structure -- by "fluidizing" or expanding neuronal and other membranes [1 - 6]. This effect results when ethanol, a somewhat hydrophobic molecule, intercalates between some fatty acid chains of membranes, reducing the degree of order of their alignment and increasing the lateral mobility of some membrane components. Decrease in the order of the fatty acid chains results in a measurable expansion of the membrane; and the change in fluidity is reflected in the change in mobility of appropriate probes that can be dissolved in the membrane [6, 7]. It is presumed that the physiological effects of ethanol are consequences of its fluidization of some critical membrane components; and that tolerance to ethanol is based on some form of resistance to this fluidization...


Regulation of ornithine decarboxylase activity in rat ovarian cells in vitro. Incubation of rat ovarian cell suspension with human choriogonadotropin (hCG) caused a marked enhancement of ornithine decarboxylase (EC 4.1.1.17) activity after a lag period of several hours. Even though ovarian ornithine decarboxylase could be induced in minimum essential medium by the hormone alone, supplementation of the medium with various sera greatly enhanced the stimulation of the enzyme activity. All the sera tested (human, fetal calf and horse) were able to stimulate ornithine decarboxylase activity even in the absence of hCG. Maximum stimulation of the enzyme activity by hCG and/or serum occurred in ovarian cell suspensions prepared from 30 to 33-day-old rats. There was a close correlation between the stimulation of ornithine decarboxylase activity and the accumulation fo cyclic AMP in response to the administration of the hormone (in the presence or absence of serum). However, while various sera alone markedly enhanced ovarian ornithine decarboxylase activity in vitro they, if anything, only marginally stimulated the accumulation of cyclic AMP and the secretion of progesterone in ovarian cells in the absence of gonadotropin. A similar dissociation of the stimulation of ornithine decarboxylase activity from the production of cyclic AMP and progesterone was likewise found when the ovarian cells were incubated in an enriched medium (M199) supplemented with albumin and lactalbumin hydrolysate in the absence of the hormone. Under these culture conditions ornithine decarboxylase activity was strikingly enhanced, greatly exceeding the stimulation obtained with various sera, while the accumulation of cyclic AMP and the secretion of progesterone remained virtually unchanged. Specific inhibition (up to 90%) of gonadotropin-induced ornithine decarboxylase activity by difluoromethyl ornithine or 1,3-diamino-2-propanol had little effect on the ability of the ovarian cells to respond to the hormone with increasing production of cyclic AMP and progesterone. While showing that rat ovarian ornithine decarboxylase can be induced in vitro by choriogonadotropin or various sera, our results indicate that the activation of the enzyme involves at least two different mechanisms: (i) One (in response to gonadotropin) involving a prior stimulation of cyclic AMP production, and (ii) another (in response to serum) that is not associated with increases in the accumulation of the cyclic nucleotide.


Plasma immunoreactive calcitonin in lung cancer. We have measured plasma calcitonin in 135 untreated eucalemic men with lung cancer and a control/smoker population. Calcitonin levels were determined by radioimmunoassay and validated by immunoextraction. Plasma immunoreactive calcitonin moieties were purified by immunoadsorbent chromatography, treated with mercaptoethanol and urea, and characterized by gel filtration. Artifacts in human calcitonin radioimmunoassays of cancer-patient plasmas were detected by parallel plasma incubations in a salmon calcitonin radioimmunoassay system which does not detect human calcitonin and by immunoprecipitation of tracer at the end of radioimmunoassay incubations. Heating fresh plasmas to 65 degrees C for 1.5 hours reduced radioimmunoassay artifacts without loss of calcitonin moieties. Such characterization of hypercalcitoninemia in each of the histopathological types of lung cancer has raised some important questions about the interpretation of plasma calcitonin radioimmunoassay measurements in lung cancer. Based on inhibition of tracer-antibody binding, plasma calcitonin seemed to be elevated in 18% (14/80) of basal plasma samples obtained from patients with epidermoid or with anaplastic lung cancer. Unequivocal hypercalcitoninemia (heat stable, causing no inhibition of antibody-tracer binding in the salmon calcitonin radioimmunoassays, and immunoextractable with human calcitonin antibodies) was not found in any of the apparently hypercalcitoninemic plasmas from persons with epidermoid or anaplastic lung cancer. By contrast, unequivocal hypercalcitoninemia was found in 27% (15/55) of plasmas from patients with small cell carcinoma or adenocarcinoma. Most of the immunoreactive calcitonin recovered from small cell and adenocarcinoma lung cancer plasmas with unequivocally elevated calcitonin is much larger than calcitonin monomer.


Rat hyperplastic hepatic nodules and primary hepatomas retaining abnormally high pyruvate kinase L type activity. As reported in our previous paper (Sato et al., Cancer Res., 38: 3086-3093, 1978), most of the hyperplastic hepatic nodules and primary hepatomas induced by N-2-fluorenylacetamide (2-FAA), diethylnitrosamine (DENA), and 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) showed that pyruvate kinase liver (L) type decreases and the prototypic M2 type increases concomitantly with dedifferentiation of tissues. However, at least 14 samples among 120, mostly hyperplastic nodules and highly differentiated hepatomas induced by 2-FAA or DENA, retained exceptionally high activities of the L type, while other enzymes of carbohydrate metabolism in these tissues deviated toward a common pattern similar to that in the fetal liver. Individual patterns of 9 enzymes including pyruvate kinase of carbohydrate metabolism in these samples are arranged and discussed as examples of unbalanced enzyme deviation in hepatocarcinogenesis.


Influence of coenzyme on the refolding and reassociation in vitro of glyceraldehyde-3-phosphate dehydrogenase from yeast. Kinetic analysis of the reactivation in vitro of glyceraldehyde-3-phosphate dehydrogenase from yeast in the presence of NAD+ suggested that transconformation reactions of inactive monomers and their subsequent association to native tetramers are responsible for the sigmoidal relaxations [R. Rudolph et al. (1977) Eur. J. Biochem. 81, 563-570]. Comparison with the reactivation behaviour in the absence of coenzyme was not feasible at this stage due to the instability of the apoenzyme. In the present study, solvent conditions were established which allowed both apoenzyme and holoenzyme to exhibit high stability. The apoenzyme is stable in phosphate buffer; but if excess NAD+ and phosphate are present (both of which stabilize the enzyme if applied separately), destabilization occurs. Protection of functional groups against oxidation by addition of a reducing agent and by degassing and preventing contact with air, increase the stability. Only partial stabilization can be achieved in the presence of NADH. Comparing the kinetics of reactivation in the presence and absence of coenzymes shows that both oxidized and reduced coenzyme enhance the rate of reactivation significantly, and to the same extent. The kinetic effect of coenzyme binding to the refolding polypeptide chain is discussed in terms of the stabilization of intermediates or end products of reconstitution on the one hand, and acceleration of folding and association reactions, on the other.


Crystal and molecular structure of adenosine 5'-O-phosphorothioate O-p-nitrophenyl ester (Sp diastereomer). Substrate stereospecificity of snake venom phosphodiesterase. Phosphorylation of 2',3'-O-methoxymethylidene adenosine with bis(p-nitrophenyl) O,O-phosphorochloridothioate, followed by alkaline and acidic deprotection, afforded a mixture of the Rp and Sp diastereomers of adenosine 5'-O-phosphorothioate O-p-nitrophenyl ester in good yield. Only one of these diastereomers is a substrate for snake venom phosphodiesterase. The other diastereomer, remaining after complete enzymatic digestion of the substrate, was crystallized as the triethylammonium salt in the triclinic space group P1. The structure was solved from three-dimensional X-ray data and refined to an R value of 5.2%. The asymmetric cell unit contains two independent molecules with almost identical conformations. The ribose is puckered C(2')-endo, the heterocycle is in anti position and the C(5')-O(5') bond gauche, gauche. The triethylammonium cation is coordinated to the free oxygen of the phosphorothioate group and the P-S bond has double bond character. The absolute configuration of the phosphorous is Sp. This is in agreement with an earlier stereochemical study of the hydrolysis of the diastereomers of 5'-O-adenosyl 3'-O-uridyl phosphorothioate by snake venom phosphodiesterase.


Purification and primary structure of cytochrome c-552 from the cyanobacterium, Synechococcus PCC 6312. Cytochrome c-552 (soluble 'cytochrome f') from the unicellular cyanobacterium Synechococcus PCC 6312 (ATCC 27167) was purified and the primary structure determined. The proposed sequence consists of one polypeptide chain of 87 residues. The sequence was determined by a combination of chemical and enzymatic cleavage, manual and automatic sequencing and mass spectroscopy. This is the first amino acid sequence of this cytochrome from a unicellular cyanobacterium to be determined in a study of the variation in primary structure between phylogenetically distant cyanobacteria. The sequence is compared to the primary structures of the cytochrome from filamentous cyanobacteria and from eukaryotic algae. The significance of these sequence comparisons to the current hypotheses concerning the origin of eukaryotic cells and their chloroplasts is discussed.


Liver phosphorylase b kinase. Cyclic-AMP-mediated activation and properties of the partially purified rat-liver enzyme. Phosphorylase b kinase was extensively purified from rat liver. It was located in a form which could be activated 20--30-fold by a preincubation with adenosine 3':5'-monophosphate (cyclic AMP) and ATP-Mg. This activation was time-dependent, and was paralleled by a simultaneous incorporation of 32P from [gamma-32P]ATP into two polypeptides which comigrated in sodium dodecyl sulfate gel electrophoresis with the alpha and beta subunits of rabbit skeletal muscle phosphorylase b kinase. The liver enzyme was eluted from Sepharose 4B and Bio-Gel A-50m columns at the same place as muscle phosphorylase b kinase, which is indicative of a molecular weight of 1.3 x 10(6). After activation, the most purified liver preparation had a specific activity about 10-fold less than the homogeneous muscle enzyme at pH 8.2. The inactive enzyme form had a pronounced pH optimum around pH 6.0, whereas the activated form was mostly active above neutral pH. The activation of the enzyme reduced the Km for its substrate phosphorylase b severalfold. Liver phosphorylase b kinase was shown to be partially dependent on Ca2+ ions for its activity: addition of 0.5 mM [ethylenebis-(oxoethylenenitrilo)]tetraacetic acid (EGTA) to the phosphorylase b kinase assay increased the Km for phosphorylase b about twofold for both the inactive and the activated form of liver phosphorylase b kinase, but affected the V of the inactive species only.


Ascorbate oxidase from Cucurbita pepo medullosa. New method of purification and reinvestigation of properties. 1. Ascorbate oxidase has been isolated from the green squash Cucurbita pepo medullosa by a new purification method. Furthermore a low-molecular-weight copper protein containing one type-1 copper/20000 Mr could be separated during the purification of the oxidase. The six-step procedure developed improved the yield of ascorbate oxidase by a factor of 2.5. The method is well reproducible and a constant value of 8 Cu (7.95 +/- 0.1/140000 Mr) has been established. By ultracentrifugal and electrophoretic criteria the enzyme preparations have been found to be homogeneous. They exhibited a specific activity of 3930 +/- 50 units/mg protein or 1088 +/- 15 units/microgram copper. 2. The pure enzyme is characterized by the following optical purity indices: A280/A610 = 25 +/- 0.5, A330/A610 = 0.65 +/- 0.05 and A610/A500 = 7.0 +/- 0.25. The molar absorption coeffient of the characteristic absorption maximum at 610 nm (oxidized minus reduced) amounts of 9700 M-1 cm-1 . 3. Computer simulations of the electron paramagnetic resonance (EPR) spectra of the oxidized enzyme reveal the following parameters: for the type-1 (blue) copper gz = 2.227, gy = 2.058, gx = 2.036; Az = 5.0 mT, Ay = Ax = 0.5 mT, for the type-2 (non-blue) copper g parallel to = 2.242, g perpendicular = 2.053; A parallel to = 19.0 mT, A perpendicular 0.5 mT. Out of the eight copper atoms present in the oxidase four are detectable by EPR. Of these, three belong to the type-1 class, and one to the type-2 class, as demonstrated by computer simulations of the EPR spectra. 4. To achieve full reduction of the enzyme, as measured by bleaching of the blue chromophore, four equivalents of L-ascorbate or reductase must be added in the absence of molecular oxygen. Upon reduction of the enzyme the fluorescence at 330 nm (lambda max ex = 295 nm) is enhanced by a factor of 1.5 to 1.75. The reduced enzyme is readily reoxidized by dioxygen, ferricyanide or hydrogen peroxide. It binds two molecules of hydrogen peroxide in the oxidized state (1/type-3 Cu pair), which can be monitored by a characteristic increase of the absorbance around 310 nm (delta epsilon = 1000 +/- 50 M-1 cm-1). Corresponding changes in EPR and fluorescence spectra have not been detected.


Mavicyanin, a blue copper protein from Cucurbita pepo medullosa. Purification and characterization. 1. The copper protein mavicyanin has been isolated and purified from the green squash Cucurbita pepo medullosa. 2. Mavicyanin contains one type-1 copper/18000 Mr, which can be characterized by: intense absorption maximum at 600 nm (epsilon = 5000 M-1 cm-1/Cu, A280/A600 = 8.0 +/- 0.5, A600/A403 = 7.0 +/- 0.25, maximum of fluorescence emission at 335 nM. 3. In the oxidized state the copper of mavicyanin is 100% detectable by electron paramagnetic resonance (EPR). Computer simulation of the rhombic EPR signal gives gz = 2.287, gy = 2.077, gx = 2.025, Az = 3.5 mT, Ay = 2.9 mT and Ax = 5.7 mT. 4. Like other simple type-1 copper proteins, such as stellacyanin, azurin or plastocyanin, mavicyanin is readily reduced by hydroquinone or L-ascorbic acid. Its midpoint potential E'm was determined to be + 285 mV. The reduced protein reacts rather slowly with dioxygen, but is rapidly reoxidized by ferricyanide.


A possibe alpha-adrenergic mechanism for drug (CRL 40028)-induced hyperactivity. CRL 40028 (benzhydryl sulfinyl acetohydroxamic acid)-induced stimulation in mice, measured as an increase in locomotor activity was studied following an intraperitoneal injection of drugs known to block alpha-adrenergic receptors. Phenoxybenzamine (20 mg/kg), prazosin (0.5-1 mg/kg) and yohimbine (2 mg/kg) prevented the development of CRL 40028-induced hyperactivity. These results suggest that stimulation of an alpha-adrenergic postsynaptic receptor is of importance for the stimulant effect of CRL 40028.


Separation of human brain angiotensin-converting enzyme from enkephalin-degrading activity. Angiotensin-converting enzyme and enkephalin-degrading enzyme activities were solubilized and purified from a particulate fraction of human diencephalon. Converting enzyme activity and enkephalin-degrading activity elute in different fractions following ion exchange chromatography on DEAE-cellulose, suggesting that they are different enzymes. Both enzymes were purified further by ion exchange chromatography on hydroxylapatite and by gel filtration on Sephadex G-200. The purified enzymes had markedly different sensitivities to known inhibitors of angiotensin-converting enzyme. The data do not support the hypothesis that angiotensin-converting enzyme and enkephalin degrading-enzyme are identical.


Cyclic AMP in the rat cerebral cortex after stimulation of the locus coeruleus: decrease by antidepressant drugs. The study concerned the effect of repeated treatment with antidepressant drugs on the elevation of cyclic AMP levels in the rat cerebral cortex following electrical stimulation of the locus coeruleus. Some of the tricyclic and tetracyclic antidepressant drugs inhibited the cyclic AMP response. Desmethylimipramine was the most potent (effective when given 5 mg/kg/day for 2 weeks). Imipramine and nomifensine (daily dose 10 mg/kg for 2 weeks) produced slight decreases, while iprindol and clomipramine were ineffective. After 6 weeks of treatment (daily 10 mg/kg) iprindol, clomipramine and mianserin were without effect. The cyclic AMP response was suppressed by higher doses of the latter two drugs (2 weeks, 20 mg/kg/day). These results indicate that tricyclic and tetracyclic antidepressant drugs are able to decrease cerebral noradrenergic neurotransmission of locus coeruleus neurons, as far as this is mediated by cyclic AMP. It is not clear, however, whether such modification is related to the therapeutic action of antidepressant drugs.


Pathogenicity of Mengo virus to mice. III. Potentiation of infection by immunosuppressants. Mengo virus has been described to cause, in dependence on the virus dose, lethal panencephalomyelitis and exocrine pancreatitis in mice after i.p. inoculation. Two immunosuppressive agents, cyclophosphamide and 1,3-bis(piperidinomethyl)-5-ethyl-5-phenyl-barbituric acid (ZIMET 176/68), were shown to potentiate Mengo virus infection as demonstrated by histopathology and enhanced mortality. Organotropism of Mengo virus did not change under the drug treatment. However, the histological lesions in brain, spinal cord and pancreas failed to exhibit any inflammatory reaction in case of cyclophosphamide, due to its antiphlogistic properties. Considering the mode of action of the drugs employed and the pathogenesis of Mengo virus infection in mice, it is concluded that in the system used both cyclophosphamide and ZIMET 176/68 exert their potentiating effects by interfering with primary virus-macrophage interaction.


Level of marker enzymes in spermatogenesis on administration of PGF2 alpha in rats. Intraperitoneal administration of PGF 2 alpha in rats significantly increased testicular acid phosphatase (p less than 0.05), decreased hyaluronidase (p less than 0.05), whereas the activities of 5'-nucleotidase, N-acetyl-B-glucosaminidase, B-galactosidase and uridine diphosphatase remained unaffected.


Effects of several preoperative medications on fat cell lipolysis, and activity of adipose tissue cyclic AMP phosphodiesterase. Effects were examined of atropine, diazepam, pethidene, promethazine, scopolamine, omnopon and papaverine on basal and noradrenaline-stimulated lipolysis in rat isolated fat cells and on rat adipose tissue cyclic AMP phosphodiesterase activity. Papaverine at high concentration (1 mM) inhibited both basal and hormone-stimulated lipolysis, whereas diazepam enhanced basal lipolysis. At a 'clinical dose', omnopon increased both basal and noradrenaline-stimulated lipolysis. Adipose tissue cAMP phosphodiesterase activity was strongly inhibited by 1 mM diazepam, papaverine, promethazine and omnopon (280 microgram ml-1). Lack of enhancement of lipolysis by the established cAMP phosphodiesterase antagonist papaverine, is compatible with simultaneous inhibition also of adipose adenyl cyclase. Diazepam-stimulated lipolysis is compatible with its phosphodiesterase inhibitory activity. It is proposed that papaverine-containing omnopon may offer some survival advantages during surgical stress by facilitating a caloric supply.


Specific inhibition of a calcium dependent activation of brain cyclic AMP phosphodiesterase activity by vinblastine. Vinblastine selectively inhibits the activation of brain cyclic AMP phosphodiesterase activity by Ca++-protein activator (50% inhibition by 2 x 10(-5) M). This inhibitory effect was reversed by excessive amounts of the activator, whereas large quantities of Ca++ caused only a slight suppression of the vinblastine effect. This result of vinblastine suggests a new site of its action and also suggests the possible role of protein activator, phosphodiesterase proteins or cyclic nucleotides in the previously known effects of vinblastine in vivo and in vitro.


Alteration of early T lymphocyte count in patients with herpes genitalis. Peripheral blood samples from 52 women, including 16 with herpes genitalis and 36 healthy persons, were studied to enumerate subpopulations of lymphocytes. It was found that the mean percentage of 'active' T lymphocytes was significantly less in the patients with herpes genitalis than in the controls.


Corticosteroidogenesis by isolated human adrenal cells: effect of serotonin and serotonin antagonists. The direct effect of serotonin and antiserotonin agents on adrenal steroid biosynthesis was studied in isolated adrenal cells derived from patients with Cushing's syndrome. The results indicate that serotonin increases corticosterone production, while the serotonin antagonists cyproheptadine and methysergide depress adrenal steroid - particularly cortisol and aldosterone - biosynthesis.


Tangential flow filtration of Bordetella pertussis submerse cultures. A procedure is reported for the large scale separation of Bordetella pertussis microorganisms from liquid culture media by tangential flow filtration (cross flow filtration) using anisotropic membranes with a cut-off limit of 1 x 10(6) daltons, and microporous membranes with a pore size of 0.22 micrometer.


Retrovirus expression in normal and pathogenic processes of man. C-type viruses have been isolated from primates only on rare occasions. To date, an endogenous C-type virus of man has not been isolated. Nevertheless, the evidence for C-type viral expression in human tissue increases. Evidence for viral antigen expression during human gestation and the pathogenesis of systemic lupus erythematosus (SLE) is summarized. The data are discussed in connection with the current hypotheses that retroviruses may participate in both normal and abnormal biologic processes.


[Role of the fusimotor system in regulating shivering]. Spontaneously active gamma-motoneurons were found in muscles of the cat during shivering. The activity of these gamma-motoneurons could be increased by stimulation of thermoreceptors of the subcutaneous vessels and nociceptors. Administration of oxotremorine, nicotin, phentolamine, isoprenaline, and seduxene into the 3rd brain ventricle suppressed the shivering but did not change the firing rate of the gamma-motoneurons. The fusomotor system seems to participate in the segmentary activation of shivering, but not in its supraspinal control.


[Studies on the radioreceptor assay of TSH: the binding of 125I-TSH to the human thyroid receptor and the interaction of LATS-IgG (author's transl)]. The radioreceptor assay system for TSH is considered to be useful in quantitating the hormone and analyzing the mechanism of its action. The assay was established, and the interaction of abnormal thyroid stimulators in Graves' patients was evaluated in this assay system. A 10,000xg fraction of human thyroid homogenates was used as the receptor. Human TSH supplied from NIH was iodinated by using lactoperoxidase. The binding of 125I-TSH to the receptor was small, and 125I-TSH was further purified by the receptor binding. The receptor (25mg equivalent), purified 125I-TSH, and standard TSH or a sample were incubated at 37 degrees C for 60 min in a final volume of 300 microliters. The binding of 125I-TSH to the receptor was time- and temperature-dependent with optimal binding under the conditions described above. The binding was completely inhibited by the addition of human, bovine and ovine TSH and partially inhibited by high concentrations of HCG, FSH-LH. However, there was no cross reactivity with insulin, prostaglandin E1, E2, T3, T4 and Nal. The assay was sensitive enough to detect 5 to 50 microU of TSH. The amount of TSH bound to the receptor was almost parallel to the TSH concentration which is necessary to stimulate human thyroid adenylate cyclase activity. Studies of dissociation kinetics and Scatchard plot indicated that there were two classes of TSH receptors in the human thyroid. A higher association constant was calculated as 1.5 x 10(8)M-1. LATS-IgG from a patient with Graves' disease completely inhibited the binding of 125I-TSH to the receptor, and studies of Lineweaver-Burk, plot suggested that TSH and LATS-IgG shared common binding sites. The radioreceptor assay of TSH appears to be useful in evaluating the abnormal thyroid stimulators present in Graves' disease.


[Studies on the radioreceptor assay of TSH: the properties of TSH-binding inhibitor immunoglobulins (TBII) in patients with Graves' disease (author's transl)]. In the radioreceptor assay system for TSH, serum immunoglobulin G (IgG) from some patients with Graves' disease has been shown to inhibit the binding of labelled TSH to its receptor sites. In order to clarify the properties of these TSH-binding inhibitor immunoglobulins (TBII) in patients with Graves' disease, TBII were measured in sera from 31 untreated and 51 131I-treated patients, and their relation to clinical and laboratory findings was studied. TBII were detected in 18 (60%) out of 31 patients with untreated Graves' disease. TBII levels in these patients correlated well with thyroidal 99mTc uptake at 30 min and also with the grade of epithelial hyperplasia of thyroid follicles. There was no significant correlation between TBII and serum T3, serum T4, free T4 index, antibody titers against thyroglobulin and microsomes, or association of exophthalmos. There were many patients with Graves' disease whose sera contained high TBII levels but no detectable bioassayable thyroid-stimulating activity (LATS), and in these patients a close correlation was observed between serum levels of TBII and bioassayable LATS-protector activity. In patients with Graves' disease who had been treated by 131I from 5 to 17 years before, the incidence of TBII was very low at 20% (10/51). All except two cases having TBII were found to be still thyrotoxic. Thus, TBII were detected in 8 out of 10 thyrotoxic patients and in only 2 out of 18 euthyroid and none of 23 hypothyroid patients. These findings suggest that TBII in patients with Graves' disease were in close association with human thyroid stimulating activity, and that TBII might be useful as an indicator for checking the effectiveness of the treatment.


[Studies on the radioreceptor assay of TSH: the properties of TSH-binding inhibitor immunoglobulins (TBII) in patients with Hashimoto's thyroiditis (author's transl)]. TSH-binding inhibitor immunoglobulins (TBII) have been detected not only in patients with Graves' disease but also in those with Hashimoto's thyroiditis by using the radioreceptor assay of TSH. In the present study, the properties of TBII in patients with Hashimoto's thyroiditis are discussed. Two (7%) of 29 patients with Hashimoto's thyroiditis had detectable levels of TBII in their gamma-globulin fractions. Both patients were untreated and clinically hypothyroid. One of them had no goiter, and her thyroidal 99mTc uptake was 0% (normal range: 0.4 approximately 3.0%). Despite having potent TSH-binding inhibitor activity in the TSH radioreceptor assay, her serum or its IgG fraction (H-IgG) did not contain any significant anti-TSH antibody, LATS, LATS-protector or human thyroid adenylate cyclase (AC) stimulating activity. This H-IgG inhibited both human thyroid AC stimulation and c-AMP increase in mice thyroid glands induced by TSH or LATS. Furthermore, her serum caused significant inhibition of 131I-release by LATS in a McKenzie mouse bioassay. The present study demonstrates that the serum of one patient with Hashimoto's thyroiditis contained antibodies which 1) blocked the binding of lablled TSH to the receptor, 2) had no thyroid-stimulating activity by themselves, and 3) inhibited AC stimulation by TSH. Such antibodies may cause unresponsiveness to TSH stimulation, hypothyroidism, and, if this state persists for a long time, eventually may result in atrophy of the thyroid tissue. Further, these data indicate that TBII detected by the TSH radioreceptor assay did not always show thyroid stimulating activities.


[A comparison of various indicators of murine thyroid stimulation by LATS (author's transl)]. To obtain an in vitro method of LATS assay which is equally sensitive to a McKenzie bioassay, we compared the quality of cAMP generation-stimulation, thyroidal adenyl cyclase stimulation, and the stimulation of T3-release from mouse thyroid slices. Among those indicators of thyroid stimulation, stimulation by IgG of the release of T3 from incubated thyroid slices is the most sensitive, reproducible and reliable indicator, reflecting faithfully the results of a McKenzie bioassay. Stimulation of cAMP generation in slice manifested less sensitivity, but reasonable reproducibility. However, adenyl cyclase stimulation by IgG of patients with Graves' disease was erratic in two successive experiments, probably due to a non-specific effect of IgG. When adenyl cyclase stimulation was adopted as a sole indicator of thyroid stimulation, the results must be interpreted cautiously.


Generalized eruptive histiocytoma. A case report with a review of the literature. A case of generalized eruptive histiocytoma in a 52-year-old Indian male is described. This is probably the tenth case in the world and the first from this country. Histopathological features are discussed in detail and the literature is reviewed.


Parallel release of ACTH, beta-endorphin, alpha-MSH and beta-MSH-like immunoreactivities in rat anterior pituitary cells in culture. Specific radioimmunoassays (RIAs) for ACTH, beta-endorphin, alpha-MSH and beta-MSH were used to identify the immunoreactive components released during incubation of rat anterior pituitary cells in primary culture. Such measurements were performed after separation of the incubation media by chromatography on Sephadex G-50 or G-75. The ACTH-RIA measured approximately equal amounts of 13 and 4.5K ACTH while equal proportions of components migrating at the position of beta-LPH and beta-endorphin were measured in the beta-endorphin RIA system. Immunoreactive components migrating at the position of gamma-LPH and alpha-MSH were measured in the beta-MSH and alpha-MSH RIA systems, resp. 3 purified corticotropin-releasing fractions (CRF) prepared from porcine hypothalami and increasing concentrations of N6,O2'-dibutyryl cyclic AMP and theophylline led to parallel release of ACTH, beta-endorphin, beta-MSH and alpha-MSH immunoreactivities while preincubation with dexamethasone led to a 30-60% inhibition of the release of all peptides. The present data show that the release of ACTH, beta-LPH, beta-endorphin, gamma-LPH and alpha-MSH-like immunoeactivities occurs in parallel in anterior pituitary cells in culture both under basal and acute stimulatory or inhibitory conditions of release.


Interaction between the hepatic growth hormone receptor and concanavalin A. The interaction between the plant lectin concanavalin A (Con A) and hepatic receptors for human growth hormone (GH) has been studied in particulate and soluble microsomal membrane preparations from rabbit and rat liver. Con A shows a dose-dependent, partial (30%) inhibition of 125I-human GH binding which is reversed by the Con A competitor, alpha-methyl mannoside. The Con A effect is dependent on the receptor concentration. The inhibition by Con A in rabbit liver is a reflection of a decreased number of available binding sites--there is no effect on binding affinity. It would appear that Con A binds directly to the GH-binding protein and not to an adjacent membrane glycoprotein. The GH receptor may consist of more than one molecular species, differing only in the carbohydrate type or content.


Chronic hyperinsulinemia in the fetal rhesus monkey: effects on hepatic enzymes active in lipogenesis and carbohydrate metabolism. Hyperinsulinemia was produced in fetal rhesus monkeys for 21 days in the last third of gestation by subcutaneous pork insulin injected at 19 U a day. Plasma insulin concentrations in treated fetuses (N = 4) were 3525 microU/ml. There was no difference in paired pre- and post-treatment fetal plasma glucose concentration. Activity of the hepatic enzymes that promote glucose utilization (glucokinase and hexokinase) and glycolysis (phosphofructokinase, pyruvate kinase, and pyruvate dehydrogenase) was unaffected. Similarly, glycogen metabolism enzymes (active and inactive synthase and phosphorylase) were unaltered. Two gluconeogenic enzymes (PEPCK and glucose-6-phosphatase) were diminished in the treated group compared with controls. Fetal hyperinsulinemia enhanced lipogenic and NADPH-producing enzyme activities, as evidenced by a twofold increase in fatty acid synthase and in citrate cleavage enzyme activity. Malic enzyme was absent. Hyperinsulinemia with euglycemia (1) increases the activity of enzymes that participate in lipogenesis, (2) decreases some of those controlling gluconeogenesis, and (3) has no effect on the enzymes of glycolysis.


Intracisternal A-particles in genetically diabetic mice: identification in pancreas and induction in cultured beta cells. Sucrose density gradient analysis of purified pancreatic homogenates from glycaemic C57BL/Ks diabetes (db/db) mice and their normoglycaemic controls have revealed the presence in the diabetics of increased Mg++-dependent RNA-directed DNA polymerase activity sedimenting with a density of approximately 1.21 g/cm3. Electron microscopy revealed that this fraction contained typical intracisternal A-particles. Purified cultures of pancreatic islet cells 4--7 day old postnatal "misty diabetic" mice and normal siblings were established and then maintained in Eagle's minimal essential medium without serum. Under these conditions, the presence of intracisternal A-particles in beta cells of both mutant and control genotypes was very rare. No change in numbers of intracisternal A-particles was seen after 2--4 days of incubation in Dulbecco's-modified minimal essential medium containing 5.5 mmol/l glucose. However, when the glucose concentration of Dulbecco's medium was elevated to 16.5 mmol/l, ultrastructural changes specific to the beta cell population occurred that were reminiscent of those alterations observed in situ. Intracisternal A-particles were commonly seen in cultured beta cells showing hypersecretion-stress morphology. Since equal numbers of intracisternal A-particles were present in cultured beta cells from normal and mutant mice, it was concluded that the db gene itself was not required for intracisternal A-particle expression. The cell culture results suggest that elevated intracisternal A-particle activity observed in vivo may be produced directly or indirectly by the ambient high blood glucose levels characteristic of this mutant.


Acceleration of mammary cancer development by grafting of fetal mammary mesenchymes in C3H mice. Transplantation of fetal mammary gland mesenchyme into mammary glands of 2-month-old syngeneic virgin mice resulted in focal re-enactment of events that normally occur probably during fetal and early postnatal development of the mammary gland. Portions of the recipient's mammary duct system in contact with the fetal mammary mesenchyme underwent branching and proliferation in a pattern resembling that of rudimentary mammary gland development. This process occurred in C3H mice regardless of whether or not the milk-transmitted mammary tumor virus (MTV-S) was present. In mice carrying MTV-S, mammary cancers of Types A and B appeared earlier and more frequently in the mammary glands that had received transplants of fetal mammary mesenchyme, compared with those in the glands that received no fetal mesenchyme. Some of the smaller cancers were shown to develop directly from portions of the mammary gland interacting with fetal mammary mesenchyme, without preformation of typical hyperplastic alveolar nodules. In C3H mice not carrying MTV-S, cancers did not appear in the similarly treated mammary glands. These facts suggest that non-hormonal and probably nonviral factors that stimulate focal proliferation in the mammary duct system resulting from transplantation of fetal mesenchymes eventually accelerate local development of mammary cancers.


Induction of tumor resistance in mice by L1210 leukemia cells persistently infected with HVJ (Sendai virus). A temperature-sensitive strain of Sendai virus, HVJ-pi, showed little or no cytopathic effect and led to establishment of carrier cultures in several cell lines. By the use of this characteristic, L1210 leukemia cells persistently infected with HVJ-pi (L1210/c--HVJ-pi) was established, almost all of which were positively stained with fluorescent HVJ antibody. They are viable and grow almost equally as uninfected L1210 leukemia cells in vitro. Athymic nude mice (BALB/c, nu/nu), deficient of T-cells, died from intraperitoneal inoculation of L1210/c--HVJ-pi cells as well as by uninfected L1210 leukemia cells. However, viable L1210/c--HVJ-pi cells showed lower transplantability in normal syngeneic mice. This immunological mechanism of rejection was explained by the modification of cell surface membrane due to HVJ-pi infection. The mice which survived the inoculation of 10(5) L1210/c--HVJ-pi cells were able to reject 10(5) uninfected L1210 leukemia cells challenged subsequently. The induction of immune resistance was more prominent in (C57BL/6 x DBA/2)F1 mice or (BALB/c x DBA/2)F1 mice than in DBA/2 mice.


Cytostatic activity of peripheral blood monocytes against bronchogenic carcinoma cells in patients with lung cancer. Cytostatic acitivity of peripheral blood monocytes against cultured cell lines of bronchogenic carcinoma was examined in patients with lung cancer. Cytostatic activity in lung cancer patients with neither augmented nor suppressed as compared with that of controls such as normal healthy persons, patients with malignancies other than lung cancer, and patients with benign respiratory diseases. There was no correlation between the cytostatic activity of monocytes and the advance of clinical stages of the disease. Conventional modalities of anticancer treatments such as surgery, radiotherapy, chemotherapy, and their combination therapy had no effect on cytostatic activity of peripheral blood monocytes. However, adequate immunotherapy with nocardia rubra cell-wall skeleton augmented the cytostatic activity of peripheral blood monocytes, although adequate immunotherapy with Mycobacterium bovis BCG cell-wall skeleton had no effect.


Lack of participation of murine myeloma cells in the fusion of cocultured XC cells. Electron microscopy of thin sections of syncytia, formed by fusion of homologous XC cells, clarified that these heterologous cells were simply invaginated by syncytia and that they were clearly separated by the limiting cytoplasmic membranes. The C particles found at the boundary showed no sign of connection with the surrounding syncytial cell membrane, while some were seen budding from the surface of the producer cells. These results strongly suggest that the XC cells, even when cocultured with the C-type virus-producing myeloma cells, fuse homologously among themselves, and the virus-producing heterologous cells do not participate in the event.


In vivo enhancement of general and specific transcription in Escherichia coli by DNA gyrase activity. The effect of drugs which inhibit DNA gyrase, including nalidixic acid, oxolinic acid and coumerycin, on transcription of Escherichia coli bacteria, phage and plasmid genomes was studied. Quantitative estimates of the synthesis of RNA under drug-treatment conditions showed that synthesis of many RNA species, including trp mRNA, was subject to inhibiton by the drug. Transcription directed by the lambda promoter pR was selectively less sensitive to the drug action than transcription initiated at the lambda promoter pL. Evidence was obtained showing that diminished transcription resulted from less frequent RNA chain initiation rather than a premature arrest of the chain elongation. Inhibiton of transcription by these DNA gyrase inhibitors was observed even in the absence of DNA replication. The inhibition by oxolinic acid or coumerycin was not observed in an E. coli strain bearing a nalAr mutation or a cour mutation, respectively. The reduction of trp mRNA synthesis in oxolinic acid-treated cells cannot be attributed to the increase in the rate of nascent mRNA degradation. These results indicate that DNA gyrase is generally required for intracellular RNA synthesis, and suggest that the supercoiling of DNA by this winding enzyme enhances the initiation of transcription.


Injury to cultured endothelial cells: the role of lipoproteins and thrombo-active agents. Thrombin and low density lipoproteins (LDL) are capable of inducing damage to human endothelial cells in primary culture. These substances both induce endothelial contraction as first recognizable morphological change. However, the effect of thrombin appeared early, was reversible and prevented by hirudin, while LDL acted over hours and the injury was progressive. Addition and high density lipoprotein (HDL), together with LDL, inhibited the cellular injury induced by LDL.


Modulation of platelet function by prostaglandins: characterization of platelet receptors for stimulatory prostaglandins and the role of arachidonate metabolites in platelet degranulation responses. The effects of PGG2 and PGH2 on platelets are mimicked by synthetic PG analogues in which the nature and specificity of the substituents on carbons 11 and 15 (or 16) are important determinants of reactivity. Arachidonic acid and stimulatory PGs induce secretion of platelet dense granule and alpha granule constituents, but not lysomal constituents, although arachidonate metabolism is necessary for collagen-induced release of lysosomal enzymes. NO164 acts on platelets as an endoperoxide antagonist: Trimethoquinol acts as an endoperoxide and TxA2 antagonist. PGs induce platelet aggregation by combining with a specific (endoperoxide) receptor.


The estimation of alcohol dehydrogenase activity in aerobic and anaerobic "permeabilised" baker's yeast cells. The in vivo and in vitro activity of alcohol dehydrogenase from baker's yeast maintained under aerobic and anaerobic conditions was measured. In vivo measurements were made in cells "permeabilised" with toluene. Michaelis constants (NAD+ as substrate) were found to be almost identical as those reported for purified preparations. In addition the Km of the enzyme from cells incubated under anaerobic conditions was virtually identical to that from cells from aerobic conditions. The activity of the enzyme was found to be greater (in both "permeabilised" cells and extracts) in cells maintained under nitrogen than air. Cells metabolizing glucose in N2 produced greater levels of ethanol than in air and the rate of NAD+ reduction was also found to be greater in N2 than in air. The results indicate that it was feasible to determine rates of this enzyme in vivo and that the difference in activity of alcohol dehydrogenase under N2 and air may conceivably account for differences in rates of glucose utilisation, ethanol production and NAD+ reduction in air and nitrogen.


Studies on cytochrome c oxidase, VII. Isolation and chemical characterization of polypeptide VII. Polypeptide VII of cytochrome c oxidase was isolated and purified by gel filtration on Bio-Gel P-10 in 10% acetic acid. Automatic Edman degradation of this peptide chain was not successful, because it is blocked at the N-terminus. The amino acid analysis shows a relatively high content of hydrophilic residues (54%). On the basis of this analysis and the apparent molecular weight by sodium dodecyl sulfate gel electrophoresis and gel filtration, a chain length of about 80 residues was calculated. Among the tryptic peptides one blocked heptapeptide was found. Cleavage of this peptide with thermolysin gave two peptide fragments, one of which was not retained on a cation exchange resin. Mass spectrometric sequence determination of this peptide revealed the structure Ac-Ala-Glu-Asp for the N-terminus of polypeptide VII. Treatment with carboxypeptidase A at two different pH values showed that the C-terminal amino acid is isoleucine and the penultimate amino acid is lysine.


Studies on cytochrome c oxidase, VIII. The amino acid sequence of polypeptide VII. The sequence determination of polypeptide VII from beef heart cytochrome c oxidase is described. The amino acid sequence is deduced from overlapping tryptic peptides and peptides obtained after cleavage with Staphylococcus aureus protease. The protein consists of 85 amino acids corresponding to a Mr of 10026, in agreement with a value of 9500 obtained by sodium dodecyl sulfate gel electrophoresis. The amino acid sequence around the only methionine present is very similar to sequences around the invariant heme binding methionine of the cytochrome c family. This similarity suggests that the protein is one of the heme bindings subunits of the oxidase.


Studies on the protein composition of human serum very low density lipoproteins: demonstration of the beta 2-glycoprotein-I. Human serum VLDL isolated by polyanion precipitation and ultracentrifugation have been delipidated with ethanal/diethyl ether. By electrophoresis in 10% polyacrylamide gels containing 8M urea, we found a protein which comigrated with apolipoprotein E. This protein was purified by column chromatography and turned out to be identical with beta 2-glycoprotein-I, the serum factor which is necessary for the precipitation of triglyceride-rich lipoproteins with sodium decyl sulfate or sodium dodecyl sulfate. Upon analytical isoelectric focusing, beta 2-glycoprotein-I gave four major bands in the pH region 5.7--6.6. All four bands gave an immunochemical reaction of identity with a monospecific antiserum. From its unique amino acid composition we conclude that beta 2-glycoprotein-I is distinct from all apolipoproteins described previously in the literature.