Effect of metabolic inhibitors on vasopressin-stimulated transport systems in the toad bladder.

Vasopressin increases the permeability of receptor cells to water and, in tissues such as toad bladder, to solutes such as urea. While cyclic AMP appears to play a major role in mediating the effects of vasopressin, there is evidence that activation of the water permeability system and the urea permeability system involves separate pathways. In the present study, we have shown that inhibitors of oxidative metabolism (rotenone, dinitrophenol, and methylene blue) selectively inhibit either vasopressin-stimulated water flow or vasopressin-stimulated urea transport. There was no inhibition, however, when exogenous cyclic AMP was substituted for vasopressin, and little to no inhibition when the potent analogue 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) was employed. Rotenone had no effect on adenylate cyclase activity or cyclic AMP levels within the cell; dinitrophenol decreased adenylate cyclase activity minimally. Additional studies with vinblastine and nocodazole, inhibitors of microtubule assembly, demonstrated an inhibition of vasopressin and cyclic AMP-stimulated water flow but showed no effect on urea transport. We would conclude that water and urea transport, as examples of hormone-stimulated processes, have different links to cell metabolism, and that in addition to cyclic AMP, a non-nucleotide pathway may be involved in the action of vasopressin.


Predictive value of the analogy between hormone-sensitive adenylate cyclase and light-sensitive photoreceptor cyclic GMP phosphodiesterase: a specific role for a light-sensitive GTPase  as a component in the activation sequence.

We report experiments which involve a light sensitive GTPase in the light dependent activation of retinal rod 3'5'-cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE). The data suggest that the light activated GTPase is intermediate between rhodopsin and PDE in the light-dependent activation sequence. We list the many striking similarities between hormone sensitive adenylate cyclase and light activated PDE in order to emphasize that the findings presented herein may have predictive value for ongoing studies of the hormone sensitive adenylate cyclase specifically regarding the role of the hormone activated GTPase in the activation sequence.


Interaction between glycophorin and phospholipids in recombined systems.

Both the MN-glycoprotein from human erythrocytes and the hydrophobic fragment from the protein isolated with trypsin treatment, T(is), have been recombined with egg phosphatidylcholine in bilayers at various phospholipid/protein ratios. In order to investigate the effect of the protein on the phospholipid headgroups, 31P nuclear magnetic resonance spectra were obtained with the MN-glycoprotein recombined with egg phosphatidylcholine, which revealed two classes of phospholipid environments, one immobilized and one not immobilized. Electron spin resonance (ESR) of fatty acid methyl ester spin labels provided supporting evidence. Computer analysis of the ESR spectra indicate that 4-5 moles of phospholipid are immobilized per mole of protein over a wide range of lipid-to-protein ratios. The immobilization of the phospholipids appears mediated by both the polar headgroups and the hydrocarbon tails of the phospholipid.


Erythrocyte spectrin alteration induced by low-density lipoprotein.

Addition of human plasma low-density lipoproteins (LDL) to intact human erythrocytes induces the erythrocytes to undergo morphologic transition from biconcave disks to echinocytes and spherocytes. The transformation is time-dependent. Two hours are required before echinocytes are detected by scanning electron microscopy. After two hours, LDL also decrease the phosphate content of spectrin by 40% relative to the control, suggesting that these lipoproteins modulate cell shape by influencing phosphorylation-dephosphorylation of a membrane-associated cytoskeletal protein. LDL do not induce depletion of intracellular adenosine triphosphate (ATP), nor do they inhibit cyclic adenosine monophosphate-independent protein kinases which phosphorylate spectrin. LDL stimulate membrane-bound phosphatases by a factor of two, thereby reducing the amount of phosphate covalently bound to membrane proteins. The observed effects are specific for LDL. High-density lipoproteins (HDL) do not stimulate dephosphorylation of spectrin or alter erythrocyte morphology. However, HDL protect the erythrocytes against LDL-induced alterations. These data suggest that the circulating lipoproteins have a role in maintaining erythrocyte morphology by regulating the extent of phosphorylation of spectrin.


Cancer of the breast. Gross and histologic pathlogy.

Increased demands are made upon the pathologist to work closely with the surgeon and the mammographer in the interest of early detection. The smallest of cancers and the very earliest phases of neoplastic development are being detected, necessitating fine discriminations between cancer and noncancer. Agreement is not always uniform at these new frontiers of diagnosis, and accuracy is paramount. Tumors are populations of heterogeneous cells. Their morphology lends itself poorly to simple categorization, and their biology is not always accurately reflected in their gross and histologic appearances. Clearly evident to the pathologist are the limitations of morphology, of the light microscope and of routine techniques for examining surgical specimens. Paradoxically, "noninvasive" cancers occasionally metastasize, and lymph nodes originally "free" of cancer are found to contain metastases on more meticulous re-examination. Notwithstanding these limitations a prognostic statement can be made with relative confidence with regard to certain morphologic types of carcinoma. Pure intraductal carcinoma and lobular carcinoma in situ entail negligible threat to normal life expectancy if the breast is thoroughly removed. Other types with a favorable prognosis are intracystic papillary carcinomas and pure mucinous carcinomas. Tumors classified as well differentiated or tubular adenocarcinoma infrequently metastasize and have excellent prospects for cure. Unfortunately, these and other favorable histologic types comprise less than one quarter of all mammary carcinomas. Most carcinomas are without such distinctive features. In this category the degree of anaplasia and the nature of the tumor borders, as well as the presence or absence of blood vessel invasion, dermal lymphatic invasion and metastases in regional lymph nodes, are of major importance in arriving at an estimate of prognosis.


Developmental aspect of phenylalanine hydroxylase in the rat -- hormonal influences.

Interpretations of the development of phenylalanine hydroxylase (PAH) in rat liver have been controversial, and the mechanism of ontogenic changes have not yet been elucidated. Fetal PAH activity at a gestational age of 21 days appeared to reach 32% that of adult male level at birth. The in vivo effectiveness of fetal PAH activity was correlated with enhancement of blood tyrosine, while amino-transferase activity appeared only after birth. No sex difference was noted in weaning rats, whereas, in adult females, PAH activity was only 42% that of males. Investigating hormonal influences on liver PAH activity we noted no change of enzyme activity following hydrocortisone, ACTH and estradiol treatment. However, 4 days of testosterone treatment in weaning female rats increased PAH activity (X1.7). Therefore, testosterone could explain increased PAH activity in adult males.


Hormone action during aging: alterations and mechanisms.

The ability to respond to certain hormones is altered during the aging process. Attempts to elucidate the mechanisms involved in such changes have focused primarily on the various molecular components which mediate hormone actions. These include chromatin, the nucleus, cytoplasmic factors, adenylate cyclase and hormone receptors. Age related changes have been observed at all of these levels, and in some cases have been correlated with changes in biological responsiveness to particular hormones. This review attempts to summarize the data obtained in these studies. Possible interpretations are discussed and future directions for such research are suggested.


[Hormonal multiplicity of an apudoma of the lung and pancreas. Characterization of the different peptides in the tumoral extracts (author's transl)].

The capacity which the cells of some tumors have of synthesizing, storing, and releasing hormonal polypetides constitutes the basic characteristic of the neoplasms of the APUD system. On many occasions these polypeptides are released as hormonal precursors of high molecular weight, with a minimal biological action in comparison with the real hormone (big ACTH, big gastrin, etc.), and they have no clinical expressivity. On other occasions they reproduce, however, the clinical syndrome of the hormone released in excess. The production of multiple hormones by a single tumor is not a common event. Here we present the case of a patient with an oat-cell carcinoma of the lung and a carcinoma of the pancreas, both histopathologically primitive. In this patient a syndrome of inadequate secretion of antidiuretic hormone was detected. By means of radioimmunoassay techniques, the existence of antidiuretic hormone, ACTH with a predominance of the components of high molecular weight (big ACTH and beta-LPH) and MSH was demonstrated in the tumoral extracts from the lung, pancreas, and from a mediastinal metastatic lymph node. While the concentrations of ACTH were much greater in the lung than in the pancreas, the opposite occurred for the antidiuretic hormone. The synthesis of MSH by the hypophyseal gland or by tumors is not at present recognized, but rather is considered as a degradation product during the process of extraction. The APUD system makes up the morphologic substrate of the syndromes of familiar multiple endocrine adenomatosis. The present case could represent a variant of sporadic multiple endocrine neoplasms which would have the same anatomical basis.


[Ectopic ACTH syndrome (author's transl)].

Ectopic ACTH syndrome is a clinicopathologic condition produced by certain tumors which release hormone that is indistinguishable from ACTH. It orginates the chemical and clinical anomalies characteristic of Cushing's syndrome by its action on the adrenal glands. The tumors may be present in any organ, though they are most frequently found in the lungs, thymus, pancreas or gastrointestinal tract. They may be benign or malignant, though usually the latter. Secretion of the hormone is completely autnomous; it is release in a way similar to that of the hypophysis. Not infrequently other hormones besides ACTH are also produced, such as MSH, serotonin, and CRF. Ectopic ACTH is of higher molecular weight than hypophyseal ACTH, which suggest it may be comprised of the latter bounded covalently to a peptide. The clinical course is rapid, so that not all of the symptoms of Cushing's syndrome develop. Moon face, osteoporosis, and obesity are typically lacking; melanodermia and hypokalemic alkalosis ofter appear. Laboratory data include an increase in ACTH and cholesterol concentrations, disappearance of the nictameral rhythm, and an increase in urinary excretion of 17-hydroxycorticoids and 17-ketosteroids. Stimulation and supression tests are abnormal. The prognosis is poor and the only possible treatment is a complete surgical removal of the tumor. Irradiation or chemotherapy could be applied as well as the correction of the adrenal hyperfunction by the administration of drugs or by total bilateral adrenalectomy.


[Investigation of the hypoosmolal syndrome in carcinomas of the lung (author's transl)].

A study was performed on 30 patients diagnosed antomopathologically for malignant neoplasia of the lung (epidermoid carcinomas, adenocarcinomas, oat-cell carcinomas, and neoplasias which could not be definitely classified). The following parameters for blood and urine were determined: osmolality, sodium, potassium, urea, and creatinine. Osmotic free water, creatinine, sodium, and potassium clearances were also calculated, as well as the plasma osmolality/urinary osmolality ratio. The basic aim of our study was to investigate for the presence of disturbances in the metabolism of water and alterations in plasmatic and urinary osmolality in this type of tumor. These could appear as complete inadequate ADH secretion syndromes as discovered by Bartter and Schwartz or as incomplete syndromes (hypoosmolality and/or hyponatremia). Among the more significant results was the tendency toward oliguira seen in 44% of the patients and the high incidence of plasmatic hypoosmolality (31%). In three patients plasmatic hypoosmolality and hyponatremia were concommitant in repeated observations. A complete inadequate ADH secretion syndrome was discovered in another patient with an oat-cell carcinoma. He presented plasmatic hypoosmolality, hyponatremia, relative urinary hypertonia, and oliguria but not renal, suprarenal, or hepatic pathology.


Tympanometry in the evaluation of vascular lesions of the middle ear and tinnitus of vascular origin.

The occurrence of vascular perturbations in the tympanogram in association with glomus tumors has previously been noted in the literature. We have broadened the scope of this method of diagnosis in the study of 3 glomus tympanicum and 3 glomus jugulare tumors, 1 dehiscent high jugular bulb, 1 aberrant carotid artery in the middle ear and 2 cases of pulsatile tinnitus of vascular origin. Analysis of illustrative tympanograms at standard and at increased sensitivity (x 20) obtained in these cases are presented. The following factors as they relate to the mechanism of recording the vascular perturbations are presented: air pressure and/or presence of fluid in the middle ear; compliance of the tympanic membrane and ossicular chain as affected by the mass, size and degree of vascularity of the mass and presence of extraneous sounds. A new method of recording the vascular perturbations utilizing a time-base generator affixed to an X-Y plotter is described. It is the purpose of this study to demonstrate the utility of obtaining tympanograms at standard and at increased sensitivity in the evaluation of vascular middle ear lesions and tinnitus of vascular origin.


The composition and metabolism of high density lipoprotein subfractions.

The composition and metabolism of high density lipoprotein (HDL) subfractions were investigated in seven normal individuals. Mean HDL2 (d, 1.063-1.125 g/ml) composition (by weight) was 43% protein, 28% phospholipid, 23% cholesterol, and 6% triglyceride, and mean HDL3 (d, 1.125-1.21 g/ml) composition was 58% protein, 22% phospholipid, 14% cholesterol, and 5% triglyceride. The mean apoA-I; apoA-II weight ratio was 4.75 for HDL2 and 3.65 for HDL3. HDL2 protein was proportionally slightly richer in C apolipoproteins and higher molecular weight constituents (including apoE) than HDL3. Kinetic studies utilized radiolabeled HDLA (d, 1.09-1.21 g/ml), HDL2, and HDL3 demonstrated rapid exchange of apoA-I and apoA-II radioactivity among HDL subfractions, similar fractional rates of catabolism of apoA-I and apo A-II within HDL, and similar radioactivity decay within HDL subfractions. Mean plasma residence time  was 5.74 days for radiolabeled HDL2 and 5.70 days for radiolabedled HDL3. Differences in HDL protein mass among individuals were largely due to alterations in catabolism, and in general both HDL2 and HDL3 were catabolized via a plasma and a nonplasma pathway. Data from simultaneous radiolabeled very low density lipoprotein and HDL studies in 2 individuals are consistent with the concept that apoC-II and apoC-III are catabolized at a different rate than are apo A-I and apo A-II within the HDL density range.


Plasma lipoprotein changes in suckling and weanling rabbits fed semipurified diets.

The distribution and composition of the plasma lipoproteins were studied in suckling rabbits and in rabbits either weaned to or fed as young adults cholesterol-free, semipurified diets containing protein of animal (casein) or plant (soy protein isolate) origin. The raised cholesterol levels of the suckling period resulted in an increase of very low density and intermediate density lipoproteins in a making similar to that seen in adult rabbits fed a high fat diet supplemented with cholesterol. Young rabbits fed the cholesterol-free, semipurified, diet containing casein also became hypercholesterolemic but, in this case, the increased cholesterol was found primarily in lipoproteins of intermediate density. Cholesterol levels remained low in those rabbits fed the soy protein isolate diet, and the lipoprotein distribution was somewhat similar to that of chow-fed animals. It appears that the raised cholesterol levels during the suckling period result in different lipoprotein patterns to those produced in young adult animals by a cholesterol-free, semipurified diet.


A common pathophysiology for sudden cot death and sleep apnoea. "The vacuum-glossoptosis syndrome".

Several congenital and acquired conditions, characterized by upper airway narrowing, may result in respiratory, cardiac, and sleep disturbances. In all these conditions the leading clinical feature is the occurrence of cyclic obstructive apnoea, mainly during sleep. The common pathogenic mechanism for the airway occlusion seems to be a backward displacement of the tongue and mandible, favoured by the muscle relaxation which occurs during sleep and by gravity in the supine position. A constant factor determining the glossoptosis is the pharyngeal vacuum occurring in these conditions as a result of inspiratory efforts in face of a narrowed upper airway. The response to this type of obstruction is age-dependent, since only in early infancy may these apnoeic spells be life-threatening. A similar pathophysiology is suggested for sudden cot death, which could be considered as a peculiar presentation of this "vacuum-glossoptosis syndrome". This hypothesis could explain why sudden cot death is often associated with nasopharyngitis and occurs always in infancy, almost invariably during sleep.


Theoretical mechanisms for synthesis of carcinogen-induced embryonic proteins: IV. The viruses.

Speculations are developed for a mechanism by which oncogenic viruses can induce alterations in cells allowing them to express embryonic genes. It is suggested that if viral deoxyribonucleic acid, directly or via ribonucleic acid directed deoxyribonucleic acid polymerase activity becomes inserted at particular euchromatin - heterochromatin junctions of quasidifferentiated stem-like cells, then deheterochromatization may result, causing in turn derepression of genes for acidic protein phosphokinases. This sets into motion a series of events including altered acid protein repressors of embryonic genes which are repressed by uniquely weak type repressors. This explains how viruses can act as specific embryonic gene-inducing agents similar to chemical inducing agents such as the hepatocarcinogen ethinine.


The cause of species differences in mammary tumourigenesis: significance of mammary gland DNA synthesis.

The causes of species differences in mammary tumourigenesis are briefly discussed from the genetical and environmental points of view, although no firm conclusions can be drawn. Distinct differences in mammary gland DNA synthesis were found between female mice and rats, which behave very differently in mammary tumourigenesis. Based on the importance of mammary gland DNA synthesis in mammary tumourigenesis, the differences in mammary gland DNA synthesis between species is hypothesized to be a major cause of the species differences in mammary tumourigenesis.


Plasma protein regulation of platelet function and metabolism.

This reviews summarizes our evidence suggesting that the plasma protein enviroment influences platelet aggregation potential and metabolic activity. Cationic proteins are capable of restoring the aggreation potential of washed human platelets. The aggregation restoring effect of gamma globulin is inhibited by more anionic proteins in subfractions of Cohn fraction IV and fractions V and VI. Artificial enhancement of the net negative charge of plasma proteins through acylation produces derivatives capable of inhibiting platelet rich plasma. The oxygen consumption of washed human platelets is lower than in platelet rich plasma while the lactate production is identical. Autologus plasma, albumin or IgG immunoglobulin restores the oxygen consumption of washed platelets to values comparable to those obtained for platelet rich plasma, while the lactate production is unaffected. Fibrinogen on IgA myeloma protein increases the lactate production, but not the oxygen consumption. Cyclic AMP levels are considerably lower in washed platelets than in platelet rich plasma. Gamma globulin and albumin causes a futher decrease, which is progressive with time. Fibrinogen causes no change in platelet cyclic AMP content. It is suggested that these observations may in part be explained by the equilibriun between anionic and cationic proteins in the platelet microenvironment. This hypothesis appears applicable in certain situations.


Cytochrome P450 in adrenocortical mitochondria.

Cytochrome P450 in the mitochondria of the adrenal cortex functions in the monooxygenation reactions for the biosynthesis of various steroid hormones, such as cholesterol side chain cleavage, hydroxylation at 11 beta-position and that at 18-position of the steroid structure. The cytochrome is firmly associated with the mitochondrial membrane and therefore can be isolated only by the aid of ionic or non-ionic detergent. Recently, two cytochromes P450 each catalyzing a specified reaction have been purified to a homogeneous state, that is, P450scc having cholesterol side chain cleavage activity and P45011 beta having 11 beta-hydroxylation activity. The properties of these purified P450's as well as the other components of the monooxygenase system, adrenodoxin and adrenodoxin reductase, are, therefore, summarized and compared to those of P450 in the mitochondrial preparation in situ. Among many findings, both purified cytochromes P450 were revealed to be a low-spin type hemoprotein and their spin states were changed to a high-spin state by being complexed with the corresponding substrate. The binding of a substrate also facilitated the reduction of the cytochrome and appeared to increase the stability of the oxygenated form of cytochrome P450. These effects are important from the point of view that the primary role of the heme of cytochrome P450 is the activation of molecular oxygen. In addition, the results of our detailed kinetic studies on the transfer of electrons from adrenodoxin to cytochrome P450 in the reconstituted system have also been described. Finally, the topology of adrenodoxin and the reductase were shown to be on the inner mitochondrial membrane by a peroxidase-labeled antibody method.


mRNP proteins, initiation factors and phosphorylation.

Information has been collected to stimulate interest regarding the nature and the possible role of mRNP protein phosphorylation in a cytoplasmic control mechanism for protein synthesis. It does not imply a direct relationship between mRNP protein and initiation factors. These proteins have some properties in common (e.g. molecular weight, phosphorylation, protein kinase, mRNA binding activity). We emphasize that some free mRNP may be translatable after modification of their protein by interference factors belonging to other cellular compartments. Thus, some mRNP proteins might possess initiation factor or protein synthetic activity if we accept Spirin's theory, i.e., "Eukaryotic messenger RNA and informosomes omnia mea mecum porto.


[Virus-specific proteins associated with components of protein-synthesizing systems in Krebs II ascites carcinoma cells infected with encephalomyocarditis virus].

Extracts from encephalomyocarditis (EMS) virus infected Krebs II ascites carcinoma cells pulse-labeled during the active virus-specific synthesis and then chased were fractionated in a sucrose concentration gradient. It has shown that some radioactivity was detectable in the polysome region as well as in the regions of ribosome monomers and ribosomal subunits. An analysis of the radioactive material in a CsCl density gradient and by polyacrylamide gel electrophoresis has shown that components of the protein-synthesizing system in infected cells are bound to some proteins, the electrophoretic mobility of which corresponds to that of polypeptides found in the infected cells, namely, polypeptides G 16 (18 kdalton) and 22 (22 kdalton). The ribosomes from normal cells were also found to be associated with three labeled polypeptides, their molecular weight (89-90, 43-48 and 39-40 kdalton) being different from those of the polypeptides bound to the ribosomes from the infected cells. Thus, the presence of polypeptides G and 22 is specific for ribosomes isolated from the infected cells. The possible significance of this binding is discussed.


[Interaction of steroids with human serum albumin by spin echo technique and paramagnetic sound methods].

The rate of water proton relaxation of the solutions of human serum albumin (HSA) modified by different spin labels has been investigated by spin echo technique. The rate of proton relaxation in the presence of the labeled HSA is higher than that in water solutions of spin labels. The proton relaxation data of the protein solutions can be explained by local properties of the water, bound with the protein (in the regions of spin labels) which seem to resemble those of water-glycerol solutions. The rate of proton relaxation was decreased in the presence of steroids that can be explained by a decrease of the local microviscosity of the water-protein matrix due to interaction of steroids with HSA.


[Study of DNA-dye interaction by spin-labels].

The binding of ethidium bromide and acriflavin dyes with DNA modified with a spin-labelled analogue of ethylene imine has been studied. These spin-labels were shown to bind covalently to DNA, at the same time the number of the dye molecules bound is decreased without any changes in the binding constant. Analysis of ESR spectra of the samples in the frozen 50% water-glycerol solution at 77 degrees K for spin-labelled DNA has shown that addition of the dyes increases distance between the labels. This fact might be explained by an increase in DNA length upon formation of the complex with dye molecules.


[Macromolecule rotative correlation time measurement by ESR for covalently bound spin label].

The dependence from temperature and viscosity of the shifts of the internal and external wide extremums in the ESR spectra of spin labelled bovine serum albumin has been studied. 2,2,6,6-tetramethylpiperidine-NI-oxyl-4-iodacetamide was used as a spin label. The obtained dependences was shown to be a consequence of the label participation in two types of rotations: an anisotropic fast rotation with tau less than 10(-9) sec relatively to a macromolecule, and the isotropic one with tau greater than 10(-8) sec due to rotation of the macromolecule itself. These conclusions were done on the basis of a model for complex rotation of the spin label. Comparison of theoretical and experimental data makes it possible to determined the correlation time for the protein moiety, to evaluate quantitatively the polarity of surroundings of the iminoxyl and to introduce a numerical parameter for the degree of mobility of the spin label relatively to protein molecule.


[Influence of histone kinase phosphorylating lysine-rich histones, on the physico-chemical properties of normal hepatocyte chromatin and after partial hepatectomy].

The influence of a specific histone kinase, phosphorylating lysin-rich histone H1, H2a, H2b on the physico-chemical properties of chromatin from hepatocytes of normal and hepatectomized guinea pigs has been investigated. A cytochemical method has been used which permits to obtain information about the physico-chemical properties of the chromatin in situ, i.e. without its isolation. This approach allows us to evaluate changes in chromatin properties in cell cultures as well as in the intact organism. It is found that the specific histone kinase changes the properties of chromatin in non-dividing cells bringing about an increase of acridine orange binding to the level characteristic for hepatocytes after partial hepatectomy. At the same time the chromatin properties in activated hepatocytes are not changed under the action of the histone kinase. It is concluded that the specific histone kinase, phosphorylating lysine-rich histones can play an important role in the course of chromatin activation in cells stimulated to proliferation.


[Investigation of myxovirus structure by spin probes. I. Thermostability of the lipid membrane of influenza and Sendai viruses].

Electron spin resonance (ESR) spectra of the spin probes C5 and C16, the iminoxyl derivatives of stearic acids, have been investigated after their incorporation into lipid membranes of influenza and Sendai viruses. At room temperature the spectra of both probes coincided indicating the similar composition and origin of lipid viral membranes. Similar to influenza virus, Sendai virus was shown to possess the external lipid bilayer. Investigation of the rigidity of the lipid phase of myxoviruses as a function temperature has shown that the structure of the lipid external layer (no less than 8 A in thickness) is due to surface glycoproteins incorporated into the viral membrane. The structural transition of the lipid phase of influenza and Sendai viruses 8 A apart from the viral surface was observed at 50 degrees C. The surface glycoproteins do not probably penetrate deep in the lipid membrane of myxoviruses and do not condition its structure at long distances (22 A) from the viral surface. The completely reversible structural transition of lipid phase at this distance was observed at temperatures 17--20 degrees C.


[Photoreduction of hemoglobin in erythrocytes].

The ability of the erythrocyte membrane to transfer the reducing equivalent from the outer solution into the cells was studied. Erythrocyte hemoglobin transformed into the metstate serves as the electron acceptor. The donors develop during illuminating with visible light the outer solution, containing NADH and eosin. Some precautions were made to inhibit the migration of the donor molecules through the membrane. The photoreduction of hemoglobin in erythrocytes in such conditions can be attributed to the diffusion of some lipophilic electron carriers if they exist in the membrane, or to the ability of the transmembrane proteins to mediate the electron transfer from definite donors to the acceptors.


[Structure of higher plant chloroplast membranes as studied by paramagnetic probes].

A study of interaction between ten different spin labels and bean chloroplast membranes was carried out. It was found that spin labels were localized in three characteristic regions of the membrane. These regions differ in density and mode of the lipids package. The processes of light--induced reduction (with loss of paramagnetism) and reoxidation of spin labels are due to the interaction with the components of electron-transport chain between photosystems I and II. The kinetics of redox-changes of spin labels depends on the site of the paramagnetic fragment location in membrane.


[Nuclear magnetic resonance study of the conformation in nucleotides, oligonucleotides, and their analogs. I. Conformation of adenosine-3',5'-cyclic phosphate and its analogs in aqueous solutions].

Conformation in aqueous solution of adenosine-3',5'-cyclophosphate, 8-(beta-aminoethylamino) adenosine-3',5'-cyclophosphate, 8-(beta-oxiethylamino) adenosine-3',5'-cyclophosphate, 8-(carboxymethylamino) adenosine-3',5'-cyclophosphate and their non-cyclic analogs has been studied by NMR spectroscopy. The conformational situation in the model of dynamic equilibrium of sin- and anti-states has been described on the basis of spinlattice relaxation times and temperature dependences of chemical shifts. Adenosine-3',5'-cyclophosphate has been demonstrated to exist mainly in anti-conformation while 8-substituted analogs -- in sin-conformation. Equilibrium constants have been calculated for the compounds under study.


[Investigation of the microstructure of biological systems by triplet label].

A method for investigating the microstruct and dynamics of biological systems by means of triplet-excited molecules is suggested. The method is based on the phenomenon of triplet excitation disactivation by exchange-resonance triplet-triplet energy transfer to the acceptor or by intercombination conversion induced by interaction of an excited molecule with a paramagnetic center. The disactivation efficiency was measured by registrating the phosphorescense decay kinetics. The interaction of the triplet label eosin isothiocyanate, covalently coupled with albumine, lysozyme, sarcoplasmic reticulum membrane and Ca-Mg-dependent sarcoplasmic reticulum ATPase, with O2, the stable nitroxide radicals and ions of Mn2+ was investigated to analyse the potentialities of this method. As a model system the eosin phosphorescence quenching by the same quenchers in glycerine-aguaous solutions was studied. The method permits to investigate the microviscosity and microstructure of biological objects in the label attached region on interaction of the label with a sound-quencher with constants being 10(4) divided by 10(9) M-1 sec-1 and to measure the lateral diffusion of molecules in highly viscosity media (10 divided by 10(5) santypuas).


[Effectivess of synaptic influences of the ascending tracts of different spinal cord funiculi on reticulospinal neurons in cats].

Extracellular recording of the responses of the reticulospinal neurons evoked by stimulation of the dorsal, dorsolateral, ventral and ventrolateral spinal funiculi were performed in cats under chloralose anesthesia. It is found that dorsal funiculi of the spinal cord are an effective source of ascending influences on the reticulospinal neurons. Correlation of synaptic responses of the reticulospinal neurons with antidromatic responses in the peripheral nerves set up by stimulation of the dorsal funiculi shows that the former can be evoked by low threshold muscle (groups I and II) and cutaneous (group II) afferent fibres. The role of the ascending tracts in the transmission of the spino-bulbo-spinal activity is discussed.


Action of harmaline and diazepam on the cerebellar content of cyclic GMP and on the activities of two endogenous inhibitors of protein kinase.

The rat cerebellum contains a significant amount of cGMP-dependent protein kinase, cAMP-dependent and cyclic nucleotide-independent protein kinase, and a large concentration of protein kinase inhibitors. These inhibitors are thermostable proteins which can be separated by gel chromatography into two molecular forms: the type 1 and type 2 inhibitors of protein kinase (14). The type 1 inhibitor blocks the rat cerebellar cAMP-dependent protein kinase activity while the type 2 inhibitor blocks the cGMP-dependent protein kinase, the cAMP-dependent protein kinase, and the cyclic nucleotide-independent protein kinases. The activity of the type 2 inhibitor increased or decreased in opposite direction to changes of cerebellar cGMP content generated by injection of 10 mg/kg harmaline 2.5 mg diazepam. No changes of type 1 inhibitor were observed under these conditions. The drug-induced shift of type 2 inhibitor of protein kinase was not mediated by changes in protein synthesis because it persisted after pretreatment with cycloheximide. These results are compatible with the hypothesis that cGMP modulates phosphorylation in cerebellum by changing the relationship between cGMP-dependent protein kinase and type 2 inhibitor content.


High-affinity uptake of gamma-aminobutyric acid in cultured glial and neuronal cells.

Both glial and neuronal cells maintained in primary culture were found to accumulate [3H]GABA by an efficient "high-affinity" uptake system (apparent Km = 9 muM, Vmax = 0.018 and 0.584 nmol/mg/min, respectively) which required sodium ions and was inhibited by 1 mM ouabain. Strychnine and parachloromercuriphenylsulfonate (pCS) (both at 1mM) also strongly inhibited uptake of [3H]GABA, but metabolic inhibitors (2,4-dinitrophenol, potassium cyanide, and malonate) were without effect. Only three structural analogs of GABA (nipecotate, beta-alanine, and 2,4-diaminobutyrate) inhibited uptake of [3H]GABA, while several other compounds with structural similarities to GABA (e.g. glycine, L-proline, and taurine) did not interact with the system. The kinetic studies indicated presence of a second uptake (Km = 92 muM, Vmax = 0.124 nmol/mg/min) in the primary cultures containing predominantly glioblasts. On the other hand, only one of the neuronal cell lines transformed by simian virus SV40 appeared to accumulate [3H]GABA against a concentration gradient. Apparent Km of this uptake was relatively high (819 muM), and it was only weakly inhibited by 1 mM ouabain and 1 mM pCS. The structural specificity also differed from that of the uptake observed in the primary cultures. Significantly, non of the nontransformed continuous cell lines of either tumoral (glioma, C6; neuroblastoma, M1; M1NN) or normal (NN;I6) origin actively accumulated [3H]GABA. It is suggested that for the neurochemical studies related to GABA and requiring homogeneous cell populations, the primary cultures offer a better experimental model than the continuous cell lines.


Bovine dihydropteridine reductase: purification by affinity chromatography and comparison of enzymes from liver and adrenal medulla.

Dihydropteridine reductase has been purified to homogeneity from bovine liver and bovine adrenal medulla by precipitation with polyethylene glycol, ion exchange chromatography, gel filtration, and affinity chromatography on 5'-AMP-Sepharose 4B. The enzymes from the two tissues seem identical by the criteria of gel filtration chromatography, affinity chromatography, polyacrylamide gel electrophoresis in the presence and absence of dodecyl sulfate, isoelectric focusing, amino acid analysis, and binding of NADH. Fluorescence studies show two independent binding sites for NADH and a dissociation constant of 10 nM at pH 6.8. Isoelectric focusing of the enzyme as purified in the presence of NADH revealed three different bands, which by removal of this coenzyme were converted into a single band, corresponding to pI 5.7. The enzyme contains no carbohydrate or zinc.


Distribution of opiate-like substances in rat tissues.

Rat tissues were tested for their ability to inhibit the binding of [3H]dihydromorphine or [3H]naloxone to membrane-bound opiate receptors. By this criterion, morphine-like substances were found in lung, heart, liver, and kidney as well as in brain. The relative activity of the extracts, based on initial tissue weight, differed with the radioactive lignand employed. With dihydromorphine, the order was as above. With naloxone, lung was most active, followed by heart, brain, liver, and kidney. The ability of all tissue extracts to inhibit opiate binding was reduced by 100 mM NaC1 and slightly reduced by 1 mM MnC1(2). Gel filtration using Sephadex G-25 indicated that the inhibitory substances were heterogeneous in molecular weight. Only with brain and kidney extracts was there significant activity at the elution volume where enkephalins would be expected. Fractionation using Amberlite XAD-2, a resin which selectively absorbs hydrophobic materials, again indicated that the major protion of activity in all tissue extracts was due to substances other than enkephalins.


Peripheral nerve injection injury: an experimental study.

In an attempt to answer questions regarding nerve injection injuries, we injected 11 agents in current use and commonly administered by intramuscular injection into the sciatic nerves of adult Wistar rats. Equal volumes of normal saline were used as control. We harvested the sciatic nerves at various times after injection and examined them by both light and electron microscopy. We performed myelinated nerve fiber counts and constructed histograms. Any impairment of motor function was also noted. We gave injections to 79 animals a total of 158 times; 116 injections were directly into the nerve fascicle (intrafascicular) and 42 were into the epineural tissue (extrafascicular). The results revealed considerable variation in the degree of nerve fiber injury according to the agent injected. Minimal damage resulted from the injection of iron-dextran, meperidine, and cephalothin, and maximal nerve injury followed the injection of penicillin, diazepam, and chlorpromazine. The site of injection was crucial. Intrafascicular injection was invariably associated with severe nerve injury, but, with few exceptions, extrafascicular injection resulted in minimal damage. The quantity of drug injected was also important in determining the degree of injury. Large, heavily myelinated fibers were more susceptible to injection injury than smaller, thinly myelinated nerve fibers. The effect of the injected drug seemed to be related to injury of the nerve fiber unit--both the axon and the Schwann cell with its myelin sheath. Regeneration in damaged nerves was a constant finding; even the most severely injured nerves, with total axonal degeneration, underwent subsequent regeneration.


Suppression of foreign innervation in axolotl muscle may not be dependent on juxtaposition of native and foreign nerve terminals.

The posterior supracoracoideus nerve of the axolotl, Ambystoma mexicanum, was induced to make synapses outside its normal muscle territory. Muscle fibres with inputs from both native and foreign nerves were studied during the period of suppression of foreign transmission and in only 8% of fibres were foreign and native terminals found within 120 micrometer of each other. A combined cholinesterase/silver staining technique revealed non-innervated endplates in foreign-innervated fibres just prior to the return of the native nerve. These results suggest a mechanism enabling suppression of foreign synapses at some distance from native synapses probably beginning with the reinnervation of empty sites by the native nerve.


The effects of intranigral injections of picrotoxin and carbachol in cats with a lesioned nigrostriatal pathway.

Picrotoxin, carbachol and atropine were injected intranigrally through chronically implanted crannulae in cats. The drug-induced behavioural syndromes, mainly asymmetric behaviour, were analyzed. The changes in these syndromes after 6-hydroxydopamine (6-OHDA) lesion of the substantia nigra (SN) indicate that, in addition to the dopaminergic (DA), a non-catecholaminergic (non-CA) nigral output exists, both being responsible for asymmetric behaviour. It is further suggested that gamma-aminobutyric acid (GABA) receptors are present on both dopaminergic and non-CA nigral output neurons, while acetylcholine (ACh) receptors, responsible for asymmetric behaviour, are almost exclusively located on non-CA nigral output neurons.


Ability of calf brain synaptic membranes to bind [35S]taurine to their triton X-100 and chloroform extracts.

A protein fraction containing reducing sugars was prepared from calf brain synaptic membranes with 0.5% Triton X-100, and another fraction containing bound phosphorus with a 2 : 1 chloroform--methanol mixture. Both protein fractions bound small amounts of [35S]taurine, the first fraction about 25 pmol/g protein and the second about 220 pmol/g protein. The Triton X-100 extract represented 13.8% of the membrane proteins, but the chloroform--methanol extract only 0.9%. The binding of taurine to the Trition X-100 extract was temperature sensitive, but was only slightly inhibited by hypotaurine and beta-alanine.


Localization of opiate receptors in substantia nigra evidence by lesion studies.

From the effects of selective lesions it is concluded that opiate receptors are located on dopamine (DA) cell-bodies and/or dendrites as well as on GABA nerve-terminals in substantia nigra.


A study on the effects of interaction between naloxone and 2-Br-alpha-ergocryptine or clonidine on luteinizing hormone, follicle-stimulating hormone, prolactin and thyroid-stimulating hormone levels in normal man serum.

The effects of 2-Br-alpha-ergocryptine (2.5 mg/osM), clonidine (50 microgram, intramuscularly) and naloxone (0.4 mg, intramuscularly) as well as the interaction between naloxone and 2-Br-alpha-ergocryptine or clonidine on luteinizing hormone (LH) follicle-stimulating hormone (FSH), prolactin (PL) and thyroid-stimulating hormone (TSH) serum levels in normal man have been studied. 2-Br-alpha-ergocryptine and clonidine clearly reduce and naloxone tends to reduce PL serum levels. TSH levels are lowered by naloxone as well by clonidine plus naloxone. The results obtained point also to a possible different pattern of LH and FSH secretion after naloxone, that is after opiate receptor blockade. The clonidine effects on PL secretion are discussed in the frame of a possible adrenergic control of the release of this hormone.


Prostaglandin E1 raises the cAMP content of peripheral nerve tissue.

Prostaglandins of the E type have been found to increase the cAMP content in peripheral nerve tissue of several species. In rabbit vagus nerve, for example, the effect is rapid (1--5 min), it can be elicited by 10(-6) M PGE1 and it is maximal at 10(-5) M. In this preparation the increase of the cAMP content caused by PGE1 is about 4-fold, whereas it is about 2-fold in the vagus of calf. PGE1 also causes an increase in cAMP level in the superior cervical ganglion of calf, rat and rabbit. In these tissues, however, the presence of a phosphodiesterase inhibitor is required for cAMP accumulation.


Persistence of virus shedding in asymptomatic women after recovery from herpes genitalis.

One hundred seventeen women were reexamined within 6 months after presenting with genital herpes. Swabs for virus isolation were taken from the cervix whether or not lesions were observed. Herpesvirus type II was isolated from 30 of 67 patients with recurrent disease, but also from 5 of 50 women during periods when there were no signs or symptoms of illness. The epidemiologic importance of virus shedding from asymptomatic women is discussed.


Herpes virus infection as a cofactor in carcinogenesis: supernatants of herpes virus type 1- and type 2-infected cell cultures contain a cell growth-stimulating factor.

Some cell cultures synthesize a mitogenic factor (DNASF) following their lytic infection with either HSV-1 or HSV-2 which is shed into the medium. Of six cell lines permissive to HSV infection tested, three produce DNASF (CV1, HF, MCA) and a significantly higher (3H)-TdR incorporation was obtained with indicator cells growing in supernatants of virus-infected cells (SupV+) than in the corresponding supernatants of sham-treated cells (SupV-). Sometimes the values obtained with SupV+ exceed those obtained with cell controls, which demonstrates that DNASF is not simply a nutritional factor. Other cell lines do not produce DNASF (HeLa, Wistar and probably REF) and SupV+ frequently inhibit cell growth. DNASF is genetically nonspecific and all indicator cells tested were stimulated by supernatants which contain the mitogen, including the nonpermissive DC-3F cells. The quantity of DNASF induced is directly proportional to the number of cells in the culture and its production peaks when the lysis of the cells is complete. It is not accumulated inside the cells.


[Catecholamine sensitivity of the hypophyseal-adrenal system in the early ontogeny of rats].

The dynamics of the hypophyseal-suprarenal system reaction to catecholamines of different type of effect was studied in the Wistar rats during the first 16 days of life. The reaction to epinephrine was noted in 4 days old and to novodrin in 8 days old rats. Since the reaction to epinephrine acting on alpha- and beta-receptors appears ahead of that to novodrin acting selectively on beta-receptors, a suggestion was put forward to the effect that alpha-receptors matured more rapidly that beta-receptors. The increase of the system reaction to the stimulation of recepors sensitive to catecholamines was noted on the 12-14th days of life and appears to be related to the formation of central adrenergic mechanisms.


A testicular tumor with gingival metastasis.

Testicular tumors comprise approximately 1 percent of all malignant neoplasms in the male. However, they are one of the more common malignant diseases of adolescent and young adult males. An unusual case of a testicular neoplasm in a 23-year-old man exhibiting gingival metastasis is presented.


Inhibition of the spinal transmission of nociceptive information by supraspinal stimulation in the cat.

In cats anaesthetized with alpha-chloralose or sodium pentobarbitone a study was made of the effects of supraspinal electrical stimulation on the excitation of dorsal horn neurones by noxious and non-noxious cutaneous stimuli. Stimulation near the dorsal raphe of the midbrain non-selectively reduced the responses of neurones to both noxious and non-noxious stimuli. Intravenous naloxone (0.3-0.6 mg/kg) had no effect on this inhibition. Electrical stimulation near the medullary raphe selectively reduced the excitation of dorsal horn neurones by noxious cutaneous stimuli. Excitation of neurones by deflection of hairs was unaffected. The inhibition of nociceptive responses outlasted the period of raphe stimulation by up to 6 min. Intravenous naloxone (0.6-1.0 mg/kg) also failed to affect this selective inhibition.


[Tumoural calcinosis in a white male (author's transl)].

A case of tumoural calcinosis in a 61-years-old man. This is a rare condition (60 published cases), affecting above all black subjects. It takes the form of hard rounded masses which progressively increase in size and vary in their location (hips, elbows, shoulders, periscapular region). On X-rays, the tumour is rounded, consisting of a mass of several calcified small nodules. On technetium pyrophosphate isotope scanning, there is very marked and periarticular uptake. There is often hyperphosphoraemia associated with normal blood calcium levels, without any abnormality in the hormones controlling calcium and phosphate metabolism. Treatment usually consists of the excision of the tumour masses. More recently, the reduction of hyperphosphoraemia by the prescription of aluminium salts and a reduction in phosphorus intake has been suggested.


[Acute pancreatitis during "diabetic lipemia": unusual disclosure of insulin-dependent diabetes (author's transl)].

A case of diabetic lipemia is reported in a 27 year-old man admitted for acute pancreatitis. Initial investigations revealed gross hyperlipoproteinemia and ketoacidosis. Hyperlipoproteinemia was progressively corrected up to normalization in four weeks under insulin-therapy; the metabolic control of diabetes was obtained in parellel. This feature is caracteristic of "diabetic lipemia". The following sequence could be suggested: onset of diabetes, occurence of diabetic lipemia and then acute pancreatitis.


[The treatment of advanced malignant melanoma with vaccina oncolysates (author's transl)].

Ten patients with recurrent stage II or III malignant melanoma received a live vaccinia virus augmented malignant melanoma cell vaccine. No patient had adverse reactions. The 3 patients surviving (10, 18 and 19 months) had: positive skin reactions to the vaccine (as they had previous positive skin reactions to common recall antigens), elevated vaccinia antibody titers, and they showed lack of progression of the disease with immunotherapy.


[Water and electrolytes intestinal secretion (author's transl)].

This paper reviews methods used for studying intestinal secretion in man and animals, fasting and post prandial intestinal secretion, as well as that induced by bacterial enterotoxins, hormonal stimuli, some endocrine tumours and various intraluminary agents; finally the possible mechanisms and sites of intestinal secretion are discussed. That the intestine secretes fluid under physiological conditions is not proven but seems very likely. Several mechanisms seem to be involved in the production of fluid by the intestine. The adenylate-cyclase-cyclic adenosine monophosphate (AMPc) system is currently thought to play a major role in endotoxins and some hormone-induced secretions. The site of intestinal secretion remains a matter of discussion: both crypts and villi seem to participate in the secretory process.


Direct detection of methylated cytosine in DNA by use of the restriction enzyme MspI.

The extent of methylation of the internal C in the sequence CCGG in DNA from various eukaryotic sources has been determined using the restriction enzyme MspI known to be specific for this sequence. The methylation of the CCGG sequence is reflected in the restriction pattern obtained by DNA treated with MspI and its isoschizomer HpaII and analyzed by gel electrophoresis. A direct method for detection 5-methylcytosine in the sequence CCGG has been deviced. DNA fragments obtained with MspI were radioactively labeled at their 5' ends and subsequently degraded to the corresponding 5'-deoxyribonucleoside monophosphates. 5 methylcytidylic acid has been found in most of the 5' ends of MspI fragments of calf thymus DNA (about 90%) indicating heavy methylation of the sequence CCGG in calf thymus DNA. The results also reveal a symmetric methylation of both strands at this sequence in calf thymus DNA. In contrast, the CCGG sequence in other eukaryotic DNAs from organisms like Neurospora, Drosophila and Herpes virus proved to be undermethylated at this sequence.


Isolation and characterization of rabbit anti-m7G-5'-P antibodies of high apparent affinity.

Antibodies highly specific for intact pm7G (7methylguanosine-5'-mono-phosphate) were induced by immunization of rabbits with a pm7G-BSA conjugate. Since the nucleotide is six-fold more stable than m7G (7-methylguanosine) to alkali-catalyzed fission of the imidazole ring, it is a more desirable antigen for obtaining antibodies capable of binding caps on eukaryotic mRNA. UV spectra demonstrated that the nucleotide in the conjugate was predominantly the intact form. Competition radioimmunoassay showed 1) high apparent affinities for pm7G, on the order of 10(-8)M, 2) low competition by the ring-opened form of the homologous hapten (*pm7G), and by m7G, 3) little or no competition by AMP, GMP, CMP, UMP or m6A, and 4) high apparent affinities for m7GpppAm, m7GpppN6MAm, m7GpppGm, m7GpppA.


Fluoride ion catalyzed alkylation of nucleic acid derivatives using trialkyl phosphates, dialkyl sulfates and alkyl methanesulfonates.

Trimethyl phosphate, dimethyl and diethyl sulfate and methyl and ethyl methanesulfonate all give high yields of alkylation on purines and pyrimidines in the presence of tetrabutylammonium fluoride. Trimethyl phosphate produces near quantitative yields of diesters of nucleic acids but gives virtually no triester formation. The alkyl sulfates produce very high yields of triesters of nucleic acids including cyclic phosphates while the alkyl methanesulfonates are intermediate in reactivity. It was observed that in the absence of fluoride ion the dialkylsulfates gave reasonable yields of thymidine monosulfates.


Sequence and location of the poly C tract in aphtho- and cardiovirus RNA.

The poly C tract in the RNA of the aphtho- and cardio viruses has been examined in several isolates of foot-and-mouth disease virus (FMDV) and encephalomyocarditis (EMC) virus. The length of the tract is variable, containing 100 to 170 bases in the FMDV isolates and 80 to 250 bases in the EMC virus isolates. Each poly C tract contains c. 10% A and U residues, located at the 5' end, i.e. most of the tract is a continuous run of C residues. The position of the tract on the genome was the same in each of the FMDV isolates, about 400 bases from the 5' end, whereas in the EMC virus isolates it was about 150 bases from the 5' end.


Evidence for removal at different rates of O-ethyl pyrimidines and ethylphosphotriesters in two human fibroblast cell lines.

The potent carcinogen, ethylnitrosourea, has been shown to ethylate oxygens, in preference to nitrogens, in the DNA of cultured cells. We have now studied the removal of seven ethyl derivatives in replicating cells. The following findings are reported. 1) The absolute amounts of 02-EtT, 04-EtT and 02-EtC are decreased in cellular DNA after correction for cell growth. However the rate of decrease diminishes after approximately 20 hr and after more than two cell doublings 20--40% of each derivative persists. This decrease is presumed to be due to enzymes since these derivatives are stable in isolated DNA. 2) The amount of ethyl phosphotriesters remains almost unchanged during 72 hr of cell culture. 3) The unstable purine derivatives, 7-EtG and 3-EtA, are both removed from cellular DNA with a rate faster than can be accounted for by the lability of the glycosyl bond. 4) Both GM 637 fibroblasts and Xeroderma pigmentosum fibroblasts (12-RO) (XP-12) have similar ability to remove ethyl products, except for O6-ethyl G which persists to a greater extent in XP12 cells. 5) The implications of the in vivo persistence of ethylated bases is discussed in regard to recent demonstrations that O2-EtT, O4-ET, O2-EtC and O6-EtG are all mutagenic.


Identification and mapping of N6-methyladenosine containing sequences in simian virus 40 RNA.

Late SV40 16S and 19S mRNAs were found to contain an average of three m6A residues per mRNA molecule. The methylated residues of both the viral and cellular mRNAs occur in two sequences; Gpm6ApC and (Ap)nm6ApC, where n = 1-4. More than 60% of the m6A residues in SV40 16S and 19S mRNAs occur in Gpm6ApC even though there are twice as many (A)nAC than GAC sequences in these messengers. The m6A containing oligonucleotides of late SV40 MRNAs were localized in the viral messengers. In the 16S mRNA two m6A oligonucleotides were located at the 5' coding region between 0.95--0.0 map units. The third m6A residue was mapped between 0.0--0.14 map units in the translated portion of this mRNA. The overall pattern of internal methylation in the 19S mRNA is similar. However, some differences between 16S and 19S mRNAs were observed in both the content and location of the longer (Ap)n m6AC nucleotides. These results provide the first example of precise localization of internal methylation sequences in mRNA species with defined coding specificity. It implies that a) location of m6A residues is not random but specific to a particular region of the RNA, b) apart from sequence specificity other structural features of the mRNA may influence internal methylation and c) m6A residues are present in coding regions of SV40 mRNAs.


Transcription of nucleosomal DNA in SV40 minichromosomes by eukaryotic and prokaryotic RNA polymerases.

SV 40 minichromosomes can be transcribed by prokaryotic and eukaryotic RNA polymerases. Size analysis of transcripts indicated that DNA in nucleosomes was accessible for transcription by both enzymes. Sedimentation of the transcription complex showed that minichromosomes which were being transcribed had a full complement of nucleosomes. Strand selection by E. coli RNA polymerase was reduced by the presence of nucleosomes. No region of SV 40 DNA was preferentially transcribed on minichromosomes by either enzyme.


Unusual silica granulomas of the skin with massive involvement of axillary lymph nodes.

Silica granulomas are rare in the skin. Involvement of the regional lymph nodes seems to be unusual. The case of a 35-year-old man with massive diffuse fibrosis of the axilla with multiple foreign body granulomas and involvement of axillary lymph nodes is presented. The lymph nodes exhibit multiple epithelioid cell granulomas and silicotic nodules which are indistinguishable from similar nodules in mediastinal lymph nodes in pulmonary silicosis. It is assumed that the numerous silica particles which were identified by X-ray microanalysis under the scanning electron microscope and recovered from the mineral ash after incineration were accidentally implanted into the skin at the time of an injury 26 years previously. The long delay between silica exposure and granuloma development is a typical feature of these lesions. The possible mechanisms operative in this process are discussed.


Myelolipoma in adenoma of accessory adrenal gland.

A tumor was incidentally found at autopsy in the vicinity of the right adrenal gland of a 69-year-old man who had died of liver cirrhosis with hepatoma. Microscopic examination disclosed a myelolipoma associated with a cortical adenoma occurring in an accessory adrenal gland. No evidence of hormonal abnormalities was found in the clinical record of the patient. The association of myelolipoma with cortical adenoma occurring in an accessory adrenal gland seems very unusual, and the present case is believed to be the first reported of this type of association.


A mode of incipient growth in chemically induced signet ring cell carcinoma of the canine stomach.

A 31-month-old female mongrel dog was orally administered with 50 mg or 100 mg of N-nitrosobutylurea (NBU) in gelatin-capsule 3 times per week for 19 months with interposing periods of complete suspension. Thirty-four foci of signet ring cell carcinoma were found in the antral region of the stomach. The majority of the foci (31 foci) were early cancer, and the remaining foci were invasive cancer. In addition to these lesions, there was "a single gland cancer" in which a row of cancer cells was confined to a single gland. The whole gland was composed of two cell layers; the inner layer facing the lumen was normal gastric cells and the outer layer was atypical or neoplastic cells underlaid by the basement membrane. Mitosis was frequently observed on the bottom of the gland. Atypical or neoplastic cells seemed to mature gradually through a process of upward migration with increase in cytoplasmic Alcian blue-PAS and HID-AB positive mucin. Some of the cells rich in mucin moved into the lamina propria. The other cells remained in the flow of the regular cell renewal system of the normal gastric cells and reached the top of the gland. This observation revealed a mode of incipient gastric cancer growth, which starts and spreads within a single gland, before it invades the surrounding lamina propria.


Elevated early gastric carcinoma. Differential diagnosis as regards adenomatous polyps.

Differential diagnostic problems between gastric carcinomas and precancerous lesions with severe dysplasia have become more perceptible with the increasing number of resected early carcinomas. Although such problems come up for all macroscopic and histologic types of gastric cancer they are particularly marked between early carcinomas of the elevated type and adenomatous polyps. Elevated early carcinomas are usually highly differentiated adenocarcinomas with a morphology which often reminds of of adenomas. But sometimes the carcinomas also demonstrate convincing signs of being developed from adenomas. The criterion of distinction between intramucosal carcinomas and adenomas is invasion through the basal membrane, often difficult to evaluate. The morphological relation between elevated early gastric carcinomas and adenomas and the criterion of distinction between them were studied in 20 early gastric carcinomas of the Japanese types I and IIa, 6 intramucosal and 14 submucosal all highly differentiated adenocarcinomas, and in 42 polyps, of which 5 were of the adenomatous type. All lesions were taken from resection specimens. Among the carcinomas 5 demonstrated convincing signs of being malignant transformed adenomas. In addition, 6 carcinomas had a morphology which more or less reminded of adenomas, but their genetic origin was more uncertain. Nine carcinomas revealed no sign of an adenomatous origin. Among the 5 polyps diagnosed as adenomas 2 revealed an extraordinary degree of severe dysplasia which caused uncertainty on the benign diagnosis. The rest of the polyps were without dysplasia. The significance of invasion through the basal membrane as an indispensable factor of distinction between adenoma and carcinoma in the stomach is discussed. It is concluded that the degree of dysplasia can be so severe and the invasion so difficult to evaluate that the classification of some few tumours depends on the subjectivity of the single pathologist. Four of the tumours, 2 adenomas and 2 intramucosal carcinomas, having a remarkable macroscopic appearance like a large mucosal fold are especially mentioned. Their relation to gastric mucosal prolaps is discussed. Furthermore, a tumour apparently demonstrating only a moderate degree of dysplasia, but even so setting up metastases is mentioned in detail.


Multifocal early gastric cancer of mixed type.

A report of three selected gastric resection specimens presents the problems of histological classification of individual early gastric cancer, illustrated in discrepancies between macroscopic or endoscopic, and histologic identification. Multifocal origin and different types of gastric cancer are demonstrated in all three cases. Problems related to exact histologic classification of this mixed type are discussed together with the prognosis and surgical consequences.


The pathogenesis of respiratory viral infection.

Sendai virus is used as a model to study the histological response and the mechanism of recovery of mice from infection. Although mice infected intranasally do not appear ill, the ciliated columnar cells of the bronchial epithelium undergoes rapid necrosis followed later by repair. Interferon is produced at an early stage of infection and antibody formation follows later. Cyclophosphamide given to mice infected with Sendai virus abolishes the humoral antibody response but not interferon production; the mice develop pneumonic lesions. They cannot eliminate virus and they die. On the other hand, deprivation of 'T' lymphocytes by thymectomy, irradiation and bone marrow reconstitution renders mice more susceptible to Sendai virus but they do not die. The humoral response is thus apparently helped by 'T' lymphocytes and interferon production is not vital for recovery.


Interferon induction by adenoviruses.

All human, simian, bovine and avian adenovirus types tested so far and the canine hepatitis virus induce interferon production in chick cells. This finding indicated this property to be characteristic for viruses belonging to the adenovirus group. Trypsin treatment, which had no effect upon the infectivity, diminished or eliminated the interferon-inducing abilities of crude adenoviruses, and thus the need for a trypsin-sensitive protein in interferon induction was suggested. T antigen and interferon were formed simultaneously in chick embryo fibroblast cells infected with human adenovirus type 12, and therefore the adenovirus-specific T antigen was resistant to the action of endogenous interferon synthetized by the same cells. In chicks inoculated with human types, the appearance of interferon was biphasic: an 'early' and a 'late' interferon could be demonstrated with maximum titre 4 and 10 hr, respectively, after virus infection. In chicks infected with adenoviruses, first interferon production and then a decreased primary immune response to sheep red blood cells was observed. It was assumed that in adenovirus-infected chicks the interferon produced by viral stimulus resulted in a transient immunosuppression.


Slow viruses and chronic disease of the central nervous system.

Although recognized since the 1930s, slow infections have only recently received considerable attention. There are three types, group A (related to the type C RNA viruses), group B (bizarre agents such as scrapie and kuru) and group C (viruses such as measles which normally produce acute infections but which are behaving here in an unusual fashion). These viruses are only united in that they produce disease with excessively long incubation periods. Many slow infections result in neurological diseases and these will be discussed, together with some possible explanations of their action.


Some aspects of the role of viruses in cancer.

The cells of tumours induced by many oncogenic DNA viruses, or cells transformed in vitro, contain virus-specific T and transplantation antigens; these have been described for SV40 virus, polyoma virus and adenoviruses. The investigation of viruses as causes of malignant disease in man has sought to establish whether tumour cells possess these virus-specific proteins; however, to date and with the limitations of present techniques, this enquiry has not demonstrated the above viruses as causal of human cancer. More recent studies with herpesvirus type 2 (HSV-2) have shown this virus to transform animal and human cells in culture, and induce cancer in experimental animals: for these reasons, many researchers have suggested that this agent may be an agent of some forms of cancer, in particular carcinoma of the cervix. The possible association of HSV-2 with human malignant disease is discussed.


Cytomegalovirus-associated gastric ulcer simulating malignancy.

A case is reported of cytomegalovirus (CMV) infection with radiological, gastroscopic and histological appearances which led to a mistaken diagnosis of carcinoma of the stomach.


[Determination of organic elements in histological sections (author's transl)].

In the microscopic differential diagnosis of fibrosing lung processes organic deposits are often encountered which defy identification by histological methods. Assessment of the aetiological significance of these deposits depends on an accurate analysis. Energy-dispersive X-ray microanalysis with an electron microscope provides the means to identify organic deposits in the tissues. Such analyses, which do not require elaborate techniques, show up the type and structure of the deposits and the topographical relation to the lung lesion. They provide detailed information for assessing pulmonary lesions caused by fibrosis-inducing dusts.


Enzyme activities in endothelial cells and smooth muscle cells from swine aorta.

In populations of cultured arterial endothelial and smooth muscle cells grown under the same conditions, we have measured the total activity per cell of 10 enzymes commonly used as "markers" for subcellular organelles: NADH: ferricyanide reductase, NADH:cytochrome c reductase (rotenone insensitive). NADPH:cytochrome c reductase, alpha-glucosidase, 5'-nucleotidase, alkaline phosphodiesterase I, cytochrome oxidase, monoamine oxidase, cathepsin D, and N-acetyl-beta-glucosaminidase. Significant differences between the cell types were found for 7 of the 10 enzymes tested. The total activity of 5'-nucleotidase in cultured smooth muscle cells was 17 times that of cultured endothelial cells. Comparison of the activities in the two cell types freshly collected and in culture showed that the difference in 5'-nucleotidase in cultured cells is due principally to loss of activity from endothelial cells, suggesting that this activity is regulated differently in the two cell types. In both cell types cathepsin D activity rose during culture.


[Localization of retinylphosphatase activity in subcellular fractions of rat liver].

Enzymic hydrolysis of retinyl phosphate in subcellular fractions of the rat liver has been investigated. A quantitative method of measuring activity of retinyl phosphatase has been developed. The method is based on the isolation of labeled retinol, the substance formed from chemically synthesized labeled retinyl phosphate through enzymatic hydrolysis. It has been shown that the major quantity of retinyl phosphatase activity (75%) with a specific activity of 80 micrograms retinyl phosphate hydrolyzed for 30 min/mg protein at 30 degrees C occurs in plasmatic membranes.


Phosphorylation and inactivation of glycogen synthase by phosphorylase kinase.

Skeletal muscle glycogen a4-synthase (EC 2.4.1.11) has been purified free of all synthase kinase and phosphatase activities by chromatography on a Glc-N-6-P-Sepharose affinity column and then on a phosphocellulose column. This preparation of glycogen synthase was tested as a substrate for purified skeletal muscle phosphorylase kinase (ATP:phosphorylase-b phosphotransferase, EC 2.7.1.38). Phosphorylase kinase (1-10 microgram/ml or 0.03-0.3 microM) catalyzes rapid phosphorylation of glycogen synthase (4.5 microM) associated with conversion of the active a form to the less active b form. In the reaction, greater than 95% of the 32P incorporation from [gamma-32P]ATP goes into the synthase subunit almost exclusively in the trypsin-insensitive region which is responsible for synthase a-to-b conversion. Synthase phosphorylation or inactivations catalyzed by phosphorylase kinase is blocked by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, is ATP dependent, is 10-fold more rapid at pH 8.6 than at pH 6.8, and is increased 10-fold by prior activation of the phosphorylase kinase with MgATP and cyclic AMP. With activated phosphorylase kinase at pH 8.2 the apparent Km and Vmax are approximately 70 microM and 4 mumol/min per mg with glycogen synthase and 70 microM and 9 mumol/min per mg with phosphorylase as substrate. It is concluded that glycogen synthase is a substrate in vitro for phosphorylase kinase, a Ca2+-dependent enzyme. The possible physiological significance of this reaction is discussed.


[Regulation of the activity of 3', 5'-cyclic AMP phosphodiesterase by pancreatic hormones].

The character of the insulin effect on the activity of phosphodiesterase (PDE) cAMP of various subcellular localization was studied in the liver of normal and diabetic rats. As a result of kinetic investigations for PDE cAMP preparations of the plasma membranes there were obtained kinetic characteristics of two PDE forms, differing by their affinity to the substrate. For the both PDE cAMP forms stimulation of the activity with insulin was indicated. In examining the insulin effect on the activity of PDE cAMP of the liver of rats with streptozotocin-induced diabetes there was revealed a significant effect of the hormone on the kinetics of the enzyme with a high affinity to cAMP only.


Segments of simian virus 40 DNA spanning most of the leader sequence of the major late viral messenger RNA are dispensable.

A highly specific procedure for the isolation of deletion mutants is described. The size and location of the deletions can be predetermined. By this method a series of deletion mutants mapping within and near the untranslated 5' leader sequence of the late 16S mRNA of simian virus 40 have been isolated. The boundaries of the deletions have been accurately determined by DNA sequence analysis. The deletions range from 20 to 223 nucleotides. All these deletions mutants are viable and grow without helper virus. The largest of these deletions removes the entire leader sequence except for six nucleotides at the 3' end that are probably involved in covalent linkage with the 5' end of the body of the mRNA located 937 nucleotides away on the genome. Three of the deletion mutants remove the 5' end of the leader that normally bears the cap structure of the mRNA. A large segment immediately preceding the leader sequence is also removed in one of these mutants, ruling out the generation of the 5' end of the mRNA via initiation of transcription at this point. The circularization of linear infecting DNA producing the DNA of the deletion mutants proceeds mainly by way of blunt end ligation in vivo.


Cyclic nucleotide phosphodiesterase activity in 10-nm filaments and microtubule preparations from bovine brain.

Cyclic nucleotide phosphodiesterase activity (3':5'-cyclic-AMP 5'-nucleotidohydrolase, EC 3.1.4.17), which is activatable by Ca(2+)-dependent regulator protein (CDR), has been identified in cycled microtubule preparations from bovine brain. By using various methods to fractionate the microtubule preparation into subfractions (e.g., phosphocellulose chromatography to obtain purified 6S tubulin and soluble microtubule-associated proteins, and gel exclusion chromatography on Bio-Gel A-150m to obtain 10-nm filaments), we found that all the fractions exhibited some enzymic activity, but that most of the phosphodiesterase activity was localized in the 10-nm filament fraction. By using cyclic GMP as substrate, a specific activity of 921 +/- 168 pmol/mg of filament protein.min was determined. Also, 10-nm filaments were prepared directly from brain homogenates by differential centrifugation and gel exclusion chromatography. This fraction also contained phosphodiesterase activity but of slightly lower specific activity (752 +/- 9 pmol/mg of protein.min). The filament-associated enzymic activity was stable during storage (-70 degrees C) and to several salt extractions at moderate ionic strength (0.5 M); the latter finding indicates that the phosphodiesterase is not adsorbed to the filaments via nonspecific electrostatic interactions. Although a chelating agent was present in the initial homogenization buffer and generally in all buffers used in preparing fractions, an activator of a smooth muscle phosphodiesterase was released upon boiling the 10-nm filaments. This activator obtained in the boiled supernatant was Ca(2+)-sensitive, trifluoperazine-sensitive, and stimulated smooth muscle phosphodiesterase to nearly the same extent as purified (exogenous) CDR; thus, it probably represents filament-associated CDR.


Mechanism for discrimination between viral and host mRNA in interferon-treated cells.

In extracts of interferon-treated HeLa cells, RNA covalently linked to double-stranded RNA (dsRNA) is preferentially degraded compared with mRNA not linked in dsRNA. This was established by following the degradation of poly(A)-containing mRNA annealed with poly(U), of poly(C)-containing encephalomyocarditis virus RNA annealed with poly(I), and of the replicative intermediate of the virus isolated from infected cells. In extracts of interferon-treated cells, dsRNA promotes the synthesis of a series of oligonucleotides, designated (2'-5')oligo(A), which in turn activate an endonuclease. Several lines of evidence suggest that the (2'-5')oligo(A) polymerase/endonuclease system is involved in the preferential degradation of mRNA linked to dsRNA. Conditions that prevent synthesis of (2'-5')oligo(A) prevent this preferential degradation, whereas addition of (2'-5')oligo(A) or conditions that favor its synthesis result in degradation of mRNA both linked and not linked to dsRNA. These results are best explained by a localized activation of the endonuclease near the dsRNA region of our model substrates. We propose that in infected cells activation of the endonuclease takes place near the replicative intermediates of RNA viruses. The replicative intermediates of encephalomyocarditis virus promote synthesis of (2'-5')-oligo(A) in extracts of interferon-treated cells and are degraded to a 20S "core" resistant to digestion with RNase A. This mechanism may be responsible for discrimination between viral and cellular mRNA in interferon-treated cells.


Multistep regulatory system for activation of a cyclic AMP-independent eukaryotic initiation factor 2 kinase.

Three functionally related components that block peptide initiation have been identified in lysates of rabbit reticulocytes. The components function consecutively in a cascade type sequence of reactions to cause phosphorylation of eukaryotic peptide initiation factor 2 (eIF-2). The eIF-2 kinase activated as part of this sequence has been tentatively identified as the same protein kinase that is activated by heme deficiency as part of the hemin-controlled repressor (HCR) system. The first component in the sequence is heat stable and can be reversibly activated by heat or pressure. It activates a second, heat-labile, component that in turn directly or indirectly activates the hemin-controlled eIF-2 kinase. This heat-labile component appears to function through proteolysis. This reaction sequence is not detectably affected by heme or cyclic AMP and thus appears to provide an alternative mechanism, independent of heme, for activation of the cyclic AMP-independent eIF-2 kinase of the HCR system.


Protein kinase activity associated with simian virus 40 T antigen.

Incubation of simian virus 40 (SV40) tumor (T) antigen-containing immunoprecipitates with [gamma-32P]ATP results in the incorporation of radioactive phosphate into large T antigen. Highly purified preparations of large T antigen from a SV40-transformed cell line, SV80, are able to catalyze the phosphorylation of a known phosphate acceptor, casein. The kinase activity migrates with large T antigen through multiple purification steps. Sedimentation analysis under non-T-antigen-aggregating conditions reveals that kinase activity and the immunoreactive protein comigrate as a 6S structure. The kinase activity of purified preparations of large T antigen can be specifically adsorbed to solid-phase anti-T IgG, and partially purified T antigen from a SV40 tsA transformation is thermolabile in its ability to phosphorylate casein when compared to comparably purified wild-type T antigen. These observations indicate that the SV40 large T antigen is closely associated with protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) activity.


DNA endonucleases associated with the avian myeloblastosis virus DNA polymerase.

A DNA endonuclease, Endo-I, which cleaves superhelical DNAs, has been isolated from avian myeloblastosis virions stripped of their coats by mild detergent treatment. The enzyme has a broad pH optimum around 7.5-8.0 and requires Mg2+ for activity. A second endonuclease, Endo-II, with a requirement for Mn2+, also present in viral cores, copurified with avian myeloblastosis virus alpha beta DNA polymerase (reverse transcriptase, RNA-dependent DNA nucleotidyltransferase) and similarly cleaved superhelical DNAs. Heat denaturation and sodium fluoride and N-ethylmaleimide inhibition studies were carried out to demonstrate a possible relationship between the two endonucleases and the viral DNA polymerase and RNase H activities. It appears that Endo-II may be an intrinsic activity of the polymerase.


Ion-channel component of cytochrome oxidase.

Cytochrome oxidase is resolvable into an electron transfer complex and an ion transfer complex. The ion transfer complex has been shown to have the capability for inducing nonspecific ion transport into liposomes. Subunit I of cytochrome oxidase has been identified as an ion-channel-forming protein.


Poliovirus replicase: a soluble enzyme able to initiate copying of poliovirus RNA.

The soluble phase of the cytoplasm of poliovirus-infected cells contains an enzymatic activity able to copy RNA without an added primer. This replicase activity has been purified 60-fold; it is absent from uninfected cells. Poly(U) polymerase activity copurifies with replicase activity. Although less pure replicase fractions copy a variety of RNAs, purer fractions respond better to poliovirus RNA than to other viral RNAs. Even the less pure fractions make a specific copy of the added template, as shown by hybridization of the product to its template RNA but not to other RNAs. Among homopolymers only poly(A)-oligo(U) was copied by the replicase; other primed homopolymer templates were inactive.


Physical modifications of rhodopsin boundary lipids in lecithin-rhodopsin complexes: a spin-label study.

The microviscosity of rhodopsin boundary lipids was studied with a spin-labeled fatty acid covalently attached to rhodopsin, in rhodopsin-egg lecithin vesicles. When the lipid-to-protein ratio was high (500:1, mole to mole), only narrow peaks were visible in electron paramagnetic resonance spectrum at 37 degrees C. This enabled us to show that, under these conditions, not more than 10% of the probes have their motion strongly restricted by the proximity of the protein. When the temperature was reduced, a second component characteristic of strong immobilization appeared. It corresponds to 50% of the signal at -5 degrees C. At all temperatures reduction of the lipid-to-protein ratio also resulted in an increase of the amount of immobilized lipid. These results show that the rhodopsin boundary layer under physiological conditions is associated with low microviscosity. However, low temperatures, low lipid-to-protein ratios, or combinations of the two can induce dramatic modifications of the physical state of the boundary lipids, which under these conditions may no longer be representative of the functional biological system. These results are relevant to the general theory of lipid-protein interaction.


Stimulation of sugar uptake and glycolysis in chicken embryo fibroblasts by the major glycoprotein from avian myeloblastosis virus.

Addition of purified major glycoprotein from avian myeloblastosis virus to growing or quiescent chicken embryo fibroblasts rapidly stimulates the rate of hexose transport and increases the lactic acid production. These stimulatory effects are dependent on the time of exposure and the dose of viral glycoprotein. In contrast, the glycoprotein only marginally affects hexose transport in chicken cells transformed by Rous sarcoma virus. Some effects of the glycoprotein on serum-starved quiescent cells were similar to those observed upon  re-addition of serum; however, the viral glycoprotein did not stimulate DNA synthesis. Quiescent cells stimulated by saturating levels of serum showed little further stimulation of hexose uptake upon exposure to viral glycoprotein for 3 hr. This behavior suggests that the glycoprotein may be acting on a system that is also a target for serum action.


Functional characteristics of dispersed rat submandibular cells.

Rat submandibular gland cells have been obtained through enzymatic dispersion using chromatographically purified collagenase (EC 3.4.24.3) and hyaluronidase (EC 3.2.1.35) and gentle mechanical force. The recovery of viable cells after the isolation procedure was 59% on the basis of total glandular DNA content. Approximately 60% of the total cell population consisted of acinar cells; less than 8% were immature granular duct cells; and the remainder were intercalated duct, striated duct, and myoepithelial cells. Most of the acinar cells were in acinar-intercalated duct complexes. The integrity of the isolated cells was substantiated by their exclusion of trypan blue, intracellular electrolyte composition, incorporation of [14C]glucosamine into trichloroacetic acid + phosphotungstic acid precipitable material at a linear rate for 1.5 hr, secretory responses to parasympathomimetic and sympathomimetic stimulation, and morphologic integrity as determined by light and electron microscopy. The cholinergic receptors were characterized through investigation of the net transmembrane flux of K+ in response to carbamoylcholine. The alpha-adrenergic receptors were characterized by investigating the net transmembrane flux of K+ in response to norepinephrine stimulation and the beta-adrenergic receptors were characterized by determining the rate of secretion of 14C-labeled mucin after isoproterenol stimulation. A high degree of sensitivity to both cholinergic and adrenergic secretagogues was observed.


Sites of sequence variability in Epstein-Barr virus DNA from different sources.

The intracellular Epstein-Barr virus (EBV) DNA present in virus-transformed cells was partly purified from 23 cell lines or biopsies of Burkitt lymphoma, nasopharyngeal carcinoma, infectious mononucleosis, or healthy carrier origin. Such DNA was cleaved in fragments (A-K) of molecular weights between 1 x 10(6) and 30 x 10(6) with restriction enzyme EcoRI, and these fragments were analyzed by standard methods involving agarose gel electrophoresis, transfer to nitrocellulose filters, and hybridization with radioactive EBV DNA or complementary RNA. Sequence variability among different EBV DNA isolates was largely confined to the A, C, and I fragments. These results are discussed in relation to the linkage map of the EcoRI fragments of EBV DNA. The EcoRI cleavage pattern of intracellular viral DNA of an EBV-like virus from baboon cells, Herpesvirus papio, was entirely different from that of human EBV isolates.


Vp1 affects intracellular localization of Vp3 polypeptide during simian virus 40 infection.

In order to understand the functions of simian virus 40 genes, permissive cells (TC7) were infected with mutants temperature sensitive in the complementation groups A, B, C, BC, and D at permissive and nonpermissive temperatures. Cells were examined for the localization of viral polypeptide antigens by immunofluorescent staining with monospecific antibodies. The results are as follows: (i) The appearance of Vp1 antigen in cells infected by tsB, C, or BC mutants was not affected appreciably by the mutations. (ii) The appearance of Vp3 antigen was affected by the mutations in B, C, or BC. Vp3 antigen is confined to the nuclei in cells infected by wild-type virus. With mutant virus infection, Vp3 antigen is found in the cytoplasm, perinuclear region, and nucleoli. (iii) The tsD mutants and the tsA mutants did not express either Vp1 or Vp3 antigens at the nonpermissive temperature. (iv) Nucleoli seem to play an essential role in the biosynthesis and assembly of viral polypeptides. Thus, mutations in any one of complementation groups B, C, or BC, which are within the structural gene for Vp1, cause an alteration of intracellular distribution of another late gene product, Vp3. These results suggest that the amino acid sequences of Vp1 polypeptide play a role(s) in the transport of viral antigens across internal membranes or in virus assembly processes or in both.


Demonstration of low density lipoprotein receptors in mouse teratocarcinoma stem cells and description of a method for producing receptor-deficient mutant mice.

Familial hypercholesterolemia, a widespread human genetic disorder implicated in vascular and coronary disease, has had no laboratory animal counterpart that would enable the pathogenesis to be analyzed and drugs to be tested in vivo. The primary lesion in some patients is known to occur in the cells' initial handling of the major cholesterol-carrying lipoprotein of plasma. It entails a deficiency in the specific cell surface receptor that binds low density lipoprotein (LDL), with a consequent alteration in the control of cholesterol metabolism. The present study was undertaken to devise a practical scheme for producing, from developmentally versatile mouse teratocarcinoma stem cells, whole-animal models with a comparable genetic lesion. This requires first learning whether the tumor stem cells in culture express LDL receptors, and next establishing a selection or screening procedure to identify receptor-deficient mutants in mutagenized cell cultures. The results show that the teratocarcinoma cells do in fact have specific high-affinity LDL receptors which are similar to those reported for fibroblasts and the parenchymal cells of specialized tissues and different from those of phagocytic cells. Sterols suppressed the otherwise efficient binding, internalization, and degradation of LDL ((125)I-labeled) by the cells. Acetylation of LDL blocked the binding. Only LDL and not high density lipoprotein (HDL) was bound. After LDL uptake and degradation, the liberated cholesterol led, as expected, to increased cholesteryl ester formation; it also suppressed activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase [HMG CoA reductase; mevalonate: NADP(+) oxidoreductase (CoA-acylating), EC 1.1.1.34], the rate-limiting step in cholesterol biosynthesis. Cells with LDL receptors were readily visualized by administering a fluorescent derivative of LDL; in the fluorescence microscope, labeling was seen in all cells. Cells with experimentally depressed receptors, yielding little fluorescence, were separable from those with normal fluorescence in the fluorescence-activated cell sorter. Thus, two methods for isolating receptor-deficient cells from mutagenized cultures are now available, either by visual recognition of low-fluorescing or nonfluorescing colonies in culture plates or by electronic cell sorting. Such mutants in an appropriate line of teratocarcinoma cells can then be passaged into blastocysts for full somatic tissue differentiation and germ-line development into mice.


Simian virus 40 recombinants are produced at high frequency during infection with genetically mixed oligomeric DNA.

Classical approaches to analysis of mitotic recombination by use of simian virus 40 (SV40) are limited in usefulness because of low frequencies of recombination. To bypass the apparent rate-limiting step in normal SV40 recombination, oligomeric SV40 was constructed in vitro by ligation of mixtures of pairs of linear DNAs carrying genetically distinct temperature-sensitive mutations. Cultured monkey cells infected with the unfractionated ligation products yielded frequencies of nonparental recombinant progeny that were increased up to 500-fold relative to cells infected with a mixture of the untreated circular molecules. Pairwise crosses were performed with tsB4, tsB8, and tsBC11 DNAs, using unfractionated oligomers constructed from linear molecules cleaved by EcoRI or BamHI. In each cross the fraction of progeny with nonparental genotypes was roughly proportional to the physical distances between the mutant sites. These results suggest a random, rather than site-specific, conversion of oligomers to monomers. Somewhat surprisingly, nonligated mixtures of linear tsB4 and tsB8 DNAs, created by EcoRI digestion, produced a 40-t to 100-fold increase in the frequency of nonparental progeny. These results indicate that intermolecular associations must occur with fairly high efficiency between these linear molecules.


(E)-5-(2-Bromovinyl)-2'-deoxyuridine: a potent and selective anti-herpes agent.

Of a series of five newly synthesized 2'-deoxyuridine derivatives, including 5-vinyl-dUrd, 5-ethynyl-dUrd, 5-(1-chlorovinyl)-dUrd, (E)-5-(2-bromovinyl)-dUrd, and (E)-5-(2-iodovinyl)-dUrd, the last two compounds were found to exert a marked inhibitory effect on the replication of herpes simplex virus type 1 [ID50 (mean inhibitory dose), 0.004-0.02 microgram/ml]. Both (E)-5-(2-bromovinyl)-dUrd and (E)-5-(2-iodovinyl)-dUrd were highly selective in their anti-herpes activity in that they did not affect the growth or metabolism of the host (primary rabbit kidney) cells unless drug concentrations were used that were 5,000- to 10,000-fold greater than those required to inhibit virus multiplication. In this sense (E)-5-(2-bromovinyl)-dUrd and (E)-5-(2-iodovinyl)-dUrd proved more selective in their activity against herpes simplex virus type 1 than all other anti-herpes compounds that have been described so far. In animal model systems (namely, cutaneous herpes infections of athymic nude mice), (E)-5-(2-bromovinyl)-dUrd suppressed the development of herpetic skin lesions and mortality therewith associated, whether the compound was administered topically or systemically. Under the same conditions, the standard anti-herpes drug 5-iodo-dUrd (Idoxuridine) offered little, if any, protection. Although the precise mechanism of action of (E)-5-(2-bromovinyl)-dUrd and (E)-5-(2-iodovinyl)-dUrd remains to be established, preliminary findings indicate that they do not specifically act at the thymidylate synthetase step.


Murine cytomegalovirus infects spermatogenic cells.

Murine cytomegalovirus replicated in reproductive tissue of male mice infected with the virus. We examined three strains of mice latently infected by injection at birth with 100 plaque-forming units of the virus. As adults, these mice contained within their testes 4--6 viral genomic equivalents per 100 cells, as tested by hybridization between mouse DNA and cytomegalovirus DNA. Acutely infected male adult CBA mice homozygous for the nude gene (athymic: nude/nude) produced infectious virus in their testes, the amounts of which varied according to the animal's age at the time of infection. Heterozygous (nude/+) litter mates contained significantly less virus than nude/nude mice. At the peak of virus replication hybridization between virus DNA and mouse DNA indicated the presence of 3.3 viral genome equivalents per testicular cell. Both in situ hybridization studies and phenol emulsion reassociation of virus DNA to DNA from purified spermatozoa localized this viral DNA to immature and mature sperm cells. Hence, murine cytomegalovirus can be harbored in testes during both acute and latent infections and can replicate in male germ-line cells.


Virus-specified protease in poliovirus-infected HeLa cells.

Previous studies have shown that primary cleavages in nascent picornavirus precursors are accomplished by cellular proteases. This study has characterized the enzyme in infected cells that produces the capsid polypeptides by secondary cleavages of viral precursors. The kinetics of the production of protease activity correlate with the time course of virus protein synthesis, and the new enzyme has characteristic pH and temperature optima. Guanidine and cycloheximide, which are inhibitors of virus RNA and protein synthesis, prevent production of the protease. As determined by introduction of amino acid analogs into the protease or inhibition by a leucyl chloromethyl ketone, the enzyme is synthesized at a time of infection when host cell proteins are not produced, and the enzyme copurified with a 40,000-dalton virus polypeptide present in the cytoplasm of infected cells. Wild-type levels of protease activity are produced by viral mutants that are defective in coat protein synthesis. The conclusion is that a non-structural poliovirus gene product participates in protein cleavages that produce the viral coat proteins.