Regulation of glycoprotein D synthesis of herpes simplex virus 1 by α 4 protein,the major regulatory protein of the virus,in stably transformed cell lines: effect of the relative gene copy numbers

Summary Earlier studies concerning 1 gene regulation by the 4 protein, the major regulatory protein of herpes simplex virus 1 (HSV-1), in stably transformed cell lines, reported conflicting results, i.e., 4 protein positively regulated the ₁ gB gene in 4/gB cells, while it negatively regulated the ₁ gD gene in 4/BJ cells. Both cell lines were derived from a common parental cell line 4/c 113 that contains 1 copy of the 4 gene, and the only apparent difference between them was the relative copy number of the gB and gD sequences (1 and 30–50, respectively) resident in the cell genome. We investigated this disparity by constructing a cell line (BA 4) that contains one copy each of the 4 and ₁ gD sequences, by fusion of 4/c 113 and BJt cells, containing and expressing respectively 1 copy of the 4 and gD genes. BA 4 cells constitutively expressed both the 4, gD genes inherited from the parental cell lines ( 4/c 113 and BJt). In BA 4 cells the 4 protein positively regulates the gD gene as evidenced from (i) higher levels of gD expression than the parental BJt cells lacking the 4 gene, and (ii) significant decrease in gD expression under conditions that render the 4 protein produced in BA 4 cells non-functional. In addition the ₂gG gene contained within the DNA fragment encoding the gD gene, is also expressed in BA 4 cells. On the basis of these data, we propose that gene regulation by the 4 protein is affected by the relative copy number of these genes, resident in the cell genome.     

Über Fortschritte der Naturwissenschaften und Technik aus der Sicht Eines Klinikers

Ohne Zusammenfassung1. Vorsitzender der Gesellschaft Deutscher Naturforscher und Ärzte 1956/58Festrede aus Anlaß der Eröffnung der 100. Tagung der Gesellschaft Deutscher Naturforscher und Ärzte, Wiesbaden 28. 9. 58.     

Distribution of degenerated fibers in the cortex of the medial and lateral surfaces of the dog's brain after destruction of the anterior limbic and motor areas

Summary Damage to the anterior limbic and motor regions of the brain cortex results in two types of distribution of degenerated fibers over the brain cortex in dogs. The first lateral type is characterized by a predominant fragmentation of a large number of thick horizontal or diagonal fibers of the lower cortical layers on the lateral surface; the second type, known as medial, by the fragmentation of single slender radial fibers and horizontal fibers of the first cortical layer on the medial surface.The lateral-type degenerated fibers are distributed over the cortex of the sigmoid, presplenial, suprasplenial and ectolateral convolutions (motor, parietal and occipital fields). They are most numerous in the motor (4) and optic (19, 18) areas. The medial-type degeneration is characteristic of the cingulum (gyrus cinguli) (limbic fields).An injury to the anterior and posterior portions of the sigmoid convolution causes a less extensive lateral-type degeneration than an injury to the genual convolution.Presented by Academician V. N. Chernigovskii Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 59, No. 3, pp. 18–22, March, 1965     

Isoenzymes of γ-glutamyl transpeptidase in liver diseases

Summary A new method for the separation of isoenzymes of-glutamyl-transpeptidase is described, using electrophoresis on acetate cellulose gel and a developing solution composed by-glutamyl-naphthylamide, and a colored diazonium compound.The method permits the separation of up to four different isoenzymes, which we called-GT₁,-GT₂,-GT₃,-GT₄, the first two showing an electrophoretic migration similar to that of ₁- and ₂-globulins and the other two to that of-globulins.The present technique has proved its usefulness in detecting isoenzymes in serum with values of total-glutamyl-transpeptidase higher than 80 U/L.The application of this method in 52 patients with different types of biliary obstruction and hepatocellular damage has shown that it provides new possibilities in differential diagnosis.     

Time constants of facilitation and depression in Renshaw cell responses to random stimulation of motor axons

Summary In 9 adult anaesthetized cats, 22 lumbosacral Renshaw cells recorded with NaCl-filled micropipettes were activated by random stimulation of ventral roots or peripheral nerves. The stimulus patterns had mean rates of 9.5–13 or 20–23 or 45 pulses per second and were pseudo-Poisson; short intervals below ca. 5 ms (except in two cases) were excluded. The Renshaw cell responses were evaluated by two kinds of peristimulus-time histograms (PSTHs). Conventional PSTHs were calculated by averaging the Renshaw cell discharge with respect to all the stimuli in a train. These PSTHs showed an early excitatory response which was often followed by a longer-lasting slight reduction of the discharge probability. These two response components were positively correlated. Conditional PSTHs were determined by averaging the Renshaw cell discharge with respect to the second (test) stimulus in pairs of stimuli which were separated by varied intervals, . The direct effect of the first conditional response was subtracted from the excitation following the second (test) stimulus so as to isolate the effect caused by the second stimulus per se. After such a correction, the effect of the first conditioning stimulus showed pure depression, pure facilitation or mixed facilitation/depression. Analysis of such conditioning curves yielded two time constants of facilitation (ranges: ca. 4–35 ms and 93–102 ms) and two of depression (ranges: ca. 7–25 ms and 50–161 ms). It is concluded that these time constants are compatible with processes of short-term synaptic plasticity known from other synapses. Other processes such as afterhyperpolarization and mutual inhibition probably are of less importance.     

The problem of detection of tissue antigens

Summary Utilization of the anaphylactic reaction for distinguishing tumor antigens from antigens of normal tissues requires the preliminary investigation of 2 problems: 1) how should desensitization against major antigens be performed in order to be sure of its completeness, and 2) how does such desensitization affect the sensitivity of the animal to minor antigens? Experiments were carried out on guinea pigs which were sensitized by mixtures of two antigens (foreign serums, tissue nucleoproteins) mixed in different proportions. It was demonstrated that the desensitizing dose of a major antigen is several times as great as the shocking dose, and therefore that repeated injection is necessary to bring about complete desensitization. This causes a pronounced decrease of the sensitivity to the minor antigen. Therefore it is necessary to increase the dose of the preparation containing the mixture of antigens (for example, a tumor extract) in order to obtain a definite anaphylatic reaction. These findings should be taken into consideration in employing anaphylaxis for the demonstration of antigenic differences between tumors and normal tissues.     

Spontaneous and agonist-induced openings of an acetylcholine receptor channel composed of bovine muscle α-, β and δ-subunits

During the development of mammalian muscle the -subunit of the nicotinic acetylcholine receptor (AChR) is replaced by the -subunit to produce well-defined alterations in the conductance and gating of the channel. To gain a better unterstanding of the functional role of the and -subunits, we have studied the properties of an AChR channel lacking these subunits. The AChR expressed in Xenopus oocytes injected with the bovine -, and -subunit-specific mRNAs (referred to as -AChR) is unusual in that its channel opens spontaneously at a high frequency in the absence of agonist. From a comparison of the -AChR with complete receptors containing either the or -subunit, we conclude that the and -subunits influence most channel properties, including agonist binding, and are especially important for stabilizing the closed state of the unliganded receptor channel. The -AChR can form when a complete set of four subunit-specific mRNAs is injected. The ease with which it is assembled raises the possibility that the - AChR contributes to some of the variations in receptor properties that occur during development.     

A T cell/B cell/epithelial cell internet for mucosal inflammation and immunity

Conclusions Mucosal immune responses are strongly regulated by CD4⁺ T cells and their derived cytokines. In this regard, IFN-^–/– mice (i.e., which lack Th1 and have elevated Th2 cells) showed strong mucosal Th2-type responses together with S-IgA production, while IL-4^–/– (e.g., dominant Th1 and lack of Th2 cells) mice had impaired mucosal Th2 and IgA responses following oral delivery of TT and CT. However, when rSalmonella or radenovirus were used for antigen delivery, significant levels of mucosal IgA responses were induced in both IFN-^–/^– and IL-4^–/^– mice. The choice of the antigen delivery system which leads to optimal Th and B cell interactions are important for the induction of effective IgA responses, even in situations where the immune system is compromised. It is clear that Th2-type cytokines are important in mucosal IgA responses; however, other cytokine combinations can compensate for mucosal immunity in situations in which Th2 cell responses are absent. Mucosally induced tolerance may be one approach to prevent several systemic immune disorders; however, the mechanism of this phenomenon still needs to be elucidated. Our recent findings have suggested that IFN- may play an important role in induction of systemic unresponsiveness since oral tolerance was not induced in IFN-^–/^– mice.Our studies as well as those of others indicated that at least two phases of a triad of cell interactions are important for the mucosal immune system. First, it has been shown that epithelial cell-produced IL-7 and SCF and T cell-derived IL-2 are essential activation and growth signals for intestinal T cells. Second, our studies with TCR knockout mice have suggested that mucosal T cells also play a critical role in the regulation of mucosal IgA responses. Thus, a mucosal internet among T cells, T cells, and IgA B cells appear critical for mucosal homeostasis and for regulation of specific mucosal immune responses.     

Expression of the L-type calcium channel with two different β subunits and its modulation by Ro 40-5967

The smooth muscle 1_Cb subunit of the L-type calcium channel was expressed alone (CHO ₁ cell) or together with the skeletal 1 (CHO1 cell) subunit or smooth muscle 3 (CHO ₁ 3 cell) subunit in Chinese hamster ovary (CHO) cells. The interaction of the expressed calcium channels with the non-dihydropyridine calcium channel blocker Ro 40-5967 was studied. Ro 40-5967 decreased isradipine binding by an apparent allosteric interaction and blocked the barium inward currents (I _Ba) in a voltage- and use-dependent manner in all cells. The steady-state inactivation curves were shifted to hyperpolarizing potentials in the presence of Ro 40-5967. The rate of channel inactivation was increased in CHO ₁ and CHO ₁ 3 cells. The shift in the steadystate inactivation curve and the increase in channel inactivation were less pronounced in CHO ₁ 1 cells than in the other cell lines. Low concentrations of Ro 40-5967 increased I _Ba by up to 198% in 33% of the CHO ₁ 1 cells. In addition, higher concentrations of Ro 40-5967 were required to inhibit I _Ba in 60% of the CHO ₁ 3 cells. These results suggest that the subunits modify the interaction of the non-dihydropyridine Ro 40-5967 with the expressed calcium channel ₁ subunit.Dedicated to the late Professor Dr. W. Osterrieder     

Interferon-γ-like immunoreactivity in developing rat spinal ganglia neuronsin vivo andin vitro

Summary Interferon--like immunoreactivity was observed in a subpopulation of 16-day-old embryonic rat spinal ganglion neurons using two monoclonal antibodies directed against different epitopes of recombinant interferon-. During ontogenesis bothin vivo andin vitro, it was found that the strong immunoreactivity was confined to small neurons when neurons become morphologically distinct on the basis of size.In vivo, the interferon--immunoreactive neurons started to express major histocompatibility complex class I antigens after the first postnatal week, whilein vitro no such antigen could be detected. A quantitative Elisa method was developed to determine the levels of major histocompatibility complex class I and interferon-in vitro, whereby increased amounts of major histocompatibility complex class I antigen was detected after exposing the cultures to recombinant interferon- and Sendai virus. Sendai virus also caused a small increase in interferon- with a peak about 12 hours after infection. Thein vitro system will be used to study further the role of the putative neuronal interferon--like molecule in the regulation of cell growth, for induction of major histocompatibility complex antigens and in virus infection of sensory neurons.     

Recognition by peptide mapping of three different structural groups of outer membrane protein YOP-1 of Yersinia enterocolitica and Yersinia pseudotuberculosis

The structural relation of YOP-1 of european and american Yersinia enterocolitica serotypes O3, O9, O5, 27, and O8 and O20, respectively, and Y. pseudotuberculosis serotypes I, II, and III was compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis and peptide mapping using Staphylococcus aureus protease V8. Apparent molecular weights of YOP-1 ranged from 206,000 (O3) to approx. 180,000 (O8). According to their respective peptide maps YOP-1 of the european and american Y. enterocolitica serotypes and Y. pseudotuberculosis serotypes could be assigned to three different groups. Evaluation of several isolates of Y. enterocolitica serotypes O3, O9, and O8 by peptide mapping indicated that YOP-1 is conserved within a serotype. However, one serotype O8 isolate differed from the consensus peptide pattern of the other serotype O8 and O20 isolates. The similarity of the peptide patterns of Yersinia serotypes which predominate in certain geographical locations, i. e., european and american Y. enterocolitica serotypes, suggest common evolution of YOP-1 of these serotypes independent of the evolution of the other serotypes.     

Detection and quantitation of cell-surface sugar receptor(s) ofLeishmania donovani by application of neoglycoenzymes

Promastigote culture forms of the log growth phase ofLeishamania donovani stock LRC L 51 were investigated for expression of cell-surface carbohydrate-binding sites using 15 types of a chemically glycosylated enzyme termed neoglycoenzyme. Carbohydrate conjugation and coupling yield were kept constant to ensure that the type of carbohydrate moiety, was the only variable feature of the applied tools. Para-aminophenyl derivatives of the following carbohydrate residues were used for the glycosylation of -galactosidase fromEscherichia coli: -d-lactose, -d-thiogalactose, -d-mannose, -l-rhamnose, -d-N-acetylgalactosamine, -d-N-acetylgalactosamine, -d-N-acetylgalactosamine, -d-N-acetylgalactosamine, -d-N-acetylglucosamine, the - and -glucosides maltose and cellobiose, -d-xylose, -d-mannose-6-phosphate, the -galactoside melibiose, -l-fucose, and -d-glucuronic acid as well as sialic acid. Only melibiose, fucose, and glucuronic acid showed no binding affinity for the cultured flagellates; this served as an internal control reaction to exclude any binding to the linker group. This result demonstrates that many but not all sugar types can be recognized by appropriate receptor structure(s) on the surface of the promastigoteLeishmania. Transformation of the binding data for neoglycoenzymes exposing lactose, mannose, rhamnose, andN-acetylated hexose residues, which was carried out to obtain the dissociation constants and to estimate the number of binding sites at saturation, revealedK _D values of around 100mm and around 10⁴ binding sites for the polyvalent ligands.     

The ultrastructure of the “excretory” system of ascaris suum larvae

Summary Electron microscope observations of the excretory cell of the infective larva reveal that it contains a large nucleus surrounded by cytoplasm containing numerous organelles, multi-granular bodies, vesicles and granules typical of glandular cells. The proximal region of the excretory duct bears a number of scattered microvilli, on its adluminal surface, and the distal region is lined with a thin multilayered cuticle.In the liver stage larva 2 days after infection, 2 lateral excretory columns are present. These arise from the excretory cell body and extend posteriorly for about half the length of the intestine. Each column contains a narrow longitudinal canal surrounded by cytoplasm rich in mitochondria, granular endoplasmic reticulum, free ribosomes and large vacuoles. Evidence was obtained of the passage of substances through the wall of the canal but their chemical nature was not determined.Further extension of the lateral columns is seen in the 8-day, lung-stage larva, the columns now extending for more than two-thirds the length of the intestine. Their diameter is also increased but their internal structure is essentially similar to that of the 2-day liver-stage larva.The excretory duct which arises immediately anterior to the nucleus has a structure similar to that of the lateral columns for the first half of its length, the microvilli described in the infective larva being absent at this stage. The distal half of the duct is lined with cuticle.     

The role ofγδ T cells in the normal and disordered immune system

Summary A small population of T cells does not express the conventional T cell receptor characterized by the and polypeptide chains (TCR) but instead, two polypeptides termed and (TCR). This alternative receptor is able to recognize antigen. It appears early in T cell ontogeny, but its role in the thymus prior to the availability of TCR remains unclear. In selected sites such as skin or gut TCR predominates in mice which might suggest a role of T cells in the first line of defense against infection, T cells secrete lymphokines and display cytotoxic activity. However, their activation requirements may differ from what is known for T cells since MHC-nonrestricted and also CD4 and CD8 negative T cells have been described. Preferential activation by mycobacterial antigens possibly indicates a special repertoire of the T cells. In various diseases slightly increased numbers of T cells were found, but these preliminary studies have not yet provided evidence for a major pathogenetic role of T cells.List of abbreviations C constant region (immunoglobulin or TCR gene segment) - CD4 cluster of differentiation 4 (mainly on helper cells) - CD8 cluster of differentiation 8 (mainly on cytotoxic cells) - D diversity region (immunoglobulin or TCR gene segment) - DNA desoxyribonucleic acid - IL2 interleukin 2 - J joining region (immunoglobulin or TCR gene segment) - kD kiloDalton - MHC major histocompatibility complex - NK natural killer (cells) - RA rheumatoid arthritis - TCR T cell receptor - V variable region (immunoglobulin or TCR gene segment)     

Enhanced stability of YEp plasmids in lager brewing yeasts is related to lager brewing yeast 2-μm DNA

Summary YEp plasmid stability in the presence of either Saccharomyces cerevisiae laboratory strain 2-m DNA, or lager brewing yeast 2-m DNA in the same genetic background, was compared under non-selective culture conditions. It was found that YEp plasmids were more stably maintained in the presence of lager 2-m DNA under these conditions. By construction of laboratory-lager 2-m DNA hybrid plasmids, an 867 bp StuI fragment of lager 2-m DNA was shown to be responsible for the enhanced stability of the YEp plasmid. Nucleotide substitutions at two sites were found by sequencing this region. It was also confirmed that increasing cell ploidy enhanced YEp stability under non-selective conditions.     

Electron microscopic study of the formation of African horse-sickness virus

Summary BHK 21/13 cells infected with African horse-sickness virus (strain 13/63, type 3) for varying periods of time have been studied with the electron microscope. Evidence is presented to show that the virus does not multiply in the nucleus, but that replication occurs in the cytoplasmic matrix. It is also indicated that the particle often leaves the host cell with an envelope derived from the cell membrane. The diameter of the particle is roughly estimated to be 70 m., excluding the envelope.     

Flow Cytometric Analysis of In Vitro Proinflammatory Cytokine Secretion in Peripheral Blood from Multiple Sclerosis Patients

The cytokines, interferon- (IFN-), tumor necrosis factor- (TNF-rpar;, and interleukin-2 (IL-2) are important endogenous proinflammatory proteins and have been linked to disease activity in multiple sclerosis. In this study, we use flow cytometric methodology to compare the secretion of IFN-, IL-2, and TNF- from peripheral blood-derived T cells of multiple sclerosis patients to the secretion in healthy controls. The percentages of IFN-, IL-2, and TNF- secreting cells are not significantly different between multiple sclerosis patients and controls. However, the TNF- secreting CDS cell percentage is correlated with the IFN- and IL-2 secreting CD3 cell percentages in multiple sclerosis patients. In the controls, only the TNF- secreting CD3 cell percentage is correlated with IFN-. These findings show that correlated secretion of cytokines occurs in multiple sclerosis and suggest that concerted intercytokine interactions may play an important role in the disease.     

”Reflexes of purpose and freedom” in the comparative physiology of higher nervous activity

The most complex unconditioned reflexes of aim and freedom, discovered by I. P. Pavlov, are compared with the competence drive and the motivation of the resistance to coercion, respectively, described by contemporary ethologists. On the basis of the unconditioned reflex of purpose, conditioned reflexes were developed in which positive emotions arising in connection with the perfection of a skill, irrespective of its pragmatic significance at a given moment, serve as the reinforcement. The unconditioned reflex of freedom is regarded as a phylogenetic precursor of the will, and its acute extinction as the physiological mechanism of hypnosis. It was demonstrated experimentally that the appearance of the state of animal hypnosis (immobilization catatonia) in rabbits is accompanied by the predominance of electrical activity and heat production in the right hemisphere, i. e., by symptoms which are found in hypnosis in man.Translated from Zhurnal Vysshei Nervnoi Deyatel'nosti imeni I. P. Pavlova, Vol. 39, No. 3, pp. 415–420, May–June, 1989.     

Die Vergrösserung des funktionellen und absoluten Totraumes und dessen Beziehung zur Gasstoffwechselstörung bei obstruktivem Lungenemphysem

Zusammenfassung Die Bestimmung der verschiedenen Totraumgrößen bei Patienten mit obstruktivem Lungenemphysem gibt die Möglichkeit, die Ursachen für die Vergrößerung des funktionellen Totraumes zu erfassen. Der absolute Totraum ist bei diesen Patienten nur mäßig vergrößert. Die Vergrößerung des Mischluftanteiles und die Entwicklung von Paralleltoträumen wird diskutiert. Als im Vordergrund stehend wird die ungleichmäßige Ventilierbarkeit des Alveolarraumes angesehen, die alle funktionellen Symptome, wie sie bei obstruktivem Lungenemphysem zu beobachten sind, einschließlich des reversiblen arterio-venösen Kurzschlusses erklären kann. Die nach der Bohrschen Formel zu errechnende Vergrößerung des funktionellen Totraumes entspricht nicht der Vergrößerung von Toträumen im anatomischen Sinne. Die Vergrößerung des funktionellen Totraumes ist ebenfalls aus der ungleichmäßigen Ventilation mit dem Auftreten von alveolär-arteriellen Kohlensäuredruckgradienten abzuleiten.     

Immunoglobulin and T cell receptor gene rearrangements and in situ immunophenotyping in lymphoproliferative disorders

Summary We investigated for rearrangements of the immunoglobulin (Ig) heavy and light chain genes and of the T cell receptor (TCRT) and (TCr) genes 45 biopsy samples from a variety of lymphoproliferative disorders. They were diagnosed histopathologically and immunophenotypically as non-Hodgkin's lymphomas (NHLs) of the B cell type (19 cases), NHLs of the T cell type (3 cases), NHLs of undetermined cell type (3 cases), atypical lymphoid proliferation (1 case) and AIDS-related lymphadenopathies with florid polyclonal follicular hyperplasia (19 cases). A monoclonal proliferation of B cells was shown by DNA analysis in all 19 B cell NHLs. In two immunohistologically determined T cell NHLs (both diagnosed as mycosis fungoides) the cells had rearrangements of TCr gene, whereas in the third case (lymphoblastic NHL) the cells had rearrangements of Ig heavy chain and TCr and TCr genes. None of the B cell NHLs exhibited TCrand TCr gene rearrangement bands. All the undetermined cell NHLs demonstrated rearrangements of Ig heavy chain gene associated with the germ line TCrand TCr genes; in two cases light chain gene rearrangements were also found. The atypical lymphoid proliferation, in which the differential diagnosis was between a reactive or malignant process, and two out of 19 cases of florid polyclonal follicular hyperplasia showed a clonal B cell population by DNA analysis. This study indicates that there was a strong correlation between the rearrangements of specific genes and the immunophenotype of the NHL; moreover, DNA analysis of tissue biopsy specimens from phenotypically undetermined cell NHLs and from equivocal lymphoid proliferation using Ig and TCR gene probes yelded an answer in the cases analyzed. The significance of clonal B cell expansions found in two AIDS-related lymphadenopathies should be interpreted with caution.This work was supported in part by a Grant No 86.00644.44 from the Consiglio Nazionale delle Ricerche, Progetto Finalizzato Oncologia, Rome, and by the Associazione Italiana per la Ricerca sul Cancro, Milan, Italy