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.     

Expression of estrogen receptors-α and -β in primary breast neoplasms and tumors exposed to neoadjuvant hormonal therapy

We studied the content and expression of mRNA for estrogen receptors receptors- and - in breast tumors before and after 3-month neoadjuvant hormone therapy with antiestrogen tamoxifen and/or aromatase inhibitors. Expression of estrogen receptors- and - was most often detected in ER⁺PR⁺ tumors and most significantly decreased in these neoplasms after exemestane therapy. Immunocytochemical and radioligand assays showed that tamoxifen and anastrozole have little effect on the number of estrogen receptors- The number of progesterone receptors in tumors decreased by the end of anastrozole therapy. Estrogen receptors- were immunocytochemically revealed in 50% primary breast tumors. Anastrozole slightly decreased, while tamoxifen increased the incidence of these receptors. Interruption of signaling through estrogen receptors and suppression of estrogen biosynthesis had different effects on the receptor status of neoplasms and distribution of estrogen receptors- and -.Translated from Byulleten Eksperimentalnoi Biologii i Meditsiny, Vol. 138, No. 11, pp. 559–562, November, 2004     

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.     

On the terms “reticulosis” and “reticulum cell sarcoma” with regard to the modern concept of the monocyte macrophage system

Summary The terms reticulosis and reticulum cell sarcoma (= malignant lymphoma, histiocytic type) are discussed regarding the modern concept of the monocyte macrophage system which today has replaced the ancient theory of the reticuloendothelial system. The monocyte macrophage system is not independent, but closely related to the myeloid system. Thus, a third blood forming system as was believed in the case of RES does not exist. Phagocytic reticulum cells of the various hematopoietic organs are highly activated monocyte-derived macrophages. All those conditions formerly termed reticuloses have been found to belong either to the myeloid or to the lymphatic system. Considering the reticulum cell sarcomas or malignant histiocytic lymphomas, most of them seem to be of lymphatic rather than of macrophage origin, representing highgrade malignant lymphomas, possibly immunoblastic sarcomas. No relationship between these tumours and the monocyte macrophage system has been established, so far. Therefore, the terms reticulosis and reticulum cell sarcoma should be no longer used in order to avoid confusion, in order to stimulate sufficient diagnostic efforts which will really clarify such cases, and in order to give full credit to modern results of hematopathology.     

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.     

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.     

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)     

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.     

Immunocytochemical characterization of porcine zona pellucida during follicular development

Sections of bovine ovaries fixed in Bouin's fluid or methanol-acetic acid and embedded in paraffin were incubated with chicken polyclonal antibodies to HPLC-purified zona glycoproteins ZP3 and ZP3. Oocytes of primordial follicles as well as of primary follicles showed weak labelling with anti-ZP3 and anti-ZP3. No immunostaining could be observed in the follicle cells. The ZP of primary follicles displayed distinct immunoreactivity for both ZP3 and ZP3. In secondary follicles, distinct labelling with anti-ZP3 and weak labelling with anti-ZP3 could be seen in the oocyte. The ZP showed immunoreactivity with antibodies to ZP3 and ZP3. Both antibodies labelled single follicle cells. In tertiary follicles, the oocytes were weakly labelled with anti-ZP3 and anti-ZP3. Some granulosa cells showed staining for ZP3 and ZP3. The ZP displayed strong immunoreactivity for ZP3 and ZP3. Cells of the corona radiata were strongly immunopositive for ZP3 and ZP3. Similar histotopography of immunoreactive cells could be seen in preovulatory follicles. The characteristic pattern observed for the distribution of ZP3 and ZP3 strongly suggests that in the porcine ovary both the oocyte and the follicle cells contribute to the synthesis of the ZP, perhaps in sequence.     

In vitro susceptibility of“Campylobacter upsaliensis” to twenty-four antimicrobial agents

The in vitro susceptibility of 41 strains ofCampylobacter upsaliensis to 24 antimicrobial agents was determined using a broth microdilution procedure. Most isolates were susceptible to the fluoroquinolones and -lactam antibiotics tested, but all strains were resistant to trimethoprim (MBCs 128 µg/ml) and teicoplanin (MBCs 32 µg/ml). These agents may be useful in a selective isolation medium forCampylobacter upsaliensis.     

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     

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     

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.     

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.     

Modulation of hematopoietic colony formation of stem cells in peripheral blood by anti-TGF-β in patients with severe immunosuppression

Summary The influence of transforming growth factor- (TGF-) on hematopoiesis has been evaluated by adding blocking antibodies against TGF- to colony forming assays (CFU-c). When optimum concentrations of recombinant growth factors, granulocyte-macrophage colony stimulating factor (GM-CSF), and interleukin-3 (IL-3) were added to stem cells from the peripheral blood of healthy individuals and certain patients with tumors or HIV infection, the anti-TGF- capable of blocking 5 ng/ml of active TGF- had no significant influence on erythroid or myeloid colony formation. However, in certain immunosuppressed individuals, anti-TGF- resulted in a significant decrease of erythroid colony formation and slight suppression of myeloid colony formation. The significant inhibition of hematopoiesis by plasma of HIV patients could be due to the presence of active forms of TGF-. The results of the blocking experiments are consistent with the concept that TGF- in low concentrations is essential for erythropoiesis and myelopoiesis but that higher levels of TGF- primarily inhibit erythropoiesis in vitro. TGF- serves as a coordinating factor when efficient recruitment of granulocytes and monocytes is more essential than erythropoiesis and stem cell growth.Abbreviations BFU-E burst forming unit-erythroid - CFC colony forming cells - CFU-GEMM colony forming unit-granulocyte/erythroid/macrophage/megacaryocyte - CFU-GM colony forming unit-granulocyte/macrophage - EPO erythropoietin - GM-CSF granulocyte/macrophage-colony stimulating factor - HIV human immunodeficiency virus - IL-1 interleukin-1 - IL-3 interleukin-3 - IMDM Iscove's Modified Dulbecco's medium - PBS phosphate buffered saline - TGF- transforming growth factor- - TNF- tumor necrosis factor-     

Effects of C5a and FMLP on Interleukin-8 Production and Proliferation of Human Umbilical Vein Endothelial Cells

Interleukin-8 (IL-8), C5a and N-formyl-methionyl-leucyl-phenylalanine (fMLP) are chemotactic peptides with predominant effects on leukocytes during inflammation. With emphasis on C5a we studied the regulation of the production of IL-8 by human umbilical vein endothelial cells (HUVEC) in vitro. Primary HUVEC cultures were incubated with IL-1, TNF, C5a and fMLP for 24 h and 48 h prior to measurement of IL-8 in supernatants of the cells by an enzyme immunoassay. Whereas IL-1 and TNF significantly increased the levels of IL-8, C5a decreased the IL-8 production after 48 h. In addition, the ability of IL-1, TNF, C5a, fMLP and IL-8 to induce cell proliferation was compared by means of a ³H-thymidine incorporation assay. In contrast with IL-1 and TNF, both C5a and fMLP increased cell proliferation of HUVEC. This increase occurred with increasing concentrations of C5a contrary to IL-8, which showed increased cell proliferation at low, but not high IL-8 concentrations.     

Binocular interactions and disparity coding in area 21a of cat extrastriate visual cortex

We have examined, using both qualitative and quantitative techniques, binocular interactions of extracellularly recorded single neurons in the extrastriate cortical area 21a of anaesthetized and paralysed cats. Consistent with previous reports we have found that: (a) all area 21a neurons were orientation-selective, with about 65% of them preferring orientations within 30° of the vertical; and (b) over 75% of area 21a cells could be activated through either eye. Furthermore, a significant minority (4 cells; about 10%) of a subpopulation of 39 neurons in which binocular interactions were examined quantitatively, were obligatory binocular neurons, that is, they responded very weakly, if at all, to the monocular stimuli presented through either eye but responded vigorously to simultaneous stimulation through both eyes. Almost 70% (27/39) of neurons tested quantitatively for binocular interaction have shown significant modulation (over 50%) of their peak responses in relation to binocular positional retinal disparities. The majority of neurons sensitive to binocular positional disparities resembled either tuned excitatory (22 cells; 56.5% of the sample) or tuned inhibitory (2 cells; 5% of our sample) cells. In particular, they gave, respectively, maximal or minimal responses to optimally oriented, moving photic stimuli when the receptive fields plotted through each eye completely or partially overlapped. Although neurons recorded in area 21a have relatively large receptive fields (mean width 3.3±1.1°; range 2.0–5.6°), the mean width of the disparity tuning curve (2.8±1.0°; range 1.3–4.8°) for our sample of area 21a neurons was similar to those of neurons with significantly smaller receptive fields, recorded in areas 17 and 18 of cat's primary visual cortex. We conclude that area 21a of the cat, like areas 17 and 18 of primary visual cortex, is likely to play an important role in binocular depth discrimination and might constitute a higher order area for stereoscopic binocular vision.     

Antigenic similarity between the protein neurotoxin α-bungarotoxin and neuromuscular blocking drugs

The snake neurotoxins -bungarotoxin (-BGT) and -bungarotoxin (-BGT) which act at the neuromuscular junction, were found to bind to IgE antibodies directed against neuromuscular blocking (NMB) drugs in the sera of two patients who had experienced lifethreatening anaphylactic reactions to succinylcholine. -BGT inhibited IgE-binding to a choline-Sepharose solid support in one patient better than the NMB drug alcuronium, choline, triethylcholine and -BGT. IC₅₀s for -BGT and succinylcholine were 16 and 10 nmol respectively for one patient and 34 and 6.0 nmol for the other.Recognition of the NMB drugs and -BGT by the same antibody is the first demonstration of an antigenic similarity between these drugs and the protein toxin.     

Enhancement of antimicrobial effects by glucocorticoids

In the past few years a body of evidence has accumulated showing that stimulation of human astrocytes and microvascular endothelial cells with IFN- induces a potent antibacterial and anti-parasitic effect. We have found that the IFN--mediated activation of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) is, at least in part, responsible for this antimicrobial activity. Glucocorticoids are frequently used in inflammatory central nervous system diseases to reduce the inflammatory reaction and cerebral edema. Since in many inflammatory conditions infection is either a primary or secondary factor, steroids are administered, in these circumstances, during infection. We investigated whether steroids could affect the antimicrobial effect of IFN--induced IDO activation. We found that hydrocortisone and dexamethasone enhance IFN--mediated IDO activity in both human astrocytoma cells and native human astrocytes. Furthermore, we found that the amounts of IDO mRNA and of IDO protein are enhanced in cells treated with IFN- and glucocorticoids. In addition, we were able to demonstrate that both steroids enhance the IFN--mediated antimicrobial activity against Toxoplasma gondii, Staphylococcus aureus and group B streptococci. The enhanced antimicrobial effect of IFN- in the presence of glucocorticoids is due to the enhancement of the IDO-mediated tryptophan degradation, demonstrated by the complete abrogation of this antimicrobial effect by tryptophan resupplementation. These data show that glucocorticoids, which were often used to inhibit proinflammatory processes, do not decrease IDO-mediated antimicrobial effects. In contrast, high doses of steroids were able to enhance the IFN--induced antimicrobial activity.