Biochemical consequences of kainic acid injection into the lateral brain ventricle in rat

Effects of a single intraventricular injection of kainic acid (KA) in a dose of 0.1 microgram per rat on the activity of different brain neurotransmitter systems were investigated. A decreased level of norepinephrine at 3 and 24 h and acceleration of its utilization at 3 h after application of KA were observed. These changes were also accompanied by a decreased level of dopamine at 24 h, increased utilization of dopamine at 3 h, increased levels of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid at 3 and 24 h, as well as by shortened time of the turnover of 5-hydroxytryptamine. No disturbances in the function of the aminergic systems were noted at 120 h after injection of KA. Lowered activity of glutamic acid decarboxylase in the striatum, hippocampus, hypothalamus and cerebellum was observed at 24 h after administration of KA. At 480 h following application of KA, this lowering persisted in the hippocampus only. The most prominent changes in the level of gamma-aminobutyric were observed at 120 h in the striatum, hippocampus and cerebellum. A decreased level of gamma-aminobutyric acid was found in the striatum and cerebellum at 480 h following injection of KA. The observed changes in the dynamic equilibrium between various neurotransmitter systems may be a consequence of the direct or indirect influence of KA.     

Malignant transformation of hamster cells following infection with bovine herpesvirus (infectious bovine rhinotracheitis virus.

Hamster embryo cells, following infection with IBR virus, showed malignant transformation. Hamsters of all ages, inbred or random bred, inoculated with two of the transformed cell lines developed solid tumors. Preliminary characterization of the tumors induced by one of the cell lines has indicated undifferentiated sarcomas. Viral specific antigen was detected in about 5% of the transformed cells and 10% of primary tumor cells in culture. Viral specific antibody was detected in the serum of tumor-bearing hamsters by the indirect immunofluorescent method, but no neutralizing antibodies were found. Infectious virus has not been recovered from either the transformed or tumor cells by cocultivation with bovine embryonic kidney cells.     

Immunocytochemical characterisation of the wall of the bovine lateral ventricle

The cytoarchitecture of the walls of the bovine lateral ventricles was investigated by the use of immunocytochemistry. We defined three types of walls. Type 1 lined regions of white matter and had ciliated cuboidal ependyma, a few subependymal cells and a narrow subjacent glial layer. Type 2 lined the striatum and possessed ependymal cells with conspicuous basal processes that extended through a wide subependyma containing many subependymal cells and a wide subjacent glial network. Type 3 lined the rostral horn and displayed ependymal cells with the longest basal processes and wider subependymal and glial layers. Ependymal cells of type 2 and 3 walls were labelled with antibodies against S-100 protein, vimentin, GFAP, BLBP and nestin. Anti-III-tubulin stained small cells in the subependyma and inside the GFAP- and vimentin-positive subjacent glial network. Anti-PCNA-positive nuclei were abundant in the subependymal and glial layers of type 2 and 3 walls. DiI in vitro tracing studies revealed small bipolar cells in the glial layer at a distance from the site of the label deposit. These results suggest that neurogenesis takes place in adult bovine subependyma mostly in the walls of the striatum and the rostral horn, and that young neuroblasts may migrate in a rostro-ventral direction through the glial network.This work was supported by DGICYT (BFI2000-1360), FIS (01-0948, PI021517) and ISCIII (red CIEN, nodo Fundación Carlos Haya)     

Changes in ovarian follicles following acute infection with bovine viral diarrhea virus

Bovine viral diarrhea virus (BVDV) has been associated with several reproductive problems in cattle, including poor fertility, early embryonic deaths, abortion and congenital anomalies. Little is known about the cause of poor fertility in cows acutely infected with BVDV. The purpose of this study was to identify changes in ovarian function following acute infection with noncytopathic BVDV. The ovaries of 5 BVDV sero-negative and virus-negative pubertal heifers were monitored daily for 4 consecutive estrous cycles. The position and diameter of all follicles (> 5 mm) and luteal structures were recorded. Daily plasma samples were collected to measure peripheral progesterone and estradiol levels. Each heifer was infected intranasally with noncytopathic BVDV following ovulation of the second estrous cycle. The maximum diameter and growth rate of dominant anovulatory and ovulatory follicles were significantly reduced following acute BVDV infection. Similarly, the number of subordinate follicles associated with both the anovulatory and ovulatory follicle was reduced following infection. There were no significant differences in other follicle or luteal dynamic parameters or in peripheral progesterone or estradiol levels. Ovarian follicular growth was different during the first 2 estrous cycles following acute infection with BVDV when compared with the 2 estrous cycles preceding infection. These differences may be important in explaining reduced fertility in herds with acute BVDV infection.     

Characterization of the bovine herpesvirus 4 major immediate-early transcript.

The major immediate-early (IE) RNA of bovine herpesvirus 4 (BHV-4) has been identified and characterized by analyzing cytoplasmic polyadenylated RNA isolated from Madin-Darby bovine kidney cells infected with BHV-4(DN-599) in the presence of cycloheximide. Hybridization of cDNA to Southern blots of viral DNA, Northern (RNA) blot analysis, and S1 nuclease analyses showed that the major BHV-4 IE RNA is a spliced, 1.7-kb RNA, which is transcribed from right to left on the restriction map of the BHV-4 genome from DNA contained in the 8.3-kb HindIII fragment E. The major IE RNA contains three small exons at its 5' end, spliced to a 1.3-kb 3' exon. This RNA is present in much-reduced amounts when cells are infected in the absence of cycloheximide. However, late in infection, the major IE RNA gene region encodes abundant RNAs which differ in structure from the major IE RNA. Nucleotide sequence analysis of the gene encoding the major IE RNA revealed an open reading frame encoding 284 amino acids. A homology search of amino acid sequence data bases showed that a 141-amino-acid region near the amino terminus of the predicted amino acid sequence is similar to sequences near the amino terminus of herpes simplex virus type 1 IE110. This region of homology includes CXXC pairs, which could be involved in zinc finger structures. The region encoding this putative zinc finger domain is also found in RNAs transcribed from this IE region late in infection, but it is spliced to different sequences than those used in IE RNA. Thus, the major IE region of the BHV-4 genome could encode a family of proteins sharing a zinc finger domain.     

Increased susceptibility to pentobarbital following mouse cytomegalovirus infection: relative roles of viral-induced interferon and viral infection of the liver

The purpose of this study was to determine the relative roles of viral-induced interferon (IFN) and viral infection of the liver in mouse cytomegalovirus (MCMV)-induced depression of cytochrome P-450 (cyt P-450) levels and enhancement of pentobarbital-induced sleeping time (PEN-ST). This was done by establishing the temporal relationship among the IFN response, viral infection of the liver, suppression of cyt P-450 levels, and enhancement of PEN-ST, by determining the effect of anti-IFN antibody treatment on all of these responses, and by manipulating factors known to influence viral pathogenesis and host response to virus such as animal age, virulence of the virus, and dose of virus. In general, manipulation of these factors toward increased stimulation of host immune responses resulted in greater depression of cyt P-450. The data are consistent with the hypothesis that some IFN-dependent mechanism may have contributed to the effects of MCMV infection on both cyt P-450 levels and PEN-ST; however, the temporal relationship among the various responses measured following viral infection suggested that the effect of the IFN response may be indirect and due to modulation of other host defense mechanisms. Use of anti-IFN antisera to definitively establish a role for IFN in the effects observed here proved unsuccessful. Effects on PEN-ST and cyt P-450 levels did not appear to be related to the magnitude of infection in the liver.(ABSTRACT TRUNCATED AT 250 WORDS)     

Accumulation,elimination, release and metabolism of pipecolic acid in the mouse brain following intraventricular injection

Following i.c.v. (intracerebral ventricular) injections ofd,l-[³H]pipecolic acid (PA), it is reabsorbed from the ventricles and redistributed to various brain regions. The highest accumulation is found in three brain regions ipsilateral to the injection site, hippocampus, neocortex, striatum, and in the diencephalon. Following preloading in vivo, the radioactivity is released from hippocampus slices in the perfusion medium after depolarization induced by high K⁺. During perfusion with a Ca⁺⁺ free medium containing EGTA, a significant reduction of release is observed.The radioactivity ofd,l-[³H]PA in the brain shows a more rapid phase of decrease from 0 to 2 hours and a slower phase from 2 to 5 hours. At 5 hours, only 28% radioactivity, represented mainly by PA, is left in the brain. Kidney secretion represents the major route of elimination of the injected PA. The presence of -aminoadipic acid both in brain and urine was observed. Probenecid (200 mg/kg) significantly increases the accumulation of i.c.v. injectedd,l-[³H]PA in brain and kidney. The presence of a regional accumulation of PA in certain brain regions, its metabolism in brain, its enhanced retention following probenecid administration and its Ca⁺⁺ dependent release following high K⁺ stimulation, all constitute indirect evidence for a neuronal localization of this brain endogenous iminoacid.     

The bovine herpesvirus 4 Bo10 gene encodes a nonessential viral envelope protein that regulates viral tropism through both positive and negative effects

All gammaherpesviruses encode a glycoprotein positionally homologous to the Epstein-Barr virus gp350 and the Kaposi's sarcoma-associated herpesvirus (KSHV) K8.1. In this study, we characterized the positional homologous glycoprotein of bovine herpesvirus 4 (BoHV-4), encoded by the Bo10 gene. We identified a 180-kDa gene product, gp180, that was incorporated into the virion envelope. A Bo10 deletion virus was viable but showed a growth deficit associated with reduced binding to epithelial cells. This seemed to reflect an interaction of gp180 with glycosaminoglycans (GAGs), since compared to the wild-type virus, the Bo10 mutant virus was both less infectious for GAG-positive (GAG(+)) cells and more infectious for GAG-negative (GAG(-)) cells. However, we could not identify a direct interaction between gp180 and GAGs, implying that any direct interaction must be of low affinity. This function of gp180 was very similar to that previously identified for the murid herpesvirus 4 gp150 and also to that of the Epstein-Barr virus gp350 that promotes CD21(+) cell infection and inhibits CD21(-) cell infection. We propose that such proteins generally regulate virion attachment both by binding to cells and by covering another receptor-binding protein until they are displaced. Thus, they regulate viral tropism both positively and negatively depending upon the presence or absence of their receptor.     

Beta-endorphin administered into the lateral ventricle of the brain causes pulmonary platelet trapping in rabbits

Human beta-endorphin was injected into the cerebrospinal fluid in rabbits by means of a needle inserted into the lateral ventricle of the brain. Control rabbits received an equal amount of saline. beta-Endorphin induced a significant pulmonary platelet trapping compared to control. beta-Endorphin had no effect on arterial blood pressure, heart rate, platelet aggregability ex vivo or fibrinolytic activity (fibrinolytic plates). The plasma activity of antithrombin III, kallikrein-like activity and kallikrein inhibitor determined by means of chromogenic substrates was not influenced by beta-endorphin.     

Seroconversion of calves following intravenous injection with embryos exposed to bovine viral diarrhea virus (BVDV) in vitro

Two recent studies demonstrated that a high-affinity isolate of BVDV (SD-1), remained associated with a small percentage of in vivo-derived bovine embryos following artificial exposure to the virus and either washing or trypsin treatment. Further, the embryo-associated virus was infective in an in vitro environment. Therefore, the objective of this study was to determine if the quantity of a high-affinity isolate of BVDV associated with single-washed or trypsin-treated embryos could cause infection in vivo. Twenty zona-pellucida-intact morulae and blastocysts (MB) were collected on day 7 from superovulated cows. After collection, all MB were washed according to International Embryo Transfer Society (IETS) standards, and all but 4 MB (negative controls) were exposed for 2 h to 10(5)-10(6) cell culture infective doses (50% endpoint) per milliliter (CCID(50)/mL) of viral strain SD-1. Following exposure, according to IETS standards, one half of the MB were washed and one half were trypsin treated. All MB were then individually sonicated, and sonicate fluids were injected intravenously into calves on day 0. Blood was drawn to monitor for viremia and(or) seroconversion. Seroconversion of calves injected with sonicate fluids from washed and trypsin-treated embryos occurred 38% and 13% of the time, respectively. Therefore, the quantity of a high-affinity isolate of BVDV associated with single-washed or trypsin-treated embryos was infective in vivo.     

Improved high-performance liquid chromatographic determination of debrisoquine and 4-hydroxydebrisoquine in human urine following direct injection

A sensitive and specific reversed-phase high-performance liquid chromatographic assay was developed for the determination of debrisoquine and 4-hydroxydebrisoquine in urine. The urine samples were directly injected following an ether clean-up step which eliminated interference. Separation of the analytes was achieved using a mobile phase consisting ofacetonitrile-methanol-0.02 M heptane sulfonic acid (pH 3.0) (6:37:57) and a μBondapak C₁₈ analytical column. The assay utilizes fluorescence detection at 208 nm (ex) and 562 (em). The within-day and between-day coefficients of variation wered10% for both components and accuracy was within 12%. The method is suitable for pharmacogenetic studies utilizing debrisoquine.     

Intrinsic functional dysregulation of CD4 T cells occurs rapidly following persistent viral infection

Effective T-cell responses are critical to eradicate acute viral infections and prevent viral persistence. Emerging evidence indicates that robust, early CD4 T-cell responses are important in effectively sustaining CD8 T-cell activity. Herein, we illustrate that virus-specific CD4 T cells are functionally inactivated early during the transition into viral persistence and fail to produce effector cytokines (i.e., interleukin-2 and tumor necrosis factor alpha), thereby compromising an efficient and effective antiviral immune response. Mechanistically, the inactivation occurs at the cellular level and is not an active process maintained by regulatory T cells or antigen-presenting cells. Importantly, a small subpopulation of cells is able to resist inactivation and persist into the chronic phase of infection. However, the virus-specific CD4 T-cell population ultimately undergoes a second round of inactivation, and the cells that had retained functional capacity fail to respond to rechallenge in an acute time frame. Based on these results we propose a biological mechanism whereby early CD4 T-cell inactivation leads to a subsequent inability to sustain cytotoxic T-lymphocyte function, which in turn facilitates viral persistence. Moreover, these studies are likely relevant to chronic/persistent infections of humans (e.g., human immunodeficiency virus, hepatitis C virus, and hepatitis B virus) by providing evidence that a reservoir of virus-specific CD4 T cells can remain functional during chronic infection and represent a potential therapeutic target to stimulate the immune response and establish control of infection.     

Interleukin-12 gene expression after viral infection in the mouse.

Interleukin-12 is a lymphokine that triggers gamma interferon secretion by various cells and differentiation of T-helper lymphocytes towards the Th1 subtype. Since viruses are potent inducers of gamma interferon production and elicit immune responses most probably mediated by Th1 cells, like B-cell immunoglobulin G2a secretion, we analyzed interleukin-12 message expression after infection of mice with lactate dehydrogenase-elevating virus, mouse hepatitis virus, and mouse adenovirus. Our results indicated that the message for the p40 component of interleukin-12 was transiently increased shortly after infection. Interleukin-12 was expressed mainly by macrophages. Therefore, production of interleukin-12 might constitute the initial event that would determine the subsequent characteristics of the immune response elicited by viral infections.     

Evaluation of chromosomal risk following intracytoplasmic sperm injection in the mouse

To investigate whether cytogenetic risks occur using the mouse intracytoplasmic sperm injection (ICSI) technique, the incidence of chromosome aberrations was compared in one-cell embryos produced by ICSI technique and those by conventional in vitro fertilization (IVF) technique. Spermatozoa were incubated in TYH medium for 1.5-2 h before IVF insemination. For the ICSI technique, spermatozoa were incubated in five different media: TYH, Hepes-buffered TYH (H-TYH), modified CZB (mCZB), Hepes-buffered mCZB (H-mCZB), and PB1 for 0.5 h, 2-2.5 h, and 6 h before injection into metaphase II oocytes. The incidence of IVF embryos with structural chromosome aberrations was 2%, whereas the occurrence of structural chromosome aberrations in ICSI embryos was dependent on the kind of medium and sperm incubation time. When spermatozoa were incubated in TYH medium for 2 h or more, the aberration rates in the resultant ICSI embryos (4%) were not significantly different from that of IVF embryos. However, there was a significant increase in aberration rates in ICSI embryos derived from spermatozoa that were incubated in other culture conditions (6%-28%). In addition, a time-dependent increase in aberration rates was found in ICSI embryos when H-TYH, H-mCZB, and PB1 were used for sperm incubation. There was no significant difference in incidence of aneuploidy between IVF and ICSI embryos. The chromosome analysis results of one-cell embryos were reflected by the performance of postimplantation embryo development. The causal mechanism of chromosome damage in ICSI embryos was discussed in relation to the plasma membrane cholesterol, the acrosome, and in vitro aging of spermatozoa.