Nuclear transfer of blastomeres expressing EGFP-reporter gene may improve the efficiency of transgenic cattle.

The effect of timing of microinjection of DNA constructs on the efficiency of transgenic embryo production and improved efficiency and quality through combining EGFP as a reporter gene with nuclear transfer techniques were examined. From 12 to 24 h after insemination, constructs of pCXNeo-EGFP were microinjected into a pronucleus of bovine IVM-IVF zygotes. Due to the difficulty in visualizing pronuclei, the incidence of successful injection of linear DNA was higher when zygotes were injected between 20 and 24 h, as compared with an early period between 12 and 16 h after insemination. However, developmental competence of DNA-injected zygotes and the EGFP expression rate were not affected by the injection time. A majority of the embryos expressing EGFP signal were mosaic. Following nuclear transfer of blastomeres expressing EGFP, 4.5% of morulae that developed from the NT eggs had a strong EGFP signal in all live blastomeres. In other embryos, EGFP signal had been lost. When cells derived from the EGFP-positive NT morulae were subcultured, all the cells expressed strong EGFP signal at the second passage and demonstrated neomycin resistance. These results show that transient expression of nonintegrated EGFP appears frequently in EGFP-positive bovine embryos and that additional selection of EGFP-positive morulae after nuclear transfer of EGFP-positive blastomeres would facilitate selection of transgenic embryos.

Both arachidonic acid and 1-oleoyl-2-acetyl glycerol in low magnesium solution induce long-term potentiation in hippocampal CA1 neurons in vitro.

The effects of phospholipase blockers on tetanus-induced long-term potentiation (LTP) and of diacylglycerol (DG) and arachidonic acid (AA) on synaptic transmission were studied in CA1 neurons of guinea pig hippocampal slices to evaluate the role of protein kinase C (PKC) and AA on the maintenance of LTP. Tetanus-induced LTP was suppressed by perfusion with neomycin (1 mM) or 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (NCDC, 0.1 mM), blockers of phospholipase. 1-Oleoyl-2-acetyl-glycerol (OAG, 100 micrograms/ml) and AA (100 microM) produced a temporal increase in both the amplitude of the population spike (PS) and the slope of the field excitatory postsynaptic potentials (EPSPs) but failed to produce LTP. Application of OAG or AA in low-Mg2+ (0.1 mM) solution induced LTP. OAG- and AA-induced LTP was blocked by DL-2-amino-phosphopentanoic acid (AP5; 50 microM). The administration of a potent activator of PKC, phorbol-12,13-dibutyrate (PDBu), in low-Mg2+ (0.1 mM) solution enhanced the PS and EPSPs for 2 or 3 h but this enhancement did not persist. These results suggest that PKC activation is not as important as AA for the maintenance of LTP and that OAG and AA play important roles in the maintenance of LTP in synergy with the influx of Ca2+ through NMDA receptor-coupled channels.

Effects of bicuculline and baclofen on paired-pulse depression in the dentate gyrus of epileptic patients.

Paired-pulse field responses were recorded from the granule cell layer of the dentate gyrus in brain slices from temporal lobe epileptic patients. Paired-pulse depression (PPD) was examined using perforant path stimulation of low to moderate intensity at an inter-stimulus interval (ISI) of 20 ms. The paired-pulse ratio (PS2/PS1) was expressed as the population spike amplitude of the second response (PS2) relative to that of the first response (PS1). Representative tissue response from each patient biopsy were divided into two groups that were significantly different based on the magnitude of the highest paired-pulse ratio recorded for each biopsy specimen: the strong paired-pulse depression group (PS2/PS1 = 0.12 +/- 0.03; n = 15) and the weak paired-pulse depression group (PS2/PS1 = 0.68 +/- 0.06; n = 13). Paired-pulse ratios from the strong PPD group were relatively independent of stimulus intensity, whereas, PPD was dependent on stimulus intensity in the weak PPD group; i.e., PPD was greatest at the lowest intensity and reached a plateau at higher intensities. Bicuculline (20 microM) and low concentrations of baclofen (0.1-0.2 microM) reduced paired-pulse depression in the strong PPD group, but did not significantly change the paired-pulse ratio in the weak PPD group. Paired-pulse facilitation was observed in some cases after inhibition was blocked pharmacologically. The number of population spikes was increased in the presence of bicuculline but was unchanged by baclofen. In the strong PPD group, baclofen significantly altered the EPSP-population spike (E-S) relationship by increasing the slope of the relationship for the second response, without having an effect on the slope of the first response. Baclofen had no effect on the E-S relationship of either response in the weak PPD group. The data are consistent with (1) less inhibition in the weak PPD group compared to the strong PPD group, (2) reduction of feedback inhibition in the strong PPD group by bicuculline and by low concentrations of baclofen, and (3) the occurrence of paired-pulse facilitation when inhibition was pharmacologically reduced in the dentate gyrus of temporal lobe epileptic patients. The results are also consistent with the presence of GABAB receptors on human inhibitory interneurons that, when activated by baclofen, result in disinhibition of granule cells through feedback circuits. Although inhibition may be compromised in some epileptic human biopsy specimens, the presence of strong inhibition in other patients' biopsy material suggest the re-evaluation of the role of inhibition in epilepsy.

Prolonged field potentials evoked by 1 Hz stimulation in the dentate gyrus of temporal lobe epileptic human brain slices.

An abnormal electrophysiological response in brain slices of the dentate gyrus from biopsy material from patients surgically treated for intractable epilepsy (46/57), exhibited characteristics similar to the physiological hallmark of epilepsy, the paroxysmal discharge, a prolonged (30-600 ms) and often large amplitude field potential. The most striking feature of the prolonged response to a single perforant path stimulus was a predominantly biphasic field potential (23/46 cases). The biphasic response was characterized by a negative field potential of substantial duration exceeding 180 ms which followed an initial shorter duration positive field potential. Multiple population spikes occurred during both phases of the response. During a 1 Hz stimulus train applied to the perforant path, the magnitude and duration of the negative component of the field response was significantly increased. Approximately half of the cases (Group 1; 30/57) exhibited potentiation of the biphasic response, while the remaining cases (Group 2; 27/57) exhibited no negative field component during 1 Hz stimulation trains. This repetitive stimulation, in general, increased the area of the field response in a large majority of cases (44/57) regardless of the sign of the field potential. The number of population spikes following 1 Hz stimulation increased significantly for cases in both groups, although the increase was greater for those in Group 1 than in Group 2. Paired pulse depression (20 ms ISI) was reduced in cases that exhibited potentiated biphasic responses during 1 Hz stimulation (Group 1) in comparison to cases that exhibited no negative field potentials (Group 2). Paired pulse depression at a 200 ms ISI was not significantly different between the groups. During a single stimulus, bicuculline disinhibition (20 microM) resulted in either a prolonged positive or biphasic field potential. Intracellularly recorded responses to single perforant path stimuli also exhibited prolonged and large depolarizations that were comparable in time course to the duration of field potentials recorded in the same area whether generated in the absence or presence of bicuculline. The prolonged field potential after bicuculline was reduced by APV (20 microM). We suggest that the prolonged field response, whether biphasic or monophasic when generated by either 1 Hz stimulation or bicuculline disinhibition, may be due directly or indirectly to an increase in membrane depolarization mediated by activation of the NMDA receptor.

The functional relationship between antidromically evoked field responses of the dentate gyrus and mossy fiber reorganization in temporal lobe epileptic patients.

Field recordings from the dentate granule cell layer of in vitro brain slices of temporal lobe epileptic patients were evoked by antidromic stimulation. Tissue from the same specimen was stained by the Timm-sulfide method to assess the pattern and degree of mossy fiber reorganization into the supragranular layer. A wide range of physiological responses and Timm staining patterns was present across patients. A significant correlation was observed between the abnormality of antidromic responses, reflected by multiple secondary population spikes, and the degree of Timm staining of the supragranular layer. This relationship lends support to the hypothesis that mossy fiber synapses located in the supragranular layer may have functional implications for granule cell excitability in human epileptic tissue.

Longitudinal variation in cell density and mossy fiber reorganization in the dentate gyrus from temporal lobe epileptic patients.

Variation in cell loss and mossy fiber reorganization was examined along the longitudinal axis of the dentate gyrus from temporal lobe epileptic (TLE) patients. Previous evidence has indicated that the anterior hippocampus is prone to seizure activity. We compared granule and hilar cell number in addition to Timm stain density of the molecular layer and hilus in more anterior and more posterior specimens of hippocampus obtained from patients surgically treated for intractable epilepsy by the removal of the anterior half of the hippocampus. Granule cells/mm in the more anterior specimen were less than or equal to those in the more posterior specimen locations in 77% of the patients, while there was no significant difference in hilar neuron density between the two blocks. These results demonstrate a significantly greater pathology in the granule cell layer in more anterior specimens and no difference in pathology for hilar neurons. Molecular layer Timm stain density was significantly greater in the more anterior specimen of 71% of the patients. The molecular layer Timm stain density ratio was inversely related to hilar cell density in more anterior specimens, whereas in more posterior specimens there was no significant relationship with hilar cell density. Our observations show that although differences exist among TLE patients for these neuroanatomic measures, pathology was greater in more anterior specimens. The latter result is consistent with the conclusion that seizure activity may originate in the anterior region of the hippocampus in a majority of patients.

Mossy fiber reorganization and its possible physiological consequences in the dentate gyrus of epileptic humans.

It is unlikely that MF reorganization is the cause of epilepsy, but it may affect the progression of the disease, i.e., the frequency or severity of seizures. We propose that early events, yet undiscovered, lead to an increased likelihood of excitability. This hyperexcitability, which initially may not be manifested in overt seizures, may erode vulnerable hilar neurons that serve an important inhibitory function, as illustrated in Fig. 6-15. As inhibition is lost, hyperexcitability reaches the level of clinically manifested seizures that are severe enough to lead to substantial loss of hilar neurons. When the loss of these cells is sufficiently high, MF reorganization occurs, first to neighboring hilar neurons and later to dendrites of granule cells (Fig. 6-15). Thus, the functional consequence of MF reorganization may provide a compensatory form of inhibition, as well as a circuit for feedback excitation. Although definitive evidence indicating that MF reorganization contributes to the acceleration or progression of epilepsy is missing, the findings to date are consistent with this hypothesis. In the event that reorganization contributes to the epileptic condition, treatments that reduce indicators of neuropathology may lead to a reduction of seizure frequency and severity. Evidence suggests that reorganization in the dentate gyrus may follow the pathways of neuronal processes of hilar neurons that have died. Thus, further study of the events that guide MF reorganization may hold important clues for developing methods for targeting regenerating axons following central nervous system injury.

Physiological and anatomical correlates of the human dentate gyrus: consequences or causes of epilepsy.

The presence of paroxysmal discharges in the epileptic human dentate gyrus provides a physiologic basis for hyperexcitability that may initiate seizure discharges during the development of epilepsy. Although these responses can occur with single orthodromic stimulation, data obtained under conditions that weaken synaptic inhibition (e.g., 1 Hz stimulation or bicuculline disinhibition) suggest that paroxysmal discharges may be a more common feature of tissue from temporal lobe epileptic patients than has been reported previously. Hilar cell loss and weakened synaptic inhibition may provide conditions favorable for the activation of N-methyl-D-aspartate acid (NMDA) receptors that would allow triggering of paroxysmal discharges that normally never are evoked in dentate granule cells in nonepileptic humans. As the dentate gyrus in normal animal tissue is not susceptible to intrinsic bursting behavior and is characterized by a relatively short duration excitatory postsynaptic potential even under pharmacologic disinhibition, paroxysmal discharges in the epileptic human dentate gyrus may provide an important clue to understanding the prerequisite conditions for seizure discharge.

Isolation of porcine epidemic diarrhea virus in porcine cell cultures and experimental infection of pigs of different ages.

This paper describes the isolation of porcine epidemic diarrhea (PED) virus in Vero and porcine cell cultures, and the influence of age on disease in experimental infection. PED virus was isolated from the small intestine of piglets inoculated with PED samples and cultured in Vero, porcine bladder and kidney cells propagated in collagen-coated tissue culture plates in maintenance medium (MM) containing trypsin. In porcine bladder and kidney cell cultures inoculated with isolated PED virus, cytopathic effects (CPE) including cell fusion were detected. Specific brilliant fluorescence was observed in the cytoplasm of these cells. Two- and 7-day old, and 2-, 4-, 8- and 12-week old specific pathogen-free (SPF) pigs were orally inoculated with PED virus isolated from an outbreak. All 2- and 7-day old pigs inoculated developed severe watery diarrhea from post-inoculation day (PID) 1 and died between PID 3 and 4. Although three of five 2-week old pigs developed diarrhea on PID 1-4, they eventually recovered. In the 4-week old group, three of five pigs had mild diarrhea for 1-2 days. None of the 8- and 12-week old pigs showed any clinical signs. Antibodies against PED virus were detected in all surviving pigs by virus neutralization (VN) test and immunofluorescence assay (IFA). Therefore, there is an age-dependent resistance to pathogenic PED virus infection in pigs.

Elevated serum haptoglobin in pigs infected with porcine reproductive and respiratory syndrome virus.

We examined the two acute phase proteins, alpha (alpha)-1 acid glycoprotein (AGP) and haptoglobin (HP), in serum of pigs following experimental porcine reproductive and respiratory syndrome (PRRS) virus infection. Increased levels of serum HP, but not AGP, were observed from 7 to 21 days post-inoculation in the infected pigs. Furthermore, serum IL-6 increased in the infected pigs, but TNF-alpha did not. The increase of serum IL-6 in pigs following PRRS virus infection may induce production of HP. Also, in the field investigation, serum HP in pigs was dramatically increased after exposure to the PRRS virus.

The role of quisqualate receptors in the induction of hippocampal long-term potentiation.

Long-term potentiation (LTP) is the persistent facilitation of synaptic responses induced by a brief tetanic stimulation (tetanus). From our observations and the findings of others, we hereby propose a novel hypothesis of the cellular mechanisms involved in the induction of hippocampal LTP. Quisqualate receptors are first activated during tetany and this stimulates the turnover of phosphatidyl inositol (Pl). The increased turnover activates protein kinase C and this results in the induction of LTP. The influx of Ca++ through N-methyl-D-aspartate-linked receptor mechanisms may participate in this process but is not the main factor for the activation of protein kinase C. With this hypothesis, the role of quisqualate receptors associated with the turnover of Pl is emphasized, and its relevance is discussed to the input specificity of LTP and to the modulation of LTP by certain drugs.

Vitamin E binding proteins in human serum.

The distribution of vitamin E in human serum lipoprotein fraction was investigated by measuring the amount of vitamin E in various protein fractions prepared by ultracentrifugation, gel filtration and electrophoresis. The larger part of the total serum vitamin E (up to 44%) was present in the alpha1-lipoproteins, while the beta-lipoproteins contained smaller concentrations (up to 26%).

Vitamin E and lipoprotein levels in the sera of pregnant women.

The serum levels of vitamin E, cholesterol and lipoproteins, alpha1-lipoproteins and beta-lipoproteins in pregnant women were investigated. It was found that serum vitamin E, cholesterol and the lipoproteins had a tendency to increase throughout gestation. The mean serum alpha1-lipoprotein concentration was higher in pregnant women than in healthy control women. The ratios of the vitamin E concentration to the sum of alpha1-lipoproteins and beta-lipoproteins concentration in sera was constant. Furthermore, it was found that serum vitamin E was correlated closely to the lipoprotein content (r = 0.814, p less than 0.001), except in a few subjects with abnormally high vitamin E levels.

Relationship between serum vitamin E levels and serum cholesterol in normal adults in rural regions of Yamagata Prefecture, Japan.

Serum vitamin E and cholesterol levels were determined for the residents in rural regions of Yamagata Prefecture, Japan. The average serum vitamin E were 0.99 +/- 0.25 mg/100ml in 166 men, and 1.06 +/- 0.26 mg/100ml in 178 women. Only 0.6% of the subjects were below 0.5 mg/100 ml. There was a significant difference between the serum vitamin E levels of men and women. However there were no statistically significant differences among serum vitamin E of various age groups. The best correlation for serum vitamin E was found with serum cholesterol.

Serological property and replication in cell cultures of porcine reproductive and respiratory syndrome viruses isolated in Japan.

The serological property and replication in swine alveolar macrophages (SAM) and MARC-145 cell cultures of 35 porcine reproductive and respiratory syndrome (PRRS) viruses isolated in SAM were investigated. All the isolates were reacted almost equally with antisera against three Japanese isolates including EDRD-1 strain (American type) in immunoperoxidase monolayer assay (IPMA), but weakly or did not react with antiserum against Lelystad virus (European type) indicating that the Japanese isolates are more closely related to an American type virus. Twenty two of 35 isolates replicated with CPE both in SAM and MARC-145 cells, whereas remaining 13 isolates did not show CPE in MARC-145 cells. The antigenicity of the isolates did not relate to the virus origin and the replication in the cell cultures.

Experimental infection of maternally immune pigs with porcine reproductive and respiratory syndrome (PRRS) virus.

To investigate the influence of maternal antibody to porcine reproductive and respiratory syndrome (PRRS) virus infection, the following examination was done using conventional and SPF pigs. Ten 17-day-old conventional pigs with maternal antibody against PRRS virus and 6 44-day-old SPF pigs seronegative were inoculated intranasally with 10(5.0) TCID50 of PRRS virus. Two conventional and 4 SPF pigs were served as non-inoculated control. In conventional pigs, coughing and febrile response were observed after inoculation, and mean rate of weight gain reduced. One of the inoculated conventional pigs died on post-inoculation-day (PID) 28 and Haemophilus parasuis was isolated from the lung. Although febrile response was also observed in the inoculated SPF pigs, reduction in weight gain rate was not recognized. Virus was isolated from all the sera of inoculated conventional and SPF pigs except one conventional pig between PID 7 and 49, and between PID 7 and 28, respectively. Onset of viremia in the several conventional pigs delayed. Virus was isolated from the tissues of the 5 conventional pigs on PID 65 and from the tissues of the dead pig. On the other hand, virus was not isolated from the tissues of non-inoculated conventional pigs, and inoculated and non-inoculated SPF pigs. At the virus inoculation, antibodies by the indirect fluorescent antibody (IFA) assay against PRRS virus were detected in the sera of conventional pigs with antibody titers of 1:20. Antibody titers gradually decreased after inoculation and rose from PID 21 or 28 and were between 1:160 and 1:640 on PID 63. Virus neutralization (VN) antibody titers were 1:2 or 1:4 at the inoculation and gradually decreased. Apparent rise in VN antibody titer was not observed after the inoculation. In the sera of control pigs, both antibody titers gradually decreased and did not rise. In the sera of the SPF pigs, antibodies by the IFA assay were first detected on PID 7 or 14. The titers of antibodies rose and reached their maximum with 1:320 to 1:2,560 on PID 21 to 35. VN antibodies were first detected in PID 42 to 56 and thereafter, the titers ranged between 1:1 to 1:4. Control SPF pigs were free of antibody throughout the examination. Antigenic variability was not recognized between the inoculated and recovered viruses by the VN test. The prolonged duration of viremia and virus isolation from the tissues on PID 65 in conventional pigs with low maternal antibody might support the present of antibody-dependent enhancement activity of PRRS virus infection.

Detection of bovine viral diarrhea virus (BVDV) using reverse transcription polymerase chain reaction assay.

The reverse transcription polymerase chain reaction (RT-PCR) was applied to detect bovine viral diarrhea virus (BVDV) for the rapid diagnosis. The primers were selected from the p80 region of BVDV gene. The RT-PCR assay detected all of the 17 BVDV strains tested including cytopathogenic and non-cytopathogenic strains, while specific amplification was not observed from 17 bovine viruses other than BVDV. Detection limit of the assay was 10(1.5) TCID50/ml. Sera and organ samples were collected from four field bovine viral diarrhea-mucosal disease (BVD-MD) cases; mucosal disease, abortion, diarrhea and persistent infection. The RT-PCR assay detected BVDV from those samples more than conventional virus isolation method.

Distinction between Aujeszky's disease virus-infected and vaccinated pigs by hemagglutination-inhibition test.

A hemagglutination-inhibition (HI) test was applied to distinguish virulent Aujeszky's disease virus infected pigs from those immunized with a glycoprotein gIII deletion vaccine. The vaccine strain, dlg92/dltk, did not have hemagglutination activity with mouse erythrocytes and the pigs vaccinated five times with the dlg92/dltk strain failed to develop HI antibody, although they developed neutralizing antibody with 128 to 512 titers to Aujeszky's disease virus. On the other hand, these pigs produced HI antibody 1 to 2 weeks after virulent virus inoculation. Thus the animals infected with virulent strain were easily differentiated from the animals immunized with the gIII deletion vaccine.

Replication of virulent and attenuated strains of Aujeszky's disease virus in swine alveolar macrophages.

In vitro and in vivo replication of Aujeszky's disease virus (ADV) in swine alveolar macrophages (AM) was studied using two virulent strains and a vaccine strain with deletions in the thymidine kinase and gIII genes. In vitro, AM were highly permissive to virulent ADV infection. Virus progeny titers of virulent strains in the cell phase and in the fluid phase were higher than 10(7.3) TCID50/ml at 84 hr post-inoculation (PI). For vaccine strain infection, AM were less permissive, yielding virus titers of 10(2.3) TCID50/ml at 84 hr PI. To study in vivo replication of ADV in AM, virus isolations were made from AM collected at intervals from pigs inoculated intranasally with both the virulent and vaccine strains. Virus was isolated from AM samples collected from all pigs infected with the virulent strain from days 2 to 22 PI. On the contrary, no virus was detected in AM samples collected from pigs infected with the vaccine strain. The results presented suggested that in vivo virulent ADV replicates for a relatively long period in swine alveolar macrophages.

In vivo replication of porcine reproductive and respiratory syndrome virus in swine alveolar macrophages and change in the cell population in bronchoalveolar lavage fluid after infection.

Replication of porcine reproductive and respiratory syndrome (PRRS) virus in swine alveolar macrophages (AM) and cell population in broncho-alveolar lavage fluid (BALF) obtained from PRRS virus-infected pigs were investigated. BALF samples were periodically collected from 6 pigs infected with PRRS virus and 3 non-inoculated control pigs by means of fiber-optic bronchoscope between post-inoculation day (PID) 0 and 56. The mean ratio of macrophages in BALF collected from infected group was 92.7 +/- 3.2% before inoculation and gradually decreased from PID 14. On the other hand, the ratio of lymphocytes was 4.8 +/- 3.2% before inoculation and increased from PID 21 and indicated 41.8 +/- 9.1% on PID 28. After that, they decreased gradually and that of macrophages correspondingly increased. The ratio of neutrophils maintained between 0.7% and 5.1%. The ratios of macrophages, lymphocytes and neutrophils collected from control group were almost stable through the examination. Intracellular PRRS virus antigens in AM were detected from PID 2 by indirect immunofluorescence assay (IIFA). PRRS virus was first isolated from BALF samples collected from inoculated group between PID 2 and 49. From serum, virus was isolated between PID 2 and 21. Antibodies in sera measured by IIFA to PRRS virus were first detected on PID 14 and the antibody titer rose to 1:640 or 1:1,280. The results suggested that PRRS virus replicates in swine AM for a relatively long period.