Monoclonal antibodies to canine intra-acrosomal sperm proteins recognizing acrosomal status during capacitation and acrosome reaction

Summary.  Monoclonal antibodies Ds-1 and Ds-2 specifically labelling dog sperm acrosome were prepared by immunization of mice with acetic acid extracts of dog spermatozoa. Electron microscopy and indirect immunofluorescence localized the site of Ds-1 and Ds-2 proteins inside the acrosomal vesicle. Ds-1 antibody detected 55, 76, 115, 120 and 190kDa proteins under non-reducing conditions, and 73 kDa and 54 kDa proteins after reduction (p73/Ds-1 and p54/Ds-1). 92 kDa and 40 kDa proteins recognized by Ds-2 (p92/Ds-2 and p40/Ds-2) migrated at > 200 kDa in the absence of reducing agent. In vivo , p73/Ds-1 and p54/Ds-1 are therefore likely to be present both in free and complexed form, while all of p92/Ds-2 and p40/Ds-2 form disulfide-bonded complexes. Decrease in the rate of acrosomes stained with Ds-1 and Ds-2 was correlated with the progress of capacitation resulting in the increased rate of spontaneous acrosome reactions, as suggested by a dramatic effect of A23187. Monoclonal antibody to boar acrosin (ACR-2) recognized dog sperm acrosin homologue. A higher rate of ACR-2-negative spermatozoa was observed after capacitation and A23187 treatment compared to Ds-1 and Ds-2, indicating that proteins recognized by Ds-1 and Ds-2 are localized in a specific compartment of acrosome, distinct from acrosin and possibly representing fraction of acrosomal matrix.     

Simple reaction time event-related potentials: Effects of alcohol and diazepam

1. 1. Acute effects of alcohol and diazepam on reaction time (RT) and event-related potential (ERP) measures were examined in 108 healthy male volunteers.

2. 2. The subjects engaged in a simple RT task at two levels of stimulus intensity during baseline and treatment sessions.

3. 3. Lower stimulus intensity produced increased RTs, increased ERP peak latencies, and suppression of peak amplitudes.

4. 4. Moderate and high doses of alcohol, and high doses of diazepam produced increased RTs. Alcohol suppressed P100 and N100 amplitudes, while diazepam suppressed P100 amplitudes only. P100 amplitudes were correlated to RTs under baseline and treatment conditions.

5. 5. These results were taken as evidence for impaired stimulus detection during alcohol and diazepam intoxication, with both drugs influencing sensory-perceptual processes and alcohol alone influencing the degree of attentiveness.

Comparison of the effect of intravenous ketoprofen, ketorolac and diclofenac on platelet function in volunteers

Background : Nonsteroidal anti–inflammatory drugs (NSAIDs) inhibit prostaglandin synthesis which may result in impaired platelet function. Because NSAIDs have different abilities to inhibit cyclo–oxygenases we compared the effect of intravenous ketoprofen, ketorolac and diclofenac on platelet function in volunteers. Methods : Ten healthy male volunteers were given ketoprofen 1.4 mg kg^-1, ketorolac 0.4 mg kg^-1 and diclofenac 1.1 mg kg^-1 in saline i.v. on three different occasions, at more than one–week intervals, in a randomized double–blind crossover study. Platelet function was evaluated before (sample 0), 2 (sample 2) and 24 h (sample 3) after the beginning of the infusion. Results : Two of the volunteers had no secondary platelet aggregation in their aggregation curves before the experiment (sample 0, studied three times) and their results were excluded from the final analysis. Diclofenac inhibited adrenaline (0.9 μg–ml^-1) induced platelet aggregation less (median maximal aggregation 22.5%) than ketoprofen (18.3%) and ketorolac (15.7%) (P<0.05) in sample 2. In the ketorolac group in sample 3 an impairment of adrenaline (0.9 ng ml^-1) induced platelet aggregation was still seen (26.7%) (P<0.05) but not in the other groups. Diclofenac did not affect adenosine diphosphate (ADP) induced platelet aggregation. However, ketorolac caused an impairment in ADP (3 μM and 6 μM) induced platelet aggregation and ketoprofen in ADP (6 μM) induced platelet aggregation in sample 2. Bleeding time was prolonged (P<0.05) after ketoprofen and ketorolac (sample 2) but not after diclofenac. Platelet retention on glass beads was unaffected by the tested drugs. Conclusion : Ketoprofen, ketorolac and diclofenac caused a reversible platelet dysfunction. Diclofenac had the mildest effect, while platelet dysfunction was still seen 24 h after the beginning of ketorolac.     

T Cell Receptor Vβ Repertoires of Angioimmunoblastic Lymphadenopathy-like T Cell Lymphoma

To elucidate the pathogenesis of angioimmunoblastic lymphadenopathy-like T cell lymphoma (AILD-T) we investigated the T cell receptor Vβ gene repertoires of four AILD-Ts and compared them with those of other histological types of lymphomas and three cases with reactive disorders. All lymphoma patients had rearrangement bands detected by Southern blot analysis. Only 1 of the 4 cases of AILD-T showed a single predominant usage of Vβ 20 gene by PCR with 20 different Vβ specific primers and the others had repertoires somewhat restricted but similar to reactive lesions. Subsequent sequencing of this PCR product revealed that only 2 of 7 clones were identical. These results suggest the monoclonal malignant cells in AILD-T are scant and that the infiltrating T cells show a reactive pattern. In the only AILD-T case with a single dominant Vβ usage, the relationships of this repertoire and lymphoma cells seems to be of some consequence.     

Indirect detection of anti-acetylcholinesterase compounds in microcolumn liquid chromatography using packed bed reactor with immobilized human red blood cell acetylcholinesterase and choline oxidase

The inhibiting compounds were separated by micro-column liquid chromatography in the mobile phase containing the natural substrate acetylcholine. A home-made packed bed microbioreactor system containing immobilized enzyme acetylcholinesterase (ACHE) in human red blood cell membrane and choline oxidase (CHO) from alcaligenes was used for the post-column conversion of acetylcholine to hydrogen peroxide which was detected by an electrochemical detector. The inhibition effect of the solutes caused a decrease in the acetylcholinesterase activity, a decrease in the formation of hydrogen peroxide and also a decrease in the response corresponding to the concentration of the solutes. The rate of the enzyme regeneration was also recorded. The micro-system was compared with a conventional LC system comprising commercially prepared enzyme reactor. The stability of the enzymes is at least 3 weeks at ambient temperature. The limit of detection depends on biological activity of inhibition and for galanthamine was 1 pmol.     

Association analysis of CA repeat polymorphism of the endothelial nitric oxide synthase gene with essential hypertension in Japanese

The nitric oxide synthase (NOS) gene is thought to be associated with essential hypertension (EH), because NO is implicated in endothelium-mediated vasodilation. We investigated the possible association between the alleles of simple tandem repeat DNA polymorphism of the endothelial constitutive NOS (cNOS) gene and EH in Japanese subjects. In all, 100 patients with EH and 123 subjects with normal blood pressure were studied. Polymerase chain reaction was used to amplify the CA repeat site in the endothelial cNOS gene and alleles based on the CA repeat number were determined. The allele frequencies in the hypertensive group and normotensive group were then compared. Twenty-three alleles were identified in this study of Japanese subjects. The overall distributions of allele frequencies in the two groups were not significantly different. However, comparing the allele frequencies in the EH group without left ventricular hypertrophy (LVH) and the normotensive group, the overall distributions were significantly different (p = 0.019). The 33-repeat allele was found more frequently in the EH group without LVH than in the normotensive group (p = 0.000047, Odds ratio = 3.71). In conclusion, the 33-repeat allele of the endothelial cNOS gene is associated with EH without LVH, and may be a genetic marker of EH in Japanese subjects.     

Lymphokine activated killer cells in murine coxsackievirus B3 myocarditis

The purpose of the present study was to determine whether lymphokine activated killer (LAK) cells were involved in the development of coxsackievirus B3 (CB3) myocarditis in both the acute viremic (Experiment I) and the subacute aviremic (Experiment II) stages. To induce LAK cells, recombinant human interleukin-2 (IL-2) was administered to CB3-infected mice subcutaneously daily, starting on day 0 in Experiment I and on day 7 in Experiment II for 7 days, respectively. The treated groups were compared to infected controls. Splenic lymphocytes of IL-2 treated mice were further cultured in vitro in IL-2 containing medium for 7 days, and LAK cell activity, i.e., cytotoxic activity of the lymphocytes against EL-4 tumor cells and against cultured fetal myocytes, was assayed by⁵¹Cr-release method. In Experiment I, histologic scores, myocardial virus titers, and LAK cell activity did not differ significantly between IL-2 treated and untreated groups. In contrast, in Experiment II, there were more cellular infiltration associated with severe necrosis and higher LAK cell activity against EL-4 cells and cultured myocytes in IL-2 treated than in untreated groups. The presence of LAK cells was demonstrated in the subacute stage of murine CB3 myocarditis. Thus, the behavior of LAK cell activity may vary with the course of myocarditis, and enhanced LAK cell activity may be involved in the development of the disease.This work was supported by research grants from the Conference on Coronary Artery Disease, Japanese Education of Science and Walfare (Nos. 08877110 and 09470164), Kanae Shinyaku Foundation, and Japan Cardiovascular Research Foundation.     

Giardia muris infection in mice with concurrent graft-versus-host reaction

The effects of concurrent graft-versus-host reaction (GvHR) on the course of Giardia infection in CBA x BALB/c F1 mice have been examined, to test the hypothesis that T-cell-mediated immunity, in the form of a local DTH reaction, alters the host-parasite relationship in favour of the host by changing the physical environment of the parasite. GvHR did not enhance immunity, indeed mice infected with Giardia at a late stage of GvHR had significantly higher faecal cyst excretion and prolongation of the plateau phase of infection, indicating a degree of immunodeficiency.