Characterization of a monoclonal antibody to human proacrosin and its use in acrosomal status evaluation.

Among the monoclonal antibodies (MAb) selected after immunization of mice with a detergent-insoluble fraction from human spermatozoa, MAb 4D4 was found to stain in immunofluorescence the principal part of the acrosome of human spermatozoa. Acrosome reaction induced decreased and spotty 4D4 immunofluorescence staining. Immunoelectron microscopy before or after embedding revealed that the epitope defined by MAb 4D4 was sequestered in the anterior acrosomal matrix and, after the acrosome reaction, remained partly bound on matrix elements attached to the inner acrosomal membrane. Western blot analysis of sperm extracts showed that the epitope defined by MAb 4D4 was located on a 55 KD polypeptide in whole cells and on 55 and 50 KD polypeptides in non-ionic detergent fractions. Human proacrosin-enriched fraction obtained by FPLC purification exhibited several proteolytic activities against gelatin in gel enzymography: a 50 KD major band and two minor bands in the 20-30 KD area; the 50 KD polypeptide reacted with MAb 4D4 in Western blots. Furthermore, the 4D4-immunoprecipitated polypeptide from sperm extract showed that the 50 KD band exhibited proteolytic activity with an optimal pH at 8.0 that was strongly inhibited by soybean trypsin inhibitor and ZnCl2. MAb 4D4 also reacted with the acrosome of the monkey Macaca fascicularis but not with the acrosome of any of the other non-primate mammalian species examined so far. Various shape defects of the acrosomal principal region were revealed by 4D4 labeling of spermatozoa with head anomalies from infertile patients. MAb 4D4 also recognized proacrosin in paraffin-embedded human testis sections. These data make the monoclonal antiproacrosin antibody 4D4 an efficient tool for evaluation of the acrosomal status of human spermatozoa and spermatids.     

Molecular cloning and expression of human trophoblast antigen FDO161G and its identification as 3 beta-hydroxy-5-ene steroid dehydrogenase

The monoclonal antibody FDO161G reacts with a 43-kDa protein found in human extravillous trophoblast, syncytiotrophoblast, adrenal cortex, interstitial cells of the testis and ovarian follicle cumulus cells. cDNAs for this protein have been isolated from the lambda gt11 library, sequenced, and expressed in COS-7 cells. The protein was identified as 3 beta-hydroxy-5-ene steroid dehydrogenase (HSD). The sequence of the HSD protein raises questions about its association with cell membrane systems. The lack of reactivity of FDO161G with other tissues suggests that HSD has a limited tissue distribution and that other enzymes may exist in peripheral tissues, which can convert delta 5 3-hydroxysteroids to delta 4 3-ketosteroids.     

Measurement of extracellular space in the rabbit AV node

We used quantitative histochemistry to measure the size of the extracellular space (ESC) in various regions of the rabbit heart. When inulin, sucrose, and sorbitol were used as ECS markers, the ECS of the AV-nodal tissue was found to be, respectively, 2.4, 2.2, and 2.5 times larger than that of left ventricular muscle. Glucose was also measured over a 50-fold serum concentration range as an extracellular marker for AV-nodal tissue, left ventricular muscle, and Purkinje fibers. Measurements with glucose also revealed that the ECS of the AV node was 2.5-2.8 times larger than that of ventricular muscle. In contrast, the ECS of the AV node was the same as that of Purkinje fibers when glucose was used as an extracellular marker. ATP content, measured as an intracellular marker, was similar in both AV-nodal and contractile tissue. Collectively, the data obtained with all extracellular markers indicate that the ECS of the AV-nodal region is approximately 2.5 times larger than that of adjacent contractile tissue. Differences in the size of the ECS in various regions of the heart probably have functional significance and should be considered appropriately during the interpretation of data obtained by biochemical and densitometric approaches.     

Chitin biosynthesis during pupal development of Drosophila melanogaster and the effect of the lethalcryptocephal mutation

Estimation of chitin deposition in the pupal and adult cuticles of adult Drosophila melanogaster during the pupal period is described. The timing of the periods of chitin deposition is compared with that deduced by previous workers using electron microscopy. The hypothesis that lethalcryptocephal mutant homozygotes are unable to evert their cephalic complexes at pupation because of excess chitin deposition is examined. The data obtained show no evidence that the mutation has any effect on chitin deposition.     

Current aspects of the short-term prognosis of meningococcal infection morbidity for the sake of epidemiological surveillance

The short-term prognosis of the epidemic situation requires the establishment of the system of constant surveillance, which takes into account the data on morbidity rates for several years, both total and in various age groups, the data on the morbidity level and its changes, induced by meningococci of the epidemic group, in the IV quarter of the year, the data on the level of carriership among the indicator groups of the population in this period, as well as the data on changes in antibody titers in the sera of donor blood samples taken in summer and in December.     

Apical Scaffolding Protein NHERF2 Modulates the Localization of Alternatively Spliced Plasma Membrane Ca2+ Pump 2B Variants in Polarized Epithelial Cells

The membrane localization of the plasma membrane Ca²⁺-ATPase isoform 2 (PMCA2) in polarized cells is determined by alternative splicing; the PMCA2w/b splice variant shows apical localization, whereas the PMCA2z/b and PMCA2x/b variants are mostly basolateral. We previously reported that PMCA2b interacts with the PDZ protein Na⁺/H⁺ exchanger regulatory factor 2 (NHERF2), but the role of this interaction for the specific membrane localization of PMCA2 is not known. Here we show that co-expression of NHERF2 greatly enhanced the apical localization of GFP-tagged PMCA2w/b in polarized Madin-Darby canine kidney cells. GFP-PMCA2z/b was also redirected to the apical membrane by NHERF2, whereas GFP-PMCA2x/b remained exclusively basolateral. In the presence of NHERF2, GFP-PMCA2w/b co-localized with the actin-binding protein ezrin even after disruption of the actin cytoskeleton by cytochalasin D or latrunculin B. Surface biotinylation and fluorescence recovery after photobleaching experiments demonstrated that NHERF2-mediated anchorage to the actin cytoskeleton reduced internalization and lateral mobility of the pump. Our results show that the specific interaction with NHERF2 enhances the apical concentration of PMCA2w/b by anchoring the pump to the apical membrane cytoskeleton. The data also suggest that the x/b splice form of PMCA2 contains a dominant lateral targeting signal, whereas the targeting and localization of the z/b form are more flexible and not fully determined by intrinsic sequence features.     

Radioprotection après injection thérapeutique de [131I]-mIBG : données préalables à l’ouverture du protocole MIITOP-0607

IntroductionThe MIITOP-0607 protocol, studying the efficiency of administration of topotecan and myelosupressive [¹³¹I]-mIBG therapy in children affected by neuroblastoma, needed to assess irradiation risks on staff and family of children to obtain the agreement of the Autorité de sûreté nucléaire (ASN). Our aim was to quantify irradiation of the staff during preparation of the mIBG and to assay the irradiation and contamination of the accompanying persons.Patient and methodsRadiation exposure of the staff was measured during the preparation, transport and administration of the first treatment. Salivary and urinary excretions were monitored well as the atmospheric radioactivity. Radiation exposure and contamination of the accompanying persons were also measured.ResultsFinger dose of 3 mSv and whole body dose of 50 μSv were estimated for preparation of an 11.1 GBq syringe. Irradiation from urinary activity can be as low as 100 μSv if a dedicated device is used. Salivary excretion decreased rapidly during the first 24 hours. Atmospheric contamination always remained below 25 Bq m^?3. Total irradiation of the accompanying persons is about 2.35 mSv for the two consecutive injections (9,3 and 11,1 GBq). Internal contamination occurred only once and corresponded to a 27 μSv whole body irradiation and 670 μSv thyroid irradiation.ConclusionThis study shows the safety of [¹³¹I]-mIBG treatments using high activities. The involved dose is not negligible but seems to be acceptable in the specific paediatric oncology context if radioprotection instructions are met and if optimization of protocols is performed.