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.     

DNA typing of HLA-DR β chain genes can discriminate between undetected alleles and real homozygotes

The polymorphism of HLA-DR antigens has been studied by Southern blot hybridization under conditions specific for the detection of the DR chain genes. Haplotype-specific patterns were defined with DNA from DR1, 2, 3, 4, 7, w8, w11, w12, and W13 homozygous typing cells, with restriction enzymes Eco RI, Bgl I, and Pvu II. Certain serological specificities, such as DR2, DR3, and DR7, can be encoded by distinct allelic forms of DR chain genes. The procedure of DNA typing was applied to family analysis of individuals expressing only a single DR specificity upon serological typing. Three cases are described here: (1) in family GR, phenotypic DR 7 homozygotes correspond to genomic heterozygotes, and a novel DR7 allele is described: (2) in family RU, the genes corresponding to a serologically undetected (blank) DR allele were identified by restriction fragment length polymorphism (RFLP); this novel DR haplotype has an RFLP pattern similar to those of the DRw52 family, even though this specificity was not expressed on the DR-blank lymphocytes; (3) in family RG, there is no blank allele, but a homozygote RFLP situation at the DR subregion.     

Nasal allergy to fungi in guinea pigs

Sensitized guinea pigs produced specific IgG and IgE antibodies toward Cladosporium and Alternaria. In presence of fungal extracts, nasal mast cells degranulate. Ultrastructural modifications of the cells during degranulation have been established. The ciliary epithelium and the ciliary beating are not affected by fungal allergens.     

Effects of castration and testosterone treatment on the activity of testosterone-metabolizing enzymes in the brain of male and female zebra finches

Recently, we described the distribution of testosterone-metabolizing enzymes (i.e., aromatase, 5 alpha- and 5 beta-reductases) in the zebra finch (Taeniopygia guttata) brain using a sensitive radioenzyme assay combined to the Palkovits punch method. A number of sex-differences in the activity of these enzymes were observed especially in nuclei of the song-control system. The hormonal controls of these differences have now been analyzed by gonadectomizing birds of both sexes and by giving them a replacement therapy with silastic implants of testosterone (T). Five nuclei of the song system (Area X [X], nucleus magnocellularis of the anterior neostriatum [MAN], nucleus robustus archistriatalis [RA], nucleus intercollicularis [ICo], hyperstriatum ventrale, pars caudalis [HVc]) and three preoptic-hypothalamic areas (preoptic anterior [POA], periventricular magnocellular nucleus [PVM], and posterior medial hypothalamic nucleus [PMH]) were studied as well as other limbic and control non-steroid-sensitive areas. The activity of the 5 alpha-reductase was higher in males than in females for the five song-control nuclei and was not affected by the hormonal treatments. The overall activity of this enzyme was not sexually dimorphic in POA and PVM. It was higher in males than in females in intact birds only, and was reduced by gonadectomy and enhanced by T. The activity of the 5 beta-reductase was higher in females than in males in all nuclei of the song system and in POA, but was not influenced by the changes in T level. Both sex and treatment effects were observed in the control of aromatase. The production of estrogens was dimorphic (females greater than males) in RA and PMH. It was increased by T in POA, PVM, and PMH, and also in RA. These data show that some of the sex differences in T-metabolizing enzymes result from the exposure to different levels of T in adulthood (e.g., 5 alpha-reductase in POA and PVM or aromatase in PVM), whereas others persist even if birds are exposed to the same hormonal conditions. These are presumably the result of organizational effects of steroids. The steroid modulation of the aromatase might be related directly to the activation of sexual, aggressive, and nest-building behaviors, whereas the stable dimorphism in 5 alpha- and 5 beta-reductase observed in the nuclei of the song system might be one of the neurochemical bases of the sex differences in the vocal behavior of the zebra finch.     

Inhibition of DNA-ethidium bromide intercalation due to free radical attack upon DNA

Summary The fluorescent intercalation complex of ethidium bromide (ETB) with DNA was used as a probe to compare the effects of various radicals with respect to impairment of the DNA base-pair region.OH radicals inhibit up to 0.7 dye intercalations perOH at low salt concentration, and for various oxidizing species the effect decreases in the orderOH > Br ₂ ^– > N ₃ > $$ " align="middle" border="0"> (SCN) ₂ ^–. DNA impairment by theOH product of Met-Gly is comparable to that of N ₃ , but no effect was found due to the interaction between DNA and Lys-Tyr-Lys phenoxyl radicals. The reducing speciese _aq ^– , H, O ₂ ^–, and CO ₂ ^– hardly affect the DNA-ETB intercalation.     

Stem cells from peripheral blood and bone marrow: a comparative evaluation of the hemopoietic potential in the dog

The kinetics and pattern of hemopoietic recovery after supralethal total-body irradiation (TBI) were compared after transfusion of cryopreserved autografts derived from peripheral blood and bone marrow. Fractionated TBI was given in three doses of 6 Gy each at intervals of 48 h. Grafts of peripheral blood mononuclear cells (MNC) were collected by means of continuous-flow centrifugation and by using the mobilizing agent, dextran sulphate. Autografts were adjusted to contain equal numbers of committed progenitor cells (CFU-GM). Dogs grafted with blood-derived MNC (group A) and with MNC from bone marrow (group B) all received about 1 X 10(5) CFU-GM per kg body weight. In all dogs consistent hemopoietic engraftment was achieved. Comparing the pattern of regeneration of the granulocytes, group A dogs showed a significant regeneratory advantage over group B dogs, particularly during the first 20 days after transplantation. Lymphoid recovery was more rapid in group A until day 14. In both groups, blood lymphocytes remained below normal values beyond day 100. The regeneration patterns of the platelets and reticulocytes revealed no significant differences. These results are in agreement with the hypothesis that there are differences in the relationship between CFU-GM content and hemopoietic potential of autografts from different sources.