Evaluation of the human sperm acrosome reaction using a monoclonal antibody, GB24, and fluorescence-activated cell sorter

GB24 is a mouse monoclonal antibody raised against human trophoblast microvilli, which recognizes an antigenic determinant on the acrosomal region of the human sperm head. By indirect immunofluorescence, reactivity of GB24 could not be detected on freshly ejaculated spermatozoa but was strongly positive after sperm permeabilization with acetone. On viable, motile spermatozoa, reactivity appeared after induction of the acrosomal reaction with the calcium ionophore A23187. These results suggest that the antigen recognized by GB24 is present on the inner acrosomal membrane. A quantitative evaluation assay of the acrosome reaction on viable spermatozoa by flow cytometry using GB24 and indirect immunofluorescence is proposed.     

Lipoproteins: carriers of dehydroepiandrosterone fatty acid esters in human serum

The association between lipoproteins, cholesterol and cholesteryl esters is very well known to facilitate both the transport in plasma and the entry of these non-polar compounds into the cellular compartment. However, recent observations suggest that in addition to cholesterol, lipoproteins contain several other steroids in their lipoidal metabolite forms which may be transported in the very low, low and high density lipoproteins in human serum. Using the important androgen and oestrogen precursor, dehydroepiandrosterone (DHEA), the biosynthetic formation of lipoidal DHEA was demonstrated in human serum. Serum was also fractionated into its lipoprotein components during the course of its incubation with tritiated DHEA. A progressive movement of the label from the fraction containing the conventional steroid binding-proteins to the lipoproteins was observed with the fraction containing the low density lipoproteins demonstrating the greatest incorporation of the label. This displacement occurred simultaneous to an extensive esterification of the labelled DHEA in serum. After 6 h of incubation, approx. 90% of the radioactivity in all the lipoprotein fractions was in the lipoidal form. Very little labelled lipoidal DHEA was associated with the serum protein fraction throughout the duration of incubation. These data suggest that lipoproteins act as the carriers of lipoidal DHEA following its formation from the non-conjugate parent steroid in serum.     

Two nuclear oncogenic proteins, P135gag-myb-ets and p61/63myc, cooperate to induce transformation of chicken neuroretina cells

Several studies have shown that full transformation of primary rodent fibroblasts can be achieved in vitro through the cooperation of two oncogenes (usually one nuclear and one cytoplasmic) classified on the basis of different complementation groups. We have shown previously that cooperation between v-mil (cytoplasmic, serine-threonine kinase product), and v-myc (nuclear, DNA-binding product) is required to transform 7-day-old chicken neuroretina cells, which in usual culture medium do not rapidly proliferate. v-mil induces sustained growth of chicken neuroretina cells without transformation; v-myc fails to stimulate the proliferation of chicken neuroretina cells but is required to achieve transformation of the proliferating cells. Here, we present results indicating that the P135gag-myb-ets nuclear protein of avian erythroblastosis virus E26 is able to induce proliferation but not transformation of chicken neuroretina cells. v-myc is required in addition to P135gag-myb-ets to achieve chicken neuroretina cell transformation. In contrast, we found that the P135gag-myb-ets and P100gag-mil proteins are not able to cooperate in this system.     

Phenotypic hiding: the carryover of mutations in RNA viruses as shown by detection of mar mutants in influenza virus.

When influenza virus monoclonal antibody-resistant (mar) mutants are selected by incubation in vitro with excess antibody, 90 to 99% of the mutants are not detectable. This observation may be explained by encapsidation of mar mutant RNAs within phenotypically wild-type envelopes. This phenotypic hiding can be revealed by selection of mar mutants in vivo after virus uncoating. Using experimental procedures appropriate to detect all viable mar mutants in a virus population, we determined precisely the mutation rates to the mar genotype by the fluctuation test for two nonoverlapping monoclonal antibodies.     

Different pituitary beta-endorphin and adrenal cortisol response to ethanol in individuals with high and low risk for future development of alcoholism

The purpose of the present studies was to investigate the activity of the adrenal gland and the pituitary beta-endorphin system in individuals from families with a 3 generation history of alcoholism, High Risk group, or from families without history of alcoholism, Low Risk group. All subjects had a medical examination, a drinking behavior personal interview and the Michigan Alcoholism Screening Test. Individuals with medical problems or excessive drinking were not included in the study. On the day of testing, a blood sample was taken at 9:00 a.m., then the subject drank a placebo drink or an ethanol solution (0.5 g ethanol/kg B.Wt.). Additional blood samples were taken at 15, 45 and 120 minutes post-drink. Results indicated that individuals of the High Risk group had lower basal levels of beta-endorphin like immunoreactivity (beta-EPLIR) than individuals of the Low Risk group. The dose of 0.5 g ethanol/kg B.Wt. induced an increase in the plasma content of beta-EPLIR of the High Risk group, but not of the Low Risk group. In the Low Risk group ethanol did not induce an increase above the 9:00 a.m. levels, however, it attenuated the beta-endorphin decrease overtime, observed following the placebo drink. Analysis of beta-endorphin-like peptides in the plasma of the High Risk group, with Sephadex G-75 chromatography indicated that the major component of the plasma beta-EPLIR was beta-lipotropin. Plasma cortisol levels, following ethanol intake, presented a small increase in the High Risk group but not in the Low Risk group. Both groups presented similar blood alcohol levels. The basal levels of immunoreactive cortisol and beta-endorphin in the plasma of individuals who were alcoholics, but had been abstinent for at least six months prior to testing were similar to the levels of the High Risk group. Thus there are differences both in the basal levels and in the response of the cortisol and the pituitary beta-endorphin system to an acute ethanol challenge between the two groups.     

Melanin concentrating hormone. V. Isolation and characterization of alpha-melanocyte-stimulating hormone from frog pituitary glands

The structure of alpha-melanocyte-stimulating hormone (alpha-MSH) has been determined in the pars intermedia of the frog Rana ridibunda. Pulse-chase labeling of frog neurointermediate lobes with selective amino acids revealed that the composition of frog alpha-MSH is similar to that of alpha-MSH from all mammalian species yet studied. Tryptic mapping of nexly synthetized alpha-MSH generated two fragments with the following amino acid composition: (T1) Trp, Pro, Lys, Gly, Val and (T2) Tyr, Arg, Phe, His, Ser, Glu. Concurrently, alpha-MSH was purified from 100 neurointermediate lobes to apparent homogeneity by reverse-phase HPLC. The sequence of the peptide determined by automated Edman degradation was Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val. The structure of frog alpha-MSH is thus identical to mammalian des-N alpha-acetyl alpha-MSH and differs from the sequence of toad (Xenopus laevis) alpha-MSH only by the first residue (Ser instead of Ala). These results confirm that the sequence of alpha-MSH has been highly preserved during evolution.     

Prenatal diagnosis of Niemann-Pick type C disease: current strategy from an experience of 37 pregnancies at risk.

Thirty-seven pregnancies at risk for Niemann-Pick type C disease were monitored by study of cultured amniotic fluid cells (8 cases) or chorionic villus cells (29 cases) in 23 couples over the period 1984-91. An early protocol combined determination of sphingomyelinase activity with electron microscopy. The current strategy, based on the demonstration of specific abnormalities in intracellular processing of exogenous cholesterol, combines the study of the early phase (first 6 h) of LDL-induced cholesteryl ester formation and the histochemical evaluation (filipin staining after 24 h of LDL uptake) of the LDL-induced accumulation of unesterified cholesterol. Thirteen fetuses were predicted to be affected. Confirmation of the diagnosis was made by study of cholesterol processing in fetal skin fibroblast cultures and/or by demonstration of a characteristic lipid storage in fetal liver, already present at 14 w gestation. Definition of the biochemical phenotype (classical, variant, or intermediate) of the index case, with regard to cholesterol-processing abnormalities, is an absolute prerequisite to adequate genetic counseling in a given family. Prenatal diagnosis has now proved a safe procedure in the predominant (approximately 85%) group of families with the classical phenotype.     

Fine needle aspiration of peripheral neuroepithelioma of soft tissues.

Peripheral neuroepithelioma of soft tissue is a malignant primitive neuroectodermal tumor that appears both in children and adults and usually has a poor outcome. Fine needle aspiration on two patients with tumors in the lower limbs showed small round cells with unipolar processes and a tendency to form Homer-Wright rosettes. The cells had a round to oval nucleus with fine chromatin, up to four small, conspicuous nucleoli and vacuolated, periodic acid-Schiff-positive cytoplasm. The diagnosis was supported by electron microscopic study of the aspirate, which showed features of neuroblastic differentiation (i.e., neurosecretory granules), and by histologic, immunohistochemical and cytogenetic study of the resected tumors.     

Purification and characterization of glutathione reductase from Rhodospirillum rubrum.

Glutathione reductase (NAD(P)H:GSSG oxidoreductase EC 1.6.4.2.) was purified 1160-fold to homogeneity from the nonsulfurous purple bacteria Rhodospirillum rubrum (wild type). Specific activity of the pure preparation was 102 U/mg. The enzyme displayed a typical flavoprotein absorption spectrum with maxima at 274,365, and 459 nm and an absorbance ratio A280/A459 of 7.6. The amino acid analysis revealed an unusually high content of glycine and arginine residues. Titration of the enzyme with 5,5'-dithiobis(2-nitrobenzoic acid) showed a total of two free thiol groups per subunit, one of which is made accessible only under denaturing conditions. An isoelectric point of 5.2 was found for the native enzyme. Km values, determined at pH 7.5, were 6.1 and 90 microM for NADPH and GSSG, respectively. NADH was about 2% as active as NADPH as an electron donor. The enzyme's second choice in disulfide substrate was the mixed disulfide of coenzyme A and glutathione, for which the specific activity and Km values were 5.1 U/mg and 3.4 mM, respectively. A native molecular weight of 118,000 was found, while denaturing electrophoresis gave a value of 54,400 per subunit, thus suggesting that R. rubrum glutathione reductase exists as a dimeric protein. Other physicochemical constants of the enzyme, such as Stokes radius (4.2 nm) and sedimentation coefficient (5.71 S), were also consistent with a particle of 110,000.