Protein micelles of human histocompatibility antigens (HLA-A and HLA-B)

Human histocompatibility antigens (HLA-A and -B) are membrane proteins which have large hydrophilic domains outside the cell membrane and a small hydrophobic portion in the lipid bilayer. In this paper we describe optimal conditions for preparing micelles of detergent-solubilized HLA-A2 and -B7 antigens. These homogeneous protein aggregates are water soluble and free of detergent and lipid. Hydrophobic interactions between the intramembraneous portions of the HLA antigens are the driving forces in the formation of these protein micelles. The papain-solubilized fragment of the HLA antigens is not included in the micelle. The average molecular weight of the HLA micelles is around 9 × 10⁵ daltons, which suggests sixteen HLA-A2 and/or HLA-B7 antigenic molecules per protein aggregate. Electron microscopic studies revealed that the most frequent size of the micelles is 12 mm and that HLA-micelles are similar but not identical to micelles from Sindbis Virus glycoproteins (E1 and E2) The HLA-A2 and -B7 micelles retained full antigenic activity as judged by precipitations with allo- and heteroantisera. Such micelles will no doubt be important tools in further studies of the role of histocompatibility antigens.     

Identification of parasite proteins in a membrane preparation enriched for the surface membrane of erythrocytes infected with Plasmodium knowlesi

A subcellular fraction enriched in erythrocyte membranes has been isolated from rhesus monkey erythrocytes infected with Plasmodium knowlesi. Infected cells were lysed by centrifugation through a zone of hypotonic buffer and membranes isolated by equilibrium density gradient centrifugation in the same tube. The purified membrane fraction was shown to include the erythrocyte surface membrane by several methods: electron microscopy, identification of Coomassie Blue stained erythrocyte membrane proteins, identification of band 3 with a monoclonal antibody, and identification of radioiodinated cell surface proteins. The resulting ghosts were shown to be specifically reactive with monkey sera against the variant surface antigens of P. knowlesi by indirect immunofluorescence and membrane agglutination. No reactivity was seen with a monoclonal antibody (13C11) against the intracellular schizont surface. A number of metabolically labelled parasite proteins were enriched in this membrane function, including peptides of 277, 208, 173, 153, 134, 109, 80, 60 and 48 kDa and the variant surface antigens of variable molecular mass (180-207 kDa). These proteins were distinct from the major parasite proteins of total infected erythrocytes and isolated merozoites. The major glucosamine labelled glycoprotein of the internal schizont (230 kDa) was not found in this fraction. Moreover, no fragment of this parasite glycoprotein was found in this membrane fraction, indicating that no part of this molecule is transported to the erythrocyte surface. In contrast, the variant antigen of P. knowlesi, known to be on the erythrocyte surface, could be readily identified as peptides unique to specific cloned parasite lines. We propose that the other nine parasite proteins found within this membrane fraction represent a starting point for the identification of other parasite proteins transported to the surface membrane of the infected erythrocyte.     

Genetic analyses of gustation in the fowl

Preferences towards quinine sulfate (QS) and dextrose (DEX) were tested in purelines and reciprocal crosses of two lines of chickens that had undergone 22 generations of selection for high and low juvenile body weight. Parental line-F₁ comparisons provided evidence for non-additive genetic variation for hedonic sensitivity towards QS and DEX, though in opposite directions. Additive genetic variation appeared to influence the preference ratios for both stimuli at super-threshold concentrations. These results are discussed with regard to their evolutionary implications.     

Monoclonal antibodies to three distinct epitopes on human IgE: their use for determination of allergen-specific IgE

Three different monoclonal antibodies (MAb) against human immunoglobulin E have been obtained which specifically bind to human myeloma and polyclonal IgE. The antibodies showed high avidities for soluble IgE (0.7 X 10(9) to 3.3 X 10(9) M-1). These MAb defined three distinct epitopes on IgE. A mixture of these antibodies in combination with an 125I-labelled anti-mouse Kappa chain MAb has been used to measure allergen-specific IgE. This determination was performed by a solid-phase radioimmunoassay using allergen extracts coated to either chemically activated paper discs or to polyvinyl chloride wells. This method is 4-10 times more sensitive than other previously reported procedures. A similar technique has also been applied to detect individual allergens in immunoblots of allergen extracts.     

Chromosomal factors of infertility in candidate couples for ICSI: an equal risk of constitutional aberrations in women and men

To assess the frequency of chromosomal aberrations in French candidates for intracytoplasmic sperm injection (ICSI), and to explore the existence of a female chromosomal factor in some cases of couple infertility, a collaborative retrospective clinical and cytogenetic study was performed, launched by the Association des Cytogénéticiens de Langue Franciaise (ACLF). The karyotypes of 3208 patients [2196 men (68.4%), 1012 (31.6%) women] included in ICSI programmes over a 3-year period in France were collected. A total of 183 aberrant karyotypes was diagnosed, corresponding to an abnormality frequency of 6.1% (134/2196) for men and 4.84% (49/1012) for women. The following frequencies of abnormalities were observed respectively for men and women: 1.23% (n = 27) and 0.69% (n = 7) for reciprocal translocations, 0.82% (n = 18) and 0.69% (n = 7) for Robertsonian translocations, 0.13% (n = 3) and 0.69% (n = 7) for inversions, 3.32% (n = 73) and 2.77% (n = 28) for numerical sex chromosome aberrations, and 0.59% (n = 13) and 0% for other structural aberrations. Among the male patients of this latter group, 0.40% (n = 9) had a Y chromosome abnormality. Among the male patients with numerical sex chromosome abnormalities, 2.23% (n = 49) were 47,XXY, 0.32% (n = 7) were 47,XYY, and 0.77% (n = 17) had a mosaicism for numerical sex chromosome anomalies. All the female patients with sex chromosome abnormalities (2.77%, n = 28) had mosaicism for numerical sex chromosome anomalies. Even if these cases-the significance of which was sometimes questioned-were disregarded in the analysis, 2.08% (21/1012) of abnormal karyotypes remained in women. An overall increased frequency of chromosomal aberrations was found, and this confirmed that in some cases of poor reproductive outcome there may be a contribution of maternal chromosome aberrations. Indeed, the existence of a chromosome abnormality in the female partner was associated with the group of infertile men in which there was no apparent cause of infertility.     

Sperm plasma membrane damage prior to intracytoplasmic sperm injection: a necessary condition for sperm nucleus decondensation

In the present study we investigated the relevance of spermimmobilization prior to intracytoplasmic sperm injection (ICSI)in the fertilization process. Using supravital staining of thespermatozoa with eosin and studying sperm decondensation with2 mM dithiothreitol (DTT) in conditions imitating sperm handlingduring ICSI, we demonstrated that immobilization of the spermatozoonby squeezing its tail between the glass pipette and the bottomof the dish damages the sperm plasma membrane. Polyvinylpyrrolidone(PVP), which is usually present in the drop with the spermatozoonto facilitate its handling, was found to impede the access ofboth eosin and DTT to the sperm nucleus. We conclude that (i)sperm immobilization prior to ICSI damages the sperm plasmamembrane, that (ii) this damage is sufficient for thiol-reducingagents to gain access to the sperm nucleus, and finally that(iii) PVP possibly interferes with sperm nucleus decondensation.     

Labeling of cells with ferumoxides-protamine sulfate complexes does not inhibit function or differentiation capacity of hematopoietic or mesenchymal stem cells

Two FDA-approved agents, ferumoxides (Feridex), a suspension of superparamagnetic iron oxide (SPIO) nanoparticles and protamine sulfate, a drug used to reverse heparin anticoagulation, can be complexed and used to label cells magnetically ex vivo. Labeling stem cells with ferumoxides-protamine sulfate (FePro) complexes allows for non-invasive monitoring by MRI. However, in order for stem cell trials or therapies to be effective, this labeling technique must not inhibit the ability of cells to differentiate. In this study, we examined the effect of FePro labeling on stem cell differentiation. Viability, phenotypic expression and differential capacity of FePro labeled CD34 + hematopoietic stem cells (HSC) and mesenchymal stem cells (MSC) were compared with unlabeled control cells. Colony-forming unit (CFU) assays showed that the capacity to differentiate was equivalent for labeled and unlabeled HSC. Furthermore, labeling did not alter expression of surface phenotypic markers (CD34, CD31, CXCR4, CD20, CD3 and CD14) on HSC, as measured by flow cytometry. SDF-1-induced HSC migration and HSC differentiation to dendritic cells were also unaffected by FePro labeling. Both FePro-labeled and unlabeled MSC were cultured in chondrogenesis-inducing conditions. Alcian blue staining for proteoglycans revealed similar chondrogenic differentiation for both FePro-labeled and unlabeled cells. Furthermore, collagen X proteins, indicators of cartilage formation, were detected at similar levels in both labeled and unlabeled cell pellets. Prussian blue staining confirmed that cells in labeled pellets contained iron oxide, whereas cells in unlabeled pellets did not. It is concluded that FePro labeling does not alter the function or differentiation capacity of HSC and MSC. These data increase confidence that MRI studies of FePro-labeled HSC or MSC will provide an accurate representation of in vivo trafficking of unlabeled cells.     

Dependence of the anterior olfactory area self-stimulation upon the lateral hypothalamic area

The functional relation between the anterior olfactory area (AO) and the lateral hypothalamic area (LH) was examined in a self-stimulation situation. Bar-pressing responses for AO sitmulation were suppressed by unilateral injection of procaine, and enhanced by glutamate, into LH. Neither procaine nor glutamate injected into AO had any influence upon LH self-stimulation. It is unlikely that the procaine effect was due to motor disturbance because similar injection of procaine into LH did not disturb the performance of a one-way avoidance task. It appears that the rewarding effect of AO stimulation is dependent upon the excitation of the more caudal structures including LH.