Polarized distribution of binding sites for concanavalin A and wheat-germ agglutinin in the zona pellucida of goodeid oocytes (teleostei)

Summary Zonae pellucidae of the viviparous goodeid teleosts Girardinichthys viviparus, Xenoophorus captivus, and Xenotoca eiseni were investigated ultrastructurally, and binding sites for ConA and WGA were localized on cross-sections using a colloidal gold technique. In late stages of development, the oocytes are surrounded by a three-zonated acellular matrix multiply perforated by pore canals allowing long microvilli of the oocyte to penetrate interstices of the follicle epithelium. Together, the surface of the microvilli and zona pellucida is coated by a thin layer of homogeneous slightly electron-dense material. In early oogenesis, the thin acellular layer is entirely packed with binding sites for WGA, whereas those for ConA occur only sparsely. Three-zonated zonae pellucidae amply contain both WGA and ConA receptors. The asymmetric labelling pattern obtained with both lectin protein gold preparations indicates a polarized organization of the different glycoconjugates. WGA receptors are concentrated within the outer region of the zona pellucida. Labelling with ConA-HRP-Au complexes produced heavy deposits of marker beads within the inner two thirds of the zona pellucida and weak labelling of the superficial coat. After prolonged digestion with neuraminidase, WGA binding sites were no longer detectable.Supported by the Deutsche Forschungsgemeinschaft (Schi 268/1-1)     

Ultrastructural localization of dentine phosphoprotein in rat tooth germs by immunogold staining

Summary Dentine phosphoprotein (DPP) was localized on thin frozen sections of fixed rat tooth germs by indirect immunogold staining. Antisera were directed against DPP and against glutaraldehyde-treated DPP and were characterized by immuno-electroblotting. In odontoblasts, DPP was found to be localized in the cisternae of the rough endoplasmic reticulum (RER) and the Golgi apparatus and in Golgi-associated vesicles. Odontoblastic processes were moderately positive for DPP and dentine was intensely labeled on frozen sections of unfixed tissue. Predentine showed a slight immunoreactivity. These results indicate the synthesis of DPP in the RER, its accumulation in the Golgi apparatus and its vesicular transport and secretion via the odontoblastic processes into dentine. The close association of the gold particles with the dentinal collagen fibres makes a role of DPP in linking mineral to collagen conceivable. Matrix vesicles were negative for DPP, suggesting that the protein is not present at the sites of matrix vesicleassociated nucleation.     

Declining incidence of AIDS dementia complex after introduction of zidovudine treatment.

OBJECTIVE--To assess the incidence of the AIDS dementia complex and the presence of HIV I p24 antigen in cerebrospinal fluid in relation to zidovudine treatment. DESIGN--Retrospective study of a consecutive series of patients with AIDS from 1982 to 1988. SETTING--An academic centre for AIDS. PATIENTS--196 Patients with AIDS and neurological symptoms examined from 1982 to 1988. INTERVENTIONS--Zidovudine treatment, which was introduced to The Netherlands on 1 May 1987 for patients with severe symptoms of HIV infection (Centers for Disease Control groups IVA, B, C, and D). MAIN OUTCOME MEASURES--Diagnosis of AIDS dementia complex and presence of HIV I p24 antigen in cerebrospinal fluid. RESULTS--The AIDS dementia complex was diagnosed in 40 of the 196 (20%) patients with AIDS. Thirty eight of 107 patients with AIDS (36%) not taking zidovudine developed the AIDS dementia complex compared with two of the 89 (2%) taking the drug (p less than 0.00001). The incidence of the AIDS dementia complex increased to 53% in the first half of 1987, after the introduction of zidovudine in May 1987, decreasing to 10% in the second half of 1987 and to 3% in 1988. Dementia was diagnosed before definition of the AIDS dementia complex (1986) according to DSM-III criteria and there was good agreement between diagnosis before and after 1986. Sixteen of 61 samples of cerebrospinal fluid (26%) from patients with AIDS (10 with the AIDS dementia complex) not taking zidovudine were positive for HIV I p24 antigen, whereas none of 37 cerebrospinal fluid samples from patients with AIDS (two with the AIDS dementia complex) taking zidovudine were positive. CONCLUSIONS--The incidence of AIDS dementia complex in patients with AIDS declined after the introduction of systematic treatment with zidovudine; the AIDS dementia complex might be prevented by inhibiting viral replication in the central nervous system.     

Treatment of amiodarone induced hyperthyroidism with potassium perchlorate and methimazole during amiodarone treatment.

To exploit the antiarrhythmic effect of amiodarone when patients develop the side effect of thyrotoxicosis three patients with hyperthyroidism induced by amiodarone were given simultaneously 1 g potassium perchlorate a day for 40 days and a starting dose of 40 mg methimazole a day while they continued to take amiodarone. As hyperthyroidism might have recurred after potassium perchlorate treatment was stopped the dose of methimazole was not reduced until biochemical hypothyroidism (raised thyroid stimulating hormone concentrations) was achieved. The patients became euthyroid (free triiodothyronine concentration returned to normal values) in two to five weeks and hypothyroid in 10 to 14 weeks. One patient became euthyroid while taking 5 mg methimazole a day and 600 mg amiodarone weekly; the two others required substitution treatment with thyroxine sodium while taking 5 mg methimazole or 50 mg propylthiouracil (because of an allergic reaction to methimazole) and 2100 or 1400 mg amiodarone weekly. Hyperthyroidism induced by amiodarone may be treated with potassium perchlorate and methimazole given simultaneously while treatment with amiodarone is continued.     

Evolution of eye lens crystallins: the stress connection

Crystallins, the structural proteins of the eye lens, ensure the transparency and integrity of the lens throughout life. Recent sequence comparisons have shown that evolution has recruited crystallins among already existing heat-shock proteins and stress-inducible enzymes.     

Structure and diversity of HLA-B27-specific T cell epitopes. Analysis with site-directed mutants mimicking HLA-B27 subtype polymorphism.

HLA-B27 subtype polymorphism is amenable to differential recognition by CTL. Site-directed mutagenesis was used to construct a series of HLA-B27 mutants reproducing most of the changes occurring in the natural subtypes. The reactivity of 21 anti-HLA-B27 CTL clones was examined with these mutants to address three issues concerning the alloreactive response against HLA-B27: 1) diversity of clonotypic specificities, 2) structural features of the epitopes recognized by these clones, and 3) role of individual positions in the differential recognition of HLA-B27 subtypes. Virtually all CTL clones displayed unique reaction patterns with the mutants, indicating a corresponding diversity of epitopes. However, these share some molecular features, such as certain amino acid residues and related locations. Individual mutations induced complex effects on multiple B27-specific CTL epitopes, revealing some of their very precise stereochemical constrains. An important feature of HLA-B27 subtype polymorphism is that every individual change was relevant, altering recognition by many CTL clones. Although the specific set affected by each mutation was partially different, the global number of clones affected by most changes was very similar. This suggests that the antigenic profile of any given subtype is not dominated by one particular change but is uniquely defined by its corresponding set of changes. An exception was the change at position 152, which totally abrogated recognition by all 20 anti-B*2705 CTL clones. This effect decisively influences the profound differences in T cell recognition between B*2705 and the two subtypes, B*2704 and B*2706, carrying this change. The results are compatible with the idea that HLA-B27 allorecognition may involve multiple peptides bound to the alloantigen on the cell surface.     

Molecular mapping of interactions between a Mycobacterium leprae-specific T cell epitope, the restricting HLA-DR2 molecule, and two specific T cell receptors.

A systematic series of 89 single residue substitution analogs of the Mycobacterium leprae 65-kDa protein-derived peptide LQAAPALDKL were tested for stimulation of two HLA-DR2 restricted 65 kDa-reactive T cell clones from a tuberculoid leprosy patient. Some analogs with substitutions outside a "core" region showed enhanced stimulation of the T cell clones. This core region of seven or eight residues was essential for recognition, whereas substitution of amino acids outside this region did not affect T cell recognition although these residues could not be omitted. Thus these core residues interact directly with the presenting HLA-DR2 molecule and/or the TCR. Except for analogs of position 419 for clone 2B6, the majority of the nonstimulatory substitution analogs did not inhibit the presentation of LQAAPALDKL and thus probably failed to bind to the HLA-DR2 molecule. Unless all of the core residues are physically involved in binding to DR2, substitution at a position not directly involved in binding appears to have an influence on other residues that do bind to the DR2 molecule. Active peptide analogs with two or more internal prolines suggest that not all analogs need be helical for activity with clone 2F10.     

Effect of protease inhibitors on human monocyte IgG Fc receptor II. Evidence that serine esterase activity is essential for Fc gamma RII-mediated binding

We have shown previously that certain proteases can modulate the affinity of human Fc gamma RII for IgG. To study whether proteolytic events not only increase FcR affinity, but are essential for Fc gamma R functioning, we evaluated the effect of different protease inhibitors on binding mediated by two classes of human monocyte IgG FcR. These R, Fc gamma RI and Fc gamma RII, can be analyzed selectively in rosetting assays by employing E sensitized by either human IgG or mouse IgG1. Rosetting by both classes of R was inhibited profoundly by incubation of monocytes with different types of serine protease inhibitors such as diisopropylfluorophosphate, PMSF, or N alpha-tosyl-L-lysyl-chloromethylketone. The type II Fc gamma R was much more sensitive to inhibition than Fc gamma RI. We, therefore, studied these effects in more detail by using cell line K562, which expresses only Fc gamma RII. PMSF, diisopropylfluorophosphate, and N alpha-tosyl-L-lysyl-chloromethylketone were, again, inhibiting Fc gamma RII-mediated binding dose-dependently, whereas several inhibitors of metal, aspartic, or thiol proteases proved ineffective. Furthermore, Fc gamma RII-mediated rosetting on both cell types was profoundly inhibited by the addition of different small synthetic substrates of serine esterases. In an attempt to discriminate whether the proteolytic event is an intra- or extracellular process, macromolecular antiproteases such as soybean or ovomucoid trypsin inhibitor or alpha 1-antiprotease were tested. Fc gamma RII-mediated binding by K562 cells was not susceptible to macromolecular antiproteases, in contrast to monocytes. In the presence of drugs which interfere both with receptor recycling and intracellular traffic between endosomal compartments (e.g., primaquine or monensin), the effects of inhibitors were largely abrogated. This showed that endocytosis of inhibitors might be essential, indicating the proteolytic event to be intracellular. Our findings suggest that human monocyte Fc gamma RII-mediated functioning is dependent upon the action of one or more serine proteases.     

5S rRNA sequences of representatives of the genera Chlorobium, Prosthecochloris, Thermomicrobium, Cytophaga, Flavobacterium, Flexibacter and Saprospira and a discussion of the evolution of eubacteria in general.

5S rRNA sequences were determined for the green sulphur bacteria Chlorobium limicola, Chlorobium phaeobacteroides and Prosthecochloris aestuarii, for Thermomicrobium roseum, which is a relative of the green non-sulphur bacteria, and for Cytophaga aquatilis, Cytophaga heparina, Cytophaga johnsonae, Flavobacterium breve, Flexibacter sp. and Saprospira grandis, organisms allotted to the phylum 'Bacteroides-Cytophaga-Flavobacterium' and relatives as determined by 16S rRNA analyses. By using a clustering algorithm a dendrogram was constructed from these sequences and from all other known eubacterial 5S RNA sequences. The dendrogram showed differences, as well as similarities, with respect to results obtained by 16S RNA analyses. The 5S RNA sequences of green sulphur bacteria were closely related to one another, and to a cluster containing 5S RNA sequences from Bacteroides and its relatives, including Cytophaga aquatilis. 5S RNA sequences of all other representatives of the 'Bacteroides-Cytophaga-Flavobacterium' phylum as distinguished by 16S RNA analysis failed to group with Bacteroides and related clusters. On the basis of 5S RNA sequences, Thermomicrobium roseum clustered with Chloroflexus aurantiacus, as was expected from 16S RNA analysis.