Infection of BALB/cByJ mice with the JHM strain of mouse hepatitis virus alters in vitro splenic T cell proliferation and cytokine production.

Earlier studies from this laboratory showed that infection of BALB/cByJ mice by a natural route with mouse hepatitis virus, strain JHM (MHV-JHM), results in functional splenic T cell suppression in vitro. This was evidenced by reduced concanavalin A-driven spleen cell proliferation and interleukin (IL)2 production measured after conventional intervals of cell culture (72 and 24 h, respectively). The purpose of the present work was to determine whether MHV-induced T cell dysfunction is kinetic or absolute and whether production of other T-cell derived cytokines is defective. Bioassays revealed that production of IL2, gamma interferon, and IL4 by spleen cells from acutely infected mice is suppressed and that some of the defects are kinetic as well as absolute. Proliferative responses of both CD4+ and CD8+ T cells were depressed, but neither cell type contained infectious virus. Cells that proliferated poorly in response to concanavalin A were fully capable of responding to specific virus stimulation. These results further emphasize the potential complications that MHV infection may pose to immunologic research using mice.     

Structure of the HLA-A*0204 antigen,found in South American Indians. Spatial clustering of HLA-A2 subtype polymorphism

The primary structure of the HLA-A2 subtype A^*0204 (isoelectric focusing variant A2.A) has been determined. cDNA encoding this subtype was amplified by the polymerase chain reaction. Four independent full-lenght cDNA clones encoding A^*0204 were analyzed to obtain a consensus sequence for this subtype. A^*0204 differs from A^*0201 by a single nucleotide change of G to T through the coding regions, resulting in an Arg to Met change at position 97. This substitution accounts for the isoelectric focusing pattern of the subtype. The same change occurs in other HLA-A specificities in association with other changes in its vicinity. The absence of additional substitutions in A^*0204 suggests that it could have arisen from A^*0201 by point mutation, and that recurrent mutations may take place during HLA diversification. The spatial location of this change implies that A^*0204 must be a functional variant. Comparison of its sequence with other HLA-A2 subtypes reveals that much of the HLA-A2 subtype polymorphism is generated by variations in four neighboring positions, including position 97, which are located in two adjacent -strands on the floor of the peptide binding site of the molecule.The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the accession number X57954. Address correspondence and offprint requests to: J. A. López de Castro.     

Lectin binding sites on head structures of the spermatid and spermatozoon of the mosquito Culex quinquefasciatus (Diptera,Culicidae)

The presence of intranuclear and acrosomal lectin binding sites in spermatids and spermatozoa of the mosquito Culex quinquefasciatus was analysed. Direct and indirect lectin-gold techniques were used on LR White-embedded cells. The nuclear compartment was the structure most intensely labelled. Early spermatid nucleus showed moderate labelling for peanut agglutinin (PNA), Griffonia simplicifolia IB4 (GS-IB4) and Ricinus communis agglutinin (RCA), and light labelling for the other lectins tested. The sperm nucleus was intensely labelled by all lectins. The acrosome, an enzyme-containing structure, was labelled by some lectins. The anterior acrosomal region was labelled by PNA, while the proximal acrosomal region was labelled by PNA and G. simplicifolia II (GS II) lectins, and showed the presence of fucose residues with the use of Ulex europaeus I (UEA-I) lectin. The spermatozoa stored in the spermatheca showed the same pattern of labelling as that observed in spermatozoa localized in testis and seminal vesicles for all lectins tested. Carbohydrate residues in the nuclear compartment may be involved with the process of chromatin condensation. In the acrosomal region these residues may play a role in the process of spermoocyte interaction.     

Surface spreading of synaptonemal complexes in three isopod crustacean species

The technique of whole mount spreading is used to investigate the SC of three species of Asellidae (isopod crustaceans), Asellus aquaticus, Proasellus coxalis and Proasellus meridianus, which display considerable differences in genomic DNA content.The three species, originally considered to belong to the same genus Asellus, were subsequently assigned to two separate genera: Asellus and Proasellus. The SCs of the three species differ in morphological details related to the shape of the centromere region, the attachments to the nuclear envelope, the width of the central region and the presence of twists of the lateral elements. Furthermore, they display some differences in the degree of compaction of genomic DNA in the mitotic chromosomes. The greatest differences are found between A. aquaticus and P. coxalis, while P. meridianus has several features in common with either species.     

A Study of Magnetic Properties of Magnetotactic Bacteria

The first direct measurements of magnetic properties of magnetotactic bacteria from natural samples are presented. Measurements were made at 4.2 K, using a Superconducting Quantum Interfering Device (SQUID) magnetometer. From the magnetization results an anisotropy is obtained that is typical of magnetized ferro- or ferri-magnetic materials. The average magnetic moment of the bacteria determined from the results is in good agreement with the estimated moment from electron microscopy.     

Production and response to interleukin 2 in vitro and in vivo after bone marrow transplantation in mice

Lymphoid cells from bone marrow radiation chimeras do not produce normal levels of IL 2 but are capable of responding to IL 2 in mitogenic and cytotoxic assays in vitro. The administration of recombinant human IL 2 into host mice that have received allogeneic, H-2-compatible marrow did not enhance mortality.     

Histochemical changes of substance P, FRAP, serotonin and succinic dehydrogenase in the spinal cord of rats with adjuvant arthritis

Various histochemical changes were found in spinal segments L4-L5 of rats with adjuvant arthritis, predominantly 30 days after inoculation. A slight to marked increase of substance P immunoreactivity occurred in laminae I, II and X. FRAP activity was enhanced in lamina II. Serotonin immunoreactivity was heavier in laminae I, VIII and IX in a few animals. The intensity of the histoenzymological reaction for succinic dehydrogenase increased in certain laminae VIII and X neurons. At day 15 of the disease the increase of substance P and FRAP activities was chiefly restricted to the medial portion of the superficial dorsal horn. There was a significant positive correlation between the scratching behaviour of arthritic rats and the substance P immunoreactivity in laminae X and I. If one accepts that scratching is pain-related, the data are consistent with a possible role of substance P in the chronic pain associated with adjuvant arthritis. They leave undetermined the significance of the other histochemical changes.     

Genetic mapping of Vibrio cholerae enterotoxin structural genes

The structural genes which constitute the cholera toxin operon, ctxAB, were genetically mapped in the Vibrio cholerae El Tor strain RV79. This strain of V. cholerae contains two copies of the ctx operon located on a 7-kilobase-pair tandemly duplicated region. We began by isolating a vibriophage VcA1 insertion mutation in one of the two ctxA genes located in this region. The mutant carrying this ctxA::VcA1 insertion, DC24, was converted to a VcA1-facilitated donor by introduction of the conjugal plasmid pSJ15, which carries an inserted copy of a defective VcA1-like prophage. The donor characteristics of DC24(pSJ15) indicated that the ctxA::VcA1 insertion mutation was near the trp region of the V. cholerae chromosome. Subsequent RV79 three-factor crosses were performed between VcA1-facilitated donors and recipient strains carrying one of two structural gene mutations in ctx, either delta ctxA23P Kmr or delta ctx-7922. The former was constructed by an in vivo marker exchange procedure and could be scored either by its kanamycin resistance phenotype or by its lack of DNA sequences homologous to the ctxA region. The delta ctx-7922 mutation is a total deletion of both ctx copies of strain RV79. The three-factor cross data strongly suggest that the two ctx loci of RV79 map between the nal and his genes of V. cholerae in the trp nal his linkage group. Physical analysis and heterologous crosses between an RV79 El Tor donor and a 569B classical recipient indicates that one of the two 569B ctx operon copies maps in the same region as the RV79 ctx loci (i.e., linked to nal). Together with previously published observations, these data show that the ctx structural genes are not closely linked to other genes known to affect toxin production in V. cholerae.     

Variability and conformation of HLA class I antigens: a predictive approach to the spatial arrangement of polymorphic regions

Comparison of available sequences of HLA-A and HLA-B antigens shows that variable positions are predominantly localized in four segments spanning residues 63-85, 105-116, 138-156, and 177-194. The fourth segment is unique in that it contains no differences between antigens of the same locus. Secondary folding of HLA heavy chain was estimated by three independent predictive methods and areas of defined structure were correlated with the distribution of local hydrophobicity to outline putative internal and external portions. The three analyses each independently predict a high probability for beta structure in the alpha 1, alpha 2, and alpha 3 domains. A single alpha-helix is predicted within residues 146-160, a segment of likely importance in cytotoxic T cell recognition and graft rejection. Substitutions within this segment are spatially related by the helical turn. Variable residues usually lie in areas of high local hydrophilicity, and therefore they are probably on the surface of the molecule. The model predicts that they are frequently located in beta strands, beta-turns, or the above-mentioned alpha-helix, so that most substitutions would be accommodated within rigid frameworks that may impose structural constraints to variability. The secondary structure of alpha 1, alpha 2, and alpha 3 domains presents some analogies that suggest that they might share common features in their tertiary folding. The predicted structure of alpha 3 is strongly reminiscent of that of immunoglobulin constant domains. Possible arrangements of elements of secondary structure are discussed, as an attempt to situating the polymorphic regions of HLA class I antigens in a spatial context.