T Cell receptor clonotype influences epitope hierarchy in the CD8+ T cell response to respiratory syncytial virus infection

CD8+ T cell responses are important for recognizing and resolving viral infections. To better understand the selection and hierarchy of virus-specific T cell responses, we compared the T cell receptor (TCR) clonotype in parent and hybrid strains of respiratory syncytial virus-infected mice. K(d)M2(82-90) (SYIGSINNI) in BALB/c and D(b)M(187-195) (NAITNAKII) in C57Bl/6 are both dominant epitopes in parent strains but assume a distinct hierarchy, with K(d)M2(82-90) dominant to D(b)M(187-195) in hybrid CB6F1/J mice. The dominant K(d)M2(82-90) response is relatively public and is restricted primarily to the highly prevalent Vβ13.2 in BALB/c and hybrid mice, whereas D(b)M(187-195) responses in C57BL/6 mice are relatively private and involve multiple Vβ subtypes, some of which are lost in hybrids. A significant frequency of TCR CDR3 sequences in the D(b)M(187-195) response have a distinct "(D/E)WG" motif formed by a limited number of recombination strategies. Modeling of the dominant epitope suggested a flat, featureless structure, but D(b)M(187-195) showed a distinctive structure formed by Lys(7). The data suggest that common recombination events in prevalent Vβ genes may provide a numerical advantage in the T cell response and that distinct epitope structures may impose more limited options for successful TCR selection. Defining how epitope structure is interpreted to inform T cell function will improve the design of future gene-based vaccines.     

GALNT11 as a new molecular marker in chronic lymphocytic leukemia

Aberrant mucin O-glycosylation often occurs in different cancers and is characterized by immature expression of simple mucin-type carbohydrates. At present, there are some controversial reports about the Tn antigen (GalNAcα-O-Ser/Thr) expression and there is a great lack of information about the [UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase (GalNAc-Ts)] expression in chronic lymphocytic leukemia (CLL). To gain insight in these issues we evaluated the Tn antigen expression in CLL patient samples using two Tn binding proteins with different fine specificity. We also studied the expression from 14 GalNAc-Ts genes in CLL patients by RT-PCR. Our results have provided additional information about the expression level of the Tn antigen, suggesting that a low density of Tn residues is expressed in CLL cells. We also found that GALNT11 was expressed in CLL cells and normal T cell whereas little or no expression was found in normal B cells. Based on these results, GALNT11 expression was assessed by qPCR in a cohort of 50 CLL patients. We found significant over-expression of GALNT11 in 96% of B–CLL cells when compared to normal B cells. Moreover, we confirmed the expression of this enzyme at the protein level. Finally we found that GALNT11 expression was significantly associated with the mutational status of the immunoglobulin heavy chain variable region (IGHV), [?²(1) = 18.26; P < 0.0001], lipoprotein lipase expression [?²(1) = 13.72; P = 0.0002] and disease prognosis [?²(1) = 15.49; P < 0.0001]. Our evidence suggests that CLL patient samples harbor aberrant O-glycosylation highlighted by Tn antigen expression and that the over-expression of GALNT11 constitutes a new molecular marker for CLL.     

Genes and sites involved in replication and incompatibility of an R100 plasmid derivative based on nucleotide sequence analysis

Summary The nucleotide sequence of the entire region required for autonomous replication and incompatibility of an R100 plasmid derivative, pSM1, has been determined. This region includes the replication region and all plasmid encoded information required for replication. Numerous reading frames for possible proteins can be found in this region. The existence of one of these proteins called RepA1 (285 amino acids; 33,000 daltons) which is encoded within the region known by cloning analysis to be required for replication is supported by several lines of evidence. These include an examination of the characteristic sequences on the proximal and distal ends of the coding region, a comparison of the sequence of the replication regions of pSM1 and the highly related R1 plasmid derivative Rsc13 as well as other biochemical and genetic evidence. The existence of two other proteins, RepA3 (64 amino acids; 7000 daltons) and RepA2 (103 amino acids; 11,400 daltons) is also consistent with most of the criteria mentioned above. However, the region encoding RepA3, which by cloning analysis is within the region responsible for both replication and incompatibility, has never been demonstrated to produce a 7,000 dalton polypeptide. Since a large secondary structure can be constructed in this region, it is possible that the region contains structure or other information that is responsible for incompatibility. RepA2, encoded entirely within the region identified by cloning analysis to be responsible for incompatibility but not for replication can be visualized in vivo and in vitro. However, the nucleotide sequence of the region encoding RepA2 is completely different in mutually incompatible plasmid derivatives of R1 and R100. It is therefore unlikely that RepA2 plays a major role in incompatibility. Thus, we predict that RepA1 is required to initiate DNA synthesis at the replication origin and that the region proximal to RepA1 either encodes a gene product or structure information that is responsible for incompatibility.     

Isocitrate dehydrogenase 1 R132C mutation occurs exclusively in microsatellite stable colorectal cancers with the CpG island methylator phenotype

The CpG Island Methylator Phenotype (CIMP) is fundamental to an important subset of colorectal cancer; however, its cause is unknown. CIMP is associated with microsatellite instability but is also found in BRAF mutant microsatellite stable cancers that are associated with poor prognosis. The isocitrate dehydrogenase 1 (IDH1) gene causes CIMP in glioma due to an activating mutation that produces the 2-hydroxyglutarate oncometabolite. We therefore examined IDH1 alteration as a potential cause of CIMP in colorectal cancer. The IDH1 mutational hotspot was screened in 86 CIMP-positive and 80 CIMP-negative cancers. The entire coding sequence was examined in 81 CIMP-positive colorectal cancers. Forty-seven cancers varying by CIMP-status and IDH1 mutation status were examined using Illumina 450K DNA methylation microarrays. The R132C IDH1 mutation was detected in 4/166 cancers. All IDH1 mutations were in CIMP cancers that were BRAF mutant and microsatellite stable (4/45, 8.9%). Unsupervised hierarchical cluster analysis identified an IDH1 mutation-like methylation signature in approximately half of the CIMP-positive cancers. IDH1 mutation appears to cause CIMP in a small proportion of BRAF mutant, microsatellite stable colorectal cancers. This study provides a precedent that a single gene mutation may cause CIMP in colorectal cancer, and that this will be associated with a specific epigenetic signature and clinicopathological features.     

Comparison of theα-globin gene cluster structure in Perissodactyla

Summary To investigate molecular evolution in a mammalian order with a comprehensive fossil record, we have constructed-globin-like gene cluster maps for members of the order Perissodactyla. Although the arrangement of genes is the same in the five Equidae examined, the tapir and rhinoceros differ from each other and the horse in the position and number of their genes, but not in the arrangement of their and genes. In contrast to morphological work, a dendrogram derived from restriction site maps associates the tapir with the horse rather than with the rhinoceros; however, this phylogeny is not statistically significant. Among the Equidae,Equus caballus emerges as an outgroup, in agreement with data from other disciplines.     

The slug foot as a site of uptake of copper molluscicide

Copper has been demonstrated in the feet of slugs exposed to the molluscicide copper sulfate. Using the scanning electron microscope coupled with X-ray microanalysis, copper was localized to the epithelium of the foot. Under the transmission electron microscope the subcellular distribution of copper was obtained by precipitating the molluscicide with potassium ferrocyanide. The reaction product was both electron opaque and insoluble in the preparative media. The precipitate was localized in the zonula adhaerens and septate junctions between adjacent epithelial cells. Reaction product was also present in some of the pinocytotic vesicles of the epithelial cells. The identity of the reaction product was confirmed using X-ray microanalysis.     

The inhibitory activity of a peptide derivative against the growth of simian immunodeficiency virus in C8166 cells.

The peptide derivative Ro 31-8959 is a potent and selective inhibitor of the aspartic proteinases encoded by HIV-1 and HIV-2 and it arrests the growth of both viruses in cell culture. We have demonstrated similar effects against the simian immunodeficiency virus SIVmac251 in the human T-cell line, C8166 (ED50 = 6nM) with a therapeutic index of 4,500. The antiviral activity of Ro 31-8959 was 250 and 22 times greater than that of ddI and ddC, respectively. The mode of action was confirmed by accumulation of the polyprotein p55 with concomitant reduction of the cleavage product, p27, and by the production of immature virions.     

Kinetic studies of human polymorphonuclear leukocyte phosphofructokinase.

Phosphofructokinase from human polymorphonuclear leukocytes has low cooperativity and high affinity for its substrate, F-6-P. It is resistant to ATP inhibition at pH 8; however, at pH 7.1 it becomes sensitive to the effect of this compound. It is activated by F-1, 6-P2; it is not very sensitive to citrate inhibition and F-2, 6-P2 has no effect on its activity. With these kinetic characteristics we assume that perhaps the predominant L-type subunit is accompanied by an F-type component.     

RFLP and RAPD markers linked to the rosy leaf curling aphid resistance gene (Sd1) in apple

 Sd ₁ is a dominant gene for resistance to biotypes 1 and 2 of the rosy leaf curling aphid, Dysaphis devecta Wlk., which can cause economic damage to apple trees. This report describes the identification of three RFLP and four RAPD markers linked to Sd ₁ in a cross between the D. devecta susceptible variety ‘Prima’ (sd ₁ sd ₁) and the resistant variety ‘Fiesta’ (Sd ₁ sd ₁). Potted trees were artificially infested in the glasshouse, and the ratio of resistant:susceptible plants supported the hypothesis that the resistance was under the control of a single dominant gene. The position of the gene was mapped to a single locus on a ‘Fiesta’ chromosome, within 2 cM of three tightly linked RFLP markers (MC064a, 2B12a and MC029b); the four RAPD markers were located further away (between 13 and 46 cM). This is the first report of molecular markers for an aphid resistance gene in tree fruit crops. The potential application of these markers in a marker-assisted resistance breeding programme is discussed. Received: 1 July 1996/Accepted: 23 August 1996