Low level expression of human papillomavirus type 16 (HPV16) E6 in squamous epithelium does not elicit E6 specific B- or T-helper immunological responses, or influence the outcome of immunisation with E6 protein

Mice transgenic for E6/E7 oncogenes of Human Papillomavirus type 16 display life-long expression of E6 in lens and skin epithelium, and develop inflammatory skin disease late in life, which progresses to papillomata and squamous carcinoma in some mice. We asked whether endogenous expression of E6 induced a specific immunological outcome, i.e. immunity or tolerance, or whether the mice remained immunologically naive to E6. We show that prior to the onset of skin disease, E6 transgenic mice did not develop a spontaneous E6-directed antibody response, nor did they display T-cell proliferative responses to dominant T-helper epitope peptides within E6. In contrast, old mice in which skin disease had arisen, developed antibodies to E6. We also show that following immunisation with E6, specific antibody responses did not differ significantly among groups of EB-transgenic mice of different ages (and therefore of different durations and amounts of exposure to endogenous E6), and non-transgenic controls. Additionally, E6 immunisation-induced T-cell proliferative responses were similar in E6-transgenic and non-transgenic mice. These data are consistent with the interpretation that unimmunised Eb-transgenic mice that have not developed inflammatory skin disease remain immunologically naive to E6 at the B- and Th levels. There are implications for E6-mediated tumorigenesis in humans, and for the development of putative E6 therapeutic vaccines. (C) 2001 Elsevier Science B.V. All rights reserved.

Proteomic analysis reveals that 14-3-3 sigma in downregulated in human breast cancer cells

The class of molecular chaperones known as 14-3-3 is involved in the control of cellular growth by virtue of its apparent regulation of various signaling pathways, including the Raf/mitogen-activated protein kinase pathway. In breast cancer cells, the sigma form of 14-3-3 has been shown to interact with cyclin-dependent kinases and to control the rate of entry into mitosis. To test for a direct role for 14-3-3 in breast epithelial cell neoplasia, me have quantitated 14-3-3 protein levels using a proteomic approach based on two-dimensional electrophoresis and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF). We show here that 14-3-3 sigma protein is strongly down-regulated in the prototypic breast cancer cell lines MCF-7 and MDA-MB-231 and in primary breast carcinomas as compared with normal breast epithelial cells. In contrast, levels of the alpha, beta, delta, or zeta isoforms of 14-3-3 mere the same in both normal and transformed cells. The data support the idea that 14-3-3 sigma is involved in the neoplastic transition of breast epithelial cells by virtue of its role as a tumor suppressor; as such, it may constitute a robust marker with clinical efficacy for this pathology.

TTYH2, a human homologue of the Drosophila melanogaster gene tweety, is located on 17q24 and upregulated in renal cell carcinoma

Using differential display PCR, we identified a novel gene upregulated in renal cell carcinoma. Characterization of the full-length cDNA and gene revealed that the encoded protein is a human homologue of the Drosophila melanogaster Tweety protein, and so we have termed the novel protein TTYH2. The orthologous mouse cDNA was also identified and the predicted mouse protein is 81% identical to the human protein. The encoded human TTYH2 protein is 534 amino acids and, like the other members of the tweety-related protein family, is a putative cell surface protein with five transmembrane regions. TTYH2 is located at 17q24; it is expressed most highly in brain and testis and at lower levels in heart, ovary, spleen, and peripheral blood leukocytes. Expression of this gene is upregulated in 13 of 16 (81%) renal cell carcinoma samples examined. In addition to a putative role in brain and testis, the overexpression of TTYH2 in renal cell carcinoma suggests that it may have an important role in kidney tumorigenesis.

Modulation of ligand selectivity by mutation of the first extracellular loop of the human C5a receptor

The cyclic C5a receptor antagonist, phenylalanine [L-ornithine-proline-D-cyclohexylalanine-tryptophan-arginine] (F-[OPchaWR]), has similar to 1000-fold less affinity for the C5a receptor (C5aR) on murine polymorphonuclear leukocytes than on human. Analysis of C5aR from different species shows that a possible cause of this difference is the variation in the sequence of the first extracellular loop of the receptor. The mouse receptor contains Y at a position analogous to P-103 in the human receptor, and D at G(105). To test this hypothesis, we expressed human C5aR mutants ((PY)-Y-103, G(105)D and the double mutant, (PY)-Y-103/G(105)D) in RBL-2H3 cells and investigated the effects of these mutations on binding affinity and receptor activation. All three mutant receptors had a higher affinity for human C5a than the wild-type receptor, but showed no significant difference in the ability of F-[OPchaWR] to inhibit human C5a binding. However, all of the mutant receptors had substantially lower affinities for the weak agonist, C5a des Arg(74) (C5adR(74)), and two altered receptors (G(105)D and (PY)-Y-103/G(105)D) had much lower affinities for the C-terminal C5a agonist peptide analogue, L-tyrosine-serine-phenylalanine-lysine-proline-methionine-proline-leucine-D-alanine-arginine (YSFKPMPLaR). Although it is unlikely that differences at these residues are responsible for variations in the potency of F-[OPchaWR] across species, residues in the first extracellular loop are clearly involved in the recognition of both C5a and C5a agonists. The complex effects of mutating these residues on the affinity and response to C5a, C5adR(74), and the peptide analogues provide evidence of different binding modes for these ligands on the C5aR. (C) 2001 Elsevier Science Inc. All rights reserved.

MUC13, a novel human cell surface mucin expressed by epithelial and hemopoietic cells

Transmembrane mucins are glycoproteins involved in barrier function in epithelial tissues. To identify novel transmembrane mucin genes, we performed a tblastn search of the GenBank(TM) EST data bases with a serine/ threonine-rich search string, and a rodent gene expressed in bone marrow was identified. We determined the cDNA sequence of the human orthologue of this gene, MUC13, which localizes to chromosome band 3q13.3 and generates 3.2-kilobase pair transcripts encoding a 512-amino acid protein comprised of an N-terminal mucin repeat domain, three epidermal growth factor-like sequences, a SEA module, a transmembrane domain, and a cytoplasmic tail (GenBank(TM) accession no. AF286113), MUC13 mRNA is expressed most highly in the large intestine and trachea, and at moderate levels in the kidney, small intestine, appendix, and stomach, In situ hybridization in murine tissues revealed expression in intestinal epithelial and lymphoid cells. Immunohistochemistry demonstrated the human MUC13 protein on the apical membrane of both columnar and goblet cells in the gastrointestinal tract, as well as within goblet cell thecae, indicative of secretion in addition to presence on the cell surface. MUC13 is cleaved, and the beta -subunit containing the cytoplasmic tail undergoes homodimerization, Including MUC13, there are at least five cell surface mucins expressed in the gastrointestinal tract.

Estrogen receptors in human preadipocytes

Estrogen influences regional adipose tissue distribution and the accompanying cardiovascular disease risk. To elucidate the mechanisms of this link further, we assessed whether human preadipocytes (PAs) expressed estrogen receptors (ERs) and whether there were any regional or gender differences in ER complement. Human PAs expressed the ER alpha gene but not ERP by reverse transcriptase-polymerase chain reaction, possessed ER alpha protein on Western blotting, and displayed specific 17 beta -estradiol (E-2) binding with calculated dissociation constants of 0.78 nM, 0.96 nM, and 1.19 nM and maximal binding capacities of 9.3 fmol/mg, 14.6 fmol/ mg, and 18.2 fmol/mg from three whole cell binding assays. There were no regional differences in ER alpha complement for males or females. There were no gender differences in ER alpha complement for subcutaneous or visceral samples. We conclude that ER alpha but not ERP is present in human PAs. This suggests that the effect of estrogen on adipose tissue deposition has a contribution from the direct effect of estrogen on human PAs via ER alpha.

Characterization of the mouse hnRNP A2/B1/B0 gene and identification of processed pseudogenes

The mouse hnRNP A2/B1/B0 gene has been cloned using a PCR-based strategy and sequenced. Analysis of this sequence showed that the gene organization closely follows that of the human orthologue with 12 exons and 11 introns. The hnRNP A2/B1/B0 gene gives rise to four splice variants through alternative splicing of exons 2 and 9. RT-PCR assays indicated that all splice variants were expressed in mouse brain, skin, and stomach tissues of varying ages, although their ratios to one another varied with age and tissue type. We also identified a small subset of all polyadenylated splice variants that included intron 11, which shows 94% sequence identity between human and mouse. Several processed pseudogenes were identified in the mouse genome. A search of the mouse genome databases located five pseudogenes, four of. which are presumed to be non-functional because of the presence of premature stop codons, large deletions or rearrangements within the coding region. The fifth, which possesses putative promoter elements and has a coding sequence identical to that of the hnRNP A2 mRNA, variant, may be functional. (C) 2002 Elsevier Science B.V. All rights reserved.

Comparison between human and animal isolates of Shiga toxin-producing Escherichia coli O157 from Australia

There is very little human disease associated with enterohaemorrhagic Escherichia coli O157 in Australia even though these organisms are present in the animal population. A group of Australian isolates of E. coli O157:H7 and O157:H- from human and animal sources were tested for the presence of virulence markers and compared by XbaI DNA macrorestriction analysis using pulsed-field gel electrophoresis (PFGE). Each of 102 isolates tested contained the gene eae which encodes the E. coli attaching and effacing factor and all but one carried the enterohaemolysin gene, ehxA, found on the EHEC plasmid. The most common Shiga toxin gene carried was stx(2c), either alone (16%) or in combination with stx(1) (74%) or stx(2) (3%) PFGE grouped the isolates based on H serotype and some clusters were source specific. Australian E. coli O157:H7 and H- isolates from human, animal and meat sources carry all the virulence markers associated with EHEC disease in humans therefore other factors must be responsible for the low rates of human infection in Australia.

Metabolism of tamoxifen by recombinant human cytochrome P450 enzymes: Formation of the 4-hydroxy, 4′-hydroxy andN-desmethyl metabolites and isomerization oftrans-4-hydroxytamoxifen

The cytochrome P450 (P450)-mediated biotransformation of tamoxifen is important in determining both the clearance of the drug and its conversion to the active metabolite, trans-4-hydroxytamoxifen. Biotransformation by P450 forms expressed extrahepatically, such as in the breast and endometrium, may be particularly important in determining tissue-specific effects of tamoxifen. Moreover, tamoxifen may serve as a useful probe drug to examine the regioselectivity of different forms. Tamoxifen metabolism was investigated in vitro using recombinant human P450s. Forms CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5, and 3A7 were coexpressed in Escherichia coli with recombinant human NADPH-cytochrome P450 reductase. Bacterial membranes were harvested and incubated with tamoxifen or trans-4-hydroxytamoxifen under conditions supporting P450-mediated catalysis. CYP2D6 was the major catalyst of 4-hydroxylation at low tamoxifen concentrations (170 +/- 20 pmol/40 min/0.2 nmol P450 using 18 muM tamoxifen), but CYP2B6 showed significant activity at high substrate concentrations (28.1 +/- 0.8 and 3.1 +/- 0.5 nmol/120 min/0.2 nmol P450 for CYP2D6 and CYP2B6, respectively, using 250 muM tamoxifen). These two forms also catalyzed 4'-hydroxylation (13.0 +/- 1.9 and 1.4 +/- 0.1 nmol/120 min/0.2 nmol P450, respectively, for CYP2B6 and CYP2D6 at 250 muM tamoxifen; 0.51 +/- 0.08 pmol/40 min/0.2 nmol P450 for CYP2B6 at 18 muM tamoxifen). Tamoxifen N-demethylation was mediated by CYP2D6, 1A1, 1A2, and 3A4, at low substrate concentrations, with contributions by CYP1B1, 2C9, 2C19 and 3A5 at high concentrations. CYP1B1 was the principal catalyst of 4-hydroxytamoxifen trans-cis isomerization but CYP2B6 and CYP2C19 also contributed.

Promiscuous CTL recognition of viral epitopes on multiple human leukocyte antigens: Biological validation of the proposed HLA A24 supertype

Multiple HLA class I alleles can bind peptides with common sequence motifs due to structural similarities in the peptide binding cleft, and these groups of alleles have been classified into supertypes. Nine major HLA supertypes have been proposed, including an A24 supertype that includes A*2301, A*2402, and A*3001. Evidence for this A24 supertype is limited to HLA sequence homology and/or similarity in peptide binding motifs for the alleles. To investigate the immunological relevance of this proposed supertype, we have examined two viral epitopes (from EBV and CMV) initially defined as HLA-A*2301-binding peptides. The data clearly demonstrate that each peptide could be recognized by CTL clones in the context of A*2301 or A*2402; thus validating the inclusion of these three alleles within an A24 supertype. Furthermore, CTL responses to the EBV epitope were detectable in both A*2301(+) and A*2402(+) individuals who had been previously exposed to this virus. These data substantiate the biological relevance of the A24 supertype, and the identification of viral epitopes with the capacity to bind promiscuously across this supertype could aid efforts to develop CTL-based vaccines or immunotherapy. The degeneracy in HLA restriction displayed by some T cells in this study also suggests that the dogma of self-MHC restriction needs some refinement to accommodate foreign peptide recognition in the context of multiple supertype alleles.

Ex vivo profiling of CD8(+)-T-cell responses to human cytomegalovirus reveals broad and multispecific reactivities in healthy virus carriers

Human cytomegalovirus (HCMV) can establish both nonproductive (latent) and productive (lytic) infections. Many of the proteins expressed during these phases of infection could be expected to be targets of the immune response; however, much of our understanding of the CD8(+)-T-cell response to HCMV is mainly based on the pp65 antigen. Very little is known about T-cell control over other antigens expressed during the different stages of virus infection; this imbalance in our understanding undermines the importance of these antigens in several aspects of HCMV disease pathogenesis. In the present study, an efficient and rapid strategy based on predictive bioinformatics and ex vivo functional T-cell assays was adopted to profile CD8(+)-T-cell responses to a large panel of HCMV antigens expressed during different phases of replication. These studies revealed that CD8(+)-T-cell responses to HCMV often contained multiple antigen-specific reactivities, which were not just constrained to the previously identified pp65 or IE-1 antigens. Unexpectedly, a number of viral proteins including structural, early/late antigens and HCMV-encoded immunomodulators (pp28, pp50, gH, gB, US2, US3, US6, and UL18) were also identified as potential targets for HCMV-specific CD8(+)-T-cell immunity. Based on this extensive analysis, numerous novel HCMV peptide epitopes and their HLA-restricting determinants recognized by these T cells have been defined. These observations contrast with previous findings that viral interference with the antigen-processing pathway during lytic infection would render immediate-early and early/late proteins less immunogenic. This work strongly suggests that successful HCMV-specific immune control in healthy virus carriers is dependent on a strong T-cell response towards a broad repertoire of antigens.

Identification of a novel protein kinase mediating Akt survival signaling to the ATM protein

We identified a novel human AMP-activated protein kinase (AMPK) family member, designated ARK5, encoding 661 amino acids with an estimated molecular mass of 74 kDa. The putative amino acid sequence reveals 47, 45.8, 42.4, and 55% homology to AMPK-alpha1, AMPK-alpha2, MELK and SNARE respectively, suggesting that it is a new member of the AMPK family. It has a putative Akt phosphorylation motif at amino acids 595600, and Ser(600) was found to be phosphorylated by active Akt resulting in the activation of kinase activity toward the SAMS peptide, a consensus AMPK substrate. During nutrient starvation, ARK5 supported the survival of cells in an Akt-dependent manner. In addition, we also demonstrated that ARK5, when activated by Akt, phosphorylated the ATM protein that is mutated in the human genetic disorder ataxia-telangiectasia and also induced the phosphorylation of p53. On the basis of our current findings, we propose that a novel AMPK family member, ARK5, is the tumor cell survival factor activated by Akt and acts as an ATM kinase under the conditions of nutrient starvation.

Identification of residues which regulate activity of the STE20-related kinase hMINK

Activity of the STE20-related kinase hMINK was investigated. hMINK was expressed widely, though not ubiquitously, in human tissues: highest levels being found in haematopoietic tissues but also in brain, placenta, and lung. Mutagenesis revealed that T-191. and Y-193 in the substrate recognition loop of the catalytic domain were critical for kinase activity against exogenous substrates and autophosphorylation. A mutation on T-187 showed reduced enzymatic activity against exogenous substrates but retained autophosphorylationactivity. Phosphorylation was confirmed by the use of a phospho-specific T-187 antibody. hMINK activated the JNK signal transduction pathway and optimal JNK activation occurred when the C-terminus was deleted. In addition, overexpression of the C-terminal domain devoid of kinase activity also resulted in significant activation of the JNK pathway. These data suggest that hMINK requires an activation step that dissociates the C terminal, thereby freeing the catalytic domain to interact with substrates. Models for receptor-mediated activation of hMINK are discussed. (C) 2002 Elsevier Science (USA). All rights reserved.

Alternatively spliced products of the human kinesin light chain 1 (KNS2) gene

Conventional kinesin is a microtubule-based molecular motor involved in the transport of membranous and non-membranous cargoes. The kinesin holoenzyme exists as a heterotetramer, consisting of two heavy chain and two light chain subunits. It is thought that one function of the light chains is to interact with the cargo. Alternative splicing of kinesin light chain pre-mRNA has been observed in lower organisms, although evidence for alternative splicing of the human gene has not been reported. We have identified 19 variants of the human KNS2 gene (KLC1) that are generated by alternative splicing of downstream exons, but calculate that KNS2 has the potential to produce 285919 spliceforms. Corresponding spliceforms of the mouse KLC1 gene were also identified. The alternative exons are all located 3' of exon 12 and the novel spliceforms produce both alternative carboxy termini and alternative 3' untranslated regions. The observation of multiple light chain isoforms is consistent with their proposed role in specific cargo attachment.

Identification and characterization of the hepatic stellate cell transferrin receptor

Activated hepatic stellate cells have been implicated in the fibrogenic process associated with iron overload, both in animal models and in human hemochromatosis. Previous studies have evaluated the role of ferritin/ferritin receptor interactions in the activation of stellate cells and subsequent fibrogenesis; however, the role of transferrin in hepatic stellate cell biology is unknown. This study was designed to identify and characterize the stellate cell transferrin receptor and to evaluate the influence of transferrin on stellate cell activation. Identification and characterization of the stellate cell transferrin receptor was determined by competitive displacement assays. The effect of transferrin on stellate cell activation was assessed using western blot analysis for alpha-smooth muscle actin expression, [H-3]Thymidine incorporation, and real-time RT-PCR for procollagen 1(I) mRNA expression. A specific receptor for rat transferrin was observed on activated but not quiescent stellate cells. Transferrin significantly increased the expression of alpha-smooth muscle actin, but caused a decrease in proliferation. Transferrin induced a significant increase in procollagen alpha1(I) mRNA expression. In conclusion, this study has demonstrated for the first time a specific, high affinity receptor for rat transferrin on activated hepatic stellate cells, which via interaction with transferrin regulates stellate cell activation. This suggests that transferrin may be an important factor in the activation of hepatic stellate cells in conditions of iron overload.