Identification of new human cadherin genes using a combination of protein motif search and gene finding methods

We have combined protein motif search and gene finding methods to identify genes encoding proteins containing specific domains. Particularly, we have focused on finding new human genes of the cadherin superfamily proteins, which represent a major group of cell-cell adhesion receptors contributing to embryonic neuronal morphogenesis. Models for three cadherin protein motifs were generated from over 100 already annotated cadherin domains and used to search the complete translated human genome. The genomic sequence regions containing motif "hits" were analyzed by eukaryotic GeneMark.hmm to identify the exon-intron structure of new genes. Three new genes CDH-J, PCDH-J and FAT-J were found. The predicted proteins PCDH-J and FAT-J were classified into protocadherin and FAT-like subfamilies, respectively, based on the number and organization of cadherin domains and presence of subfamily-specific conserved amino acid residues. Expression of FAT-J was shown in almost all tested tissues. The exon-intron organization of CDH-J was experimentally verified by PCR with specifically designed primers and its tissue-specific expression was demonstrated. The described methodology can be applied to discover new genes encoding proteins from families with well-characterized structural and functional domains.     

Induction of autoantibodies to CCR5 in macaques and subsequent effects upon challenge with an R5-tropic simian/human immunodeficiency virus

Antibodies against CCR5, the major coreceptor for human immunodeficiency virus type 1 (HIV-1), may have antiviral potential as viral fusion inhibitors. In this study, we generated a virus-like particle (VLP)-based vaccine that effectively breaks B-cell tolerance and elicits autoantibodies against CCR5 in pig-tailed macaques. Initial studies in mice identified a polypeptide comprising the N-terminal domain of pig-tailed macaque CCR5 fused to streptavidin that, when conjugated at high density to bovine papillomavirus major capsid protein L1 VLPs, induced high-titer immunoglobulin G (IgG) that bound to a macaque CCR5-expressing cell line in vitro. In macaques, CCR5 peptide-conjugated VLP preparations induced high-avidity anti-CCR5 IgG autoantibody responses, and all five immunized macaques generated IgG that could block infection of CCR5-tropic simian/human immunodeficiency virus SHIV(SF162P3) in vitro. Although the anti-CCR5 IgG titers declined with time, autoantibody levels were boosted upon revaccination. Vaccinated macaques remained healthy for a period of over 3 years after the initial immunization, and no decline in the number of CCR5-expressing T cells was detected. To test the prophylactic efficacy of CCR5 autoantibodies, immunized macaques were challenged with SHIV(SF162P3). Although the plasma-associated virus in half of six control macaques declined to undetectable levels, viral loads were lower, declined more rapidly, and eventually became undetectable in all five macaques in which CCR5 autoantibodies had been elicited. In addition, in the four vaccinated macaques with higher autoantibody titers, viral loads and time to control of viremia were significantly decreased relative to controls, indicating the possibility that CCR5 autoantibodies contributed to the control of viral replication.     

Investigation of the importance of the C-2 oxygen function in the transformation of stemodin analogues by Rhizopus oryzae ATCC 11145

A new stemodinoside, stemodin-alpha-L-arabinofuranoside (5), was isolated from the plant Stemodia maritima. Incubation of stemodin (2) with Rhizopus oryzae ATCC 11145 gave 2 alpha,7 beta,13(S)-trihydroxystemodane (17) and 2 alpha,3 beta,13(S),16 alpha-tetrahydroxystemodane (18) whilst stemodinone (8) afforded 6 alpha,13(S)-dihydroxystemodan-2-one (19). The bioconversion of 2 beta,13(S)-dihydroxystemodane (10) by the fungus yielded 2 beta,7 beta,13(S)-trihydroxystemodane (20) whereas stemod-12-en-2-one (9) provided 7 beta,17-dihydroxystemod-12-en-2-one (21). The results provide useful information about the relationship between the functional groups of the substrates and their potential for bioconversion.     

Diterpenes from the brown algae Dictyota dichotoma and Dictyota linearis

Nineteen secondary metabolites of the brown alga Dictyota dichotoma (Huds.) Lam. and fifteen metabolites of the brown alga D. linearis (Ag.) Grev. were isolated and their chemical structures were elucidated on the basis of their NMR and mass spectral data. The diterpenes isopachydictyolal (1) from D. dichotoma and 4alpha-acetyldictyodial (2) from D. linearis are new natural products. The antiviral activity of metabolites isolated in adequate amounts was evaluated in laboratory assays against Herpes simplex virus I (HSV I) and Poliomyelitis Virus I, using Vero cells as hosts.     

C20orf9-003 (ACI-1), a gene localized on chromosome 20q13.12 encoding for a 49 kD cytoplasmic protein with a putative nucleotide binding site.

Murine NGD5 is a gene identified from NG108-15 cells which is postulated to be involved in opioid receptor function. Here we report the cloning and characterization of a cDNA C20orf9-003 (ACI-1) encoding the human orthologue of the mouse NGD5. Analysis of the genomic structure revealed that C20orf9-003 (ACI-1) contains 13 exons and 12 introns, spanning 52.5kb of genomic DNA and is a variant of C20orf9. Chromosomal localization of human C20orf9-003 (ACI-1) assigned this gene to chromosome 20q13.12. Genes at this locus have been associated with the progression and possibly the development of various cancers. In addition several linkage studies support the possibility that one or more genes affecting obesity are located in 20q13. No function can be clearly assigned to C20orf9-003 (ACI-1), however, the protein has a cytoplasmic subcellular location and the secondary structure contains a Rossman fold like feature which is found in many nucleotide binding proteins.     

Structure of the bovine natural resistance associated macrophage protein (NRAMP 1) gene and identification of a novel polymorphism.

The NRAMP 1 gene is a major candidate gene influencing the outcome of infections with intracellular pathogens in numerous species. NRAMP 1 is highly conserved in many mammalian species and the NRAMP 1 gene shows considerable conservation in structure between mice and humans. The association of NRAMP 1 gene polymorphisms with disease in cattle has been limited to a single microsatellite located within the 3'-non coding region of the bovine NRAMP 1 gene. In order to facilitate further studies on this important gene, we now report the nearly complete structure of the bovine NRAMP 1 gene, including sizes and positions of 13 introns relative to the bovine NRAMP 1 gene coding sequence and the DNA sequence of intron-exon junctions. Comparison of the bovine, murine and human NRAMP 1 gene structures revealed a high degree of conservation in intron placement, though the lengths of several introns were less-well conserved. In general, the greatest divergence in intron lengths occurred in regions of the NRAMP 1 gene displaying the lowest coding sequence conservation. In addition, mutations near intron-exon junctions could account for 25 of the 75 total amino acid differences between murine and bovine NRAMP 1. Using information gained through this study, it was possible to rapidly identify a novel polymorphism within the bovine NRAMP 1 gene intron X. This polymorphism was shown by direct DNA sequence analysis to consist of insertion of three guanine nucleotides at positions 37,40 and 98 relative to the intron X start point. Initial scans of several cattle breeds suggest that the two intron X alleles identified here are stable and widespread in the Bos taurus population.     

Mitochondrial respiratory chain dysfunction, a non-receptor-mediated effect of synthetic PPAR-ligands: biochemical and pharmacological implications

Peroxisome proliferator activated receptors (PPARs) are a class of nuclear receptors involved in lipid and glucidic metabolism, immune regulation, and cell differentiation. Many of their biological activities have been studied by using selective synthetic activators (mainly fibrates and thiazolidinediones) which have been already employed in therapeutic protocols. Both kinds of drugs, however, showed pharmacotoxicological profiles, which cannot be ascribed by any means to receptor activation. To better understand these non-receptorial or extrareceptorial aspects, the effect of different PPAR-ligands on the metabolic status of human HL-60 cell line has been investigated. At this regard, NMR analysis of cell culture supernatants was accomplished in order to monitor modifications at the level of cell metabolism. Cell growth and chemiluminescence assays were employed to verify cell differentiation. Results showed that all the considered PPAR-ligands, although with different potencies and independently from their PPAR binding specificity, induced a significant derangement of the mitochondrial respiratory chain consisting in a strong inhibition of NADH-cytochrome c reductase activity. This derangement has been shown to be strictly correlated to the adaptive metabolic modifications, as evidenced by the increased formation of lactate and acetate, due to the stimulation of anaerobic glycolysis and fatty acid beta-oxidation. It is worthy noting that the mitochondrial dysfunction appeared also linked to the capacity of any given PPAR-ligand to induce cell differentiation. These data could afford an explanation of biochemical and toxicological aspects related to the therapeutic use of synthetic PPAR-ligands and suggest a revision of PPAR pathophysiologic mechanisms.     

Isotopic tracking of foraging and long-distance migration in northeastern Pacific pinnipeds

We investigated the impact of foraging location (nearshore vs offshore) and foraging latitude (high vs middle) on the carbon (δ¹³C) and nitrogen (δ¹⁵N) isotope compositions of bone collagen of northern fur seals (Callorhinus ursinus), harbor seals (Phoca vitulina), California sea lions (Zalophus californianus), and northern elephant seals (Mirounga angustirostris). Nearshore-foraging harbor seals from California had δ¹³C values 2.0‰ higher than female northern elephant seals foraging offshore at similar latitudes. Likewise, nearshore-foraging harbor seals from Alaska had values 1.7‰ higher than male northern fur seals, which forage offshore at high latitudes. Middle-latitude pinnipeds foraging in either the nearshore or offshore were ¹³C enriched by ∼1.0‰ over similar populations from high latitudes. Male northern elephant seals migrate between middle and high latitudes, but they had δ¹³C values similar to high-latitude, nearshore foragers. Female northern fur seal δ¹³C values were intermediate between those of high- and middle-latitude offshore foragers, reflecting their migration between high- and middle-latitude waters. The δ¹³C values of California sea lions were intermediate between nearshore- and offshore-foraging pinnipeds at middle latitudes, yet there was no observational support for the suggestion that they use offshore food webs. We suggest that their “intermediate” values reflect migration between highly productive and less-productive, nearshore ecosystems on the Pacific coasts of California and Mexico. The relative uniformity among all of these pinnipeds in δ¹⁵N values, which are strongly sensitive to trophic level, reveals that the carbon isotope patterns result from differences in the δ¹³C of organic carbon at the base of the food web, rather than differences in trophic structure, among these regions. Finally, the magnitude and direction of the observed nearshore-offshore and high-to middle-latitude differences in δ¹³C values suggest that these gradients may chiefly reflect differences in rates and magnitudes of phytoplankton production as well as the δ¹³C value of inorganic carbon available for photosynthesis, rather than the input of ¹³C-enriched macroalgal carbon to nearshore food webs. Received: 8 September 1998 / Accepted: 24 February 1999     

The short 5′ untranslated region of the βA3/A1-crystallin mRNA is responsible for leaky ribosomal scanning

Leaky ribosomal scanning allows the expression of multiple proteins from a single mRNA by occasionally skipping the first start codon, and initiating translation at a subsequent one. A3- and A1-crystallin, two members of the -crystallin family of vertebrate eye lens proteins, are produced via this mechanism, of which, until now, only very few examples have been found in eukaryotic genes. Since the two start codons on the A3/A1 messenger lie in the same reading frame, the two translated proteins are identical, except for the 17 residues shorter N-terminal extension of A1-crystallin. It has been suggested that the very short leader (5–7 nucleotides) of the A3/A1 messenger might cause slippage at the first start codon, although the unfavorable context of this start codon might also be responsible. Using transient transfections, we now demonstrate that increasing the length of the leader sequence to 67 nucleotides indeed completely abolishes translation initiation at the second start codon, and thus expression of the A1-crystallin protein. Messengers having a leader of 5, 7 or 14 nucleotides all express both A3- and A1-crystallin at very similar relative levels.     

Influence of White Clover Mosaic Potexvirus Infection on the Endogenous Cytokinin Content of Bean

The cytokinin content in the primary leaves of bean (Phaseolus vulgaris) was monitored for 10 d after inoculation with white clover mosaic potexvirus. The cytokinins were isolated, purified, separated by high-performance liquid chromatography, and quantified by radioimmunoassay. The cytokinins detected at the time of inoculation (d 0) were: (a) the free bases, zeatin (Z), dihydrozeatin (DZ), and isopentenyladenine; (b) the riboside, DZ riboside (DZR); (c) the O-glucosides of DZ, DZR, and Z riboside; (d) the nucleotides, Z riboside-5′-monophosphate and isopentenyladenosine-5′-monophosphate; and (e) trace amounts of Z-9-glucoside and DZ-9-glucoside. During the 10 d after inoculation with white clover mosaic potexvirus, marked quantitative changes in this cytokinin profile were observed. The concentration of the free bases and DZR decreased, accompanied by an increase in the 9-glucosides and the nucleotides. Virus titer increased rapidly 3 d after inoculation, attaining a maximum level at d 5. This increase coincided with the increases in the 9-glucosides and the nucleotides. We propose that the decline in the cytokinin free bases and riboside may allow the increase of virus titer in bean and lead to the senescence of infected leaves.