Sequence analysis of the granulysin and granzyme B genes in familial hemophagocytic lymphohistiocytosis

Familial hemophagocytic lymphohistiocytosis (FHL) is an autosomal recessive disorder of immune regulation. Mutations in the gene encoding perforin were previously identified in a subset of FHL patients. The present analysis of two novel candidate genes, granzyme B and granulysin, by direct sequencing in a total of 16 FHL families, disclosed several sequence variations. However, none of these sequence variations were associated with the manifestations of FHL. These data do not support the notion that granulysin and granzyme B are candidate genes for FHL.     

Evolution of primate α and θ defensins revealed by analysis of genomes

Defensins are endogenous peptides with cysteine-rich antimicrobial ability that contribute to host defence against bacterial, fungal and viral infections. There are three subfamilies of defensins in primates: α, β and θ-defensins. α-defensins are most present in neutrophils and Paneth cells; β-defensins are involved in protecting the skin and the mucous membranes of the respiratory, genitourinary and gastrointestinal tracts; and θ-defensins are physically distinguished as the only known fully-cyclic peptides of animal origin, which are first isolated from rhesus macaques. All three kinds of defensins have six conserved cysteines, three intramolecular disulfide bonds, a net positive charge, and β-sheet regions. α and θ-defensins are closely related, comparative amino acid sequences showed that the difference between them is that θ-defensins have an additional stop codon limits the initial defensin domain peptides to 12 residues. Humans, chimpanzees and gorillas do not produce θ-defensin peptides due to a premature stop codon present in the signal sequence of all θ-defensin pseudogenes. By using comprehensive computational searches, here we report the discovery of complete repertoires of the α and θ-defensin gene family in ten primate species. Consistent with previous studies, our phylogenetic analyses showed all primate θ-defensins evident formed one distinct clusters evolved from α-defensins. β-defensins are ancestors of both α and θ-defensins. Human has two copies of DEFA1 and DEFT1P, and two extra DEFA3 and DEFA10P genes compared with gorilla. As different primates inhabit in quite different ecological niches, the production of species-specific α and θ-defensins and these highly evolved θ-defensins in old world monkeys would presumably allow them to better respond to the specific microbial challenges that they face.     

A new polymorphic marker of the T-cell antigen receptor α chain genes in man

The restriction fragment length polymorphism of the unrearranged T-cell antigen receptor (Tcr) chain gene was investigated. Taq I digests, when probed with a Tcr chain cDNA probe, revealed polymorphic bands of 7.0, 2.0, and 1.4 kb, due to variations around the C gene, and the V gene cluster. Family studies confirmed the segregation of these polymorphic bands as allelic markers. These polymorphisms provide a new marker for the analysis of genetic variation of the Tcr a chain, and the influence of variation of the Tcr genes on the immune response.     

Soret Spectral and Bioinformatic Approaches Provide Evidence for a Critical Role of the α -Subunit in Assembly of Tetrameric Hemoglobin

Soret spectral contributions of the α-subunit heme pocket have been evaluated by performing static titrations of apohemoglobin A with CNProtohemin under varied experimental conditions. Increasing the temperature from 5 to 30°C in 0.05 M potassium phosphate buffer, pH 7, resulted in a decreasingly prominent hypsochromic shifts reflecting altered the vinyl–globin interactions. Studies at 10°C in over pH range of 6.7–8.0 revealed a profile for the spectral shifts approximating the side chain pK value (7.4) a histidyl residue. These overall spectral changes correspond to ΔE of ≤7 kJ/mol indicative of electrostatic noncovalent interactions. Further our current molecular modeling studies indicate that the spatial arrangement and critical noncovalent interactions of tyrosine 42 and histidine 45 (aromatic residues unique to the α-subunit) make significant contribution to the Soret spectra. Most interestingly, phylogenetic analyses have revealed the presence of a histidyl triad in the α-chain of all vertebrates that form heterotetramers.     

Dihydropteridine reductase variation in man and the characid fish “Cheirodon axelrodi”: Evidence for a dimeric enzyme structure

Summary Genetic evidence for a dimeric structure of dihydropteridine reductase in man and in the fish species Cheirodon axelrodi and Salmo irideus is presented. A single locus in man and two loci in the fishes examined encode this enzyme. Zymograms revealed two alleles for the locus in man and two alleles for each locus in the fish Cheirodon axelrodi. The liver homogenate of a patient with dihydropteridine reductase deficiency showed no detectable activity in the gel, while his parents showed the normal electrophoretic phenotype.     

Western lowland gorilla density and nesting behavior in a Gabonese forest logged for 25 years: implications for gorilla conservation

The tropical forests of the Congo Basin constitute biodiversity refuges that still hold large numbers of species, including endemic and endangered vertebrates. Along with several key species, the critically endangered western lowland gorilla (WLG) potentially contributes to forest dynamics through seed dispersal. Considering the extensive influence of timber harvesting on tropical forest ecosystems, the survival of gorilla populations in logged forests might prove critical for forest ecosystem conservation. We estimated WLG density, through a nest count survey, in a forest in southeast Gabon that has been logged for 25 years. Nesting behavior and habitat use were described and we applied generalized linear models to identify the factors that influence gorilla day and night habitat use. The estimated density of weaned gorillas, 1.5 gorillas km^?2, is comparable with estimates from some protected areas and other sustainably managed sites within their range. Habitat type had the greatest influence on nest site distribution. We observed a preference for nesting in open terra firma forest, and open habitats in general, which supports the findings of previous studies. Habitat use during the day was strongly influenced by habitat type and human activities, and to a lesser degree by functional and non-functional roads, and rivers. Our results support the suggestion that logged forests are suitable habitats for WLG if hunting and poaching are controlled. We recommend collaborations between timber operators and scientists to improve the conservation potential of tropical forests and enhance the wildlife-management aspects of logging practices.     

Comprehensive analysis and characterization of the TCR α chain sequences in the common marmoset

The common marmoset (Callithrix jacchus) is useful as a nonhuman primate model of human diseases. Although the marmoset model has great potential for studying autoimmune diseases and immune responses against pathogens, little information is available regarding the genes involved in adaptive immunity. Here, we identified one TCR α constant (TRAC), 46 TRAJ (joining), and 35 TRAV (variable) segments from marmoset cDNA. Marmoset TRAC, TRAJ, and TRAV shared 80%, 68–100%, and 79–98% identity with their human counterparts at the amino acid level, respectively. The amino acid sequences were less conserved in TRAC than in TCRβ chain constant (TRBC). Comparative analysis of TRAV between marmosets and humans showed that the rates of synonymous substitutions per site (d _S ) were not significantly different between the framework regions (FRs) and complementarity determining regions (CDRs), whereas the rates of nonsynonymous substitutions per site (d _N ) were significantly lower in the FRs than in CDRs. Interestingly, the d _N values of the CDRs were greater for TRBV than TRAV. These results suggested that after the divergence of Catarrhini from Platyrrhini, amino acid substitutions were decreased in the FRs by purifying selection and occurred more frequently in CDRβ than in CDRα by positive selection, probably depending on structural and functional constraints. This study provides not only useful information facilitating the investigation of adaptive immunity using the marmoset model but also new insight into the molecular evolution of the TCR heterodimer in primate species.     

Chromosomal localization of genes required for the terminal steps of oxidative metabolism: α and γ subunits of ATP synthase and the phosphate carrier

The terminal steps of oxidative phosphorylation include transport of phosphate and ADP into the mitochondrial matrix, synthesis of ATP in the matrix, and transport of the product ATP into the cytosol where it can be utilized to perform cellular work. Three nuclear genome encoded membrane proteins, namely, the phosphate carrier (PHC), the adenine nucleotide carrier (ANT), and the ATP synthase complex, consisting of at least 13 individual subunits, catalyze these reactions. The locations of the and subunits of the mitochondrial ATP synthase complex and the mitochondrial phosphate carrier, PHC, on human chromosomes were determined using cloned rat liver cDNA as probes. Human homologues of the subunit are on chromosomes 9 and 18, the subunit are on chromosomes 10 and 14, and the PHC was localized to chromosome 12.     

Interleukin-1 α and β genes: linkage on chromosome 2 in the mouse

Two interleukin-1 polypeptides, and , are known, and cDNAs corresponding to each have been described. Genomic cloning and Southern blotting experiments suggest that in the mouse each is encoded by a gene present in one copy per haploid genome. Analysis of a panel of somatic cell hybrids carrying various mouse chromosomes on a constant Chinese hamster background indicates that both genes map to mouse chromosome 2. Further, analysis of the inheritance of DNA restriction fragment length polymorphisms associated with each gene in recombinant inbred strains of mice shows the two loci to be tightly linked to one another, and to lie approximately 4.7 centimorgans distal to B2m (beta-2 microglobulin). We have named the locus encoding IL-1 Il-1 and the locus encoding IL-1 Il-1b.     

Sequence analysis of the upstream regions of Xenopus laevis β-globin genes and arrangement of repetitive elements within the globin gene clusters

The globin gene clusters of Xenopus laevis are interspersed by various different repetitive DNA elements. A specific repeat, the JH12 element, has been mapped by Southern analysis and some of its locations have been subsequently confirmed by nucleotide sequencing. JH12 family members seem to represent mobile genetic elements and display a high degree of divergence. The nucleotide sequences upstream to the adult _I-globin gene and to the two coordinately expressed larval _I- and _II genes have been determined and compared to those of the adult -genes. Besides some repetitive DNA elements and a short sequence of rather weak homology we have found no characteristic sequence motifs to be common to the adult - and -genes. The two larval -genes share one short sequence element being absent from the adult genes. This might reflect completely different sequence requirements for protein interactions and for the regulation of adult and larval globin gene expression.     

Genomic analysis of immunity in a Urochordate and the emergence of the vertebrate immune system: “waiting for Godot”

Genome-wide sequence analysis in the invertebrate chordate, Ciona intestinalis, has provided a comprehensive picture of immune-related genes in an organism that occupies a key phylogenetic position in vertebrate evolution. The pivotal genes for adaptive immunity, such as the major histocompatibility complex (MHC) class I and II genes, T-cell receptors, or dimeric immunoglobulin molecules, have not been identified in the Ciona genome. Many genes involved in innate immunity have been identified, including complement components, Toll-like receptors, and the genes involved in intracellular signal transduction of immune responses, and show both expansion and unexpected diversity in comparison with the vertebrates. In addition, a number of genes were identified which predicted integral membrane proteins with extracellular C-type lectin or immunoglobulin domains and intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and immunoreceptor tyrosine-based activation motifs (ITAMs) (plus their associated signal transduction molecules), suggesting that activating and inhibitory receptors have an MHC-independent function and an early evolutionary origin. A crucial component of vertebrate adaptive immunity is somatic diversification, and the recombination activating genes (RAG) and activation-induced cytidine deaminase (AID) genes responsible for the Generation of diversity are not present in Ciona. However, there are key V regions, the essential feature of an immunoglobulin superfamily VC1-like core, and possible proto-MHC regions scattered throughout the genome waiting for Godot.     

Evidence for a non-antioxidant,dose-dependent role of α -lipoic acid in caspase-3 and ERK2 activation in endothelial cells

Endothelial cell apoptosis contributes to atherosclerosis and may be exacerbated by oxidative stress. Results from clinical trials using antioxidant supplementation are equivocal and could be enhanced by antioxidants with additional non-antioxidant properties such as -lipoic acid and -tocopherol. The aim of this study was to investigate the effects of these antioxidants on cytoprotective pathways and endothelial apoptosis. Endothelial cells were incubated with -lipoic acid and -tocopherol, alone or in combination, prior to incubation with H₂O₂ or staurosporine. -lipoic acid pre-treatment alone increased caspase-3 activity in a dose-dependent manner. Both H₂O₂ and staurosporine increased DNA fragmentation and caspase-3 activity and pre-treatment of cells with -lipoic acid and/or -tocopherol failed to prevent stress-induced apoptosis. Neither antioxidant treatments nor apoptotic inducers alone altered expressions of Bcl-2, Bax, HSP70 or pERK1/2 or pJNK. -lipoic decreased pERK2 in staurosporine-treated cells in a dose-dependent manner. These findings indicate that pre-incubation with -lipoic acid and -tocopherol, alone or in combination, does not protect against oxidative- or non-oxidative-induced apoptosis in endothelial cells. Moreover, we have demonstrated a non-antioxidant, dose-dependent role of -lipoic acid in caspase-3 and ERK2 activation. These data provide an insight and indicate caution in the use of high doses of -lipoic acid as an antioxidant.     

Molecular evidence for five distinct MHC class II α genes in the rabbit

Human HLA class 11 gene probes were used to identify five distinct genes encoding the class 11 heavy chain (a chain) in the rabbit. The rabbit genes were defined by both mapping data and hybridization studies of genomic clones derived from the inbred B/J rabbit strain. Analysis of the clones by hybridization at graded stringencies indicated that one group of clones corresponded to HLA-DR, one group to HLA-DQ, and two groups to HLA-DP. Clones within a fifth group, designated DN, hybridized weakly to HLA-DR and may carry a fourth species of class II genes in the rabbit. Clones within the group showing high homology to HLA-DR were found to also contain sequences hybridizing with a probe for HLA-DR . No HLA-DP, -DQ, or -DR sequences were detected in any of the other class II clones. Distinct banding patterns observed in Southern blot analyses using either human or rabbit class II probes revealed restriction fragment length polymorphism for the different rabbit haplotypes studied. TheDN, DQ, andDR genes appear to be present as single copies whereas there are two distinctDP-like genes in the rabbit.Abbreviations used in this paper RLA major histocompatibility complex of the rabbit - RFLP restriction fragment length polymorphism - RL-5 rabbit T-cell line - SSC 0.15 M sodium chloride, 0.015 M sodium citrate     

Wild barley: a source of genes for crop improvement in the 21st century?

The development of new barleys tolerant of abiotic and biotic stresses is an essential part of the continued improvement of the crop. The domestication of barley, as in many crops, resulted in a marked truncation of the genetical variation present in wild populations. This process is significant to agronomists and scientists because a lack of allelic variation will prevent the development of adapted cultivars and hinder the investigation of the genetic mechanisms underlying performance. Wild barley would be a useful source of new genetic variation for abiotic stress tolerance if surveys identify appropriate genetic variation and the development of marker-assisted selection allows efficient manipulation in cultivar development. There are many wild barley collections from all areas of its natural distribution, but the largest are derived from the Mediterranean region. The results of a range of assays designed to explore abiotic stress tolerance in barley are reported in this paper. The assays included; sodium chloride uptake in wild barley and a mapping population, effects for delta 13C and plant dry weight in wheat aneuploids, effects of photoperiod and vernalization in wild barley, and measurements of root length in wild barley given drought and nitrogen starvation treatments in hydroponic culture. There are examples of the use of wild barley in breeding programmes, for example, as a source of new disease resistance genes, but the further exploration of the differences between wild barley and cultivars is hampered by the lack of good genetic maps. In parallel to the need for genetic studies there is also a need for the development of good physiological models of crop responses to the environment. Given these tools, wild barley offers the prospect of a 'goldmine' of untapped genetic reserves.     

Evolution of the human sarcomeric-actin genes: Evidence for units of selection within the 3′ untranslated regions of the mRNAs

Summary The complete 3 untranslated region (3UTR) sequence of the human skeletal-actin gene has been compared with the corresponding regions of the rat and chicken skeletal-actin genes. This comparison reveals that the skeletal-actin 3UTR is composed of conserved and nonconserved segments. By using genomic Southern transfer blots and thermal stability (T_m) measurements, we found that the cardiac-actin gene 3UTR also consists of conserved and nonconserved segments. Comparison of human andXenopus laevis cardiac-actin mRNA sequences confirms the presence of a region of high similarity in the 3UTR. We conclude that subsegments of the 3UTRs of both skeletal- and cardiac-actin genes of birds and mammals are under considerable selective pressure. This suggests that these conserved sequences may have functional roles in actin-gene expression or regulation, and that these roles might be different for each actin isoform.