Comparative genomic identification and validation of ß-defensin genes in the Ovis aries genome.

BACKGROUND: ß-defensins are small, cationic, antimicrobial peptides found in species across the plant and animal kingdoms. In addition to microbiocidal activity, roles in immunity as well as reproduction have more recently been documented. ß-defensin genes in Ovis aries (domestic sheep) have been poorly annotated, having been identified only by automatic gene prediction algorithms. The objective of this study was to use a comparative genomics approach to identify and characterise the ß-defensin gene repertoire in sheep using the bovine genome as the primary reference. RESULTS: All 57 currently predicted bovine ß-defensin genes were used to find orthologous sequences in the most recent version of the sheep genome (OAR v4.0). Forty three genes were found to have close genomic matches (>70% similarity) between sheep and cattle. The orthologous genes were located in four clusters across the genome, with 4 genes on chromosome 2, 19 genes on chromosome 13, 5 genes on chromosome 20 and 15 genes on chromosome 26. Conserved gene order for the ß-defensin genes was apparent in the two smaller clusters, although gene order was reversed on chromosome 2, suggesting an inversion between sheep and cattle. Complete conservation of gene order was also observed for chromosome 13 ß-defensin orthologs. More structural differences were apparent between chromosome 26 genes and the orthologous region in the bovine reference genome, which is known to be copy-number variable. In this cluster, the Defensin-beta 1 (DEFB1) gene matched to eleven Bovine Neutrophil beta-Defensin (BNBD) genes on chromosome 27 with almost uniform similarity, as well as to tracheal, enteric and lingual anti-microbial peptides (TAP, EAP and LAP), suggesting that annotation of the bovine reference sequence is still incomplete. qPCR was used to profile the expression of 34 ß-defensin genes, representing each of the four clusters, in the ram reproductive tract. Distinct site-specific and differential expression profiles were detected across the reproductive tract of mature rams with preferential ß-defensin gene expression in the epididymis, recapitulating observations for orthologous genes in other species. CONCLUSIONS: This is the first comprehensive analysis of ß-defensin genes encoded by the ovine reference sequence, and the first report of an expanded repertoire of ß-defensin genes in this species. The preferential expression of these genes in the epididymis suggests a role in fertility, possibly providing immunoprotection for sperm within the female reproductive tract.

Suicide, adolescents and Puerto Rico.

Suicide is a multifactoral phenomena. This article reviews the recent literature and attempts to identify those factors which have particular relevance for Puerto Rican adolescents. Risk factors that correlate highly with the Puerto Rican experience include homosexuality, due to the hostility that the person may experience, depression, gender, prevalence of psychiatric disorders, lack of social integration and social skills, military experience, cultural and religious factors, alcoholism, substance abuse and unemployment/poverty. The literature reviewed indicates that the Puerto Rican adolescent male is in a high risk group for suicide and that the risk increases with age, sexual preference, dysfunction in the family and substance abuse.

Adolescent suicide: a review of the literature.

This article reviews the literature on the risk factors related to teen suicide in the United States and Puerto Rico. Findings indicate the interplay of multifactors including depression, homosexuality--due to the hostility that is often experienced by the person--, sexual abuse, lack of coping, social and problem-solving skills stemming from family dysfunction, feelings of isolation and helplessness, contagion, gender differences, alcohol and drug abuse, psychiatric disorders, biological factors, as well as natural disasters. Included in this report are some statistics on the prevalence of suicide among teens and in the military.

Avoidance of phrenic nerve paresis during continuous supraclavicular regional anaesthesia.

This study sought to determine whether it is possible reliably to avoid phrenic nerve block using the bent needle technique for continuous supraclavicular brachial plexus anaesthesia. In a prospective study, 100 patients undergoing a variety of upper extremity surgical procedures were studied. Ultrasound examinations of the patients' diaphragms were performed after insertion of an anaesthetic block, and were repeated a day later with the analgesic block working. Three phrenic nerve blocks were detected in different patients. Factors were identified in all three cases that we think contributed to the phrenic nerve blocks. We think it is possible to provide continuous supraclavicular regional anaesthesia and analgesia for a wide range of upper extremity operations without phrenic nerve blockade.

Osteoporosis risk assessment and treatment intervention after hip or shoulder fracture. A comparison of two centres in the United Kingdom.

This study compares the investigation of and treatment for osteoporosis in two groups of fracture patients at two orthopaedic centres in the UK. One centre had a formal fracture liaison service (FLS) responsible for screening fracture patients for osteoporosis. The other centre relied upon individual clinicians to initiate investigation or treatment for osteoporosis in patients following fracture. Patients who had been treated in either centre for a proximal humeral or hip fracture during a 6-month period were followed up 6 months later to identify how many had received screening or treatment for osteoporosis. Information was retrieved from a prospectively compiled database or by postal questionnaire. The study revealed that in the centre with an FLS 85% of patients with a proximal humeral fracture and 20% with a hip fracture had been offered a dual-energy X-ray absorptiometry (DEXA) scan. Approximately 50% and 85%, respectively, were receiving treatment for osteoporosis 6 months following their fracture. This compared with DEXA being offered to only 6% and 9.7% of humeral and hip fracture patients, respectively, and 20% (hip) and 27% (proximal humerus) receiving osteoporosis treatment in the other centre. The presence of an FLS resulted in a considerably higher proportion of patients receiving investigation and treatment for osteoporosis following a hip or proximal humeral fracture.

Comparison of total cellular DNA, mRNA, and rRNA levels between normals and Down syndrome patients.

Two recent studies have suggested that the absolute cellular RNA and/or DNA levels in Down syndrome (DS) may be unusual compared to normals and may be linked to phenotypic expression (Pash et al., 1990; Dahl et al. 1988). We have extended these two studies by directly quantitating and comparing the total cellular mRNA, rRNA, and DNA levels in fibroblasts and lymphocytes derived from normal and DS individuals. The assay methods used, which allowed us to present the various nucleic acids levels in picograms per cell, did not reveal any significant difference between the two groups.

A homologous subfamily of satellite III DNA on human chromosomes 14 and 22.

We describe a new subfamily of human satellite III DNA that is represented on two different acrocentric chromosomes. This DNA is composed of a tandemly repeated array of diverged 5-base-pair monomer units of the sequence GGAAT or GGAGT. These monomers are organised into a 1.37-kilobase higher-order structure that is itself tandemly reiterated. Using a panel of somatic cell hybrids containing specific human chromosomes, this higher-order structure is demonstrated on chromosomes 14 and 22, but not on the remaining acrocentric chromosomes. In situ hybridisation studies have localised the sequence to the proximal p-arm region of these chromosomes. Analysis by pulsed-field gel electrophoresis (PFGE) reveals that 70-110 copies of the higher-order structure are tandemly organised on a chromosome into a major domain which appears to be flanked on both sides by non-tandemly repeated genomic DNA. In addition, some of the satellite III sequences are interspersed over a number of other PFGE fragments. This study provides fundamental knowledge on the structure and evolution of the acrocentric chromosomes, and should extend our understanding of the complex process of interchromosomal interaction which may be responsible for Robertsonian translocation and meiotic nondisjunction involving these chromosomes.

Identification of DNA sequences flanking the breakpoint of human t(14q21q) Robertsonian translocations.

We have employed molecular probes and in situ hybridization to investigate the DNA sequences flanking the breakpoint of a group of t(14q21q) Robertsonian translocations. In all the families studied, the probands were patients with Down syndrome who carried a de novo t(14q21q) translocation. The DNA probes used were two alphoid sequences, alphaRI and alphaXT, which are specific for the centromeres of chromosomes 13 and 21 and of chromosomes 14 and 22, respectively; a satellite III sequence, pTRS-47, which is specific for the proximal p11 region of chromosomes 14 and 22; and a newly defined satellite III DNA, pTRS-63, which is specific for the distal p11 region of chromosome 14. The two alphoid probes detected approximately the same amount of autoradiographic signal on the translocated chromosomes as was expected for chromosomes 14 and 21 of the originating parent, suggesting that there has been no loss of these centromeric sequences during the translocation events. Results with the two satellite III probes indicated that the domain corresponding to pTRS-47 was retained in the translocated chromosomes, whereas the domain for pTRS-63 was lost. These results have allowed us to place the translocation breakpoint between the pTRS-47 and pTRS-63 domains within the p11 region of chromosome 14.

Molecular cloning and sequence analysis of the cDNA for small proteoglycan II of bovine bone.

The cDNA for the full-length core protein of the small chondroitin sulphate proteoglycan II of bovine bone was cloned and sequenced. A 1.3 kb clone (lambda Pg28) was identified by plaque hybridization with a previously isolated 1.0 kb proteoglycan cDNA clone (lambda Pg20), positively identified previously by polyclonal and monoclonal antibody reactivity and by hybrid-selected translation in vitro [Day, Ramis, Fisher, Gehron Robey, Termine & Young (1986) Nucleic Acids Res. 14, 9861-9876]. The cDNA sequences of both clones were identical in areas of overlap. The 360-amino-acid-residue protein contains a 30-residue propeptide of which the first 15 residues are highly hydrophobic. The mature protein consists of 330 amino acid residues corresponding to an Mr of 36,383. The core protein contains three potential glycosaminoglycan-attachment sites (Ser-Gly), only one of which is within a ten-amino-acid-residue homologous sequence seen at the known attachment sites of related small proteoglycans. Comparisons of the published 24-residue N-terminal protein sequence of bovine skin proteoglycan II core protein with the corresponding region in the deduced sequence of the bovine core protein reveals complete homology. Comparison of the cDNA-derived sequences of bovine bone and human embryonic fibroblast proteoglycans shows a hypervariable region near the N-terminus. Nucleotide homology between bone and fibroblast core proteins was 87% and amino acid homology was 90%.

Identification and characterization of satellite III subfamilies to the acrocentric chromosomes.

The centromeres and the short arms of the five pairs of acrocentric chromosomes in humans are composed of tandemly ordered repetitive DNA. Previous studies have suggested that the exchanges between acrocentric chromosomes have resulted in concerted evolution of different DNA sequences in their short arms. The acrocentric chromosomes are clinically relevant since they are involved in Robertsonian translocation formation and non-disjunction resulting in aneuploidy. Here we have identified seven new satellite III repetitive DNA subfamilies, determined their nucleotide sequences and established their chromosomal distributions on the short arms of the acrocentric chromosomes. Knowledge of these related sequences may help to elucidate the molecular basis of Robertsonian translocation formation.

Isolation of the osteonectin gene: evidence that a variable region of the osteonectin molecule is encoded within one exon.

A complementary DNA clone for bovine osteonectin was used to isolate the osteonectin gene from two libraries of bovine genomic DNA fragments. Two overlapping clones were obtained whose relationship was determined by restriction mapping and sequence analysis. The two clones contain the entire osteonectin coding region spanning approximately 11 kilobases of genomic DNA. The coding region of the gene was determined, by electron microscopy and DNA sequencing, to reside in nine exons. In addition, there is at least one 5' exon interrupted by an intron in the 5'-nontranslated sequence of the gene. Excluding this 5' exon and the 3'-terminal exon, the exons are small and approximately uniform in size, averaging 130 +/- 17 base pairs. Three of the exons at the 5' end of the gene were sequenced and appear to encode discrete protein domains. For example, the putative exon 2 contains the coding region for the leader peptide of the molecule. The amino-terminal protein sequence was determined for osteonectin extracted from human, rabbit, and chicken bone and compared with those for bovine, mouse, and pig osteonectin. These data suggest that osteonectin is highly conserved between species, interspecies changes being seen primarily at the amino terminus of the protein and specifically in the region encoded by putative exon 3 in the bovine gene.

Structure and expression of osteonectin mRNA in human tissue.

A cDNA encoding osteonectin was isolated from a human bone cell cDNA library and used to examine osteonectin protein structure, mRNA structure and expression in human tissue. The deduced protein sequence shows complete identity with a recently isolated placental form and extensive homology to mouse and bovine counterparts. The protein is rich in cysteine residues, which are conserved between species except for cys 194 which is only present in the bovine. In the human, osteonectin mRNA is of two sizes, 2.3 and 3.0 kb, the former being dominant in all tissues studied. Human mRNA was detected in the Ewing sarcoma and in non-bone cell and tissue sources. The potential folded structure of osteonectin mRNA was estimated, based on computer predictions, and indicates the presence of a bulge at the 5' end of the message which includes the start of translation. Southern analysis of human genomic DNA using radiolabeled osteonectin cDNA as probe demonstrates a simple banding pattern confirming earlier studies that the osteonectin gene is present in one copy per haploid human genome.

Osteonectin promoter. DNA sequence analysis and S1 endonuclease site potentially associated with transcriptional control in bone cells.

To understand the basis of osteonectin (SPARC) transcriptional regulation, we have isolated a bovine genomic clone (lambda Og15) encoding exon 1 and 15 kilobase pairs (kb) of flanking DNA. Direct RNA sequencing of the 5' end of the osteonectin message showed it contained a sequence identical to that of a 2.4-kb EcoRI-BamHI fragment located midway in the clone lambda Og15. The results indicate exon 1 is located 10 kb away from exon 2 in the bovine genome. The DNA sequence unit CCTG is repeated five times in exon 1 which is composed exclusively of untranslated sequence. Sequence analysis of the 5'-flanking DNA revealed the presence of many regulatory motifs including a "GC" box with four overlapping SP1 consensus sequences. Immediately downstream from the GC box is a 72-base pair purine-rich stretch composed primarily of direct repeats of the sequence motifs GGGGA and GGA (GAGA box). Digestion of the flanking DNA in vitro with S1 endonuclease showed a site for the enzyme at position -55 which is just 3' to the GAGA box. Chimeric chloramphenicol acetyltransferase constructs were prepared containing the S1-sensitive site and showed substantial transcriptional activity in UMR-106 and fetal and adult human bone cells which are known to be high producers of the protein. The results indicate a potential regulatory activity of the S1 site in osteonectin gene activation.

Adolescent suicide: a review of the literature

This article reviews the literature on the risk factors related to teen suicide in the United States and Puerto Rico. Findings indicate the interplay of multifactors including depression, homosexuality--due to the hostility that is often experienced by the person--, sexual abuse, lack of coping, social and problem-solving skills stemming from family dysfunction, feelings of isolation and helplessness, contagion, gender differences, alcohol and drug abuse, psychiatric disorders, biological factors, as well as natural disasters. Included in this report are some statistics on the prevalence of suicide among teens and in the military

Suicide, adolescents and Puerto Rico

Suicide is a multifactoral phenomena. This article reviews the recent literature and attempts to identify those factors which have particular relevance for Puerto Rican adolescents. Risk factors that correlate highly with the Puerto Rican experience include homosexuality, due to the hostility that the person may experience, depression, gender, prevalence of psychiatric disorders, lack of social integration and social skills, military experience, cultural and religious factors, alcoholism, substance abuse and unemployment/poverty. The literature reviewed indicates that the Puerto Rican adolescent male is in a high risk group for suicide and that the risk increases with age, sexual preference, dysfunction in the family and substance abuse