Treatment of osteoporosis using a selective androgen receptor modulator ostarine in an orchiectomized rat model.

PURPOSE: The selective androgen receptor modulator ostarine has been shown to have advantageous effects on skeletal tissue properties, reducing muscle wasting and improving physical function in males. However, data on effects in male osteoporosis remain limited. In this study, the effects of ostarine on osteoporotic bone were evaluated in a rat model of male osteoporosis and compared with those of testosterone treatments. METHODS: Eight-month-old male Sprague-Dawley rats were either non-orchiectomized to serve as a healthy control (Non-Orx, Group 1) or orchiectomized (Orx, Groups 2-6) and then grouped (n = 15/group): (1) Non-Orx, (2) Orx, (3) Ostarine Therapy, (4) Testosterone Therapy, (5) Ostarine Prophylaxis and (6) Testosterone Prophylaxis. Prophylaxis treatments started directly after orchiectomy and continued for 18 weeks, whereas Therapy treatments were initiated 12 weeks after Orx. Ostarine and Testosterone were applied orally at daily doses of 0.4 and 50 mg/kg body weight, respectively. The lumbar vertebral bodies and femora were analyzed using biomechanical, micro-CT, ashing, and gene expression analyses. RESULTS: Ostarine Prophylaxis showed positive effects in preventing osteoporotic changes in cortical and trabecular bone (femoral trabecular density: 26.01 ± 9.1% vs. 20.75 ± 1.2% in Orx and in L4: 16.3 ± 7.3% vs 11.8 ± 2.9% in Orx); biomechanical parameters were not affected; prostate weight was increased (0.62 ± 0.13 g vs 0.18 ± 0.07 g in Orx). Ostarine Therapy increased solely the cortical density of the femur (1.25 ± 0.03 g/cm3 vs. 1.18 ± 0.04 g/cm3 in Orx); other bone parameters remained unaffected. Testosteron Prophylaxis positively influenced cortical density in femur (1.24 ± 0.05 g/cm3 vs. 1.18 ± 0.04 g/cm3 in Orx); Test. Therapy did not change any bony parameters. CONCLUSION: Ostarine Prophylaxis could be further investigated as a preventative treatment for male osteoporosis, but an androgenic effect on the prostate should be taken into consideration, and combination therapies with other anti-osteoporosis agents could be considered.

Misidentification as Pseudomonas aeruginosa in hospital water supply samples.

Drinking water in hospitals is often tested for Pseudomonas aeruginosa because of its virulence potential. This article describes a case where, based on EN ISO 16266, seven of 11 (64%) samples taken simultaneously from the drinking water system at a single hospital tested positive for P. aeruginosa. This resulted in extensive investigations and interventions, and a number of measures were implemented. However, supplementary analyses with more discriminatory power (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, 16S-rRNA sequencing) ruled out P. aeruginosa completely. The authors wish to raise awareness of this problem, and suggest that diagnostic uncertainty of results obtained by EN ISO 16266 should be indicated on laboratory reports. Wrongly assuming the presence of P. aeruginosa in hospital water supply systems can lead to unnecessary control measures, as analytical uncertainty massively influences the health risk assessment and the remediation measures initiated in medical environments.

A combined treatment with selective androgen and estrogen receptor modulators prevents bone loss in orchiectomized rats.

PURPOSE: Enobosarm (EN), a selective androgen receptor modulator and raloxifene (RAL), a selective estrogen receptor modulator, have been shown to improve bone tissue in osteoporotic males. The present study evaluated the effects of a combination therapy of EN and RAL on bone properties in orchiectomized rats compared to the respective single treatments. METHODS: Eight-month-old male Sprague-Dawley rats were either left intact (Non-Orx) or orchiectomized (Orx). The Orx rats were divided into four groups (n = 15 each): 1) Orx, 2) EN treatment (Orx + EN), 3) RAL treatment (Orx + RAL), 4) combined treatment (Orx + EN + RAL). EN and RAL (0.4 mg and 7 mg/kg body weight/day) were applied immediately after Orx with a soy-free pelleted diet for up to 18 weeks. The lumbar spine and femora were examined by micro-CT, biomechanical, histomorphological, ashing, and gene expression analyses. RESULTS: EN exhibited an anabolic effect on bone, improving some of its parameters in Orx rats, but did not affect biomechanical properties. RAL exhibited antiresorptive activity, maintaining the biomechanical and trabecular parameters of Orx rats at the levels of Non-Orx rats. EN + RAL exerted a stronger effect than the single treatments, improving most of the bone parameters. Liver weight increased after all treatments; the kidney, prostate, and levator ani muscle weights increased after EN and EN + RAL treatments. BW was reduced due to a decreased food intake in the Orx + RAL group and due a reduced visceral fat weight in the Orx + EN + RAL group. CONCLUSION: The EN + RAL treatment appeared to be promising in preventing male osteoporosis, but given the observed side effects on liver, kidney, and prostate weights, it requires further investigation.

Marsupial uncoupling protein 1 sheds light on the evolution of mammalian nonshivering thermogenesis.

Brown adipose tissue expressing uncoupling protein 1 (UCP1) is responsible for adaptive nonshivering thermogenesis giving eutherian mammals crucial advantage to survive the cold. The emergence of this thermogenic organ during mammalian evolution remained unknown as the identification of UCP1 in marsupials failed so far. Here, we unequivocally identify the marsupial UCP1 ortholog in a genomic library of Monodelphis domestica. In South American and Australian marsupials, UCP1 is exclusively expressed in distinct adipose tissue sites and appears to be recruited by cold exposure in the smallest species under investigation (Sminthopsis crassicaudata). Our data suggest that an archetypal brown adipose tissue was present at least 150 million yr ago allowing early mammals to produce endogenous heat in the cold, without dependence on shivering and locomotor activity.

Architecture and anatomy of the genomic locus encoding the human leukemia-associated transcription factor RUNX1/AML1.

The RUNX1 gene on human chromosome 21q22.12 belongs to the 'runt domain' gene family of transcription factors (also known as AML/CBFA/PEBP2alpha). RUNX1 is a key regulator of hematopoiesis and a frequent target of leukemia associated chromosomal translocations. Here we present a detailed analysis of the RUNX1 locus based on its complete genomic sequence. RUNX1 spans 260 kb and its expression is regulated through two distinct promoter regions, that are 160 kb apart. A very large CpG island complex marks the proximal promoter (promoter-2), and an additional CpG island is located at the 3' end of the gene. Hitherto, 12 different alternatively spliced RUNX1 cDNAs have been identified. Genomic sequence analysis of intron/exon boundaries of these cDNAs has shown that all consist of properly spliced authentic coding regions. This indicates that the large repertoire of RUNX1 proteins, ranging in size between 20-52 kDa, are generated through usage of alternatively spliced exons some of which contain in frame stop codons. The gene's introns are largely depleted of repetitive sequences, especially of the LINE1 family. The RUNX1 locus marks the transition from a ~1 Mb of gene-poor region containing only pseudogenes, to a gene-rich region containing several functional genes. A search for RUNX1 sequences that may be involved in the high frequency of chromosomal translocations revealed that a 555 bp long segment originating in chromosome 11 FLI1 gene was transposed into RUNX1 intron 4.1. This intron harbors the t(8;21) and t(3;21) chromosomal breakpoints involved in acute myeloid leukemia. Interestingly, the FLI1 homologous sequence contains a breakpoint of the t(11;22) translocation associated with Ewing's tumors, and may have a similar function in RUNX1.

The DNA sequence of human chromosome 21.

Chromosome 21 is the smallest human autosome. An extra copy of chromosome 21 causes Down syndrome, the most frequent genetic cause of significant mental retardation, which affects up to 1 in 700 live births. Several anonymous loci for monogenic disorders and predispositions for common complex disorders have also been mapped to this chromosome, and loss of heterozygosity has been observed in regions associated with solid tumours. Here we report the sequence and gene catalogue of the long arm of chromosome 21. We have sequenced 33,546,361 base pairs (bp) of DNA with very high accuracy, the largest contig being 25,491,867 bp. Only three small clone gaps and seven sequencing gaps remain, comprising about 100 kilobases. Thus, we achieved 99.7% coverage of 21q. We also sequenced 281,116 bp from the short arm. The structural features identified include duplications that are probably involved in chromosomal abnormalities and repeat structures in the telomeric and pericentromeric regions. Analysis of the chromosome revealed 127 known genes, 98 predicted genes and 59 pseudogenes.

Characterization of a novel gene, C21orf6, mapping to a critical region of chromosome 21q22.1 involved in the monosomy 21 phenotype and of its murine ortholog, orf5.

Phenotypic and molecular analyses of patients with partial chromosome 21 monosomy enabled us to define a region, spanning 2.4 Mb between D21S190 and D21S226, associated with arthrogryposis, mental retardation, hypertonia, and several facial anomalies. The markers of the region were used to screen a total human PAC library (Ioannou, RZPD). We isolated 57 PACs, which formed primary contigs. EST clusters (UNIGENE collection) located in a 6-Mb interval, between D21S260 and D21S263, were mapped in individual bacterial clones. We mapped the WI-17843 cluster to the PAC clone J12100, which contains the two anchor markers LB10T and LA329. The open reading frame extends over 960 bp, with three putative start codons. The 1695-bp cDNA containing a polyadenylation signal should correspond to the full-length cDNA. From the genomic sequence, we deduced that the gene contained five exons and that there was a putative promoter sequence upstream from exon 1. In silico screening of DNA databases revealed similarity with a murine EST. The corresponding cDNA (1757 bp) sequence was very similar (>85%) to the human cDNA and had an open reading frame of 876 nucleotides. Somatic hybrid mapping localized the cDNA to mouse chromosome 16. EST analyses and RT-PCR indicated that the third exon in the human gene (exon 2 in the mouse) undergoes alternative splicing. Northern blot hybridization showed that the gene was ubiquitously expressed in humans and mice. The longest mouse clone was used to generate riboprobes, which were hybridized to murine embryos at stages E-9.5, E-10.5, E-12.5, E-13.5, and E-14.5-15, to study the pattern of expression during development. Ubiquitous labeling was observed, with strong signals restricted to limited areas of the telencephalon, the mesencephalon, and the interrhombomeric regions in the central nervous system, and other regions of the body such as the limb buds, branchial arches, and somites.

Genomic organization of a 225-kb region in Xq28 containing the gene for X-linked myotubular myopathy (MTM1) and a related gene (MTMR1).

MTM1 is responsible for X-linked recessive myotubular myopathy, which is a congenital muscle disorder linked to Xq28. MTM1 is highly conserved from yeast to humans. A number of related genes also exist. The MTM1 gene family contains a consensus sequence consisting of the active enzyme site of protein tyrosine phosphatases (PTPs), suggesting that they belong to a new family of PTPs. Database searches revealed homology of myotubularin and all related peptides to the cisplatin resistance-associated alpha protein, which implicates an as yet unknown function. In addition, homology to the Sbf1 protein (SET binding factor 1), involved in the oncogenic transformation of fibroblasts and differentiation of myoblasts, was also evident. We describe 225 kb of genomic sequence containing MTM1 and the related gene, MTMR1, which lies 20 kb distal to MTM1. Although there is only moderate conservation of the exons, the striking similarity in the gene structures indicates that these two genes arose by duplication. Calculations suggest that this event occurred early in evolution long before separation of the human and mouse lineages. So far, mutations have been identified in the coding sequence of only 65% of the patients analyzed, indicating that the remaining mutations may lie in noncoding regions of MTM1 or possibly in MTMR1. Knowledge of the genomic sequence will facilitate mutation analyses of the coding and noncoding sequences of MTM1 and MTMR1.

The neural cell adhesion molecule L1: genomic organisation and differential splicing is conserved between man and the pufferfish Fugu.

The human gene for the neural cell adhesion molecule L1 is located on Xq28 between the ALD and MeCP2 loci. Mutations in the L1 gene are associated with four related neurological disorders, X-linked hydrocephalus, spastic paraplegia (SPG1), MASA syndrome, and X-linked corpus callosum agenesis. The clinical relevance of L1 has led us to sequence the L1 gene in human and to investigate its conservation in the vertebrate model genome of the pufferfish, Fugu rubripes (Fugu), a species with a compact genome of around 40Mb. For this purpose we have sequenced a human and a Fugu cosmid clone containing the corresponding L1 genes. For comparison, we have also amplified and sequenced the complete Fugu L1 cDNA. We find that the genomic structure of L1 is conserved. The human and Fugu L1 gene both have 28 exons of nearly identical size. Differential splicing of exons 2 and 27 is conserved over 430 million years, the evolutionary time span between the teleost Fugu and the human L1 gene. In contrast to previously published Fugu genes, many introns are larger in the Fugu L1 gene, making it slightly larger in size despite the compact nature of the Fugu genome. Homology at the amino acid and the nucleotide level with 40% and 51%, respectively, is lower than that of any previously reported Fugu gene. At the level of protein structure, both human and Fugu L1 molecules are composed of six immunoglobulin (Ig)-like domains and five fibronectin (Fn) type III domains, followed by a transmembrane domain and a short cytoplasmic domain. Only the transmembrane and the cytoplasmic domains are significantly conserved in Fugu, supporting their proposed function in intracellular signalling and interaction with cytoskeletal elements in the process of neurite outgrowth and fascicle formation. Our results show that the cytoplasmic domain can be further subdivided into a conserved and a variable region, which may correspond to different functions. Most pathological missense mutations in human L1 affect conserved residues. Fifteen out of 22 reported missense mutations alter amino acids that are identical in both species.