Novel transcribed sequences represented in the complex genomic region 5q13.

YACs from the complex repetitive human genomic region 5q13, spanning the spinal muscular atrophy (SMA) locus, have been searched for transcribed sequences using the method of End Ligation Coincident Sequence Cloning. Six transcripts (PT1-6) have been identified, three of which (PT4, PT5 and PT6) are novel. Five of these elements hybridise to multiple loci in 5q13, but PT5 is single copy and maps very close to markers that show linkage disequilibrium with SMA.

Detection of germ cell-derived proteins in testicular interstitial fluid: potential for monitoring spermatogenesis in vivo.

The aim of the present study was to assess whether proteins secreted by the seminiferous tubules (ST) can be detected in testicular interstitial fluid (IF) and testicular (TV), spermatic (SV), and peripheral venous (PV) plasma from adult rats. An antiserum was raised against seminiferous tubule-conditioned medium (STCM) prepared from adult rats and used in conjunction with Western blot analysis to screen IF and blood samples resolved by one-dimensional (1-D) and two-dimensional (2-D) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Samples of IF and PV were analyzed from control adult rats and rats exposed to scrotal heating (43 degrees C for 30 minutes) 24 hours earlier to ascertain whether damage to spermatogenesis would affect 'leakage' of proteins from the seminiferous tubules. In all control rats, the STCM antiserum specifically detected three proteins in testicular IF with molecular weights of 24, 16, and 14 kDa, respectively. Heat treatment increased the abundance of these proteins and induced the appearance of several other less-abundant proteins, all with molecular masses below 25 kDa. Two of the proteins present in IF were identified, the 24-kDa protein as phosphatidylethanolamine-binding protein (PEBP), and the 14-kDa protein as an androgen-regulated protein (ARP-2). Both of these proteins have been shown in previous studies to be secreted by round spermatids. Our results suggest that germ cell secretory products can gain access to the interstitium under both normal physiological conditions and more easily after induction of damage to spermatogenesis. The antiserum was unable to detect any ST-derived proteins in blood, although it is likely that this result may be due to insensitivity of the presently used techniques. The development of specific immunoassays for germ cell-secreted proteins (e.g., PEBP and ARP-2) should enable more definitive assessment of whether proteins secreted by the seminiferous epithellum can be measured in blood and thus provide a potential means of monitoring spermatogenesis.

The dissemination of the British Guideline on the Management of Asthma 2003.

The BTS/SIGN British Guideline on the Management of Asthma was published in February 2003 (4). If health outcomes are to be influenced successfully, dissemination of the guideline and implementation of recommendations is essential. We report the dissemination activities undertaken during the 18 weeks following the guideline launch. To facilitate implementation a range of educational materials were produced reflecting the key messages from the guideline. In addition to postal mailing of the guideline to appropriate healthcare professionals, both educational materials and the guidelines were made freely available from the BTS and SIGN websites. In total, 135,710 copies of the guideline and 90,198 copies of the Quick Reference Guide were downloaded in the first 18 weeks, representing a considerable increase over the number of copies of the 1997 guidelines disseminated by mailing alone. Large quantities of educational materials were downloaded with many used for teaching purposes. An on-line survey suggested that most respondents rated the materials as useful or very useful. Using websites to disseminate guidelines is a cost-effective method of informing health professionals of their content and is a more active process than the passive receipt of mailed copies. The availability of interactive educational materials for use in teaching appears to have been popular.

Evaluation of possible determinants and consequences of Leydig cell heterogeneity in man.

Leydig cells in the human testis are highly heterogeneous, consisting of variably staining light and dark cells. The basis for this difference is unknown. The present study has assessed whether differing numbers or proportions of dark and light Leydig cells are related: (1) to the pronounced inter-individual variation in testosterone production by isolated Leydig cells, and (2) to differences in structural composition of the testis. Testes (paired weight 6.6-59.48 g) were obtained from 27 men aged 72.9 +/- 9.5 years (range 54-89 years) undergoing orchidectomy as primary treatment for prostatic cancer. Leydig cells were isolated by Percoll-purification and cultured for 20 h under basal and hCG-stimulated conditions. The proportion of light and dark Leydig cells isolated by this method was shown to reflect their proportions in situ, based on the morphometric analysis of fixed testicular tissue from the same men. Leydig cells isolated from all testes produced testosterone in vitro and responded to stimulation by hCG, though the amounts of testosterone produced varied widely between subjects. Because of the latter, samples were grouped into 'low' (n = 9), 'medium' (n = 11) and 'high' (n = 7) groups on the basis of their testosterone production. These groups did not differ in their age, testicular size or gross testicular morphology, though men in the 'high' group tended to have more total Leydig cells per testis. However, there was no overall correlation between testosterone production by isolated Leydig cells and the numbers of light or dark Leydig cells or their ratio or the total number of Leydig cells per testis. The relationship between the volume of light and dark Leydig cells and testicular composition was also assessed. The volume of both types of Leydig cells was strongly correlated (p < 0.001) with the volume of germ cells, but otherwise light and dark Leydig cells correlated positively with different structures. Thus, the volume of light Leydig cells correlated (p < 0.001) with the volume of blood vessels and of peritubular tissue whereas the volume of dark Leydig cells correlated (p < 0.01) with that of the tubular lumen. These differences could indicate differences in regulation and/or function of light and dark Leydig cells. However, the present data do not support the idea that light and dark Leydig cells may differ in their steroidogenic capacity.

Alu-based vectorettes and splinkerettes. More efficient and comprehensive polymerase chain reaction amplification of human DNA from complex sources.

Alu-polymerase chain reaction (PCR) is widely used to amplify human specific fragments from complex heterologous DNAs, such as somatic cell hybrids or yeast artificial chromosome (YAC) recombinants, but the fragments amplified are limited in number and are nonrepresentative. This report describes a modified one-sided alu-PCR technique, which offers better representation of amplified sequences while maintaining human specificity. The method relies on the ligation of partially mismatched double-stranded oligonucleotides (vectorettes or splinkerettes) to endonuclease-restricted DNA and universal priming with a single alu-consensus primer, the complement to which is the unpaired region. Alu-vectorette and alu-splinkerette-PCR of two somatic cell hybrids results in a greater complexity of products than alu-PCR alone. The advantage of alu-splinkerette over alu-vectorette-PCR is the elimination of nonspecific priming owing to the presence of the vectorette primer and to an increase in the product size range, a consequence of the difference in the splinkerette design. Alu-splinkerette-PCR is a useful technique for generating new and more comprehensive markers of the human sequences contained in somatic cell hybrids and YACs.

Comparative analysis of proteins secreted in vitro by isolated seminiferous tubules from man and the rat.

The aims of this study were to provide an overall comparison of the in-vitro protein secretory profile of seminiferous tubules (ST) isolated from man and the rat, and to identify specific proteins secreted by both species. Two-dimensional SDS PAGE was used to compare the profile of proteins secreted by cultured ST from 15 men undergoing orchidectomy with those secreted by ST isolated at stages VI-VIII of the spermatogenic cycle from normal adult rats, and by ST at the same stages isolated from rats pretreated with methoxyacetic acid (MAA) in order to deplete both pachytene spermatocytes and round spermatids. Two abundant groups of proteins not present in the medium of rat ST were secreted consistently by human ST, though the profile of proteins secreted by human ST was otherwise more variable than in the rat. Twelve proteins secreted by ST isolated from humans were identified as possible homologues of rat ST-secreted proteins, including the major rat Sertoli cell products SGP-1 and SGP-2, and an androgen-regulated protein which derives in the rat from round spermatids. Otherwise, the majority of the 12 potential homologues corresponded to proteins which in the rat are secreted by ST from both normal and germ cell-depleted testes or by ST from germ cell-depleted testes only, suggesting that they are probably secreted by Sertoli cells and/or peritubular cells. Based on the potential homology of the proteins identified, our results suggest, first, that these proteins may play an important role in spermatogenesis and second, that the profile of proteins secreted by human ST is more akin to that secreted by ST isolated from germ cell-depleted rats.

Polymerase chain reaction screening for Y chromosome microdeletions: a first step towards the diagnosis of genetically-determined spermatogenic failure in men.

Overall, approximately 11% of men attending infertility clinics suffer unexplained oligo- or azoospermia. Cytogenetic observations of loss of the distal portion of the Y chromosome long arm (Yq) were found to be associated with disrupted spermatogenesis. The existence of a gene locus involved in the regulation of spermatogenesis, the azoospermia factor (AZF), was thus postulated. It is suggested that microdeletions, or mutations, at the AZF locus could result in impaired spermatogenesis in chromosomally normal men. In order to test this hypothesis we have carried out Y chromosome genetic screening of 100 oligo- or azoospermic 46XY patients. We have also assessed phenotype/genotype relationships in those patients whose infertility has an underlying genetic aetiology. Patients were screened by polymerase chain reaction (PCR) with a set of Y chromosome-specific sequence tagged sites (STS) for submicroscopic deletions of their Y chromosome. Our results show that as many as 8% of cases of unexplained male infertility may have an underlying genetic aetiology related to microdeletions in two specific regions of the Y chromosome. Positive results from such a screen will be important when deciding the suitability of a patient for assisted conception schemes such as intracytoplasmic sperm injection.

Cloning the shared components of complex DNA resources.

The complex and repetitive nature of mammalian genomes limits the ability of conventional molecular techniques to recover sequences of interest. Here we describe a rapid and simple procedure for the direct cloning of sequences which are coincident between DNA mixtures of whole genome complexity. The system, called end ligation coincident sequence cloning (EL-CSC), can enrich coincident DNA by greater than 10(6)-fold and overcomes problems associated with repetitive elements. Applying EL-CSC to various paired DNA resources enables the facile cloning of both genomic markers and novel genes. To demonstrate the power of the method we have i) selectively purified single copy sequences from a complete genome, and ii) isolated gene fragments from 260 kb of cloned genomic DNA.

Mapping of retrotransposon sequences in the unstable region surrounding the spinal muscular atrophy locus in 5q13.

The mutation that underlies the autosomal recessive disorder spinal muscular atrophy (SMA) is located on chromosome 5q13. Recent studies show that SMA patients frequently have deletions and rearrangements in this region compared to normal controls. During the isolation of candidate cDNAs for the disease, we identified a sequence that shows high homology to the THE-1 retrotransposon gene family. Using YAC fragmentation techniques, we have refined the localization of this sequence to the domain known to show instability in SMA patients. The implication of these results for the mechanism of the mutation in SMA is discussed.