The roles of RNA-binding proteins in spermatogenesis and male infertility.

RNA-binding proteins are essential for spermatogenesis: they are required in the nucleus of germ cells, for the production of specific mRNA isoforms, and in the cytoplasm - where proteins required for chromatin condensation and for changes in cell morphology are translated long after transcription ceases. Some of the RNA targets and the RNA-binding proteins themselves have been identified recently. Both nuclear and cytoplasmic proteins are affected in examples of azoospermia in men.

The tumour-suppressor protein ASPP1 is nuclear in human germ cells and can modulate ratios of CD44 exon V5 spliced isoforms in vivo.

The ASPP1 (Apoptosis Stimulating Protein of p53) protein is an important tumour-suppressor. We have detected a novel protein interaction between the human ASPP1 (hASPP1) protein and the predominantly nuclear adaptor protein SAM68. In the human testis, full-length endogenous hASPP1 protein is located in the nucleus like SAM68, predominantly within meiotic and postmeiotic cells. Mouse ASPP1 (mASPP1) protein is mainly expressed in the brain and testis. The interaction with nuclear SAM68 is likely to be restricted to human germ cells, since endogenous mASPP1 protein is exclusively cytoplasmic. The C-terminal region of hASPP1 efficiently targeted a fused GFP molecule to the nucleus, whereas the N-terminus of hASPP1 targeted GFP to the cytoplasm. In the context of the full-length molecule this cytoplasmic targeting sequence is dominant in HEK293 and Saos-2 cells, since full-length hASPP1-GFP is almost exclusively cytoplasmic. Despite its predominantly cytoplasmic location, we show that ASPP1-GFP expression in HEK293 cells can regulate the ratio of alternative spliced isoforms derived from a pre-mRNA regulated downstream of cytoplasmic signalling pathways, and our data suggest that ASPP1 may operate in this case downstream or parallel to RAS signalling pathways.

Nucleation and growth on defect sites: experiment-theory comparison for Pd/MgO(001).

It is well established that nucleation of metal clusters on oxide and halide surfaces is typically dominated by defect sites. Rate equation models of defect nucleation have been developed and applied to these systems. By comparing the models with nucleation density experiments, energies for defect trapping, adsorption, surface diffusion and pair binding have been deduced in favourable cases, notably for Pd deposited on Ar-cleaved MgO(001). However, the defects responsible remain largely unknown. More recently, several types of ab initio calculation have been presented of these energies for Pd and related metals on MgO(001) containing several types of surface defect; these calculated values are surveyed, and some are widely divergent. New rate equation nucleation density predictions are presented using the calculated values. Some calculations, for some defect types, are much closer to experiment than others; the singly charged F(s)(+) centre and the neutral divacancy emerge as candidate defects. In these two cases, the Pd/MgO(001) nucleation density predictions agree well with experiment, and the corresponding surface defects deserve to be taken seriously. Energy and entropy values are discussed in the light of differences in calculated charge redistribution between the metal atoms, clusters and (charged) surface defects, and (assumed or calculated) cluster geometries.

The RNA-binding specificity of the mouse Dazl protein.

DAZ is an RNA-binding protein encoded by a region on the Y chromosome implicated in infertility, and DAZ-like (Dazl) proteins are master regulators of germ line gene expression in all animals. In mice Dazl is only expressed in germ cells and is necessary for meiosis. A dual approach was taken to understand the RNA-binding specificity of the Dazl protein: (i) traditional SELEX and (ii) a novel tri-hybrid screen. Both approaches led to the same conclusion, namely that Dazl binds oligo(U) stretches interspersed by G or C residues. In a directed tri-hybrid assay the strongest interaction was with the consensus (GUn)n. This motif is found in the 5' UTR of CDC25C whose homologue is thought to be the target of Boule, the Dazl homologue in flies. CDC25C 5' UTR also interacted specifically with Dazl in vitro. The tri-hybrid screen retrieved UTRs of known genes that may be physiological substrates of Dazl.

From the Journals.

Bacterial infection in chronic wounds The care of patients with atopic eczema Venous incompetence and the results of foot volumetry Mortality and surgical wound infection The use of interleukin-1ß in the treatment of pressure sores.

The management and treatment of eczema.

This article outlines the principles of the treatment and management of eczema including the practical aspects of nursing patients with eczema. Awareness of basic dermatological nursing skills is important for all nurses as is an understanding of how to provide support and advice to patients and their families.

Knowledge needed to educate patients about psoriasis.

Psoriasis is a common skin disorder: two out of every 100 people in Europe have the condition. Nurses will encounter people with psoriasis in their work and everyday lives. This paper gives an overview of the knowledge nurses need in order to educate patients about psoriasis. Many of the lifestyle changes vary depending on the individual patient, the extent of the condition, the patient's ability to cope, and the support he or she receives from family and friends.

Kinetically suppressed ostwald ripening of Ge/Si(100) hut clusters.

Low area density Ge/Si(100) hut cluster ensembles are stable during days-long growth temperature anneals. Real-time scanning tunneling microscopy shows that all islands grow slowly at a decreasing rate throughout the anneal. Island growth depletes the Ge supersaturation that, in turn, reduces the island growth rate. A mean-field facet nucleation and growth model quantitatively predicts the observed growth rate. It shows that Ostwald ripening is kinetically suppressed for Ge supersaturations high enough to support a critical nucleus size less than the smallest facet.

The Wilms tumour suppressor protein WT1 (+KTS isoform) binds alpha-actinin 1 mRNA via its zinc-finger domain.

Mutations in WT1 are associated with developmental syndromes that affect the urogenital system and neoplasms, including Wilms tumour, acute myeloid leukemia, and breast and prostate cancers. The WT1 protein belongs to the early growth response family of zinc-finger transcription factors. Uniquely to WT1, an evolutionarily conserved alternative splice event inserts the tripeptide KTS, between zinc fingers 3 and 4. Whereas -KTS isoforms bind DNA and activate or repress transcription, +KTS isoforms bind DNA less efficiently and interact with splice factors and RNA in vitro and in vivo. Although candidate DNA targets have been found, physiological mRNA targets are yet to be defined. We examined the distribution of WT1 in ribonucleoprotein (RNP) complexes in nuclear extract prepared from M15 cells, a mouse mesonephric fetal kidney cell line. WT1 cofractionated with the splice factor PSF in large RNP particles >or=2 MDa. We also found that PSF co-immunoprecipitated with WT1, suggesting a functional interaction between these 2 multifunctional proteins. Using yeast three-hybrid library constructed from the co-immunoprecipitated RNA we found that WT1 (+KTS) binds close to or at the start codon of alpha-actinin 1 (ACTN1) mRNA. A band shift assay confirmed the ability of the WT1 zinc-finger domain (+KTS) to bind this sequence in vitro. ACTN1 is the first likely physiological mRNA target of WT1.

The MIDAS project at ASU: John Cowley's vision and practical results.

An overview of the conception and development of the MIDAS system at Arizona State University is given: a Microscope for Imaging, Diffraction and Analysis of Surfaces. John Cowley's vision in the early 1980s was ambitious and far-reaching, and it was because of him the authors came to ASU. We were centrally involved in the design and implementation of MIDAS from the mid 1980s onwards; the novel design features are briefly reviewed. Practical results obtained using this instrument are listed, and the scope for future development and applications are indicated. While it is clear that many new results have been demonstrated, even more possibilities still remain to be explored. Some comments are made about the feasibility of such developments in the light of competing instrumentation.

Home-based care for orphaned children infected with HIV/AIDS in Uganda.

The primary aim of this paper is to describe an outreach programme from a main state hospital in sub-Saharan Africa, which has been running for three years. This programme is based in Mulago Hospital, Kampala, Uganda and cares for up to 200 children infected with HIV/AIDS in their home. We describe the clinic and how we meet the families and enrol them, the infrastructure of the programme and the personnel involved. Children and their families receive physical, psychological and social care and we describe each aspect of this. The knowledge base about older children with AIDS in Africa is scarce and the secondary aim of this paper is to publish observations that were made while providing care. This includes demographics and the health problems encountered among children living with HIV/AIDS in a resource-poor setting who do not receive antiretroviral medication. Finally, we discuss the strengths and weaknesses of this model of care and the prerequisites to setting up a similar model.

Bone density at the os calcis: reference values, reproducibility, and effects of fracture history and physical activity.

AIMS: To establish reference values for bone mineral density (BMD) measured at the os calcis (OC) in healthy UK Caucasian children. Secondary objectives were to assess the reproducibility of the measurement and the effects of fracture history and habitual physical activity. METHODS: A total of 403 children aged 5-18 were studied. Main outcome measures were: BMDoc measured by peripheral DXA, total BMD measured by whole body axial scanner, age, anthropometry, pubertal status, self-reported fracture history, and physical activity (PA) expressed as a three point score. RESULTS: Complete data were available on 171 girls and 123 boys free of a history of fracture. BMDoc was related positively to age, body size, and total BMD, and could be predicted using a proportional model based on height alone (R2: 65% girls, 77% boys). Mean BMDoc appears to plateau in girls at 15 years and attain a value that concurs with the mean peak value in adult women. The 95% limits of agreement in repeated measures were -0.029 to 0.029 g/cm2 (n = 53). Compared with sedentary children, those doing regular sports or PA for more than five hours a week had an increased BMDoc (by about 0.03 g/cm2 or about 7% of the overall mean). A history of fracture (n = 81) was associated with a reduced BMDoc in boys but not in girls, though our study may have been underpowered for a subgroup analysis. CONCLUSIONS: BMDoc can be measured easily and quickly in children older than 5 years and provides an objective measure of areal bone density for clinical and research studies using a reference range derived from its relation to height.

Lessons from knockout and transgenic mice for infertility in men.

This review concentrates on the clear cases where knocking out a gene in mice has caused male infertility and thus comes near to proving that the gene plays a role in the development of sperm. Knockout mice have been created with primary defects at every stage of spermatogenesis thus creating a framework for decoding the genetic hierarchy that causes male germ cell differentiation. As well as defining essential genes in vivo experiments have defined promoter and untranslated sequences responsible for the expression of proteins at all the spermatogenic stages. In conclusion knockout mice remain the ultimate test of spermatogenic hypotheses as well as providing detailed information about this complex process.

Island nucleation in a reactive two-component system.

The flux and temperature dependence of titanium silicide islands formed by reactive deposition near 500 degrees C indicate a critical nucleus containing 2 Ti atoms and a single activation energy of E(d) + 1/2E(2) = 1.4 +/- 0.2 eV, where E(d) and E(2) are the surface diffusion and cluster binding energies, respectively. These values are not consistent with STM observations of Ti dimer-vacancy hopping at lower temperatures and show that silicide island nucleation involves a different, highly mobile Ti species.

Selective nucleation and controlled growth: quantum dots on metal, insulator and semiconductor surfaces.

Nucleation and growth models are well developed for nucleation on homogeneous substrates, and they can typically be described in terms of three energy parameters. Nucleation on substrates containing point-defect traps has been investigated, at the cost of introducing more energy parameters. This paper outlines the quantitative description of such growth models, using rate and rate-diffusion equations, in terms of energies for individual surface processes, with examples taken from metal-metal, metal-insulator and semiconductor growth. The challenge to modelling is to describe the large range of length and time-scales in thin-film fabrication and degradation, without relying on too many (unknown) material parameters, which often occur in combination. Separating them into elementary processes often proves to be a challenge. One typically requires selective nucleation using patterned substrates, in combination with controlled, self-organized, growth for reliable nanotechnology. Reconstructed semiconductor surfaces offer both a further challenge to modelling and an opportunity for future technology; these paradoxes are discussed briefly.

RBMY, a probable human spermatogenesis factor, and other hnRNP G proteins interact with Tra2beta and affect splicing.

The RBMY gene family is found on the Y chromosome of all mammals, and microdeletions are strongly associated with infertility in men. RBMY expresses RBM only in the nuclei of germ cells, whereas its X chromosome homologue, RBMX, expresses hnRNP G ubiquitously. We show here that RBM, hnRNP G and a novel testis-specific relative, termed hnRNP G-T, interact with Tra2beta, an activator of pre-mRNA splicing that is ubiquitous but highly expressed in testis. Endogenous hnRNP G and Tra2beta proteins are associated in HeLa nuclear extracts. RBM and Tra2beta co-localize in two major domains in human spermatocyte nuclei. Phosphorylation enhanced the interaction and reduced competing RNA binding to the interaction domains. Incubation with the protein interaction domain of RBM inhibited splicing in vitro of a specific pre-mRNA substrate containing an essential enhancer bound by Tra2beta. The RNA-binding domain of RBM affected 5' splice site selection. We conclude that the hnRNP G family of proteins is involved in pre-mRNA splicing and infer that RBM may be involved in Tra2beta-dependent splicing in spermatocytes.

T-STAR/ETOILE: a novel relative of SAM68 that interacts with an RNA-binding protein implicated in spermatogenesis.

RBM is an RNA-binding protein encoded on the Y chromosome in mammals and is expressed only in the nuclei of male germ cells. Genetic evidence from infertile men implicates it in spermatogenesis, but its function is unknown. Of a number of potential partners for RBM identified by a yeast two-hybrid screen with testis cDNA, the most frequent isolates encoded a novel RNA-binding protein, termed T-STAR, that is closely related to SAM68, an Src-associated protein of unknown function. The mouse homologue was also cloned and designated étoile. It mapped to chromosome 15, while T-STAR mapped to the syntenic region on human chromosome 8. T-STAR/étoile is expressed primarily in the testis; in rat germ cells, the expression of both T-STAR/étoile and SAM68 is regulated during meiosis. Transfection of T-STAR/étoile fused with green fluorescent protein into HeLa cells caused an accumulation of protein in a novel compartment of the nucleus, adjacent to the nucleolus but distinct from the peri-nucleolar compartment. RBM and other hnRNP G family members are candidate downstream targets for regulation by T-STAR/ETOILE and SAM68.