Differentiation of adult-type Leydig cells occurs in gonadotrophin-deficient mice

During mammalian testis development distinct generations of fetal and adult Leydig cells arise. Luteinising hormone (LH) is required for normal adult Leydig cell function and for the establishment of normal adult Leydig cell number but its role in the process of adult Leydig cell differentiation has remained uncertain. In this study we have examined adult Leydig cell differentiation in gonadotrophin-releasing hormone (GnRH)-null mice which are deficient in circulating gonadotrophins. Adult Leydig cell differentiation was assessed by measuring expression of mRNA species encoding four specific markers of adult Leydig cell differentiation in the mouse. Each of these markers (3β-hydroxysteroid dehydrogenase type VI (3βHSD VI), 17β-hydroxysteroid dehydrogenase type III (17βHSD III), prostaglandin D (PGD)-synthetase and oestrogen sulphotransferase (EST)) is expressed only in the adult Leydig cell lineage in the normal adult animal. Real-time PCR studies showed that all four markers are expressed in adult GnRH-null mice. Localisation of 3βHSD VI and PGD-synthetase expression by in situ hybridisation confirmed that these genes are expressed in the interstitial tissue of the GnRH-null mouse. Treatment of animals with human chorionic gonadotrophin increased expression of 3βHSD VI and 17βHSD III within 12 hours further indicating that differentiated, but unstimulated cells already exist in the GnRH-null mouse. Thus, while previous studies have shown that LH is required for adult Leydig cell proliferation and activity, results from the present study show that adult Leydig cell differentiation will take place in animals deficient in LH.     

Extracting information from imaging cytometry: a review

The extraction of statistically meaningful quantitative information from microscopy images is increasingly important for modern biological research. Obtaining accurate, quantitative information from biological specimens, however, is a complex process that requires optimization of several parameters. One must consider the number of probes, fluorescent channels required, type of plate to be used, number of fields to be acquired and optimal resolution for image acquisition. The extraction of information from images is dependent on and can be aided greatly by careful consideration of the factors involved in the image acquisition process. I summarize here the general principles behind the imaging and software technology that is used to quantify images and highlight particular issues of concern for critically applying image quantitation techniques for research.     

The European Renal Genome Project: An Integrated Approach Towards Understanding the Genetics of Kidney Development and Disease

Rapid progress in genome research creates a wealth of information on the functional annotation of mammalian genome sequences. However, as we accumulate large amounts of scientific information we are facing problems of how to integrate and relate the data produced by various genomic approaches. Here, we propose the novel concept of an organ atlas where diverse data from expression maps to histological findings to mutant phenotypes can be queried, compared and visualized in the context of a three-dimensional reconstruction of the organ. We will seek proof of concept for the organ atlas by elucidating genetic pathways involved in development and pathophysiology of the kidney. Such a kidney atlas may provide a paradigm for a new systems-biology approach in functional genome research aimed at understanding the genetic bases of organ development, physiology and disease.Key Words: EuReGene, kidney, genome, development, pathophysiology, genetics     

A covariotide model explains apparent phylogenetic structure of oxygenic photosynthetic lineages

The aims of the work were (1) to develop statistical tests to identify whether substitution takes place under a covariotide model in sequences used for phylogenetic inference and (2) to determine the influence of covariotide substitution on phylogenetic trees inferred for photosynthetic and other organisms. (Covariotide and covarion models are ones in which sites that are variable in some parts of the underlying tree are invariable in others and vice versa.) Two tests were developed. The first was a contingency test, and the second was an inequality test comparing the expected number of variable sites in two groups with the observed number. Application of these tests to 16S rDNA and tufA sequences from a range of nonphotosynthetic prokaryotes and oxygenic photosynthetic prokaryotes and eukaryotes suggests the occurrence of a covariotide mechanism. The degree of support for partitioning of taxa in reconstructed trees involving these organisms was determined in the presence or absence of sites showing particular substitution patterns. This analysis showed that the support for splits between (1) photosynthetic eukaryotes and prokaryotes and (2) photosynthetic and nonphotosynthetic organisms could be accounted for by patterns arising from covariotide substitution. We show that the additional problem of compositional bias in sequence data needs to be considered in the context of patterns of covariotide/covarion substitution. We argue that while covariotide or covarion substitution may give rise to phylogenetically informative patterns in sequence data, this may not always be so.      

Inhibition of the acyl-CoA desaturases involved in the biosynthesis of Spodoptera littoralis sex pheromone by analogs of 10,11-methylene-10-tetradecenoic acid

The desaturase inhibitory activity of the cyclopropenyl alcohols 9,10-methylene-9-tetradecen-1-ol (9-MTOL), 10,11-methylene-10-tetradecen-1-ol (10-MTOL) and 11,12-methylene-11-tetradecen-1-ol (11-MTOL), which are structural analogs of 10,11-methylene-10-tetradecenoic acid (10-MTA), is reported. At equimolar ratios with respect to the different substrates, the three compounds completely inhibited the three desaturation reactions involved in the biosynthesis of Spodoptera littoralis sex pheromone. The dose-dependence of inhibition was determined for 10-MTA and its alcohol derivative. Both compounds inhibited the transformation of perdeuterated palmitic acid into perdeuterated (Z)-11-hexadecenoic acid and that of (E)-11-tridecenoic acid into (Z,E)-9,11-tridecadienoic acid with similar IC(50) values. The overall results presented in this work support scattered data that neither the free carboxyl groups nor their acyl-CoA esters are a requisite for inhibition of desaturases. Since the synthesis of cyclopropenols is much more convenient than that of cyclopropene fatty acids, this finding is of economical relevance regarding the putative use of cyclopropene derivatives in pest control.     

In vitro generation of murine myeloid dendritic cells from CD34-positive precursors

Dendritic cells (DCs) link the innate and adaptive immune system. Currently, murine DCs for cell biology investigations are developed from MHC class II-negative bone marrow (BM) precursor cells, non-depleted BM cells or BM monocytes in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). Here we demonstrate an isolation procedure of functionally intact myeloid CD11c⁺ CD11b⁺ DCs derived from murine CD34-positive precursors. DCs derived from CD34⁺ cells show functional internalization, maturation, cytokine secretion, MHC-restricted antigen presentation, and MHCII retrograde transport of antigens from the lysosomes to the cell surface. In comparison to the established method, the advantages of this isolation procedure are a shorter cultivation period, a superior transfection efficiency, the yield of a purer and more homogeneous population of immature DCs, and less consumption of cell culture medium and GM-CSF. The new isolation procedure and the functional quality of CD34⁺ cell-derived murine myeloid DCs make them ideally suited for immunology and cell biology studies.     

Electron microscopy evidence that cytoplasmic localization of the p16INK4A "nuclear" cyclin-dependent kinase inhibitor (CKI) in tumor cells is specific and not an artifact. A study in non-small cell lung carcinomas

It is well established that p16^INK4A protein acts as a cell cycle inhibitor in the nucleus. Therefore, cytoplasmic localization of p16 ^INK4A usually is disregarded by investigators as nonspecific. Three recent studies reported findings that differ from the current view concerning p16^INK4A immunohistochemical localization. All three demonstrated that breast and colon cancers expressing cytoplasmic p16^INK4 represent distinct biological subsets. We previously detected in a percentage of non-small cell lung carcinomas simultaneous nuclear and cytoplasmic p16^INK4A staining. In view of the reports concerning breast and colon carcinomas, we conducted an ultrastructural re-evaluation of our cases to clarify the specificity of p16^INK4A cytoplasmic expression. We observed p16 ^INK4A immunolocalization in both the nucleus and the cytoplasm of a proportion of tumor cells. Diffuse dense nuclear staining was detected in the nucleoplasm, whereas weaker granular immunoreactivity was observed in the cytoplasm near the rough endoplasmic reticulum. Negative tumor cells also were visible. In the tumor-associated stromal, cells p16^INK4A immunoreactivity was detected only in the nuclei. We have demonstrated that p16^INK4A cytoplasmic staining is specific and suggest that it represents a mechanism of p16^INK4A inactivation similar to that observed in other tumor suppressor genes.     

A Dutch guideline for the treatment of scoliosis in neuromuscular disorders

Background

Children with neuromuscular disorders with a progressive muscle weakness such as Duchenne Muscular Dystrophy and Spinal Muscular Atrophy frequently develop a progressive scoliosis. A severe scoliosis compromises respiratory function and makes sitting more difficult. Spinal surgery is considered the primary treatment option for correcting severe scoliosis in neuromuscular disorders. Surgery in this population requires a multidisciplinary approach, careful planning, dedicated surgical procedures, and specialized after care.

Methods

The guideline is based on scientific evidence and expert opinions. A multidisciplinary working group representing experts from all relevant specialties performed the research. A literature search was conducted to collect scientific evidence in answer to specific questions posed by the working group. Literature was classified according to the level of evidence.

Results

For most aspects of the treatment scientific evidence is scarce and only low level cohort studies were found. Nevertheless, a high degree of consensus was reached about the management of patients with scoliosis in neuromuscular disorders. This was translated into a set of recommendations, which are now officially accepted as a general guideline in the Netherlands.

Conclusion

In order to optimize the treatment for scoliosis in neuromuscular disorders a Dutch guideline has been composed. This evidence-based, multidisciplinary guideline addresses conservative treatment, the preoperative, perioperative, and postoperative care of scoliosis in neuromuscular disorders.