Cloning of breakpoints of a chromosome translocation identifies the AN2 locus.

Chromosome translocations involving 11p13 have been associated with familial aniridia in two kindreds highlighting the chromosomal localization of the AN2 locus. This locus is also part of the WAGR complex (Wilms tumor, aniridia, genitourinary abnormalities, and mental retardation). In one kindred, the translocation is associated with a deletion, and probes for this region were used to identify and clone the breakpoints of the translocation in the second kindred. Comparison of phage restriction maps exclude the presence of any sizable deletion in this case. Sequences at the chromosome 11 breakpoint are conserved in multiple species, suggesting that the translocation falls within the AN2 gene.

Analysis of WT1 target gene expression in stably transfected cell lines.

The Wilms' tumour suppressor gene WT1 encodes a zinc finger protein that is mutated in a subset of Wilms' tumours. Mutation screening and animal studies revealed essential roles during development and later function of the kidneys and the entire genitourinary system. Sequence similarity suggested a possible role for WT1 as a transcription factor. Indeed, sequence specific DNA binding and transcriptional activation or repression potential could be demonstrated in transient transfection assays with various reporter constructs. To identify endogenous WT1 target genes we established HEK293 cell lines expressing the different WT1 isoforms in a tetracycline dependent manner. Differential display PCR (ddPCR) was performed on RNA from stable WT1 transfected HEK293 cell lines and two other WT1 transfected lines (G401 and Saos-2). In an extended survey of several thousand ddPCR bands only few differences in intensity were seen and none of these could unambiguously be verified as being WT1 regulated by subsequent Northern blot analysis. In addition, almost none of the WT1 target genes identified to date in transient co-transfection assays could be confirmed by either ddPCR or Northern hybridization in the three stable transfected cell lines. Among the nine genes expressed, the only exceptions were CSF1 and to a lesser extent IGF1R being induced in Saos-2/G401 and HEK293 cells, respectively. At least two of the cell lines tested had previously shown clear biological effects though -- either WT1 dependent apoptosis (Saos-2) or greatly reduced tumorigenicity (G401). This suggests that WT1 may regulate only a very small set of genes that escape the detection methods used or it may not act as a transcription factor that influences steady state levels of mRNA.

Developmental expression patterns of mouse sFRP genes encoding members of the secreted frizzled related protein family.

Development of the metanephric kidney is an experimental model system to analyze interactions between mesenchymal and epithelial cells and mesenchymal-epithelial transition. To study the underlying genetic mechanisms we employed organ culture and differential display PCR to identify genes regulated upon induction of mesenchymal cells. One of the genes found encodes the secreted frizzled related protein 2 (sFRP2) that is upregulated within 2 days of in vitro development. In vivo sFRP2 expression was likewise found in mesenchymal condensates and subsequent epithelial structures. Detailed in situ hybridization analysis revealed sFRP2 expression during development of the eye, brain, neural tube, craniofacial mesenchyme, joints, testis, pancreas and below the epithelia of oesophagus, aorta and ureter where smooth muscles develop. In a comparative analysis transcripts of the related sFRP1 and sFRP4 genes were frequently found in the same tissues as sFRP2 with their expression domains overlapping in some instances, but mutually exclusive in others. While sFRP1 is specifically expressed in the embryonic metanephros, eye, brain, teeth, salivary gland and small intestine, there is only weak expression of sFRP4 except for the developing teeth, eye and salivary gland. The interpretation of the highly specific spatial and temporal expression patterns of sFRP genes will partly depend on a better functional understanding of the interaction between wnt, fz and sFRP family members. Nevertheless, sFRP genes must play quite distinct roles in the morphogenesis of several organ systems.

Hey genes: a novel subfamily of hairy- and Enhancer of split related genes specifically expressed during mouse embryogenesis.

We have identified a novel subfamily of mammalian hairy/Enhancer of split (E(spl))-related basic helix-loop-helix (bHLH) genes together with a putative Drosophila homologue. While hairy/E(spl) proteins are characterized by an invariant proline residue in the basic domain and a carboxyterminal groucho-binding WRPW motif, our genes encode a carboxyterminal KPYRPWG sequence and were thus designated as Hey genes (Hairy/E(spl)-related with YRPW motif). Furthermore, they bear a unique C-terminal TE(I/V)GAF motif and the characteristic proline is changed in all Hey family members to glycine. RNA in situ hybridization analysis revealed specific expression of Hey1 during development of the nervous system, the somites, the heart and the craniofacial region. Hey2 is similarly expressed in the somites whereas it shows a complementary expression in the heart, the craniofacial region and the nervous system. The diversity of expression patterns implies unique functions in neurogenesis, somitogenesis and organogenesis.

Analysis of HeyL expression in wild-type and Notch pathway mutant mouse embryos.

In vertebrates Notch signaling regulates cell fate decisions and boundary formation and it underlies several murine and human diseases. Gene targeting experiments point to key roles of Notch receptors, ligands, modulators and downstream targets in somitogenesis, neurogenesis and vascular development. Here we report the embryonic expression of the hairy-related basic helix-loop-helix gene HeyL in wild-type and Notch pathway mutant mice. We show that HeyL is strongly expressed in the presomitic mesoderm, the somites, the peripheral nervous system and smooth muscle of all arteries. Loss of HeyL expression at the level of nascent somites in Notch1 and Delta-like1 knockout mutants implicates HeyL as a Notch effector during somite formation. Furthermore, HeyL expression in vascular smooth muscle cells and in the thymus strikingly overlaps with that of Notch3, mutations of which underlie the CADASIL vascular disorder.

Fjx1, the murine homologue of the Drosophila four-jointed gene, codes for a putative secreted protein expressed in restricted domains of the developing and adult brain.

The Drosophila gene four jointed (fj) codes for a secreted or cell surface protein important for growth and differentiation of legs and wings and for proper development of the eyes. Here we report the cloning of the mouse four-jointed gene (fjx1) and its pattern of expression in the brain during embryogenesis and in the adult. In the neural plate, fjx1 is expressed in the presumptive forebrain and midbrain, and in rhombomere 4, however a small rostral/medial area of the forebrain primordium is devoid of expression. Expression of fjx1 in the neural tube can be divided into three phases. (1) In the embryonic brain fjx1 is expressed in two patches of neuroepithelium: in the midbrain tectum and the telencephalic vesicles. (2) In fetal and early postnatal brain fjx1 is expressed mainly by the primordia of layered telencephalic structures: cortex (ventricular layer and cortical plate), olfactory bulb (subependymal layer and in the mitral cell layer). In addition expression is observed in the superior colliculus. (3) In the adult, fjx1 is expressed by neurones evenly distributed in the telencephalon (isocortex, striatum, hippocampus, olfactory bulb, piriform cortex), in the Purkinje cell layer of the cerebellum, and numerous medullary nuclei. In the embryo, strong expression can further be seen in the apical ectodermal ridge of fore- and hindlimbs and in the ectoderm of the branchial arches.

Local analgesia by percutaneous electrical stimulation of sensory nerves.

Experimental C-fiber pain caused by radiant heat was applied to the skin area supplied by the left sural nerve of 20 subjects. Percutaneous electrical stimulation (PNS) was performed on the left sural nerve, the left superficial peroneal nerve and the right superficial radial nerve. Stimulation frequencies were: 3, 50, 100, 300, 500 and 1000 Hz. The analgesia resulting at the different stimulation sites was recorded according to a preset scale of estimation. Without considering the influence of the different frequencies, the best analgesic effects were reached if noxious heating and PNS were both performed on the left sural nerve; the anatomical conditions prevented us from distinguishing between the effects of possible peripheral blockade or spinal modification of pain. PNS of the superficial peroneal nerve seems to indicate spinal, possibly polysegmental, interactions between C-fiber pain and electrical stimulation of thick myelinated fibers. However, long loop effects may also play a part in local analgesia as demonstrated by PNS of the right radial nerve.

Identification of optimized target sequences for the GLI3 zinc finger protein.

GLI3 represents an important control gene for development and differentiation of several body structures. Reduction in gene dosage already leads to severe perturbation, especially of limb morphogenesis. The gene encodes a zinc finger protein that likely functions as a transcriptional modulator. Because the five zinc fingers should be capable of recognizing an extended stretch of genomic DNA, we sought to identify sequences bound by GLI3 that may facilitate the search for target genes acting downstream of GLI3. Starting from the nonamer DNA binding sequence of the highly related GLI protein, we employed an oligonucleotide selection protocol to determine an optimized binding sequence for the GLI3 protein. The resulting sequence bound by the GLI3 zinc fingers consists of 16 nucleotides and shows a high degree of similarity to sequences bound by the GLI and tra-1 proteins. Comparison with protein-DNA interactions in the known crystal structure of the GLI-DNA complex suggests relevant interactions of additional amino acids of GLI3 with its target site. The newly identified GLI3 target sequence should prove very useful for both the structural analysis of the protein-DNA complex and the search for genes whose expression is subject to regulation by the GLI3 gene product.

Screening Tanzanian medicinal plants for antimalarial activity.

Forty-three different plant species commonly used in traditional medicine for the treatment of malaria were selected and screened for their antimalarial activity against Plasmodium falciparum in vitro. Thirteen of the 43 species were obtained directly from traditional healers who use these plants for the treatment of malaria. The other plant species were collected on the basis of ethnomedicinal information in the literature. The plant material was collected from Morogoro, Dar es Salaam and Kagera regions in Tanzania. Fifty-eight plant samples from these 43 plant species, including leaves, roots and stem bark, were investigated. Apart from the crude EtOH extracts, petroleum ether (PE), ethyl acetate (EtAc) and H2O fractions of these extracts were also tested. The in vitro testing revealed that 37% of the investigated plants showed strong antimalarial activity with IC50 values below 10 micrograms/ml. The four most active plants included Cissampelos mucronata, Maytenus senegalensis, Salacia madagascariensis and Zanthoxylum chalybeum.

Differential expression of the cellular oncogenes c-src and c-yes in embryonal and adult chicken tissues.

The cellular onc-genes c-src and c-yes are expressed very differently during chicken embryonic development. The c-src mRNA and its translational product are detectable at high levels in brain extracts of chicken embryos and adult chickens, whereas muscle extracts show an age-dependent decrease in the amounts of c-src-specific mRNA and pp60c-src kinase activity. In contrast, the levels of c-yes mRNA in brain, heart, and muscle are relatively low in early embryonic stages and increase later on to values comparable to those found for liver, while in adult animals the pattern of c-yes expression is similar to that of the c-src gene. From the close correlation between the levels of pp60c-src, its enzymatic activity, and its corresponding mRNA at a given stage of development and in given tissues, it appears that the expression of pp60c-src is primarily controlled at the level of transcription. It is suggested that because of the different patterns of expression, the two cellular oncogenes, c-src and c-yes, play different roles in cell proliferation during early embryonic stages as well as in ensuing differentiation processes.

[Relations between fox populations and halting the rabies in North Rhine-Westphalia]. / Relations entre population de renards et limitation de la propagation de la rage en Rhénanie-Westphalie.

Since Sylvatic rabies started in North Rhine-Westphalia in 1953 many epidemics have occurred, only in mid-mountain areas. As a characteristic, epidemics halted, in each case at the limit between big game (in south-east) and small game (in the north-west) hunting areas. The fact that rabies died out when reaching these limits is surprising for, according to the number of foxes killed in small game hunting areas, there was a fox density which usually does not allow the epidemic to stop. Explanation of this phenomenon could be the different hunting methods. Indeed in small game hunting areas 80% of killed foxes are cubs or sub-adults. Which means that most of the growing population is destroyed early enough, in contrast with big game hunting areas where less than half of the killed foxes are cubs or sub-adults. This is why in big game hunting areas, better conditions are found for rabies appearance than in the other type of hunting area. Studying the number of foxes killed, these relations are described for three regions of North Rhine-Westphalia. It is obviously very important in rabies control that, besides other measures, growing foxes are killed in priority.

Follow-up studies of three subtypes of acute postinfectious glomerulonephritis ascertained by renal biopsy.

Quantitative correlative investigations by means of light, immunofluorescence and electron microscopy carried out in the early phase of the disease on 58 patients (children and adults) with acute postinfectious glomerulonephritis (APGN) formed the basis of subtyping APGN into a starry sky type, a mesangial type and a garland type [Sorger et al. 1982 and 1983]. The subtypes also showed differences in the clinical picture. The garland type was of special interest since most patients had severe proteinuria. This caused us to follow-up the patients with these three subtypes (up to 10 years and 7 months). Proteinuria proved to be the most reliable follow-up parameter. A comparison of the three groups showed that proteinuria rapidly declined as a rule in the patients with the starry sky and the mesangial patterns. In the garland pattern there were also cases with a complete disappearance of proteinuria, especially in younger patients, but other patients still had a distinct proteinuria after months to years indicating a protracted or chronic course. The morphological findings of the rebiopsies correlated with the clinical courses, especially with the course of proteinuria. The three morphological subtypes are thus significant for estimating the prognosis of APGN, which is favorable as a rule in patients with the starry sky and mesangial types, but much more unfavorable in patients with the garland type. Even if fewer cases with demonstrated streptococcal etiology were found in the garland pattern group, i.e., among patients with the most uncertain prognosis, than in the remaining groups, these differences were not statistically significant. Therefore, our investigations do not provide any indications that different etiological factors are responsible for the three subtypes. The individual immune response of the host body is likely to be very much more decisive.

Traditional healers in Tanzania: the perception of malaria and its causes.

The coordination of traditional and western medicine is still in its infancy in most African countries. Although there is much discussion about the contribution of traditional medicine and its practitioners, especially on the primary health care level, it has rarely be done in practice. This is probably due to the lack of knowledge of how to do it, because a serious attempt to include traditional medicine in health planning would presuppose that it is known what traditional medicine has specifically to offer for certain diseases/illnesses and how traditional healers manage such conditions. The aim of this study was to investigate the management of malaria by traditional healers in different areas in Tanzania. This included looking at the perception, the causation concepts and the knowledge about prevention of the disease/illness of malaria. For this purpose traditional healers were interviewed in different rural and urban places in Tanzania: in the Kilombero valley (Kilombero/Ulanga District), on the main island of Ukerewe (Ukerewe District), in the region near Bukoba town (Bukoba District) and in the settlement of Dar es Salaam (largest town of Tanzania). The results of the study show that most of the interviewed traditional healers were very familiar with the signs and symptoms relating to malaria, as it is defined by western medicine. Many healers were aware of different manifestations of malaria and attributed to them different local names, which match the scientific terms which describe the different types of Plasmodium falciparum malaria, such as cerebral malaria, clinical malaria or febrile type, and gastrointestinal type, respectively. Differences compared to western medical knowledge were found for concepts of causation, and in the fact that severe malaria in children may not be perceived as being associated with malaria.

Traditional healers in Tanzania: sociocultural profile and three short portraits.

Traditional healers are an important part of African societies, but unfortunately the knowledge of the extent and character of traditional healing and the people involved in the practice is limited and impressionistic. They are frequently ignored in studies of user/provider patterns, although they cover the health needs of a substantial proportion of the population. For future health planning it is necessary to know what the reasons are that even in big cities, where western health care services are available, traditional healers flourish, and even compete with each other for certain aspects. The aim of this study was to investigate certain aspects of the profession of traditional healing in general in different areas in Tanzania in order to get an idea about the kind of traditional medical services which are available, and about the people who provide such services. For this reason traditional healers were interviewed with a semi-structured questionnaire in different rural and urban places: in the Kilombero valley (Kilombero/Ulanga district), on the main island of Ukerewe (Ukerewe District), and in the region near Bukoba town (Bukoba District), and in the settlement of Dar es Salaam (largest city of Tanzania). The results of the study show that traditional healers are a very heterogeneous group of persons not having much in common relating to their religion, sex and level of education. The traditional practice is very often taken over from a family member, but also other reasons for becoming a healer, like initiation through ancestor spirits, are very frequently given. More than 50% of the respondents practice full time. These full time practitioners are mainly found among men and in the younger age group. Treatment of in-patients, who can stay in special patient-houses, is offered by half of the traditional healers. Divination used as a diagnostic tool was found mainly among men. Referral of patients to the hospital was mentioned by almost all respondents in cases where they failed with their own treatment or when they knew that the patient would be better treated in the hospital or dispensary.

PIP: While traditional healers are an important part of African societies, not enough is known about the extent and character of traditional healing and the people involved in the practice. The authors interviewed 23 male and 8 female traditional healers in the Kilombero Valley, on the main island of Ukerewe, in the region near Bukoba town, and in the settlement of Dar es Salaam to gain insight into what kind of traditional medical services are available and the people who provide such services. The healers are a very heterogenous group of persons without much in common with regard to their religion, sex, and educational level. The traditional practice is often taken over from a family member. Other reasons for becoming a healer, such as initiation by ancestral spirits, were also frequently given. More than 50% of the respondents practice full time; these healers are mainly male and younger. Inpatient treatment is offered by half of the healers, and divination was used in diagnosis mainly by male practitioners. Almost all healers reported referring patients to hospitals when traditional treatment failed to work or when they knew that the patient would be better treated at a hospital or dispensary.

Traditional healers in Tanzania: the treatment of malaria with plant remedies.

In order to collect ethnobotanical information about antimalarial plants which is essential for the further evaluation of the efficacy of plants an antimalarial remedies, we investigated the management of malaria with traditional herbal remedies, including the use, preparation and administration, by traditional healers in Tanzania. Interviews with traditional healers were conducted in different rural and urban places in Tanzania: in the Kilombero valley (Kilombero/Ulanga District), on the main island of Ukerewe (Ukerewe District), in the region near Bukoba town (Bukoba district), and in the settlement of Dar es Salaam (largest city in Tanzania). The results of the study show that all traditional healers treat malaria with herbal remedies consisting of one to five different plants. The list of plants which they use for antimalarial treatment contains a large number of species from different families. Multiple citations of plants by different healers were rare. Most of the respondents attributed to the plants mentioned, or to the remedies made from them, specific effects and sometimes side effects, explaining and illustrating their use or non-use for different patients or manifestations of the disease/illness.

[Parasitological and immunological progress control during and after chemotherapy of canine leishmaniasis]. / Parasitologische und immunologische Verlaufskontrollen Während und nach Chemotherapie der Leishmaniose des Hundes.

Promastigote Leishmania-organisms were diagnostically cultivated in vitro from popliteal lymph node aspirates obtained from 32 of in total 36 dogs returning from endemic areas. Isoenzyme analysis (glucosephosphate-isomerase (GPI), phosphoglucomutase (PGM) and glutamate-oxaloacetate-transaminase (GOT) resulted in the identification of Leishmania infantum (syn. Leishmania (L.) infantum) for all 18 isolates investigated. Parasites were still able to be cultivated in vitro in 79% of 28 biopsies (from 15 dogs) even following chemotherapy by Glucantime, independent of the time of sampling and the course of disease after treatment. Dogs with a progressive form of disease (despite chemotherapy) showed only a minor or no reduction (between 0 and 4.8%) of the relative antibody concentration (determined by ELISA), whereas regressive forms of disease (without recurrences observed in the period of 10 to 37 months after therapy) demonstrated a marked reduction of the relative antibody concentration (between 6.7 and 16.2%) within the first 5 to 8 months; thereafter the decrease diminished and changed to a persistent low relative antibody concentration.

[Adjustable transobturatoric sling system in men : diagnosis and therapy recommendations to persistent pain]. / Adjustierbare transobturatorische Schlingensysteme beim Mann : Diagnostik und Therapieempfehlungen bei persistierenden Schmerzen.

BACKGROUND: In addition to artificial sphincters, male slings are recommended in the current guidelines for the treatment of persistent male stress incontinence. Today, several sling systems are available. Well-known complications of all sling systems are infections, erosion, residual urine/urinary retention, de novo urgency, and postoperative pain. DISCUSSION: Compared to retropubic implanted adjustable sling systems or functional slings, pain is more common after transobturatoric implantation of adjustable sling systems. Early postoperative pain is very common. In contrast, persistent pain is rare. However, the treatment of persistent pain is a large challenge for urologists and patients. There are no recommendations for diagnostic workup or treatment. RESULTS: After pain classification, pain management should be started with nonsteroidal anti-inflammatory drugs and/or tricyclic antidepressive agents, if necessary treatment escalation with a weak opioid and if not effective interventional procedures should be performed. Sling explantation is only necessary in rare cases.

[Structure, use, and risks of biomaterial repositories of embryonal tumors]. / Biomaterialbanken für embryonale Tumoren: Struktur, Nutzen und Risiken.

Availability of statistically sufficient numbers of tumor samples and other biomaterials in high quality together with corresponding clinical data is crucial for biomedical research. Tumor repositories from individual scientists are mostly not sufficient to satisfy these criteria, especially since pediatric tumors are rare. In 2000 three centralized tumor repositories (neuroblastoma in Cologne, nephroblastoma in Würzburg, hepatoblastoma, brain tumors in Bonn) have been established by the "German Competence Net Pediatric Oncology und Hematology". The aim was to collect biomaterial including tumor samples, normal tissue, and blood in high quality for research and diagnostic purposes at a central institution. Informed consent of the parents or patients is a prerequisite for scientific use of the samples and is requested by the therapy trial. The samples are collected according to accepted standards and shipped in the specially designed Tumorbox. The tumor repository organizes the distribution of the samples to the cooperating diagnostic laboratories. The number of collected tumor samples has increased over the years. In 2000, samples from 200 patients were collected while the patient number increased to 321 in 2005. Over the years the tumor repositories collected more than 7,150 samples (fresh frozen tumor, fresh frozen normal tissue, and blood). Through links with clinical trial databases the samples can be connected with clinical data. 12 of 14 applications for tumor material to be used in specific scientific projects have been approved by an independent supervisory board. The establishment of central tumor repositories represents a major step for biomedical research activities and quality control in pediatric oncology.

Expression profiling of Wilms tumors reveals new candidate genes for different clinical parameters.

Wilms tumor is the most frequent renal neoplasm in children, but our understanding of its genetic basis is still limited. We performed cDNA microarray experiments using 63 primary Wilms tumors with the aim of detecting new candidate genes associated with malignancy grade and tumor progression. All tumors had received preoperative chemotherapy as mandated by the SIOP protocol, which sets this study apart from related approaches in the Unites States that are based on untreated samples. The stratification of expression data according to clinical criteria allowed a rather clear distinction between different subsets of Wilms tumors. Clear-cut differences in expression patterns were discovered between relapse-free as opposed to relapsed tumors and tumors with intermediate risk as opposed to high risk histology. Several differentially expressed genes, e.g.TRIM22, CENPF, MYCN, CTGF, RARRES3 and EZH2, were associated with Wilms tumor progression. For a subset of differentially expressed genes, microarray data were confirmed by real-time RT-PCR on the original set of tumors. Interestingly, we found the retinoic acid pathway to be deregulated at different levels in advanced tumors suggesting that treatment of these tumors with retinoic acid may represent a promising novel therapeutic approach.