Evidence for Parapatric Speciation in the Mormyrid Fish, Pollimyrus castelnaui (Boulenger, 1911), from the Okavango–Upper Zambezi River Systems: P. marianne sp. nov., Defined by Electric Organ Discharges, Morphology and Genetics

We report on parapatric speciation in the mormyrid fish, Pollimyrus castelnaui (Boulenger, 1911), from the Okavango and the Upper Zambezi River systems. We recognise samples from the Zambezi River as a distinct species, P. marianne, displaying an eastern phenotype of electric organ discharge (EOD) waveform (Type 3) that is distinct from the western EOD phenotype (Type 1) observed in P. castelnaui samples from the neighbouring Okavango. Samples from the geographically intermediate Kwando/Linyanti River (a tributary of the Zambezi that is also intermittently connected to the Okavango) presented a more variable third EOD phenotype (Type 2). In 13 out of 14 morphological characters studied, the Zambezi River samples differed significantly from P. castelnaui. Morphologically and in EOD characters, the Kwando/Linyanti fish are distinct from both P. castelnaui and P. marianne. Sequence analysis of the mitochondrial cytochrome b gene unambiguously reveals that specimens from the Zambezi River System form a well supported taxon which clearly differs from P. castelnaui from the Okavango (1.5–2.5% sequence divergence). Within specimens from the Kwando–Zambezi System some geographic differentiation can be detected (nucleotide substitutions up to 0.6%); but groups cannot be resolved with certainty. Significant allozyme differences were found between the Okavango and all other EOD types from the Upper Zambezi System, and, within the Zambezi System, between the Kwando (Type 2) and Zambezi (Type 3) individuals. The low Wright's fixation index values, the lack of fixed allele differences, and small genetic distances provide little evidence for speciation between groups within the Zambezi System, but moderate to great fixation index values and significant allele frequency differences were observed between the Okavango and the other fishes. It is concluded that within the Zambezi System, differentiation between Kwando/Linyanti and Zambezi populations (as revealed by morphology and EOD waveform comparisons) is so recent that substantial genetic (allozyme and mitochondrial sequence) differences could not have evolved, or were not detected.     

Autoantibody systems in rheumatoid arthritis: specificity, sensitivity and diagnostic value

The diagnosis of rheumatoid arthritis (RA) is primarily based on clinical symptoms, so it is often difficult to diagnose RA in very early stages of the disease. A disease-specific autoantibody that could be used as a serological marker would therefore be very useful. Most autoimmune diseases are characterized by a polyclonal B-cell response targeting multiple autoantigens. These immune responses are often not specific for a single disease. In this review, the most important autoantibody/autoantigen systems associated with RA are described and their utility as a diagnostic and prognostic tool, including their specificity, sensitivity and practical application, is discussed. We conclude that, at present, the antibody response directed to citrullinated antigens has the most valuable diagnostic and prognostic potential for RA.     

Splinting or surgery for carpal tunnel syndrome? Design of a randomized controlled trial [ISRCTN18853827]

Background  

Carpal tunnel syndrome is a common disorder, which can be treated with surgery or conservative options. However, there is insufficient evidence and no consensus among physicians with regard to the preferred treatment for carpal tunnel syndrome. Therefore, a randomized controlled trial is conducted to compare the short- and long-term efficacy of surgery and splinting in patients with carpal tunnel syndrome. An attempt is also made to avoid the (methodological) limitations encountered in earlier trials on the efficacy of various treatment options for carpal tunnel syndrome.     

Molecular and Functional Characterization of the Four-Transmembrane Molecule L6 in Epidermal Keratinocytes

In normal human epidermal keratinocytes (NHEK) proteolytic detachment from the substrate induces a complex activation cascade including expression of new proteins, morphological alterations, and the onset of migration for epidermal regeneration. By subtractive cloning we have shown that L6, a four-transmembrane protein, is newly expressed after proteolytic keratinocyte detachment. In this study, we have generated a novel anti-L6 antibody (clone HD-pKe#104-1.1) and investigated L6 expression regulation in vitro and in vivo as well as L6 function in keratinocyte migration. Dispase-mediated detachment induced L6 expression in NHEK at the mRNA and protein level. Immunohistology of skin biopsies displayed a strong expression of L6 in follicular epidermis and epidermolytic lesions of autoimmune bullous dermatoses (bullous pemphigoid, pemphigus vulgaris), but not in normal interfollicular epidermis. In contrast to normal keratinocytes, HaCaT cells showed constitutive L6 expression, indicating a constitutively active phenotype. After artificial wounding of confluent HaCaT cultures, anti-L6 antibody strongly impaired cell migration velocity and migratory reepithelization of the defect, indicating L6 involvement in keratinocyte migration. These findings suggest that L6 is an important activation-dependent regulator of keratinocyte function and epidermal tissue regeneration.     

Molecular mapping of the Oregon Wolfe Barleys: a phenotypically polymorphic doubled-haploid population

A phenotypically polymorphic barley (Hordeum vulgare L.) mapping population was developed using morphological marker stocks as parents. Ninety-four doubled-haploid lines were derived for genetic mapping from an F₁ using the Hordeum bulbosum system. A linkage map was constructed using 12 morphological markers, 87 restriction fragment length polymorphism (RFLP), five random amplified polymorphic DNA (RAPD), one sequence-tagged site (STS), one intron fragment length polymorphism (IFLP), 33 simple sequence repeat (SSR), and 586 amplified fragment length polymorphism (AFLP) markers. The genetic map spanned 1,387 cM with an average density of one marker every 1.9 cM. AFLP markers tended to cluster on centromeric regions and were more abundant on chromosome 1 (7H). RAPD markers showed a high level of segregation distortion, 54% compared with the 26% observed for AFLP markers, 27% for SSR markers, and 18% for RFLP markers. Three major regions of segregation distortion, based on RFLP and morphological markers, were located on chromosomes 2 (2H), 3 (3H), and 7 (5H). Segregation distortion may indicate that preferential gametic selection occurred during the development of the doubled-haploid lines. This may be due to the extreme phenotypes determined by alleles at morphological trait loci of the dominant and recessive parental stocks. Several molecular markers were found to be closely linked to morphological loci. The linkage map reported herein will be useful in integrating data on quantitative traits with morphological variants and should aid in map-based cloning of genes controlling morphological traits. Received: 23 August 2000 / Accepted: 15 December 2000     

Cross-reactivity of myelin basic protein-specific T cells with multiple microbial peptides: experimental autoimmune encephalomyelitis induction in TCR transgenic mice.

Activation of autoreactive T cells is a crucial event in the pathogenesis of autoimmune diseases. Cross-reactivity between microbial and self Ags (molecular mimicry) is one hypothesis that could explain the activation of autoreactive T cells. We have systematically examined this hypothesis in experimental autoimmune encephalomyelitis using mice bearing exclusively myelin basic protein (MBP)-specific T cells (designated T+ alpha-). A peptide substitution analysis was performed in which each residue of the MBPAc1-11 peptide was exchanged by all 20 naturally occurring amino acids. This allowed the definition of the motif (supertope) that is recognized by the MBPAc1-11-specific T cells. The supertope was used to screen protein databases (SwissProt and TREMBL). By the search, 832 peptides of microbial origin were identified and synthesized. Of these, 61 peptides induced proliferation of the MBPAc1-11-specific transgenic T cells in vitro. Thus, the definition of a supertope by global amino acid substitution can identify multiple microbial mimic peptides that activate an encephalitogenic TCR. Peptides with only two native MBP-residues were sufficient to activate MBPAc1-11-specific T cells in vitro, and experimental autoimmune encephalomyelitis could be induced by immunizing mice with a mimic peptide with only four native MBP residues.     

Comparative proteome analysis identified CD44 as a possible serum marker for docetaxel resistance in castration‐resistant prostate cancer

Baseline or acquired resistance to docetaxel (DOC) represents a significant risk for patients with metastatic prostate cancer (PC). In the last years, novel therapy regimens have been approved providing reasonable alternatives for DOC‐resistant patients making prediction of DOC resistance of great clinical importance. We aimed to identify serum biomarkers, which are able to select patients who will not benefit from DOC treatment. DOC‐resistant PC3‐DR and DU145‐DR sublines and their sensitive parental cell lines (DU145, PC3) were comparatively analyzed using liquid chromatography‐coupled tandem mass spectrometry (LC‐MS/MS). Results were filtered using bioinformatics approaches to identify promising serum biomarkers. Serum levels of five proteins were determined in serum samples of 66 DOC‐treated metastatic castration‐resistant PC patients (mCRPC) using ELISA. Results were correlated with clinicopathological and survival data. CD44 was subjected to further functional cell culture analyses. We found at least 177 two‐fold significantly overexpressed proteins in DOC‐resistant cell lines. Our bioinformatics method suggested 11/177 proteins to be secreted into the serum. We determined serum levels of five (CD44, MET, GSN, IL13RA2 and LNPEP) proteins in serum samples of DOC‐treated patients and found high CD44 serum levels to be independently associated with poor overall survival (p = 0.001). In accordance, silencing of CD44 in DU145‐DR cells resulted in re‐sensitization to DOC. In conclusion, high serum CD44 levels may help identify DOC‐resistant patients and may thereby help optimize clinical decision‐making regarding type and timing of therapy for mCRPC patients. In addition, our in vitro results imply the possible functional involvement of CD44 in DOC resistance.     

Lack of direct effect of 2,3,7,8-tetrachlorodibenzo-P-dioxin (TCDD) on protein kinase C activity in EL4 cells

In vivo administration of TCDD produces an increase in the level of Protein Kinase C in the hepatic plasma membrane. We have studied the direct effects of TCDD on cultured EL4 thymoma cells, which contain a large amount of Protein Kinase C and respond to phorbol esters with rapid translocation of the kinase to the membrane, followed by growth inhibition, adherence to substrate and production of interleukin 2. TCDD (10-1000 nM) did not compete with 3H-phorbol dibutyrate for binding to cytosolic Protein Kinase C, and had no effect on Protein Kinase C activity in vitro. TCDD did not stimulate translocation of Protein Kinase C to the membrane, and did not affect phorbol ester-stimulated translocation. TCDD did not inhibit EL4 cell growth or affect phorbol ester induced growth inhibition, and failed to stimulate production of interleukin 2. Thus, TCDD does not appear to activate Protein Kinase C in EL4 cells.     

A fast-acting cytotoxin derived from Con A-activated porcine leukocytes. I. Biochemical characteristics and target cell specificity

We here describe a new cytotoxin which was detected in serum-free culture supernatants of mitogen stimulated porcine leukocytes. The factor was precipitated at 35-45% (NH4)2 SO4 concentration, sensitive to heat, low pH, and trypsin, thus indicating its protein nature. Column chromatography on hydrophilic or hydrophobic matrices revealed amphiphilic properties. The smallest unit which was sufficient to induce complete target cell lysis had a molecular weight of 33K. This porcine cytotoxin (PCT) could be distinguished functionally from lymphotoxin (LT) and tumor necrosis factor (TNF). It is a fast acting mediator which exerts strong cytostatic and cytotoxic anti-tumor activity using a panel of T lymphoma target cells.