Methodological Standards and Problems in Preclinical Homoeopathic Potency Research.
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Journal Article↗
Biomedical subjects
S Baumgartner
Publications and source records attributed to S Baumgartner.
A Drosophila neurexin is required for septate junction and blood-nerve barrier formation and function.
Septate and tight junctions are thought to seal neighboring cells together and to function as barriers between epithelial cells. We have characterized a novel member of the neurexin family, Neurexin IV (NRX), which is localized to septate junctions (SJs) of epithelial and glial cells. NRX is a transmembrane protein with a cytoplasmic domain homologous to glycophorin C, a protein required for anchoring protein 4.1 in the red blood cell. Absence of NRX results in mislocalization of Coracle, a Drosophila protein 4.1 homolog, at SJs and causes dorsal closure defects similar to those observed in coracle mutants. nrx mutant embryos are paralyzed, and electrophysiological studies indicate that the lack of NRX in glial-glial SJs causes a breakdown of the blood-brain barrier. Electron microscopy demonstrates that nrx mutants lack the ladder-like intercellular septa characteristic of pleated SJs (pSJs). These studies identify NRX as the first transmembrane protein of SJ and demonstrate a requirement for NRX in the formation of septate-junction septa and intercellular barriers.
Amino Acid Sequence↗
Re-isolated extra sex combs (esc) gene of Drosophila.
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Animals↗
The Polycomb-group gene, extra sex combs, encodes a nuclear member of the WD-40 repeat family.
We have delimited the extra sex combs (esc) gene to < 4 kb that include a single transcript and are able to rescue both the maternal and zygotic esc phenotypes. Several mutations have been identified within the esc transcript. In agreement with earlier genetic studies, esc is expressed maternally and its product is most abundant during the early embryonic stages. It encodes a protein of the WD-40 repeat family, which localizes predominantly to the nucleus. During germ band extension, it is expressed in a stereotypic pattern of neuroblasts. We propose a model in which Esc is recruited by gap proteins both to act as a corepressor that competes with the TAFII80 coactivator to block transcription and also to mediate the transition to permanent repression by Polycomb-group proteins.
Amino Acid Sequence↗
The HEM proteins: a novel family of tissue-specific transmembrane proteins expressed from invertebrates through mammals with an essential function in oogenesis.
We report the identification of a new family of proteins, termed the HEM family, which show distinct expression patterns in blood cells and the central nervous system. Through the isolation and characterization of the corresponding brain-specific Drosophila (dhem-2) and rat orthologues (Hem-2), and through the detection of the Caenorhabditis elegans Hem-2 orthologue in the database, we show that this family is conserved throughout evolution. HEM proteins show a conserved length ranging from 1118 to 1126 amino acid residues. Moreover, they are at least 35% identical with each other and harbour several conserved membrane-spanning domains, indicative for their location on the cell surface. One of the members, the Drosophila orthologue dhem-2, was analysed in detail for its spatial expression pattern during development and for its mutant phenotype. dhem-2 is expressed maternally in the oocyte and shows uniform expression during the first half of embryogenesis, but becomes restricted to the brain and the nervous system during late embryogenesis, consistent with the expression of its vertebrate orthologue in the brain. One P-element insertion, located 39 base-pairs downstream from the dhem-2 transcription start site, causes female sterility, due to the fact that developmental processes in the oocyte are disturbed. Of the vertebrate HEM family members, the mammalian Hem-1 gene is expressed only in cells of hematopoietic origin, while Hem-2 is preferentially expressed in brain, heart, liver and testis.
Adaptor Proteins, Signal Transducing↗
Purification of exo- and endoinulinase from crude inulinase extract for the analysis of fructans.
This paper describes a technique for rapid and easy separation of a crude inulinase extract into exo- and endoinulinase from a commercially available inulinase stock (Novozym 230 from Novo Nordisk A/S) by means of non-denaturating polyacrylamide gel electrophoresis. The purified enzymes were tested with inulin. Characterization of the reaction products was done by high-performance thin-layer chromatography, size exclusion chromatography and semi-preparative reverse-phase high-performance liquid chromatography.
Chromatography, High Pressure Liquid↗
Tenm, a Drosophila gene related to tenascin, is a new pair-rule gene.
We describe the molecular characterization of the Drosophila gene tenm, a large transcription unit spanning > 110 kb of DNA. tenm encodes a large extracellular protein of 2515 amino acids related to the extracellular matrix molecule tenascin. The Tenm protein is found in seven stripes during the blastoderm stage, and each stripe overlaps with the even-skipped stripes. tenm mutants show a phenotype resembling that of odd-paired (opa), a member of the pair-rule class of segmentation genes. Thus, Tenm is the first example of a pair-rule gene product acting from outside the cell. While the Tenm protein is under the control of fushi tarazu and even-skipped, but not of opa, at least two pair-rule genes, paired (prd) and sloppy paired (slp), and all segment-polarity genes analysed to date are under the control of tenm. Our data suggest that Tenm initiates a signal transduction cascade which acts, via or in concert with opa, on downstream targets such as prd, slp, gooseberry, engrailed and wingless, leading to an opa-like phenotype.
Amino Acid Sequence↗
Tenascin-C expression by fibroblasts is elevated in stressed collagen gels.
Chick embryo fibroblasts cultured on a collagen matrix exert tractional forces leading to the contraction of unrestrained, floating collagen gels and to the development of tension in attached, restrained gels. On a restrained, attached collagen gel the fibroblasts synthesize large quantities of tenascin-C, whereas in a floating, contracting gel tenascin-C synthesis is decreased. This regulation of tenascin-C synthesis can be observed by the secretion of metabolically labeled tenascin-C into the conditioned medium, as well as by the deposition of tenascin-C into the collagen matrix as judged by immunofluorescence. Regulation appears to occur at the transcriptional level, because when cells on attached or floating collagen gels are transfected with promoter constructs of the tenascin-C gene, luciferase expression driven by the tenascin-C promoter parallels the effects measured for endogenous tenascin-C synthesis, whereas luciferase expression under the control of the SV40 promoter does not depend on the state of the collagen gel. The promoter region responsible for tenascin-C induction on attached collagen gels is distinct from the region important for the induction of tenascin-C by serum, and may define a novel kind of response element. By joining this tenascin-C sequence to the SV40 promoter of a reporter plasmid, its activity can be transferred to the heterologous promoter. We propose that the tenascin-C promoter is directly or indirectly activated in fibroblasts generating and experiencing mechanical stress within a restrained collagen matrix. This may be an important aspect of the regulation of tenascin-C expression during embryogenesis as well as during wound healing and other regenerative and morphogenetic processes.
Animals↗
Novel tenascin variants with a distinctive pattern of expression in the avian embryo.
Previous studies have shown that several forms of the glycoprotein tenascin are present in the embryonic extracellular matrix. These forms are the result of alternative splicing, which generates tenascin variants with different numbers of fibronectin type III repeats. We have used degenerate primers and PCR to isolate a novel tenascin exon from an avian genomic library. Genomic clones contained a sequence encoding a fibronectin type III repeat that corresponds to repeat 'C' from the variable domain of human tenascin. To demonstrate that tenascin containing repeat 'C' is actually synthesized by avian cells, a monospecific antiserum was raised against a repeat 'C' fusion protein. This antiserum recognized a novel high-molecular-weight variant on immunoblots of tenascin isolated from chicken embryo fibroblast-conditioned medium, and stained tendons on frozen sections of chicken embryos. A cDNA probe specific for mRNA encoding repeat 'C' was used for in situ hybridization. This probe hybridized in a subset of the embryonic tissues labelled with a universal tenascin probe, including tendons, ligaments and mesenchyme at sites of epithelial-mesenchymal interactions. Finally, we provide evidence that additional fibronectin type III repeats, one corresponding to a recently discovered human repeat as well as one entirely novel sequence, also exists in chicken tenascin mRNA. These data indicate that tenascin is present in the embryonic matrix in a multitude of forms and that these forms have distinctive distributions that may reflect more than one function for tenascin in development.
Amino Acid Sequence↗
Drosophila ribosomal protein S19 cDNA sequence.
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Amino Acid Sequence↗
The 55 kd regulatory subunit of Drosophila protein phosphatase 2A is required for anaphase.
The gene encoding the Drosophila protein phosphatase 2A 55 kd regulatory subunit (PR55) is located at 85F and directs the synthesis of differentially spliced transcripts. Maternal RNAs are present at very high levels in early embryos and decline around cellularization. Zygotic transcripts are present mainly in the developing embryonic nervous system and gonads. Transcripts are uniformly distributed in third instar larval discs and testes and at lower levels in the proliferative centers of the brain. Mutations in abnormal anaphase resolution (aar) are rescued by the wild-type gene for PR55. aar mutants display intact lagging chromatids that have undergone separation from their sisters, but that remain at the position formerly occupied by the metaphase plate, as well as anaphase figures that show bridging chromatin having two centromeric regions.
Alternative Splicing↗
Tena, a Drosophila gene related to tenascin, shows selective transcript localization.
We report the identification and molecular characterization of tena*, a Drosophila gene located at 11A6-9 on the X-chromosome. The deduced protein of 782 amino acids contains eight tenascin-type EGF-like repeats not described in Drosophila before, but lacks the fibronectin type III repeats and the fibrinogen homology present in the vertebrate tenascin molecules. Tena codes for a large transcript which exhibits extremely long 5' and 3' untranslated regions. Tena transcripts show a specific perinuclear localization within cells and are mainly expressed in the central nervous system, in the brain and near muscle attachment sites during embryogenesis. During pupal stages, tena is detected in the eye. These expression patterns are reminiscent of those of vertebrate tenascin. Tenascin-type EGF-like sequences are also detected in other loci of Drosophila and in various other organisms, indicating the existence of a family of genes related to tenascin.
Amino Acid Sequence↗
Molecular cloning and developmental expression of the catalytic and 65-kDa regulatory subunits of protein phosphatase 2A in Drosophila.
cDNA clones encoding the catalytic subunit and the 65-kDa regulatory subunit of protein phosphatase 2A (PR65) from Drosophila melanogaster have been isolated by homology screening with the corresponding human cDNAs. The Drosophila clones were used to analyze the spatial and temporal expression of the transcripts encoding these two proteins. The Drosophila PR65 cDNA clones contained an open reading frame of 1773 nucleotides encoding a protein of 65.5 kDa. The predicted amino acid sequence showed 75 and 71% identity to the human PR65 alpha and beta isoforms, respectively. As previously reported for the mammalian PR65 isoforms, Drosophila PR65 is composed of 15 imperfect repeating units of approximately 39 amino acids. The residues contributing to this repeat structure show also the highest sequence conservation between species, indicating a functional importance for these repeats. The gene encoding Drosophila PR65 was located at 29B1,2 on the second chromosome. A major transcript of 2.8 kilobase (kb) encoding the PR65 subunit and two transcripts of 1.6 and 2.5 kb encoding the catalytic subunit could be detected throughout Drosophila development. All of these mRNAs were most abundant during early embryogenesis and were expressed at lower levels in larvae and adult flies. In situ hybridization of different developmental stages showed a colocalization of the PR65 and catalytic subunit transcripts. The mRNA expression is high in the nurse cells and oocytes, consistent with a high equally distributed expression in early embryos. In later embryonal development, the expression remains high in the nervous system and the gonads but the overall transcript levels decrease. In third instar larvae, high levels of mRNA could be observed in brain, imaginal discs, and in salivary glands. These results indicate that protein phosphatase 2A transcript levels change during development in a tissue and in a time-specific manner.
Amino Acid Sequence↗
Epithelial synthesis of tenascin at tips of growing bronchi and graded accumulation in basement membrane and mesenchyme.
The extracellular matrix protein, tenascin, has been proposed as mediator in epithelial-mesenchymal interactions because of its characteristic distribution during embryogenesis. Here we compared the accumulation of tenascin and laminin in the early chicken lung bud. Laminin is deposited in the basement membrane, starting at the tips and increasing along the shafts of growing primary and secondary bronchi. In contrast, tenascin accumulation is highest in basement membranes and mesenchyme at sites where new bronchial branches are formed. By in situ hybridization, tenascin mRNA was found to be produced exclusively by the epithelium at sites of active growth of bronchial tubes.
Animals↗
Nerve-dependent and -independent tenascin expression in the developing chick limb bud.
The extracellular matrix protein, tenascin, appears in a restricted pattern during organ morphogenesis. Tenascin accumulates along developing peripheral nerves as they leave the spinal cord and enter the limb mesenchyme (Wehrle and Chiquet, Development 110, 401-415, 1990). Here we found that most but not all tenascin deposited along growing nerves is of glial origin. By in situ hybridization with a tenascin cDNA probe, we determined the site of tenascin mRNA accumulation both in normal and nerve-free limbs. In normal wing buds, tenascin mRNA was first detected within the developing limb nerves. Vinculin-positive glial precursor cells, which comigrate with the axons, are the likely source of this tenascin message. In nerveless wing grafts, tenascin was first expressed in tendon primordia in the absence, and thus independently, from innervation. In contrast to normal limbs, grafted wing buds neither contained vinculin-positive glial precursor cells, nor expressed tenascin in regions proximal to tendon primordia. In normal wing buds, tenascin deposited by tendon primordia transiently parallels and surrounds certain developing nerves. After the major nerve pattern is established, tenascin mRNA disappears from nerves in the upper limb, but is expressed in perichondrium and tendons. We propose that glial tenascin facilitates the penetration of axons into the limb bud and is important for nerve fasciculation. In some places, early tendon primordia might help to guide the migration of axons and glial precursor cells towards their target.
Animals↗
Network of interactions among pair-rule genes regulating paired expression during primordial segmentation of Drosophila.
A model of the trans-regulation of the Drosophila pair-rule segmentation gene, paired (prd), has been derived from the observed alterations in the distribution of prd transcripts during early embryogenesis in single and double pair-rule mutants. Important aspects of the model include the following features and implications: (i) The regulation of prd is subject to a regulatory hierarchy among pair-rule genes. In particular, it shows that prd is at the bottom of this hierarchy, mediating the transition from pair-rule to segment-polarity genes. The transition of the early 'pair-rule' to the 'segment-polarity' pattern of prd expression is regulated by the secondary pair-rule genes opa and odd. The model predicts the distributions of pair-rule gene products regulating prd at late syncytial blastoderm, e.g., that of opa. (ii) The initial activation of prd is independent of pair-rule genes. (iii) The regulation of prd is combinatorial and hence probably cooperative. Combination of one pair-rule gene product with different pair-rule proteins may lead to opposite regulatory effects on prd. Furthermore, we discuss a two-step conversion of the initial analogue specification of position along the anteroposterior axis into a digital code specified by combinations of active segment-polarity and homeotic genes.
Animals↗
Isolation of two tissue-specific Drosophila paired box genes, Pox meso and Pox neuro.
Two new paired domain genes of Drosophila, Pox meso and Pox neuro, are described. In contrast to the previously isolated paired domain genes, paired and gooseberry, which contain both a paired and a homeo-domain (PHox genes), Pox meso and Pox neuro possess no homeodomain. Evidence suggesting that the new genes encode tissue-specific transcriptional factors and belong to the same regulatory cascade as the other paired domain genes includes (i) tissue-specific expression of Pox meso in the somatic mesoderm and of Pox neuro in the central and peripheral nervous system, (ii) nuclear localization of their proteins, (iii) dependence on prd activity and (iv) presence of the paired domain in genes of known regulatory activity. While no mutant phenotypes of Pox meso and Pox neuro have yet been discovered, a murine gene with a paired domain closely homologous to that of Pox meso has recently been identified with the undulated mutant. Both Pox meso and undulated are expressed in tissues derived from the somatic mesoderm. The five known Drosophila paired domains fall into three classes: (i) the prd,gsb-class, (ii) the Pox meso, undulated-class and (iii) the Pox neuro-class which probably includes the paired domain of the murine gene Pax 2.
Amino Acid Sequence↗
Molecular characterization of spalt, a homeotic gene required for head and tail development in the Drosophila embryo.
The isolation, identification and structure of the spalt gene is described. This novel homeotic gene of Drosophila is required for the establishment of the posterior-most head and the anterior-most tail segments of the embryo. It encodes a small mRNA of 0.8 kb which is under the control of over 15 kb of upstream sequences as indicated by the phenotype of transformed embryos. The putative spalt protein contains internal repeats and other interesting structural motifs but no homeo box. The spalt transcript accumulates motifs but no homeo box. The spalt transcript accumulates to high levels in the segmental anlagen affected in mutant embryos but is also found in regions of the embryo where no functional requirement has been demonstrated.
Journal Article↗