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Biomedical subjects

M Saarma

Publications and source records attributed to M Saarma.

At least 73 records · Page 4Linked to original sources
PubMed1997

Cloning, mRNA distribution and chromosomal localisation of the gene for glial cell line-derived neurotrophic factor receptor beta, a homologue to GDNFR-alpha.

Glial cell line-derived neurotrophic factor (GDNF) is a potent survival factor for central dopaminergic neurons, motor neurons and several other populations of neurons in the central and peripheral nervous system. GDNF and its receptor complex of c-RET tyrosine kinase and a glycosyl-phosphatidylinositol linked protein GDNFR-alpha are of great interest due to their potential use in the therapy of Parkinson's and motoneuron diseases. We have cloned the human and rat cDNA sequences of GDNFR-beta, a new gene encoding for a 464 amino acid long homologue of GDNFR-alpha, and assign the locus of this new gene to human chromosome 8p21-22 and mouse chromosome 14D3-E1. Similarly to GDNFR-alpha, GDNFR-beta mediates GDNF-induced Ret autophosphorylation in transfected cells. By northern hybridisation we show that the transcript level of human GDNFR-beta mRNA is high in the adult brain, intestine and placenta and in fetal brain, lung and kidney. Studied by in situ hybridisation, GDNFR-beta mRNA shows in E17 rat embryo different distribution to that of GDNFR-alpha mRNA, especially, in adrenal gland, kidney and gut. In the developing nervous system, GDNFR-beta mRNA expression is restricted to certain neuronal populations, while GDNFR-alpha mRNA is widely expressed also in non-neuronal cells. The distinct tissue distribution of GDNFR-beta mRNA and its ability to mediate GDNF signal in transfected cells suggest a role in signal transduction of GDNF and, possibly, related neurotrophic factors in vivo.

Amino Acid Sequence↗
PubMed1997

Exclusion of the p75 neurotrophin receptor gene as a candidate gene for Meckel syndrome.

Nerve growth factor receptor p75 (NGFR) gene was investigated as a potential candidate gene in Meckel syndrome (MKS) because of its important role in embryonic development, chromosomal localization adjacent to the MKS locus and Meckel syndrome-resembling findings in knock-out mice phenotype. The sequence analysis of the coding region of the gene revealed one polymorphism but no potential disease mutation. Physical mapping of the critical chromosomal region finally showed that the NGFR gene lies outside the MKS locus.

Abnormalities, Multiple↗
PubMed1997

Monoclonal antibodies specific for Semliki Forest virus replicase protein nsP2.

A panel of monoclonal antibodies (MAbs) was raised against Semliki Forest virus (SFV) nonstructural protein nsP2, which is a protease, an NTPase, a putative RNA helicase, and a regulator of the synthesis of the subgenomic 26S mRNA encoding the structural proteins. nsP2, used for immunization, was expressed as a histidine fusion protein in Escherichia coli and purified by metal affinity chromatography. Dot-blot assay, using a membrane fraction from SFV-infected cells as antigen, gave 33 positive clones. Of these, 30 MAbs recognized nsP2 in Western immunoblotting, and 25 showed positive indirect immunofluorescence (IFAT) in SFV-infected cells; 15 MAbs stained the cytoplasmic vacuoles (CPVI), which are the sites of viral RNA synthesis in alphavirus-infected cells. MAb 3B5 recognized only CPVIs, as shown by double immunofluorescence staining with polyclonal anti-nsP3 antiserum. Most of the MAbs (20/33) recognized the nuclear form of nsP2, which may be associated with SFV neurovirulence. Immunoprecipitation with MAbs revealed that the SFV nonstructural proteins are associated with each other. None of the MAbs recognized Sindbis virus nsP2 in immunoblotting, indicating that they were directed to non-conserved sequences specific for SFV. Interestingly, these epitopes were located mostly within the N-terminal half of nsP2. Unexpectedly, the anti-nsP2 MAb 1E9 cross-reacted strongly with a host protein of 78 kDa from uninfected human, murine, avian and insect cells. This protein was identified as the immunoglobulin binding protein, BiP, by 2-D gel mapping and reaction with anti-BiP antiserum.

Animals↗
PubMed1997

Localization of nerve cells in the developing rat tooth.

Earlier studies have shown that mammalian tooth formation can take place in the absence of peripheral nerve fibers. This has been taken to indicate that neurons are not needed for mammalian tooth development. However, our recent localization of peripherin, which is a neuronal cell marker, has suggested that neuronal cell bodies may be associated with developing teeth. In this study, we have analyzed in vivo and in vitro the presence of neuronal cells in developing rat tooth germs. When E14 and E16 rat first molars (thickening of presumptive dental epithelium and bud-stage tooth germ, respectively) were cultured in vitro, peripheral trigeminal axons degenerated. However, with antibodies against peripherin and L1 neural cell adhesion protein, we detected neuronal cell bodies and their axons in the explants. Next, the expression of neurofilament light-chain (NF-L) mRNAs was studied by in situ hybridization of embryonic E12 first branchial arches and tooth germs from initiation to completion of crown morphogenesis (E13, five-day post-natal teeth). NF-L transcripts were first seen at the bud stage (E15) next to the dental epithelium at the buccal side of the tooth germ. At the cap stage (E18), NF-L mRNAs were located under the oral epithelium at some distance from dental epithelium. These expression patterns correlate to the previous localization of peripherin-positive cells and suggest that NF-L expression also revealed neuronal cells. Taken together, these results demonstrate that, in addition to projections of peripheral neurons, neuronal cells are associated with the developing teeth. Hence, it is possible that neuronal cells may participate in the regulation of mammalian tooth formation.

Animals↗
PubMed1997

Glial-cell-line-derived neurotrophic factor is required for bud initiation from ureteric epithelium.

The shapes of different organs can be explained largely by two fundamental characteristics of their epithelial rudiments - the pattern of branching and the rate of proliferation. Glial-cell-line-derived neurotrophic factor (GDNF) has recently been implicated in the development of metanephric ureteric epithelium (Pichel, J. G., Shen, L., Sheng, H. Z., Granholm, A.-C., Drago, J., Grinberg, A., Lee, E. J., Huang, S. P., Saarma, M., Hoffer, B.J., Sariola, H. and Westphal, H. (1996). Nature 382, 73-76; Sánchez, M.P., Silos-Santiago, I., Frisén, J., He, B., Lira, S.A. and Barbacid, M. (1996). Nature 382, 70-73; Vega, Q.C., Worby, C.A., Lechner, M.S., Dixon, J.E. and Dressler, G.R. (1996). Proc. Nat. Acad. Sci. USA 93, 10657-10661). We have analysed the target cells of GDNF and the manner in which it controls ureteric development, and have compared it with other growth factors that have been associated with the regulation of branching morphogenesis, namely hepatocyte growth factor (HGF) and transforming growth factor-beta1 (TGFbeta1). We show that GDNF binds directly to the tips of ureteric bud branches, and that it has the ability to promote primary ureteric buds from various segments of Wolffian duct and to attract ureteric branches towards the source of GDNF. It increases cell adhesion, but is not obviously mitogenic for ureteric cells. The data indicate that GDNF is required primarily for bud initiation. Comparison of GDNF, HGF and TGFbeta1 suggests that the latter act later than GDNF, and may represent a partially redundant set of mesenchyme-derived growth factors that control ureteric development. Thus, GDNF is the first defined inducer in the embryonic metanephric kidney.

Animals↗
PubMed1996

mRNAS for one, two or three members of trk receptor family are expressed in single rat trigeminal ganglion neurons.

We studied the expression of mRNAs of neurotrophin (NTF) receptors trkA, trkB and trkC in single rat trigeminal ganglion neurons at embryonic days 12 and 16 to determine, whether single trigeminal ganglion neurons express one trk family member or coexpress several of them. For that purpose we elaborated a sensitive technique of reverse transcriptase-polymerase chain reaction to detect all neurotrophin receptors in a single neuron. Expression of neurofilament light chain mRNA was used as a positive marker to confirm the recovery of mRNAs from single neurons. Neurofilament-positive samples were subsequently analyzed for the expression of mRNAs for catalytic trkA, trkB, and trkC, and in some cases, low-affinity neurotrophin receptor (p75). We found neurons expressing one, coexpressing two, or even all three trk receptors. In many neurons analyzed, p75 mRNA was coexpressed with trks, but we also found neurons expressing only trks without p75, and a neuron expressing p75 alone. There were also neurons containing neither trk receptors nor p75. We provide here first direct evidence that single sensory neurons can simultaneously express three or even four neurotrophin receptors.

Animals↗
PubMed1996

Induced expression of neurotrophins in transgenic mice overexpressing ornithine decarboxylase and overproducing putrescine.

Transgenic mice overexpressing ornithine decarboxylase (ODC) were recently generated (Halmekytö et al.: Biochem Biophys Res Commun 180:262-267, 1991). The dramatic ODC overexpression resulted in a very high accumulation of the polyamine putrescine in the brain. As elevated polyamine levels in the brain are believed to be associated with neuronal damage, we studied whether enhanced putrescine accumulation in the brain of these mice affects the expression of neurotrophins and their high affinity receptors. Northern blot analysis indicated that mRNA levels of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and neurotrophin-3 (NT-3) were significantly elevated about 1.5-fold in the hippocampus of ODC transgenic mice as compared with control animals. The levels of BDNF, NGF and NT-3 mRNA were also elevated in the kidneys of the transgenic mice. In eight other tissues examined there were no significant differences. The expression pattern of BDNF mRNA and of trkB and trkC (high affinity receptors for BDNF and NT-3 respectively) immunoreactivity in the hippocampal formation did not reveal significant differences. The induction of the expression of neurotrophins could belong to neuroprotective measures triggered by ODC overexpression.

Animals↗
PubMed1996

The rho 1 GABA receptor cloned from rat retina is down-modulated by protons.

We have cloned and sequenced the full-length cDNA of the rat GABA (gamma-aminobutyric acid) rho 1 receptor subunit. The deduced amino acid sequence (474 amino acids) of the rat rho 1 receptor is 95% homologous to the previously cloned human rho 1 receptor. The rho 1 cDNA includes a 5' 129 bp and a 3' 2.6 kb untranslated region, which contains a sequence homologous to the human medium reiteration frequency repetitive sequence, MER18. The rat rho 1 receptor shows 45-50% similarity to the GABAA receptor beta subunits. This similarity is among the highest between all GABAA receptor subunit classes, giving no support at the molecular level to the classification of the rho subunits to a novel GABAC receptor class. The rat rho 1 cDNA formed functional GABA receptors insensitive to bicuculline, but sensitive to blockade by picrotoxin, when transiently expressed in the human embryonic kidney cell line HEK 293. We studied the sensitivity of the rho 1-mediated GABA current to variations in extracellular pH (pH0), and found that these receptors are strongly down-modulated by H+ ions. A decrease in pH0 from 7.4 to 6.4 decreased the GABA current by 51 +/- 8%, whereas an increase in pH0 from 7.4 to 8.4 increased the current by 77 +/- 8%. In view of the up-regulatory effect of protons on the GABA current observed in a number of preparations, the rho 1 receptors may contain a novel down-regulatory binding site for protons characteristic to these receptors.

Amino Acid Sequence↗
PubMed1996

Defects in enteric innervation and kidney development in mice lacking GDNF.

Glial-lial-cell-line-derived neurotrophic factor (GDNF) has been isolated as neurotrophic factor for midbrain dopaminergic neurons. Because of its neurotrophic activity on a wide range of neuronal populations in vitro and in vivo, GDNF is being considered as a potential therapeutic agent for neuronal disorders. During mammalian development, it is expressed not only in the nervous system, but also very prominently in the metanephric kidney and the gastrointestinal tract, suggesting possible functions during organogenesis. We have investigated the role of GDNF during development by generating a null mutation in the murine GDNF locus, and found that mutant mice show kidney agenesis or dysgenesis and defective enteric innervation. We demonstrate that GDNF induces ureter bud formation and branching during metanephros development, and is essential for proper innervation of the gastrointestinal tract.

Animals↗
PubMed1996

GDNF signalling through the Ret receptor tyrosine kinase.

Mutational analysis in humans and mice has demonstrated that the Ret, the product of the c-ret proto-oncogene, a member of the receptor tyrosine kinase (RTK) superfamily, is essential for development of the enteric nervous system and kidney. Despite the established role of Ret in mammalian embryogenesis, its cognate ligand(s) is currently unknown. Here we demonstrate, by using a Xenopus embryo bioassay, that glial-cell-line-derived neurotrophic factor (GDNF), a distant member of the transforming growth factor (TGF)-beta superfamily, signals through the Ret RTK. Furthermore, using explant cultures from wild-type and Ret-deficient mouse embryos, we show that normal c-ret function is necessary for GDNF signalling in the peripheral nervous system. Our data strongly suggest that Ret is a functional receptor for GDNF, and that GDNF, in addition to its potential role in the differentiation and survival of central nervous system neurons, has profound effects on kidney organogenesis and the development of the peripheral nervous system.

Animals↗
PubMed1996

Expression of neurotrophin receptors during rat tooth development is developmentally regulated, independent of innervation, and suggests functions in the regulation of morphogenesis and innervation.

Low-affinity neurotrophin receptor (LANR) and trk receptor tyrosine kinases (trks) serve as low- and high-affinity receptors for neurotrophins. Besides promoting the development and maintenance of the mammalian nervous system, it has been suggested that neurotrophins may have broader functions in the development of non-neuronal tissues. To evaluate the possible roles of neurotrophic factors in tooth development, we performed a detailed examination of the expression patterns of neurotrophin receptors during development of the rat tooth from initiation to completion of crown morphogenesis. mRNA expression was studied by in situ hybridisation and LANR protein was localised by immunohistochemistry. Furthermore, dissected tooth germs were cultured in vitro to examined the role of trigeminal innervation in the expression of neurotrophin receptors. mRNAs for LANR, trkB, and trkC, but not trkA, were detected in developing teeth. LANR and the truncated form of trkB, which lacks the intracellular tyrosine kinase domain, were expressed throughout tooth morphogenesis and their expression patterns were largely non-overlapping and changed spatio-temporally. trkC was expressed after birth, and it was restricted to dental papilla mesenchyme. The expression of all receptors correlated with the development of innervation, but, in addition, the expression of LANR and trkB appeared to be associated with cell differentiation and epithelial-mesenchymal interactions. The patterns of LANR, trkB, and trkC in teeth which underwent morphogenesis in organ culture were similar to those in vivo, which indicates that the expression of these neurotrophin receptors is not regulated by and does not depend on trigeminal innervation. The data suggest that neurotrophin receptors have roles in the development of tooth innervation, but that they also have non-neuronal, organogenetic functions.

Aging↗
PubMed1996

Infectious in vitro transcripts from cloned cDNA of the potato A potyvirus.

A full-length cDNA copy of potato virus A RNA was constructed downstream from the bacteriophage T7 RNA polymerase promoter. A single extra guanosine not present in PVA RNA was added to the 5'-end of the cDNA. The capped in vitro transcripts from cloned cDNA were infectious in the mesophyll protoplasts and intact plants of Nicotiana tabacum. PVA coat protein accumulated in transcript-inoculated tobacco protoplasts and infected systematically intact plants producing high PVA titres according to the ELISA and immunoblot assay. The infected tobacco plants produced similar mild mosaic symptoms in the systematically infected leaves, irrespective of whether the RNA transcripts or parental virus were used as inoculum; the virus particles were also morphologically similar according to immunosorbent electron microscopy.

Base Sequence↗
PubMed1996

Localization of glial cell line-derived neurotrophic factor (GDNF) mRNA in embryonic rat by in situ hybridization.

The localization of glial cell line-derived neurotrophic factor (GDNF) mRNA was studied by in situ hybridization in rat from embryonic (E) day E10 to E15. At E10, GDNF mRNA is found in the urogenital field and the cranial part of the gut. At E11, the most abundant expression of GDNF mRNA is seen in the epithelial cells of the second, third and fourth pharyngeal pouches, the third and fourth pharyngeal arches and pharynx. Also mesenchymal cells of the gut and mesonephric tubules contain GDNF mRNA. At E13, expression is observed in the mesenchymal cell layers of the oesophagus, intestine and stomach, the mesenchymal cells around the condensing cartilages and metanephric kidney mesenchyme. Also, the epithelia of Rathke's pouch and pharynx are intensely labelled. High expression of GDNF mRNA continues at E15 in kidney, gastrointestinal tract and cartilage. At that stage, GDNF mRNA is seen also in whisker pad and skeletal muscles. The distribution of GDNF mRNA in embryonic rat suggests important roles for GDNF in the early differentiation of the kidney tubules, the innervation of the gastrointestinal tract and the differentiation process of the cartilage and muscle. Our results indicate novel functions for GDNF outside the nervous system.

Animals↗
PubMed1996

Expression of mRNAs for neurotrophins and their receptors in developing rat heart.

Because the neurotrophic system has not been systematically studied in developing heart, we studied the expression of mRNAs for neurotrophins and their high- and low-affinity receptors by radioactive in situ hybridization in the rat heart from embryonic day 9 (E9) to parturition. The neurotrophin-3 (NT-3) transcripts were seen in the group of Leu-7 immunoreactive cells in the ventricular region from E11 to parturition, suggesting that NT-3 is expressed in the part of the developing conduction system, mRNAs for truncated trk receptors, trkC.TK- and trkB.T1, were expressed in the outflow tract at E12 and in the walls of developing aorta and pulmonary trunk from E13 to parturition, whereas the mRNA for catalytic trkC.TK+ was revealed in the walls of aorta and pulmonary trunk from E13 to parturition and in the cardiac ganglion neurons from E14 to adult stage. Transcripts for low-affinity neurotrophin receptor (p75) were transiently seen in the distal outflow tract from E11 to E13, declining by E14. At E18, p75 transcripts were also seen in the cardiac ganglia. Transcripts for nerve growth factor, neurotrophin-4/5, trkA, or trkB.TK+ were not detected. Expression of NT-3 mRNA in the developing conduction system and of trkC.TK + mRNA in the cardiac neurons suggests a role for NT-3 in the innervation of the conduction system. Expression of trkC.TK+ in the wall of aorta and pulmonary trunk suggests that NT-3 also may affect the development of the smooth muscle cells.

Aging↗
PubMed1995

Neurotrophin 3 rescues neuronal precursors from apoptosis and promotes neuronal differentiation in the embryonic metanephric kidney.

We analyzed the developmental regulation and role of the neurotrophins during metanephric kidney morphogenesis. RNase protection assay revealed the presence of nerve growth factor, neurotrophin 3 (NT-3), and brain-derived neurotrophic factor mRNAs and the regulation of their expression during embryonic development of rat metanephros. NT-3 induced differentiation (neurite outgrowth) and survival (inhibition of apoptosis) of the neuronal precursors in cultured nephrogenic mesenchymes and neuronal differentiation in cultured whole kidneys, whereas NT-4/5, brain-derived neurotrophic factor, and nerve growth factor were without effect. The neurotrophins did not trigger tubular differentiation of isolated nephrogenic cells, which underwent apoptosis when cultured with or without the neurotrophins. NT-3 is thus an inducer of differentiation and a survival factor for renal neuronal cells, but none of the neurotrophins is a morphogen in kidney tubule induction.

Animals↗
PubMed1995

Sequencing of the rat light neurofilament promoter reveals differences in methylation between expressing and non-expressing cell lines, but not tissues.

The DNA methylation pattern of the promoter (pNF-L) region of the rat light-neurofilament-encoding gene (NF-L), a neuron-specific gene, was assessed in NF-L expressing and non-expressing cell lines and tissues by genomic sequencing using PCR amplification of bisulfite-modified DNA. We analysed twenty-five potential CpG methylation sites between nucleotide (nt) positions -311 and +103 of pNF-L, containing Sp1- and AP-2-binding sites, a CGCCCCCGC box and a cAMP-responsive element. Six out of 25 possible CpG methylation sites are within these elements. The pNF-L promoter was unmethylated in NF-L-expressing rat brain, as well as in liver not expressing NF-L. In NF-L-expressing PC12 cells, the promoter was unmethylated, whereas in non-expressing glioma C6 cells intensive methylation occurred. A cluster of methylated CpG dinucleotides spanned the region from nt -176 to -67 bp. Thus, methylation of this promoter region could play a role in silencing NF-L in the glioma cell line in vitro, but not in liver tissue in vivo. In a non-CpG sequence, in the CpApG trinucleotide at nt position -114, cytosine was found to be partially methylated. It is thus possible to describe the methylation state of each cytosine present in the area of genomic DNA of interest.

Animals↗
PubMed1995

The putative replicase of the cocksfoot mottle sobemovirus is translated as a part of the polyprotein by -1 ribosomal frameshift.

The polyprotein of cocksfoot mottle sobemovirus (CfMV) is encoded by two overlapping open reading frames (ORF). The ORF 2a codes for the putative VPg and serine protease and the ORF 2b codes for the putative replicase. The consensus signals for a -1 ribosomal frameshifting event are found at the very beginning of the overlapping region of these ORFs. The shifty heptanucleotide in CfMV is UUUAAAC, and the secondary structure after the shifty sequence is predicted to be a stem-loop. In vitro translation of the CfMV RNA in wheat germ extract produced proteins of several sizes, including one of 100 kDa. According to the nucleotide sequence data, no single ORF is capable of directing the synthesis of a 100-kDa protein. A chimeric beta-glucuronidase-CfMV cDNA containing the entire ORF 2a and 2b overlap region including frameshift signals was constructed. A trans-frame protein of 108 kDa was produced from this construct with an efficiency of 26-29% by in vitro translation in wheat germ extract. CfMV is the first sobemovirus in which the putative replicase is reported to be produced as a part of a polyprotein by a -1 frameshift event. The replicases of the sobemoviruses are related to the luteovirus subgroup II replicases, which are known to be produced by -1 ribosomal frameshift. The reported amino acid sequences of the putative replicases of sobemo- and subgroup II luteoviruses were compared to that of the putative replicase of CfMV. This comparison revealed more extensive homology between these groups than previously reported.

Amino Acid Sequence↗