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PubMed2001

Mouse nestin cDNA cloning and protein expression in the cytoskeleton of transfected cells.

Complementary DNA (cDNA) corresponding to mouse nestin intermediate filament protein, a specific marker for neural stem cells, was isolated and characterized. The complete sequence comprised 5983 base pairs encoding 1821 amino acids, and the deduced polypeptide was similar to rat (84%), hamster (73%), and human (62%) nestin. Southern blots showed that mouse nestin was a single-copy gene, and Northern blots detected a 6.0 kilobase mRNA transcript. When the cDNA was overexpressed as an enhanced green fluorescent fusion protein in COS7 cells, nestin immunoreactivity appeared in the filamentous cytoskeletal network. Accordingly, biologically active mouse nestin cDNA may offer an important new tool for stem cell research.

Amino Acid Sequence↗

PubMed1997

Cloning and tissue distribution of human membrane-bound aminopeptidase P.

Complementary DNA clones encoding human membrane-bound aminopeptidase P (AmP) were isolated by reverse transcription-polymerase chain reaction (RT-PCR) of human kidney and lung poly (A)+ RNA. Comparison of the human AmP sequence to that of the pig shows significant evolutionary divergence with only 83% amino acid sequence identity between the two species. Northern hybridization analysis and RT-PCR suggests that the soluble and membrane-bound forms of human AmP are products of two distinct genes or, through alternative splicing, have different C-terminal sequences.

Alternative Splicing↗

PubMed2000

Complete sequence and characterization of chick ventricular myosin heavy chain in the developing atria.

We isolated five complementary DNA (cDNA) clones, encoding the chick ventricular myosin heavy chain (MyHC) by reverse transcription polymerase chain reaction (RT-PCR). The entire cDNA consists of 5995 nucleotides with the 52 bp 5'-untranslated region and the 129 bp 3'-untranslated region. The complete cDNA encodes 1937 amino acids. Expression of the chick ventricular MyHC gene was also studied by Northern blot analysis. This gene continued to be strongly expressed in the ventricle during cardiac development. On the other hand, its expression was moderate in the early embryonic atria, and was down-regulated during development. In the adult atria, this gene was expressed at very low levels. To determine the localization of the ventricular MyHC protein, an immunohistochemical study was performed. The ventricular MyHC was present in early embryonic atrial myocytes. During development, the expression of this protein in the atrial myocytes was down-regulated, but continued to be present in the atrial conduction system. Our results indicate that the ventricular MyHC appears in the primary atrial myocardium and is then localized in the conduction cells of the atria.

Animals↗

PubMed1996

Cloning and expression of pyranose oxidase cDNA from Coriolus versicolor in Escherichia coli.

Complementary DNA encoding pyranose oxidase (PROD) was cloned and sequenced for the first time from Coriolus versicolor. The nucleotide sequence revealed an open reading frame encoding a polypeptide composed of 623 amino acid residues. Compared with the experimentally determined N-terminal sequence of the PROD from C. versicolor. 38 amino acids from the N-terminus of the protein appeared to be eliminated during protein maturation. The cDNA was successfully expressed under the control of lacUV5 promoter in Escherichia coli at 25 degrees C, which will be beneficial in industrial production.

Amino Acid Sequence↗

PubMed2000

Partial cloning of constitutive and inducible nitric oxide synthases and detailed neuronal expression of NOS mRNA in the cerebellum and optic tectum of adult Atlantic salmon (Salmo salar).

Studies of different species have implicated nitric oxide (NO) synthase (NOS) in various physiological and pathological events. Three major NOS isoforms are present in the brain of mammals; endothelial NOS (eNOS), neuronal NOS (nNOS) and inducible NOS (iNOS). Little is known about the significance of the presence of these proteins in the brain. We report the first investigation into the presence of nNOS and iNOS isoforms in a teleost, adult Atlantic salmon (Salmo salar). Complementary DNA was synthesized from cerebellum and thymus mRNA using RT-PCR techniques with primers against conserved regions of NOS. Cloning and sequencing revealed a partial gene sequence of 560 bp corresponding to mammalian nNOS from cerebellum cDNA. The predicted protein sequence of identified salmon nNOS possessed 85% identity to that of mammalian nNOS. Northern blot analysis of different tissues revealed expression in brain and heart, and indicated expression of three different nNOS mRNAs in the brain. In addition, a 389 bp sequence corresponding to iNOS was identified in thymus cDNA. Salmon iNOS is almost identical to rainbow trout iNOS (95%), but shows much less amino acid identity to goldfish (65%) and mammalian (52%) iNOS. Phylogenetically, all vertebrate nNOS and iNOS homologues are clustered separately. Expression studies by means of in situ hybridization revealed abundant nNOS mRNA transcripts in distinct neuronal populations throughout the Purkinje cell layer of the corpus cerebellum and the periventricular layer of the optic tectum. Our data show that adult Atlantic salmon possess a gene encoding an nNOS isoform and putative alternatively spliced forms, which are expressed in distinct neuronal populations in the cerebellum and optic tectum, and in yet unidentified cell types in the heart. The data suggest that the arising of different vertebrate NOS isoforms is an evolutionary old event. The well conserved sequences present in salmon and mammalian nNOS may reflect their importance in protein function, whereas interspecies distributional differences in cellular expression of nNOS and sequence differences of iNOS may reflect variations and specializations in routes of NO action in the vertebrate phylogeny.

Age Factors↗

PubMed2000

Identification of differentially expressed genes in the visual structures of brain using high-density cDNA grids.

The hybridization patterns of 18,371 high-density-grid-arrayed non-redundant complementary DNA (cDNA) clones were examined using three different sources of cDNA probes. The first set of probes was synthesized from mRNA isolated from visual brain areas MT and V4 of Vervet monkey. The second set of probes was derived from cDNA libraries constructed from two micro dissected sets of layers of the monkey Lateral Geniculate Nucleus layers within the visual pathway, namely the magnocellular and parvocellular layers. The third set of cDNA probes was synthesized from the subtracted fractions of the cDNAs enriched for either the magnocellular or the parvocellular layers of the Lateral Geniculate Nucleus. Software, linked directly to the Genbank database, was developed to aid in the rapid identification of both expressed and differentially expressed genes. Our results indicate that both the cDNA probes synthesized from mRNA and cDNA libraries can identify similar fractions of expressed genes. However, the subtracted cDNA probes improve the efficiency of detection for those genes that are expressed at much lower abundance. Analyses of these results for the differential expression patterns of these genes were validated by semi-quantitative PCR on the DNA derived from the whole tissue cDNA libraries. A list of some known genes that are statistically differentially expressed within the magnocellular layers of the LGN and area MT in the primate visual areas is derived.

Animals↗

PubMed2000

Cloning, localisation and functional expression of a novel human, cerebellum specific, two pore domain potassium channel.

We have isolated, by degenerate PCR, a complementary DNA encoding a novel two pore domain potassium channel. This is the 7th functional member of the human tandem pore domain potassium channel family to be reported. It has an open reading frame of 1.125 kb and encodes a 374 amino acid protein which shows 62% identity to the human TASK-1 gene: identity to other human members of the family is 31-35% at the amino acid level. We believe this gene to be human TASK-3, the ortholog of the recently reported rat TASK-3 gene: amino acid identity between the two is 74%. 'Taqman' mRNA analysis demonstrated a very specific tissue distribution pattern, showing human TASK-3 mRNA to be localised largely in the cerebellum, in contrast rat TASK-3 was reported to be widely distributed. We have shown by radiation hybrid mapping that human TASK-3 can be assigned to chromosome 8q24.3. Human TASK-3 was demonstrated to endow Xenopus oocytes with a negative resting membrane potential through the presence of a large K(+) selective conductance. TASK-3 is inhibited by extracellular acidosis with a mid-point of inhibition around pH 6. 5, supporting the predictions from the sequence data that this is a third human TASK (TWIK-related acid sensitive K(+) channel) gene.

Amino Acid Sequence↗

PubMed1998

Synaptic activity-dependent modulation of mitochondrial gene expression in the rat hippocampus.

In order to identify genes that may underlie the maintenance of long-term potentiation (LTP) at perforant path synapses, complementary DNA libraries were synthesised from dentate gyrus total RNA extracts prepared 48 h after the induction of LTP and from control dentate gyrus extracts. Through differential screening of the LTP library we have identified the mitochondrial 12S rRNA (mt12SrRNA) as a transcript that was elevated at this late time. Northern blot analyses showed that the elevation in mt12SrRNA expression began around 8 h and persisted for at least 2 weeks post-tetanus. We then examined the expression patterns of other mitochondrially-encoded genes and demonstrated a similar elevation in their expression. mt12SrRNA levels were also elevated in other hippocampal regions, including areas CA3 and CA1 and were elevated following low-frequency stimulation or in the presence of an N-methyl-D-aspartate receptor antagonist where induction of LTP was precluded. Taken together, these observations suggest that a long-lasting up-regulation of energy production may be triggered by synaptic activity and this activity need not be of sufficient strength to induce LTP, but may be related to the induction of a metaplastic state.

Animals↗

PubMed2000

Molecular cloning and expression of cDNA encoding a brain bombesin/gastrin-releasing peptide-like peptide in goldfish.

A complementary DNA (cDNA) of 928 bp encoding a bombesin (BBS)/gastrin-releasing peptide (GRP) precursor was identified from goldfish brain. Goldfish BBS/GRP messenger RNA (mRNA) encodes a 157 amino acid precursor, which contains a signal peptide sequence, the 22 amino acid putative BBS/GRP-like peptide, and a carboxy-terminal extension peptide. Reverse transcription-polymerase chain reaction (PCR) (RT-PCR) demonstrated that the mRNA for this precursor has a widespread distribution in goldfish brain, and is also present in skin, gastrointestinal tract, gonad, and gill. Phylogenetic analysis of BBS/GRP-like peptide precursors in vertebrates shows that goldfish BBS/GRP is more closely related to the known GRP precursors than to BBS precursors.

Amino Acid Sequence↗

PubMed2000

Changes of cytokine profiles during peritonitis in patients on continuous ambulatory peritoneal dialysis.

Continuous ambulatory peritoneal dialysis (CAPD) has emerged as an important dialysis treatment modality worldwide. One of the major complications is bacterial peritonitis, which may result in subsequent technique failure because of loss of peritoneal clearance or peritoneal fibrosis. Bacterial peritonitis leads to the release of proinflammatory cytokines from resident and infiltrating cells in the peritoneal cavity. We studied 35 patients undergoing CAPD with acute bacterial peritonitis. All patients treated with antibiotics for 2 weeks after the clinical diagnosis of peritonitis had a good recovery. Peritoneal dialysate effluent (PDE) was collected on days 1, 3, 5, 10, 21, and 42 after the start of treatment. Cell populations were monitored by flow cytometry. PDE levels of interleukin-1beta (IL-1), IL-6, transforming growth factor-beta (TGF-beta), and basic fibroblast growth factor (FGF) were measured by enzyme-linked immunosorbent assay. Gene transcription of TGF-beta in macrophages from PDE was measured by quantitative polymerase chain reaction. Bacterial peritonitis was associated with a sharp increase in total cell and neutrophil counts (400-fold) in PDE up to 3 weeks after peritonitis despite clinical remission (P < 0.0001). There was an increased absolute number of macrophages during the first 3 weeks despite the reduced percentage of macrophages among total cells in PDE compared with noninfective PDE. There was a progressive increase in the percentage of mesothelial cells or dead cells in the total cell population in PDE over the entire 6-week period. PDE levels of IL-1, IL-6, TGF-beta, and FGF increased markedly on day 1 before their levels decreased gradually. PDE levels of these cytokines or growth factors were significantly greater than those in noninfective PDE (n = 76) throughout the study period (P < 0.01). Similarly, TGF-beta complementary DNA (cDNA) molecules per macrophage were significantly greater than those of macrophages in noninfective PDE throughout this period (P < 0.01). There was no significant correlation between PDE levels of TGF-beta and TGF-beta cDNA molecules per macrophage, suggesting that peritoneal macrophages are not the only source of TGF-beta in PDE. We conclude there is an active release of proinflammatory cytokines and sclerogenic growth factors through at least 6 weeks despite apparent clinical remission of peritonitis. The peritoneal cytokine networks after peritonitis may potentially affect the physiological properties of the peritoneal membrane.

Bacterial Infections↗

PubMed1997

Polyadenylated mRNA in Escherichia coli: modulation of poly(A) RNA levels by polynucleotide phosphorylase and ribonuclease II.

The effect of 3'-exoribonucleases on the polyadenylation of mRNA in Escherichia coli was studied by comparing the synthesis and levels of poly(A) RNA in wild-type E coli and mutant strains defective in the two major 3'-exoribonucleases: polynucleotide phosphorylase and ribonuclease II. Mutations which substantially reduced the activity of these 3'-exonucleases caused a 10-fold increase in pulse-labeling of total poly(A) RNA in intact cells. When the net rate of RNA synthesis was measured in permeabilized cells, the mutant with defective 3'-exonucleases showed 20- to 60-fold increased synthesis of total poly(A) RNA as well as of specific polyadenylated mRNAs, with less than two-fold changes in non-poly(A) RNA. Measurement of mRNA polyadenylation in permeable cells under conditions when 3'-exoribonucleases were inactive showed a 6-fold higher rate of poly(A) synthesis in the exonuclease-deficient mutant strain, suggesting a higher concentration of mRNA 3'-ends amenable to polyadenylation. Steady-state levels of poly(A) RNA, measured by the ability to serve as template for oligo(dT)-dependent complementary DNA synthesis, also increased more than 40-fold when the 3'-exonucleases were inactivated. Monitoring of the length of the poly(A) tracts by denaturing polyacrylamide gel electrophoresis showed chain lengths of up to 45 residues in the 3'-exonuclease-deficient mutant, whereas most of the poly(A) tracts in the parent strain were shorter than 12 residues. These results show that 3'-exonucleases reduce the level of polyadenylated mRNA in E coli not merely by causing its degradation but also by reducing its rate of synthesis, presumably by competing with poly(A) polymerase for the 3'-ends of mRNA.

Cell Membrane Permeability↗

PubMed1996

Identification of cytokeratin subspecies altered in rat experimental esophageal tumors by subtractive cloning.

By using the subtractive hybridization method, two complementary DNA clones differently expressed in rat normal esophageal epithelium and squamous cell carcinoma induced by administration of precursors of N-nitrososarcosine ethyl ester were isolated. A rat homologue of the human 50-kDa type I cytokeratin 14 was cloned for the first time and shown to be expressed preferentially in squamous cell papillomas and carcinomas, whereas it was weakly expressed or absent in normal squamous epithelial cells and in hyperplastic lesions. A rat homologue of the mouse 57-kDa type II cytokeratin showed strong expression in both normal and tumor tissues. These results are well consistent with the reported alteration of keratin subspecies in human esophageal cancers, therefore, encouraging us to use this experimental system as a model for human esophageal carcinogenesis.

Animals↗

PubMed1998

Cloning and characterization of a cDNA fragment coding beta-casein-like protein preferentially expressed in cervical adenocarcinoma cell line CAC-1.

We generated new murine monoclonal antibodies against bovine beta-casein. One of eight new monoclonal antibodies generated, 12G2, specifically reacted not only with beta-casein, but also with human cervical adenocarcinoma cell line CAC-1. To identify the gene encoding the putative protein (BCLP) defined by monoclonal antibody (mAb) 12G2, a complementary DNA library was constructed. We obtained the inserted cDNA fragment that showed high homology to reported DNA sequences of unknown function in normal human placenta. We think that BCLP was overexpressed or changed its structure by carcinogenesis.

Adenocarcinoma↗

PubMed1997

Molecular cloning and characterization of the epididymis-specific glutathione peroxidase-like protein secreted in the porcine epididymal fluid.

The epididymis-specific glutathione peroxidase was purified from the porcine cauda epididymal fluid in order to analyze its enzymatic activity and roles in the epididymis. The purified protein was found to consist of four identical 23 kDa subunits. The complementary DNA encoding the 23 kDa subunit was cloned from the cDNA library of the porcine proximal caput epididymis, only where the 23 kDa subunit is expressed. Although the selenocysteine codon (TGA) is contained in the cDNA of the other cytosolic type of glutathione peroxidases, it is replaced by cysteine codon (TGT) in the 23 kDa subunit cDNA, similarly to the results previously obtained for cDNAs encoding the epididymis-specific form of the secreted glutathione peroxidases of mouse, rat and monkey. By the direct analysis of the selenium, the purified protein was proved to contain no selenium atom in the molecule. The activities of the purified epididymis-specific glutathione peroxidase toward hydrogen peroxide or organic hydroperoxides were by far lower than the activity of cytosolic selenium-dependent glutathione peroxidase (less than 0.1%). In addition, the concentration of glutathione in the porcine epididymal fluids was about 20 microM, which is much lower than the optimal concentration for the glutathione peroxidase activity of the purified protein. These results strongly suggest that this protein is enzymatically quiescent at least in the porcine epididymal fluid. An immunocytochemical study showed that this protein was found to bind to the acrosomal region of the epididymal sperm and to disappear during the acrosome reaction. Furthermore, this protein significantly retarded the acrosome reaction induced in vitro. The possibilities have been discussed that it protects sperm from the premature acrosome reaction and maintains sperm fertilizing ability in the epididymis.

Amino Acid Sequence↗

PubMed2001

Spinal cord genes enriched in rat dorsal horn and induced by noxious stimulation identified by subtraction cloning and differential hybridization.

Persistent nociceptive input increases neuronal excitability and induces a program of gene expression in the dorsal spinal cord. The alteration in gene expression commences with phosphorylation and induction of immediate early genes and proceeds to target genes. Only a few target genes have been identified as yet. The present report uses a polymerase chain reaction-based subtraction cloning procedure to obtain an "anatomically focused" complementary DNA library enriched in transcripts related to sensory spinal cord (rat dorsal horn minus ventral horn). A subset of clones from this library (n=158) was screened to verify dorsal horn enrichment and to identify those regulated by carrageenan-induced peripheral inflammation. Molecular classes which displayed enriched expression included a proto-oncogene not previously associated with sensory processes, two regulators of the Rho/Rac pathway which controls cell shape, and three genes involved in cytoskeletal regulation and scaffolding. Additional transcripts coded for proteins involved in intercellular communication or intracellular function. Within the set of 158 transcripts, one known and two unknown genes were induced by persistent noxious input. The known gene codes for the secreted cysteine proteinase inhibitor, cystatin C, suggesting that modulation of extracellular proteolytic activity occurs. Since it is secreted, cystatin C may also provide a cerebrospinal fluid bio-marker for persistent pain states. Using a combined anatomical and functional approach, we have extended the molecular repertoire of genes expressed and induced in second-order neurons or supporting glial cells in several new directions, with particular emphasis on regulation of cell morphology and plasma membrane dynamics. Some of these proteins reveal new pathways for information signaling in the sensory half of the spinal cord and require further research to understand their role in the adult spinal cord. The induced genes may provide new molecular targets for therapeutic development and provide new probes for investigating the dynamic state of cellular activity that occurs during persistent pain states.

Animals↗

PubMed1997

Brain cytochrome oxidase subunit complementary DNAs: isolation, subcloning, sequencing, light and electron microscopic in situ hybridization of transcripts, and regulation by neuronal activity.

The goal of the present study was to isolate, for the first time, cytochrome oxidase subunit genes from murine brain complementary DNA library and to characterize the expression of these genes from mitochondrial and nuclear sources at both light and electron microscopic levels. Brain subunit III (mitochondrial) shared 100% identity with that of murine L cells. Subunit VIa (nuclear) was known to have tissue-specific isoforms in other species: the ubiquitous liver isoform and the heart/muscle isoform. Our brain subunit VIa shared 93% homology with that of the rat liver and 100% identity with the recently reported murine liver isoform, which is only 62% identical to that of the rat heart isoform. In situ hybridization with riboprobes revealed messenger RNA labelling that was similar, though not identical, to that of cytochrome oxidase histochemistry. Monocular enucleation in adult mice induced a significant down-regulation of both subunit messages in the contralateral lateral geniculate nucleus. However, the decrease in subunit III messenger RNAs surpassed that of subunit VIa at all time periods examined, suggesting that mitochondrial gene expression is more tightly regulated by neuronal activity than that of nuclear ones. At the electron microscopic level, subunit III messenger RNA was localized to the mitochondrial compartment in both cell bodies and processes, while that of nuclear-encoded subunit VIa was present exclusively in the extramitochondrial compartment of somata and not of dendrites or axons. Surprisingly, the message was primarily associated with the rough endoplasmic reticulum, suggesting a novel pathway for its synthesis and trafficking. Our results indicate that the unique properties of neurons impose special requirements for subunits of a single mitochondrial enzyme with dual genomic origins. At sites of high energy demands (such as postsynaptic dendrites and some axon terminals), mitochondrial-encoded cytochrome oxidase subunits can be locally transcribed and translated, and they provide the framework for the subsequent importation and incorporation of nuclear-encoded subunits, which are strictly synthesized in the cell bodies. Dynamic local energy needs are met when subunits from the two genomic sources are assembled to form functional holoenzymes.

Animals↗

PubMed1999

Differential expression of bcl-w and bcl-x messenger RNA in the developing and adult rat nervous system.

The bcl-2 family of proteins comprises both anti-apoptotic and pro-apoptotic members, which play a pivotal role in regulating cell death. Bcl-w is a recently identified member of this family, which was shown to inhibit apoptosis in haemopoietic cell lines. However, the function and expression patterns of bcl-w in the nervous system have so far not been described. We have cloned complementary DNA corresponding to rat bcl-w and analysed the expression of bcl-w messenger RNA during rat brain development, using RNA blotting and in situ hybridization techniques. We also compared the expression patterns of bcl-w with those of bcl-xL. During brain development, the levels of bcl-w messenger RNA were found to increase, with highest expression located to specific regions of the mature brain, such as hippocampus, cerebellum, piriform cortex and locus coeruleus. Bcl-w messenger RNA was expressed by neurons, as shown with double labeling with neuronal markers. In contrast to bcl-w, bcl-xL messenger RNA expression levels were highest during early development, particularly in cortex, hippocampus, thalamus, spinal cord and dorsal root ganglia. During postnatal development the expression of bcl-xL messenger RNA decreased and were only detected at low levels in the adult nervous system. As shown earlier for bcl-2, the expression of bcl-w and bcl-x messenger RNA in cultured cerebellar granule cells was not altered by the deprivation of neurotrophic factors. The present results suggest that bcl-w may play an important role in the mature nervous system.

Aging↗

PubMed1997

Cloning and characterization of human cDNAs encoding a protein with high homology to rat intestinal development protein OCI-5.

We constructed a lambda complementary DNA expression library from the mitoxantrone-resistant human gastric carcinoma cell line EPG85-257RNOV. The library was screened by differential hybridization (resistant cell line against non-resistant cell variant). By this procedure we found five independent cDNA clones representing one single gene that has much higher expression in the mitoxantrone-resistant cell line EPG85-257RNOV than in the non-resistant variant EPG85-257P. One of the cDNA clones (MXR7) contains a complete open reading frame (ORF) encoding a 580-amino acid polypeptide. Amino acid and nucleotide sequence analysis revealed that this gene codes the human variant of a rat intestinal development protein OCI-5.

Amino Acid Sequence↗