Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

RNA amplification with transcript sequencing (RAWTS) is a rapid and sensitive method of direct sequencing that involves complementary DNA synthesis, polymerase chain reaction (PCR) with a primer or primers containing a phage promoter, transcription from the phage promoter, and reverse transcriptase-mediated sequencing. By means of RAWTS, it was possible to sequence each of four tissue-specific human messenger RNAs (blue pigment, factor IX, phenylalanine hydroxylase, and tyrosine hydroxylase) in four cell types examined (white blood cells, liver, K562 erythroleukemia cells, and chorionic villus cells). These results indicate that there is a basal rate of transcription, splicing, and polyadenylation of tissue-specific mRNAs in adult and embryonic tissues. In addition to revealing sequence information, it is possible to generate a desired in vitro translation product by incorporating a translation initiation signal into the appropriate PCR primer. RAWTS can be used to obtain novel mRNA sequence information from other species as illustrated with a segment of the catalytic domain of factor IX. In general, the ability to obtain mRNA sequences rapidly across species boundaries should aid both the study of protein evolution and the identification of sequences crucial for protein structure and function.
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PMID:Access to a messenger RNA sequence or its protein product is not limited by tissue or species specificity. 279 88

Disseminating disease in neuroblastoma is of considerable clinical importance. Detection of circulating neuroblastoma cells using tyrosine hydroxylase (TH) as a tissue-specific target for reverse transcriptase-polymerase chain reaction has proved to be a sensitive and specific method for the detection of contaminating tumour cells in peripheral blood. The aim of this study was to report the early clinical observations made using this technology in neuroblastoma patient blood samples. A strong association was found between the detection of neuroblastoma cells in circulation with the detection of neuroblastoma in bone marrow. This method may be of use to monitor disease status and identify early signs of relapse in clinically disease-free patients. These results show that RT-PCR detection of TH mRNA is a relatively noninvasive, sensitive method for the detection of circulating tumour cells in neuroblastoma patients.
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PMID:Early clinical evaluation of neuroblastoma cell detection by reverse transcriptase-polymerase chain reaction (RT-PCR) for tyrosine hydroxylase mRNA. 757 66

Peripheral nerve crush induces novel projections from noradrenergic sympathetic neurons to sensory ganglia, and it has been suggested that these projections provide an anatomical substrate for chronic pain syndromes that occur after nerve injury. The present study demonstrates that novel sympathetic projections to sensory neurons are also induced in transgenic mice that overexpress nerve growth factor (NGF) in the skin. Specifically, a large proportion of trigeminal neurons in NGF transgenic mice were innervated by tyrosine hydroxylase (TH)-positive pericellular arborizations that were seen only rarely in controls. Electron microscopic analysis of NGF transgenic mice revealed that trigeminal neurons were surrounded by numerous axonal varicosities containing synaptic specializations. Removal of the superior cervical ganglion abolished TH-immunoreactive arborizations in the ipsilateral trigeminal ganglion confirming that these fibers were sympathetic axons. A two-site enzyme-linked immunosorbent assay revealed that transgenic ganglia contained a tenfold increase in NGF peptide compared to controls. However, reverse transcriptase polymerase chain reaction analysis showed no apparent expression of transgene mRNA in sensory ganglia, suggesting that the additional NGF was derived from increased NGF expression in the skin. These results indicate that NGF can induce novel sympathetic projections to sensory neurons in vivo and suggests a model in which increased NGF expression plays a role in the development of sympathetic hyperalgesia after nerve injury.
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PMID:Overexpression of nerve growth factor in transgenic mice induces novel sympathetic projections to primary sensory neurons. 785 36

Previous studies [Czyzyk-Krzeska et al.: J Neurochem 1992;58:1538] demonstrated the relationship between low O2 breathing and tyrosine hydroxylase (TH) gene expression in chemosensory type I cells of the carotid body. In the present study, we have exposed carotid bodies in vitro to hypoxic superfusion media, and subsequently used the reverse transcriptase-polymerase chain reaction technique to measure relative changes in the TH transcript in an effort to elucidate the cellular mechanisms which regulate TH gene expression. Carotid bodies and superior cervical ganglia (SCG) were exposed for 3 h to superfusion media equilibrated with either 10% O2 or 100% O2 and then rapidly frozen on dry ice prior to extraction of total RNA. Hypoxia elevated TH mRNA in the carotid body 3.63 +/- 0.84-fold (mean +/- SEM), while in contrast, these parameters were unchanged in SCG similarly exposed to hypoxic media. Incubation of carotid bodies in zero Ca2+ superfusates greatly attenuated the increase in TH mRNA evoked by hypoxia (1.39 +/- 0.34-fold increase; p < 0.025 compared to normal Ca2+ group). Likewise, exposure to the guanylate cyclase activator, atriopeptin III (100 nM), attenuated the TH mRNA hypoxic response (p < 0.005), while activation of adenylate cyclase with forskolin (10 microM) tended to elevate the response to low O2. Our data suggest that hypoxia, independent of circulating hormones, induces TH gene expression in the carotid body, and that multiple factors, including [Ca2+] and cyclic nucleotides, may be important components of the signal transduction pathway.
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PMID:Second messenger regulation of tyrosine hydroxylase gene expression in rat carotid body. 870 28

By using a semi-quantitative reverse transcriptase-PCR assay (RT-PCR) we have analyzed dopamine transporter (DAT), tyrosine hydroxylase (TH) and synaptic vesicle monoamine transporter (VMAT2) gene expression in rat mesencephalic (MES) primary cultures. Consistent with previous data obtained during rat MES ontogeny, the onset of DAT transcription in vitro is delayed in embryonic day (E)13, but not in E16, MES neurons when compared to that of TH and VMAT2. In co-culture, the addition of target striatal cells (STR) to E13 MES selectively increases DAT mRNA level in DA neurons during the first 3 days in vitro; cortical cells are ineffective. On the contrary, DAT gene does not appear up-regulated in E16 MES co-cultured with target STR cells, indicating that MES DA neurons respond to STR stimulation only at defined developmental stages. Up-regulation of DAT mRNA level by STR in E13 MES seems to require direct cell interactions since target cells do not exert their effect on DAT transcription when are separated from MES cells by a porous barrier, which only allows diffusion of soluble molecules. Thus maturation of DA neurotransmission in vitro appears to follow a developmental program which can be specifically modulated by their target STR cells.
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PMID:Dopamine transporter gene expression in rat mesencephalic dopaminergic neurons is increased by direct interaction with target striatal cells in vitro. 880 24

The mammalian testis is innervated by extrinsic catecholaminergic nerves and responds to catecholamines with steroid secretion. Although the primate testis has also been shown to be innervated, potential differences in the density of this innervation between immature and sexually developed individuals have not been described. A recent study demonstrated that the primate ovary contains a network of neuron-like cells and that some of these cells are catecholaminergic. It is thus possible that the male gonad is also endowed with a similar intragonadal source of catecholamines. The present study addresses these two issues. Catecholaminergic nerves were identified as such by their content of immunoreactive tyrosine hydroxylase (TH; the rate-limiting step in catecholamine biosynthesis), and in some cases by glyoxylic acid histochemistry. Fibers containing TH were abundant in testes from juvenile animals (1-2 yr of postnatal life), but the density of this innervation was not maintained in adult animals, whose testis showed only a few TH-positive fibers scattered in the interstitial tissue. Testicular norepinephrine (NE) concentration was much lower in adult than in juvenile animals, suggesting that the marked increase in testicular weight that occurs with the attainment of sexual maturity is not accompanied by corresponding changes in NE content. At the ultrastructural level, testicular nerve fibers contained pleiomorphic, dense-core and clear vesicles, suggesting the presence of catecholamines and other neurotransmitters. In addition to this extrinsic catecholaminergic innervation, prepubertal testes, but not adult gonads, contained an intrinsic population of TH-immunopositive neuron-like elements, identified as cells by confocal scanning laser microscopy. To determine whether the prepubertal monkey testis indeed expresses the TH gene, testicular RNA was subjected to reverse transcriptase polymerase chain reaction to amplify the 5' end of TH mRNA, which encodes the regulatory domain of the enzyme. The cDNA that was obtained predicts an amino acid sequence similar, but not identical, to that encoded by the alternatively spliced type 1 TH mRNA form present in the adrenal gland. These results indicate 1) that the primate testis receives a dual catecholaminergic input, one provided by the extrinsic innervation and the other by neuron-like cells located within the gonad itself, and 2) that the influence exerted by both sources on testicular function may be more prominent during the prepubertal period than in adulthood. The presence in the testis of a TH mRNA variant encoding amino acid substitutions in its 5' end suggests that regulation of testicular TH enzyme activity may include a gonad-specific component.
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PMID:Testis of prepubertal rhesus monkeys receives a dual catecholaminergic input provided by the extrinsic innervation and an intragonadal source of catecholamines. 886 66

The highly sensitive method to detect neuroblastoma (NB) cells using reverse transcriptase polymerase chain reaction (RT-PCR) was applied in the practical clinics, and its efficacy was assessed in the present study. Human tyrosine hydroxylase (TH), a rate-limiting enzyme in the catecholamine biosynthesis, was used as the marker for NB cells, and the expression of THmRNA was examined in 13 samples (four peripheral blood and nine bone marrow) harvested from seven patients (four with stage IV, one with stage III, two with stage II) using RT-PCR with our original primers. The positive signals for NB cells were detected in four samples (one peripheral blood and three bone marrow) by the PCR method, but were undetectable by the conventional histological examinations. In the present series, a case that showed a positive signal for NB cells in the peripheral blood showed a remarkably unfavorable clinical course, indicating that the circulating NB cells detected by the PCR method can be a sign of the progressively advanced NB, and may define a new prognostic factor suggesting higher risk. In another case, the PCR detection for the residual NB cells in the bone marrow provided important supporting evidence to determine the necessity of the additional chemotherapy and the suitable timing for bone marrow transplantation. This detection also guaranteed the safety of the bone marrow for transplantation. The PCR method was considered to be very beneficial in the selected cases. However, some problems such as the false-negative results in the negative urinary vanillylmandelic acid secretor were also highlighted in the present study.
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PMID:Clinical application of minimal residual neuroblastoma cell detection by reverse transcriptase-polymerase chain reaction. 902 73

While not all circulating tumor cells survive, one could postulate that patients with circulating tumor cells might be candidates for adjuvant chemotherapy because of the risk of relapse. Recently, reverse transcriptase-polymerase chain reaction (RT-PCR) for the detection of circulating tumor cells has been suggested as a potential technique for staging of cancer. In malignant melanoma, the detection of circulating melanoma cells by tyrosinase RT-PCR correlated with the clinical stage of patients and was an independent prognostic factor for recurrence. A strong association was found between the detection of neuroblastoma cells in circulation by tyrosine hydroxylase RT-PCR with bone marrow micrometastasis. This method may be of use to identify early signs of relapse in disease-free patients. In prostatic cancer, the frequency of positivity for detection of circulating tumor cells in peripheral blood by PSA RT-PCR increased with tumor stages but a significant proportion of patients with metastatic disease were negative. The prognostic significance of the detection of tumor cell in blood in other epithelial tumors is not established and will require further long-term follow-up study.
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PMID:[Molecular diagnostic detection of circulating tumor cells and their prognostic implications]. 905 Nov 25

We used a catecholaminergic neuron-like cell line (CATH.a cells) as a model system to investigate the likelihood that pituitary adenylate cyclase-activating polypeptide (PACAP) may participate in the regulation of specific gene expression in catecholaminergic neurons. Analysis by reverse transcriptase-PCR amplification revealed the presence in these cells of type I PACAP receptors, with a short isoform, together with a heavier so-called Hop splice variant. PACAP38 and PACAP27 enhanced, in a dose-dependent manner, both cyclic AMP formation and phosphoinositide breakdown, with EC50 values of, respectively, 0.6 x 10(-10) and 2 x 10(-9) M. These peptides, in addition, also elevated [Ca2+]i by mobilizing intracellular calcium pools. Vasoactive intestinal peptide (VIP) was approximately 1,000-fold less potent in stimulating cyclic AMP (with EC50 = 2 x 10(-7) M) and failed to change the turnover of phosphoinositides and to alter [Ca2+]i. Both forms of PACAP, as well as forskolin, stimulated transcriptional induction of tyrosine hydroxylase (TH) and c-fos promoters fused to a chloramphenicol acetyltransferase (CAT) reporter gene in transiently transfected cells (p < 0.01 vs. controls). Induction of CAT activity linked to both TH and c-fos promoters was obliterated upon coexpression of a dominant inhibitory mutant (Mt-RAB) of cyclic AMP-dependent protein kinase. We conclude that CATH.a cells do express functional PACAP type I receptors, the activation of which impinges on TH and c-fos transcription according to a process that is primarily dependent on the cyclic AMP-PKA pathway.
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PMID:Pituitary adenylate cyclase-activating polypeptide triggers dual transduction signaling in CATH.a cells and transcriptionally activates tyrosine hydroxylase and c-fos expression. 908 43

Transgenic mice that overexpress nerve growth factor (NGF) in cells producing glial fibrillary acidic protein were used to determine whether sympathetic axons will invade the undamaged, postnatal mammalian brain. By using reverse transcriptase-polymerase chain reaction, NGF mRNA transgene expression was detectable in the hippocampi and cerebella of transgenic mice but not in age-matched, wild type mice. Elevated levels of NGF protein were detected in the hippocampi and cerebella of postnatal and adult transgenic animals as well as in conditioned media from transgenic cerebellar astrocytes in culture. The brains of these transgenic mice were found to contain postganglionic sympathetic fibers, as identified by their immunohistochemical staining for tyrosine hydroxylase and by their disappearance following superior cervical ganglionectomy. In the cerebellum, a robust plexus of sympathetic fibers was evident in the deep white matter and in the inferior cerebellar peduncles. These axons within the cerebellum were observed as early as 14 days after birth and dramatically increased in number with age. Sympathetic axons were also associated with the large blood vessels of the hippocampal fissure and were present within the hilar region of the dentate gyrus. NGF immunoreactivity was present within the sympathetic axons as well as within glial cells in the transgenic cerebellum and hippocampus. Wild type mice, however, lacked similar patterns of immunostaining. These results demonstrate that elevated expression of NGF in the intact mammalian brain results in the growth of sympathetic axons into the central nervous system in the absence of injury.
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PMID:Sympathetic axons invade the brains of mice overexpressing nerve growth factor. 918 86


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