Reverse Transcriptase And Polymerase Chain Reaction Research Articles

MTOR: a possible Signaling Intermediate in the Leptin‐Kisspeptin Pathway during the Pubertal Transition in Female Rats

While leptin and kisspeptin have both been implicated in the onset of puberty in mammals, the pathway linking leptin and kisspeptin is still not completely understood. Such knowledge could play an important role in treating or preventing infertility, both in humans, for the purpose of child bearing, and in other mammals, for agricultural reasons. Recent studies have suggested that mTOR, a ubiquitous serine/threonine kinase involved in cell and tissue growth and also a target of leptin signaling in the paraventricular and arcuate nuclei of the hypothalamus, may act as a signaling intermediate in the leptin‐kisspeptin pathway. Therefore, increases in mTOR in the hypothalamus may correlate with increases of leptin and kisspeptin in the hypothalamus as well. In this experiment, the gene expression of leptin, kisspeptin, and mTOR were examined using reverse‐transcriptase and polymerase chain reaction (RT‐PCR) from the hypothalami of female rats in ten day intervals beginning on postnatal day‐10 through postnatal day‐60. An increase in mTOR expression at day 40 correlated with increased expression in both leptin and kisspeptin. These findings suggest that mTOR could be involved in the leptin‐kisspeptin pathway and, therefore, the onset of puberty. This research was supported by a Lander University Foundation Grant.

Detection of GBV-C/HGV RNA in cervico-vaginal smears from healthy individuals

The purpose of the present study was to evaluate the sexual transmission of GBV-C/HGV, through RNA detection in cervicovaginal smears. Therefore the GBV-C/HGV RNA in cervicovaginal smears from apparently healthy women was investigated using routine proceedings for prophylactic screening to cervical cancer. GBV-C/HGV RNA was detected by reverse transcriptase and polymerase chain reaction (RT-PCR). Only one woman presented co-infection with human papilloma virus (HPV). The GBV-C/HGV RNA was detected in 13/73 (17.57%) healthy women and it's prevalence in participating women between 28-43 years old was 53.85%. No association was found with GBV-C/HGV for the age of first sexual intercourse and number of pregnancies. In GBV-C/HGV RNA positive women, 69.23% were married. In conclusion, the present findings show that cervical and vaginal specimens could contain the GBV-C/HGV RNA.

Periodic Monitoring of Commercial Turkeys for Enteric Viruses Indicates Continuous Presence of Astrovirus and Rotavirus on the Farms

A longitudinal survey to detect enteric viruses in intestinal contents collected from turkeys in eight commercial operations and one research facility was performed using molecular detection methods. Intestinal contents were collected from turkeys prior to placement, with each flock resampled at 2, 4, 6, 8, 10, and 12 wk of age. The samples were screened for astrovirus, rotavirus, reovirus, and turkey coronavirus (TCoV) by a reverse transcriptase and polymerase chain reaction (RT-PCR), and for groups 1 and 2 adenovirus by PCR. Rotavirus was the only virus detected prior to placement (7 of 16 samples examined). All of the commercial flocks were positive for rotavirus and astrovirus from 2 until 6 wk of age, and most were intermittently positive until 12 wk of age, when the birds were processed. Of the 96 samples collected from birds on the farms, 89.5% were positive for astrovirus, and 67.7% were positive for rotavirus. All flocks were negative for TCoV, reovirus, and group 1 adenovirus at all time points, and positive for group 2 adenovirus (hemorrhagic enteritis virus) at 6 wk of age. All the flocks monitored were considered healthy or normal by field personnel. Turkeys placed on research facilities that had been empty for months and thoroughly cleaned had higher body weights and lower feed conversion rates at 5 wk of age when compared to turkeys placed on commercial farms. Intestinal samples collected at 1, 2, and 3 wk of age from these turkeys were free of enteric viruses. This report demonstrates that astroviruses and rotaviruses may be present within a turkey flock through the life of the flock. Comparison of infected birds with one group of turkeys that were negative for enteric viruses by the methods used here suggests that astrovirus and/or rotavirus may affect production. The full impact on flock performance needs to be further determined.

Tight junctions and compression therapy in chronic venous insufficiency

Tight junctions (TJs) provide a barrier function, inhibiting solute and water flow through the paracellular space. There had been no analysis until now as to how tight junction molecules could be involved in the pathology of patients with chronic venous insufficiency. The aim of the study was to analyse the expression pattern of TJ-molecules occludin (OCLN), claudin-1 (CLDN-1), claudin-3 (CLDN-3) and claudin-5 (CLDN-5) on mRNA and protein level in patients with edema, venous leg ulcers and healthy controls. Biopsy specimens were taken in healthy individuals and in patients before, and four weeks after compression therapy. mRNA-expression was determined by using reverse-transcriptase and polymerase chain reaction (RT-PCR) and the protein-expression was determined by Western blotting from tissue specimens. Quantification performed determining the expression for TJ-molecules displayed diminished expression for CLDN-1 (p<0.01) and CLDN-5 (p<0.01) in patients with chronic venous insufficiency in comparison with healthy controls on mRNA as well as protein level. No statistical differences were detected for OCLN and CLDN-3 between the edema group and healthy controls. There was a significantly elevated expression (p<0.01) on mRNA and protein level between the leg ulcer group and healthy controls for OCLN and CLDN-3. Densitometric evaluation revealed a more significantly elevated expression (p<0.01) for CLDN-1 and CLDN-5 on mRNA and protein level after four weeks of compression therapy in comparison with prior to treatment for the edema as well as the leg ulcer group. Compression therapy tightens the paracellular barrier via elevated expression of specific TJs and prevents thereby the progression of chronic venous insufficiency due to inhibited permeability of fluid into the perivascular tissue.

Hcc-2, a novel mammalian ER thioredoxin that is differentially expressed in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the most common primary cancer of the liver. Thus there is great interest to identify novel HCC diagnostic markers for early detection of the disease and tumour specific associated proteins as potential therapeutic targets in the treatment of HCC. Currently, we are screening for early biomarkers as well as studying the development of HCC by identifying the differentially expressed proteins of HCC tissues during different stages of disease progression. We have isolated, by reverse transcriptase and polymerase chain reaction (RT-PCR), a 1741 bp cDNA encoding a protein that is differentially expressed in HCC. This novel protein was initially identified by proteome analysis and we designate it as Hcc-2. The protein is upregulated in poorly-differentiated HCC but unchanged in well-differentiated HCC. The full-length transcript encodes a protein of 363 amino acids that has three thioredoxin (Trx) (CGHC) domains and an ER retention signal motif (KDEL). Fluorescence GFP tagging to this protein confirmed that it is localized predominantly to the cytoplasm when expressed in mammalian cells. Protein alignment analysis shows that it is a variant of the TXNDC5 gene, and the human variants found in Genbank all show close similarity in protein sequence. Functionally, it exhibits the anticipated reductase activity in the insulin disulfide reduction assay, but its other biological role in cell function remains to be elucidated. This work demonstrates that an integrated proteomics and genomics approach can be a very powerful means of discovering potential diagnostic and therapeutic protein targets for cancer therapy.

Expression of cytochrome P450scc mRNA and protein in the rat kidney from birth to adulthood

The expression of cytochrome P450scc, encoded by the CYP11A gene, was investigated in the rat kidney from birth to adulthood. In the male and female rat kidneys, the corresponding mRNA was detected by semi-quantitative reverse transcriptase and polymerase chain reaction (RT-PCR) analysis with specific primers, resulting in higher levels of expression during the first 15 days from birth. RT-PCR and sequence analysis showed that the P450scc mRNA coding region was the same for both kidney and testis, whereas 5′-RACE analysis (rapid amplification of cDNA ends) demonstrated that the renal transcription utilizes a distal transcription start site (TSS) located 76 b upstream of that used in ovarian and testicular P450scc mRNA expression, which is placed 43 b upstream of the first ATG. The 5′-UTR sequence of renal P450scc cDNA exactly matched the contiguous upstream untranslated region of the gene, suggesting that alternative splicing was not involved in the synthesis of this transcript. Northern hybridization detected a specific transcript only in the newborn male, but not in adult rat kidney, confirming the higher levels of expression in the first days of the rat’s life. Positive immunodetections of cytochrome P450scc were found in renal cortical distal tubules and the results were confirmed by Western blotting analysis. As demonstrated by semi-quantitative RT-PCR, the male kidney also expresses the messengers corresponding to the steroidogenic acute regulatory (StAR) and steroidogenic factor 1 (SF-1) proteins, which are normally required for steroidogenesis in steroidogenic tissues, such as gonads and adrenal cortex. These studies suggest that the rat kidney has the capability for local steroid hormone production, although the physiological significance of the pregnenolone eventually produced remains to be established.

Molecular Investigation of GB Virus C RNA in Hemodialysis and Thalassemics Patients from Brazil

The GB virus C (GBV-C)/hepatitis G virus (HGV) is a member of the Flaviviridae family. Based on the clinical and epidemiological profiles, this virus could be acquired mainly by parenteral transmission through contaminated blood. We therefore investigated the presence of GBV-C/HGV and its relation with the other blood borne viruses as hepatitis B and C viruses (HBV, HCV) in hemodialysis and thalassemic individuals and blood donors from Ribeirão Preto–-Brazil. Detection of blood borne virus markers including HBV surface antigen (HbsAg), HBV core antibody (anti-Hbc) and HCV antibody was carried out. HIV-1, HIV-2, HTLV-1 and HTLV-2 were also investigated. GBV-C/HGV RNA was detected by reverse transcriptase and polymerase chain reaction (RT-PCR). Ninety-four serum samples from patients with chronic renal failure were analyzed. GBV-C/HGV RNA was identified in 12 (12.8%) patients, anti-HCV antibodies in 28 (29.8%), anti-Hbc in 9 (9.6%), anti-HIV in 1 (1%), HBsAg in 33 (35.1%), and HBsAg/anti-HBc was observed in 2 (2.1%) patients. Thirty-six (38.3%) samples were non-reactive. Seven of the 12 GBV-C/HGV RNA infected samples were co-infected with other viruses: 3 (25%) with HBsAg, 2 (16.7%) with anti-HCV and 2 (16.7%) with anti-HBc/anti-HCV/HBsAg. Among the 42 thalassemic patients, GBV-C/HGV RNA was detected in 6/42 patients (14.2 %). Three patients presented GBV-C/HGV, with other blood borne markers. We also detected GBV-C/HGV in 6/50 (12%) blood donors. In these GBV-C/HGV positive thalassemics patients, 50% (3/6) were young individuals (lesser 15 years old) and 67% (4/6) were female patients. The presence of GBV-C RNA in the absence of hepatitis B and C infection in the young patients and healthy donors could be indicate that this virus is capable of independent transmission and does not contribute to liver disease.

Rat cytochrome P450c17 gene transcription is initiated at different start sites in extraglandular and glandular tissues

In the male rat, the mRNA of the steroidogenic cytochrome P450c17 is expressed extraglandularly in the stomach, duodenum, kidney and liver, throughout the animal’s lifespan, as demonstrated by reverse transcriptase and polymerase chain reaction (RT-PCR) analysis with specific primers. Northern analysis indicated that all tissues, except the kidney, contain high levels of such mRNA, but the relative mobility of liver mRNA is slightly less than that of the testis and other tissues. Thus, we analysed their 5′- and 3′-untranslated terminal regions (UTRs) by means of 5′- and 3′-rapid amplification of cDNA ends (RACE) techniques. All tissues utilised the same polyadenylation site as the testis. In the 5′-UTR of liver mRNA, however, we found a distal transcription start site (TSS) located 252 b upstream of that used in testicular P450c17 mRNA, which is placed 41 b upstream of the first ATG. The 5′-UTR sequence of liver P450c17 cDNA exactly matched the contiguous upstream untranslated region of the gene, suggesting that alternative splicing was not involved in the synthesis of liver P450c17 transcript. The other tissues used the same TSS present in the testis. Nevertheless, a second TSS located 125 b upstream of the first ATG was found in the stomach and duodenum. These results show that the transcriptional regulation of the CYP17 gene in the rat is complex and differs between tissues in the use of TSSs.

Molecular screening of genes mapped in POF loci

Objective: Premature ovarian failure (POF) - amenorrhea with elevated gonadotropins occurring before the age of 40 - with normal karyotype may be secondary to Xq chromosome defect. Two specific regions on Xq have been defined as POF loci: POF1 Xq26-qter and POF2 Xq13-Xq22 (Am J Med Genet; 1999, 89: 186–200) The POF1 segment, the QM or RPL10 (Ribosomal protein L10) gene, which maps to Xq28, is a novel gene that belongs to a class of transcription regulatory proteins and is highly conserved throughout eukaryotic evolution. There is strong evidence in Drosophila and suggestive evidence in humans that deficiencies of ribosomal proteins may yield viable but abnormal phenotypes. The Minute phenotype from Drosophila (reduced body size and infertility) results from deficiencies at any one of the ribosomal loci spread across the genome (Genome Res; 1998, 8: 509–523). Its location and function makes the QM gene a candidate for ovarian failure mapped to that region. The only candidate gene that maps the POF2 segment is the human homologue of the Drosophila melanogaster diaphanous gene (DIA). Mutated alleles of this gene affect spermatogenesis and oogenesis in the fruit fly. A disruption, due to a breakpoint in the 3’ end of the DIA gene, mapped to Xq22, has been previously described in familial POF (Am J Hum Genet; 1998, 62: 533–541). To investigate whether QM and DIA genes are involved with oogenesis and infertility we analyze the expression of mRNA in human oocyte and screened for mutations in these genes are associated with POF. Design: In vitro experiment at Academic Medical Center. Materials/Methods: RNA was extracted from oocytes (n=14) and granulosa cells from IVF patients and blood samples from 27 POF patients and 100 controls were screened for mutations in the coding region. The RNA was extracted from samples for subsequent reverse transcriptase and polymerase chain reaction (RT-PCR). Amplified cDNA was analyzed by single-stranded conformational polymorphism (SSCP) and/or by DNA sequencing. Results: There was detectable QM mRNA in oocytes and granulosa cells. In one POF patient a missense mutation in the QM gene was disclosed. A TTG→TCG conversion at nucleotide 308 resulted in a serine at codon 103 in place of leucine, which represents a change in QM protein hydrophobicity. No mutations were found in the DIA gene, ruling out the last exon mutations in this gene as the cause of POF in these patients. Conclusions: According to these results it is tempting to speculate that QM gene might be involved on the genesis of oocyte development and infertility. Mutational analysis of this gene could be useful for diagnosis of infertility. Supported by: FAPESP 99/3366–7.

Cloning of two mRNA variants of brain aromatase cytochrome P450 in rainbow trout ( Oncorhynchus mykiss Walbaum)

This work describes the molecular cloning of the cDNA encoding the rainbow trout ( Oncorhynchus mykiss Walbaum) brain cytochrome P450arom by means of reverse transcriptase and polymerase chain reaction (RT-PCR) and 5′- and 3′-rapid amplification of cDNA ends (RACE) analyses. The results obtained demonstrate that, as in other teleost fishes, the trout genome contains, besides the gene previously identified in the ovary, a second CYP19 gene ( CYP19B) expressed at high level in the brain. Moreover, two P450aromB mRNAs, forms I and II, were found to be transcribed in trout. Form I (1816 sequenced nt) contains an open reading frame (ORF) of 1464 b, a 5′-untranslated terminal region (UTR) of 124 b and at least 228 b in the 3′-UTR (incomplete, as the polyadenylation signal was not determined). Form II (1930 sequenced nt) contains an ORF of 1362 b, a 5′-UTR of 340 b and the same 3′-UTR as form I. Form II lacks the first 34 amino acids of form I, corresponding to the membrane-anchoring segment, whereas the sequence of the remaining coding region is almost the same in the two forms, resulting in proteins of 454 and 488 amino acids, respectively. Whether the two transcripts derive from the same gene by alternative splicing or are encoded by different CYP19B genes remains to be clarified. On Northern blot analyses with brain and ovary specific ORF probes and poly(A) +-enriched RNAs from trout ovary and brain, a transcript of about 2.6 kb was identified in the ovary, as expected, whereas the full-length mRNA of brain P450arom is about 3.8 kb. The brain form is expressed in the brain and gonads, whereas expression in peripheral tissues is limited mostly to the gills. The two trout CYP19 genes are not equivalent in tissue-specific expression, indicating the possibility of distinct promoters and regulatory mechanisms.

Somatostatin expression in human renal cortex and mesangial cells

Somatostatin modulates important physiologic functions of the kidney, including mesangial cell contraction, glomerular prostaglandin synthesis, and phosphate, water and sodium excretion. In diabetic nephropathy, somatostatin inhibits renal hypertrophy. High affinity somatostatin receptors are expressed in the kidney. Circulating somatostatin concentrations, however, are generally well below the affinity constants of known somatostatin receptors. Thus, we hypothesized that somatostatin is produced in the kidney and released locally to act in an autocrine/paracrine manner. Using reverse transcriptase and polymerase chain reaction (RT-PCR) analysis, we found that fresh human renal cortex and cultured human mesangial cells express somatostatin mRNA. Restriction enzyme and Southern blot analysis confirmed that RT-PCR cDNA products were derived from somatostatin mRNA. Radioimmunoassay of mesangial cell culture supernatants demonstrated SS-immunoreactive peptide (87±30 pg/ml compared to 19±9 pg/ml in medium not exposed to cells; P<0.05). In contrast, renal cells did not transcribe detectable levels of vasoactive intestinal peptide (VIP) or neuropeptide Y (NPY) mRNA, nor did they synthesize measurable peptide. Our results demonstrate that renal cells produce somatostatin and suggest that kidney-derived somatostatin may regulate renal function in an autocrine/paracrine manner. Characterization of this pathway may lead to novel methods to alter the course of diabetic nephropathy and other renal diseases.

Analysis at the DNA level of human Fc gamma RII isoforms in relation to the polymorphic binding of murine IgG2b to human B cells.

Three classes of human leucocyte Fc gamma receptors (hFc gamma R) have been identified so far: hFc gamma RI, hFc gamma RII, and hFc gamma RIII. Previous studies have demonstrated that genetically determined differences between individuals exist both with respect to the binding of murine IgG1 (mIgG1) to hFc gamma receptors, and with respect to the binding of murine IgG2b (mIgG2b). The polymorphism in binding of mIgG1 could be ascribed to hFc gamma RIIA, an isoform of hFc gamma RII. The authors have now investigated whether one of the isoforms of hFc gamma RII is also responsible for the polymorphism in binding of mIgG2b. In these studies the authors used EBV-transformed human B cells that demonstrated either binding or no binding of mIgG2b in EA-rosetting assays. mRNA obtained from these cells was amplified by reverse transcriptase and polymerase chain reaction (RT-PCR). Hybridization experiments with the RT-PCR products revealed that the hFc gamma RIIB but not the hFc gamma RIIA isoform was present in these cells. DNA sequencing further demonstrated that the nucleotide sequence of both the extracellular part and the cytoplasmic moiety of hFc gamma RIIB was identical for all individuals tested, regardless of their ability to bind mIgG2b. These findings indicate that the polymorphic binding of mIgG2b cannot be ascribed to one of the isoforms of hFc gamma RII. Since hFc gamma RI and hFc gamma RIII are not present on the cell surface of these cells, the authors conclude that an Fc receptor different from the known hFc gamma receptors must be responsible for the polymorphic binding of mIgG2b. These data further expand the complexity of hFc gamma R.

Gene Expression of Mutant Erythropoietin in Hepatocellular Carcinoma

A case of hepatocellular carcinoma complicated by erythrocytosis showed an increased level of serum immunoreactive erythropoietin (EPO) and EPO bioactivity. RT-PCR (reverse transcriptase and polymerase chain reaction) of EPO mRNA extracted from a surgical specimen indicated high expression of EPO mRNA in the tumor tissue. The nucleotide sequences of PCR amplified regions of the EPO precursor mRNA in tumor tissue showed three differences to those of normal EPO cDNA. The deduced amino acid sequence of the coding region also showed three differences from that of normal EPO. The erythrocytosis improved and the high serum EPO immunoreactive and bioactive level decreased after resection of the tumor. This is the first demonstration of mutant EPO mRNA expression and bioactive mutant EPO protein in hepatocellular carcinoma tissue.

Expression of cytokine genes, including IL-6, in human malignant melanoma cell lines.

As a preliminary to transducing human melanoma cells with lymphokine genes, we sought for constitutive gene expression and production of eight interleukins, tumour necrosis factors and granulocyte-colony stimulating factor in 19 human melanoma cell lines. Conversion of RNA into cDNA by reverse transcriptase and polymerase chain reaction (RT-PCR) were employed to evaluate gene expression while enzyme-linked immunosorbent assays (ELISA) or biological assays were used to assess the presence of proteins. No expression of interleukins (IL) 3, 4, and 5 or interferon-gamma RNA was found, while the other cytokines were variably expressed in melanoma lines, with IL-1 alpha, IL-1 beta, IL-6, IL-8, being detectable in most of the lines. At protein level, 10 melanoma cells were tested with ELISA and all were found to produce IL-8, five produced IL-6, two tumour necrosis factor (TNF)-alpha, one IL-1 alpha and two TNF beta. The levels of TNF beta were at the limit of test sensitivity. The amount of various cytokines released by the different lines varied widely. Biological assay with the D10-G4 clone confirmed the presence of IL-1 alpha in the supernatant of melanoma (ME) 10221 and revealed an IL-1 activity in the supernatant of Me 4024/1. The proliferating activity of melanoma supernatants on D10-G4 was inhibited by treatment with polyclonal antibodies against IL-1 alpha but not with antibodies against IL-1 beta. TNF biological activity was tested against the TNF-susceptible fibrosarcoma WEHI 164 clone 13.(ABSTRACT TRUNCATED AT 250 WORDS)