Quantitative Competitive Reverse Transcriptase PCR Research Articles

Prolactin and Insulin Ultradian Secretion and Adipose Tissue Lipoprotein lipase Expression in Severely Obese Women After Bariatric Surgery

Hyperprolactinemia is associated with obesity. Furthermore, in human adipose tissue cultured in vitro, prolactin (PRL) inhibited lipoprotein lipase (LPL) activity via functional PRL receptors. To study PRL and insulin ultradian rhythm and subcutaneous adipose tissue LPL mRNA and protein expressions in severely obese women before and after malabsorptive bariatric surgery. Seven severely obese, fertile women were studied twice, once before and the second time 1 year after bilio-pancreatic diversion (BPD), when the weight was stable for at least 3 months. Metabolizable energy intake and 24-h energy expenditure (EE) were measured. Fourier and PULSEFIT analyses were applied to 24-h hormonal time-series to study daily fluctuations and hormonal clearance. Insulin sensitivity was assessed by euglycemic-hyperinsulinemic clamp. Quantitative-competitive reverse transcriptase-PCR and western blot analysis were used to measure LPL gene expression. Spontaneous 24-h PRL secretion was significantly reduced after BPD (mean-daily release, 128.4 +/- 28.1 microg/l vs. 67.2 +/- 9.2 microg/l distribution volume (Vd/l.24 h), P = 0.02); insulin secretion also was significantly reduced (499.9 +/- 204.0 microg/Vd/l.24 h vs. 85.6 +/- 21.0 microg/Vd/l.24 h, P = 0.0001). Metabolizable energy/kg(FFM) did not change significantly after BPD. Twenty-four-hour EE, but not 24-h EE/FFM, was significantly decreased after BPD (P < 0.05). Insulin sensitivity significantly (P < 0.0001) increased after BPD from 21.41 +/- 1.92 to 68.62 +/- 5.03 micromol/kg(FFM)/min. LPL mRNA concentration (from 42.63 +/- 4.21% to 19.00 +/- 2.74% of cyclophilin mRNA, P = 0.001) as well as LPL protein level (from 8.94 +/- 2.73 to 3.16 +/- 1.05 as ratios of protein of interest vs. housekeeping protein, P = 0.038) significantly decreased after BPD. The major determinant of PRL secretion was insulin secretion, whereas the best predictors of LPL expression were insulin and PRL secretion rates. The restriction of lipid metabolizable energy rather than weight loss seems to be responsible for both reduction in PRL circulating levels and normalization of its secretion rhythm after bariatric surgery. Furthermore, the reduced adipose tissue LPL expression, being significantly correlated with the decrease in insulin and PRL, suggests a role of hyperinsulinemia and hyperprolactinemia in inducing and sustaining obesity.

A quantitative assay method of Toxoplasma gondii HSP70 mRNA by quantitative competitive-reverse transcriptase-PCR

Toxoplasma gondii ( T. gondii)-derived heat shock protein 70 ( T.g.HSP70) has been identified as a virulent molecule expressing only in T. gondii tachyzoites during lethal acute infection. Therefore, it is of importance to determine the expression of T.g.HSP70 mRNA in a quantitative manner for analysis of virulence of T. gondii in tissues. We have constructed a competitor T.g.HSP70 and have successfully established a quantitative competitive-reverse transcriptase–polymerase chain reaction (QC-RT-PCR) targeting T.g.HSP70 gene. By using the established QC-RT-PCR method, we have demonstrated that the copy number of T.g.HSP70 mRNA per T. gondii tachyzoite was highest in the lung among the organs examined in interferon-γ knockout (GKO) mice.

Transcription Factor NFIC Undergoes N-Glycosylation during Early Mammary Gland Involution

Expression of a 74-kDa nuclear factor I (NFI) protein is triggered in early involution in the mouse mammary gland, and its expression correlates with enhanced occupation of a twin (NFI) binding element in the clusterin promoter, a gene whose transcription is induced at this time (Furlong, E. E., Keon, N. K., Thornton, F. D., Rein, T., and Martin, F. (1996) J. Biol. Chem. 271, 29688-29697). We now identify this 74-kDa NFI as an NFIC isoform based on its interaction in Western analysis with two NFIC-specific antibodies. A transition from the expression of a 49-kDa NFIC in lactation to the expression of the 74-kDa NFIC in early involution is demonstrated. We show that the 74-kDa NFIC binds specifically to concanavalin A (ConA) and that this binding can be reversed by the specific ConA ligands, methyl alpha-D-mannopyranoside and methyl alpha-D-glucopyranoside. In addition, its apparent molecular size was reduced to approximately 63 kDa by treatment with the peptide N-glycosidase. The 49-kDa lactation-associated NFIC did not bind ConA nor was it affected by peptide N-glycosidase. Tunicamycin, a specific inhibitor of N-glycosylation, blocked formation of the 74-kDa NFI in involuting mouse mammary gland in vivo when delivered from implanted Elvax depot pellets. Finally, the production of the ConA binding activity could be reiterated in "mammospheres" formed from primary mouse mammary epithelial cells associated with a laminin-rich extracellular matrix. Synthesis of the 74-kDa NFIC was also inhibited in this setting by tunicamycin. Thus, involution triggers the production of an NFIC isoform that is post-translationally modified by N-glycosylation. We further show, by using quantitative competitive reverse transcriptase-PCR, that there is increased expression of the major mouse mammary NFIC mRNA transcript, mNFIC2, in early involution, suggesting that the involution-associated change in NFIC expression also has a transcriptional contribution.

Expression of efflux pump gene pmrA in fluoroquinolone-resistant and -susceptible clinical isolates of Streptococcus pneumoniae.

Thirty-four ciprofloxacin-resistant (MIC > or = 2 microg/ml) and 12 ciprofloxacin-susceptible clinical isolates of Streptococcus pneumoniae were divided into four groups based upon susceptibility to norfloxacin and the effect of reserpine (20 microg/ml). The quinolone-resistance-determining regions of parC, parE, gyrA, and gyrB of all ciprofloxacin-resistant clinical isolates were sequenced, and the activities of eight other fluoroquinolones, acriflavine, ethidium bromide, chloramphenicol, and tetracycline in the presence and absence of reserpine were determined. Despite a marked effect of reserpine upon the activity of norfloxacin, there were only a few isolates for which the activity of another fluoroquinolone was enhanced by reserpine. For most isolates the MICs of acriflavine and ethidium bromide were lowered in the presence of reserpine despite the lack of effect of this efflux pump inhibitor on fluoroquinolone activity. The strains that were most resistant to the fluoroquinolones were predominantly those with mutations in three genes. Expression of the gene encoding the efflux pump PmrA was examined by Northern blotting (quantified by quantitative competitive reverse transcriptase PCR) and compared with that of S. pneumoniae R6 and R6N. Within each group there were isolates that had high-, medium-, and low-level expression of this gene; however, increased expression was not exclusively associated with those isolates with a phenotype suggestive of an efflux mutant. These data suggest that there is another reserpine-sensitive efflux pump in S. pneumoniae that extrudes ethidium bromide and acriflavine but not fluoroquinolones.

The expression of the Helicobacter pylori genes ureA and nap is higher in vivo than in vitro as measured by quantitative competitive reverse transcriptase-PCR

The expression of the virulence-associated genes ureA, encoding the urease subunit A, and nap, encoding the neutrophil activating protein, in Helicobacter pylori grown both in the stomach of C57/Bl6 mice and in Brucella broth was quantified by quantitative competitive reverse transcriptase-PCR using a homologous RNA standard (competitor) and an external standard (16S rRNA). The results showed that the ureA and nap transcripts were increased up to 15 and 80 times, respectively, in vivo compared to in vitro. The transcription of ureA and nap also differed in that ureA showed highest expression early in infection in mice whereas nap transcription was variable throughout the 18-week infection period.

The expression of the Helicobacter pylori genes ureA and nap is higher in vivo than in vitro as measured by quantitative competitive reverse transcriptase-PCR.

The expression of the virulence-associated genes ureA, encoding the urease subunit A, and nap, encoding the neutrophil activating protein, in Helicobacter pylori grown both in the stomach of C57/Bl6 mice and in Brucella broth was quantified by quantitative competitive reverse transcriptase-PCR using a homologous RNA standard (competitor) and an external standard (16S rRNA). The results showed that the ureA and nap transcripts were increased up to 15 and 80 times, respectively, in vivo compared to in vitro. The transcription of ureA and nap also differed in that ureA showed highest expression early in infection in mice whereas nap transcription was variable throughout the 18-week infection period.

Simian Immunodeficiency Virus Replicates to High Levels in Naturally Infected African Green Monkeys without Inducing Immunologic or Neurologic Disease

African green monkeys can maintain long-term persistent infection with simian immunodeficiency viruses (SIVagm) without developing AIDS and thus provide an important model for understanding mechanisms of natural host resistance to disease. This study assessed the levels and anatomic distribution of SIVagm in healthy, naturally infected monkeys. Quantitative competitive reverse transcriptase PCR assays developed to measure SIVagm from two African green monkey subspecies demonstrated high levels of SIV RNA in plasma (>6 x 10(6) RNA copies/ml) in sabaeus and vervet monkeys. Infectious virus was readily recovered from plasma and peripheral blood mononuclear cells and shown to be highly cytopathic in human cell lines and macrophages. SIVagm DNA levels were highest in the gastrointestinal tract, suggesting that the gut is a major site for SIVagm replication in vivo. Appreciable levels of virus were also found within the brain parenchyma and the cerebrospinal fluid (CSF), with lower levels detected in peripheral blood cells and lymph nodes. Virus isolates from the CSF and brain parenchyma readily infected macrophages in culture, whereas lymph node isolates were more restricted to growth in human T-cell lines. Comparison of env V2-C4 sequences showed extensive amino acid diversity between SIVagm recovered from the central nervous system and that recovered from lymphoid tissues. Homology between brain and CSF viruses, macrophage tropism, and active replication suggest compartmentalization in the central nervous system without associated neuropathology in naturally infected monkeys. These studies provide evidence that the nonpathogenic nature of SIVagm in the natural host can be attributed neither to more effective host control over viral replication nor to differences in the tissue and cell tropism from those for human immunodeficiency virus type 1-infected humans or SIV-infected macaques.

Chemokine mRNA expression in the cauda equina of Lewis rats with experimental allergic neuritis

Chemokines play an important role in the migration of leukocytes to inflammatory sites. In this study, using the quantitative competitive reverse transcriptase PCR method, we analyzed sequential expression of certain chemokine mRNAs in the cauda equina (CE) of rats with experimental allergic neuritis (EAN). Interferon-γ-inducible protein (IP)-10, monocyte chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-1α, the regulated upon activation normal T cell expressed and secreted chemokine (RANTES), and lymphotactin were analyzed on days 0 (pre-immunization), 7 (preclinical stage), 10 (disease onset), 13 (clinical progression), 17 (disease peak), as well as on days 20, 24, and 34 post-immunization (p.i.) (recovery). MCP-1 message increased at the preclinical stage and peaked at day 17 p.i. The increase in the early stage was not detected in other tissues, indicating peripheral nerve-specific upregulation. MIP-1α and IP-10 messages surged at day 13, then returned to low in the recovery stage. RANTES message increased at day 13 and peaked at day 17 p.i.; however, unlike other chemokines, it showed a second peak of expression on day 24. Lymphotactin message was undetectable at any time point. MCP-1 protein was detected immunohistologically in endothelial cells at day 7 p.i. The sequential expression of these chemokines in relation to the inflammatory process in the nerve leading to demyelination is discussed.

The expression of cytokine mRNA in the cauda equina of Lewis rats with experimental allergic neuritis

Autoreactive CD4+ T cells can transfer experimental allergic neuritis (EAN) to naive recipients. In order to further analyze the role of these T cells and their corresponding cytokines in EAN, we studied the expression of mRNA for IFN- γ, IL-4, and IL-10 in the cauda equina of rats with EAN using a quantitative competitive reverse transcriptase PCR method. Nerves were studied on days 0 (pre-immunization), 10 (disease onset), 13 (clinical progression), 16 (disease peak), as well as 20, 24, and 34 post immunization (recovery). IFN- γ messages increased at disease onset and peaked at day 13 p.i. IL-10 message remained at a very low level at disease onset and surged at day 16. Both messages were low in recovery stage. IL-4 message was undetectable at any time point. These data suggest a pro-inflammatory role of IFN- γ and anti-inflammatory role of IL-10 in EAN lesions. It is also possible that a clonal switch from Th1 to Th2 occurs in EAN lesions during the disease course.

A novel highly reproducible quantitative competitive RT PCR system

Small changes in plasma fibrinogen concentration are often clinically important. To detect changes in fibrinogen mRNA level, we have designed a novel quantitative competitive reverse transcriptase (RT) PCR system, based on our recent understanding that an important factor causing imprecision with RT PCR is a change in the initial ratio of target to standard RNA templates during reverse transcription. The system comprises: (1) titration of a fixed amount of standard RNA with four different amounts of total RNA in each tube during cDNA synthesis; (2) amplification of each of the four cDNAs with four consecutive PCR cycles; and (3) quality control reactions in which synthesised quality control RNA (rather than total RNA) is added to the standard RNA under the same RT PCR conditions as the assay reaction. To obtain reproducible data, we suggest three criteria for analysis of RT PCR results. Employing these criteria, reproducibility of RT PCR was markedly improved with an inter-assay CV of 6.5% ( n = 5). Using this system, we are able to detect reduction in mRNA levels of all fibrinogen genes caused by dietary protein restriction in rats after weaning (α 39%, p = 0.028; β 55%, p = 0.017; γ 35%, p = 0.038), with a parallel reduction of plasma fibrinogen concentration (mean (±SD), 157(±19) versus 130(± 14) mg/dl, p = 0.006).

Two variants of quantitative reverse transcriptase PCR used to show differential expression of alpha-, beta- and gamma-fibrinogen genes in rat liver lobes.

Quantitative reverse transcriptase PCR (RT-PCR) is a sensitive method for the measurement of mRNA copy number. However, the methodology has gained a reputation for poor reproducibility, leading to concern over the validity of much of the data generated using this technique. We have developed two variants of quantitative competitive RT-PCR using a synthesized RNA as an internal standard to measure precisely the relative levels of alpha-, beta- and gamma-fibrinogen mRNAs in the four lobes of the rat liver. In the first of these variants we altered only the amount of total RNA in the RT-PCR reaction, keeping the amount of internal standard RNA and the number of PCR cycles constant. In the second variant only the number of PCR cycles was altered, and the amounts of total RNA and standard RNA were kept constant. Both variants of RT-PCR allowed calculation of the number of mRNA copies, which did not differ significantly between the two techniques. Of the two variants, the second gave better reproducibility, and the intra-assay coefficient of variation for this technique was 14% (n = 20). Using these two variants we have shown that there are different numbers of fibrinogen mRNAs in the four liver lobes for each of the three genes (alpha-fibrinogen F = 14.64, P = 0.0003; beta-fibrinogen F = 3.74, P = 0.04; gamma-fibrinogen F = 3.75, P = 0.04). In conclusion, by using two variants of quantitative competitive RT-PCR we have shown that this technique can be used to give reproducible results, and the low intra-assay coefficient of variation suggests that quantitative RT-PCR should be the technique of choice for accurate measurement of mRNA copy number.

Longitudinal assessment of feline immunodeficiency virus kinetics in plasma by use of a quantitative competitive reverse transcriptase PCR.

Cats infected with feline immunodeficiency virus (FIV) develop a disease syndrome similar to that caused by human immunodeficiency virus type 1 (HIV-1) infection in humans. HIV-1 replication has been shown to correlate with the disease stage and progression. To assess replication kinetics and disease progression in early FIV infection, we developed a quantitative competitive reverse transcriptase PCR to measure the plasma virus load at serial time points after virus exposure. We found that an early peak viremia immediately preceded the onset of acute-phase symptoms in infected cats. Plasma virus levels remained high throughout the symptomatic phase of infection, which lasted for 8 to 10 weeks, and then declined as clinical symptoms resolved; however, all cats maintained significant plasma virus titers through 36 weeks postinfection. Early peak viral replication coincided with the initial precipitous decline in circulating CD4+ T lymphocytes. These results indicate that FIV kinetics are similar to those of HIV-1 during the acute and secondary phase of infection and that the plasma FIV load correlates with the disease stage. These results serve to further develop the FIV model and to enhance its usefulness for pathogenesis, vaccine development, and therapeutic studies related to HIV.