Quantitative Competitive Reverse Transcription Polymerase Chain Reaction Research Articles

The Adverse Effect of Air Pollution with Polycyclic Aromatic hydrocarbon (PAH) on 8-OXO-DG and gene expression (HOGG1) in Midland Refineries Company-Daura Refinery Workers

Introduction: Polycyclic aromatic hydrocarbons (PAHs) are a group of different kind of hazardous organic chemicals which are considered to be the top of pollutants that released by petroleum industries exploration activities to the environment. PAH Metabolites that affect initiation of cancer by reaction with DNA are practically modified chemically by enzymes. PAH Mutagenic metabolites include radical PAH cations, diol epoxides, and quinones. Reactive oxygen species (ROS) mostly produce by routine oxidation processes in mitochondria is responsible for many types of DNA damage. The aim of study identify the DNA alteration due to air pollution by modulation of gene expression and regulatory gene Human 8-oxoguanine DNA Glycosylase (HOOG1) and association with 7,8-dihydro-8-oxoguanine (8-oxo-dg). Methods: there were 168 participants included in this study divided in three groups (control, office workers and in field workers) .PAH, 8-OXO-DG and gene expression (HOGG1) detected for each participant by GC/MS, competitive ELISA and quantitative-competitive reverse transcription-PCR respectively. Results: PAH were not detected in the blood of control group, and there was significant difference in the concentration of PAH between office and field workers. The concentration of (8-oxo DG) and (hOGG1) was significantly higher in field worker than in office worker and control group, significant difference was found between office workers and control. Discussion: It is observed that increased levels of PAH and its metabolites when exposed to polluted air. PAHs metabolism is associated with production of ROS as well as oxidative damaged. Among this damage, the most common lesion is (8-OHdG) .Increase in (8-OHdG) levels associated with increase oxidative stress and (HOGG1) gene. Conclusion: (a) increased PAH and its metabolites levels in serum causing high 8-OHdG and (HOGG1) expression levels in refineries workers (b) there is a correlation between 8-OHdG and hOGG1 expression with PAH.

Effect on Clinical Outcome and Growth Factor Synthesis With Adjunctive Use of Pulsed Electromagnetic Fields for Fifth Metatarsal Nonunion Fracture: A Double-Blind Randomized Study.

Electromagnetic bone growth stimulators have been found to biologically enhance the bone healing environment, with upregulation of numerous growth factors. The purpose of the study was to quantify the effect, in vivo, of pulsed electromagnetic fields (PEMFs) on growth factor expression and healing time in fifth metatarsal nonunions. This was a prospective, randomized, double-blind trial of patients, cared for by 2 fellowship-trained orthopedic foot and ankle surgeons. Inclusion criteria consisted of patients between 18 and 75 years old who had been diagnosed with a fifth metatarsal delayed or nonunion, with no progressive signs of healing for a minimum of 3 months. Eight patients met inclusion criteria and were randomized to receive either an active stimulation or placebo PEMF device. Each patient then underwent an open biopsy of the fracture site and was fitted with the appropriate PEMF device. The biopsy was analyzed for messenger-ribonucleic acid (mRNA) levels using quantitative competitive reverse transcription polymerase chain reaction (QT-RT-PCR). Three weeks later, the patient underwent repeat biopsy and open reduction and internal fixation of the nonunion site. The patients were followed at 2- to 4-week intervals with serial radiographs and were graded by the number of cortices of healing. All fractures healed, with an average time to complete radiographic union of 14.7 weeks and 8.9 weeks for the inactive and active PEMF groups, respectively. A significant increase in placental growth factor (PIGF) level was found after active PEMF treatment (P = .043). Other factors trended higher following active PEMF including brain-derived neurotrophic factor (BDNF), bone morphogenetic protein (BMP) -7, and BMP-5. The adjunctive use of PEMF for fifth metatarsal fracture nonunions produced a significant increase in local placental growth factor. PEMF also produced trends toward higher levels of multiple other factors and faster average time to radiographic union compared to unstimulated controls. Level I, prospective randomized trial.

A combined phenotypic and genotypic method for the detection of Mex efflux pumps in Pseudomonas aeruginosa

Mex efflux pumps contribute to multidrug resistance in Pseudomonas aeruginosa. Evidencing their expression in clinical isolates would help in rationalizing antibiotic selection. We have developed a combined phenotypic and genotypic approach for the differential diagnosis of resistance mediated by four major transporters (MexAB-OprM, MexCD-OprJ, MexEF-OprN, MexXY-OprM). The methodology was validated using reference strains harbouring only one specific transporter and its applicability evaluated towards seven selected clinical isolates, the resistance mechanisms of which could not be assigned by conventional techniques. Phenotypic detection used MIC measurements with reporter antibiotics [carbenicillin (MexAB-OprM); erythromycin (MexCD-OprJ); norfloxacin and imipenem (MexEF-OprN); gentamicin (MexXY-OprM)] with and without Phe-Arg-beta-naphthylamide. Genotypic detection was made by semi-quantitative reverse transcription PCR (RT-PCR) for mexC and mexE, and by quantitative competitive RT-PCR and real-time PCR for mexA and mexX (correlation between both methods: > 88 % ; overexpression levels ranging between 4.8 and 8.1). Convergence between phenotypic and genotypic methods was observed in control strains for all pumps. For clinical isolates, convergence was obtained in 6 of 7 strains for MexXY-OprM and MexEF-OprM, and in 5 of 7 for MexAB-OprM and MexCD-OprJ, mostly due to hard to interpret phenotypic data. The data plead for combining phenotypic and genotypic approaches in the diagnosis of efflux-mediated resistance in P. aeruginosa.

Messenger RNA transcripts of the meiotic regulator BOULE in the testis of azoospermic men and their application in predicting the success of sperm retrieval

Testicular sperm retrieval can lead to paternity for azoospermic patients with spermatogenic failure. The human BOULE gene, a meiotic regulator of germ cells, is a gene whose altered expression may be associated with sterility. We determined the levels of BOULE transcripts in the testes of azoospermic patients, and evaluated the relationship between BOULE transcript levels and patients' testicular phenotypes, clinical parameters and sperm retrieval results. BOULE transcript levels in the testes of 41 azoospermic patients were examined by quantitative competitive-reverse transcription-polymerase chain reaction. A significant decrease in BOULE transcript levels was detected in patients with spermatogenic failure, and BOULE transcript levels progressively decreased with increasing severity of testicular failure. BOULE transcript levels did not correlate with the serum hormone parameters measured. Significantly higher BOULE transcript levels were detected in 19 patients with successful sperm retrieval than in 12 patients with failed sperm retrieval. When using a cut-off value of 0.5 for BOULE transcript ratio to predict the success of sperm retrieval, both the sensitivity and specificity value were 100%. We suggest the BOULE transcript plays an important role in human spermatogenesis and that the levels may predict the presence of testicular sperm in patients with spermatogenic failure.

Increased Expression of Two Multidrug Transporter-Like Genes Is Associated with Ethidium Bromide and Ciprofloxacin Resistance in Mycoplasma hominis

Two genes, md1 and md2, coding for multidrug resistance ATP-binding cassette transporters were identified in Mycoplasma hominis PG21. Expression of these two genes, quantified by quantitative competitive reverse transcription-PCR, was significantly increased in ethidium bromide-resistant strains of M. hominis compared to that in M. hominis PG21.

Expression profiles of the DAZ gene family in human testis with and without spermatogenic failure

Objective To determine the expression profiles of the DAZ gene family in men with and without spermatogenic failure. Design Prospective case study. Setting University-based reproductive clinics and genetics laboratory. Patient(s) Thirty-four infertile men presenting with azoospermia. Intervention(s) The mRNA transcript concentrations of the DAZ family genes (BOULE, DAZL, DAZ) and the housekeeping GAPDH gene in the testes of azoospermic men were examined by quantitative competitive–reverse transcription–polymerase chain reaction (QC-RT-PCR). The steady-state concentrations of mRNA encoding for each gene in each testicular sample were normalized by the amounts of GAPDH. Main outcome measure(s) Transcript ratios (gene/GAPDH) of BOULE, DAZL, and DAZ. Result(s) The transcript ratios for BOULE, DAZL, and DAZ were significantly decreased in tissues with spermatogenic failure (hypospermatogenesis, maturation arrest, and Sertoli cell–only). However, the ratios of BOULE/DAZL and DAZ/DAZL did not reveal any significant difference in all tissues. Three patients with DAZ deletion possess lower transcripts of BOULE and DAZL. Conclusion(s) All members of the DAZ gene family play important roles in human spermatogenesis. Decreased concentrations of DAZ family members in men with spermatogenic failure may be due to the secondary effect of germ cell loss, and transcriptional control of BOULE, DAZL, and DAZ are not altered in the various degrees of spermatogenic failure. Although the sample size is limited, no compensatory increase of DAZL or BOULE transcription was found in men with DAZ deletion.

Mitosis increases levels of secretory leukocyte protease inhibitor in keratinocytes

Chronic wounds are a major health care burden. Multiple factors produced by healing wounds play important roles in efficient and orderly wound healing. Secretory leukocyte protease inhibitor (SLPI) is constitutively expressed in epithelial cells, and its expression is increased by inflammation. SLPI has antimicrobial activities and improves wound healing. We hypothesized that SLPI expression correlates with keratinocyte growth rate and is increased by epidermal growth factor (EGF). Keratinocytes were isolated from neonatal foreskin. Subconfluent and confluent culture conditions were used. SLPI-specific primers were designed for use in quantitative-competitive reverse-transcription polymerase chain reaction assays to detect and quantify SLPI mRNA expression levels. SLPI levels were increased 2-fold in subconfluent cultures and 1.6-fold in confluent cultures to which EGF had been added. Confluent cultures also demonstrated 7-fold (growth factor depleted) and 6-fold (EGF) increase in SLPI RNA copy number, compared to subconfluent cultures. The results indicate that EGF increases SLPI expression.

Development and evaluation of a quantitative competitive reverse transcription polymerase chain reaction (RT-PCR) for hepatitis C virus RNA in serum using transcribed thio-RNA as internal control

A method for quantitation of hepatitis C virus (HCV) RNA was developed based on competitive reverse transcription polymerase chain reaction (RT-PCR) using in vitro transcribed mutated thio-RNA as a competitor template. The thio-RNA is more resistant to RNAse and is stable over a year. This assay was compared with the commercially available Roche Amplicor HCV Monitor assay V 2.0 and real time PCR using SYBR green 1 dye method. A total of 18 pre-therapy serum samples from chronic hepatitis C cases were tested in parallel by the three assays. All samples could be quantitated using the in-house competitive RT-PCR and real time PCR and there was a significant correlation in the virus titer ( P<0.05). However, 8 (44%) samples could not be quantified by Amplicor HCV Monitor assay, which has a lower detection range (10 2 to 10 5.5 copies/ml). The in-house method of competitive RT-PCR showed a detection range of 10 3 to 10 10 copies/ml. In the patients the mean viral titer was found to be (9.66±9.3)×10 6 copies/ml. Ten (55%) of the samples, assessed by the Amplicor HCV Monitor assay showed a mean viral titre of (1.13±0.75)×10 6 copies/ml, which was lower than the other two tests. The competitive PCR method and real time PCR could amplify all prevalent genotypes. This in-house quantitative competitive RT-PCR method is simple, cheap, reproducible and useful for estimation of HCV RNA load.

Identification and characterization of the CYP52 family of Candida tropicalis ATCC 20336, important for the conversion of fatty acids and alkanes to alpha,omega-dicarboxylic acids.

Candida tropicalis ATCC 20336 excretes alpha,omega-dicarboxylic acids as a by-product when cultured on n-alkanes or fatty acids as the carbon source. Previously, a beta-oxidation-blocked derivative of ATCC 20336 was constructed which showed a dramatic increase in the production of dicarboxylic acids. This paper describes the next steps in strain improvement, which were directed toward the isolation and characterization of genes encoding the omega-hydroxylase enzymes catalyzing the first step in the omega-oxidation pathway. Cytochrome P450 monooxygenase (CYP) and the accompanying NADPH cytochrome P450 reductase (NCP) constitute the hydroxylase complex responsible for the first and rate-limiting step of omega-oxidation of n-alkanes and fatty acids. 10 members of the alkane-inducible P450 gene family (CYP52) of C. tropicalis ATCC20336 as well as the accompanying NCP were cloned and sequenced. The 10 CYP genes represent four unique genes with their putative alleles and two unique genes for which no allelic variant was identified. Of the 10 genes, CYP52A13 and CYP52A14 showed the highest levels of mRNA induction, as determined by quantitative competitive reverse transcription-PCR during fermentation with pure oleic fatty acid (27-fold increase), pure octadecane (32-fold increase), and a mixed fatty acid feed, Emersol 267 (54-fold increase). The allelic pair CYP52A17 and CYP52A18 was also induced under all three conditions but to a lesser extent. Moderate induction of CYP52A12 was observed. These results identify the CYP52 and NCP genes as being involved in alpha,omega-dicarboxylic acid production by C. tropicalis and provide the foundation for biocatalyst improvement.

Differential Expression of the Lactose Transporter Gene Affects Growth of Staphylococcus aureus in Milk

In this study, differential display polymerase chain reaction (PCR) was used to search for unique or enhanced expression of genes in prevalent bovine mastitis-causing Staphylococcus aureus strains. Comparison of a pair of prevalent and rare strains revealed the differential expression of several genes. The lactose-specific permease, enzyme II (EII), was highly expressed in the prevalent strain. This gene was selected for further study due to its potential influence on bacterial growth, because lactose is the primary carbohydrate in milk. Growth analysis illustrated that prevalent strains reach significantly higher growth densities sooner than rare strains. Quantitative competitive reverse transcription PCR (QC RT-PCR) revealed increased EII mRNA expression in prevalent strains as compared to rare strains. Mutation of the EII gene resulted in abrogated growth and decreased EII mRNA expression in media containing lactose. These data suggest that increased EII expression may facilitate the pathogenesis of S. aureus mastitis by enhancing growth. This study is the first to implicate EII as a potential virulence factor in mastitis, and therefore may be useful in the development of novel therapeutic strategies against S. aureus mastitis.

Loss of the perforin cytotoxic pathway predisposes mice to experimental cytomegalovirus retinitis.

AIDS-related human cytomegalovirus (HCMV) retinitis continues to be a chronic ophthalmologic problem among human immunodeficiency virus type 1 (HIV-1)-infected patients who do not respond to highly active antiretroviral therapy. Although HCMV retinitis occurs during HIV-1-induced immunosuppression, the precise effector mechanism(s) that fails during the immunopathogenesis of AIDS to allow onset and progression of HCMV retinal disease remains unclear. We therefore performed a series of experiments to explore the relative roles of distinct pathways of lymphocyte-mediated cytotoxicity in either resistance or susceptibility to experimental murine cytomegalovirus (MCMV) retinitis in mice. Whereas mutant C57BL/6 mice deficient in the Fas/FasL cytotoxic pathway (gld mice) were identical to normal C57BL/6 mice and exhibited absolute resistance to retinal necrosis following subretinal MCMV inoculation, knockout C57BL/6 mice deficient in the perforin cytotoxic pathway (PKO mice) were susceptible to MCMV retinitis. Susceptibility of PKO mice to MCMV retinitis correlated with increased ocular MCMV titers when compared with ocular MCMV titers of gld and normal mice. Since mice with retrovirus-induced immunodeficiency syndrome (MAIDS) exhibited a frequency and severity of MCMV retinitis that were equivalent to those observed in PKO mice, we hypothesized that susceptibility to MCMV retinitis during MAIDS correlates with a decrease in the perforin cytotoxic pathway. To test this hypothesis, we developed a quantitative competitive reverse transcription-PCR assay to measure mouse perforin mRNA levels in the splenic T lymphocytes and MCMV-inoculated eyes of normal mice or mice with MAIDS. Perforin mRNA levels in splenic T lymphocytes were significantly decreased during MAIDS, by approximately 100-fold, from perforin mRNA levels in normal mice. Moreover, MCMV-inoculated eyes destined to develop retinitis during MAIDS also showed a significant decrease in perforin mRNA levels from the perforin mRNA levels of MCMV-inoculated eyes of normal mice destined to be resistant to retinitis. As expected, perforin mRNA could not be detected in unmanipulated and uninfected eyes of normal mice. These results provide the first evidence that the perforin cytotoxic pathway is more important than the Fas/FasL cytotoxic pathway in providing resistance to experimental MCMV retinitis and that loss of the perforin cytotoxic pathway predisposes to MCMV retinitis.

Matrix metalloproteinase inhibition modulates fibroblast-mediated matrix contraction and collagen production in vitro.

To investigate the effect of matrix metalloproteinase (MMP) inhibition on fibroblast-mediated matrix contraction and production. Free-floating fibroblast-populated type I collagen lattices were prepared with human Tenon's capsule fibroblasts. Lattice areas were photographed and digitally analyzed to indicate the degree of lattice contraction. Quantitative competitive reverse transcription-polymerase chain reaction (QCRT-PCR) and enzyme-linked immunosorbent assay (ELISA) were used to quantify mRNA and protein respectively for MMP-1, -2, and -3 by fibroblasts during lattice contraction. Gelatin zymography demonstrated activity of MMPs released into the conditioned medium of contracting lattices. Concentrations of the broad-spectrum MMP inhibitors ilomastat, CellTech (Slough, UK), and BB-94 were added to the contracting fibroblast-populated collagen lattices. Secreted C-terminal propeptide of type I collagen was measured in conditioned medium of contracting lattices by ELISA. Fibroblast proliferation in the presence of concentrations of ilomastat was measured by using the reagent water-soluble tetrazolium-1 (WST-1). During contraction of type I collagen lattices, Tenon's capsule fibroblasts expressed MMP-1, -2, and -3 mRNA and protein. Zymography demonstrated the release of four gelatinolytic species into the conditioned medium of contracting lattices (57, 72, 91, and 100 kDa). Inclusion of MMP inhibitors in the zymogram-developing buffer reduced the proteolytic activity of the detected bands. MMP inhibition (1-100 microM) significantly reduced fibroblast-mediated collagen lattice contraction (P < 0.05), and this effect was found to be reversible. Ilomastat also significantly inhibited production of collagen in a dose-dependent manner (P < 0.05). No effect on fibroblast proliferation was found in the presence of ilomastat. MMPs are produced during Tenon's capsule fibroblast-mediated collagen lattice contraction. Broad-spectrum MMP inhibition significantly reduced matrix contraction and production without cell toxicity. Future clinical use of MMP inhibitors may be possible, because MMP inhibition significantly reduces fibroblast functions associated with contractile scarring.

Increased expression of cytotoxic effector molecules: different interpretations for steroid-based and steroid-free immunosuppression.

Cytotoxic T lymphocyte (CTL) effector molecules have been studied as markers of acute rejection in renal allograft recipients on steroid-based immunosuppression. We hypothesized that basal CTL gene expression may vary with time post-transplantation as well as with different immunosuppression protocols (steroid-based or steroid-free). Variations in CTL gene expression may thus impact on the ability to predict acute allograft rejection. We used the non-invasive method of quantitative competitive-reverse transcription-polymerase chain reaction (QC-RT-PCR) to quantify the amounts of CTL effector molecules (granulysin, GL; perforin, P; granzyme B, GB) in serial peripheral blood lymphocyte (PBL) samples from steroid-free and steroid-based adult and pediatric renal allograft recipients. Patients on both protocols were clinically monitored by protocol biopsies at 1, 3, 6, and 12 months post-transplantation and for graft function at 1 yr post-transplantation in a separate clinical study. Steroid-free patients with stable graft function showed an increase in GL, P, and GB gene expression over time post-transplantation with the increase being seen largely by the first post-transplant month. A further increase in GL expression was noted at the end of the first post-transplant year in the absence of acute rejection, whereas GB and P levels were unchanged. At comparative time-points post-transplantation, CTL genes were found to be higher in steroid-free patients with stable graft function, compared to steroid-based recipients with either clinically stable graft function or acute rejection. This study suggests that levels of CTL gene expression, although important in a steroid-based regimen to monitor the risk of acute rejection, may not be similarly applied in patients on steroid-free immunosuppression. The early increase in levels seen in steroid-free patients appears to correlate with the total absence of steroids. As steroid-free patients seem to have a lower incidence of acute rejection and better long-term graft function at 1 yr, the early increase in CTL genes in the absence of acute rejection may suggest an early adaptive immune activation response, promoting early graft acceptance in this protocol.

Effect of superovulation on the expression of tissue inhibitor of metalloproteinases-3 (TIMP-3) in murine endometrium

Objective: Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are important in endometrial remodeling process during embryonic trophoblast invasion. Previous studies, using in situ hybridization, revealed that the predominant proteinase expressed by the implanting mouse embryo is matrix metalloproteinase-9 (MMP-9), and the major tissue inhibitor of metalloproteinases (TIMP) produced in murine endometrium during this period is TIMP-3. This study was performed to evaluate the expression of TIMP-3 mRNA during periimplantation period in the hyperstimulated murine endometrum.Design: Laboratory research.Materials/Methods: Quantitative, competitive-reverse transcription-polymerase chain reaction (QC-RT-PCR) was performed to evaluate the expression of TIMP-3 mRNA during periimplantation period in the hyperstimulated murine endometrum from pregnant uteri of gestation day (g.d.) 6.0 and 7.0 after injection of pregnant mare serum gonadotropin (PMSG) 5 IU and 10 IU. TIMP-3 mRNA expression was corrected by the simultaneous transcript level of matrix metalloproteinases-9 (MMP-9) which is mainly expressed by mouse embryos, since TIMP-3 is expressed mainly in the murine endometrium adjacent to implanting mouse embryos.Results: In both PMSG 5 IU and 10 IU groups, the expression of TIMP-3 mRNA showed an increase while MMP did not show a significant difference compared to control group (natural coitus without PMSG injection), on g.d. 6.0 and 7.0. Corrected TIMP-3 mRNA expression for MMP-9 also showed a high level of transcription in murine endometrium in the superovulation groups.Conclusions: This study suggests that ovarian hyperstimulation by gonadotropin, which produces many oocytes and embryos, may have a detrimental effect on embryonic implantation via its relevant endometrial remodeling process by the increase in expression of TIMP-3 in murine endometrium.Supported by: None. Objective: Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are important in endometrial remodeling process during embryonic trophoblast invasion. Previous studies, using in situ hybridization, revealed that the predominant proteinase expressed by the implanting mouse embryo is matrix metalloproteinase-9 (MMP-9), and the major tissue inhibitor of metalloproteinases (TIMP) produced in murine endometrium during this period is TIMP-3. This study was performed to evaluate the expression of TIMP-3 mRNA during periimplantation period in the hyperstimulated murine endometrum. Design: Laboratory research. Materials/Methods: Quantitative, competitive-reverse transcription-polymerase chain reaction (QC-RT-PCR) was performed to evaluate the expression of TIMP-3 mRNA during periimplantation period in the hyperstimulated murine endometrum from pregnant uteri of gestation day (g.d.) 6.0 and 7.0 after injection of pregnant mare serum gonadotropin (PMSG) 5 IU and 10 IU. TIMP-3 mRNA expression was corrected by the simultaneous transcript level of matrix metalloproteinases-9 (MMP-9) which is mainly expressed by mouse embryos, since TIMP-3 is expressed mainly in the murine endometrium adjacent to implanting mouse embryos. Results: In both PMSG 5 IU and 10 IU groups, the expression of TIMP-3 mRNA showed an increase while MMP did not show a significant difference compared to control group (natural coitus without PMSG injection), on g.d. 6.0 and 7.0. Corrected TIMP-3 mRNA expression for MMP-9 also showed a high level of transcription in murine endometrium in the superovulation groups. Conclusions: This study suggests that ovarian hyperstimulation by gonadotropin, which produces many oocytes and embryos, may have a detrimental effect on embryonic implantation via its relevant endometrial remodeling process by the increase in expression of TIMP-3 in murine endometrium. Supported by: None.

Comparison and verification of quantitative competitive reverse transcription polymerase chain reaction (QC-RT-PCR) and real time RT-PCR for avian leukosis virus subgroup J

Avian leukosis virus subgroup J (ALV-J) infections cause significant economic losses because of increased mortality, tumor production, decreased production, and cost for eradication. Current quantification methods for ALV-J expressed by TCID 50 are difficult to determine because of the lack of cytopathic effect in cell cultures and non-specificity of currently available antigen-capture ELISA tests. In this study, a one-tube fluorescent probe based real time RT-PCR method was developed for quantification of ALV-J and compared with available quantification methods. Cell lysates with different TCID 50s determined by cell culture and antigen capture ELISA (ag-ELISA) were used for one-tube real time RT-PCR using fluorogenic probe and quantitative competitive RT-PCR (QC-RT-PCR). The results of QC-RT-PCR and real time RT-PCR were highly correlated to the TCID 50s determined by conventional culture methods. They were also very specific, sensitive, easy to perform, reproducible, and rapid compared with conventional methods. These RT-PCR based quantification methods of ALV-J viral RNA will be useful for virological and pathogenesis studies.

Heparan sulfate (HS)/heparan sulfate proteoglycan (HSPG) and bikunin are up-regulated during calcium oxalate nephrolithiasis in rat kidney.

We reported that expression of both HSPG and of bikunin are increased in calcium oxalate (CaOx) nephrolithic rat kidneys (lida et al., J. Am. Soc. Nephrol. 1999, Urol. Res. 1997). However, these findings were obtained from separate experiments. The present study evaluates whether levels of HSPG and bikunin expression differ in the rat kidney during calcium oxalate nephrolithiasis. Twenty-four male Wistar rats weighing 200-250 g were assigned to one of four groups (n = 6 each group) and administered with 0.5% ethylene glycol daily and 0.5 microgram of 1 alpha-OH-D3 every other day to induce CaOx nephrolithiasis. Animals were sacrificed 1 or 2 weeks later and both kidneys were excised. The cortex was separated from the medulla and papillary tips in the right kidney, then stored in liquid nitrogen for quantitative competitive-reverse transcription-polymerase chain reaction (QC-RT-PCR). The left kidney was fixed in 10% buffered formalin for histochemical studies. We assessed the variable gene expression of both HSPG and bikunin by QC-RT-PCR. Immunohistochemical analyses of left kidney tissue samples determined the localization of HSPG and bikunin. Normal rats serving as controls (n = 6 each) were also sacrificed and processed in the same manner as the experimental groups. QC-RT-PCR confirmed that HSPG and bikunin mRNA expression is significantly increased in nephrolithic kidneys (p < 0.05; Mann-Whitney test), and that medulla and papillary tips tended to express more mRNA of both. Immunohistochemical studies revealed that the production of HS and bikunin was increased in both the distal and proximal tubules of nephrolithic kidneys. These findings suggest that the increased expression of both HSPG and bikunin play an important role during calcium oxalate stone formation. In addition, this phenomenon might be associated with the progression of urothelial damage.

Expression and postnatal changes of adrenergic receptor subtype mRNA in rat submandibular glands

Adrenergic receptors (ARs) are involved in regulating saliva secretion and composition in salivary glands. Nine AR subtypes, including three α 1-ARs (α 1a-, α 1b- and α 1d-ARs), three α 2-ARs (α 2A-, α 2B- and α 2C-ARs) and three β-ARs (β 1,β 2- and β 3-ARs), have been identified through molecular cloning. The five subtype genes, α 1a-, α 1b-, α 2A-, β 1-, and β 2-ARs, were expressed in rat submandibular glands. In contrast, the other four subtype mRNAs, α 1d-, α 2B-, α 2C- and β 3-ARs, were not detected by reverse transcription-polymerase chain reaction (RT-PCR). The steady-state mRNA expression for the five AR subtypes in rat submandibular glands was measured by quantitative competitive RT-PCR using synthetic DNA as internal standard at different stages of postnatal development. The relative rank order of AR subtype mRNA expression was α 1a>β 2>β 1>α 2A>α 1b at all stages except that β 1- and α 2A-subtypes were reversed at 2 weeks of age. The gene expression of α 1a-AR subtype relative to total AR was low at 2 weeks of age and increased and reached a maximum at 6 weeks of age, whereas those patterns of α 2A-, β 1- and β 2-AR subtypes were similar to each other and their gene expressions were high at 2 weeks of age and then decreased. On the other hand, the gene expression of α 1b-AR subtype did not change over the different stages in relation to that of a housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase, and to total AR. Although rat submandibular glands contain the five AR subtype mRNAs, distinct subtype-specific expression is evident.

Evidence for an efflux pump mediating multiple antibiotic resistance in Salmonella enterica serovar Typhimurium.

The mechanism of multiple antibiotic resistance in six isolates of Salmonella enterica serovar Typhimurium recovered from a patient treated with ciprofloxacin was studied to investigate the role of efflux in the resistance phenotype. Compared to the patient's pretherapy isolate (L3), five of six isolates accumulated less ciprofloxacin, three of six isolates accumulated less chloramphenicol, and all six accumulated less tetracycline. The accumulation of one or more antibiotics was increased by carbonyl cyanide m-chlorophenylhydrazone to concentrations similar to those accumulated by L3 for all isolates except one, in which accumulation of all three agents remained approximately half that of L3. All isolates had the published wild-type sequences of marO and marR. No increased expression of marA, tolC, or soxS was observed by Northern blotting; however, three isolates showed increased expression of acrB, which was confirmed by quantitative competitive reverse transcription-PCR. However, there were no mutations within acrR or the promoter region of acrAB in any of the isolates.

Quantification of VEGF mRNA Expression in Non-Small Cell Lung Cancer Using a Real-Time Quantitative Reverse Transcription-PCR Assay and a Comparison with Quantitative Competitive Reverse Transcription-PCR

A reliable quantitative method for measuring gene product expression is important in investigating the relationship between growth factors and tumor biological behavior. In this study, we quantified the expression of vascular endothelial growth factor (VEGF) mRNA in 104 paired samples of lung cancer tissue and the surrounding nontumorous lung tissue using a new kinetic quantitative polymerase chain reaction (PCR) method, ie, real-time quantitative reverse transcription-PCR (RTQ RT-PCR). The samples consisted of 46 squamous cell carcinomas, 50 adenocarcinomas, and 8 undifferentiated cell carcinomas. In 17 cases, the results obtained were compared with those obtained using quantitative competitive RT-PCR (QC RT-PCR). Using RTQ RT-PCR, VEGF mRNA expression was detected and quantified in all 104 (100%) lung cancer samples and their normal counterparts. VEGF mRNA expression in the lung cancer tissue was significantly higher than in the normal counterparts (95% CI: 0.575 ∼ 1.727, p < 0.001; paired t test). In 68 (65.4%) cases, VEGF mRNA expression was higher in the cancer tissue than normal tissue. VEGF mRNA expression was higher in nonsquamous cell carcinoma (p = 0.02), and higher in tumor with hilar or mediastinal lymph node metastasis (p = 0.024). QC RT-PCR was able to detect and quantify VEGF mRNA expression in 15/17 (88%) lung cancer samples and 12/17 (70.6%) normal tissue samples. The values for VEGF mRNA expression were higher in the tumor in 13 (76%) cases. Comparison of the values of the VEGF mRNA expression quantified using RTQ RT-PCR or QC RT-PCR showed a good correlation between results obtained using these two methods, both in cancer tissue (r = 0.68, p = 0.0025) and normal counterpart (r = 0.53, p = 0.027). Agreement between the results for the relative expression of VEGF mRNA expression in cancer and normal tissue obtained using these two methods was seen in 14/16 cases (88%). We conclude that RTQ RT-PCR is as accurate as QC RT-PCR and is more sensitive than QC RT-PCR in detecting and quantifying VEGF mRNA expression in lung cancer and normal tissues, and both methods reveal that the VEGF mRNA expression is higher in lung cancer tissue than in healthy lung tissue.

Increased expression of osteopontin in activated Kupffer cells and hepatic macrophages during macrophage migration in Propionibacterium acnes-treated rat liver.

Osteopontin is an extracellular matrix component that can act as a chemokine to induce macrophage migration. The significance of osteopontin in macrophage infiltration into the liver was examined in rats given heat-killed Propionibacterium acnes. In normal rats, osteopontin mRNA expression in the liver was minimal, determined by quantitative-competitive reverse transcription-polymerase chain reaction (RT-PCR) assay. Northern blot analysis revealed that osteopontin mRNA was not expressed in Kupffer cells isolated from normal rats. When rats received heat-killed P. acnes intravenously, marked macrophage accumulation, forming granulomas, developed in the liver later than 3 days after the injection and its extent became maximal between 5 and 7 days. In these rats, osteopontin mRNA expression was increased in the liver later than 1 day (with its peak at 3 days after the injection), and the mRNA expression was increased markedly in Kupffer cells and hepatic macrophages isolated at 7 days. The mRNA expression of monocyte chemotactic protein-1 (MCP-1) and macrophage inflammatory protein-1alpha (MIP-1alpha), chemokines for monocytes and macrophages, was also increased in the liver of P. acnes-treated rats, with peak expression at 3 days. We conclude that osteopontin derived from Kupffer cells and hepatic macrophages may contribute to the infiltration of monocytes and macrophages into the liver cooperatively with the actions of MCP-1 and MIP-1alpha in P. acnes-treated rats.