Competitive RT-PCR Research Articles

A simple competitive RT-PCR assay for quantitation of HIV-1 subtype B and non-B RNA in plasma

An easy, inexpensive competitive RT-PCR assay for HIV-1 RNA quantitation was constructed. A 138-bp sequence in the HIV-1 gag p24 region was selected as the target and co-amplified with competitor RNA containing an internal 44-bp deletion. Quantitation of serial dilutions of control RNA samples prepared from the LAI isolate demonstrated a good linearity ( R 2 = 0.991) within the range between 10 and 250 copies/sample. The detection limit of the assay was determined to be 3.8 copies/sample by Probit analysis and corresponded to 110 copies/ml in plasma. The intra-assay CV value was 9.1%, and the inter-assay value was 25.9%. Both were comparable to those obtained with commercially available HIV-1 RNA quantitation kits. The correlation efficient for the results obtained in 47 plasma samples from HIV-1-infected individuals (subtype A in 1, subtype B in 25, subtype C in 4, subtype F in 1, and CRF01_AE in 16) with the competitive RT-PCR and Cobas Amplicor HIV-1 Monitor test v1.5 was 0.956 for subtype B and 0.947 for subtype non-B. The assay devised is a good alternative for monitoring antiretroviral therapy in resource-poor countries.

SGNE1/7B2 is epigenetically altered and transcriptionally downregulated in human medulloblastomas

In a genome-wide screen using differential methylation hybridization (DMH), we have identified a CpG island within the 5' region and untranslated first exon of the secretory granule neuroendocrine protein 1 gene (SGNE1/7B2) that showed hypermethylation in medulloblastomas compared to fetal cerebellum. Bisulfite sequencing and combined bisulfite restriction assay were performed to confirm the methylation status of this CpG island in primary medulloblastomas and medulloblastoma cell lines. Hypermethylation was detected in 16/23 (70%) biopsies and 7/8 (87%) medulloblastoma cell lines, but not in non-neoplastic fetal (n=8) cerebellum. Expression of SGNE1 was investigated by semi-quantitative competitive reverse transcription-polymerase chain reaction and found to be significantly downregulated or absent in all, but one primary medulloblastomas and all cell lines compared to fetal cerebellum. After treatment of medulloblastoma cell lines with 5-aza-2'-deoxycytidine, transcription of SGNE1 was restored. No mutation was found in the coding region of SGNE1 by single-strand conformation polymorphism analysis. Reintroduction of SGNE1 into the medulloblastoma cell line D283Med led to a significant growth suppression and reduced colony formation. In summary, we have identified SGNE1 as a novel epigenetically silenced gene in medulloblastomas. Its frequent inactivation, as well as its inhibitory effect on tumor cell proliferation and focus formation strongly argues for a significant role in medulloblastoma development.

Expression of MMPs and TIMPs in liver fibrosis – a systematic review with special emphasis on anti-fibrotic strategies

In liver tissue matrix metalloproteinases (MMPs) and their specific inhibitors (tissue inhibitors of metalloproteinases, TIMPs) play a pivotal role in both, fibrogenesis and fibrolysis. The current knowledge of the pathophysiology of liver fibrogenesis with special emphasis on MMPs and TIMPs is presented. A systematic literature search was conducted. All experimental models of liver fibrosis that evaluated a defined anti-fibrotic intervention in vivo or in vitro considering MMPs and TIMPs were selected. The methodological quality of all these publications has been critically appraised using an objective scoring system and the content has been summarized in a table.

Age-related changes of P2X 1 receptor mRNA in the bladder detrusor from men with and without bladder outlet obstruction

The urinary bladder purinergic system is reported to change with age and with bladder dysfunction. Here, we examined the expression of purinergic P2X 1 receptors in detrusor and mucosa (urothelium + lamina propria) from male control bladder and investigated age-related P2X 1 receptor mRNA expression in control and obstructed detrusor. Biopsy specimens were obtained at cystoscopy from control patients ( n = 46, age range 30–86 years) and patients diagnosed with outlet obstruction ( n = 29, 46–88 years). Calponin expression (measured by RT-PCR) was similar in control and obstructed detrusor and did not change with age. Quantitative competitive RT-PCR was used to measure P2X 1 receptor and GAPDH mRNA in control and obstructed detrusor. P2X 1 receptor mRNA expression was 9-fold ( p < 0.0001) higher in the detrusor than in the mucosa. Expression of mRNA for the internal control GAPDH remained stable with age and across control and obstructed detrusor. No difference in P2X 1 receptor expression was observed between control and obstructed detrusor ( p = 0.35). However, an age-related decrease in P2X 1 mRNA expression was observed in control ( n = 27; p = 0.0054; Spearman coefficient r = −0.520) but not obstructed detrusor ( n = 19; p = 0.093; r = −0.396). Downregulation of P2X 1 mRNA expression might occur as a result of an increased component of neural ATP release in the aging bladder.

Tumor susceptibility and prognosis of breast cancer associated with the G870A polymorphism of CCND1

We aimed to investigate the role of CCND1 G870A polymorphism genetic and transcriptomic effects susceptibility in association with breast cancer carcinogenesis and clinical prognosis. A case-control study was conducted with the enrollment of 992 sporadic breast cancer patients and the corresponding 960 normal controls from routine mammographic or sonographic screening for breast cancer between 1995 and 2003 in Taiwan. The 167 fragment spanning the G870A polymorphism in exon 4-intron 4 boundary was amplified to identify genotype of CCND1 (G870A) polymorphism. Competitive RT-PCR were further performed to investigate alternative transcript in four different specimens in association with immunohistochemistry markers. The results showed that AG and AA subgroup were at increased risk for developing breast cancer compared with the GG genotype by 19% (OR 1.19 (0.85-1.67)) and by 34% (OR 1.34 (0.04-1.74)), respectively. A870 allele revealed a recessive tendency while GG and AA/AG subgroup was compared (OR 1.35 (1.07-1.70)). AA genotype also had a higher risk in premenopausal women than postmenopausal ones. The recurrence-free survival was longer in patients with GG+AG than that in patients with AA (P = 0.034). A870 allele produced more transcript b both in malignant. There were significant correlations between several immunohistochemistry markers (such as Ki-67) and cyclin D1 or CDk4. We concluded CCND1 G870A polymorphism make significant contribution to breast cancer in the country with the preponderance of breast cancer in young women. The role of G870A polymorphism in alternative transcript was not only implicated in CCND1 alternative splicing but also correlated with immunohistochemistry markers.

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.

Suppressive effects of Chelidonium majus methanol extract in knee joint, regional lymph nodes, and spleen on collagen-induced arthritis in mice

Chelidonium majus L. has multiple applications in Korean traditional medicine because of its anti-tumoral, cytotoxic, anti-inflammatory and anti-microbial activities and has long been known to have anti-inflammatory effects. However, no study on the anti-arthritic activity of Chelidonium majus has been reported in vivo. Rheumatoid arthritis (RA) is a systemic autoimmune disease with chronic inflammation characterized by hyperplasia of synovial cells in affected joints, which ultimately leads to the destruction of cartilage and bone. Cytokine production and gene expression were assessed during CIA (collagen-induced arthritis) model mice in knee joint, lymph node (LN), and spleen, using ELISA and competitive RT-PCR. DBA/1J mice were immunized with bovine type II collagen. After a second collagen immunization, mice were treated with CME orally at 400, 40 mg/kg once a day for 4 weeks. The severity of arthritis within the knee joints was evaluated by histological assessment of cartilage destruction and pannus formation. Administration of CME significantly suppressed the progression of CIA and inhibited the production of TNF-α and IL-6 in spleen and lymph node. The erosion of cartilage was dramatically reduced in mouse knees after treatment with CME. In conclusion, our results demonstrates that CME significantly suppressed the progression of CIA and that this action was characterized by the decreased production of TNF-α, IL-6, IFN-γ, B cells, γδ T cells (in spleen) and increased proportion of CD4+CD25+ regulatory T cells in vivo. In the serum of CME-treated mice, the levels of IgG and IgM RA factor were decreased.

Selective epigenetic alteration of layer I GABAergic neurons isolated from prefrontal cortex of schizophrenia patients using laser-assisted microdissection

Among the most consistent results of studies of post-mortem brain tissue from schizophrenia patients (SZP) is the finding that in this disease, several genes expressed by GABAergic neurons are downregulated. This downregulation may be caused by hypermethylation of the relevant promoters in affected neurons. Indeed, increased numbers of GABAergic interneurons expressing DNA methyltransferase 1 (DNMT1) mRNA have been demonstrated in the prefrontal cortex (PFC) of SZP using in situ hybridization. The present study expands upon these findings using nested competitive reverse transcription-polymerase chain reaction combined with laser-assisted microdissection to quantitate the extent of DNMT1 mRNA overexpression in distinct populations of GABAergic neurons obtained from either layer I or layer V of the PFC of SZP. In a cohort of eight SZP and eight non-psychiatric subject (NPS) post-mortem BA9 tissue samples, DNMT1 mRNA was found to be selectively expressed in GABAergic interneurons and virtually absent in pyramidal neurons. DNMT1 mRNA expression was approximately threefold higher in GABAergic interneurons microdissected from layer I of SZP relative to the same neurons microdissected from NPS. GABAergic interneurons obtained from layer V of the same samples displayed no difference in DNMT1 mRNA expression between groups. In the same samples, the GABAergic neuron-specific glutamic acid-decarboxylase(67) (GAD(67)) and reelin mRNAs were underexpressed twofold in GABAergic interneurons isolated from layer I of SZP relative to GABAergic interneurons microdissected from layer I of NPS, and unaltered in GABAergic interneurons of layer V. These findings implicate an epigenetically mediated layer I GABAergic dysfunction in the pathogenesis of schizophrenia, and suggest novel strategies for treatment of the disease.

Krox20 is down-regulated following triazole in vitro embryonic exposure: A polycompetitor-based assay

This study was conducted in order to analyse gene-expression alterations in rat embryos following exposure to triazoles, using an easy-handling approach. Triazole derivatives have been shown to alter the morphology of cranio-facial structures and to induce abnormalities in hindbrain patterning and neural crest cell migration. Specification of hindbrain segments is regulated by retinoic acid and the hox code. Krox20 was chosen as molecular marker for its specific distribution in the anterior neural tube. In fact, this zinc-finger protein is expressed in rhombomere 3 and 5. Mis-regulation of Krox20 levels have shown to induce severe alterations in the correct patterning of the rhomboencephalon and the derived structures. In order to analyse Krox20 mRNA levels in rat embryos exposed in vitro to the triazole derivative triadimefon, a semi-quantitative approach utilising the competitive RT-PCR was chosen. A lambda phage-based plasmid construct that could compete with target and internal standard gene at the same time during enzymatic reaction was generated. Results were confirmed by real-time RT-PCR analysis on the same samples. Our data show a down-regulation of Krox20 transcript levels after exposure to the triazole derivative, implying a key role of this molecule in the pathogenic pathway induced by triazole exposure.

Retinoic Acid Enhances the Production of IL-10 While Reducing the Synthesis of IL-12 and TNF-α from LPS-Stimulated Monocytes/Macrophages

Vitamin A and its metabolites, e.g., all trans-retinoic acid (atRA) and 9-cis-retinoic acid have attracted considerable attention as compounds that have a broad range of immune modulating effects on both humoral and cellular immune responses. The cellular and molecular mechanisms that underlie the effects of retinoids on the immune system remain to be more clearly defined. These immune modulating effects of atRA may be mediated by cytokines elaborated by monocytes and other cell types. To further understand the mechanism(s) by which retinoids affect the immune response, we examined the effects of atRA on several proinflammatory and immune modulating cytokines produced by monocytes. The effects of atRA on LPS-induced mRNA expression of IL-10, IL-12p40, TNF-alpha, IL-18, and TGF-beta in the THP-1 monocyte/macrophage cell line and in cord blood mononuclear cells were measured by competitive RT-PCR. The ELISPOT was employed to evaluate IL-10 and TNF-alpha protein production enumerating the number of IL-10 and TNF-alpha producing cells. The addition of atRA to cell cultures potentiated the LPS-induced IL-10 mRNA expression and the number of IL-10 secreting cells from THP-1 cells and cord blood mononuclear cells. In contrast, the addition of atRA inhibited the LPS-induced TNF-alpha and IL-12p40 mRNA expression, and the number of ELISPOT positive cells for TNF-alpha. atRA did not change the LPS-induced mRNA expression of IL-18 and TGF-beta. These results suggest that atRA may have multiple effects on LPS-induced monocyte/macrophage derived cytokines. While atRA downregulated the proinflammatory cytokines, e.g., IL-12 and TNF-alpha, the production of an immune modulating cytokine, IL-10 was enhanced by atRA. The effects of atRA on these cytokines may play an important role in the modulation of the immune and inflammatory responses.

Stability of Potato virus X expression vectors is related to insert size: implications for replication models and risk assessment

We investigated the stability of expression constructs based on Potato virus X (PVX) as a function of insert length. Five different inserts ranging in length from 261 to 1,758 bp (human proinsulin, murine interleukin-10, HIV-1 nef, petunia expansin-1 and human gad65) were expressed using a PVX vector in Nicotiana benthamiana plants for three sequential passages. Using a competitive RT-PCR approach we demonstrated that insert-deletion could occur in the first infection cycle for all inserts, but that this was much more likely to be the case for longer ones. This suggested a negative correlation between insert length and vector stability. Sequence analysis of the deleted constructs suggested that recombination usually occurred at sites close to the duplicated sub-genomic promoter, but in a smaller number of cases the foreign gene itself was probably involved, resulting in partially deleted constructs containing transgene fragments. The implications of these results in the context of recombinant protein expression and its risks are discussed.

Clinical significance of mucosal suppressors of cytokine signaling 3 expression in ulcerative colitis

To investigate the clinical significance of mucosal expression of suppressors of cytokine signaling 1 (SOCS1) and SOCS3 in human ulcerative colitis (UC). Biopsy specimens for histological analysis and mRNA detection were obtained endoscopically from the rectum of 62 patients with UC (36 men; age 13-76 years). The patients were classified endoscopically according to Matts' grade (grade 1 to 4). Expression of SOCS1 and SOCS3 mRNAs was quantified in samples by competitive reverse transcription-polymerase chain reaction (RT-PCR). GAPDH was used as an internal control for efficiency of RT-PCR and amount of RNA. SOCS3 mRNA expression was significantly higher in inflamed mucosa of UC than in inactive mucosa. The level of expression was well correlated with the degree of both endoscopic and histologic inflammation. Interestingly, among the patients in remission, the group with relatively low expression of SOCS3 showed a higher rate of remission maintenance over a 12-mo period. In contrast, SOCS1 mRNA was expressed in both inflamed and non-inflamed colonic mucosa and was not correlated with the activity of colonic mucosa or prognosis. These observations suggest that increased expression of mucosal SOCS3, but not of SOCS1, may play a critical role in the development of the colonic inflammation of UC.

Downregulation of the V2 vasopressin receptor in dehydration: mechanisms and role of renal prostaglandin synthesis

The vasopressin-aquaporin 2 system plays a key role in urine concentration in dehydration. In contrast to the upregulation of aquaporin 2, the downregulation of the vasopressin V2 receptor in dehydration is known. We investigated the mechanisms of this downregulation in dehydration using reverse transcription-competitive polymerase chain reaction (RT-competitive PCR) and Western blot analysis. The incubation of microdissected inner medullary collecting ducts (IMCDs) in a hypertonic medium or with vasopressin stimulated V2 receptor mRNA and protein expression, showing that dehydration-induced hyperosmolality in renal medulla and increased plasma arginine vasopressin (AVP) concentration should upregulate V2 receptor. The presence of inhibitory factors on the V2 receptor in dehydration was suggested. Prostaglandin E(2) (PGE(2)) is known to inhibit AVP-induced cAMP production and to increase production in dehydration. PGE(2) slightly stimulated V2 receptor mRNA expression in IMCD in vitro. However, PGE(2) inhibited V2 receptor mRNA expression in IMCD in the presence of 10(-9) M vasopressin. The blockade of PGE(2) synthesis by indomethacin in dehydrated rats increased V2 receptor protein expression after 24-48 h with an early increase in V2 receptor mRNA expression. In summary, these data suggest that increased production of PGE(2) in renal medulla plays a key role in the downregulation of V2 receptor in dehydration.

Expression of Nuclear and Mitochondrial Thyroid Hormone Receptors in Postnatal Rat Tongue Muscle

In this quantitative study, a competitive RT-PCR analysis was used to measure the level of the thyroid hormone receptors (TRs) in rat tongue muscle during the development of male Wistar rats aged 0, 5, 10, 15 and 21 postnatal days. There were differences between the expression of TR-α1 mRNA and the mRNAs for TR-β1 and TR-β2 in rat tongue muscle. Using Western blot analysis, a difference in expression between TR-α1 protein (c-ErbAα1 protein) and 43-kD c-ErbAα1 protein (T₃-binding 43-kD mitochondrial protein) was detected during the development of the rat tongue muscle. Immunohistochemical examination using electron microscopy showed that TR-α1 was found in the mitochondria and nuclei in contrast to TR-β1 detected in rat tongue muscle. In mitochondrial fractions from rat tongue muscle, the expression of 43-kD c-ErbAα1 protein was increased dramatically at 15 and 21 days, and a similar tendency was seen in cytochrome c proteins using Western blot analysis. We presume that the 43-kD c-ErbAα1 protein plays a role in regulating mitochondrial RNA synthesis during the postnatal development of rat tongue. The mRNA and protein myosin heavy chain isoforms of muscle also had a different expression during development. The slow myosin isoform protein was not found from day 10 in contrast to fast myosin isoforms. It is likely that the expression of TR-α1 mRNA from the rat tongue muscle may be related to a specific phase in muscle phenotype during the development.

Removal of Astrovirus from Water and Sewage Treatment Plants, Evaluated by a Competitive Reverse Transcription-PCR

Quantification of human astrovirus genogroups A and B was undertaken with sewage and water samples, collected from the Greater Cairo area in Egypt from November 1998 to October 1999, by a competitive reverse transcription (RT)-PCR with an internal control. The number of RNA copies of genogroup A/liter in quantifiable samples ranged from 3.4 x 10(3) to 5.6 x 10(6) in raw sewage and from 3.4 x 10(3) to 1.1 x 10(4) in treated effluents, while the number of infectious units per liter in these samples as determined by cell culture RT-PCR (CC-RT-PCR U/liter) ranged from 3.3 x 10(1) to 3.3 x 10(3) in raw sewage and was 3.3 x 10(0) in treated effluents. On the other hand, the number of RNA copies/liter in quantifiable genogroup B samples ranged from 1.1 x 10(4) to 8.7 x 10(6) in raw sewage and from 1.1 x 10(3) to 6.2 x 10(5) in treated effluents, while the number of infectious units ranged from 3.3 x 10(1) to 3.3 x 10(5) CC-RT-PCR U/liter in raw sewage and from 3.3 x 10(1) to 3.3 x 10(2) CC-RT-PCR U/liter in treated effluents. These higher numbers of both RNA copies/liter and infectious particles of genogroup B may indicate the emergence of genogroup B in the area. Additionally, genogroup B astrovirus exhibited a higher resistance to removal treatments with regard to the number of RNA copies per ml. When the equipment for real-time approaches is unavailable, a competitive PCR or RT-PCR with an internal control may be employed for virus quantification in validations of the efficiency of virus removal treatments.

DNA Methyltransferase Inhibitors Coordinately Induce Expression of the Human Reelin and Glutamic Acid Decarboxylase 67 Genes

Reelin and glutamic acid decarboxylase 67 (GAD67) mRNAs and protein levels are substantially reduced in postmortem brains of patients with schizophrenia. Increasing evidence suggests that the observed down-regulation of reelin and GAD67 gene expression may be caused by dysfunction of the epigenetic regulatory mechanisms operative in cortical GABAergic interneurons. To explore whether human reelin and GAD67 mRNAs are coordinately regulated through DNA methylation-dependent mechanisms, we studied the effects of DNA methyltransferase inhibitors on reelin and GAD67 expression in NT-2 neuronal precursor cells. Competitive reverse transcription-polymerase chain reaction with internal standards was used to quantitate mRNA levels. The data showed that reelin and GAD67 mRNAs are induced in the same dose- and time-dependent manners. We further demonstrated that the activation of these two genes correlated with a reduction in DNA methyl-transferase activity and DNA methyltransferase 1 (DNMT1) protein levels. Time course Western blot analysis showed that DNMT1 protein down-regulation occurs temporally before the reelin and GAD67 mRNA increase. In addition, chromatin immunoprecipitation assays demonstrated that the activation of the reelin gene correlates with the dissociation of DNMT1 and methyl-CpG binding protein 2 (MeCP2) from the promoter, and an increased acetylation of histones H3 in the region. Together, our data strongly imply that human reelin and GAD67 genes are coordinately regulated through epigenetic mechanisms that include the action of DNMT1. Our study also suggests that negative regulation of the reelin gene involves methylation-dependent recruitment of DNMT1, MeCP2, and certain histone deacetylases, which most likely reduce the activity of the promoter by shifting the surrounding chromatin into a more compact state.

NO‐mediated regulation of NAD(P)H oxidase by laminar shear stress in human endothelial cells

The flowing blood generates shear stress at the endothelial cell surface. In endothelial cells, NAD(P)H oxidase complexes have been identified as major sources of superoxide anion (.O(2)(-)) formation. In this study, we analysed the effect of laminar shear stress on .O(2)(-) formation by cytochrome c reduction assay and on NAD(P)H oxidase subunit expression by standard calibrated competitive reverse transcription-polymerase chain reaction and Western blot in human endothelial cells. Primary cultures of human umbilical vein endothelial cells were exposed to laminar shear stress in a cone-and-plate viscometer for up to 24 h. Short-term application of shear stress transiently induced .O(2)(-) formation. This was inhibited by NAD(P)H oxidase inhibitor gp91ds-tat, but NAD(P)H oxidase subunit expression was unchanged. Long-term arterial laminar shear stress (30 dyne cm(-2), 24 h) down-regulated .O(2)(-) formation, and mRNA and protein expression of NAD(P)H oxidase subunits Nox2/gp91(phox) and p47(phox). In parallel, endothelial NO formation and eNOS, but not Cu/Zn SOD, protein expression was increased. Down-regulation of .O(2)(-) formation, gp91(phox) and p47(phox) expression by long-term laminar shear stress was blocked by l-NAME. NO donor DETA-NO down-regulates .O(2)(-) formation, gp91(phox) and p47(phox) expression in static cultures. In conclusion, our data suggest a transient activation of .O(2)(-) formation by short-term shear stress, followed by a down-regulation of endothelial NAD(P)H oxidase in response to long-term laminar shear stress. NO-mediated down-regulation by shear stress preferentially affects the gp91(phox)/p47(phox)-containing NAD(P)H oxidase complex. This mechanism might contribute to the regulation of endothelial NO/.O(2)(-) balance and the vasoprotective potential of physiological levels of laminar shear stress.

Schwann Cells Express Motor and Sensory Phenotypes That Regulate Axon Regeneration

Schwann cell phenotype is classified as either myelinating or nonmyelinating. Additional phenotypic specialization is suggested, however, by the preferential reinnervation of muscle pathways by motoneurons. To explore potential differences in growth factor expression between sensory and motor nerve, grafts of cutaneous nerve or ventral root were denervated, reinnervated with cutaneous axons, or reinnervated with motor axons. Competitive reverse transcription-PCR was performed on normal cutaneous nerve and ventral root and on graft preparations 5, 15, and 30 d after surgery. mRNA for nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor, hepatocyte growth factor, and insulin-like growth factor-1 was expressed vigorously by denervated and reinnervated cutaneous nerve but minimally by ventral root. In contrast, mRNA for pleiotrophin (PTN) and glial cell line-derived neurotrophic factor was upregulated to a greater degree in ventral root. ELISA confirmed that NGF and BDNF protein were significantly more abundant in denervated cutaneous nerve than in denervated ventral root, but that PTN protein was more abundant in denervated ventral root. The motor phenotype was not immutable and could be modified toward the sensory phenotype by prolonged reinnervation of ventral root by cutaneous axons. Retrograde labeling to quantify regenerating neurons demonstrated that cutaneous nerve preferentially supported cutaneous axon regeneration, whereas ventral root preferentially supported motor axon regeneration. Schwann cells thus express distinct sensory and motor phenotypes that are associated with the support of regeneration in a phenotype-specific manner. These findings suggest that current techniques of bridging gaps in motor and mixed nerve with cutaneous graft could be improved by matching axon and Schwann cell properties.

Increased cardiac endothelial nitric oxide synthase expression in patients taking angiotensin‐converting enzyme inhibitor therapy

The efficacy of angiotensin-converting enzyme (ACE) inhibitors has been demonstrated in large clinical trials, but knowledge of the underlying mechanisms remains incomplete. Therefore, this study investigated the impact of ACE inhibitor therapy on cardiac nitric oxide (NO) synthases in patients with coronary artery disease (CAD) or heart failure. The mRNA expression was quantified by standard calibrated competitive RT-PCR, protein expression by Western blotting and NOS activity by monitoring the conversion of [3H]arginine to [3H]citrulline during enzymatic formation of NO in tissue homogenates of myocardium of patients with, or without, ACE inhibitor treatment before elective coronary artery bypass grafting or heart transplantation. The mRNA expression (amol microg(-1) RNA) of endothelial NO synthase (eNOS) was higher (22.5 +/- 4.8, n = 23) in the atrial myocardium of patients taking ACE inhibitor treatment, before elective coronary artery bypass grafting, compared with patients not taking this therapy (8.9 +/- 0.7, n = 33, P < 0.0001). The ACE inhibitor therapy increased eNOS protein expression from [(9 +/- 0.7) relative units (RUs) to (12 +/- 0.9) RUs, P < 0.05, respectively] and cardiac NOS activity from 17.6 +/- 1.3 to 23.7 +/- 1.1 pmol mg protein(-1) min(-1) (P < 0.001, respectively). Inducible and neuronal NO synthase expression was not changed by the ACE inhibition. A similar up-regulation of eNOS by ACE inhibition was found in the left ventricles of patients with heart failure. The augmented endothelial NOS expression and activity was not the result of differences in clinical characteristics and concomitant therapy between the patient groups. Increased eNOS expression and activity might contribute to the beneficial effects of ACE inhibitor therapy in the treatment of CAD and heart failure.

In vivo expression of the heat stable ( estA) and heat labile (e ltB) toxin genes of enterotoxigenic Escherichia coli (ETEC)

Enterotoxigenic Escherichia coli (ETEC) colonize the intestine and adhere to the epithelium by means of different host specific colonization factors (CFs). Colonizing ETEC produce one or both of two enterotoxins; the heat stable (ST) and heat labile (LT) toxins which are both able to cause diarrhoea. The regulation of virulence genes in ETEC during infection of the human intestine is mainly unknown. In this study we analysed the level of mRNA expression of estA, coding for ST, and eltB, coding for the B subunit of LT, during human infection. The expressions of the toxins in ETEC strains expressing both ST and LT were investigated in bacteria isolated directly from patient stool without sub-culturing, (in vivo) and compared to the expression pattern of the corresponding ST/LT strains grown in liquid broth (in vitro) by quantitative competitive RT-PCR using fluorescent primers. We found that estA and eltB are expressed in the in vivo samples but no significant up-or down regulation of the expression levels of either estA or eltB could be determined in vivo as compared to in vitro.