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PubMed2004

Calcium influx pathways in rat CNS pericytes.

In central nervous system (CNS), pericytes have been proposed to play a role in broad functional activities including blood-brain barrier, microcirculation, and macrophage activity. However, contractile responses and Ca2+ signaling in CNS pericytes have not been elucidated. The aim of the present study is to investigate contractility and Ca2+ influx pathway in CNS pericytes. CNS pericytes were cultured from rat brain. Contraction of the pericytes in response to various stimuli was evaluated by the change in surface area measured by a light microscope with a digital camera. Reverse transcription and polymerase chain reaction (RT-PCR) was performed to examine the expression of mRNA of alpha-smooth muscle actin. Intracellular Ca2+ was measured using fura-2 fluorescence spectroscopy. A23187 (Ca2+ ionophore), high external K+ (4 x 10(-2) mol/l), endothelin-1, and serotonin induced contraction of CNS pericytes. RT-PCR analysis revealed the expression of alpha-smooth muscle actin in CNS pericytes. Cytosolic Ca2+ ([Ca2+]i) increased after application of high concentration of external K+, tetraethylammonium, and charybdotoxin, which was inhibited by nicardipine and removal of external Ca2+. Angiotensin-II, serotonin, acetylcholine, ATP, and endothelin-1 caused biphasic response in [Ca2+]i. In response to these agents, [Ca2+]i rapidly increased and then decayed to a relatively constant Ca2+ plateau. The Ca2+ plateau was partially inhibited by nicardipine and completely abolished by omission of external Ca2+. After intracellular Ca2+ store was depleted by the removal of external Ca2+ and addition of thapsigargin, reapplication of external Ca2+ evoked increases in [Ca2+]i. These results indicate that CNS pericytes express mRNA of alpha-smooth muscle actin and possess contractile ability. In CNS pericytes, resting membrane potential is regulated by large conductance Ca2+-activated K+ channels and Ca2+ enters into the cells via L-type voltage-dependent Ca2+ channels, agonist-activated Ca2+ permeable channels, and capacitative Ca2+ entry pathways.

Acetylcholine↗
PubMed2000

Mucosal human papillomavirus types in squamous cell carcinomas of the uterine cervix and subsequently on fingers.

Human papillomavirus (HPV), especially type 16, is causally involved in the pathogenesis of anogenital cancer. There is an increasing number of reports of HPV infections in squamous cell carcinoma (SCC) of the fingers. A search of the Swedish cancer register covering the period 1958-94 inclusive for women with a history of genital and upper extremity SCC revealed 63 cases. Archival material from both cervical and cutaneous lesions was traced and analysed for the presence of HPV DNA in 32 of these patients. A newly developed 'neighbour primer' polymerase chain reaction (PCR) for HPV 16 DNA, aimed at overcoming the obstacle of cross-linked target DNA, was shown to be superior to conventional general and type-specific HPV PCR tests. HPV DNA was significantly more frequently found in digital tumours than in tumours at other cutaneous sites of the upper extremities [67% (10 of 15) vs. 7% (three of 43); P < 0.001]. Among 13 patients with a history of both cervical and finger SCC, HPV 16 was found in cervical samples from seven patients. From five of these seven patients, HPV 16 was also present in the corresponding finger lesions. The results support the hypothesis of a possible transmission of patients' genital HPV infections to fingers.

Aged↗
PubMed2002

Aldosterone synthase (CYP11B2) expression and myocardial fibrosis in the failing human heart.

The pathway of tissue aldosterone production may exist in the heart, and may be an important contributory factor to myocardial fibrosis and cardiac remodelling in the failing heart. CYP11B2 (aldosterone synthase) catalyses the final step of aldosterone production. The aim of the present study was to determine whether CYP11B2 and CYP11B1 (11beta-hydroxylase) are expressed in myocardial tissues, and whether these enzymes contribute to collagen accumulation and myocardial dysfunction in the failing human heart. Endomyocardial tissues were obtained from 23 patients with chronic heart failure (CHF) and 10 controls. CYP11B2 and CYP11B1 mRNA levels were measured by real-time quantitative reverse transcriptase-PCR. The myocardial collagen volume fraction (CVF) was determined by digital planimetry. CYP11B2 mRNA expression was greater in the CHF group than in the controls (P<0.05), while CYP11B1 mRNA was barely expressed in either group. There was a positive correlation between CYP11B2 mRNA levels and CVF (r=0.64, P=0.001). CYP11B2 mRNA was particularly highly expressed in subgroups of CHF patients with a large left ventricular end-systolic diameter (>55 mm) or a low left ventricular ejection fraction (<30%). CYP11B2 mRNA expression and CVF were lower in a CHF subgroup treated with a combination of spironolactone and angiotensin-converting enzyme inhibitors (ACEIs) than in a subgroup not treated with these drugs. In conclusion, this study has shown that increased myocardial expression of CYP11B2 mRNA is associated with increased myocardial fibrosis and with the severity of left ventricular dysfunction in human CHF. In addition, CYP11B2 expression and cardiac fibrosis are found to be decreased in CHF patients on drug therapy comprising spironolactone combined with ACEIs.

Adult↗
PubMedDate not supplied

Quantitative radiographic imaging using a photostimulable phosphor system.

We have evaluated a photostimulable phosphor x-ray imaging system [Philips Computed Radiography (PCR) system] for use in quantification of x-ray exposure in diagnostic radiography. An exponential function was fitted to data yielding quantitative x-ray exposure values as a function of digital pixel values. We investigated several factors that affect the accuracy of exposure measurement using the PCR including repeatability, background noise as a function of time delay between plate erasure and use, sensitivity variation between different plates, nonuniformity of sensitivity within a plate, decay of the latent image between time of exposure and readout (observed as a change in sensitivity), and the accuracy with which the (exponential) calibration function yields exposure values as a function of digital pixel values. The calibration was performed over the exposure range from 5.1 X 10(-9) to 2.0 X 10(-5) C/kg (0.02-75 mR). The accuracy of exposure measurements made with a single imaging plate is between 1.6% and 4.2%. If measurements from several plates are involved, the uncertainty in the final measurement will be between 5% and 5.9%.

Evaluation Studies as Topic↗
PubMed2000

Characterization of cultured smooth muscle cells obtained from the palmar digital arteries of horses.

OBJECTIVE: To develop methods to isolate, culture, and characterize smooth muscle cells (SMC) from equine palmar digital arteries. SAMPLE POPULATION: Segments of the medial or lateral palmar digital arteries from the forelimbs of 6 horses. PROCEDURE: To obtain smooth muscle explants, arterial segments were incised longitudinally. The tunica intima was gently scraped from the underlying tunica media, and explants were obtained from the tunica media. Approximately 18 to 24 explants were obtained from each palmar digital arterial segment. A substrate-attached technique was used to initiate primary culture of SMCCultured cells were identified as SMC, using light microscopy, electron microscopy, reverse transcriptase-polymerase chain reaction (RT-PCR), and northern blot analysis. The replication index and serum dependence of equine SMC in culture was characterized by use of bromodeoxyuridine. RESULTS: The SMC of equine palmar digital arteries were successfully cultured, as confirmed by RT-PCR and northern blot analysis techniques for smooth muscle alpha-actin and detection of SMC-specific organelles during electron microscopy. When characterized by light and electron microscopy, SMC were found to have undergone phenotypic modulation to a more synthetic phenotype in culture while retaining characteristics of SMC. CONCLUSIONS AND CLINICAL RELEVANCE: Culture of SMC from equine palmar digital arteries via an explant protocol is a viable technique for studying vascular biological mechanisms in horses. In vitro studies of SMC may aid investigators in determining cellular mechanisms involved in disease processes such as laminitis.

Actins↗
PubMed1995

Cellular deformation reversibly depresses RT-PCR detectable levels of bone-related mRNA.

Osteoblastic cells respond to mechanical stimuli with alterations in proliferation and/or phenotypic expression. In some cases, these responses occur within only a few applications of stimuli (i.e. 'cycle-dependent trigger response') rather than in a dose-dependent manner. To explore potential mechanisms of the cycle dependent trigger response, we raised the following questions: (1) Does strain of bone cells alter gene expression; if so, how quickly does it occur and how long does it last? (2) Are alterations in message level strain magnitude dependent? (3) Are alterations in steady-state message levels cycle dependent? Cultures were evaluated for osteocalcin mRNA one week following a daily stretch application at four stretch magnitudes and four cycle numbers and compared to nonstretched controls. Steady state mRNA message was ascertained prior to and at 10, 20, 30, 60, 120, 180, and 240 min following initiation of stretch. Following mRNA isolation, first strand cDNA synthesis was performed and fluorometrically quantitated. A reverse transcriptase based PCR (RT-PCR) approach allowed assessment of osteocalcin mRNA levels from microcultures (50,000 cells per 10 microliters culture or 5000 cells mm2) of rat calvarial osteoblasts. Optimized PCR was performed using primers to the bone specific protein, osteocalcin (OC) and two 'housekeeping' genes, beta-actin and GAP-DH. PCR products were separated on 4% agarose gels and band intensities digitized with relative quantitation based on internal standards in each gel. The lowest magnitude of stretch (- 1 KPa) at 1800 cycles per day reproducibly depressed message for osteocalcin, but not beta-actin when assayed immediately following the cessation of strain application. By three hours following the initiation of stretch, message levels returned to control values. At the time of stretch cessation, the 1800 cycle stretch regimen diminished (p < 0.0001) steady-state osteocalcin message independently of the four stretch magnitudes. Stretch for 300 cycles failed to depress (p = 0.05) osteocalcin message cultures at any time, but 600 cycles depressed message by 30 min. By one and two hours, cultures stretch 600, 900, and 1800 cycles showed similar levels of message depression. Four hours following the initiation of stretch, message levels returning to nonstrained levels in all groups. We conclude that alterations in cell response to strain are in part mediated by gene expression, that alterations last 3-4 h in this system, and that the message mechanism itself exhibits a trigger-response dependency to cycle number.

Actins↗
PubMed2002

[Studies of DNA polymorphism at D7S21 locus in Hebei Han population].

To study the polymorphism at D7S21 locus in Hebei Han population, 124 unrelated individuals were detected rapidly by Minisatellite Variant Repeat-Polymerase Chain Reaction (MVR-PCR) and polyacrylamide gradient gel electrophoresis followed by silver staining,and digital codes were obtained. About 36 digital codes were obtained from each individual. No two unrelated individuals shared the same codes. The probability of identity in 36 digital codes was 3.48 x 10(-18). The percentage of three repeat units, a-type, t-type and 0-type was 48.5%, 49.4% and 2.1% respectively. The heterozygosity (H), excluding probability of paternity (EPP)and polymorphism information content (PIC) were 0.9876, 0.9746 and 0.9872 respectively. The results suggested that D7S21 locus has highly polymorphism in Hebei Han population. The method-polyacrylamide gradient gel electrophoresis followed by silver staining was simple,rapid and practical.

English Abstract↗
PubMed2005

Expression of lysyl oxidase and transforming growth factor beta2 in women with severe pelvic organ prolapse.

OBJECTIVE: To determine the mRNA expression of lysyl oxidase (LOX) and transforming growth factor beta2 (TGF beta2) in subjects with advanced pelvic organ prolapse as compared to asymptomatic controls. STUDY DESIGN: Seventeen women undergoing vaginal hysterectomy were recruited for the study. Nine women with grade > or = 3 pelvic organ prolapse by the Baden-Walker grading scale were recruited. Eight subjects without pelvic organ prolapse or urinary incontinence undergoing hysterectomy for other indications were recruited as asymptomatic controls. A 1-cm portion of the bilateral distal uterosacral ligaments was excised and immediately frozen at -80 degrees C. Total RNA was extracted, quantified and subjected to reverse transcription for cDNA synthesis. beta-Actin was used as the reference for the semiquantitative analysis. Known volumes of the polymerase chain reaction (PCR) products for each reaction were electrophoresed in agarose gels stained with ethidium bromide and photographed with a digital camera. The optical density for each sample was measured by using computerized image analysis software. The relative abundance of each specific message was calculated as the ratio of PCR product for LOX or TGF beta2 divided by the amount of PCR product for beta-actin from the same sample. RESULTS: The mean age of the study subjects was 55.7 years (38-67) versus 47.1 for the controls (44-54). Mean parity was 4.4 for the study subjects and 2.7 for the controls. Seventy-eight percent of the study subjects were postmenopausal, and 14% of the controls postmenopausal. All postmenopausal subjects had at least 1 month of estrogen therapy prior to surgery. The mean LOX/beta-actin ratio for the study group was 0.35 as compared to 0.72 for the control group. This was statistically significant with a p value of 0.049. The mean TGF beta2/beta-actin ratio for the study group was 0.12 as compared to 0.20 for the control group. This was not statistically significant (p = 0.46). CONCLUSION: Patients with severe pelvic organ prolapse (grade > or = 3) have a significant reduction in the mRNA expression of lysyl oxidase as compared to asymptomatic controls. The mRNA expression of TGF beta2 was not statistically different between the 2 groups. These findings lend further credence to the concept of specific biochemical changes in the pelvic floor in women with pelvic organ prolapse.

Adult↗
PubMed1994

Potential forensic applications of minisatellite variant repeat (MVR) mapping using the polymerase chain reaction (PCR) at D1S8.

Minisatellite variant repeat (MVR) mapping using the polymerase chain reaction (PCR) at D1S8 (MS32) was applied to samples from various human tissues. All DNA samples obtained from an individual's organs at autopsy consistently gave the same digital diploid codes. Even 1 ng of genomic DNA was sufficient to obtain authentic diploid MVR coding ladders. MVR-PCR could be reliably applied to DNA isolated from bloodstains, saliva stains, seminal stains and plucked hair roots, and should become a powerful tool for individual identification in forensic investigations.

Base Sequence↗
PubMed2005

Association of statin use with a pathologic complete response to neoadjuvant chemoradiation for rectal cancer.

PURPOSE: To assess whether 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, or statins, might enhance the efficacy of neoadjuvant chemoradiation in rectal cancer. METHODS AND MATERIALS: Between 1996 and 2001, 358 patients with clinically resectable, nonmetastatic rectal cancer underwent surgery at Memorial Sloan-Kettering Cancer Center after neoadjuvant chemoradiation for either locally advanced tumors or low-lying tumors that would require abdominoperineal resection. We excluded 9 patients for radiation therapy dose <45 Gy or if statin use was unknown, leaving 349 evaluable patients. Median radiation therapy dose was 50.4 Gy (range, 45-55.8 Gy), and 308 patients (88%) received 5-fluorouracil-based chemotherapy. Medication use, comorbid illnesses, clinical stage as assessed by digital rectal examination and ultrasound, and type of chemotherapy were analyzed for associations with pathologic complete response (pCR), defined as no microscopic evidence of tumor. Fisher's exact test was used for categoric variables, Mantel-Haenszel test for ordered categoric variables, and logistic regression for multivariate analysis. RESULTS: Thirty-three patients (9%) used a statin, with no differences in clinical stage according to digital rectal examination or ultrasound compared with the other 324 patients. At the time of surgery, 23 nonstatin patients (7%) were found to have metastatic disease, compared with 0% for statin patients. The unadjusted pCR rates with and without statin use were 30% and 17%, respectively (p = 0.10). Variables significant univariately at the p = 0.15 level were entered into a multivariate model, as were nonsteroidal anti-inflammatory drugs (NSAIDs), which were strongly associated with statin use. The odds ratio for statin use on pCR was 4.2 (95% confidence interval, 1.7-12.1; p = 0.003) after adjusting for NSAID use, clinical stage, and type of chemotherapy. CONCLUSION: In multivariate analysis, statin use is associated with an improved pCR rate after neoadjuvant chemoradiation for rectal cancer. The low prevalence of statin use limits the power to detect a significant difference at a type I error threshold of p = 0.05 in this analysis. Although no definitive conclusions can be drawn on the basis of this retrospective study, the unusually high incidence of pCR after chemoradiation suggests that the use of statins in the treatment of rectal cancer warrants further evaluation.

Adult↗
PubMed1996

Comparison of different electrophoretic methods for digital typing of the MS32 (D1S8) locus.

Minisatellite variant repeat mapping by polymerase chain reaction (MVR-PCR) is a new and powerful approach for individual identification from human DNA. This method has been successfully applied to the hypervariable human minisatellite MS32 (locus D1S8). In this work, we further investigate this locus using different electrophoretic techniques which include: agarose gels and nonradioactive hybridization, miniaturized polyacrylamide gels, followed by silver staining, and automatic detection of fluorescent labeled alleles. The advantages and disadvantages of each method are described. Also, MVR codes from a Galician population (NW Spain) are provided.

Chromosome Mapping↗
PubMed2004

Cardiac involvement in non-human primates infected with the Lyme disease spirochete Borrelia burgdorferi.

To investigate cardiac involvement in the non-human primate (NHP) model of Lyme disease, we inoculated 39 adult Macaca mulatta with Borrelia burgdorferi sensu stricto strains N40 (BbN40) by needle (N=22, 14 immunocompetent (IC), seven permanently immunosuppressed (IS), and four transiently immunosuppressed (TISP)) or by tick-bite (N=4, all TISP) or strain 297 (Bb297) by needle (N=2 IS), or with B. garinii strains Pbi (N=4, 2 TISP and 2 IS), 793 (N=2, TISP) or Pli (N=2, TISP). Five uninfected NHPs were used as controls. Infection and inflammation was studied in the hearts and the aorta removed at necropsy 2-32 months after inoculation by (1) H&E and trichrome-staining; (2) immunohistochemistry and digital image analysis; (3) Western blot densitometry; and (4) TaqMan RT-PCR. All NHPs inoculated with BbN40 became infected and showed carditis at necropsy. The predominant cells were T cells, plasma cells, and macrophages. There was increased IgG and IgM in the heart independent of immunosuppression. The B-cell chemokine BLC was significantly increased in IS-NHPs. There was increased deposition of the complement membrane attack complex (MAC) in TISP and IS-NHPs. The spirochetal load was very high in all BbN40-inoculated IS-NHPs but minimal if any in IC or TISP NHPs. Double-immunostaining revealed that many spirochetes in the heart of BbN40-IS NHPs had MAC on their membranes. We conclude that carditis in NHPs infected with B. burgdorferi is frequent and can persist for years but is mild unless they are immunosupressed.

Animals↗
PubMedDate not supplied

Quantification of thymidylate synthase gene expression in human gastrointestinal carcinoma tissues using competitive PCR.

BACKGROUND/AIMS: Thymidylate synthase (TS) is a target enzyme for 5-fluorouracil (5-FU). It is important to know the TS expression level in tumor tissue for chemotherapy. We evaluated the TS expression level using competitive polymerase chain reaction (cPCR), and estimated the reliability and reproducibility of this method. METHODOLOGY: TS expression was assessed by both Fluorodeoxyuridine-monophosphate (FdUMP) binding assay and cPCR in 9 human adenocarcinoma cell lines and 50 human adenocarcinoma tissues obtained by surgical resection. TS expression was also assessed by cPCR in nine biopsy specimens obtained before surgery. We synthesized TS and beta-actin competitors. Following cPCR, PCR products were quantified on ethidium bromide-stained gels using a digital image analyzer. TS mRNA/beta-actin mRNA ratio was used to determine relative TS expression level. RESULTS: The number of FdUMP binding sites on TS and TS mRNA/beta-actin mRNA ratio were significantly correlated in both cell lines and surgically resected specimens (p < 0.01). A linear regression line formed from the data points was obtained for TS mRNA/beta-actin mRNA ratios in surgical specimens versus TS mRNA/beta-actin mRNA ratios in biopsy specimens (p < 0.01). CONCLUSIONS: Assessment of TS expression by cPCR using our competitors was accurate and reproducible.

Adenocarcinoma↗
PubMed1999

An alternatively spliced form of CD79b gene may account for altered B-cell receptor expression in B-chronic lymphocytic leukemia.

Several functional anomalies of B-chronic lymphocytic leukemia (B-CLL) cells may be explained by abnormalities of the B-cell receptor (BCR), a multimeric complex formed by the sIg homodimer and the noncovalently bound heterodimer Igalpha/Igbeta (CD79a/CD79b). Because the expression of the extracellular Ig-like domain of CD79b has been reported to be absent in the cells of most CLL cases, we have investigated the molecular mechanisms that may account for this defect. Peripheral blood lymphocytes (PBL) from 50 patients and two cell lines (MEC1, MEC2) obtained from the PBL of one of them were studied. MEC1, MEC2, and 75% of CLL cases did not express detectable levels of the extracellular Ig-like domain of CD79b, which was nevertheless present in greater than 80% CD19(+) cells from normal donors. In healthy subjects the expression of CD79b was equally distributed in CD5(+) and CD5(-) B-cell subsets. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of CD79b RNA from all patients and from MEC1 and MEC2 cell lines consistently yielded two fragments of different size (709 bp and 397 bp). The 709-bp band corresponds to CD79b entire transcript; the 397-bp band corresponds to an alternatively spliced form lacking exon 3 that encodes the extracellular Ig-like domain. Both fragments were also visible in normal PBL. The expression of the 397-bp fragment was increased in normal activated B cells, while no difference was seen between CD5(+) and CD5(-) B cells. To obtain a more accurate estimate of the relative proportions of the two spliced forms, a radioactive PCR was performed in 13 normal and 22 B-CLL samples and the results analyzed using a digital imager. The mean value of the CD79b to the CD79b internally deleted ratio was 0.64 +/- 0.20 SD in normal donors and 0.44 +/- 0.27 SD in B-CLL (P =.01). Direct sequencing of 397-bp RT-PCR products and of genomic DNA corresponding to exon 3 from MEC1, MEC2, their parental cells, and five fresh B-CLL samples did not show any causal mutation. Single-strand conformation polymorphism analysis of exon 3 performed in 18 additional B-CLL cases showed a single abnormal shift corresponding to a TGT --> TGC polymorphic change at amino acid 122. We propose a role for the alternative splicing of CD79b gene in causing the reduced expression of BCR on the surface of B-CLL cells. As normal B cells also present this variant, the mechanism of CD79b posttranscriptional regulation might reflect the activation stage of the normal B cell from which B-CLL derives.

Antigens, CD↗
PubMed2001

Gender-specific difference in cardiac ATP-sensitive K(+) channels.

OBJECTIVES: The main objective of this study was to establish whether gender regulates expression and/or properties of cardiac ATP-sensitive K(+) (K(ATP)) channels. BACKGROUND: Recently, evidence has been provided that differing cardiac responses in males and females to metabolic stress may result from gender-specific difference(s) in the efficiency of endogenous cardioprotective mechanism(s) such as K(ATP) channels. METHODS: A reverse transcription polymerase chain reaction (RT-PCR) using primers specific for Kir6.2, Kir6.1 and SUR2A subunits was performed on total RNA from guinea pig ventricular tissue. Western blotting using anti-Kir6.2 and anti-SUR2A antibodies was performed on cardiac membrane fraction. Whole-cell, single-channel electrophysiology and digital epifluorescent Ca(2+) imaging were performed on isolated guinea pig ventricular cardiomyocytes. RESULTS: The RT-PCR revealed higher levels of SUR2A, but not Kir6.1 and Kir6.2, messenger RNA in female tissue relative to male tissue, while much higher levels of both Kir6.2 and SUR2A proteins in cardiac membrane fraction in female tissue compared with male tissue were found. In both male and female tissue, pinacidil (100 microM), a K(ATP) channel opener, induced outward whole-cell currents. The current density of the pinacidil-sensitive component was significantly higher in female tissue than it was in male tissue, while no differences in single K(ATP) channel properties between genders were observed. Ischemia-reperfusion challenge induced significant intracellular Ca(2+) loading in male, but not female, cardiomyocytes. To test the hypothesis that SUR2A expression is the limiting factor in K(ATP) channel formation, we took different volumes of Kir6.2 and SUR2A complementary DNA (cDNA) from the same cDNA pool and subjected them to PCR. In order to obtain a band having 50% of the maximal intensity, a volume of SUR2a cDNA approximately 20 times the volume of Kir6.2 cDNA was required. CONCLUSIONS: This study has demonstrated that female tissue expresses higher levels of functional cardiac K(ATP) channels than male tissue due to the higher expression of the SUR2A subunit, which has an impact on cardiac response to ischemia-reperfusion challenge.

Adenosine Triphosphate↗
PubMed2006

Differential gene expression in abdomens of the malaria vector mosquito, Anopheles gambiae, after sugar feeding, blood feeding and Plasmodium berghei infection.

BACKGROUND: Large scale sequencing of cDNA libraries can provide profiles of genes expressed in an organism under defined biological and environmental circumstances. We have analyzed sequences of 4541 Expressed Sequence Tags (ESTs) from 3 different cDNA libraries created from abdomens from Plasmodium infection-susceptible adult female Anopheles gambiae. These libraries were made from sugar fed (S), rat blood fed (RB), and P. berghei-infected (IRB) mosquitoes at 30 hours after the blood meal, when most parasites would be transforming ookinetes or very early oocysts. RESULTS: The S, RB and IRB libraries contained 1727, 1145 and 1669 high quality ESTs, respectively, averaging 455 nucleotides (nt) in length. They assembled into 1975 consensus sequences--567 contigs and 1408 singletons. Functional annotation was performed to annotate probable molecular functions of the gene products and the biological processes in which they function. Genes represented at high frequency in one or more of the libraries were subjected to digital Northern analysis and results on expression of 5 verified by qRT-PCR. CONCLUSION: 13% of the 1965 ESTs showing identity to the A. gambiae genome sequence represent novel genes. These, together with untranslated regions (UTR) present on many of the ESTs, will inform further genome annotation. We have identified 23 genes encoding products likely to be involved in regulating the cellular oxidative environment and 25 insect immunity genes. We also identified 25 genes as being up or down regulated following blood feeding and/or feeding with P. berghei infected blood relative to their expression levels in sugar fed females.

Abdomen↗
PubMed2006

A new method of competitive reverse transcription polymerase chain reaction with SYBR Gold staining for quantitative analysis of mRNA.

There are several methods available to analyze the mRNA concentration quantitatively. Among them, the competitive reverse transcription (RT-)PCR method is very useful. For this method, Cy5-labeled primers were used, and after gel electrophoresis in 7 M urea, the Cy5-labeled single-strand DNA was measured by a fluorescence detector. However, as the equipment to measure the Cy5-labeled fluorescence is expensive, we developed a new method using SYBR Gold staining. After gel electrophoresis in 7 M urea, the single-strand PCR product DNA was stained with SYBR Gold, and photographed with a standard UV-transilluminator and a standard digital camera with a specific filter for SYBR Gold staining. The photographic image was digitized by an imaging software. We measured beta-actin and plasma glutathione peroxidase (Gpx3) mRNA concentrations of HepG2 cell cultured at 5 and 20% oxygen tension. The Gpx3 expression was increased by hypoxia. The result was equivalent to the data obtained by the real-time PCR analysis.

Actins↗
PubMed2001

Fibroblast growth factors in regenerating limbs of Ambystoma: cloning and semi-quantitative RT-PCR expression studies.

Urodele amphibians (newts and salamanders) have the ability to regenerate amputated limbs throughout their life span. Because fibroblast growth factors (Fgfs) play important roles in developing limbs, we initiated studies to investigate these growth factors in regenerating limbs. Partial cDNAs of Fgf4, 8, and 10 were cloned from both the Mexican axolotl, Ambystoma mexicanum, and locally collected spotted salamander, Ambystoma maculatum, two salamanders well recognized for their regenerative capabilities. cDNAs from the two Ambystoma species were virtually identical, ranging from 97-100% nucleotide identity. Axolotl Fgf4, 8, and 10 showed nucleotide sequence identity with chick Fgf4, 8, and 10 of 79%, 83%, and 72%, respectively. RT-PCR showed that these growth factors are expressed in regenerating axolotl limbs as well as in developing salamander larvae at the three-digit forelimb stage. Fgf8 and 10 are upregulated during regeneration and thus may be involved in distal signaling similar to that of the developing chick limb. Fgf4, however, was undetectable by RT-PCR in the distal tips of regenerates, suggesting that it does not play the same role in limb regeneration that it does in limb development. We also investigated the role these Fgfs may have in the nerve-dependence of regeneration. They were expressed similarly in aneurogenic and innervated limbs, suggesting that they are not the neurotrophic factors responsible for nerve-dependence. Denervation prevented Fgf8 and 10 upregulation, suggesting Fgf pathways are downstream of nerve-dependence. These data highlight important similarities and differences in Fgf expression between limb development and limb regeneration. J. Exp. Zool. 290:529-540, 2001.

Ambystoma↗