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PubMed2007

A UspA2H-negative variant of Moraxella catarrhalis strain O46E has a deletion in a homopolymeric nucleotide repeat common to uspA2H genes.

Moraxella catarrhalis strains can express either a UspA2 protein or a UspA2H protein. The latter protein is encoded by a gene that possesses a homopolymeric nucleotide tract containing eight adenine (A) residues [i.e., a poly(A) tract] which is located near the 5' end. A spontaneous UspA2H-negative variant of M. catarrhalis strain O46E, designated O46E.U2V, was found to have a uspA2H poly(A) tract that contained seven A residues. Northern blot analysis of total RNA from the O46E parent strain revealed a readily detectable uspA2H mRNA transcript, whereas little or no uspA2H transcript was detectable in total RNA from the UspA2H-negative variant O46E.U2V. The 5' end of the uspA2H genes from both the O46E parent strain and the O46E.U2V variant were ligated to a promoterless lacZ gene to prepare translational fusions for use as reporter constructs. The level of beta-galactosidase activity expressed by the fusion construct containing eight A residues in its poly(A) tract was 200-fold greater than that obtained with the construct that had seven A residues. Site-directed mutagenesis of the 5' end of the uspA2H gene confirmed that translation was initiated at a GTG codon located 21 nucleotides (nt) upstream of the poly(A) tract. Primer extension analysis determined that the transcriptional start site of the uspA2H gene was located 291 nt upstream from the GTG translational start codon. This poly(A) tract was also found to be present in the uspA2H genes of other M. catarrhalis strains.

Amino Acid Sequence↗
PubMed1996

Expression of the gap junction gene connexin43 (Cx43) in preimplantation bovine embryos derived in vitro or in vivo.

In this study we have examined the presence of mRNA encoding connexin 43 (Cx43) in bovine embryos derived in vivo and in vitro. Cumulus-oocyte complexes, immature and matured oocytes liberated from cumulus cells, zygotes, 2-4-cell and 8-16-cell embryos, morulae, blastocysts and hatched blastocysts were produced in vitro from ovaries obtained from an abattoir using TCM 199 supplemented with hormones and 10% oestrous cow serum for maturation. Cumulus-oocyte complexes matured for 24 h were exposed to bull spermatozoa for 19 h and then cultured in TCM 199 supplemented with 10% oestrous cow serum to the desired developmental stage. Morulae and blastocysts derived in vivo were collected from superovulated donor cows. Total RNA was extracted from pools of 60-200 bovine oocytes or embryos using a modified phenol-chloroform extraction method and analysed by reverse transcriptase polymerase chain reaction. Before reverse transcription, aliquots of DNase-digested embryonic RNA were tested by polymerase chain reaction using bovine-specific primers to control for residual genomic DNA contamination. DNA-free, total RNA was reverse transcribed after preincubation with the Cx43 specific 3'primer. The resultant cDNA was amplified by polymerase chain reaction using Cx43 specific primers that define a 516 bp fragment of Cx43. The reverse transcriptase polymerase chain reaction product was verified by restriction enzyme analysis with Alu I and sequencing. Assays were repeated at least twice for each developmental stage and provided identical results between replicates. Cx43 transcripts were detected in bovine morulae and blastocysts grown in vivo. In contrast, whereas the early in vitro stages from cumulus-oocyte complexes to morulae expressed Cx43, blastocysts and hatched blastocysts did not have detectable concentrations of mRNA from this gene. Restriction enzyme cutting revealed three fragments of the predicted size (139, 177, 200 bp). The amplified product showed 100% identity with the published bovine genomic DNA sequence. Under our in vitro conditions the Cx43 gene either had never been activated, which would require that the maternal transcript was stable through early development, or embryonic gene expression that had been active was then terminated prematurely. The differences in transcription between bovine embryos derived in vivo or in vitro indicate that culture conditions affect gene expression. This affords a tool for the further optimization of in vitro production systems for bovine embryos and contributes towards physiological characterization by definition of transcription phenotype of bovine embryos produced in vitro.

Animals↗
PubMed1991

A de novo G to T transversion in a pro-alpha 1 (I) collagen gene for a moderate case of osteogenesis imperfecta. Substitution of cysteine for glycine 178 in the triple helical domain.

Cultured fibroblasts from a patient affected with a moderate form of osteogenesis imperfecta were defective for the synthesis of type I collagen molecules; about half of the alpha 1(I) chains contained a cysteine residue in the triple helical domain and a disulfide link formed when two mutant alpha 1(I) chains were incorporated into a type I collagen heterotrimer. The proband's parents were clinically and biochemically normal. The cysteine was localized within peptide alpha 1(I)CB8 between residues 170 and 200 of the triple helical domain using a chemical procedure with 2-nitro-5-thiocyanobenzoic acid (Tenni, R., Rossi, A., Valli, M., Mottes, M., Pignatti, P. F., and Cetta, G. (1990) Matrix 10, 20-26). Type I procollagen heterotrimers containing either one or two mutant chains showed (i) a slight abnormality in secretion from cells; (ii) a low degree of post-translational overmodifications; (iii) the same, but lower than normal, thermal stability. Total RNA was isolated from the proband's dermal fibroblast cultures, and cDNAs for pro-alpha 1(I) were prepared d using total RNA. A portion of cDNA, coding for the region encompassing residues 119-193 of alpha 1(I) triple helical domain, was amplified by polymerase chain reaction. A single base pair mismatch was identified by chemical cleavage of DNA.DNA heteroduplexes, indicating a possible substitution of a guanine in the triplet coding for glycine 178 or 181. The same unique mismatch was detected by chemical cleavage in about one-half of the molecules in heteroduplexes formed between patient's pro-alpha 1(I) mRNAs and a normal cDNA probe. The amplified products were cloned and sequenced, confirming the heterozygous nature of the patient and demonstrating the presence and the location of a missense mutation; a single T for G substitution was found in the first base of the triplet coding for residue 178 of alpha 1(I) triple helical domain, leading to a cysteine for glycine substitution. Allele-specific oligonucleotide hybridization to amplified DNA confirmed a de novo point mutation in the proband's genome. The findings in this patient are in accord with the phenotypic gradient model, which correlates the localization of the structural defect with the clinical outcome of osteogenesis imperfecta. The mutant protein has some properties that differ from the caused by the cysteine for glycine 175 substitution, suggesting a direct influence of the neighboring amino acids on the effects of the mutation.

Alleles↗
PubMed1994

Entactin gene expression in normal and fibrotic rat liver and in rat liver cells.

BACKGROUND: Entactin, a constituent of basement membranes, is a sulfated glycoprotein with binding sites for laminin, collagen type IV, fibronectin, and cell surfaces. As it is known that excess matrix deposition and sinusoidal basement membrane formation is a central characteristic of liver fibrogenesis, we investigated whether the entactin gene is expressed in normal and in damaged rat liver and which cell types are able to express this gene. In addition, we were interested in the cellular origin and time course of laminin synthesis, a matrix protein closely associated with entactin. EXPERIMENTAL DESIGN: Entactin gene expression was analyzed in normal, acutely and chronically damaged rat livers (CCl4-model) by immunohistochemistry and in situ detection of specific transcripts. Rat fat-storing cells (FSC) (Ito), hepatocytes, Kupffer cells, liver endothelial cells, arterial smooth muscle cells (SMC), and skin fibroblasts (SF) were isolated according to standard techniques. Entactin gene expression in cultured cells was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of immunoprecipitates, Northern blot analysis, and immunocytochemistry. RESULTS: In normal liver, entactin was detected in the vessel walls as continuous deposits and in a spot-like fashion along the sinusoids. Entactin was detectable among the cells of the inflammatory infiltrates of acutely damaged liver and in connective tissue septa, in the walls of newly formed vessels and bile ducts of fibrotic liver. Laminin distribution in the vessels was similar, but it was additionally present in the space of Dissé of damaged liver. By in situ hybridization, few entactin-positive cells were found in normal liver sections. Strongly positive cells were scattered over the injured parenchyma of acutely and chronically damaged livers. Northern blot analysis of total RNA extracted from normal and damaged liver tissue showed a distinct increase of entactin specific transcripts during development of fibrosis. Hybridization of total RNA from cultured FSC, hepatocytes, Kupffer cells, endothelial cells, SMC, and SF revealed entactin specific mRNA in FSC, SMC, SF, and endothelial cells; laminin mRNA was found in FSC and SF. Synthesis and secretion of both proteins were found in FSC, SMC and SF. Entactin and laminin gene expression increased in parallel to FSC during time in culture. CONCLUSIONS: Among the main liver cells, FSC show the highest entactin gene expression and might therefore play the dominant role in the synthesis of this protein in normal and fibrotic liver. However, endothelial cells and liver myofibroblasts could also contribute to entactin production.

Animals↗
PubMed1993

Regulation of the gene expression of preproenkephalin in the rat brain: influence of hemorrhage.

In the present study, we examined the expression of the gene encoding for the production of the opioid peptide enkephalin in specific brain regions during hemorrhage in the conscious rat. Rats were subjected to fixed-volume (8 ml/300 g) bleeding via jugular vein within 10 min. Total RNA was isolated from rat brain tissue using the hot phenol/chloroform method. Preproenkephalin transcript levels were quantified using Northern blot hybridization of total RNA with alpha-32P-labeled cDNA probe, and autoradiograms were scanned with a densitometer. The ppENK transcript in midbrain was significantly elevated following one and two hours of hemorrhage compared to control group (P < 0.01). The ppENK transcript in spinal cord was also significantly increased after two hours of hemorrhage, when compared with control group (P < 0.01). In the brainstem, ppENK transcript was significantly decreased after both 1 and 2 hr of hemorrhage compared with control values (P < 0.01). These results indicate that hemorrhage acutely changes levels of the transcript of ppENK in specific brain regions. It is possible that the changes in brainstem ppENK transcript may be a mechanism by which certain inhibitory effects of enkephalins on important brain cardiovascular regulatory centers are mediated.

Animals↗
PubMed1996

Amplification, cloning, and sequence comparison of the growth hormone gene for carp (Cyprinus carpio) by the polymerase chain reaction.

Total RNA was isolated from carp pituitary gland. The first strand cDNA was synthesized using oligo(dT) 12-18 as a primer, the total RNA as a template, and AMV reverse transcriptase. Next the polymerase chain reaction (PCR) was performed using the first strand cDNA as a template, the synthetic 29 oligonucleotides as primer and Taq DNA polymerase (94 degrees C, 60 s; 55 degrees C, 30 s; 72 degrees C, 50 s; 35 cycles). After PCR amplification, the products were cloned into an E. coli expression vector (PBluescript II KS+/-). The result of the sequence analysis and the restriction map shows that an open reading frame of the carp growth hormone gene contains 630 base pairs which code for a polypeptide of 210 amino acids including 22 amino acids of the signal peptide and 188 amino acids of the nature growth hormone. Nucleotide sequence and amino acid sequence of our carp growth hormone gene are the same as Koren's carp GH cDNA in the coded region. Compared with Chao's carp GH cDNA, the homology of nucleotide sequence and amino acid sequence for our carp growth hormone gene is 95.6% and 96.7%, respectively, in the coded region.

Amino Acid Sequence↗
PubMed1992

Demonstration and localization of growth hormone receptor in human skin and skin fibroblasts.

Clinical evidence suggests that skin is responsive to GH status in vivo. We sought to demonstrate the presence of GH receptors in human skin and in cultured skin fibroblasts using the techniques of immunohistochemistry and northern blotting. Human foreskin was obtained at surgery for preparation of sections and primary fibroblast cultures. Skin sections and fibroblast monolayers were immunostained using a monoclonal antibody which recognizes the hGH receptor (MAb 263). Positive immunoperoxidase staining was seen in all epidermal layers except the stratum corneum, in dermal sweat and sebaceous glands, and in dermal fibroblasts. In cultured fibroblasts capping of surface GH receptor was observed after aqueous formaldehyde fixation, whereas fixation in Carnoy's solution resulted in granular cytoplasmic staining. Fibroblast poly A+ RNA was prepared from cultured skin fibroblasts, separated by denaturing agarose gel electrophoresis, blotted onto nitrocellulose, and hybridized to a 32P-labeled, 847 base pair (bp) hGH receptor complementary DNA (cDNA) clone. Human liver and non-pregnant rabbit liver total RNA were used as controls. Fibroblast poly A+ RNA contained a single hybridizing species of approximately 5.2 kilobase. Human liver total RNA also contained a single hybridizing species of 4.9 kilobase. We have demonstrated the presence of GH receptor protein in human skin and growth hormone receptor mRNA and protein in cultured human skin fibroblasts. These observations suggest that GH may indeed have a direct role in modulating keratinocyte and fibroblast function.

Adolescent↗
PubMed1998

RNA extraction from virus-diseased sweet potato for reverse transcriptase polymerase chain reaction analysis.

Sweet potato virus disease (SPVD), which causes severe crop losses in Africa, is caused by a complex of sweet potato feathery mottle potyvirus (SPFMV) and sweet potato chlorotic stunt crinivirus (SPCSV). Extraction of pure RNA (for diagnosis by reverse transcriptase polymerase chain reaction [RT-PCR] methods) from diseased sweet potato proved to be difficult using five different total-RNA extraction procedures: (a) that of Chomczynski and Sacchi (1) (b) an adaptation of Lodhi et al., (2) and three commercially available kits (c) Clonsep (Clontech, Clontech Labs Inc, Palo Alto, CA; (d) RNeasy (Qiagen, West Sussex, UK); and (e) RNA isolator (Genosys Biotechnologies). Four of these methods (b-e) generated sufficient RNA, but it was unsuitable for RT-PCR amplification of SPFMV. When these RNA samples were treated with Wizard DNA extraction resin (Promega), the inhibitors of RT-PCR were consistently removed from three (b-d) of these four samples. Another deproteinating step was needed to allow RT-PCR amplification of sample e. The Wizard DNA column purification in conjunction with one of these total RNA extraction methods facilitates quick and reliable extraction of pure RNA for diagnostic purposes and might be suitable for similar problematic plant material.

Plant Diseases↗
PubMed2003

Determination of prostate specific antigen mRNA in peripheral blood by reverse transcriptase polymerase chain reaction and a simple chemiluminometric hybridization assay in a high-throughput format.

In recent years, the mRNA for prostate-specific antigen (PSA) has been investigated as a potential marker for molecular staging of prostate cancer. We report a simple, rapid, and sensitive assay protocol for the quantification of PSA mRNA in peripheral blood by using reverse transcriptase polymerase chain reaction (RT-PCR) and a chemiluminometric hybridization assay. A recombinant RNA internal standard (IS) that has the same size and primer binding sites as the PSA mRNA is included in the RT-PCR mixture. Total RNA from the sample is coextracted with a constant amount of IS RNA and subjected to RT-PCR. Amplified sequences are labeled with biotin during PCR by using a biotinylated upstream primer. The products are heat-denatured and hybridized with oligonucleotide-specific probes (for PSA and IS) that are immobilized in microtiter wells. Immobilization of oligonucleotide probes is achieved by adsorption of their conjugates with bovine serum albumin. The hybrids are measured using alkaline phosphatase-labeled streptavidin and a dioxetane chemiluminogenic substrate. The ratio of the luminescence values obtained for the PSA mRNA and the RNA IS is a linear function of the initial amount of PSA mRNA present in the sample prior to RT-PCR amplification. The linear range extended from 50 to 500,000 PSA mRNA copies, and the overall reproducibility of the assay, including RT-PCR and hybridization, ranged from 11.5 to 14.2%. Samples containing total RNA from PSA-expressing LNCaP cells give luminescence ratios that are linearly related to the number of cells in the range of 0.04-400 cells. The method was applied to PSA mRNA determination in peripheral blood of healthy individuals, patients with benign prostate hyperplasia, patients with prostate cancer, and patients with other types of localized cancer.

Female↗
PubMed2006

Preparation of a single prostate needle biopsy specimen for histological diagnosis and RNA analysis.

PURPOSE: When a prostate needle biopsy specimen is used for molecular study, a second specimen from the same area as the first one has previously been required for pathological diagnosis. However, we developed a method for obtaining substances such as RNA and we also made pathological diagnosis possible from a single needle biopsy specimen, improving reliability. MATERIALS AND METHODS: Prostate needle biopsy specimens from 118 patients were frozen in optimal cutting temperature compound. Tissue from the upper part of the frozen specimen was sectioned longitudinally for histopathological examination. The remainder of the specimen was placed in TRIzol reagent to extract total RNA for molecular biological investigation. RESULTS: Pathological diagnosis and total RNA extraction (1.6 to 32.7 microg) could be obtained from 1 needle biopsy specimen. When 2 specimens were obtained from the same area of the prostate, pathological diagnoses were discordant in 19% of the cases. CONCLUSIONS: We established a method for histopathological diagnosis in the prostate needle biopsy specimen used for molecular investigation. This single specimen method may facilitate molecular research in prostate cancer.

Aged↗
PubMed1982

Synthesis of transfer RNA in rat liver after acute and chronic aflatoxin B1 administration.

Synthesis of rat liver tRNA following acute and chronic administration of aflatoxin B1 was compared to protein synthesis. Twenty hours after acute administration of aflatoxin B1 total RNA synthesis was inhibited by 91% and tRNA synthesis by 77% while protein synthesis as assayed in vitro was inhibited by 75%. In the chronic (long-term) experiment at 7, 18 and 30 weeks after administration of aflatoxin B1 no inhibition of total RNA and tRNA synthesis was observed but protein synthesis in the cell-free system was inhibited by 16%. Thus, the inhibitory effects on protein synthesis observed in the long-term experiments are not correlated with the inhibition of tRNA synthesis.

Aflatoxin B1↗
PubMedDate not supplied

[Changes in the concentrations and synthesis of DNA and RNA in tissues during accelerated growth and development of rats].

Daily administration of low doses of caffeine to 5-30 day old rats increases their growth, mainly due to the increase in muscle weight and results in acceleration of maturation of vegetative functions. DNA and total RNA levels in skeletal muscles and liver increase together with the increase of incorporation of labeled uridine and lysin into total RNA and proteins. These findings indicate stimulation of protein synthesis in these tissues. Significant increase in the relative weight of the thymus, its DNA and RNA contents, as well as its uridine-14C uptake may indicate anabolic effects of this gland upon growth. In control and experimental animals, the level of DNA and RNA in the thymus is 5-10 times higher as compared to that in muscles, liver and heart.

Animals↗
PubMed1970

Control of deoxyribonucleic acid and ribonucleic acid synthesis in pyrimidine-limited Escherichia coli.

The effects of pyrimidine limitation on chromosome replication and the control of ribosomal and transfer ribonucleic acid syntheses were investigated. Chromosome replication was studied by autoradiography of (3)H-thymine pulse-labeled cells. Pyrimidine limitation did not affect the fraction of cells incorporating radioactive thymine during a short pulse, indicating that when growth is limited by the supply of pyrimidine, the time required for chromosome duplication increases in proportion to the time required for cell duplication. Control of ribosomal RNA and transfer RNA syntheses was examined by chromatographing cell extracts on methylated albumin kieselguhr columns. When growth was controlled by carbon-nitrogen limitation, the ratio of tRNA to total RNA remained roughly constant at growth rates above 0.5 doublings per hour. During pyrimidine limitation, however, the control of rRNA synthesis was apparently dissociated from the control of tRNA synthesis: the ratio of tRNA to total RNA increased as the growth rate decreased.

Autoradiography↗
PubMed2000

Antenatal dexamethasone administration increases fetal lung DNA synthesis and RNA and protein content in nitrofen-induced congenital diaphragmatic hernia in rats.

Antenatal glucocorticoids treatment has been shown to correct pulmonary immaturity. The thymidine analog bromodeoxyuridine (BrdU) is incorporated into S-phase cells and used as a marker of DNA synthesis. In this study, we investigated the effect of antenatal glucocorticoid administration on DNA synthesis and RNA and protein content in nitrofen-induced congenital diaphragmatic hernia (CDH) in rats to better understand the effect of antenatal glucocorticoids on CDH lung. The CDH model was induced in pregnant rats using nitrofen. Dexamethasone (0.25 mg/kg) was given on d 18.5 and 19.5 of gestation (term = 22 d). BrdU was administered 1 h before fetuses were killed on d 21, and detected by immunohistochemistry. DNA synthesis was evaluated by percentage of BrdU-incorporated nuclei (BrdU labeling index). Total RNA and soluble protein were extracted from another set of left lungs to measure RNA and protein content. BrdU labeling index and total RNA content were significantly decreased in CDH lung compared with control rats. Antenatal dexamethasone treatment significantly increased BrdU labeling index and RNA and protein content in the left CDH lung. Our findings of decreased DNA synthesis and decreased RNA and protein content in CDH lung suggest that lung growth and development are suppressed in hypoplastic CDH lung. Increased DNA synthesis and increased RNA and protein content in dexamethasone-treated CDH lung suggest that antenatal glucocorticoids may accelerate fetal lung growth and development in CDH.

Animals↗
PubMedDate not supplied

Early effect of cyclophosphamide on oncogene expression in vivo.

Cyclophosphamide (CP) is a widely used chemotherapeutic drug, with proven carcinogenic effects. Secondary tumours induced by CP are kidney tumours in humans and haemopoietic malignancies in rodents. Previous experiments have shown its effect on H-ras, c-myc and p53 gene expression in long term in vivo experiments. Our model was developed to analyse the events in the first 24 hours after the administration of CP in short term experiments. The expression of Ha -ras, c-myc and p53 was investigated in the target organs during and up to 24 hours after the administration, at 0.25, 0.5, 1, 6, 12 and 24 h. Since the majority of CP-induced tumours are leukemias and lymphomas in the CBA/Ca mouse model, RNA was obtained from the thymus and the spleen. The results show that p53 is strongly expressed in the thymus during the focused period. On the other hand, the samples were subjected to in situ hybridisation and compared with the results of in situ hybridisation of lung and liver samples. Comparing the results of total RNA and in situ hybridisation should prove useful if the total RNA signal is too weak or not detectable at all. The in situ hybridisation picture showed many positive cells without high expression of oncogenes. Further flow-cytometric studies are necessary to provide a full explanation of the mechanism of CP induced changes.

Animals↗
PubMed2000

In vitro IL-12 treatment of peripheral blood mononuclear cells from patients with leukemia or myelodysplastic syndromes: increase in cytotoxicity and reduction in WT1 gene expression.

Interleukin-12 (IL-12) has potent antitumor activities. We examined whether IL-12 enhanced the cytotoxicity of peripheral blood mononuclear cells (PBMNC) and decreased leukemia cells in 30 patients with leukemia or myelodysplastic syndromes (MDS): 12 acute myeloid leukemia (AML) (five in complete remission (CR) and seven in non-CR); six chronic myeloid leukemia (CML); and 12 MDS (three refractory anemia (RA), eight RA with excess of blasts and one chronic myelomonocytic leukemia). PBMNC from patients and five healthy volunteers were cultured at 5 x 10(5)/ml parallel with or without 100 units/ml of IL-12 for 3 days. Cytotoxicity of PBMNC against K562 cells was assessed by flow cytometry. To quantify the amount of leukemia cells, WT1 mRNA was measured by competitive reverse transcription polymerase chain reaction (RT-PCR), since WT1 mRNA is considered as a marker of minimal residual disease (MRD) in leukemia or MDS. The cytotoxicity of non-IL-12-treated PBMNC of 30 patients was 13.4+/-9.3% at the effector to target (E:T) ratio of 20:1, and significantly lower than that of normal subjects (25.7+/-8.4%). The cytotoxicity increased to 30.6+/-17.9% in the IL-12-treated PBMNC. WT1 mRNA in PBMNC of five healthy volunteers was less than 10(3) copies/microg of total RNA. Following the 3-day IL-12 treatment, mean WT1 mRNA of PBMNC was reduced from 10(4.8) to 10(4.2) copies/microg of total RNA in six CML patients, from 10(5.4) to 10(4.8) copies/microg in 12 MDS patients and from 10(5.0) to 10(4.2) copies/microg in five AML patients in CR, but not reduced in five of seven AML in non-CR. These results showed that IL-12 significantly enhanced PBMNC cytotoxicity and decreased the quantity of leukemia cells in PBMNC of most patients with MDS, CML and AML in CR. IL-12 might be of considerable benefit in the elimination of MRD in patients with hematological malignancies.

Adult↗
PubMed2006

Reduced gene expression of intestinal alpha-defensins predicts diarrhea in a cohort of African adults.

BACKGROUND: Human defensin (HD) 5 and HD6, both Paneth cell alpha-defensins, contribute to the antimicrobial barrier against intestinal infection. We have previously demonstrated that levels of both HD5 and HD6 mRNA were reduced in adults living in urban Zambia, compared with those in adults living in London. The aim of the present study was to determine, during 2 years of follow-up, whether alpha-defensin expression in Zambian adults is related to susceptibility to diarrhea. METHODS: We analyzed intestinal biopsy samples from a longitudinal cohort study conducted in 83 Zambian adults by quantitative reverse-transcription polymerase chain reaction, Western blotting, immunohistochemistry, and in situ hybridization, and we measured the incidence of diarrhea. RESULTS: Levels of HD5 and HD6 mRNA in Paneth cells varied between participants, over time, and seasonally and were strongly correlated with mucosal architecture. Gene expression was almost exclusively restricted to Paneth cells. The median (interquartile range) HD5 mRNA level was 6.0 (5.6-6.7) log10 transcripts/microg of total RNA among 18 participants who experienced diarrhea within 2 months after biopsy-sample collection, compared with 6.8 (6.2-7.3) log10 transcripts/microg of total RNA among 94 participants who did not (P=.006). A similar observation was made for HD6. CONCLUSIONS: These data indicate that intestinal alpha-defensin expression is dynamic and seasonal and suggest that susceptibility to intestinal infection is related to alpha-defensin expression.

Adult↗
PubMed2001

Quantitative analysis of mRNA amplification by in vitro transcription.

Effective transcript profiling in animal systems requires isolation of homogenous tissue or cells followed by faithful mRNA amplification. Linear amplification based on cDNA synthesis and in vitro transcription is reported to maintain representation of mRNA levels, however, quantitative data demonstrating this as well as a description of inherent limitations is lacking. We show that published protocols produce a template-independent product in addition to amplifying real target mRNA thus reducing the specific activity of the final product. We describe a modified amplification protocol that minimizes the generation of template-independent product and can therefore generate the desired microgram quantities of message-derived material from 100 ng of total RNA. Application of a second, nested round of cDNA synthesis and in vitro transcription reduces the required starting material to 2 ng of total RNA. Quantitative analysis of these products on Caenorhabditis elegans Affymetrix GeneChips shows that this amplification does not reduce overall sensitivity and has only minor effects on fidelity.

Animals↗