Transcriptase-polymerase Chain Reaction Amplification Research Articles

Presence and Localization of Neurotrophins and Neurotrophin Receptors in Rat Conjunctiva and their Role in Stimulating Goblet Cell Secretion

Binge-like ethanol exposure on postnatal day (PN) 4 induces a concentration dependent loss of Purkinje cells in the rat cerebellum. The mechanism of this ethanol-induced Purkinje cell vulnerability is not presently understood. Nevertheless, the specific timing of this vulnerability leads us to consider the neurotrophin system crucial to the regulation of neuronal development. Differentiation, maturation, and survival of Purkinje cells are shown to involve an intimate interaction between brain-derived nerve growth factor (BDNF) and neurotrophin-3 (NT3) acting primarily through their specific tyrosine-kinase (Trk) receptors. We believe that the specific ethanol vulnerability, and the timing of this vulnerability result from alterations in the BDNF-NT3 interplay. We hypothesize that disruption of TrkB and/or TrkC mediated neurotrophin communication is, in part, responsible for the ethanol-induced loss of Purkinje cells during development. The current study was undertaken to define the impact of ethanol exposure at the onset of ethanol vulnerability on the relative concentrations of mRNA encoding the neurotrophic factor receptors TrkB and TrkC. The reverse transcriptase (RT) polymerase chain reaction (PCR) amplification technique was used to identify the relative expression levels of mRNA specific to these receptors as well as the truncated TrkB receptor isoforms. We identify a specific decrease in overall TrkB receptor mRNA expression that is primarily a function of the TrkB-T2 receptor isoform. Concurrent decreases in mRNA specific to BDNF were also identified. No significant alterations to the expression of TrkC mRNA were found indicating that ethanol-exposure appears to act selectively on the BDNF communication system.

Predominance of rotavirus G9 genotype in children hospitalized for rotavirus gastroenteritis in Belgium during 1999–2003

Group A rotavirus genotypes G1, G2, G3 and G4 are the main etiological agents of infantile diarrhea. The G9 rotavirus has recently emerged as a fifth important genotype all over the world. To characterize the VP7 gene of group A rotaviruses from gastroenteritis patients admitted to the Gasthuisberg University Hospital, Leuven, Belgium, during 1999-2003. Rotavirus antigen was detected in stool specimens using an enzyme immunoassay. G-typing was performed by reverse transcriptase polymerase chain reaction (RT-PCR) amplification and sequencing of the complete VP7 gene. The genotype distribution varied markedly over the four rotavirus years in Belgium. In the 1999-2000 rotavirus year, G1 was the predominating type (72%), and G9 was present in 5% of the rotavirus-positive patients. In the 2000-2001 and 2002-2003 years, G9 appeared as the dominating strain (45% and 53%, respectively). In the 2001-2002 year, between two G9 epidemic years, G1 was dominating (66%) but G9 was still present in 24%. All the G9 isolates were combined with P[8] and shared a high gene sequence similarity (<3% sequence divergence on the nucleotide and amino acid level). Phylogenetic analysis of the VP7 genes revealed that our Belgian G9 strains clustered together with recent G9 strains from all over the world, distinct from the prototype G9 strains isolated in the 1980s. Our study indicates that although the first introduction of G9 isolates in the Belgian population was recorded in 1997, G9 strains were able to establish themselves quickly as the predominant genotype. The emergence of G9 as an important pathogen in both developing as industrialized countries necessitates the urgent consideration of the G9 moiety in rotavirus vaccines.

National epidemic of Lordsdale Norovirus in the UK.

In early 2002 reports of outbreaks of gastroenteritis reached unprecedented levels in the UK. Forty five Norovirus outbreaks were reported in January 2002. The objective of the study was to determine whether the outbreaks were Noroviral in origin and if so whether they represented a homogeneous or heterogeneous collection of Noroviruses by applying EIA and sequence analysis to representative faecal samples. Faecal specimens were collected during the week of highest incidence from 21 outbreaks in a variety of health care settings including hospitals and nursing homes. The outbreaks occurred in geographically distinct regions of the UK and samples were collected by reference laboratories in Glasgow, Manchester, Bristol and Southampton. The samples were all positive for Noroviruses by negative stain electron microscopy (EM) and Lordsdale virus (LV) EIA, therefore reverse transcriptase polymerase chain reaction (RT-PCR) amplification and nucleotide sequencing of the Norovirus RNA polymerase gene was performed on amplicons from samples of each of the 21 outbreaks to investigate the nature and extent of diversity. All samples were very closely related to the reference Lordsdale virus genome sequence. LV was first discovered during an hospital outbreak of gastroenteritis in Southampton General Hospital in March 1993. Noroviruses are a major cause of outbreaks of gastroenteritis in health care settings. LV is the predominant Norovirus in the UK and was detected in outbreaks that occurred during the national peak of gastroenteritis reports in January 2002.

Secreted brevican mRNA is expressed in the adult rat pituitary

Brevican is the most abundant chondroitin sulfate proteoglycan in the extracellular matrix (ECM) of the adult rat brain. It has been found only in the central nervous system (CNS). In this study, we found that secreted brevican transcript was detectable in the pituitary of both male and female adult rats by reverse transcriptase-polymerase chain reaction (RT-PCR) amplification. In posterior lobe of pituitary, pituicytes were heavily labelled. In anterior and intermediate lobes of pituitary, signals for brevican transcripts were observed in cells of various sizes. These data demonstrated that secreted brevican mRNA is expressed in the adult rat pituitary and brevican might not be a CNS-specific ECM.

Noninvasive detection of Simian immunodeficiency virus infection in a wild-living L'Hoest's monkey (Cercopithecus Ihoesti).

L'Hoest's monkey's (Cercopithecus Ihoesti) are believed to be naturally infected with a simian immunodeficiency virus (SIV), termed SIVIho, but only a handful of isolates, all derived from captive animals from the Democratic Republic of Congo (DCR), have thus far been characterized. Here, we report the noninvasive detection and molecular characterization of SIVIho in a wild L'Hoest's monkey from the Nyungwe Forest in Rwanda. Screening four L'Hoest's monkey fecal samples collected opportunistically as part of a larger noninvasive survey of SIV prevalence in Nyungwe National Park we identified one to be vRNA positive. Reverse transcriptase polymerase chain reaction (RT-PCR) amplification of a subgenomic pol fragment (598 bp) identified a new SIVIho strain (RW30) that differed from previously reported SIVIho isolates in 17-22% of its nucleotide sequence. In a phylogenic tree of partial Pol protein sequences, RW30 fell well within the SIVIho radiation, but was not particularly closely related to any of the other strains. These results provide the first direct evidence that L'Hoewst's monkeys harbor SIVIho in the wild, that infection is prevalent in different areas of the species' habitat, and that geographically diverse SIVIho strains cluster in a single group according to their species of origin. L'Hoest's monkeys represent the third primate species for which the utility of noninvasive SIV testing has been documented.

Localization of RNAs in oocytes of Eleutherodactylus coqui, a direct developing frog, differs from Xenopus laevis

Eleutherodactylus coqui develops directly on land to a frog. The large 3.5-mm oocyte of E. coqui has enough yolk to allow development without a feeding tadpole. In the smaller Xenopus laevis oocyte, 1.3 mm in diameter, mRNAs involved in germ layer formation, such as VegT and Vg1, are localized to the vegetal cortex of the oocyte. We hypothesized that an animal shift has occurred in the localization of the E. coqui Orthologs of VegT and Vg1 due to the large egg size. Through a combination of degenerate reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE), we cloned 1634 bp of EcVegT and 1377 bp of EcVg1. Northern blot analysis shows that the lengths of these transcripts are 2.5 kb and 1.3 kb, respectively. This result suggests that we have obtained the complete Vg1 transcript, although this transcript has an extremely short 3' untranslated region compared with X. laevis, 256 bp and 1268 bp, respectively. Zygotic expression of EcVegT closely resembles that of VegT, supporting their orthology. Radioactive RT-PCR and in situ hybridization demonstrated the presence of EcVegT and EcVg1 predominantly near the animal pole of the oocyte. RT-PCR showed that the animal blastomeres, formed from the first horizontal cleavage, inherit half of the EcVegT and EcVg1 transcripts, although they contain only about 1% of the embryo volume. Our results indicate major differences between the molecular organization of the eggs of X. laevis and E. coqui.

Coprocytobiology: on the nature of cellular elements from stools in the pathophysiology of colonic disease.

The gastrointestinal epithelium is known to undergo constant and rapid renewal resulting in millions of cells being shed into the fecal stream every day. The conventional wisdom was that these cells disintegrate upon exfoliation and will not survive the transit through the intestinal tract. In 1990, we (P.N.) made the discovery that a significant number of these cells remain intact and viable and that they can be isolated. The implications of this important discovery became apparent when we demonstrated that these cells are exclusively of colonic origin, are anatomically representative of the entire colon, and can be used for clinical investigations of disease processes. The term coprocytobiology (CCB) was coined to encompass the broad range of applications of this new technology. The somatic cell sampling and recovery (SCSR) process involves the isolation of exfoliated colonocytes from a small sample of stool ( approximately 1 g) collected and transported in a unique medium at ambient temperature, providing cells for the detection of a number of biomarkers of disease propensity. These exfoliated colonocytes express cytokeratins indicating epithelial lineage as well as colon-specific antigen. Over the years, the study of exfoliated colonocytes has provided striking new insights into the biology of colon cancer and inflammatory bowel disease, including detection of p53 gene mutations, reverse transcriptase polymerase chain reaction amplification, and identification of CD44 splice variants, neoplasia-associated specific binding of plant lectins, and expression of COX-2, the inducible form of cyclooxygenase. The functional diversity of cells isolated by SCSR is revealed by the demonstration of cell surface markers such as secretory component, IgA, and IgG on the one hand and the amplification and cloning of the human insulin receptor and the expression of the multidrug resistance gene mdr-1 on the other hand. This review portrays the immense potential of CCB as a powerful tool for investigating the pathophysiology of disease, identifying genetic variants in pharmacogenetics, assessment of mucosal immunity, and several other applications that use somatic cells.

Expression of wnt signaling molecules in the synovial membranes of rabbit ankle joints injected with Enterococcus faecalis cell fractions

The cell adhesion molecule β-catenin mediates the transduction of wnt signals to various downstream events such as gene expression, cell proliferation, and cell adhesion. In this study, the results of reverse transcriptase polymerase chain reaction (RT–PCR) amplification showed that wnt1 and β-catenin expression increased in response to E. faecalis, and that the increases in wnt1 and β-catenin activated transcription of cyclin D1. Immunohistochemistry also showed that stimulating wnt1 with E. faecalis cell fractions leads to the stabilization and accumulation of β-catenin in the synovial membrane. On the other hand, the results of RT–PCR showed overexpression of various inflammatory cytokines, including IL-1-β, TGF-β, TNF-α, and IL-6, in the synovial membrane of joints injected with E. faecalis cell fractions. These findings suggest that expression of cyclin D1 is strongly dependent on β-catenin/Tcf and has a direct effect on the proliferation of synoviocytes, such as IL-1-β and TGF-β, that leads to inhibition of Fas-antigen-mediated apoptosis of synovial cells.

EP2 Splicing Variants in Rhesus Monkey (Macaca mulatta) Epididymis1

The expression pattern of EP2 variants was examined in the rhesus monkey (Macaca mulatta). Using reverse transcriptase-polymerase chain reaction and rapid amplification of complementary cDNA protocols, 11 message variants were identified in rhesus epididymis, only three of which (EP2B, EP2C, and EP2E) have previously been reported. The most abundant variant found in human, EP2A, was not found in rhesus. Seven of the eight new rhesus EP2 variants (EP2J-EP2Q) use previously unidentified 5'-splicing sites in exon 3, and four variants use three previously unidentified exons whose counterparts are present in the human EP2 gene. Overall, 3 of the 11 variants, EP2C, EP2E, and EP2Q, code for beta-defensin-like peptides whose probable physiological role is to protect the male reproductive tract against microbial invasions. Because of the complex splicing pattern that causes some downstream exons to be read in any of the three reading frames, the N-termini of the other eight EP2 peptide variants consist of a partial beta-defensin motif with three cysteines, followed by amino acid sequences that have no recognizable homology to known proteins.

Molecular characterization of the cDNA and localization of the mRNA encoding the prohormone convertase PC5-A in the European green frog.

The structure and distribution of PC5-A, a prohormone convertase that is thought to be involved in post-translational processing of peptide hormone and neuropeptide precursors, have not been investigated in submammalian vertebrates. In the present study, we characterized the cDNA encoding PC5-A in the European green frog Rana esculenta. The frog PC5-A cDNA encodes a 913-amino acid protein that encompasses a 28-amino acid signal peptide, the Asp/His/Ser catalytic triad found in all serine proteinases of the subtilisin family, and two potential N-linked glycosylation sites located in a C-terminal cysteine-rich domain. Reverse transcriptase polymerase chain reaction amplification showed that PC5-A mRNA is expressed in various organs including the brain, spinal cord, pituitary, lung, liver, intestine, and testis, but not in the stomach and pancreas. The distribution of PC5-A mRNA in the frog brain was studied by in situ hybridization histochemistry. Intense expression was observed in the mitral cellular layer of the olfactory bulb, the nucleus of the diagonal band of Broca, the anterior preoptic area, and the suprachiasmatic and ventral hypothalamic nuclei. The expression pattern of PC5-A mRNA in the central nervous system of anuran amphibians was consistent with the implication of this prohormone convertase in the processing of various neuropeptide precursors.

Presence of circulating prostate cancer cells in African American males adversely affects survival

African-Americans (AAM) with prostate cancer are more likely to relapse than Caucasian-Americans (CAM) despite controlling for known prognostic factors. One explanation may be that micrometastatic disease in AAM behaves more aggressively than in CAM. We tested this hypothesis by comparing the reverse transcriptase polymerase chain reaction amplification of the Prostatic Specific Antigen-mRNA (RTPCR PSA-mRNA) results from peripheral blood samples of AAM and CAM with respect to disease outcome. We evaluated the peripheral blood of 246 consecutive patients at the time of radical prostatectomy. The RTPCR PSA-mRNA test for determination of circulating prostate cancer cells was performed. The results were stratified by races and correlated with standard clinico-pathological variables and disease free survival. 27% and 23% of AAM and CAM patients were RTPCR PSA-mRNA positive, respectively. The RTPCR PSA-mRNA status correlated with the pathologic stage in CAM but not in AAM, (p=0.05). There was no association with Gleason score, PSA level, or clinical stage with the RTPCR PSA-mRNA status in either group. AAM with organ-confined prostate cancer were marginally more likely to have circulating prostate cells than similarly staged CAM (24% vs. 17%). In AAM but not CAM who had prostate cancer, the RTPCR PSA-mRNA status correlated with and was an independent predictor of disease-free survival. Our data suggests that, though the likelihood of having circulating prostate cells is the same in AAM and CAM, the presence of circulating prostate cells in AAM is predictive of a worse outcome. This may partially explain the worse prognosis in AAM vs. CAM with clinically localized prostate cancer.

Expression of the bovine high affinity IL-12 receptor β2

Four fragments of the bovine IL-12 receptor β2 were sequenced following generation by reverse transcriptase polymerase chain reaction (RT-PCR) amplification of RNA from mitogen-activated bovine peripheral blood mononuclear cells (PBMC). Primers were based on sequences within regions of the human IL-12Rβ2 gene that displayed high levels of similarity with the mouse IL-12Rβ2 gene sequence. The amplified bovine IL-12Rβ2 fragments had 82–87% similarity at the nucleotide level with human IL-12Rβ2 and 70–88% similarity at the predicted amino acid level. Bovine IL-12Rβ2 gene expression was induced following culture of PBMC with Concanavalin A (Con A), with immobilized monoclonal antibody to CD3 or with human recombinant IL-12 p70 and correlated with interferon-γ (IFN-γ) production. Expression of bovine IL-12Rβ2 by PBMC was detected by 2 h of culture with Con A and sustained for at least 5 days when cultured with rHuIL-12. Expression, however, did not require cellular proliferation since IL-12 did not induce proliferation, although both Con A and anti-CD3 monoclonal antibody did do so. Addition of rHuIL-10 inhibited IFN-γ production without abrogating bovine IL-12Rβ2 gene expression.

Early postnatal ethanol exposure selectively decreases BDNF and truncated TrkB-T2 receptor mRNA expression in the rat cerebellum

Binge-like ethanol exposure on postnatal day (PN) 4 induces a concentration dependent loss of Purkinje cells in the rat cerebellum. The mechanism of this ethanol-induced Purkinje cell vulnerability is not presently understood. Nevertheless, the specific timing of this vulnerability leads us to consider the neurotrophin system crucial to the regulation of neuronal development. Differentiation, maturation, and survival of Purkinje cells are shown to involve an intimate interaction between brain-derived nerve growth factor (BDNF) and neurotrophin-3 (NT3) acting primarily through their specific tyrosine-kinase (Trk) receptors. We believe that the specific ethanol vulnerability, and the timing of this vulnerability result from alterations in the BDNF-NT3 interplay. We hypothesize that disruption of TrkB and/or TrkC mediated neurotrophin communication is, in part, responsible for the ethanol-induced loss of Purkinje cells during development. The current study was undertaken to define the impact of ethanol exposure at the onset of ethanol vulnerability on the relative concentrations of mRNA encoding the neurotrophic factor receptors TrkB and TrkC. The reverse transcriptase (RT) polymerase chain reaction (PCR) amplification technique was used to identify the relative expression levels of mRNA specific to these receptors as well as the truncated TrkB receptor isoforms. We identify a specific decrease in overall TrkB receptor mRNA expression that is primarily a function of the TrkB-T2 receptor isoform. Concurrent decreases in mRNA specific to BDNF were also identified. No significant alterations to the expression of TrkC mRNA were found indicating that ethanol-exposure appears to act selectively on the BDNF communication system.

Identification of rab7 as a Melanosome-Associated Protein Involved in the Intracellular Transport of Tyrosinase-Related Protein 1

The melanosome is a unique secretory granule of the melanocyte in which melanin pigments are synthesized by tyrosinase gene family glycoproteins. Melanogenesis is a highly regulated process because of its inherent toxicity. An understanding of the various regulatory mechanisms is important in delineating the pathophysiology involved in pigmentary disorders and melanoma. We have purified and analyzed the total melanosomal proteins from B16 mouse melanoma tumors in order to identify new proteins that may be involved in the control of the melanogenesis process. Melanosomal proteins were resolved by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis, a predominant spot (27 kDa with isoelectric point 5.8-6.4) was excised and digested with cyanogen bromide, and the fragments were sequenced. Synthetic oligonucleotide primers were synthesized corresponding to the peptide sequences, and reverse transcriptase polymerase chain reaction amplification of total RNA from B16 cells was carried out. Sequencing of one of the polymerase-chain-reaction-mediated clones demonstrated 80%-97% sequence homology of 200 bp nucleotide with GTP-binding proteins at the 3'-untranslated region. GTP-binding assay on two-dimensional gels of melanosomal proteins showed the presence of several (five to six) small GTP-binding proteins, suggesting that small GTP-binding proteins are associated with the melanosome. Among the known GTP-binding proteins with similar molecular weight and isoelectric point ranges, rab3, rab7, and rab8 were found to be present in the melanosomal fraction by immunoblotting. Confocal immunofluorescence microscopy showed that rab7 is colocalized with the tyrosinase-related protein 1 around the perinuclear area as well as, in part, in the perikaryon, thereby suggesting that rab7 might be involved in the intracellular transport of tyrosinase-related protein 1. Tyrosinase-related protein 1 transport was blocked by the treatment of B16 cells with antisense oligonucleotide to rab7. We suggest (i) that rab7 is a melanosome-associated molecule, (ii) that tyrosinase-related protein 1 is present in late-endosome delineated granules, and (iii) that rab7 is involved in the transport of tyrosinase-related protein 1 from the late-endosome delineated granule to the melanosome.

Na+/H+ and CI-/HCO3-antiporters of bovine pigmented ciliary epithelial cells.

Medical therapy of glaucoma commonly aims at slowing aqueous humor formation by the ocular ciliary epithelial bilayer, but underlying mechanisms are poorly understood. The first step in secretion is NaCl uptake from the stroma into the pigmented ciliary epithelial (PE) cell layer by electroneutral transporters. After crossing gap junctions into the nonpigmented ciliary epithelial (NPE) cell layer, solute is released into the aqueous humor. Published data have indicated that both paired Na+/H+ and Cl-/HCO3- antiporters and the Na+-K+-2Cl- symporter are involved in net uptake. The molecular identities of the paired antiporters have not been elucidated. We have studied continuously cultured bovine PE cells. Acid-activated 22Na+ uptake was inhibited by cariporide, EIPA (ethyl-isopropyl-amiloride) and amiloride, at concentrations characteristic of the NHE-1 isoform. Videomicroscopy of BCECF-loaded PE cells verified the presence of an EIPA-inhibitable Na+/H+ antiporter. Removing external Cl- also triggered an alkalinization, which was Na+-independent and could be inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). Application of hypotonicity followed by return to isotonicity triggered a regulatory volume increase, which was pharmacologically similar to the uptake mechanisms described for intact rabbit ciliary epithelium. Reverse transcriptase polymerase chain reaction (RT-PCR) amplification of RNA from the human ciliary body detected expression of the AE2 Cl-/HCO3- exchanger, but not of AE1, cAE3 or bAE3. Immunostaining of bovine PE cells also revealed the presence of AE2 epitope. We conclude that paired NHE-1 Na+/H+ and AE2 Cl-/HCO3- antiporters are important components in the initial step in aqueous humor formation.

Expression of beta-defensin genes by human salivary glands.

This study investigated expression of genes encoding human beta-defensins 1 and 2 by human salivary glands. Tissues from surgical biopsies were collected fresh onto ice and stored in liquid nitrogen. Total RNA was extracted using Trizol reagent and human beta-defensin messenger RNA detected by reverse transcriptase polymerase chain reaction amplification. DNA sequencing of amplified fragments, after ligation into pGEM-T Easy vector and transformation of competent Escherichia coli, confirmed identities of cloned fragments. Human beta-defensin 1 messenger RNA was detected in all 25 samples that generated amplifiable cDNA, as assessed using abl-specific primers. Three of 13 submandibular gland samples (two normal, one chronically inflamed), and 2 of 2 minor salivary gland samples (one normal, one chronically inflamed) expressed human beta-defensin 2 messenger RNA. All six parotid gland samples studied were negative for human beta-defensin 2 messenger RNA. Thus, human beta-defensin 1 gene expression occurred in all human major and minor salivary glands studied, whereas human beta-defensin 2 expression occurred only in a small number of gland samples.

Cytokine expression in B-CLL in relation to disease progression and in vitro activation.

Earlier, we reported an association between low in vitro and in vivo IL-1 and IL-6 production, decreased IL-1beta and IL-10 mRNA expression and B cell chronic lymphocytic leukemia (B-CLL) disease progression. We have now further investigated cytokine mRNA transcription in B-CLL cells and cytokine serum levels in B-CLL patients. Reverse transcriptase polymerase chain reaction (RT-PCR) amplification of tumor necrosis factor (TNFalpha), IFNgamma, IL-6 and BCGF was equally often seen in non-progressive and progressive patients. However, 4 out of 23 non-progressive cases expressed mRNA for IL-12 while no IL-12 expression was seen in 15 progressive patients. No IL-12 was found in sera or supernatants from in vitro stimulated B-CLL cells, whereas TNFalpha and IL-10 were detected in sera from 51 and 31 of 65 B-CLL patients, respectively. TNFalpha values were significantly high in sera from patients in stages III and IV with disease progression. TNFalpha and IL-10 were also detected in culture supernatants from in vitro stimulated B-CLL cells, whereas IFNgamma was undetectable in these cultures and rarely positive in serum. Although further investigations are required, our data and that from previous reports indicate that B-CLL-derived cytokines are involved in B-CLL disease progression.

Identification of a Large Region of Secondary Structure in the 3′-Untranslated Region of Chicken Elastin mRNA with Implications for the Regulation of mRNA Stability

Synthesis of aortic elastin peaks in the perinatal period and then is strongly down-regulated with postnatal vascular development. Our laboratory has previously shown that changes in elastin mRNA stability contribute to this developmental decrease in elastin production. Here we identify a large region of stable secondary structure in the 3'-untranslated region (3'-UTR) of chicken elastin mRNA. Reverse transcriptase polymerase chain reaction or polymerase chain reaction amplification of the 3'-UTR consistently resulted in products with an approximately 328-bp deletion from the central region of the 3'-UTR, suggesting the presence of secondary structure. The presence of this structure was confirmed by probing the 3'-UTR with RNases with selectivity for single- or double-stranded RNA. Gel migration shift assays using cytosolic extracts from 2-day old chicken aorta demonstrate specific binding of a cytosolic protein to riboprobes containing the 3'-UTR of elastin but not to riboprobes either corresponding to other areas of the message or containing the 3'-UTR but lacking the region of secondary structure. Binding of cytosolic protein was particularly prominent in aortic extracts from 2-day old chickens, a time when elastin message is stable, as compared with 8- and 15-week old chickens, when the elastin message is relatively unstable, suggesting that this region of secondary structure may play a role in developmental regulation of stability of elastin mRNA.

Placental expression of glucose transporter proteins 1 and 3 in growth-restricted fetal rats

Objective: The purpose of this study was to determine the effect of intrauterine growth restriction on the placental expression of glucose transporter proteins. Study Design: Intrauterine growth restriction was induced by bilateral uterine artery ligation in the pregnant rat at a gestational age of 19 days (term is 21.5 days). Maternal rats were killed and fetuses were delivered by hysterotomy on gestational days 20 and 21. Control fetuses from mothers that had been subjected to a sham operation were studied simultaneously. Glucose transporter protein 1 and glucose transporter protein 3 messenger ribonucleic acid was quantified by reverse transcriptase–polymerase chain reaction amplification. Glucose transporter protein 1 and glucose transporter protein 3 densities in placental membranes were also assessed by Western blotting and by immunohistochemical analysis. Results: Glucose transporter protein 1 messenger ribonucleic acid, expressed as a multiple of the matched sham control value, was unchanged on both days 20 and 21 of gestation. Glucose transporter protein 3 messenger ribonucleic acid was also unchanged. Western blotting demonstrated no change in expression of glucose transporter protein 1 or glucose transporter protein 3 on either day 20 or 21 of gestation. Immunohistochemical staining patterns for glucose transporter protein 1 and glucose transporter protein 3 on the syncytiotrophoblastic membranes were similar between the growth-restricted group and the sham control group. Conclusion: Placental expression of glucose transporter proteins in the pregnant rat is unchanged with uteroplacental insufficiency. (Am J Obstet Gynecol 1999;180:1017-23.)

Lack of DMBT1 expression in oesophageal, gastric and colon cancers.

Loss of sequences from human chromosome 10q has been reported in several different cancers. Recently, a second candidate tumour-suppressor gene, DMBT1, was identified in this chromosomal region. We studied the mRNA expression and homozygous deletion of this gene in human oesophageal, gastric and colon cancers. Reverse transcriptase polymerase chain reaction (RT-PCR) amplification demonstrated that 23 (53.5%) of 43 oesophageal, 5 (12.5%) of 40 gastric, and 4 (16.7%) of 24 colorectal cancer cases showed an apparent reduction in DMBT1 mRNA in tumour tissues compared with paired normal tissues. Twelve out of 15 oesophageal cancer cell lines also showed no expression. We next studied homozygous deletions within the DMBT1 gene in oesophageal cancers by using duplex PCR. Consequently, it was recognized in five (11.6%) of the primary tumours and two (13.3%) of the cell lines. These findings suggest that DMBT1 may act as a tumour-suppressor gene not only in brain tumours but also in gastrointestinal cancers, especially in oesophageal cancers. © 1999 Cancer Research Campaign