Agonist-mediated destabilization of human beta1-adrenergic receptor mRNA: role of the 3'' untranslated translated region

The objective of this study was to increase our understanding of the importance of members of the Streptococcus milleri (SM) group as respiratory pathogens, by studying the epidemiological and clinical features of thoracic infections caused by this group and comparing the epidemiology and prognosis of empyema caused by SM with cases of pneumococcal aetiology. The clinical histories and microbiology reports were reviewed in 27 cases of thoracic infection caused by SM over a period of 8 yrs. Cases of pneumococcal empyema that occurred during the same period were also analysed. Diagnoses were made of cases of empyema, including six with pneumonia and one with pulmonary abscess, three cases of pneumonia and two of mediastinitis. In 17 cases, SM was the only pathogen isolated. There was a history of instrument or surgical procedures on the digestive or respiratory tract in 59%. Secondary bacteraemia was documented in three cases. The treatment administered, a combination of antibiotics and surgery, was successful in 22 of 27 (81%) of cases. All strains were susceptible to penicillin. When the characteristics of the empyemas caused by monomicrobial SM infection were compared with those of pneumococcal aetiology from the same period of study, significant differences were found with respect to age, origin of the infection and the need for surgery. In conclusion, thoracic infections caused by Streptococcus milleri are largely pleural. They are polymicrobial in one-third of cases, commonly acquired in hospital and, in most patients, associated with major surgery and/or surgical procedures of the respiratory or digestive tract. The empyema frequently requires thoracotomy for complete resolution.     

Selenium regulation of classical glutathione peroxidase expression requires the 3' untranslated region in Chinese hamster ovary cells

Classical glutathione peroxidase (GPX) mRNA levels fall dramatically in selenium (Se)-deficient animals, but it is not known whether this mechanism is related to the mRNA 3' untranslated region (3'UTR) sequences that have been shown to direct Se incorporation. In this study, we used recombinant GPX constructs to investigate the role of the GPX 3'UTR in Se regulation of GPX mRNA levels in Chinese hamster ovary (CHO) cells. The CHO cells were transfected with GPX (pRc/GPX), GPX lacking the 3'UTR (pRc/Delta3'UTR) or the pRc/CMV vector alone, and GPX activity and GPX mRNA levels were determined in stable transfectants grown in low Se basal medium with a range of added Se concentrations. We identified two pRc/GPX transfectants with significantly elevated GPX activity levels compared with pRc/CMV transfectants. The elevated GPX expression did not dramatically shift the amount of Se that was sufficient for GPX activity to reach the Se-adequate plateau level (100 nmol/L added Se). As expected, GPX activity was not significantly different when pRc/Delta3'UTR transfectants were compared with pRc/CMV control transfectants. Among the wild type and transfected CHO cells, Se-deficient GPX activity levels averaged 35 +/- 5% of Se-adequate levels. Selenium-deficient levels of endogenous GPX mRNA as well as recombinant pRc/GPX mRNA averaged 54-58% of Se-adequate levels; 3-4 nmol/L added Se was sufficient for maximal GPX mRNA levels. In contrast, pRc/Delta3'UTR mRNA levels in the unsupplemented cells remained at Se-adequate levels and showed no distinct Se regulation. These studies demonstrate that the GPX 3'UTR is necessary for Se regulation of GPX mRNA levels in addition to its role in Se incorporation.     

Genetic variation in the 3' untranslated region of the neurofibromatosis 1 gene: application to unequal allelic expression

Neurofibromatosis type 1 (NF1) is a common genetic disorder caused by inactivation of neurofibromin, a protein capable of modulating signal transduction by activating Ras-GTPase activity. We have used cDNA cloning and Northern blot analysis to confirm the NF1 gene produces alternatively polyadenylated mRNAs with 3' untranslated regions (3' UTR) that show striking evolutionary conservation. Scanning of the 3'UTRs for genetic variation revealed three common sequence polymorphisms (> 30% heterozygosity), one less informative polymorphism (approximately 5% heterozygosity) and one rare variant (1/144 chromosomes). These differences were used to examine relative levels of expression of normal and mutant NF1 alleles in lymphoblast cell lines and in one case, autopsy tissue, from patients with NF1. Unequal allelic expression (up to 4-fold) was observed in a subset of both sporadic and familial NF1 cases. Where linkage phase could be determined, the allele segregating with the disorder displayed a relative reduction in expression. However, the magnitude of this effect was variable suggesting the operation of additional, non-genetic factors in determining the degree of relative expression of the mutant allele.     

Mutational analysis of two unstructured domains of the 5'' untranslated region of HCV RNA

Substance P (SP) has been suggested to regulate gonadotroph function both directly and indirectly in different species. In pigs, the possible role of hypothalamic and pituitary SP in the control of LH release has not been examined. Here, we investigated SP effects on basal and GnRH-stimulated LH secretion by porcine gonadotrophs in vitro, in both static and dynamic culture systems. SP concentrations of 100 nM or above stimulated LH release from monolayer cultures without affecting intracellular LH content. In the same cultures, SP potentiated GnRH (10 nM)-stimulated LH release and reversed the GnRH-induced decrease of gonadotroph LH stores. The GnRH, but not the SP, effect was completely blocked by the potent GnRH-receptor antagonist antide. In superfused pituitary fragments, three successive pulses of SP or GnRH also stimulated LH release, yet the combined administration of both factors did not result in a synergistic stimulation. These results demonstrate that SP acts directly on porcine gonadotrophs to stimulate LH release, and to maintain the levels of hormonal stores, through a GnRH receptor-independent mechanism. Furthermore, our findings suggest that continuous exposure of gonadotrophs to SP would potentiate GnRH-stimulated LH secretion, thus supporting a possible role of SP as modulator of porcine gonadotroph function.     

Ongoing mutation in MALT lymphoma immunoglobulin gene suggests that antigen stimulation plays a role in the clonal expansion

Indirect antigenic stimulation by H. pylori-specific T cells is implicated in the development of low-grade gastric lymphoma of mucosa-associated lymphoid tissue (MALT), however, the role of direct antigen stimulation is unknown. To study the role of direct antigen stimulation in MALT lymphomagenesis and its relationship with the pathogenesis of distinct pathological lesions, which represent different stages of the tumour progression, we cloned and sequenced the rearranged immunoglobulin (Ig) heavy chain gene in three low-grade (two from the lung, one from the stomach) and one high-grade (from the stomach) cases. In the low-grade gastric case, we studied the Ig sequence in primary as well as its disseminated and recurrent tumours. In the high-grade gastric case, we analysed the Ig sequence in tumour cell populations microdissected from the residual diffuse low-grade lesions, diffuse high-grade areas from follicles colonized by high-grade blasts. Compared with the published germline sequences, the heavy chain variable (VH) genes of three MALT lymphomas, in which the putative germline was identified, contained frequent somatic mutations, showing a much higher ratio of replacement/silent mutations in the complementarity determining regions (CDRs) than the framework regions (FRs). Ongoing mutation as indicated by intraclonal variation of the Ig sequence clearly existed in low-grade tumour including its dissemination and recurrence, but was not evident in high-grade tumour cell populations including those microdissected from independent colonized follicles. In addition, the germlines of VH genes used by the three MALT lymphomas are frequently found in autoreactive antibodies. Our results suggest that MALT lymphoma derives from postgerminal centre memory B cells, possibly autoreactive B cell clones, and that direct antigen stimulation may play an important role in the clonal expansion of low-grade MALT lymphoma.     

Helix 2 of the paired domain plays a key role in the regulation of DNA-binding by the Pax-3 homeodomain

Pax3 contains two structurally independent DNA-binding domains, a paired domain (PD) and a homeodomain (HD). Biochemical and mutagenesis studies have shown that both domains are functionally interdependent. In particular, it has been shown that the PD can regulate the DNA-binding specificity and dimerization potential of the HD. To delineate Pax3 protein segments that are involved in the regulation of HD DNA-binding, a series of chimeric proteins were created in which the HD and linker region were gradually replaced with corresponding sequences from a heterologous HD protein, Phox. Characterization of chimeric proteins by electrophoretic mobility shift analysis (EMSA) suggests that a portion of the linker region contributes to the functional interaction between the PD and HD. In addition, stepwise removal of sequences from the Pax3 PD was used to define regions within this domain that are involved in the regulation of HD DNA-binding. EMSA of these proteins in the context of the chimeric Pax3/Phox backbone provided two key findings: (i) the C-terminal subdomain of the PD does not play a major role in the regulation of HD DNA-binding and (ii) the N-terminal subdomain and, in particular, the second alpha-helix are essential for modulation of HD DNA-binding. Significantly, deletion of helix 2 was found to be sufficient to uncouple regulation of HD DNA-binding by the PD.     

Regulation of the stability of heat-stable antigen mRNA by interplay between two novel cis elements in the 3' untranslated region

The heat-stable antigen (HSA) is a costimulatory molecule for T-cell activation. Its expression is strictly regulated during lymphocyte development and differentiation. Recent studies using HSA-transgenic mice have demonstrated that this regulated expression is critical for normal development of T and B lymphocytes. However, the mechanisms that control the expression of HSA are largely unknown. HSA mRNA is comprised of a 0.23-kb open reading frame and a 1.5-kb 3' untranslated region (3'UTR). The function of the long 3'UTR has not been addressed. Here we investigate the role of the 3'UTR of HSA mRNA. We show that a 160-bp element, located in the region of nucleotides 1465 to 1625 in the 3'UTR of HSA mRNA, promotes RNA degradation and that this effect is neutralized by a 43-bp fragment approximately 1 kb upstream of the negative cis element. Both positive and negative cis elements in the HSA mRNA are distinct from other sequences that are known to modulate mRNA stability. These results provide direct evidence that the interplay between two novel cis elements in the 3'UTR of HSA mRNA determines cell surface HSA expression by modulating its RNA stability.     

The 3' untranslated region of manganese superoxide dismutase RNA contains a translational enhancer element

A redox-sensitive protein that binds to the 3' untranslated region (UTR) of manganese superoxide dismutase (MnSOD) RNA has been described previously [Fazzone, H., Wangner, A., and Clerch, L. B. (1993) J. Clin. Invest. 92, 1278-1281; Chung, D. J., and Clerch, L. B. (1997) Am. J. Physiol. 16, L714-L719]. In the present study, cross-competition gel retardation and RNase H assays were used to identify a 41-base region located 111 bases downstream of the stop codon as the 3' UTR cis element involved in protein binding. The base sequence of this region is approximately 75% conserved among the 3' UTRs of rat, mouse, cow, and human MnSOD mRNAs at approximately the same distance downstream of the stop codon. The role of this protein-binding region in RNA translation was assessed in an in vitro rabbit reticulocyte lysate system. Translation of MnSOD RNA from which the 3' UTR element was deleted decreased 60% compared with translation of MnSOD RNA containing the 3' UTR cis element. In the presence of a specific competitor oligoribonucleotide that inhibits MnSOD RNA protein-binding activity, translation of MnSOD RNA containing the 3' UTR was decreased by 65%. Thus, both the cis element and RNA protein-binding activity were required for more efficient translation of the MnSOD. An analysis of ribosomal profiles suggests the MnSOD RNA-binding protein participates in the formation of the translation initiation complex. When MnSOD RNA-binding activity was inhibited, initiation complex formation was decreased by 50%. From the data obtained in this study, we propose that the 3' UTR cis element of MnSOD through its interaction with MnSOD RNA-binding protein may function as a translational enhancer.     

Essential promoter elements are located within the 5'' untranslated region of human insulin-like growth factor-I exon I

The connection between work-related exposures and the onset of back injury or pain is complex and not clearly understood. This paper raises design issues related to the planning and conduct of cohort studies of industrial low back pain (or injury)(LBP), with care given to definition and measurement of exposure and outcome events. These issues include sample size, outcome definition, study biases, and practical considerations when seeking and maintaining company collaboration with a research effort. Without resolving these issues, the authors conclude: (1) cohort studies of worksite-based LBP are needed to elucidate the causal associations between work tasks and LBP onset, (2) both acute and cumulative exposures should be assessed as risk factors for low back injury or pain, and (3) attention should be paid to the planning of such studies and minimization of potential biases that can limit the validity of the results. These design issues will benefit researchers and companies engaged in the planning and conduct of cohort studies of industrial LBP.     

RNA-protein interactions within the 3 ' untranslated region of vimentin mRNA

Several functions have been attributed to protein binding within the 3'untranslated region (3'UTR) of mRNA, including mRNA localization, stability, and translational repression. Vimentin is an intermediate filament protein whose 3'untranslated sequence is highly conserved between species. In order to identify sequences that might play a role in vimentin mRNA function, we synthesized32P-labeled RNA from different regions of vimentin's 3'UTR and assayed for protein binding with HeLa extracts using band shift assays. Sequences required for binding are contained within a region 61-114 nucleotides downstream of the stop codon, a region which is highly conserved from Xenopus to man. As judged by competition assays, binding is specific. Solution probing studies of 32P-labeled RNA with various nucleases and lead support a complex stem and loop structure for this region. Finally, UV cross-linking of the RNA-protein complex identifies an RNA binding protein of 46 kDa. Fractionation of a HeLa extract on a sizing column suggests that in addition to the 46 kDa protein, larger complexes containing additional protein(s) can be identified. Vimentin mRNA has been shown to be localized to the perinuclear region of the cytoplasm, possibly at sites of intermediate filament assembly. To date, all sequences required for localization of various mRNAs have been confined to the 3'UTR. Therefore, we hypothesize that this region and associated protein(s) might be important for vimentin mRNA function such as in localization.     

Genomic organization of the human dystrophin gene across the major deletion hot spot and the 3'' region

The genomic organization of most of the human dystrophin gene has not been defined at single-exon level, owing to its enormous size (2300 kb). By taking advantage of a YAC-based restriction map of the gene previously constructed, we have localized individual dystrophin exons from 42 to 79 along the central and 3' regions of the gene. These data elucidate the general organization of this large portion of the gene (1250 kb) and, in particular, characterize the genomic region most frequently involved in deletion mutations responsible for Duchenne and Becker muscular dystrophies.     

Colon carcinoma cells blocked in polarization exhibit increased expression of carcinoembryonic antigen

Carcinoembryonic antigen (CEA) is an oncofetal protein whose regulation is poorly understood, although CEA is commonly expressed on many carcinoma cell types and enhances experimental metastases. The abundance of membrane-associated CEA was increased 3-fold when HD6 colon carcinoma cells were prevented from polarizing by culture for 3 days in low calcium medium. Polarization is an early event in HD6 cell differentiation, with the polarized cells forming a tight, laterally adherent monolayer by culture in normal calcium medium. Lateral adherence can occur because 3 days of culture in normal calcium medium increases expression of calcium-dependent intercellular adhesion proteins: a 35-fold increase in membrane abundance of LCAM and a 16-fold increase in membrane abundance of the desmosomal protein desmoglein I. Polarized HD6 cells exhibit low levels of CEA only at their apical luminal surface. Rounded, unpolarized HD6 cells do not exhibit increases in either LCAM or desmoglein I membrane expression, but express increased levels of CEA molecules throughout their cell surface, where they act as intercellular adhesion molecules, allowing unpolarized cells to form random cell to cell contacts. Cells cultured in low calcium medium form calcium-independent cell aggregates whose formation can be blocked by Fab' fragments of anti-CEA monoclonal antibody col-1. The familiar pattern of random, multilayered associations of tumor cells both in vitro and in xenographs in vivo may be due to intercellular adhesion mediated by CEA which is up-regulated and expressed throughout the cell surface of unpolarized tumor cells.     

An interaction between penton base and alpha v integrins plays a minimal role in adenovirus-mediated gene transfer to hepatocytes in vitro and in vivo

Studies in cultured cell lines have shown that adenovirus infection involves binding of adenovirus fiber to its cell surface receptor and binding of penton base to alpha v integrins. However, much less is known about the role of these interactions in cells that are targets for adenovirus-mediated gene transfer. Earlier work showed that hepatocytes are readily infected by adenovirus, making them an attractive target for gene therapy in several diseases. We found that addition of fiber protein blocked adenovirus infection of primary cultures of hepatocytes. This suggests an important role for fiber and its receptor. However, mutation of the integrin-binding motif in penton base did not inhibit infection of hepatocytes, even though the mutation impaired infection of HeLa cells. Hepatocytes had undetectable amounts of alpha v integrins on their cell surface and showed no specific adherence to vitronectin, the natural substrate of alpha v integrins. Adenovirus with an intact penton base enhanced infection of liver following intravenous injection, but only by three-fold as compared with virus in which the integrin-binding motif was disrupted. These studies suggest that interactions between cell surface integrins and penton base are not required for adenovirus infection of hepatocytes in vitro, but the interaction enhances infection to a small degree in vivo.     

Innervated liver plays an inhibitory role in regulation of food intake

BACKGROUND: With the onset of eating, the associated rise of dopamine in the lateral hypothalamus (LHA-DA) is thought to regulate quantity of food consumed per meal. Early release of LHA-DA induced by eating is facilitated by oronasal stimulation; we propose that the subsequent LHA-DA response induced by nutrients in the portal vein is dampened by the innervated liver. This was tested by measuring LHA-DA in normal rats: during parenteral feeding to bypass oronasal stimulation, while eating during parenteral feeding, and while eating only. METHODS: Rats had either total liver denervation or sham operation, with placement of a jugular vein catheter and LHA-DA microdialysis cannula. After a 3-week recovery period total liver denervated rats were randomized to parenterally fed, food only, and parenteral plus food groups each with sham-operated controls in which LHA-DA was measured. RESULTS: No difference in LHA-DA release in food only groups occurred between total liver denervated or sham-operated rats. A significantly higher rise in LHA-DA was observed in total liver denervated versus sham-operated rats in parenterally fed (129% +/- 4% versus 116% +/- 2%; p < 0.05) and parenteral plus food (151% +/- 4% versus 134% +/- 4%; p < 0.05) groups. CONCLUSIONS: In total liver denervation versus sham operation, an increase in LHA-DA release occurs during parenteral feeding and eating during parenteral feeding, suggesting that innervated liver inhibits LHA-DA release.