13S mRNA Research Articles

Simple purification of small RNAs from seeds and efficient detection of multiple microRNAs expressed in Arabidopsis thaliana and tomato (Lycopersicon esculentum) seeds

MicroRNAs (miRNAs) play critical roles in the development of animals and plants. Characterizing the stage- and tissue-specific expression of miRNAs that potentially regulate target transcription factor expression is becoming more important for understanding the regulatory mechanisms of critical events during plant development. A simple method for purifying small RNAs from seeds is described, as well as an efficient non-radioactive labelling system for making miRNA probes. In Arabidopsis thaliana seed extracts, low molecular-weight (LMW) RNAs (e.g. 5S rRNA, tRNA and miRNA) were separated from high molecular-weight (HMW) nucleic acids (e.g. 28S and 18S rRNA, mRNA and genomic DNA) by fractionation using isopropanol. HMW RNAs precipitated in 20% isopropanol, while most LMW RNAs remained in the supernatant. The purified LMW RNAs were used successfully for RNA gel blotting to detect miRNAs expressed in Arabidopsis and tomato (Lycopersicon esculentum) seeds. To increase the detection sensitivity of the microRNA probes, additional digoxigenin-labelled uridine triphosphates (UTPs) were incorporated into the miRNA probes by designing template oligo DNAs with three extra adenines (A) at each end of their sequence. These DNA oligomers were used to make double-stranded DNA templates for miRNA probe synthesis. This probe (termed AAAPLUS) exhibited stronger signals than normal probes. A technique was also developed to quickly screen expressed miRNAs in seeds using a miniblot system, which enabled simultaneous examination with multiple miRNA probes. This method provides a simple alternative to microRNA microarrays to identify the major miRNAs expressed in seeds.

Translation of Sindbis Virus 26 S mRNA Does Not Require Intact Eukariotic Initiation Factor 4G

The infection of baby hamster kidney (BHK) cells by Sindbis virus gives rise to a drastic inhibition of cellular translation, while under these conditions the synthesis of viral structural proteins directed by the subgenomic 26S mRNA takes place efficiently. Here, the requirement for intact initiation factor eIF4G for the translation of this subgenomic mRNA has been examined. To this end, SV replicons that contain the protease of human immunodeficiency virus type 1 (HIV-1) or the poliovirus 2A(pro) replacing the sequences of SV glycoproteins have been constructed. BHK cells electroporated with the different RNAs synthesize protein C and the corresponding protease at late times. Notably, the proteolysis of eIF4G by both proteases has little effect on the translation of the 26S mRNA. In addition, recombinant viable SVs were engineered that encode HIV-1 PR or poliovirus 2A protease under the control of a duplicated late promoter. Viral protein synthesis at late times of infection by the recombinant viruses is slightly affected in BHK cells that contain proteolysed eIF4G. The translatability of SV genomic 49S mRNA was assayed in BHK cells infected with a recombinant virus that synthesizes luciferase and transfected with a replicon that expresses poliovirus 2Apro. Under conditions where eIF4G has been hydrolysed significantly the translation of genomic SV RNA was deeply inhibited. These findings indicate a different requirement for intact eIF4G in the translation of genomic and subgenomic SV mRNAs. Finally, the translation of the reporter gene that encodes green fluorescent protein, placed under the control of a second duplicate late promoter, is also resistant to the cleavage of eIF4G. In conclusion, despite the presence of a cap structure in the 5' end of the subgenomic SV mRNA, intact eIF4G is not necessary for its translation.

Molecular identification and characterization of novel coronaviruses infecting graylag geese (Anser anser), feral pigeons (Columbia livia) and mallards (Anas platyrhynchos)

In light of the finding of a previously unknown coronavirus as the aetiology of the severe acute respiratory syndrome (SARS), it is probable that other coronaviruses, than those recognized to date, are circulating in animal populations. Here, the results of a screening for coronavirus are presented, using a universal coronavirus RT-PCR, of the bird species graylag goose (Anser anser), feral pigeon (Columbia livia) and mallard (Anas platyrhynchos). Coronaviruses were found in cloacal swab samples from all the three bird species. In the graylag goose, 40 of 163 sampled birds were coronavirus positive, whereas two of 100 sampled pigeons and one of five sampled mallards tested positive. The infected graylag geese showed lower body weights compared with virus-negative birds, suggesting clinical significance of the infection. Phylogenetic analyses performed on the replicase gene and nucleocapsid protein sequences, indicated that the novel coronaviruses described in the present study all branch off from group III coronaviruses. All the novel avian coronaviruses harboured the conserved s2m RNA structure in their 3' untranslated region, like other previously described group III coronaviruses, and like the SARS coronavirus. Sequencing of the complete nucleocapsid gene and downstream regions of goose and pigeon coronaviruses, evidenced the presence of two additional open reading frames for the goose coronavirus with no sequence similarity to known proteins, but with predicted transmembrane domains for one of the encoded proteins, and one additional open reading frame for the pigeon coronavirus, with a predicted transmembrane domain, downstream of the nucleocapsid gene.

Prolactin receptor mRNA expression in oocytes and preimplantation mouse embryos

Prolactin was first identified as an anterior pituitary lobe hormone, responsible for the regulation of mammary gland growth and development. Prolactin receptors have been localized in a number of peripheral tissues, including tissues involved in reproduction. Studies with knockout animals have shown that prolactin receptor deficient mice present reproductive defects, whereas prolactin promotes the developmental potential of preimplantation mouse and rat embryos in vitro. To better understand the role of prolactin in the process of reproduction and early embryo development in mice, the expression of the four transcript variants of prolactin receptor was examined in the first stages of mouse embryo development. Prolactin long receptor mRNA was expressed in all stages examined, that is in cumulus cells, oocytes, zygotes, 2-cell embryos, 4-cell embryos, morulae and blastocysts. Prolactin receptor type S1 mRNA was observed only in cumulus cells, while S2 mRNA was present in cumulus cells, oocytes, zygotes and 2-cell embryos. S3 mRNA was expressed only in cumulus cells and oocytes. These results indicate that different isoforms of prolactin receptors may be present in the various stages of mouse preimplantation embryo and may play an important role in the control of its growth and development.

230 EFFECT OF IN VITRO CULTURE TREATMENT AND DONOR EWE DIET ON IGF2R EXPRESSION IN FETAL TISSUES

In separate experiments, elevation of donor ewe plasma urea levels and IVC of embryos in serum have been associated with occurrence of large offspring syndrome (LOS). Fetuses displaying LOS also have abnormal expression of the imprinted gene IGF2R. The present study, therefore, examined the effect of variation in donor ewe nitrogen metabolism and embryo culture on fetal development. Zygote-donor ewes were offered a diet with either a 0% (LN) or 3% urea supplement (HN) for two weeks during which superovulation and artificial insemination (AI) took place. Zygotes were recovered 36 h after AI and cultured for 5 days in synthetic oviductal fluid (SOF) to which either steer serum (10%; SOFS) or albumin (0.4%; SOFA) was added. In vivo control (C) blastocysts were recovered (Day 5) from superovulated ewes offered diet LN. All embryos were transferred singly to recipient ewes; fetal tissues were recovered and snap-frozen at Day 125 of gestation. IGF2R expression was measured by semi-quantitative PCR in fetal heart and kidney and expressed as the ratio to 18S mRNA. Data were analyzed by ANOVA with donor ewe plasma urea-N prior to (preUN) and mean plasma urea-N during (meanUN) feeding as covariates. Correlation analysis was used to test relationships between variables. Heart IGF2R expression (see Table 1) was lower in SOFA than in SOFS fetuses (P = 0.003; preUN, P = 0.007), while kidney IGF2R expression was lower in HN than in LN fetuses (P = 0.04; preUN, P = 0.015; meanUN, P = 0.032). Overall the range of both heart and kidney IGF2R expression was greater for fetuses from cultured (1st/3rd quartiles; heart, 0.87–1.05; kidney, 0.77–1.10) than from C embryos (heart, 1.03–1.16; kidney, 0.84–1.00). In IVC fetuses, heart IGF2R expression was negatively associated with fetal body (r = −0.31; P = 0.003) and heart weight (r = −0.35; P = 0.008) whereas kidney IGF2R expression was not associated (r = −0.23; P = 0.089) with fetal body weight and not related to kidney weight. Heart and kidney IGF2R expression were only weakly associated (r = 0.26; P = 0.054). In conclusion, changes in IGF2R expression in both fetal heart and kidney were associated with donor ewe N status and IVC conditions. In this study, decreases in heart IGF2R expression were reflected in increased heart weight, but kidney IGF2R expression was not related to kidney weight. Table 1. Fetal (kg), organ weights (g), and IGF2R expression This study was funded by Defra.

The Structure of a Rigorously Conserved RNA Element within the SARS Virus Genome

We have solved the three-dimensional crystal structure of the stem-loop II motif (s2m) RNA element of the SARS virus genome to 2.7-Å resolution. SARS and related coronaviruses and astroviruses all possess a motif at the 3′ end of their RNA genomes, called the s2m, whose pathogenic importance is inferred from its rigorous sequence conservation in an otherwise rapidly mutable RNA genome. We find that this extreme conservation is clearly explained by the requirement to form a highly structured RNA whose unique tertiary structure includes a sharp 90° kink of the helix axis and several novel longer-range tertiary interactions. The tertiary base interactions create a tunnel that runs perpendicular to the main helical axis whose interior is negatively charged and binds two magnesium ions. These unusual features likely form interaction surfaces with conserved host cell components or other reactive sites required for virus function. Based on its conservation in viral pathogen genomes and its absence in the human genome, we suggest that these unusual structural features in the s2m RNA element are attractive targets for the design of anti-viral therapeutic agents. Structural genomics has sought to deduce protein function based on three-dimensional homology. Here we have extended this approach to RNA by proposing potential functions for a rigorously conserved set of RNA tertiary structural interactions that occur within the SARS RNA genome itself. Based on tertiary structural comparisons, we propose the s2m RNA binds one or more proteins possessing an oligomer-binding-like fold, and we suggest a possible mechanism for SARS viral RNA hijacking of host protein synthesis, both based upon observed s2m RNA macromolecular mimicry of a relevant ribosomal RNA fold.

Cardiac Enlargement in a Mouse Model of Intestinal Cancer

1233 Cancer-induced cachexia is a life-threatening wasting syndrome characterized by reduced body mass and decreased antioxidant defense systems. This condition is often associated with the activation of the immune system and also heart failure. PURPOSE: To characterize cardiac muscle remodeling in a cachexic mouse model induced by intestinal tumors. The APC+/− mouse used in this study has a high incidence of intestinal tumor formation. We hypothesized that these cachexic mice would have altered heart remodeling. METHODS: Female C57BL6 (CON, n = 7) and APCmin+/− (MIN, n = 7) mice, 6–8 months of age, were monitored for daily food intake. Animals were sacrificed and liver, heart, and tibias were excised. Liver catalase (CAT) activity was measured spectrophotometrically. Cardiac α-myosin heavy chain and 18S mRNA abundance were analyzed by RT-PCR. RESULTS: CON and MIN ate similarly (p>0.05), but MIN BW was decreased by 20.5% (p<0.05). MIN CAT was decreased 54% compared to CON. The heart weight:tibia length ratio was significantly increased 27% in MIN compared to CON (p<0.05). Cardiac α-myosin heavy chain, corrected with 18S and normalized to CON was decreased 40% in MIN (p = 0.045). CONCLUSIONS: Cachexic female APCmin+/− mice, as described by decreased body weight with normal eating behavior as well as decreased catalase activity, suffer from cardiac hypertrophy with altered remodeling. While the stimulus of cardiac enlargement in this cachexic model is not certain, blood volume expansion and atrial natriuretic peptide signaling may be important mechanisms for the induction of this cardiac enlargement. This study was funded by the Colorectal Cancer COBRE program from the National Institute of Health/USC.

Cardiac Enlargement in a Mouse Model of Intestinal Cancer

1233 Cancer-induced cachexia is a life-threatening wasting syndrome characterized by reduced body mass and decreased antioxidant defense systems. This condition is often associated with the activation of the immune system and also heart failure. PURPOSE: To characterize cardiac muscle remodeling in a cachexic mouse model induced by intestinal tumors. The APC+/− mouse used in this study has a high incidence of intestinal tumor formation. We hypothesized that these cachexic mice would have altered heart remodeling. METHODS: Female C57BL6 (CON, n = 7) and APCmin+/− (MIN, n = 7) mice, 6–8 months of age, were monitored for daily food intake. Animals were sacrificed and liver, heart, and tibias were excised. Liver catalase (CAT) activity was measured spectrophotometrically. Cardiac α-myosin heavy chain and 18S mRNA abundance were analyzed by RT-PCR. RESULTS: CON and MIN ate similarly (p>0.05), but MIN BW was decreased by 20.5% (p<0.05). MIN CAT was decreased 54% compared to CON. The heart weight:tibia length ratio was significantly increased 27% in MIN compared to CON (p<0.05). Cardiac α-myosin heavy chain, corrected with 18S and normalized to CON was decreased 40% in MIN (p = 0.045). CONCLUSIONS: Cachexic female APCmin+/− mice, as described by decreased body weight with normal eating behavior as well as decreased catalase activity, suffer from cardiac hypertrophy with altered remodeling. While the stimulus of cardiac enlargement in this cachexic model is not certain, blood volume expansion and atrial natriuretic peptide signaling may be important mechanisms for the induction of this cardiac enlargement. This study was funded by the Colorectal Cancer COBRE program from the National Institute of Health/USC.

Immunohistochemical localisation and mRNA quantification of P-gp in human term placentas - no impact of corticosteroid administration for preterm labor

P-glycoprotein (P-gp) is an energy-dependent efflux pump expressed in the syncythiotrophoblast of human placenta throughout pregnancy, suggesting a potential functional importance of Pgp during pregnancy. Human data are limited and the effects of maternal treatment with corticosteroids in case of preterm labor have not known. After maternal consent, placenta from term pregnancies (n=20) or term pregnancies treated with betamethasone for preterm labor (n=43) were studied. Betamethasone (12 mg twice at 24 hour interval) was administered between 22 and 34 week's gestation, interval between end of treatment and birth was 35 ± 26 days. Immunohistochemical localisation by a sandwich staining method with Dako 3522 and F4 Kamiya monoclonal antibodies in paraffin embedded human placenta showned that P-gp is located in the endothelial cells without evidence for expression in the syncythiotrophoblast. P-gp mRNA and the constititively expressed mRNA 18S) were measured by RT-PCR and results expressed as the ratio of Pgp over 18S mRNA. PgP was highly expressed in the placenta but Pgp/18S mRNA ratio was not different at term between normal pregnancies and pregnancies treated with betamethasone. A acute effect of betamethasone on P-gp expression cannot be rulled out, as the free interval between betamethasone and birth was more than three weeks and will be investigated by additional studies. Clinical Pharmacology & Therapeutics (2004) 75, P73–P73; doi: 10.1016/j.clpt.2003.11.277

Age-Dependent Expression of Advanced Glycation End Product Receptor Genes in the Human Heart

Background: Advanced glycation end products (AGEs) are formed by the reaction of sugars and NH₂ groups of lysine and arginine residues and have been shown to accumulate in tissues, including the heart, with normal ageing. The interaction of AGEs with their receptors is known to cause changes in cell function, leading, for example, to the production of pro-inflammatory cytokines and free radicals. Objective: This study investigated the gene expression of the five known AGE receptors: AGE-R1, AGE-R2, AGE-R3, the scavenger receptor II, and the receptor for AGEs (RAGE) in human heart tissue. Methods: Tissue samples were taken from the right cardiac auricles from three patient groups: children (2.4 ± 1.1 years), adults (45.3 ± 0.8 years) and elderly subjects (76.4 ± 0.4 years). Analysis of gene expression of the five AGE receptors was performed using the reverse transcription-polymerase chain reaction (RT-PCR) and 18S mRNA levels as loading controls. Results: Our results show an age-dependent upregulation of the genes for AGE-R3 and the scavenger receptor II, but a downregulation for RAGE and no significant differences for AGE-R1 and AGE-R2. Conclusion: This study supports a pathophysiological function for AGE receptors such as AGE-R3 and RAGE in the ageing heart.

Optimization of RNA yield, purity and mRNA copy number by treatment of urine cell pellets with RNA later

Background: We have shown that measurement of mRNA for cytotoxic attack proteins perforin and granzyme B in urinary cells is a noninvasive means of diagnosing acute rejection of human renal allografts. Urinary cell mRNA studies have yielded useful information in other patient populations such as patients with cancer. The isolation of sufficient and high quality ribonucleic acid (RNA) from urinary cells however is problematic. RNA later, an RNA stabilization solution, has been reported to optimize RNA isolation from tumor tissues stored at room temperature and from pigment-rich ocular tissues. Methods: We explored whether the addition of RNA later to urine cell pellets improves RNA yield, enhances purity and facilitates measurement of low abundance mRNAs. We measured, with the use of real-time quantitative polymerase chain reaction (PCR) assay, levels of expression of a constitutively expressed gene 18S rRNA and mRNA for granzyme B and transforming growth factor-β 1 (TGF-β 1) in urine specimens and renal biopsies obtained from renal allograft recipients. Results: RNA yield ( P<0.01, Wilcoxon signed rank test) and the A260/ A280 ratio ( P<0.01) were both higher with urine cell pellets treated with RNA later prior to snap freezing compared to cell pellets that were not treated with RNA later prior to snap freezing. Levels (copy number per 1 μg of total RNA) of 18S rRNA ( P<0.02), granzyme B mRNA ( P=0.002) and TGF-β 1 ( P=0.02) were all higher with treated urine cell pellets compared to untreated cell pellets. Kruskall–Wallis one way analysis of variance and pair-wise comparisons with Student–Newman–Keuls test showed that the levels of mRNA for granzyme B ( P<0.05) and TGF-β 1 ( P<0.05) are significantly different between renal allograft biopsies and untreated urine cell pellets but not between the biopsy specimens and RNA later-treated urine cell pellets. Conclusions: The addition of RNA later to urine cell pellets improves RNA isolation from urinary cells and facilitates measurement of low abundance mRNAs.

Changes in the expression of calcitonin receptor-like receptor, receptor activity-modifying protein (RAMP) 1, RAMP2, and RAMP3 in rat uterus during pregnancy, labor, and by steroid hormone treatments.

Calcitonin gene-related peptide (CGRP) and its related peptide, adrenomedullin (AM), are potent smooth muscle relaxants in a variety of tissues. The CGRP has been reported to play an important role in maintaining uterine relaxation during pregnancy. We have previously reported that CGRP-induced uterine relaxation was gestationally regulated. Calcitonin receptor-like receptor (CRLR), a seven-domain transmembrane protein functions as CGRP-A receptor, in association with receptor activity-modifying protein (RAMP) 1, a single-domain transmembrane protein, whereas CRLR and RAMP2 or RAMP3 constitute a receptor for AM. In the present investigation, we examined the mRNA expression of CRLR, RAMP1, RAMP2, and RAMP3 in rat uterus (n = 8) by reverse transcriptional analysis and polymerase chain reaction to assess the changes in the expression of CGRP-A- and AM-receptor components during pregnancy and labor and by steroid hormone treatments in adult ovariectomized rats. The changes in mRNA are expressed relative to the 18S mRNA in the uterus of rats at various stages: nonpregnant, pregnant on Day 18, spontaneous labor at term, Day 2 postpartum, and in pregnant rats on treatment with RU486. Ovariectomized rats treated for 3 days twice daily s.c. with estradiol-17beta (2.5 microg/injection), progesterone (2 mg/injection), and the combination of estradiol-17beta and progesterone (same doses as above) were also examined for the expression of various receptor components. Results showed that mRNA expression of the receptor components was significantly higher (P < 0.001 for CRLR, P < 0.01 for RAMP1, P < 0.05 for RAMP2, and P < 0.01 for RAMP3) in pregnant compared to nonpregnant rats. Except for RAMP3, expression of all the other three genes decreased significantly (P < 0.05) during labor. A progesterone antagonist, RU486 significantly decreased (P < 0.01 for CRLR, P < 0.05 for RAMP1, RAMP2, and RAMP3) all the receptor components during pregnancy. In adult ovariectomized rats, progesterone caused significant increases in CRLR (P < 0.001), RAMP1 (P < 0.05), and RAMP2 (P < 0.01). Levels of RAMP3 were unaffected by the progesterone treatment. Estradiol-17beta treatment decreased all of the four receptor components significantly (P < 0.01 for CRLR, P < 0.05 for RAMP1, RAMP2, and RAMP3). Our results demonstrate that both CGRP and AM may play a role in uterine quiescence during pregnancy and that their receptor components are regulated by the steroid hormones.

The adenoviral E1A induces p21 WAF1/CIP1 expression in cancer cells

The adenovirus-5 E1A gene encodes two main proteins of 289 and 243 amino acid residues from 13S and 12S mRNA, respectively. The E1A gene products function as transcriptional regulators and have anti-tumor activities. Despite the fact that E1A gene therapy has been tested in clinical trials, the molecular mechanism by which it suppresses tumor cell growth is still not completely understood. Here, we show that E1A increases the expression of the cyclin-dependent kinase (CDK) inhibitor p21 WAF1/CIP1, which inhibits cell growth. We further show that 13S E1A, but not 12S E1A, can transactivate the p21 promoter through Sp1 sites. Interestingly, the E1A-induced transactivation occurs only in cancer cells, not in normal cells. This study provides new insight into the links between E1A and the CDK inhibitor and may have important clinical implications.

Identification of ribosomal proteins S2 and L10a as tumor antigens recognized by HLA-A26-restricted CTL.

Recent identification of cytotoxic T lymphocyte (CTL)-directed peptides binding to the HLA-A2 and -A24 alleles has opened the door to peptide-based cancer immunotherapies. However, subsequent studies have succeeded in identifying no more than a few CTL-directed peptides that bind to alleles other than HLA-A2 and -A24, thus hampering development of immunotherapies directed at other alleles. We have shown in this study that two genes coding for ribosomal proteins (S2 and L10a) encoded tumor antigens recognized by HLA-A26-restricted CTLs. The S2 mRNA was expressed in all of the cancer cells and non-malignant cell lines tested, but was not expressed in normal tissues except for the testis, muscle, and peripheral mononuclear leukocyte cell (PBMC). In contrast, the L10a mRNA was expressed in all of these cancer and non-malignant cell lines, and also normal tissues, although the expression levels in normal tissues were mostly low. One S2-derived peptide and two L10a-derived peptides had the ability to induce HLA-A26-restricted and peptide-specific CTLs reactive to tumor cells in PBMCs of cancer patients, respectively. These ribosomal protein-derived peptides, and particularly the S2-derived peptide, could be suitable for use in peptide-based immunotherapy for HLA-A26+ cancer patients.

Trypanosoma cruzi: allelic comparisons of the actin genes and analysis of their transcripts

Two allelic genomic fragments containing actin genes from Trypanosoma cruzi were isolated and characterized. One allele comprises two complete tandem repeats of a sequence encoding an actin gene. In the other, only one actin gene is found. Each one of these three gene copies encode for a complete and identical potential protein of 376 amino acids which is 93% similar with its homolog from Trypanosoma brucei. Northern hybridizations of both total and polysomal RNA from epimastigotes demonstrated the presence of an actin polyadenylated mRNA of about 1.6 kb. Actin transcripts processing sites were determined by 5 ′- and 3 ′-RACE. The obtained sequence data demonstrates that actin genes from both alleles are expressed. The stability of actin mRNA was found to be similar to the one exhibited by the ribosomal protein S4 mRNA as an internal reference. A time course analysis of cultured epimastigotes showed a novel behaviour in which actin mRNA steady state concentration peaks during the transition from the logarithmic to the stationary phase of growth. Index Descriptors and Abbreviations: cDNA, complementary DNA; EDTA, ethylenediaminetetraacetic acid; mRNA, messenger RNA; ORF, open reading frame; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; RNase, ribonuclease; SIRE, short interspersed element; ssrRNA, small subunit ribosomal RNA; UV, ultraviolet; UTR, untranslated region

The role of caspases 9 and 9-short (9S) in death ligand- and drug-induced apoptosis in human astrocytoma cells

Caspases are essential components of the mammalian cell death machinery. Caspase 9 is positioned at the apex of the pro-apoptotic signaling cascade induced by cytochrome c release from mitochondria. The splicing variant caspase 9-short (caspase 9S) lacks the catalytic domain and may therefore act as an endogenous inhibitor of caspase 9. Here, we report that human astrocytoma and neuroblastoma cell lines strongly express caspase 9 and 9S mRNA, whereas non-neoplastic human astrocytes show little caspase 9 and 9S mRNA expression. Transient overexpression of caspase 9S protects LN-229 astrocytoma cells from CD95 ligand (CD95L)-mediated apoptosis. However, stable overexpression of either caspase 9 or caspase 9S does not alter the sensitivity of LN-18 and LN-229 astrocytoma lines to CD95L or cytotoxic drugs. We conclude that the expression levels of caspase 9 or caspase 9S do not play a major role in determining vulnerability to apoptosis in human astrocytoma cells.

Alphavirus minus-strand RNA synthesis: identification of a role for Arg183 of the nsP4 polymerase.

A partially conserved region spanning amino acids 142 to 191 of the Sindbis virus (SIN) nsP4 core polymerase is implicated in host restriction, elongation, and promoter recognition. We extended the analysis of this region by substituting Ser, Ala, or Lys for a highly conserved Arg183 residue immediately preceding its absolutely conserved Ser184-Ala-Val-Pro-Ser188 sequence. In chicken cells, the nsP4 Arg183 mutants had a nonconditionally lethal, temperature-sensitive (ts) growth phenotype caused by a ts defect in minus-strand synthesis whose extent varied with the particular amino acid substituted (Ser>Ala>Lys). Plus-strand synthesis by nsP4 Arg183 mutant polymerases was unaffected when corrected for minus-strand numbers, although 26S mRNA synthesis was enhanced at the elevated temperature compared to wild type. The ts defect was not due to a failure to form or accumulate nsP4 at 40 degrees C. In contrast to their growth in chicken cells, the nsP4 Arg183 mutants replicated equally poorly, if at all, in mosquito cells. We conclude that Arg183 within the Pro180-Asn-Ile-Arg-Ser184 sequence of the SIN nsP4 polymerase contributes to the efficient initiation of minus strands or the formation of its replicase and that a host factor(s) participates in this event.

Initiation of tRNA maturation by RNase E is essential for cell viability in E. coli.

RNase E, an essential endoribonuclease in Escherichia coli, is involved in 9S rRNA processing, the degradation of many mRNAs, and the processing of the M1 RNA subunit of RNase P. However, the reason that RNase E is required for cell viability is still not fully understood. In fact, recent experiments have suggested that defects in 9S rRNA processing and mRNA decay are not responsible for the lack of cell growth in RNase E mutants. By using several new rne alleles, we have confirmed these observations and have also ruled out that M1 processing by RNase E is required for cell viability. Rather, our data suggest that the critical in vivo role of RNase E is the initiation of tRNA maturation. Specifically, RNase E catalytic activity starts the processing of both polycistronic operons, such as glyW cysT leuZ, argX hisR leuT proM, and lysT valT lysW valZ lysY lysZ lysQ, as well as monocistronic transcripts like pheU, pheV, asnT, asnU, asnV, and asnW. Cleavage by RNase E within a few nucleotides of the mature 3' CCA terminus is required before RNase P and the various 3' --> 5' exonucleases can complete tRNA maturation. All 59 tRNAs tested involved RNase E processing, although some were cleaved more efficiently than others.

RELATIONSHIP BETWEEN SKELETAL MUSCLE UNCOUPLING PROTEIN 3 MRNA AND AEROBIC FITNESS

Skeletal muscle uncoupling protein 3 (UCP3) is believed to have a role in energy expenditure. It has been hypothesized that the increase in energy efficiency associated with endurance training is a result of decreased skeletal muscle UCP3. If this hypothesis were to be true, then one would expect skeletal muscle UCP3 mRNA expression to be negatively correlated with markers of aerobic fitness, such as peak VO2 and citrate synthase (CS) activity. PURPOSE: To determine the relationship between skeletal muscle UCP3 mRNA expression and aerobic fitness as measured by peak VO2 and CS activity. METHODS: Twelve male Sprague Dawley rats were given a graded, maximal treadmill test using a rodent motorized treadmill from Columbus Instruments (Columbus, OH). All animals were sacrificed approximately 48 hours following the test by CO2 inhalation. The plantaris and soleus muscles were rapidly removed, frozen in liquid nitrogen and then stored at -80°C until UCP3 mRNA and CS activity could be assessed. RT-PCR was conducted to evaluate UCP3 and 18S ribosomal mRNA. UCP3 mRNA was normalized to the 18S mRNA. A Pearson Correlation test was used to determine the relationship between UCP3 mRNA, CS, and peak VO2. RESULTS: Normalized UCP3 mRNA expression was not correlated with peak VO2 (correlation coefficient = −0.30, p = 0.34) or CS activity (correlation coefficient = −0.30, p = 0.34). CONCLUSION: These data suggest that skeletal muscle UCP3 mRNA expression is not related to aerobic fitness as measured by peak VO2 or CS activity in rats.

Ontogeny and insulin regulation of fetal ovine white adipose tissue leptin expression.

Leptin, an adipocyte-derived factor, has multiple biological roles including mitogenesis. We investigated the effect of normal development, acute and chronic hyperglycemia and hypoglycemia, and selective acute hyperglycemia, or hyperinsulinemia, on fetal ovine white adipose tissue (WAT) leptin mRNA concentrations. Leptin mRNA amounts expressed as a ratio to the internal control ribosomal S2 mRNA decreased threefold with advancing gestational age (P < 0.05). This gestational decrease was opposite to the 10-fold increase in fetal body weight during the same developmental period. Chronic hyperglycemia with hyperinsulinemia led to no change in WAT leptin mRNA concentrations over a 1- to 10-day duration, but it caused a 40% increase over a 14- to 20-day duration (P < 0.05) along with an increase in fetal body weight (P < 0.05). In contrast, hypoglycemia with hypoinsulinemia, while not affecting WAT leptin mRNA from 1 to 34 days, resulted in a 50% decline over a 36- to 76-day duration along with a decline in fetal body weight (P < 0.05). Acute 24-h studies of selective hyperglycemia with euinsulinemia showed no significant change in WAT leptin mRNA, but in response to selective hyperinsulinemia with euglycemia at 24 h, a twofold increase was observed (P < 0.05). We conclude that fetal WAT leptin mRNA amounts are regulated by fetal development and circulating insulin concentrations. We speculate that chronic in utero metabolic perturbations that alter circulating insulin concentrations affect fetal leptin production that may mediate insulin's influence on fetal growth.