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At least 919 records · Page 51Linked to original sources
PubMed2006

Association of CD4+ T-lymphocyte counts and new thymic emigrants in HIV-infected children during successful highly active antiretroviral therapy.

BACKGROUND: In a cohort of children receiving highly active antiretroviral therapy (HAART) with sustained plasma HIV-1 RNA < 50 copies/mL, children who reached undetectable RNA after week 8 (slow responders, median: week 20) had higher HIV-1 intracellular DNA (HIV-1 DNA) and equal or greater CD4+ T-lymphocyte counts compared with children who reached undetectable plasma HIV-1 RNA by week 8 (rapid responders) throughout HAART. OBJECTIVE: To determine whether levels of T-cell receptor excision circles (TRECs) could explain the apparent inconsistency between the quantity of HIV-1 DNA and CD4+ T-lymphocyte counts in HIV-1-infected children receiving HAART with sustained virologic suppression. METHODS: T-cell receptor excision circles and HIV-1 DNA and plasma HIV-1 RNA were quantified longitudinally by PCR in 31 children (median age, 5.6 years) with sustained undetectable plasma HIV-1 RNA for >104 weeks of HAART. RESULTS: There was a positive correlation between TREC and HIV-1 DNA during HAART, notably at weeks 48 and 80 (P < .004). During the early stage of HAART, TREC levels positively correlated with CD4+ T-lymphocyte percentages (P < .02) and naive CD4+ T-lymphocyte counts (P < .001) and percentages (P = .05). Median TREC levels were consistently equal or higher in slow responders compared with rapid responders (P < .001) despite slow responders having consistently greater quantities of HIV-1 DNA. CONCLUSION: To maintain adequate levels of CD4+ T-lymphocytes, children with high HIV-1 DNA maintain high levels of TREC while receiving HAART. Thus, a thymic control mechanism is required to maintain new CD4+ T lymphocytes in the presence of persistent virus. CLINICAL IMPLICATIONS: The TREC level is a useful marker of thymic function in HIV-infected children.

Adolescent↗
PubMed1989

A dosage-sensitive modifier of retrotransposon-induced alleles of the Drosophila white locus.

The apricot allele of the white locus results from the insertion of the retrotransposon copia. Mutations in a newly discovered locus, the Darkener-of-apricot (Doa), suppress wa and some of its revertants. Of 44 other white alleles tested, only wsp55 is affected by Doa, although, in contrast, it is enhanced by Doa mutations. The Doa locus modulates wa and wsp55 expression as a function of its own dosage. Mutations in Doa are dominant suppressors or enhancers and are recessive lethals. Rare Doa mutant homozygotes escaping lethality demonstrate extreme phenotypic suppression of wa and enhancement of wsp55. RNA from wa is substantially wild-type in structure in escapers, although reduced in quantity.

Alleles↗
PubMed1985

Survival of Brevibacterium linens during nutrient starvation and intracellular changes.

The present work reports the survival capacity of a strain of Brevibacterium linens isolated from a French camembert cheese and the ensuing changes in cell composition. Exponentially growing cells were harvested, washed and resuspended with shaking in pH 8.0 buffer at 21 degrees C in the absence of a carbon source. The viability of this strain, assessed with slide cultures, is much less than that of coryneform bacteria isolated from soil samples, even though no cell lysis was detected. Intracellular RNA was rapidly consumed during the first few days although magnesium levels remained high. The quantity of DNA initially increased by 17% within 24 h and then remained stable during the 30 days of the experiment. During the same period, absorbance of the medium at 260 nm reached 2 absorbance units. Reserve polysaccharides in this strain are less abundant than in Arthrobacter and were rapidly consumed. Proteolysis was regular and thus maintained a pool of free amino acids which was greater than 60% of the initial value. There was a parallel accumulation of ammonia in the medium. Catalase activity decreased regularly during the first 80 h whereas the quantity of Adenosine-5'-triphosphate (ATP) dropped by 47% in 10 h, stabilizing at less than 10% of its initial value. Cell respiration declined very rapidly and was very low after 24 h.

Adenosine Triphosphate↗
PubMed1996

Dual function of the intron of the rat insulin I gene in regulation of gene expression.

Since the short intron in the 5'-untranslated region (5'-UTR) has been preserved during duplication of the insulin genes in rodents we postulated a possible involvement of these sequences in the regulation of gene expression. To examine this hypothesis we fused nested 5'-deletion fragments of the rat insulin I (rins1) promoter and sequences of the 5'-UTR up to nucleotide +170 with the reporter gene chloramphenicol acetyltransferase (CAT) and generated two series of expression constructs differing by the presence or absence of the intron (rins11VS). Transient expression of these chimeric genes in HIT M2.2.2 cells revealed a four-fold higher CAT expression in the presence of rins1IVS. Comparison of the CAT transcript quantities generated by both counterparts showed only a 1.7-fold difference in the total nuclear RNA fraction, but a four-fold difference in the fraction of nuclear polyadenylated RNA. Further analysis of cytoplasmic RNA excluded nuclear-cytoplasmic transport, RNA stability, and efficiency of translation as targets of the rins1IVS-mediated effect. The higher rate in polyadenylated CAT transcripts generated by rins1IVS-containing vectors suggests a possible coupling between splicing and polyadenylation. Transient expression studies using chimeras containing mutations or deletions between nucleotides -87 and +110 showed a reduction of expression by 30%. These data suggest a dual function of the rins1 intron on transcription initiation and transcript maturation.

Animals↗
PubMed1987

Expression of myosin heavy chain gene in the sea urchin: coregulation with muscle actin transcription in early development.

A fragment of a Strongylocentrotus purpuratus (Sp) myosin heavy chain (MHC) gene was isolated from a genomic recombinant DNA library by cross-reaction with a cloned Drosophila melanogaster (Dm) MHC probe. A portion of a 227-nucleotide Sp coding sequence that is included in this fragment predicts a peptide very closely homologous with a region of the Dm sequence. The MHC gene sequence is present in a single copy per haploid Sp genome, and the gene is utilized in adult as well as embryonic muscle. The quantity of MHC transcript was measured in embryos of various stages by single-strand RNA probe excess titration. Transcripts are not observed until postgastrular stages, after which they accumulate rapidly. The time course of accumulation closely parallels that measured earlier for muscle actin message.

Actins↗
PubMed1986

Increase of cadmium-thiolate clusters as a measure of morphological non-toxic cadmium accumulation in the rat liver.

When cadmium is chronically administered to rats, an increase by more than 10% of protein bound disulphides and cadmium-thiolate clusters appears to be an indicator for non-toxic accumulation of cadmium in liver and kidney and probably in other organs as well. Using enzyme histochemistry, no damage could be observed in these livers, on the contrary, even signs of increased cellular activity could be demonstrated with specific staining for single stranded RNA. It is clearly demonstrated that in the case of 2 livers with the same quantity of accumulated cadmium morphological damage is completely dependent on dose and schedule of administration. However, despite the fact that cadmium is retained very well in rat livers showing an increase in protein-bound disulphides and cadmium-thiolate clusters, there are still small morphological changes, especially in cells and tissues that appear to have a relatively small potency for producing cadmium-binding proteins.

Animals↗
PubMed1997

Myosin heavy chain mRNA transform to faster isoforms in immobilized skeletal muscle: a quantitative PCR study.

A quantitative polymerase chain reaction (PCR) method was used to measure the quantities of type I, IIa, IIx, and IIb myosin heavy chain (MHC) mRNA in total RNA preparations of the soleus, gastrocnemius, and plantaris muscles of normal and hindlimb-immobilized rats. Type IIx and even type IIb MHC mRNA were demonstrated at extremely low levels in normal soleus, 2.1 +/- 0.4 x 10(5) and 5.0 +/- 0.2 x 10(5) molecules of mRNA per microgram total RNA, respectively. Immobilization for 1 wk significantly altered the gene expression of MHC isoforms. In soleus, both type IIx and IIb MHC genes became significantly upregulated, 24-fold (P < 0.005) and 2.6-fold (P < 0.05), respectively. In gastrocnemius, the level of type IIa MHC mRNA decreased by 51% (P < 0.01) and the level of type IIx MHC mRNA increased by 140% (P < 0.05). In plantaris, the level of type IIa MHC mRNA decreased by 58% (P < 0.005). In conclusion, immobilization changed the MHC mRNA profile in three different types of skeletal muscle toward faster isoforms. The quantitative results permit reliable evaluation of changes in mRNA levels.

Animals↗
PubMed1975

Specific detection and semiquantitation of micro-organisms in tissue by nucleic acidhybridization. I. Characterization of the method and application to model systems.

On the basis of circumstantial evidence, a number of human diseases have been postulated to result from the presence of occult infection. To investigate this problem amethod of specifically detecting and semiquantitaing trace quantities of bacterial DNA in infected tissue has been devised. It is based on RNA-DNA hybridization using radiolabeled RNAcomplementary to the DNA of the organism being sought. The characteristics of the reaction are shown to conform to those expected for RNA-DNA hybridization and the sensitivity of the method is shown to correspond to approximately one organism per 50 mammalian cells. Specificity at this level of sensitivity is sufficient to clearly distinguish between infections with Listeria monocytogenes and Erysipelothrix insidiosa. This approach may have general applicability forthe study of diseases putatively caused by chronic infection not readily demonstrable bythe usual cultural or immunologic techniques. It may also be useful for helping distinguish contaminants from pathogens when isolates are obtained from diseases tissue. Unique to this approach is the value of negative information in excluding the presenceof an organism within its limits of sensitivity.

Animals↗
PubMed1998

The lambda holin accumulates beyond the lethal triggering concentration under hyperexpression conditions.

Most bacteriophages terminate infection by creating lesions in the cytoplasmic membrane, which not only cause immediate cell death but also allow escape of a phage-encoded endolysin. Destruction of the peptidoglycan and cell lysis follows very rapidly, allowing efficient release of the progeny virions. These membrane lesions are formed by a small integral membrane protein called a holin. Holins have highly charged carboxyl-termini that are thought to have two transmembrane alpha-helical domains. Holins are believed to oligomerize and form large holes in the inner membrane. The prototype holin is the S protein from bacteriophage lambda. Scheduling of the lytic event is determined in part by the "structure directed initiation" or sdi translational control region. Inductions of S, cloned under a variety of native and nonnative promoters but with native translational control, resulted in cell lysis at about 1000 molecules of holin per cell, and thus do not produce biochemically useful amounts of S protein. By utilizing a plasmid-based system with the T7 RNA polymerase promoter in tandem with a consensus ribosome binding site, Coomassie blue-detectable quantities of S protein were obtained upon induction, corresponding to an approximately 100-fold increase over the normal lethal level of holin. Characterization of this expression system is presented and discussed with respect to the current model of holin function.

Amino Acid Sequence↗
PubMed1989

High levels of expression of full length human pro-alpha 2(V) collagen cDNA in pro-alpha 2(V)-deficient hamster cells.

A full length cDNA encoding human pro-alpha 2(V) collagen was constructed. Partial sequencing of the cDNA and primer extension analysis of mRNA from fibroblasts found that pro-alpha 2(V) mRNA differs from the mRNAs of other fibrillar collagens in the increased length of its 5'-untranslated region. The pro-alpha 2(V) cDNA was placed downstream of the human cytomegalovirus immediate early promoter/regulatory sequences for expression studies in cultured Chinese hamster lung cells. These cells have been shown previously to synthesize large quantities of pro-alpha 1(V) homotrimers as their only collagenous product. Transfection resulted in a number of clonal cell lines that express human alpha 2(V) RNA at levels comparable to, and in some cases greater than, levels found in normal human skin fibroblasts. Pro-alpha 2(V) chains produced in the majority of clonal lines were of sufficient quantity to complex all available endogenous pro-alpha 1(V) chains. Chimeric heterotrimers, composed of hamster alpha 1(V) and human alpha 2(V) chains in a 2:1 ratio, were stable to pepsin digestion and were found predominantly associated with the cell layer. Surprisingly, pro-alpha 2(V) chains, in excess to pro-alpha 1(V) chains, were found in the extracellular matrix and, in much greater abundance, in media. These chains were pepsin sensitive, indicating that pro-alpha 2(V) chains can be secreted as nonstable homotrimers or as free chains.

Animals↗
PubMed1988

High resolution structure of the RNA duplex [U(U-A)6A]2.

RNA is involved in many biological functions, ranging from information storage and transfer to the catalysis of reactions involving both nucleic acids and proteins. Previous crystallographic studies on RNA oligomeric chains provide only averaged structures or information limited in resolution. The oligomer [U(U-A)6A]2 was chosen for the study of protein-RNA interactions in viruses. Its size and base composition mimic portions of the genomic RNA in alfalfa mosaic virus that bind to the amino terminus of the viral subunit. The actual sequence was designed to guarantee the formation of a single species of duplex and to facilitate the production of the pure oligomer in large quantities. The molecular structure, derived from the 2.25 A resolution X-ray diffraction data, allows the most detailed analysis of an A-RNA helix reported to date. Two kinks are observed that divide the duplex into three blocks, each close to a canonical A-helical conformation. A few intermolecular hydrogen bonds involving 2'-hydroxyl groups stabilize this peculiar conformation of the RNA, which may be related to the temperature used for the crystallization (35 degrees C). The structure demonstrates both the plasticity of the RNA molecule and the role of the 2'-hydroxyl groups in intermolecular interactions.

Base Sequence↗
PubMed1998

Molecular characterization and postsplicing fate of three introns within the single rRNA operon of the hyperthermophilic archaeon Aeropyrum pernix K1.

The single rRNA operon (arnS-arnL) of the hyperthermophilic archaeon Aeropyrum pernix K1 was sequenced. The DNA sequence data and detailed RNA analyses disclosed an unusual feature: the presence of three introns at hitherto undescribed insertion positions within the rRNA genes. The 699-nucleotide (nt) intron Ialpha was located at position 908 (Escherichia coli numbering [H. F. Noller, Annu. Rev. Biochem. 53:119-162, 1984]) of the 16S rRNA, while the 202-nt intron Ibeta and 575-nt intron Igamma were located at positions 1085 and 1927 (E. coli numbering), respectively, of the 23S rRNA. They were located within highly conserved sites which have been implicated as crucial for rRNA function in E. coli. All three introns were remarkably AT rich (41.5 to 43.1 mol% G+C) compared with the mature rRNAs (67.7 and 69.2 mol% G+C for 16S and 23S rRNAs, respectively). No obvious primary sequence similarities were detected among them. After splicing from rRNA transcripts in vivo, a large quantity of intronic RNAs were stably retained in the linear monomeric form, whereas a trace of topoisomeric RNA molecules also appeared, as characterized by their behavior in two-dimensional gel electrophoresis. Secondary structural models of the Ialpha-, Ibeta-, and Igamma-containing rRNA precursors agree with the bulge-helix-bulge motif. Two of the introns, Ialpha and Igamma, contained open reading frames whose protein translation exhibited no overall similarity with proteins reported so far. However, both share a LAGLI-DADG motif characteristic of homing endonucleases.

Amino Acid Sequence↗
PubMed1981

Labeling of eukaryotic messenger RNA 5' terminus with phosphorus -32: use of tobacco acid pyrophosphatase for removal of cap structures.

In recent years, there has been a growing appreciation of the potential applications of 5'-32P-end-labeled mRNA, not only for screening recombinant clones and mapping gene structure, but also for revealing possible nucleotide sequence and structural signals within mRNA molecules themselves, which may be important for eukaryotic mRNA processing and turnover and for controlling differential rates of translational initiation. Three major problems, however, have retarded progress in this area, lack of methods for efficient and reproducible removal of m7G5ppp5'-cap structures, which maintain the integrity of an RNA molecule; inability to generate a sufficient amount of labeled mRNA, owing to the limited availability of most pure mRNA species; and the frequent problem of RNA degradation during in vitro end-labeling owing to RNAse contamination. The procedures presented here permit one to decap and label minute quantities of mRNA, effectively. Tobacco acid pyrophosphatase is relatively efficient in removing cap structures from even nanogram quantities of available mRNA, and enough radioactivity can be easily generated from minute amounts ofintact mRNA with very high-specific-activity [gamma-32P]ATP and the inhibition of ribonuclease contamination with diethylpyrocarbonate. These procedures can be modified and applied to almost any other type of RNA molecule as well. In Section III of this volume, we explore in detail how effectively 5'-end-labeled mRNA can be used not only for nucleotide sequence analysis, but also for mapping mRNA secondary structure.

Adenosine Triphosphate↗
PubMed1983

Imbalanced synthesis of human choriogonadotropin alpha and beta subunits reflects the steady state levels of the corresponding mRNAs.

Previous studies have demonstrated an imbalance in placental levels of the human choriogonadotropin (hCG) alpha and beta subunits. Free alpha subunit was present in first trimester placentae, and the imbalance was accentuated as gestation approached parturition. Two sets of experiments were performed to assess the control on production levels of each subunit. Synthesis of the alpha and beta subunits was assessed by labeling the nascent chains of polysomes derived from first trimester placenta. The products of these reactions were immunoprecipitated with subunit-specific antisera and the labeled subunits were quantitated; the ratio of alpha to beta subunit synthesized was 1.7. To examine whether this imbalanced synthesis reflected differences in the amount of subunit mRNAs, or differing mRNA translational efficiencies, the ratio of the steady state levels of these mRNAs was also determined. Total first trimester placental RNA was hydrolyzed with alkali, 5'-end-labeled with 32P, and hybridized in DNA excess to cloned alpha and beta cDNAs. These experiments demonstrated the presence of twice as much hCG-alpha mRNA as hCG-beta mRNA. In term placenta, the amounts of excess alpha subunit are greater than at first trimester; the ratio of alpha to beta mRNAs in term RNA was about 12:1. Thus, the subunit mRNA levels are independently regulated and their imbalance accounts for differences in the quantities of alpha and beta subunits seen in placental tissue.

Base Sequence↗
PubMed2003

Expression of mammalian paralogues of HRAD9 and Mrad9 checkpoint control genes in normal and cancerous testicular tissue.

Human and mouse paralogues of the evolutionarily conserved mammalian HRAD9 and Mrad9 cell cycle checkpoint control genes have been isolated and called HRAD9B and Mrad9B, respectively. HRAD9B encodes a protein that is 414 amino acids long and is 55% similar and 35% identical to the HRAD9 gene product. The Mrad9B protein is 398 amino acids long and is 50% similar and 35% identical to its paralogue. We demonstrate that the encoded human protein is nuclear and can physically interact with checkpoint proteins HRAD1, HRAD9, HHUS1, and HHUS1B, much like HRAD9. Northern blot analysis to detect tissue specificity indicates that the human and mouse genes are expressed predominantly in the testis. The abundance of HRAD9B RNA, as judged by quantitative reverse transcription-PCR, is very low in most testicular tumors, particularly those of germ cell origin, i.e., seminomas, relative to normal testis control, nonseminomas, or Leydig tumor cells. RNA levels corresponding to HRAD17, another checkpoint control gene, demonstrated a similar pattern, but in general, higher quantities of this message were detected in samples. Furthermore, normal/tumor tissue differences were not as dramatic or consistent from sample to sample, especially for the seminomas. Our results demonstrate for the first time that HRAD9 and Mrad9 are part of a gene family and reveal a new genetic element encoding a product that interacts with multiple, known cell cycle checkpoint control proteins. The findings also indicate that HRAD9B can serve as a biomarker in particular for testicular seminomas and might be causally related to the disease.

Amino Acid Sequence↗
PubMed2001

Qualitative and quantitative PCR measures of cytomegalovirus in patients with advanced HIV infection who require transfusions.

The Viral Activation Transfusion Study (VATS) was a randomized trial that compared leukocyte-reduced transfusions with unfiltered red blood cell transfusions in HIV and cytomegalovirus (CMV) antibody-positive patients with anemia who were undergoing their first blood transfusion. The relations of the baseline qualitative and quantitative polymerase chain reaction (PCR) measures of plasma CMV viremia, HIV RNA, CD4(+) cell counts, and quality of life in these study subjects were examined. The 511 study subjects had a median CD4(+) cell count equal to 15 cells/mm3, and 110 (21.5%) had CMV viremia by qualitative assay. In multivariate models, frequency of positive qualitative CMV increased with decreasing CD4(+) cell counts (p =.04 trend), higher HIV RNA (p <.001), and a history of CMV disease (p <.001). Quantitative CMV PCR were performed on the 110 qualitative assay-positive study subjects. Median CMV viral load was 1780 copies/ml. In multivariate regression models, lower CD4(+) cell count (p =.03), and a history of CMV disease (p <.001) correlated with the level of CMV load. HIV RNA load and CMV load were not correlated. A lower Karnofsky score was associated with both the presence and quantity of CMV DNA.

Adult↗
PubMed1992

Persistence of viral genome into late stages of murine myocarditis detected by polymerase chain reaction.

BACKGROUND: Enteroviruses have been considered as the most common etiologic agents in clinical myocarditis and dilated cardiomyopathy; however, their pathogenetic role remains unknown. Hence, the relation of viral replication and development of cardiomyopathy has been determined in a murine model of myocarditis by evaluating the persistence of viral genome during acute and chronic stages of myocarditis by means of Northern blot hybridization and polymerase chain reaction (PCR). METHODS AND RESULTS: DBA/2 mice (n = 146) were injected peritoneally with 10 plaque-forming units of encephalomyocarditis (EMC) virus, and the control mice (n = 33) were injected with normal saline. Animals were randomly killed at 4, 7, 10, 14, 21, 28, 35, and 42 days after infection. Histology revealed acute myocardial necrosis with massive inflammatory cell infiltrate peaking on day 14 followed by increasing fibrosis and declining chronic inflammation features compatible with dilated cardiomyopathy between days 21 and 42. Northern blot analysis of control and infected hearts showed detectable viral RNA in the infected hearts initially at day 4, peaking by day 7, diminishing between day 7 and day 14, and absent at day 21 and day 28. However, potential viral remnants present in low quantities and undetectable by Northern blot were further detected by PCR followed by confirmation with an internal oligonucleotide probe after day 14 up to day 42. CONCLUSIONS: Viral RNA signals on Northern blot showed a strong correlation with massive myocyte necrosis on day 14, but the viral RNA fragment was consistently detectable into late stages of cardiomyopathy on days 21, 28, 35, and 42 by PCR. This indicated that the mature virions are fully developed early in infection and are capable of persisting in the myocardium after virus-mediated myocytolysis stage. Therefore, PCR is an extremely sensitive method for detecting residual viral genome and viral persistence in the myocardium and may offer insights into the pathogenesis of chronic myocarditis leading to dilated cardiomyopathy.

Animals↗
PubMed2005

DNA sequence analysis for structure/function and mutation studies in Becker muscular dystrophy.

We systematically screened the whole coding region of 18 male muscular dystrophy patients whose clinical, histological and laboratory findings suggest Becker muscular dystrophy (present but abnormal dystrophin). No systematic mutation study of a cohort of patients with dystrophin of normal quality but abnormal quantity has been published. The complete coding sequence of the dystrophin gene (11 kb) of each patient was subjected to an automated sequence analysis by using muscle biopsy RNA; 535 bp of the gene promoter and 5'UTR were likewise sequenced. We identified seven disease-causing mutations (40%). Six were novel, including missense, nonsense, small deletion and splice site mutations. Sixty percent (11/18) of patients with decreased quantities of normal molecular weight dystrophin showed no mutation, but most of them had a family history highly suggestive of X-linked inheritance, suggesting transcription or translational deleterious affection, i.e. outside what was screened. Quantitative multiplex fluorescence polymerase chain studies of mutation-negative patients showed normal levels of dystrophin mRNA. In three patients, there was some reduction of the transcript suggesting a deleterious undetected gene change resulted in the reduction of RNA levels. Our data address important structure/function and genotype/phenotype correlations and it suggests that dystrophin protein studies must be interpreted with caution in deletion-negative male muscular dystrophy patients.

5' Untranslated Regions↗