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At least 1,207 records · Page 67Linked to original sources
PubMed1999

Acanthocheilonema viteae: characterization of a molt-associated excretory/secretory 18-kDa protein.

Post-invasive third-stage larvae (pL3) of Acanthocheilonema viteae were labeled with [(35)S]-methionine in vivo, and proteins released into the culture supernatant before and during the third molt were analyzed. The molting supernatant (MSN) contained abundant proteins of 14, 18, 29, and 36 kDa. The 14- and 29-kDa proteins were exclusively found in the MSN, while the 18- and 36-kDa proteins were also produced by nonmolting pL3, albeit in much lower quantities. The cDNA for the most abundant protein in the MSN, an 18-kDa protein (Av18), was isolated by polymerase chain reaction (PCR) with reverse transcribed (RT) RNA of pL3, using information of the protein sequence. The Av18 full-length cDNA of 583 base pairs contained the 5' spliced leader sequence of nematodes, an open reading frame of 427 base pairs, and a poly(A) tail in typical distance to a polyadenylation signal. The deduced amino acid sequence encodes for a protein with a calculated size of 15.8 kDa. The N-terminus starts with a hydrophobic signal sequence and a predicted cleavage site after amino acid 20. The Av18 protein showed homologies to the deduced amino acid sequence of the larval transcripts Bm-alt-1 and alt-2 of Brugia malayi and to the Dirofilaria immitis proteins Di20/22 as well as to the Onchocerca volvulus proteins Ov-alt-1 and Ov-alt-2. Av18 is present in all parasite stages within the mammalian host, as determined by immunoblot with sera against the Escherichia coli-expressed protein and RT-PCR experiments. However, it was released into culture medium only by L3 and adult female worms. In female worms Av18 was localized in the cuticular region as demonstrated by immunofluorescent antibody tests using cryosections.

Amino Acid Sequence↗
PubMed1986

Regulation of ferritin synthesis in malignant and non-malignant lymphoid cells.

The different amounts of H-rich and L-rich isoferritins found in malignant and non malignant lymphoid cells are accompanied by proportional variations in the relative quantity of messenger RNAs for the H and L subunits of ferritin. The correlation between levels of messenger RNAs and proteins suggests that the amount of messenger RNA plays an important role in ferritin biosynthesis in these cells. The enhanced expression of ferritin messenger RNAs in some neoplastic cells is not caused by gross alterations in the structure of ferritin genes.

Biopsy↗
PubMed1966

Effect of interferon on early interferon production.

Chick embryo cells given prior treatment with interferon make new interferon earlier and in larger quantities upon stimulation with Chikungunya virus than cells not so treated. By the criterion of loss of sensitivity to actinomycin, the time needed for formation of messenger RNA for interferon was decreased in the primed cells. Thus interferon affects virus action within 1 hour after infection.

Animals↗
PubMed2004

Cell-free DNA and RNA in plasma as a new molecular marker for prostate cancer.

Extracellular nucleic acids could serve as molecular markers in the early detection of cancer and in the prediction of disease outcome. In this study we examined six molecular markers, such as: variations in the quantity of DNA in plasma, glutathione-S-transferase P1 (GSTP1) gene methylation status in plasma, carcinoembryonic antigen (CEA) and prostate-specific membrane antigen (PSMA) mRNA in peripheral blood mononuclear cells (PBMC), and plasma samples from prostate cancer patients in different stages. The combination of DNA load and GSTP1 promoter methylation status identified 83% (10/12) of the prostate cancer patients before therapy. This study shows that free circulating DNA can be detected in patients with prostate cancer compared with disease-free individuals, and suggests a new, noninvasive approach for early detection of prostate cancer.

Cell-Free System↗
PubMed2002

[Microflora of the peri-ulcer zone in patients with ulcer disease and its sensitivity to antibacterial agents].

The investigations carried out on 126 patients with ulcer disease has shown that from bioptates of periulcer mucosa it is possible to elicit 21 kinds of microorganisms including H. pylori, in quantity 2.8-5.76 lg KOE in 1 gr of bioptate in a combination from 2 to 6 various microbial cultures. They have hemocatheretic, lecithin, urease, gelatinous, DRNA, RNA, katalase activity and cytotoxic properties, what makes the microbiological characteristics of ulcer defect close to parameters of contaminated wound. The given circumstance allows to conclude that in treatment of ulcer relapse it is necessary to suppress the activity not only H. pylori, but also another high pathogenic microbial flora. The testing of various antibacterial medications to microorganisms of periulcer zone has shown, that Ampicillin had the greatest effect in a combination with Gentamicin or Ofloxacin.

Adult↗
PubMed1985

[Association of eukaryotic aminoacyl-tRNA-synthases with polyribosomes].

Upon fractionation of a mitochondria-free extract of rabbit reticulocytes into a ribosome-free extract and mono- and polyribosomes the bulk of the aminoacyl-tRNA synthetase activity was found in the fraction of mono- and polyribosomes. All the fifteen aminoacyl-tRNA synthetases were revealed, although in somewhat different quantities, in both fractions of the mitochondria-free reticulocyte extract. Aminoacyl-tRNA synthetases of the ribosome-free extract are found in two forms: RNA-binding one, and, the one having no affinity for high molecular weight RNAs. Aminoacyl-tRNA synthetases dissociated from the complexes with polyribosomes exist only in the RNA-binding form. All aminoacyl-tRNA synthetases can be removed from such complexes by an addition of 16S rRNA of E. coli, poly(U) or tRNA of rabbit reticulocytes. This testifies to labile association of aminoacyl-tRNA synthetases with the RNA-component of polyribosomes as well as to a rather nonspecific character of their interaction. After EDTA-induced dissociation of polyribosomes, the aminoacyl-tRNA synthetase activity was detected in the complex with both ribosomal subunits.

Amino Acyl-tRNA Synthetases↗
PubMed1995

Tutorial. Molecular biology for the clinician. Part II. Tools of molecular biology.

This is the second of a series of tutorials on molecular biology for the clinician. Part I introduced general principles of nucleic acid and gene structure. This tutorial will build on those general principles and describe some of the commonly used techniques employed in research and diagnosis to examine genes and gene function. The tools of molecular biology include recombinant DNA techniques that have revolutionized the study of DNA and genetic information. Recombinant DNA technology allows the isolation of specific regions of chromosomal DNA and the recovery of unlimited quantities of that DNA for analyses, including determination of the nucleotide sequence of genes, examination of the mechanisms of gene regulation, and the identification of gene products (RNA and protein).

Animals↗
PubMed2003

HIV type 1 DNA development during long-term supervised therapy interruption.

We studied the pattern of HIV-1 DNA development and the association to other HIV-related factors during long-term supervised therapy interruption (LT-STI). Fifteen patients were treated with long-time protease inhibitor-based antiretroviral therapy (PI-ART). They had HIV-1 RNA at <50 copies/ml over 33.4 (SD 9.5) months and CD4(+) cell counts of 875 (SD 415) x 10(6)/liter. A real-time polymerase chain reaction, amplifying fragments of the HIV-1 pol gene and the human albumin gene simultaneously, was used to quantify HIV-1 DNA molecules in CD4(+) cells. The quantity of HIV-1 DNA in CD4(+) cells increased during LT-STI in all 15 patients, with an average doubling time of 2 months. Tentatively, three patterns were observed: rapid initial increase with subsequent stabilizing levels, rapid continuous increase, and slow increase. The HIV-1 DNA slope was positively related to the HIV-1 RNA maximum and steady state level and the baseline HIV-1 DNA value. It was inversely related to the decrease in CD4(+) cells both before the start of PI-ART and during the LT-STI. To conclude, HIV-1 DNA persists in infected CD4(+) cells despite long-term effective PI-ART and will increase after therapy interruption. The most important clinical predictor of long-term STI failure was the rapid CD4(+) cell decline before PI-ART. In patients with a steep pre-PI-ART slope it may be prudent to continue treatment and not initiate therapy interruption.

Anti-HIV Agents↗
PubMed1984

Purification of monoclonal antibodies from mouse ascites eliminates contaminating infectious mouse type C viruses and nucleic acids.

We examined the fate of infectious mouse type C viruses (MuLV) and nucleic acids during the purification of monoclonal antibodies (MoAbs) from mouse ascites. Hybridoma cell lines and their cell fluids can contain infectious ecotropic and xenotropic MuLV whose presence in ascitic fluid may be masked by the presence of anti-viral factors, particularly the anti-xenotropic virus neutralizing factor. They can also contain DNA and RNA. The specific techniques we used for the purification of MoAbs from the ascitic fluid completely eliminated all detectable infectious MuLV, anti-viral factors, and nucleic acids, even when these agents were added in quantities far in excess of those normally expected. Viral proteins, potentially contaminating the MoAb preparations, were also substantially reduced by our procedures. Because infectious MuLV or nucleic acids may have adverse effects on human subjects, attention should be given to each MoAb purification process to insure their effective removal.

Animals↗
PubMed2006

Genomic DNA as a general cohybridization standard for ratiometric microarrays.

Feature variability on ratiometric microarrays is accommodated by simultaneous cohybridization of a labeled reference standard with a labeled experimental sample. An optimal reference standard would provide full and equal representation for all array features from a given genome so that it would function on any array, would represent all features with similar signal intensity, and would be highly reproducible-both technically and biologically-from preparation to preparation and laboratory to laboratory. A low cost and a good shelf life are also highly desirable. Finally, providing for straightforward recovery of RNA prevalence information and for integration of data across multiple, initially unrelated studies would be significant advances over current methods. For virtually all ratiometric array studies published to date the reference standard has been some kind of RNA sample assembled from a number of different cell lines, tissues, or experimental time points. These RNA references fall short of the desired universality, uniformity, and reproducibility criteria, which then affect data quality and integration across studies. Also, the various mixed RNA standards cannot be used to derive RNA prevalence information from an experimental sample. In contrast, genomic DNA is a natural choice to meet all the criteria, although it has not yet been widely exploited for eukaryotic array experiments. Principal stumbling blocks have been achieving high enough absolute signals for large mammalian and plant genomes and finding a way to stabilize labeled DNA so that it can be stored and used with ease. This chapter describes two genomic DNA-labeling methods that make it possible to use genomic DNA as a universal microarray cohybridization standard. The indirect labeling method permits production of a large quantity of a stable genomic DNA standard that can then be quality tested and stored frozen. This optimizes experimental consistency and significantly improves ease of use. This chapter also shows that the genomic DNA reference standard can deliver RNA prevalence measurements from ratiometric array platforms.

Animals↗
PubMed1999

Fast evaluation of internal loops in RNA secondary structure prediction.

MOTIVATION: Though not as abundant in known biological processes as proteins, RNA molecules serve as more than mere intermediaries between DNA and proteins. Research in the last 15 years demonstrates that RNA molecules serve in many roles, including catalysis. Furthermore, RNA secondary structure prediction based on free energy rules for stacking and loop formation remains one of the few major breakthroughs in the field of structure prediction, as minimum free energy structures and related quantities can be computed with full mathematical rigor. However, with the current energy parameters, the algorithms used hitherto suffer the disadvantage of either employing heuristics that risk (though highly unlikely) missing the optimal structure or becoming prohibitively time consuming for moderate to large sequences. RESULTS: We present a new method to evaluate internal loops utilizing currently used energy rules. This method reduces the time complexity of this part of the structure prediction from O(n4) to O(n3), thus reducing the overall complexity to O(n3). Even when the size of evaluated internal loops is bounded by k (a commonly used heuristic), the method presented has a competitive edge by reducing the time complexity of internal loop evaluation from O(k2n2) to O(kn2). The method also applies to the calculation of the equilibrium partition function. AVAILABILITY: Source code for an RNA secondary structure prediction program implementing this method is available at ftp://www.ibc.wustl.edu/pub/zuker/zuker .tar.Z

Algorithms↗
PubMedDate not supplied

Preparative fractionation of protein, RNA, and plasmid DNA using centrifugal precipitation chromatography with tubular dialysis membrane inside a convoluted tubing as separation channel.

Fractionation of clarified E. coli lysate components in bench-scale and preparative-scale centrifugal precipitation chromatography (CPC), using a solution of cationic surfactant cetyltrimethylammonium bromide (CTAB) containing 0.5 M NaCl as precipitant, are compared here. Step gradient of CTAB from 0.50% to 0.16% (w/v) gave a successful fractionation in bench-scale CPC; however, a linear gradient of lower CTAB concentration, 0.20-0% (w/v), was used in the preparative scale and resulted in similar fractionation. The preparative-scale CPC has a superior sample loading capacity by the use of tubular dialysis membrane inside convoluted tubing as the separation channel. In this study, the quantity of the sample loaded into the preparative CPC was about 15 times more than that in the bench scale, and in a single run the preparative CPC could prepare approximately 3 mg of plasmid DNA with about 96% of RNA removed. The higher surface area per length of the separation channel in the preparative CPC was believed to benefit mass transfer of CTAB across the membrane, leading to less CTAB being required in the process.

Centrifugation↗
PubMedDate not supplied

[Structural and functional interrelations between the liver and endocrine system of the pancreas in disorders of corticosteroid balance].

Experiments on male rats have shown that correlative interrelationships of the liver and endocrine apparatus of the pancreas depend on the content of corticosteroids in the body. The administration of a physiological dose of hydrocortisone raises the synthesis of RNA, phospholipids and glycogen in the liver accompanied by B-insulocyte hypertrophy. Large hormone doses result in the suppression of protein synthesis in hepatocytes with further development of fatty degeneration of the liver and hypofunction of pancreatic islet B-cells. Corticosteroid deficiency causes metabolic derangement of hepatocytes, a rise of the quantity and synthetic activity of pancreatic A-cells which can be corrected by means of hydrocortisone substitution therapy.

Adrenalectomy↗
PubMedDate not supplied

[Viral hepatitis C].

INTRODUCTION: Viral hepatitis type C became one of the most dangerous hepatic diseases, bearing high risk of eventually fatal complications. Now a great deal of public health funds has to be used for prevention and treatment of this serious disease. Only very detailed knowledge of the disease could help to a medical practitioner in his everyday confrontation with this serious problem. MATERIALS AND METHODS: Data, diagnostic, therapeutic and preventive suggestions given in this paper are result of a comprehensive review of relevant literature. RESULTS: The causative agent of hepatitis type C is an RNA virus with six different genotypes. It is easily transmitted from one host to the other only by transferring large amounts of body fluids (blood or plasma transfusion, or prolonged, repeated inoculations of small quantities of infected fluids intravenous drug abusers, recipients of clotting factors, accidental needle sticks). The quantification of the disease activity could be done by a numerical scoring system, originally issued by Knodell, which takes into account four categories: periportal necrosis, intralobular necrosis, portal inflammation and fibrosis. The incubation period of hepatitis C varies from 5 to 7 weeks. It starts like a relatively mild acute disease, but eventually it progresses to chronicity. About 10-20% of patients develops cirrhosis, and yet unknown percentage of patients develops hepatocellular carcinoma. On average, it takes about 30 years for chronic hepatitis C to progress to cirrhosis or cancer. DISCUSSION: Serologic testing for anti-HCV proves the existence of specific antibodies against hepatitis C virus. It becomes positive only after 5-6 weeks from clinical onset. Much more sensitive test is PCR, which proves the viral RNA in body fluids. PCR is positive as early as 2 weeks from the onset of hepatitis. Up to now, the only 100% certain way to prove existence of chronic hepatitis is liver biopsy. Interferon alpha is nowadays used for management of this serious disease. The accepted dose is 3,000,000 U three times weekly for 24 weeks. About 46% of treated patients will have both serological and histological improvement. Total liver collagen and iron staining in portal areas are significantly decreased after the treatment course, giving hope for postponing the onset of cirrhosis. However, half of the responders will experience relapse of the disease within 8 months from the end of treatment, and sustained biochemical and virological response could be seen in only 5% of patients. The sustained response rate was increased in some studies to 29% when iron reduction was undertaken along with interferon. CONCLUSION: Since there is no effective treatment for hepatitis C, much of the efforts should be directed to prevention. Since hepatitis C virus is transmitted only by parenteral route or close personal contact (sexual contact mostly), in the family environment general hygienic measures are considered sufficient. Hands should be washed properly, food, clothing, utensils, linen and excreta of the patient should be handled separately. During sexual intercourse, prophylactics should be used. The most important measure for prevention of posttransfusion hepatitis C is regular testing of all blood donors for anti-HCV antibodies.

Hepatitis C↗
PubMed1994

Models for solid-state transport: messenger RNA movement from nucleus to cytoplasm.

This paper explores the idea that mRNAs are transported between their transcription and processing sites in the nucleus, and their translation and degradation sites in the cytoplasm, by a 'solid-state' process. The underlying assumption is that negligible quantities of mRNA and of mRNA precursors are in solution in vivo. Therefore, mRNA transport cannot be considered as movement in the aqueous phase of the cell. The main lines of experimental evidence supporting this 'solid-state' concept are summarized and related controversies are outlined. Three possible models for a solid-state transport mechanism are discussed: a direct transfer model, with receptors organized analogously to the components of a multienzyme complex; a motor-driven model, analogous to synaptic vesicle transport in axons; and an assembly-driven model which assumes net movement along a fibril resulting from differential activities at the poles. Qualitative evaluation indicates that each of these models has characteristic advantages and disadvantages. The possibility that other nucleocytoplasmic transport processes might operate by solid-state mechanisms is briefly discussed.

Animals↗
PubMed1996

Epidermal growth factor and its receptor (EGF-R) in human pituitary adenomas: EGF-R correlates with tumor aggressiveness.

Epidermal growth factor (EGF) has been localized in several human neoplasms and has been shown to have a significant correlation to prognosis. We investigated the potential role of the EGF receptor (EGF-R) system in pituitary tumorigenesis by examining the expression of EGF and EGF-R in the different types of human pituitary adenomas. EGF was identified by immunohistochemistry in all cell types of the nontumorous adenohypophysis and in all types of morphologically characterized functional (n = 9) and nonfunctional (n = 17) adenomas. To confirm local EGF synthesis, ribonucleic acid (RNA) from human pituitary adenomas was reverse transcribed and PCR amplified. Transcript signals of the expected size were identified, with marked variation in 41 of 48 adenomas. To assess possible secretion in vitro, EGF was measured in pituitary tumor culture medium. No measurable quantities of EGF were present in conditioned culture medium from all 35 adenomas examined, consistent with the rapid uptake of EGF by unoccupied functional EGF-R sites. Using a specific monoclonal antibody that recognizes the extracellular ligand-binding domain of the human EGF-R, we found EGF-R in cells in the normal pituitary and in some functional and nonfunctional adenomas with extremely variable intensity. By RT-PCR, EGF-R messenger RNA (mRNA) expression was also identified, with marked variation in all 48 adenomas examined and in the nontumorous pituitary. The highest degrees of EGF-R mRNA expression were present in somatotroph adenomas and the aggressive silent subtype 3 adenomas. Tumors from patients with recurrent acromegaly demonstrated significantly higher levels of EGF-R mRNA than those from patients with nonrecurrent disease. In conclusion, EGF and EGF-R are expressed in a variable manner in all types of human pituitary adenomas. The overexpression of EGF-R in recurrent somatotroph adenomas and aggressive silent subtype 3 adenomas suggests a selective mechanism for the EGF/EGF-R family in the growth of aggressive pituitary tumors.

Adenoma↗
PubMed1999

Expression and inducibility of alpha, pi, and Mu glutathione S-transferase protein and mRNA in murine lung.

This investigation sought to establish the cellular expression and distribution of the alpha, pi, and mu classes of glutathione S-transferase (GST) enzymes in murine lung under control conditions and after treatment with tert-butyl-4-hydroxyanisole (BHA). Immunohistochemical and in situ hybridization studies were used to identify lung cells that were labeled for the GST subunits Yp, Ya, and Yb1. Immunoblotting of cytosolic proteins produced single bands of 28, 29, and 31 kD for Ya, Yp, and Yb1, respectively, in samples from untreated and BHA-treated mice. Treatment with BHA increased Ya and Yp reactivity, but this was not as marked for Yb1. Immunohistochemical staining for the Yp, Ya, and Yb1 subunits was localized in bronchioles and parenchyma of untreated and BHA-treated mice. Bronchiolar Clara and alveolar type II cells were stained to the greatest extent for all of the GST subunits. BHA treatment produced increased staining that was most pronounced in the bronchiolar epithelium. Ya and Yp were localized in the cytoplasm and nucleus, whereas Yb1 was found mainly in the cytoplasm. Immunoblots of extracted nuclear proteins revealed a band of 29 kD for Ya, with increased immunoreactivity in BHA-treated mice. In situ hybridization done with oligonucleotide probes showed abundant silver grains representing Ya, Yp, and Yb1 messenger RNA (mRNA) transcripts in the bronchioles. Grains were also localized in alveolar septa, and were most numerous in type II cells. Quantitative image analysis confirmed good agreement between relative levels of protein and mRNA transcripts. Quantities of mRNA transcripts for all subunits were increased in the parenchyma by BHA treatment, but the magnitudes of induction were most striking for Ya and Yp in the bronchioles. These results demonstrated that Ya, Yp, and Yb1 reside in specific lung areas and cells, and that in induced states, their increased expression is accompanied by increased mRNA.

Animals↗
PubMed1993

A Euglena gracilis zinc endonuclease.

A 26-kDa endonuclease has been purified to homogeneity from zinc-sufficient Euglena gracilis. The protein binds to single-stranded DNA with a higher affinity than to double-stranded DNA, but it exhibits nucleolytic activity toward both. Thus, it converts supercoiled plasmid pBR322 DNA into the linear form, a property characteristic of endonucleases, and it continues to act on the linearized DNA until it is completely degraded. It also hydrolyzes heat-denatured, single-stranded calf thymus DNA. Moreover, at amounts below 1 microgram, it enhances RNA synthesis by RNA polymerase II, a characteristic observed with other DNases. Its addition to an in vitro transcription assay increases RNA synthesis up to 3-fold. The nuclease requires two metal components to carry out its enzymatic activities. It hydrolyzes DNA only in the presence of millimolar amounts of magnesium or micromolar quantities of other activating metal ions, such as manganese, zinc, or cobalt. However, even when optimal concentrations of Mg2+ are present, micromolar amounts of the metal-chelating agents OP and HQSA completely inhibit pBR322 digestion. Transcription enhancement is also inhibited completely by both chelators at concentrations that do not affect the intrinsic polymerase II activity. By atomic absorption spectrometry, the enzyme contains 1 g-atom of Zn/mol, which is the likely target of chelator action. The nuclease protein can also be isolated from zinc-deficient E. gracilis, but remarkably it then contains 1 mol of Cu/g-atom and no zinc.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗