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Peptidyl transfer ribonucleic acid hydrolase activity of proteinase k.

Proteinase k, a seryl-protease obtained from Tritirachium album, is able to specifically hydrolyze N-blocked aminoacyl transfer ribonucleic acids (tRNAs). The blocked amino acid is released, and the tRNA molecule remains able to be recharged by its cogante amino acid. Aminoacyl-tRNAs are highly resistant to hydrolysis by the protease. This activity is not due to contamination of the protease preparation. A commercial protease from Streptomyces griseus displayed a similar activity, while trypsin, chymotrypsin, and papain unspecifically hydrolyzed all charged tRNAs tested. The characteristics of the hydrolysis performed by proteinase k closely resemble the peptidyl-tRNA hydrolase activity described in different cells as a scavenger for the peptidyl-tRNA that eventually falls from the polysomes. Out results warn about a hasty identification of any N-blocked aminoacyl-tRNA hydrolase activity in the cytoplasm as an independent peptidyl-tRNA hydrolase.

Endopeptidases↗

Unusual valyl-transfer ribonucleic acid synthetase mutant of Escherichia coli.

Escherichia coli strain NP2907 was isolated as a spontaneous mutant of strain NP29, which possesses a thermolabile valyl-transfer ribonucleic acid (tRNA) synthetase. The valyl-tRNA synthetase of the new mutant, unlike that of its immediate parent, retains enzymatic activity in vitro but differs from the wild-type enzyme in stability and apparent K(m) for adenosine triphosphate. The new mutant locus, valS-102, cotransduces with pyrB at the same frequency as does the parental locus, valS-1. Cultures of strain NP29 cease growth immediately in any medium when shifted from 30 to 40 C. The new mutant grows normally at 30 C, and upon a shift to 40 C growth quickly accelerates exactly as for normal cells. Exponential growth, however, cannot be sustained at 40 C. At a point characteristic for each medium, growth becomes linear with time. This transition occurs almost immediately in rich media and after 1.5 generations in glucose minimal medium. Net synthesis of valyl-tRNA synthetase ceases in the new mutant as soon as the temperature is raised to 40 C, irrespective of the growth medium. We conclude that it is the amount of valyl-tRNA synthetase activity that limits the rate of growth in the linear phase at 40 C. This property of the mutant makes it possible to evaluate the in vivo efficiency of this enzyme at different growth rates and thereby to determine the concentration that is necessary for a given rate of protein synthesis. The results of our measurements indicate that cells of E. coli growing in minimal medium normally possess a functional excess of valyl-tRNA synthetase with respect to protein synthesis and to repression of threonine deaminase.

Adenosine Triphosphate↗

Relationships between bactericidal effect and inhibition of ribonucleic acid nucleotidyltransferase by rifampicin in Escherichia coli K-12.

The mechanism of action of rifampicin, an antibiotic which inhibits in vitro the polycondensation of ribonucleotides by ribonucleic acid (RNA) nucleotidyltransferase, was studied in vivo in Escherichia coli. It is argued that the inhibition of RNA nucleotidyltransferase represents the primary lesion and is responsible for the bactericidal effect. This conclusion is based on (i) the correlation between concentrations of the antibiotic which block in vivo incorporation of labeled uracil and the bactericidal concentrations, (ii) the evidence that the loss of viability of the cells immediately follows the block of RNA synthesis, and (iii) the observation that the reversal of the inhibition of RNA synthesis goes together with a reversal of the loss of viability.

Bacterial Proteins↗

Regulation of follicle-stimulating hormone beta and common alpha-subunit messenger ribonucleic acid by gonadotropin-releasing hormone and estrogen in the sheep pituitary.

The effect of gonadotropin-releasing hormone (GnRH) and/or estradiol (E2) on pituitary messenger ribonucleic acid (mRNA) levels of luteinizing hormone beta (LH beta), follicle-stimulating hormone beta (FSH beta) and the common alpha-subunit were determined in anterior pituitary glands from ovariectomized (OVX) ewes. Hypothalamo-pituitary disconnected (HPD) ewes receiving appropriate hormonal treatment were used to assess the relative roles of GnRH and E2 in directly regulating FSH beta and alpha-subunit mRNA levels. Levels of LH beta mRNA were increased in OVX animals compared with intact controls, and E2 treatment of OVX animals significantly reduced mRNA levels of LH beta and FSH beta. HPD substantially reduced FSH beta and alpha-subunit mRNA levels. Treatment of OVX/HPD animals with pulses of GnRH (250 ng/2 h) for 1 week restored FSH beta and alpha-subunit mRNA to OVX levels. Combined GnRH and E2 treatment significantly lowered FSH beta mRNA levels, but resulted in a rise in alpha-subunit mRNA levels. Treatment of OVX/HPD ewes with E2 alone had no effect on FSH beta and alpha-subunit mRNA levels. These findings indicate that E2 acts directly on the pituitary to negatively regulate FSH beta mRNA levels, and to positively regulate alpha-subunit mRNA levels in the presence of GnRH.

Animals↗

Prolactin receptor messenger ribonucleic acid in normal and neoplastic human pituitary tissues.

We examined the specific cell types in normal human pituitaries that expressed PRL receptor (PRL-R) messenger ribonucleic acid (mRNA) by combined in situ hybridization and immunohistochemistry. The distribution of PRL-R mRNA in 28 pituitary adenomas was examined by in situ hybridization and reverse transcription-PCR in 12 cases of adenomas. In another set of experiments, 34 PRL adenomas from men, women, and bromocriptine-treated patients were analyzed for PRL-R by in situ hybridization. In the normal pituitary, PRL- and LH-producing cells had significantly more mean grain counts per cell and higher percentages of cells positive for PRL-R than GH and TSH cells. PRL-R mRNA was present in all groups of adenomas by in situ hybridization and reverse transcription-PCR. PRL adenomas had a significantly higher density of labeling compared to other adenoma types. Although there was no difference in the levels of PRL-R mRNA in PRL adenomas from men and premenopausal and postmenopausal women, patients treated with bromocriptine before pituitary surgery had significantly lower levels of PRL-R compared to all other groups. These results indicate that in the normal pituitary, PRL and LH cells have the highest level of PRL-R mRNA, whereas PRL adenomas have significantly higher levels of PRL-R mRNA than other types of adenomas, and bromocriptine treatment decreases the levels of PRL-R mRNA in PRL adenomas.

Adenoma↗

Detection and levels of androgen receptor messenger ribonucleic acid in the rat brain by means of reverse transcription-polymerase chain reaction.

In order to examine the existence and level of androgen receptor messenger ribonucleic acid (ARmRNA) in the rat brain, reverse transcription-polymerase chain reaction (RT-PCR)-Southern blot analysis was carried out. Total RNA extracted from each tissue was reverse transcribed, followed by PCR with two oligonucleotide primers specific for a part (458 bp) of the androgen binding domain of the rat ARcDNA. It was confirmed by direct nucleotide sequencing that the amplified fragment corresponded to part of the rat ARcDNA. To detect and quantify the amplified fragments, a Southern blot analysis was carried out. The levels of amplified fragments were calculated from the standard curve obtained from graded diluted adrenal total RNAs. In the present study, it was revealed that the RT-PCR-Southern blot analysis possessed a high-degree of sensitivity and allowed the quantitative estimation of mRNA. With this method, amplified fragments were obtained from all five brain regions examined. The results indicate that ARmRNA is widely distributed in the whole brain. Moreover, since the ARmRNA level roughly paralleled the AR protein level, it seems that the AR protein level in the brain may be primarily regulated by the ARmRNA level.

Amino Acid Sequence↗

Proopiomelanocortin, prolactin and growth hormone messenger ribonucleic acid levels in the fetal sheep pituitary during late gestation.

We have measured the relative levels of proopiomelanocortin (POMC), prolactin (PRL) and growth hormone (GH) messenger ribonucleic acid (mRNA) in the anterior and neurointermediate lobes of the fetal pituitary during the last 2-3 weeks of gestation. The mean POMC mRNA/18S RNA ratio in the fetal anterior pituitary was significantly greater (p < 0.02) at 130-136 days (0.90 +/- 0.08; N = 9) than at 141-143 days of gestation (0.67 +/- 0.07; N = 6). In contrast, the mean PRL mRNA/18S RNA ratio increased significantly (p < 0.02) between 130 and 136 days (0.31 +/- 0.05; N = 9) when compared with 141-143 days of gestation (0.58 +/- 0.10; N = 6). There was no significant difference, however, between the mean GH mRNA/18S RNA ratio in fetal anterior pituitaries at 130-136 days (0.95 +/- 0.04; N = 9) when compared with 141-143 days of gestation (1.08 +/- 0.14; N = 6). The POMC mRNA/18S RNA ratio in the neurointermediate lobes was seven-, five- and tenfold higher than anterior pituitaries at 130-134, 135-136 and 141-143 days of gestation, respectively. We hypothesize that elevated circulating cortisol levels after 140 days of gestation act in the slow time domain (i.e. over days) to suppress POMC gene expression and that the increase in fetal pituitary PRL mRNA levels may be a consequence of oestrogen stimulation in late gestation.

Animals↗

Oligonucleotide-conjugated thiazole orange probes as "light-up" probes for messenger ribonucleic acid molecules in living cells.

"Light-up" probes, icosa-alpha-thymidylate-thiazole orange conjugates, for the in situ time-resolved detection of messenger ribonucleic acid (mRNA) in living cells are evaluated. Upon annealing with polyA in aqueous solutions, the icosa-alpha-thymidylate-thiazole orange conjugates were shown to be up to 15 times more fluorescent. Microinjection of these probes into adherent fibroblasts resulted in high yields of hybridization and fluorescent signals. Incubation of cells in the presence of these probes resulted in facile internalization of the probe and similar painting of the messenger RNA in the nuclear and cytosolic regions.

Absorptiometry, Photon↗

Detection of estrogen receptor messenger ribonucleic acid in human oocytes and cumulus-oocyte complexes using reverse transcriptase-polymerase chain reaction.

OBJECTIVES: To examine the expression of estrogen receptor (ER) messenger ribonucleic acid in the human ovary. To detect ER gene transcripts in human oocytes; and to study the possible autocrine effect of estrogen (E) in granulosa/cumulus cells. DESIGN: Human ovaries from benign gynecological surgeries as well as the oocytes, cumulus-oocyte complexes, and granulosa/cumulus cells from the in vitro fertilization program were used to detect ER gene transcripts using the highly sensitive reverse transcriptase-polymerase chain reaction technique. SETTING: Molecular biology laboratory. RESULTS: The ER gene is expressed in human ovary. Estrogen receptor transcripts were detected in human cumulus-oocyte complexes and oocytes but not in granulosa/cumulus cells. CONCLUSIONS: These results suggest a lack of receptor-mediated autocrine effect of E during folliculogenesis and that E, secreted by granulosa/cumulus cells, may exert a paracrine effect to influence oocyte maturation and fertilization competence directly.

Adult↗

[Histochemical behavior of succinate dehydrogenase and lactate dehydrogenase as well as ribonucleic acid in the epithelium of lactic ducts and alveoli of cow udder].

The activities of succinate dehydrogenase (SDR) and lactate dehydrogenase (LDH) as well as the ribonucleic acid levels in the epithelium of both the lactic ducts and alveoli of lactic glands were examined in heifers, close to full term, lactating cows, some of them with subclinical mastitis lesions, and udder biopsies of two cows. The activities of SDH and LDH as well as RNA levels were pronounced in the glandular epithelium of alveoli and in the epithelium of the lesser lactic ducts. All three parameters were also pronounced but at lower levels in the cells of the apical stratum of the two-layer epithelium in the greater lactic ducts. Relationships were found to exist between the enzyme activities and RNA levels and the lactation cycle. Their patterns and manifestations were more or less typical of those areas of the lactic gland which were affected by mastitis.

Age Factors↗

Simultaneous ultrastructural identification of growth hormone and its messenger ribonucleic acid using combined immunohistochemistry and non-radioisotopic in situ hybridization: a technical note.

The present electron microscopical study is concerned with the simultaneous visualization of messenger ribonucleic acid (mRNA) and its encoded protein in the same specimen. Pre-embedding electron microscopical in situ hybridization (EM-ISH) on rat pituitary gland tissue localized growth hormone mRNA in the polysomes of the rough endoplasmic reticulum, and subsequent postembedding immunolabelling using protein A-colloidal gold particles identified growth hormone mainly in the secretory granules. We believe that our report provides the first simultaneous ultrastructural identification of mRNA and its encoded protein using combined pre-embedding EM-ISH and immunohistochemistry. In this method, the signals for mRNA were localized specifically as highly electron dense products on the polysomes of the endoplasmic reticulum, and those for its encoded protein were recognized as gold particles both in the cisternae of the reticulum and in the secretory granules. Our ultrastructural double labelling method for mRNA and protein may provide a tool to find important clues for elucidating the intracellular correlation of mRNA translation and secretion of translated protein, because of its high resolution, good morphological preservation, and the specific localization of the reaction products.

Animals↗

p53 protein and its messenger ribonucleic acid in human adrenal tumors.

The role of p53 tumor suppressor gene in the pathomechanism of adrenal tumors was investigated by measuring p53 protein and its messenger ribonucleic acid (mRNA) in 12 normal human adrenals as well as in 56 adrenal tumors (7 aldosterone-producing adenomas, 5 adrenocortical adenomas causing Cushing's syndrome, 19 non-hyperfunctioning adrenocortical adenomas, 5 adrenocortical carcinomas, 12 pheochromocytomas, 3 myelolipomas, 4 ganglioneuromas and 1 hemangioma). The p53 protein concentration was significantly increased in aldosterone-producing adenomas (394+/-36 pg/mg cytosolic protein, mean+/-SE, vs 266+/-18 in normal human adrenals), whereas the concentration of this protein in Cushing's adenomas, non-hyperfunctioning adrenocortical adenomas, pheochromocytomas, and in all but one adrenocortical carcinomas was similar to that measured in normal human adrenal tissues. One adrenocortical carcinoma tissue showed very high p53 protein content (3000 pg/mg cytosolic protein). By contrast, myelolipomas (23+/-20) ganglioneuromas (43+/-15) and a hemangioma (11 pg/mg cytosolic protein) had very low p53 protein content. Northern blot analysis revealed the presence of p53 mRNA in each adrenal tissue examined with highest levels in aldosterone-producing and Cushing's adenomas. It is possible that the differences in p53 protein and/or mRNA contents reflect corresponding differences in the pathogenetic importance of p53 alterations in these types of adrenal tumors.

Adenoma↗

Expression of interleukin-1 (IL-1) beta messenger ribonucleic acid (mRNA) and IL-1 receptor antagonist mRNA in peritoneal macrophages from patients with endometriosis.

OBJECTIVE: To investigate the expression of interleukin-1 (IL-1) beta messenger ribonucleic acid (mRNA) and IL-1 receptor antagonist (IL-1ra) mRNA in peritoneal macrophages. DESIGN, SETTING: Peritoneal fluid (PF) samples were collected from patients who underwent laparoscopy or laparotomy. Northern blot analysis was performed at the reproductive research laboratory. PATIENTS: Twenty-six patients with endometriosis, 10 patients with postinflammatory pelvic adhesion, and 12 control women with normal pelvis. MAIN OUTCOME MEASURE: Polyadenylated RNA isolated from peritoneal macrophages was analyzed on Northern blots by using synthetic oligonucleotide probes. RESULTS: The level of IL-1 beta mRNA expression was elevated in the group with stage I endometriosis, whereas the increased expression of IL-1ra mRNA was observed in the group with stages III and IV endometriosis. The level of IL-1 beta mRNA showed a positive correlation with that of IL-1 beta in PF and a negative correlation with the level of IL-1ra mRNA. CONCLUSIONS: Our results suggest that peritoneal macrophages express IL-1ra mRNA rather than IL-1 beta mRNA with the progress of endometriosis and that peritoneal macrophages may secrete IL-1ra protein that modulates the effects of IL-1.

Adult↗

Demonstration of ribosome-dependent photoinduced chain breakage of the 16S ribosomal ribonucleic acid component of the Escherichia coli 30S ribosomal subunit.

The effects of 253.7-nm radiation on the structural integrities of free and ribosome-bound 16S ribosomal ribonucleic acid (rRNA) have been elucidated. Exposure of aqueous solutions of Escherichia coli 30S ribosomal subunits to 253.7-nm radiation results in RNA-chain scission and the formation of single-stranded breaks in double-stranded regions of the ribosome-bound 16S rRNA. The minimum doses of incident 253.7-nm radiation required for the first detection of the two types of RNA chain breaks are 2 X 10(20) quanta for single-strand breaks in double-stranded regions of the ribosome-bound 16S rRNA,and at least 5 X 10(20) quanta for RNA-chain scission. In contrast to the photosensitivity of ribosome-bound 16S rRNA toward chain breakage, free 16S rRNA seems to be resistant toward photoinduced chain breakage at doses of incident 253.7-nm radiation up to at least 10(21) quanta.

Escherichia coli↗

Synthesis of ribonucleic acids during the germination of Botryodiplodia theobromae pycnidiospores.

During the initial phases of germination of Botryodiplodia theobromae pycnidiospores, protein synthesis was initiated in the absence of detectable ribonucleic acid (RNA) synthesis. The lack of RNA synthesis during the early stages of germination was not an artefact due to reduced precursor permeability or to changes in the nucleotide pool. Further studies showed that the synthesis of messenger RNA and transfer RNA began about 30 min after the initiation of germination and that of 5S RNA after 45 min, whereas ribosomal RNA synthesis was not detected until after 1 h of germination. Approximately 45 to 55% of the messenger-like RNA synthesized during the first hour of germination was devoid of polyadenylate segments.

Adenosine Triphosphate↗

Antibacterial action of primaquine: effects in vitro on polypeptide synthesis and in vivo on ribosomes and ribosomal ribonucleic acid.

Primaquine inhibited polyphenylalanine formation directed by poly(U) in a cell-free system obtained from Bacillus megaterium only when the drug was preincubated with transfer ribonucleic acid (tRNA), poly(U), or ribosomes. Considerably less inhibition was produced when the ionic strength of the preincubation mixture of tRNA or poly(U) plus primaquine was increased; with ribosomes, the extent of inhibition was only slightly reduced. In cultures of B. megaterium, primaquine induced the breakdown of ribosomes and their RNA.

Bacillus megaterium↗

Isolation and partial characterization of temperature-sensitive Escherichia coli mutants with altered leucyl- and seryl-transfer ribonucleic acid synthetases.

Two temperature-sensitive mutants of Escherichia coli have been found in which the conditional growth is a result of a thermosensitive leucyl-transfer ribonucleic acid (tRNA) synthetase and seryl-tRNA synthetase, respectively. The corresponding genetic loci, leuS and serS, cotransduce with lip and serC, respectively. As a result of the mutationally altered leucyl-tRNA synthetase, some leucine-, valine-, and isoleucine-forming enzymes were derepressed. Thus, leucyl-tRNA synthetase is involved in the repression of the enzymes needed for the synthesis of branched-chain amino acids.

Acylation↗