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PubMed1979

Development of the nitrogen-fixing and protein-synthesizing apparatus of bacteroids in pea root nodules.

Some aspects of root nodule development of Pisum sativum inoculated with Rhizobium leguminosarum were examined. 1. Nitrogenase activity (measured as acetylene reduction) appears to be preceded by leghemoglobin synthesis (measured immunologically). 2. Syntheses of component I and component II of nitrogenase are not strictly coordinated. Synthesis of component I starts before component II. 3. Plant and bacteroid protein synthesis (measured by [35S]sulfate labeling) in root nodules declines rapidly during nodule development. Corresponding with this decline is a decrease in quantity and quality of rRNA.

Fabaceae↗
PubMed1995

Structural specificity of nuclease from wheat chloroplasts stroma.

A single-strand-specific nuclease from wheat chloroplasts (ChS nuclease) was tested as a tool for RNA secondary and tertiary structure investigations, using yeast tRNA(Phe) and yeast tRNA(Asp) as models. In tRNA(Phe) the nuclease introduced main primary cleavages at positions U33, A35 and A36 in the anticodon-loop and G18 and G19 in the D-loop. In tRNA(Asp) the main primary cleavages occurred at positions U33, G34 and U35 in the anticodon-loop and the lower one at position C20:1 in the D-loop. No primary cleavages were observed within the double-stranded stems. Because ChS nuclease has (i) a low molecular weight, (ii) a wide pH range of action (5.0 to 7.5) (iii) no divalent cation requirement in the reaction mixture and (iv) can be obtained as a pure protein in rather large quantities it appeared to be a very good tool for secondary and tertiary structural studies of RNAs.

Base Sequence↗
PubMed1988

Homozygous hypobetalipoproteinemia: a disease distinct from abetalipoproproteinemia at the molecular level.

apoB DNA, RNA, and protein from two patients with homozygous hypobetalipoproteinemia (HBL) were evaluated and compared with normal individuals. Southern blot analysis with 10 different cDNA probes revealed a normal gene without major insertions, deletions, or rearrangements. Northern and slot blot analyses of total liver mRNA from HBL patients documented a normal size apoB mRNA that was present in greatly reduced quantities. ApoB protein was detected within HBL hepatocytes utilizing immunohistochemical techniques; however, it was markedly reduced in quantity when compared with control samples. No apoB was detectable in the plasma of HBL individuals with an ELISA assay. These data are most consistent with a mutation in the coding portion of the apoB gene in HBL patients, leading to an abnormal apoB protein and apoB mRNA instability. These results are distinct from those previously noted in abetalipoproteinemia, which was characterized by an elevated level of hepatic apoB mRNA and accumulation of intracellular hepatic apoB protein.

Apolipoproteins B↗
PubMed2005

[The use of in-situ hybridization for the detection of Brachyspira spp. in pigs].

Diagnosis of Brachyspira infections in swine and the differentiation of the involved bacteria is time-consuming and in most cases unsatisfactory. Detecting Brachyspira directly in the damaged Brachyspira of the large intestine could provide a direct correlation between histological lesionsa and bacterial growth. In this study we investigated whether in-situ hybridization (ISH) with a digoxigenin-labeled RNA-probe is a suitable method for detecting Brachyspira in the mucosa of the large intestine. Formalin-fixed and paraffin-embedded tissue sections of the large intestine from 78 pigs, which showed macroscopic and histological findings of Brachyspira-associated colitis, were stained with hematoxylin and eosin and Warthin-Starry silver impregnation and subjected to ISH. We used a RNA-probe with a length of 334bp, complementary to a part of the 23S rRNA of all members of the genus Brachyspira. All sections were treated with this anti-sense probe and with a sense control probe. 64 samples (82%) showed clearly positive ISH signals. Thus ISH is a suitable method for detecting Brachyspira directly within the lesions of the large intestine. The quantity of Brachyspira identified by ISH was always lower than by Warthin-Starry staining. Whether this reflects lower sensitivity of the ISH technique, or the fact that other bacteria with morphological similarities to Brachyspira were also stained by Warthin-Starry is unknown as yet. The present investigations provide a basis of further research developing specific probes to distinguish between pathogenic and non pathogenic Brachyspira species and probes detecting other bacteria with morphological similarity to Brachyspira.

Animals↗
PubMed1982

Temperature-sensitive mutant rho-115 rho-RNA binary complexes, and stabilization by substrates and analogues.

To determine the molecular basis for the temperature-sensitivity of pure rho RNA-dependent ATPase from Escherichia coli mutant rho-115 cells, we investigated mutant rho binding to [3H] polyC as measured by retention on nitrocellulose filters. Complexes of wild-type rho and polyC incubated at 37 degrees C and 45 degrees C were similarly stable. At 37 degrees C mutant rho-polyC binary complexes were inactivated at a slightly faster rate than complexes with wild-type rho. Upon shift to 45 degrees C the quantity of rho-115 bound to polyC declined immediately, resulting in one-fifth of the quantity of complexes observed at 37 degrees C. Shift back to 37 degrees C restored the level of observed complexes by two-fold. The inclusion of ATP or the analogue beta-gamma methylene ATP during 45 degrees C incubation resulted in stable mutant rho-polyC complexes. The hydrolysis product ADP was also effective in stabilizing binary complexes at 45 degrees C but this effect was observed with an order of magnitude more ADP than ATP. Adenine, adenosine, AMP or Pi had no stabilizing effect. We conclude that the mutant rho-115 protein exhibits a structural instability as a result of binding RNA. Furthermore ATP confers a wild-type phenotype upon rho-115 protein, probably as a result of conformational change due to binding of this compound. The effect of ATP on the stability of mutant rho-polyC binary complexes supports the model of ATP modulation of rho-RNA interaction proposed by Galluppi and Richardson (1980).

Adenine↗
PubMed1983

The uncoupling of macromolecular synthesis from cell division in SV3T3 cells by glucocorticoids: the imposition of a G2 block.

Through a receptor-mediated process glucocorticosteroids block cell division by 20-45 hours in SV40-transformed 3T3 (SV3T3) mouse fibroblasts growing in a low calf serum (0.30% v/v) medium containing biotin. However, the rate of DNA synthesis, determined at various times after dexamethasone addition by the incorporation of radioactive thymidine into acid-insoluble material, is not inhibited by this steroid as late as 66 hours. A modest decrease is observable by 91 hours. There is also no reduction in the uptake of exogenous thymidine into acid-soluble cellular pools. Similarly, RNA synthesis and the uptake of radioactive uridine are not affected by the glucocorticoid up to 69 hours. Measurements of the amounts of cellular DNA (by the fluorescent dye, 4', 6-diamidino-2-phenylindole) and protein revealed that both macromolecules are present in elevated quantities in steroid-treated cells. (The constancy of the protein content in the nonproliferative stage suggests that protein synthesis and degradation are occurring at equal rates.) If the steroid is removed and fresh 10% calf serum medium added, cell division commences (even if nearly 90% of protein synthesis is inhibited by cycloheximide) as early as 45 minutes later such that by 2 hours the viable cell count increases by as much as 70%. Since the growth curve after recovery resembles a step function, it appears that the cells are partially synchronized by the glucocorticoid. These results demonstrate that the glucocorticoid cytostatic effect in SV3T3 cells is the result of a block not in G1, as previously thought, but in G2.

Animals↗
PubMed1990

Genomic amplification in the cotyledon parenchyma of common bean.

The cotyledon parenchyma cells of common bean (Phaseolus vulgaris L.) produce large quantities of storage proteins during embryo maturation; throughout this period, these cells also accumulate nuclear DNA (nDNA). To investigate the basis of this nDNA accumulation, we have measured storage protein mRNA pools, nDNA mass, and gene copy number at specific stages of cotyledon development. RNA blotting and hybridization show that transcripts encoding the major embryo-specific storage proteins are present very early in cotyledon development, accumulate in coordinate fashion to peak during mid-maturation, and fall in abundance prior to the onset of dormancy. During this same period, nDNA mass per parenchyma cell nucleus, as measured by Feulgen microspectrophotometry, increases from 2C-4C to about 64C (C being the haploid germ cell genomic complement). The nDNA values do not cluster around integral multiples of the diploid 2C amount. DNA blotting and hybridization are used to evaluate the relative representations of different classes of the bean genome in DNA samples isolated from vegetative tissues, from cotyledons beginning to accumulate storage proteins, and from cotyledons of late maturation embryos entering dormancy. The results demonstrate that the observed DNA accumulation in the cotyledon parenchyma is due to overlapping rounds of replication of the complete genome and not to disproportionate amplification of specific sequences nor to random DNA synthesis.

Cell Nucleus↗
PubMed1988

A simple and efficient procedure for the isolation of high-quality phage lambda DNA using a DEAE-cellulose column.

A simple and rapid procedure for purifying large quantities of bacteriophage lambda particles and DNA is described. The procedure involves DEAE-cellulose column chromatography of the phage particles and elution of the phage particles from the column with a low-ionic-strength buffer. The resulting phage were well separated from RNA, DNA, and proteins derived from Escherichia coli host cells. The lambda DNA was prepared from the purified phage particles by the conventional method of phenol extraction and ethanol precipitation. This procedure did not use nucleases, proteases, detergents, or CsCl density gradient centrifugation. The lambda DNA obtained by this method was equivalent in purity to the material prepared by CsCl density gradient centrifugation and amenable to restriction enzyme digestion, ligation, radiolabeling, and double-stranded DNA sequencing. A detailed protocol is described for obtaining 0.5 to 1.0 mg DNA from a 1-liter liquid lysate in less than 5 h. This procedure is simple, inexpensive, and timesaving, and is particularly suitable for large-scale isolation of lambda DNA.

Bacteriophage lambda↗
PubMed2002

Regulation of translational initiation in plants.

The abundance of cytosolic mRNA does not necessarily correspond to the quantity of polypeptide synthesized in plant cells. The initiation of mRNA translation is regulated at the global and message-specific levels. mRNAs compete for discriminatory initiation factors that couple the 5'-(7m)GpppN-cap and the 3'-poly(A) tail of the RNA message. The resultant circularization of the mRNA promotes the association of the 43S pre-initiation complex that scans the 5'-leader for the initiation codon of the protein coding sequence. The physiological and developmental regulation of these events governs the level of polypeptide synthesis from endogenous and viral transcripts.

3' Untranslated Regions↗
PubMedDate not supplied

Hypertrophic scar tissues and fibroblasts produce more transforming growth factor-beta1 mRNA and protein than normal skin and cells.

Transforming growth factor-beta1 is a well-known fibrogenic cytokine produced by many types of cells including dermal fibroblasts. To investigate whether this fibrogenic cytokine is involved in development of hypertrophic scar, transforming growth factor-beta1 gene expression was evaluated in small skin samples. Because a sufficient quantity of normal skin from patients with hypertrophic scar is not readily available, a reverse transcription-polymerase chain reaction technique was used. Quantitation of gene expression by reverse transcription-polymerase chain reaction is difficult partly due to the lack of suitable complementary RNA standards. We have established a convenient, reliable procedure to construct an internal standard for transforming growth factor-beta1 starting with a gene specific polymerase chain reaction product. After digestion of the polymerase chain reaction product with endonuclease, a small piece of cDNA from human procollagen alpha1(I) cDNA with compatible ends was inserted into the polymerase chain reaction-DNA fragment. The recombinant cDNA was re-amplified by polymerase chain reaction and subcloned into a plasmid containing bacteriophage T7 and T3 promoters. Complementary RNA was prepared from the recombinant plasmid and amplified by reverse transcription-polymerase chain reaction together with the tissue or cellular RNA. After amplification, the products were electrophoresed in an agarose gel containing ethidium bromide. The bands for internal standard and transforming growth factor-beta1 mRNA were scanned, digitized, and plotted against the amount of internal standard complementary RNA added in the reverse transcription-polymerase chain reaction. The number of mRNA molecules/cell was calculated. We examined the transforming growth factor-beta1 mRNA in hypertrophic scar tissue and in normal skin and found that hypertrophic scar tissues expressed five-fold more transforming growth factor-beta1 mRNA than normal skin per unit of wet weight. We used this procedure to quantitate transforming growth factor-beta1 mRNA expression in 5 pairs of fibroblast cultures derived from hypertrophic scar and normal skin. The results showed that hypertrophic scar fibroblast cultures contain significantly more molecules of mRNA for transforming growth factor-beta1 than normal cells (116 +/- 6 vs. 97 +/- 7, p = 0.017, n = 5). These results were supported by Northern analysis for transforming growth factor-beta1 mRNA in the cells and enzyme-linked immunosorbent assay for TGF-beta1 protein in fibroblast-conditioned medium. In conclusion, hypertrophic scar tissue and fibroblasts produce more mRNA and protein for transforming growth factor-beta1, which may be important in hypertrophic scar formation. The construction of the gene specific internal standard for reverse transcription-polymerase chain reaction is a simple and reliable procedure useful to quantitate gene expression in a small amount of tissue or number of cells.

Biopsy, Needle↗
PubMed1996

[Study on porcine proteoglycans relevant to structure and function of temporomandibular joint].

The purpose of this study was to characterize the biochemical and immunological properties of the temporomandibular joint (TMJ). Proteoglycans (PGs) were extracted from the porcine TMJ retrodiscal tissue (RT) and superficial fibrous layer of condyle (SLC). The proportion of Glycosaminoglycans (GAGs) with TMJ disc. RT and SLC resembled that of disc and RT GAGs, but SLC GAGs were different from others. TMJ disc had a higher Dermatan sulfate/Chondroitin sulfate ratio than that of RT. and TMJ disc had stronger tensional load resistance than that of RT. Immunologically, a high molecular weight PG resembled Aggrecan and two low molecular weight PGs resembled Decorin and Biglycan. Total RNA was isolated from SLC, and RT-PCR was performed. These products were sequenced, and these sequences were compared to the sequences of porcine Aggrecan, human Decorin and Biglycan. These results suggested that TMJ had Decorin, Biglycan, and Aggrecan. The difference of PG quantity in each region seemed to be associated with the pathological conditions found on each component of TMJ.

Amino Acid Sequence↗
PubMed1995

Nesidioblastosis and persistent neonatal hyperinsulinism.

Neonatal hyperinsulinism is characterized by severe hypoglycaemia which can cause serious neurologic effects. Pancreatic morphological abnormalities involve either focal or diffuse lesions. The former can be cured by resection, whereas the latter, of uncertain pathogenesis, often require subtotal pancreatectomy. We investigated various hypotheses in an effort to explain the origin of this latter form of hyperinsulinism. We determined that nesidioblastosis, long considered to be the basic structural lesion of the diffuse form of hyperinsulinism, is not specific and does not correspond to a continuous proliferation of endocrine cells. We found that an increase in beta-cell mass can be excluded since the volume density of beta cells is not systematically higher in hyperinsulinemic infants than in controls. The hypothesis of a decrease in D cells is attractive but should be considered with due caution since the decrease of the D-cell volume density observed in hypoglycaemic infants is inconstant. Finally, the notion of beta-cell functional abnormality seems the most likely explanation since a higher quantity of proinsulin was detected within the Golgi area by a specific antibody and abnormal nuclei with abundant cytoplasm were observed in some cells. These histological abnormalities can be observed during intraoperative morphological examination. Functional activity might also be evaluated by studying the messenger RNA of proinsulin.

Chronic Disease↗
PubMed1989

Association of prolactin and insulin receptors with mammogenesis and lobulo-alveolar formation in pregnant ewes.

1. Pregnant, multiparous ewes were sacrificed at d 50 (n = 3), 80 (n = 4), 115 (n = 3), or 140 (n = 4) for biochemical, histological and autoradiographical quantitation of mammary growth. 2. Significant increases in concentration of mammary parenchyma DNA (0.25-24 mg/g tissue), total under DNA (57-1304 mg), and total under RNA (36-1504 mg) were observed by d 115. 3. Mammary tissue at d 115 contained the maximal percent of tissue volume occupied by epithelium (41.2%), number of cells per alveolar cross section (36.6) and percent of [methyl-3H]thymidine labeled epithelial cell nuclei (3.5%). 4. Concentration prolactin binding sites were significantly increased at d 115 of gestation, but serum prolactin and growth hormone concentrations remained low until d 140. 5. Mammary insulin binding sites (per unit membrane protein) progressively decreased during gestation. 6. Results suggest that serum growth hormone concentration and quantity of insulin receptors do not limit mammogenesis but greater concentrations of prolactin binding sites coincident with lobulo-alveolar formation, suggest a primary association with mammogenesis in the ewe.

Animals↗
PubMed2005

esiRNAs purified with chromatography suppress homologous gene expression with high efficiency and specificity.

Many preclinical studies have shown RNA interference (RNAi) as a new promising way to treat various human diseases including cancer and virus infection and there is an increasing demand for the large-scale preparation of short interfering RNAs (siRNAs) at low cost. Data are accumulating to show that endoribonuclease-prepared siRNAs (esiRNAs) are superior to chemically synthesized siRNAs in terms of expense, efficiency, and specificity. Yet all procedures available for esiRNA purification were designed to produce small amount of siRNAs for laboratory use. In this article, a new method of purification of esiRNAs based on ion exchange chromatography and size exclusion chromatography is reported. The esiRNAs prepared with this method are shown here to be of high purity and specifically suppress homologous gene expression without activating interferon response and with higher efficiency than chemically synthesized siRNAs. We can expect that the new method can be scaled up easily to provide large quantities of esiRNAs to meet the requirement of preclinical and clinical studies.

Chromatography, Gel↗
PubMed1997

Dichloromethane metabolism to formaldehyde and reaction of formaldehyde with nucleic acids in hepatocytes of rodents and humans with and without glutathione S-transferase T1 and M1 genes.

Metabolism of dichloromethane (DCM) to formaldehyde (HCHO) via a glutathione S-transferase (GST) pathway is thought to be required for its carcinogenic effects in B6C3F1 mice. In humans, this reaction is catalyzed primarily by the protein product of the gene GSTT1, a member of the Theta class of GST, and perhaps to a small extent by the protein product of the gene GSTM1. Humans are polymorphic with respect to both genes. Since HCHO may bind to both DNA and RNA forming DNA-protein crosslinks (DPX) and RNA-formaldehyde adducts (RFA), respectively, these products were determined in isolated hepatocytes from B6C3F1 mice, F344 rats, Syrian golden hamsters, and humans to compare species with respect to the production of HCHO from DCM and its reaction with nucleic acids. Only mouse hepatocytes formed detectable amounts of DPX, the quantities of which corresponded well with quantities of DPX formed in the livers of mice exposed to DCM in vivo [Casanova, M., Conolly, R.B., and Heck, H. d'A. (1996). Fundam. Appl. Toxicol. 31, 103-116]. Hepatocytes from all rodent species and from humans with functional GSTT1 and GSTM1 genes formed RFA. No RFA were detected in human cells lacking these genes. Yields of RFA in hepatocytes of mice were 4-fold higher than in those of rats, 7-fold higher than in those of humans, and 14-fold higher than in those of hamsters. The RFA:DPX ratio in mouse hepatocytes incubated with DCM was approximately 9.0 +/- 1.4, but it was 1.1 +/- 0.3 when HCHO was added directly to the medium, indicating that HCHO generated internally from DCM is not equivalent to that added externally to cells and that it may occupy separate pools. DPX were not detected in human hepatocytes even at concentrations equivalent to an in vivo exposure of 10,000 ppm; however, the possibility that very small amounts of DPX were produced from DCM cannot be excluded, since HCHO was formed in human cells. Maximal amounts of DPXliver that might be formed in humans were predicted from the amounts in mice and the relative amounts of RFA in hepatocytes of both species. With predicted DPXliver as the dosimeter, the unit risk, the upper 95% confidence limit on the cancer risk, and the margin of exposure were calculated at several concentrations using the linearized multistage and benchmark dose methods. Since the actual delivered dose is smaller than that predicted, the results suggest that DCM poses at most a very low risk of liver cancer to humans.

Animals↗
PubMed1992

Rapid increase in relaxin gene expression in early pregnancy in the pig.

Relaxin mRNA concentrations in porcine corpora lutea were examined during the peri-implantation period and throughout pregnancy using Northern and slot blot analysis. Total RNA was extracted from corpora lutea obtained from pigs of known breeding dates and pregnancy was confirmed by embryo recovery. A 32P-labelled porcine relaxin cDNA probe identified the 1.0 kilobase relaxin transcript. Slot blots were subsequently used to quantify relaxin mRNA concentrations. Relaxin mRNA was detectable in the corpus luteum of the regular cycle and was also present at similar low levels in corpora lutea of days 10, 11 and 12 of pregnancy. In corpora lutea from day 16 of pregnancy onwards 100-fold greater quantities of relaxin mRNA were observed. The intensity remained similar in samples between days 16 and 102 of pregnancy. These studies indicate that elevated relaxin gene expression commences very early in pregnancy and is first detectable in the peri-implantation period.

Animals↗
PubMed2004

Secretion of oncostatin M by neutrophils in rheumatoid arthritis.

OBJECTIVE: Neutrophils are known to express and release a large number of proinflammatory cytokines when they are stimulated by inflammatory stimuli. The objective of this study was to determine whether neutrophils express oncostatin M (OSM), a member of the interleukin-6 family of cytokines that has been implicated in the pathogenesis of inflammatory joint disease. METHODS: Neutrophils were isolated from the blood of healthy volunteer donors and from the blood and synovial fluid of patients with rheumatoid arthritis (RA). OSM levels were measured in cell extracts and in culture supernatants by Western blotting. Total RNA was isolated from control and granulocyte-macrophage colony-stimulating factor (GM-CSF)-treated neutrophils, and OSM messenger RNA levels were quantified by hybridization of a radiolabeled probe. RESULTS: GM-CSF stimulated a rapid and transient expression and release of OSM from blood neutrophils, which was more rapid than the expression and release from blood monocytes. A 28-kd protein was identified in cell extracts, but an additional 25-kd isoform was detected in culture supernatants. Synovial fluid neutrophils could not be stimulated to express OSM, but this cytokine was detected in cell-free supernatants at various levels. CONCLUSION: Blood neutrophils can be stimulated to express and rapidly release large quantities of OSM. We propose that this important cytokine is released from neutrophils as they infiltrate rheumatoid joints and, thus, contribute to the complex cytokine network that characterizes RA.

Arthritis, Rheumatoid↗