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PubMed1975

Characterization of an inhibitor causing potassium chloride sensitivity of an RNA polymerase from T4 phage-infected Escherichia coli.

The nature of the inhibition by salt (KCl) of DNA-dependent RNA polymerase from T4 phage-infected Escherichia coli (T4 enzyme) was studied using holoenzyme preparations, core enzyme and sigma fractions obtained by phosphocellulose column chromatography, and sigma fractions further purified by gradient centrifugation in the presence and absence of 6 M urea. We showed with holoenzyme preparations that salt inhibits the formation of rifampicin-resistant preinitiation complexes. The inhibition was considerably reduced when a nonionic detergent (particularly of the Triton series) was included in the reaction mixtures. With T4 core enzyme and T4 sigma fractions together with the same fractions from uninfected cells (host enzyme fractions) and different DNA templates, we showed that the T4 sigma fraction plays a role in the salt-sensitive activity with T4 DNA. The salt sensitivity of the T4 sigma fraction was antagonized by Triton; it was not a function of sigma fractions isolated from phage cultures infected in the presence of chloramphenicol. As reported previously (Stevens, A. (1973), Biochem. Biophys. Res. Commun. 54, 488), the T4 sigma fraction inhibited the activity of host sigma when they were present together in reaction mixtures, particularly in the presence of salt. T4 sigma further purified by centrifugation in glycerol gradients had the same properties as the cruder fraction, and the T4-specific polypeptide of mol wt 10000 (Stevens, A. (1972), Proc. Natl. Acad. Sci. U.S.A. 69, 603) was found in the same fractions. If the glycerol gradients contained 6 M urea, the mol wt 10000 polypeptide was separated from the salt-stimulated sigma. Fractions containing the small polypeptide could be added back to produce the salt-inhibitory effects. The inhibitory activity of both the crude sigma fraction and the fractions containing the small polypeptide was inactivated at 65 degrees C. The results suggest that the mol wt 10000 protein is a salt-promoted inhibitor, but the small amounts of it which are present in purified fractions of the T4 enzyme have not yet allowed its isolation in large enough quantities to permit a detailed study of its properties.

Coliphages↗

PubMed1984

Induction of human interleukin 1 mRNA measured by collagenase- and prostaglandin E2-stimulating activity in rheumatoid synovial cells.

Human blood peripheral monocyte/macrophages release in culture a mononuclear cell factor (MCF) which stimulates the production of collagenase and prostaglandin E2 by human rheumatoid synovial cells and dermal fibroblasts. These two products play a role in connective tissue destruction. MCF has an apparent molecular weight of approximately 15 000 and is biologically and biochemically indistinguishable from interleukin 1. MCF therefore belongs to the well-documented nonimmune biological activities attributed to interleukin 1. Studies on the mechanisms of production and action of such monokine(s) have been difficult in view of the minute quantities produced by freshly isolated cells or from human monocytic lines. Starting from lectin-stimulated human blood mononuclear cells, we have isolated poly(A)+ RNA and studied its translation following microinjection into Xenopus laevis oocytes. The mRNA translation products stimulated collagenase and prostaglandin E2 production in human rheumatoid synovial cells and dermal fibroblasts. The size of MCF-mRNA was estimated to be 10 S. The mRNA of a member of the interleukin 1 family can now be studied in a system based on a specific and direct relevant biological assay and eventually compared with those of other monokines.

Animals↗

PubMed1995

Applied molecular biology of sepsis.

The effective treatment of sepsis and septic shock has remained elusive despite intense research efforts. The tools of molecular biology have been applied to the problem of sepsis in an attempt to design more rational, directed therapy. Cellular interactions with invading microorganisms begin a series of stimulation events within the cell. One of the important interactions is the binding of lipopolysaccharide (LPS) from gram-negative bacteria to the LPS binding protein, and then this complex binding to CD14 on monocytes. Cell stimulation occurs through activation of signal transduction pathways within the cell, many of which have been defined. These include the kinases that phosphorylate proteins, and phosphatases that dephosphorylate proteins. The next step after activation of the signal transduction pathways is stimulation of nuclear regulatory factors. One of the best characterized of these is nuclear regulatory factor kappa B (NF-kappa B), which is a trans activating element that binds to specific DNA nucleotide sequences to allow transcription of downstream elements. Many inflammatory mediators are located downstream of NF-kappa B so that activation of NF-kappa B causes upregulation of the inflammatory mediators. The cytokines have been identified as a group of mediators important in the pathogenesis of sepsis, because several studies have shown that higher levels are correlated with a worse outcome in patients. Additionally, in experimental animal models, inhibition of cytokines improves survival, and administration of exogenous, recombinant cytokines reproduces many of the pathophysiologic alterations observed in sepsis. Molecular biology has played a critical role in the understanding of sepsis by providing the tools to make the recombinant cytokines of sufficient purity and quantity for infusion into experimental animals. The cellular response for the production of cytokines occurs through classic protein chemistry, with the signal transduction inducing messenger RNA (mRNA) coding for the cytokines, which are then translated and secreted. The relative contribution of local versus systemic cytokine production is beginning to be appreciated, with several diseases showing substantially higher local cytokine levels. The cytokines exert their activity on other cells by binding to their specific cytokine receptors. These receptors are part of the immune response and may be shed from the cell surface. These soluble receptors bind to and inactivate the cytokines. Inhibition of cytokine activity has been hypothesized as a potential therapy for sepsis. This inhibition has been done with antibodies directed against either the cytokines themselves or their receptors. Naturally occurring cytokine inhibitors have been cloned and expressed by molecular biologists and used for treatment of sepsis and other diseases. Using molecular biology techniques, the murine antibodies have been "humanized" to reduce their immunogenicity. The measurement of cytokines is critically important to our understanding of their role in health and disease. Cytokines may be measured by either immunologic methods or biological assays. Molecular biology has made important contributions to our understanding of sepsis by precisely identifying some of the mediators and providing reagents for therapeutic use.

Cytokines↗

PubMedDate not supplied

Rapid, high-yield recovery of a recombinant digoxin binding single chain Fv from Escherichia coli.

We have isolated milligram quantities of active single chain antibody from the insoluble fraction of Escherichia coli cultures. The system relies on high-level expression from a T7 RNA polymerase-directed gene construct, 8 M urea to dissolve the desired protein out of the insoluble fraction, presumably inclusion bodies, isolation and concentration of the desired protein by nickel chelate [IDA-Ni(II)] immobilized metal-ion affinity chromatography (IMAC), and removal of urea from column fractions by dialysis directly into storage buffer. Routinely, about 50% of the protein loaded onto an IMAC column is recovered as single chain Fv at a concentration of approximately 0.7 mg/mL. As little as 3 days are required to obtain 10 mg of final product when starting with an overnight inoculum.

Base Sequence↗

PubMed1971

3 -Hydroxy steroid dehydrogenase activity in the mitochondria of rat adrenal homogenates.

The activity of 3beta-hydroxy steroid dehydrogenase (EC 1.1.1.51) in the mitochondrial fraction of rat adrenal homogenates was approx. 31% of the total activity recovered after differential centrifugation and washing of the particulate fractions. Some 45% of the total activity was found in the microsomal fraction. The activity was assayed by a radioisotopic method devised in this laboratory for the purpose of studying small quantities of tissue and cell fractions. Satisfactory separation of the two fractions was demonstrated by electron microscopy of the pellets and by comparative recoveries of RNA, steroid 21-hydroxylase and cytochrome c oxidase in the various compartments. Analyses of the kinetics of the enzyme activity in the two fractions revealed no significant differences in apparent K(m) for pregnenolone, dehydroepiandrosterone or NAD(+), but demonstrated a distinct difference in the K(m) for NADP(+). pH optima and susceptibility to cyanoketone inhibition were similar in both fractions.

Adrenal Glands↗

PubMed1983

Development of translationally active mRNA for larval muscle acetylcholinesterase during ascidian embryogenesis.

Relative quantities of translationally active acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) mRNA present at various developmental stages were compared in embryos of the ascidian Ciona intestinalis. Purified RNA was tested for its translational capacity by microinjection into Xenopus laevis oocytes; the acetylcholinesterase produced was immunoprecipitated with antibody to Ciona acetylcholinesterase and enzyme activity was assayed radiometrically. With this protocol, enzyme synthesis was found to be directly related to the amount of RNA injected and to the oocyte incubation time. A functional template for acetylcholinesterase was first detected at 6 hr of development (late gastrula) and is probably present as early as 5 hr. The level of this template activity increased until the middle tail formation stage (11-12 hr after fertilization) and then remained constant until 16 hr of development (the final stage examined), 2 hr before hatching. These findings, and the results of previous actinomycin D inhibition experiments, indicate that mRNA for ascidian larval muscle acetylcholinesterase is first synthesized during gastrulation.

Acetylcholinesterase↗

PubMed2004

[Raman spectroscopy study of 1,3-dimethyluracil and its ab initio (DFT) calculation].

The Raman spectrum of polycrystal 1,3-dimethyluracil (DMU) was obtained and investigated. Ab initio (DFT) calculation (B3PW91/6-31G method) of DMU was performed. And the vibration modes of DMU molecular were analyzed. The results suggest that DMU molecule, as a model compound of bio-base, has some advantages in Raman analysis, such as stronger intensity of marker bands and sharper, clearer bands of double band stretching than uracil's. The ultimate goal of this research is to get large quantity of spectral information of DMU and its cyclobutane dimers, use them as model compounds to research on the Raman characteristics of DNA's or RNA's photodamage. And this work is also an essential supplement to the 1,3-dimethyluracil's Raman study.

Calcium Phosphates↗

PubMedDate not supplied

Production of radiolabeled neuropeptide precursors by in vitro transcription and translation.

Bioactive peptide hormones and neurotransmitters are required for neuroendocrine regulation of cellular functions. Importantly, proteolytic processing of inactive neuropeptide precursors is required to generate physiologically active peptide hormones and neurotransmitters. Studies of the processing enzymes require authentic neuropeptide precursors as substrates, rather than peptide substrates. This study demonstrates an efficient method to general 35S-precursors from cloned cDNAs by in vitro transcription and translation. In vitro transcription of neuropeptide cDNAs with SP6 RNA polymerase generates large amounts (micrograms) of corresponding RNAs. Subsequent in vitro translation of RNAs with wheat germ extract and 35S-methionine generates large quantities of 35S-precursors (10-25 million cpm 35S-precursor protein per reaction) with high specific radioactivity. The radiolabeled precursor substrates offer a reliable, sensitive and accurate method for detecting the proteolytic activity. Importantly, specific detection of the primary proenkephalin processing activity in chromaffin granules by 35S-enkephalin precursor as substrate, but not by peptide methylcoumarinamide (MCA) substrates, illustrates the significance of using full-length precursor to detect appropriate processing enzymes. This study demonstrates that efficient production of radiolabeled neuropeptide precursors by in vitro transcription and translation will be useful for in vitro assays of relevant processing proteases.

Animals↗

PubMedDate not supplied

[Influence of denervation of the liver on its level of nucleic acids and free adenine nucleotides].

It is established that liver denervation is accompanied with a significant increase of DNA content on the 10, 20 and 30th day after operation: 19, 22 and 25%, respectively. The 12% rise on the average in RNA content is observed on the 10th day. This level is preserved during the following days after denervation. The changes in the content of free adenine nucleotides are contrary. The decrease in ATP quantity, which is insignificant on the 10th day, makes progress and reaches 20% on the 20th day. The decrease in ADP is 13%, the content of AMP remains practically unchanged. The obtained data evidence for the participation of the nervous system in maintaining the normal level of poly- and mononucleotides, specific for the liver.

Adenine Nucleotides↗

PubMed2005

Critical role of Epstein-Barr Virus (EBV)-encoded RNA in efficient EBV-induced B-lymphocyte growth transformation.

It was demonstrated that Epstein-Barr virus (EBV)-encoded small RNAs (EBERs) were nonessential for B-lymphocyte growth transformation. We revisited this issue by producing a large quantity of EBER-deleted EBV by using an Akata cell system. Although the EBER-deleted virus efficiently infected B lymphocytes, its 50% transforming dose was approximately 100-fold less than that of the EBER-positive EBV. We then engineered the genome of EBER-deleted virus and generated a recombinant virus with the EBER genes reconstituted at their native locus. The resultant EBER-reconstituted EBV exhibited restored transforming ability. In addition, lymphoblastoid cell lines established with the EBER-deleted EBV grew significantly more slowly than those established with wild-type or EBER-reconstituted EBV, and the difference between the growth rates was especially highlighted when the cells were plated at low cell densities. These results clearly demonstrate that EBERs significantly contribute to the efficient growth transformation of B lymphocytes by enhancing the growth potential of transformed lymphocytes.

B-Lymphocytes↗

PubMed1985

Immunoactive products of placenta: VIII. Translation products of messenger RNA extracted from murine placenta or A6B9IC5 teratocarcinoma are immunosuppressive in vitro.

RNA was extracted from placentae of synpregnant C3H mice (day 14 of gestation) and from A6B9IC5 teratocarcinoma. After isolation of poly-(A)+ RNA on an oligo d(T) cellulose column, putative messenger RNA was injected into Xenopus laevi oocytes. Supernatants and homogenates from the oocytes were assayed for immunoregulatory activity in MLR, CML, and NK assays and found to be immunosuppressive. These data indicate that immunomodulators could be obtained from the placenta by molecular biology technology in sufficient quantities for eventual clinical applications.

Animals↗

PubMed2006

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

In this study, we examined several molecular markers in prostate and breast cancer patients and in normal individuals. The markers tested were: variations in the quantity of plasma DNA, glutathione-S-transferase P1 gene (GSTP1), Ras association domain family 1A (RASSF1A), and ataxia telangiectasia mutated (ATM) 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. DNA quantification in plasma was performed using real-time PCR (RT-PCR). We assessed the methylation status of GSTP1 in plasma DNA using methylation-specific PCR (MSP) assay, while the methylation status of RASSF1A and ATM genes was examined by the MethyLight technology. RT-PCR analysis was used for the detection of mRNA, PSMA, and CEA. In 58.3% of newly diagnosed prostate cancer patients and 26.7% of prostate cancer patients under therapy, plasma DNA levels were increased. Additionally, 48.5% of breast cancer patients showed plasma DNA levels above the cutoff limit. GSTP1 Promotor hypermethylation was detectable in 75% of plasma samples obtained from patients with newly diagnosed prostate cancer and in 36.8% of patients under therapy, whereas 26% and 14% of the breast cancer patients tested were positive for RASSF1A and ATM methylation, respectively. The combination of DNA load and promotor methylation status identified 88% of prostate cancer patients and 54% of breast cancer patients. This study shows that free-circulating DNA can be detected in cancer patients compared with disease-free individuals, and suggests a new, noninvasive approach for early detection of cancer.

Antigens, Surface↗

PubMed1984

Biochemical studies of the tracheobronchial epithelium.

Tracheobronchial epithelium has been a focus of intense investigation in the field of chemical carcinogenesis. We have reviewed some biochemical investigations that have evolved through linkage with carcinogenesis research. These areas of investigation have included kinetics of carcinogen metabolism, identification of carcinogen metabolites, levels of carcinogen binding to DNA, and analysis of carcinogen-DNA adducts. Such studies appear to have provided a reasonable explanation for the susceptibilities of the respiratory tracts of rats and hamsters to carcinogenesis by benzo(a)pyrene. Coinciding with the attempts to understand the initiation of carcinogenesis in the respiratory tract has also been a major thrust aimed at effecting its prevention both in humans and in animal models for human bronchogenic carcinoma. These studies have concerned the effects of derivatives of vitamin A (retinoids) and their influence on normal cell biology and biochemistry of this tissue. Recent investigations have included the effects of retinoid deficiency on the synthesis of RNA and the identification of RNA species associated with this biological state, and also have included the effects of retinoids on the synthesis of mucus-related glycoproteins. Tracheal organ cultures from retinoid-deficient hamsters have been used successfully to indicate the potency of synthetic retinoids by monitoring the reversal of squamous metaplasia. Techniques applied to this tissue have also served to elucidate features of the metabolism of retinoic acid using high pressure liquid chromatography. In brief, formidable strides have been made in biochemistry specific to this important target tissue, despite the inability to acquire tracheobronchial epithelium in large quantities.

Animals↗

PubMedDate not supplied

Secondary heterotypic versus homotypic infection by Coxsackie B group viruses: impact on early and late histopathological lesions and virus genome prominence.

The impact of prior exposure to a different or identical strain of Coxsackievirus B (CVB) on murine CVB myocarditis was studied using a susceptible murine host (A/J[H-2a]) and myocarditic CVB3 or avirulent CVB2 as primary or secondary infectants. The effects of secondary heterotypic infection (CVB2 followed by CVB3) and homotypic infection (CVB3 followed by CVB3) 28 days after primary inoculation, versus CVB2 or CVB3 alone, on injury and viral genomic replication, both early (day 7) and late (days 28 and 56), were evaluated. After the primary infection by CVB2, trivial viral RNA was present in the heart and other organs, and a substantial positivity was observed with CVB3 infection. Seven days after secondary heterotypic (CVB2-CVB3) infection, the quantity of CVB genome in heart, pancreas, liver, and spleen was increased compared with the virus genome in the CVB3-CVB3 group and in the group with primary CVB3 infection alone. This phenomenon was seen in the heart and spleen up to day 28 postsecondary infection. Tissue inflammation and necrosis in heart and pancreas were prominent 7 days postsecondary infection with CVB2-CVB3 and correlated well with an increased quantity of CVB genome. Virus genome was present in heart and spleen 28 days after CVB3 infection alone. Serum CVB3 neutralization titer was increased to 1:128 in CVB2-CVB3 group at days 7 and 28 postsecondary infection, and serum completely neutralized cytopathological effects of CVB3 in the CVB3-CVB3 group at day 7 and 28 postsecondary infection. Our results indicate that secondary heterotypic infection by CVB causes increased injury, inflammation, and CVB replication in target organs such as the heart and pancreas, as well as in immune compartments like the spleen. Compared with CVB3 alone, the intense inflammatory infiltriate in the CVB2-CVB3 group is as not due solely to postviral sensitization of the immune system, but rather to the inability of the host to eradicate the virus.

Animals↗

PubMed2005

High affinity nucleocapsid protein binding to the muPsi RNA packaging signal of Rous sarcoma virus.

The genomes of all retroviruses contain sequences near their 5' ends that interact with the nucleocapsid domains (NC) of assembling Gag proteins and direct their packaging into virus particles. Retroviral packaging signals often occur in non-contiguous segments spanning several hundred nucleotides of the RNA genome, confounding structural and mechanistic studies of genome packaging. Recently, a relatively short, 82 nucleotide region of the Rous sarcoma virus (RSV) genome, called muPsi, was shown to be sufficient to direct efficient packaging of heterologous RNAs into RSV-like particles. We have developed a method for the preparation and purification of large quantities of recombinant RSV NC protein, and have studied its interactions with native and mutant forms of the muPsi encapsidation element. NC does not bind with significant affinity to truncated forms of muPsi, consistent with earlier packaging and mutagenesis studies. Surprisingly, NC binds to the native muPsi RNA with affinity that is approximately 100 times greater than that observed for other previously characterized retroviral NC-RNA complexes (extrapolated dissociation constant K(d)=1.9 nM). Tight binding with 1:1 NC-muPsi stoichiometry is dependent on a conserved UGCG tetraloop in one of three predicted stem loops, and an AUG initiation codon controvertibly implicated in genome packaging and translational control. Loop nucleotides of other stem loops do not contribute to NC binding. Our findings indicate that the structural determinants of RSV genome recognition and NC-RNA binding differ considerably from those observed for other retroviruses.

Amino Acid Sequence↗

PubMed1986

The Drosophila melanogaster actin 5C gene uses two transcription initiation sites and three polyadenylation sites to express multiple mRNA species.

At least six mRNAs are made from the Drosophila melanogaster act5C gene. We investigated the structures of these RNAs in detail and determined that they are heterogeneous at both their 5' and 3' ends. At the 5' end there were two nonhomologous leader exons which were alternately spliced to the remainder of the gene. These leader exons mapped to 1.7 and 0.7 kilobases, respectively, upstream of a common splice acceptor site which was eight base pairs 5' to the translation initiator AUG. Exon 1 is 147 bases in length, while exon 2 is 111 bases. A consensus TATA sequence was found roughly 30 base pairs upstream from exon 1, but none was found in the analogous position upstream of exon 2. The transcript length diversity arose principally from the use of three polyadenylation sites. This gave rise to RNA molecules with 3'-untranslated regions of roughly 375, 655, and 945 base pairs. With two start sites and three termination sites, this gene has the potential to produce six different transcripts. All six possible transcripts were present in whole fly mRNA. Transcripts containing the two different leader exons were found in roughly the same relative quantities through development. In contrast, the various 3' ends were differentially represented through development.

Actins↗

PubMed1975

[The preparation and characterization of transfer factor (author's transl)].

A transfer factor (TF) was prepared and characterized from the leucocytes of Mantoux-positiv donors using a variety of techniques. The results allowed the following conclusions: Whilst extraction by dialysis and phenol (without precipitation) led to nearly the same positiv immunological activities, when assayed in the MEM-test, phenol extracts subjected to RNA-precipitation lost considerable activity. The residual activity was associated with the lower molecular fractions, and RNA itself has shown no activity. Residues after leucocyte dialysation have also shown considerable TF-activity when extracted by phenol. It was found that phenol extraction followed by gel filtration allowed recovery of considerable quantities of active TF, confined to several specific filtration fractions. Combination of active and inactive gel filtration fractions suggested that there existed an inhibitory factor within the extracted leucocyte preparations. This modified preparation and purification procedure may allow the preparation of TF for therapeutic purposes.

Chromatography, Gel↗

PubMed1987

Breda and Breda-like viruses: diagnosis, pathology and epidemiology.

Breda virus serotype 1 (Iowa) was isolated from a severe outbreak of neonatal calf diarrhoea in which 15% of the diarrhoeic animals died. Two further isolates, one from Iowa and one from Ohio, are antigenically related to the first isolate but belong to the Breda virus serotype 2 group by haemagglutination inhibition and by immuno-electron microscopy (IEM). The majority (88.5%) of cattle are serologically positive for Breda virus by enzyme-linked immunosorbent assay (ELISA). The diagnostic methods of choice should include a combination of electron microscopy, IEM, haemagglutination inhibition or ELISA, in order to identify all serotypes of Breda virus. Serotypes 1 and 2 infect and replicate in crypt and epithelial cells of the small intestine, from the mid jejunum region posteriorly through to the descending colon. Virus replicates in and is released from cells before the appearance of microscopic lesions and clinical signs. Diarrhoea starts within 24-72 hours of infection and death can occur within 36-48 hours after the onset of diarrhoea. The morphology of Breda virus, determined by negative staining and from ultrastructural studies of infected cells, is unique and probably diagnostic. Little is known about the epidemiology, other than that transmission is faecal-orally and may require close contact. Large quantities of virus are released in the faeces.

Animals↗