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Cell-free protein synthesis using cell extract of Pseudomonas fluorescens and CspA promoter.

We have modified the cell-free coupled transcription/translation system of bacteria. The cell-free extract of Pseudomonas fluorescens was used for translation instead of Escherichia coli. In addition, transcription of the target gene was regulated by CspA promoter with endogenous RNA polymerase instead of by T7 promoter with exogenous T7 RNA polymerase. We could increase the yields of soluble proteins using different combinations of the S30 extract and the promoter and different temperatures for protein synthesis. Increasing the variety of synthesis systems allows production of large quantities of soluble proteins. In order to carry out efficient cell-free protein synthesis, versatile pCop-plasmids carrying CspA promoter were constructed and these plasmids were applicable to expression of recombinant proteins in E. coli cells.

Amino Acid Sequence↗

Construction of a gene probe for detection of P virus (Reoviridae) in a marine decapod.

The construction is described of a molecular probe to P virus, a double stranded RNA virus belonging to the Reoviridiae, which is an endemic pathogen of swimming crabs in British coastal waters and the Mediterranean. The probe hybridises to the P virus genome and can be easily produced in large quantities by PCR. It may be used by dot blotting or in situ hybridisation to specifically detect P virus in tissues and cells of natural or experimentally infected animals. Analyses of tissue samples with this probe show that the virus infects connective tissues of gills and hepatopancreas. This is the first gene probe to be constructed for a native viral pathogen of temperate water brachyurans and it will be useful to study virus ecology and virus-host interactions in vivo and in vitro. An understanding of these processes is essential to control and manage disease and, ultimately, to identify immune effectors capable of destroying viral pathogens.

Animals↗

Immunoaffinity purification and properties of a high molecular weight calf thymus DNA alpha-polymerase.

A rapid, three-step purification of DNA alpha-polymerase from calf thymus is described. The key feature is immunoaffinity chromatography using a column of immobilized monoclonal immunoglobulin G (IgG) developed against human KB cell alpha-polymerase. This step is followed by preparative sucrose gradient sedimentation. The highly purified polymerase has a specific activity of 35 000 nmol of nucleotide incorporated per hour per milligram. Its molecular weight is 404 000. This molecular weight is higher than observed in some earlier purifications, possibly because salt concentrations are kept at nearly physiological levels. Also, the rapidity of purification in the presence of multiple protease inhibitors minimizes degradation. The purified enzyme is inhibited by aphidicolin, N-ethylmaleimide, and the specific monoclonal IgG, thereby identifying it as DNA alpha-polymerase. ATP at 4 mM concentration stimulates enzymatic activity up to 4-fold on calf thymus DNA templates. The enzyme is also capable of priming single-stranded DNA with RNA. The procedure represents a significant advance from purifying alpha-polymerase from calf by conventional means, since it avoids ion-exchange chromatography and harsh conditions. It also minimizes the time required to produce sufficient quantities of purified high molecular weight polymerase for analysis.

Animals↗

Penicillium stoloniferum virus: large-scale concentration and purification by polyethylene glycol.

An experimental procedure developed to concentrate fungal viruses from large volumes of homogenized mycelia with water-soluble polymers, such as polyethylene glycol, possesses advantages over more conventional methods. Concentration of the Penicillium stoloniferum fast-moving virus from mycelial homogenates after addition of polyethylene glycol was rapid and produced large quantities of pure virus.

Ammonium Sulfate↗

Ro(SS-A) and anti-Ro(SS-A): an update.

Anti-Ro/SSA antibodies are the antinuclear antibodies most commonly found in everyday clinical practice and are closely associated with Sjögren's syndrome, systemic lupus erythematosus and neonatal lupus. They play a pathogenic role in a variety of clinical manifestations, including skin lesions and neonatal lupus heart block. Autoantibodies to Ro(SS-A) recognize a ribonucleoprotein complex composed of small single-stranded RNAs (hYRNAs) and of one or more peptides. Four molecular forms of this complex have been differentiated based on the nature of the peptide: a lymphocyte and an erythrocyte Ro with a 60 kDa peptide, a lymphocyte Ro with a 52 kDa peptide and an erythrocyte Ro with a 54 kDa peptide. The Ro complex is found in most tissues and cells (erythrocytes, platelets), with differences in structure and quantity across tissues, species and embryonic development stages. Although its function remains unknown, its ability to bind nucleic acids and the fact that it shares homologies with gene regulation proteins suggest that it may participate in RNA transcription processes. A number of environmental factors (exposure to ultraviolet radiation, viral infections) may cause translocation of the Ro complex to nucleocytoplasmic and membrane sites where it is not normally found, thereby leading to the development of autoimmunity. The structure of the Ro(SS-A)-hYRNA complex and the development of autoimmunity are currently the focus of active research efforts that can be expected to improve our understanding of the clinical manifestations directly related to anti-Ro(SS-A) antibodies.

Antibodies↗

Growth velocity, fat-free mass and energy intake are inversely related to viral load in HIV-infected children.

The study objectives were to assess the relationships among human immunodeficiency virus (HIV) replication, energy balance, body composition and growth in children with HIV-associated growth failure (GF). Energy intake and expenditure, body composition and level of HIV RNA were measured in 16 HIV-infected children with growth failure (HIV+/GF+), defined as a 12-mo height velocity </= 5th percentile for age, and 26 HIV-infected children with normal rates of growth (HIV+/GF-). Energy intake was measured by repeated 24-h dietary recall, resting energy expenditure (REE) by indirect calorimetry and total energy expenditure (TEE) by the doubly labeled water method. Fat-free mass (FFM) was determined by dual X-ray energy absorptiometry and plasma HIV RNA by the polymerase chain reaction method. The mean plasma HIV RNA content among the HIV+/GF+ group was nearly 1.5 log higher than that of the HIV+/GF- group (4. 89 +/- 1.08 vs. 3.43 +/- 1.64 x10(2) copies/L, P: = 0.009). The mean daily energy intake, and age-adjusted REE and TEE were lower in HIV+/GF+ children (P: = 0.003, 0.06 and 0.16, respectively). HIV+/GF+ children had a mean daily energy deficit of 674 +/- 732 kJ/d compared with HIV+/GF- children who had a mean energy surplus of 1448 +/- 515 kJ/d (P: = 0.030). There were no differences in REE after adjustment for differences in FFM and age using multiple regression analysis (P: = 0.88). There was a significant inverse relationship between FFM and plasma HIV RNA [R:(2) = 0.64, standard error of the estimate (SEE) = 3.23] and between viral load and 12-mo growth velocity (R:(2) = 0.61, SE = 1.51). Viral load and energy intake were also inversely related (R(2) = 0.17, SEE = 573.2, P: = 0. 0125). In HIV-infected children, rate of growth, quantity of FFM and energy intake are closely related to the level of HIV replication. The energy intake of children with HIV-associated GF may not be adequate for supporting normal development of FFM and growth, despite possible decreases in total energy expenditure.

Body Composition↗

Morphological and biochemical changes in the hepatic endoplasmic reticulum and golgi apparatus of male Xenopus laevis after induction of egg-yolk protein synthesis by oestradiol-17 beta.

This report describes morphological and biochemical changes accompanying oestrogen induced synthesis of the egg-yolk protein precursor, vitellogenin, in male Xenopus liver. Extensive proliferation of the rough and smooth endoplasmic reticulum and the Golgi apparatus occurs between 3 and 9 days after administration of oestradiol-17 beta. Subcellular fractionation showed that microsomal fractions have an increased number of ribosomes available for protein synthesis, hormone treatment enhances the in vitro protein synthetic capacity per unit of RNA; both in microsome and ribosome preparations. Polypeptides synthesized in vitro by ribosome preparations show an enrichment in serine content after hormone treatment and an increased proportion of ribosomes can be immunoprecipitated by antibodies directed against vitellogenin. Our data are consistent with the proposal that vitellogenin is synthesized on the ribosomes of the rough endoplasmic reticulum and processed and packaged for secretion in the smooth endoplasmic reticulum and Golgi apparatus. Response to hormonal induction of vitellogenin involves an early phase in which membrane proliferation occurs in order to increase the cellular capacity to synthesize, process and secrete large quantities of egg-yolk protein precursor.

Animals↗

Intrahepatic cytokine expression is downregulated during HCV/HIV co-infection.

HIV co-infection is associated with reduced HCV treatment response rates and accelerated HCV-related liver disease. Cytokines play an important role in regulating hepatic inflammation and fibrogenesis during chronic HCV infection, yet the roles of HIV and/or its therapies on cytokine expression are unknown. Total RNA was extracted from liver biopsies of 12 HCV mono-infected and 14 HCV/HIV co-infected persons. We used real-time PCR to quantify cytokines that contribute to innate and adaptive immune responses, including IFNalpha, IFNgamma, TNFalpha, TGFbeta(1), IL-2, IL-4, IL-8, IL-10, and IL-12p40. Positive- and negative-strand HCV RNA levels were quantified using a molecular beacon approach. Detection of positive-strand HCV RNA was 100% in both groups; negative-strand HCV RNA was detected in four (33%) HCV mono-infected persons and in nine (64%) HCV/HIV co-infected persons. Median strand-specific HCV RNA levels were not significantly different between the two groups. Detection rates of cytokine mRNAs were lower for the HCV/HIV co-infected group compared to the HCV mono-infected group; the detection rates for TNFalpha, IL-8, and IL-10 were statistically significant. Overall, cytokine mRNA quantities were lower for HCV/HIV co-infected compared to HCV mono-infected persons, with the exception of TGFbeta1. These data suggest that a defect in cytokine activation may occur in HCV/HIV co-infected persons that limits efficient clearance of HCV from the liver.

Biopsy↗

Studies of interactions of porphyrins with transfer RNA by high-resolution NMR.

The interactions of tetra-4N-methylpyridyl porphyrin and its zinc(II), copper(II) and manganese(III) complexes with brewer's yeast type V phenylalanine specific tRNA have been evaluated by high-resolution NMR. Differences in chemical shifts have been noted for three proton resonances in response to the presence of small quantities of the free base and the zinc and copper complexes. The protons giving rise to these signals are located on bases T54 and psi 55, both of which are involved in the primary intraloop and interloop hydrogen bonds that hold the D and T psi C loops together in the tertiary structure. In addition, broadening of specific resonances due to hydrogen bonding protons in the D stem at low ratios of porphyrin to tRNA indicates that the association of porphyrins increases the rate of imino proton exchange. The titration of the tRNA with the manganese(III) complex did not reveal shifts or specific broadening comparable to the other porphyrins at low ratios. The changes induced in the NMR spectrum of tRNA by porphyrins define their site of interaction with the polynucleotide. This site, at the outside of the elbow-bend in the tRNA 'L', is different from the locus of binding in tRNA for other classical DNA intercalators. Furthermore, a new mode of binding may be involved that is neither intercalative nor simply electrostatic.

Binding Sites↗

MxA gene expression after live virus vaccination: a sensitive marker for endogenous type I interferon.

MxA gene expression is known to be regulated tightly and exclusively by type I interferons (IFNs). The kinetics of MxA gene expression was analyzed in peripheral blood mononuclear cells from 11 healthy volunteers vaccinated with the 17-D strain of yellow fever virus. A reliable induction of MxA RNA and MxA protein was found in the absence of easily detectable serum IFN activity. Thus, steady-state MxA RNA levels were elevated 8- to 30-fold above prevaccination levels on day 5 after vaccination. The average increase of MxA protein was approximately 50-fold. In contrast, no induction of MxA RNA or MxA protein was detectable in 3 similarly vaccinated controls who were immune because of previous vaccinations. The IFN marker 2'-5'-oligoadenylate (2-5A) synthetase known to react to both type I and type II IFNs showed a similar response but did not differentiate equally well between nonimmune and immune vaccinees. beta 2-microglobulin and neopterin reacted poorly, remaining at low levels within the normal range. These results demonstrate that MxA gene expression is a good marker for detecting minute quantities of biologically active type I IFN during viral infections.

Adult↗

Development of the rat conceptus in vitro and associated changed in components of culture medium.

Rat conceptuses of pregnancy day 11 (embryonic age approximately 10-5 days) with embryos within the yolk and amniotic sacs and polar allantoic placental tissues were cultured by the 'New system' utilizing homologous serum in 60 ml roller bottles with 20% O2, 5% CO2 and 75% N2 gas environment (five conceptuses/10 ml serum). Embryonic growth and development was assessed by external morphology and somite counts, and by measurement of DNA, RNA and protein contents. The embryonic growth was considerably retarded beyond 24 h when culture was extended to 48 h. During continuous culture of conceptuses for 48 h, the partial pressure of O2 in the medium decreased gradually with simultaneous increase of pCO2 and decline of pH values. Glucose level was depleted considerably and small quantities of urea, uric acid and creatinine accumulated in the medium. The concentrations of electrolytes (Na+, K+, Cl-) and total Ca and P, and lactic dehydrogenase activity in the medium increased during the latter part of the 48 h culture period. The growth and differentiation of embryos were significantly improved by transferring conceptuses to fresh serum medium with 40% O2, 5% CO2 and 55% N2 gas phase after 22-24 h culture and continuing the culture for an additional 24 h. Embryos grown in vitro for 48 h developed 39 somites and showed a tenfold increase in DNA content and a five- to seven-fold increase in RNA and protein contents, over initial values.

Animals↗

Establishment of a screening system for selection of siRNA target sites directed against hepatitis B virus surface gene.

To study the inhibitory effects of plasmid-derived small interfering RNA (siRNA) and synthetic siRNA on the expression of the hepatitis B virus surface (HBs) gene, three plasmid-derived siRNAs and one synthetic siRNA that complement the coding region of the HBs gene were prepared. The HBs expression plasmid pHBs-EGFP was also constructed. HeLa cells were co-transfected with pHBs-EGFP and the above siRNAs. The HBs mRNA quantities were measured by reverse-transcription PCR, and the level of HBs-EGFP fusion protein was quantified by fluorescent microscope. The concentrations of the hepatitis B virus surface antigen (HBsAg) derived from the culture supernatant of transfected HepG2.2.15 cells were measured by an enzyme-linked immunosorbent assay (ELISA) kit. The results showed that the three plasmid-derived siRNAs and the synthetic siRNA can effectively reduce the quantities of HBs mRNA and protein. The plasmid-derived siRNA psiRNA1 was found to be the most effective inhibitor of HBs expression. It can inhibit HBs-EGFP expression by 63.3% and suppress HBs mRNA by 75.6%. To further substantiate the above observations, psiRNA1 was transfected into HepG2.2.15 cells (an HBV secreting cell line). The transfections resulted in almost complete blockage of HBsAg production, whereas control vector-transfected cells secreted high levels of HBsAg 7 days post-transfection. In conclusion, our data suggests that RNA interference (RNAi) is an efficient approach for reducing the level of HBs transcripts and proteins and for suppressing HBsAg production.

Base Sequence↗

WT1 gene expression lowered by IL-12 In vitro in peripheral blood mononuclear cells from patients with leukemia or myelodysplastic syndromes.

Previous studies demonstrated that interleukin-12 (IL-12) enhances the non-MHC-restricted cytotoxic activity of NK cells and facilitate specific allogeneic human cytotoxic T lymphocyte responses against fresh leukemia cells and cell lines. The Wilms' tumor gene, WT1 mRNA, has been used as a marker of minimal residual disease (MRD) for evaluating therapeutic efficacy of patients with leukemia or myelodysplastic syndrome (MDS). This study was aimed to investigate whether in vitro IL-12 can lower WT1 gene expression in peripheral blood monuclear cells (PBMNC) from patients with leukemia or MDS. PBMNC from these 30 patients and 5 healthy volunteers were cultured at 5 x 10(5) cells/ml alone with or without 100 units/ml of IL-12 for 3 days. WT1 mRNA was measured by competitive reverse transcription polymerase chain reaction (RT-PCR) since WT1 mRNA is considered as a marker of minimal residual disease (MRD) in leukemia and MDS. The results demonstrated that WT1 mRNA in PBMNC of 5 healthy volunteers was less than 10(3) copies/microg of total RNA. Following the 3-day IL-12 treatment, mean WT1 mRNA of PBMNC was reduced from 10(4.8) to 10(4.2) copies/microg of total RNA in 6 CML patients, from 10(5.4) to 10(4.8) copies/microg in 12 MDS patients and from 10(5.0) to 10(4.2) copies/microg in 5 AML patients in CR, but not reduced in 5 of 7 AML in non-CR. It is concluded that IL-12 significantly decrease the quantity of leukemia cells in PBMNC of most patients with MDS, CML and AML in CR. IL-12 may be of considerable benefit in the elimination of MRD in patients with hematological malignancies.

Adult↗

Effect of protein nutrition on the mRNA content of insulin-like growth factor-binding protein-1 in liver and kidney of rats.

Effect of quantity and nutritional quality of dietary proteins on the content of mRNA of insulin-like growth factor-binding protein-1 (IGFBP-1) was studied in rat liver and kidney. IGFBP-1 mRNA content per unit RNA increased in liver and kidney of rats fed on a protein-free diet and in those of fasted rats compared with that in the rats fed on a casein diet. When rats were given a gluten diet for 7 d, IGFBP-1 mRNA content in liver did not change significantly but that in kidney increased considerably compared with that in those organs of the rats fed on the casein diet. Because IGFBP-1 mRNA has been demonstrated both in liver parenchymal and non-parenchymal cells (Takenaka et al. 1991), the effect of the protein-free diet on these two types of cells has been studied. An increase in IGFBP-1 mRNA content under protein deprivation was observed in both liver parenchymal and non-parenchymal cells, suggesting that these two types of cells are regulated in a similar mode as far as IGFBP-1 mRNA content is concerned. The physiological and nutritional significance of the previously stated results on protein anabolism are discussed when considered together with our previous observations on the plasma concentrations of IGF-1 (Takahashi et al. 1990) and IGFBP (Umezawa et al. 1991) and insulin-like growth factor-1 mRNA content in liver (Miura et al. 1991).

Animals↗

Superinduction of interferon with metabolic inhibitors: possible mechanisms and practical applications.

Ten years have passed since cycloheximide was first shown to enhance endotoxin-induced production of interferon in mice, in the first demonstration of what was later called the superinduction of interferon. Various inhibitors of protein and RNA symthesis, as well as combinations of these inhibitors, have been shown to act as superinducing agents of interferon production stimulated by polyriboinosinic-polyribocytidylic acid. The evidence that superinduction is due to the suppression of a mechanism of post-transcriptional regulation of interferon synthesis is rather convincing. The notion (first proposed about six years ago) that this modification is caused by a protein "repressor" remains the most plausible, albeit still unproved, hypothesis. The availability of systems that translate with fidelity interferon messenger RNA isolated from induced cells should prove most useful in the elucidation of post-transcriptional control mechanisms of interferon synthesis and of interferon superinduction. Meanwhile, superinduction has become a useful tool for the production of large quantities of interferon. In particular, this technique has been successfully applied to the production of interferon from human diploid fibroblasts. The clinical potential of this material remains to be critically examined.

Animals↗

Glyoxysomes in megagamethophyte of germinating ponderosa pine seeds.

Decoated ponderosa pine (Pinus ponderosa Laws) seeds contained 40% lipids, which were mainly stored in megagametophytic tissue and were utilized or converted to sugars via the glyoxylate cycle during germination. Mitochondria and glyoxysomes were isolated from the tissue by sucrose density gradient centrifugation at different stages of germination. It was found that isocitrate lyase, malate synthase, and catalase were mainly bound in glyoxysomes. Aconitase and fumarase were chiefly localized in mitochondria, whereas citrate synthase was common for both. Both organelles increased in quantity and specific activity of their respective marker enzymes with the advancement of germination. When the megagametophyte was exhausted at the end of germination, the quantity of these organelles and the activity of their marker enzymes decreased abruptly. At the stage of highest lipolysis, the isolated mitochondria and glyoxysomes were able to synthesize protein from labeled amino acids. Both organellar fractions contained RNA and DNA. Some degree of autonomy in glyoxysomes is indicated.

Journal Article↗

Soluble HLA class I antigen secretion by normal lymphocytes: relationship with cell activation and effect of interferon-gamma.

HLA class I antigens are thought to be integral membrane proteins. However, soluble forms of these molecules have been detected. Our laboratory has recently shown that the predominant form of these soluble proteins present in human serum, spleen tissue and culture supernatant of activated lymphocytes exhibits molecular weight and structure similar to classical HLA class I antigens, but lacks HLA A or B polymorphic determinants. In the present study, the secretion of such soluble proteins by lymphocytes has been further explored. Phytohaemagglutinin-stimulated normal lymphocytes secrete considerable quantities of soluble HLA (sHLA) class I proteins. This secretion seems to be a general property of lymphocytes, since activation of T as well as B cells by appropriate mitogens equally induce sHLA I secretion. Lymphocytes require RNA and protein synthesis, but not DNA synthesis, for the secretion to occur. Kinetic studies reveal that maximal sHLA I secretion precedes the peak of DNA synthesis by 24 h. In vitro stimulation with antigens or alloantigens also provokes sHLA I secretion. Moreover, this phenomenon has also been detected for in vivo-activated lymphocytes, as enhanced spontaneous sHLA I secretion was observed in cultures of low-density blastic B and T cells, and of blood lymphocytes obtained from normal subjects who had received a booster immunization 5 days earlier. Interferon-gamma (IFN-gamma) increases the expression of membrane-bound class I antigens but does not induce any sHLA I secretion, suggesting that both molecules are under different regulatory mechanisms. Our results indicate that human lymphocytes, upon stimulation, actively secrete considerable amounts of a soluble form of these biologically relevant proteins.

B-Lymphocytes↗