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PubMed2002

Sludge bed development in denitrifying reactors using different inocula-performance and microbiological aspects.

Aerobic and methanogenic consortia were evaluated as inocula for laboratory scale denitrifying reactors, fed with a synthetic wastewater with acetate as the main electron donor. The denitrifying microflora of inocula and reactors was evaluated by specific denitrifying activity, enumeration and isolation of denitrifiers, which were screened by amplified ribosomal DNA restriction analysis. Reactor performance was monitored by COD and nitrate removal efficiencies and granule size. The aerobic sludge failed to form granules, probably due to the development of a filamentous, nitrate-reducing organism which was characterised by 16SrDNA sequencing as Bacillus cereus. The methanogenic sludge showed denitrifying activity and adapted very rapidly to denitrifying conditions in the two reactors seeded with granules of different sizes. Denitrifiers grew around the granules, increasing the specific denitrifying activity of the sludge over 10-fold. Exopolymer-forming organisms, belonging to the same species, were isolated from both reactors. Granule size increased during operation, but flotation of the aggregates, related to gas retention was observed.

Acetates↗
PubMed1995

Appearance of neurons in the developing chick gut.

The enteric nervous system is formed from neural crest-derived cells. These cells enter the gut, migrate, proliferate, and ultimately differentiate into neurons and glia. We have used a human anti-neuronal autoantibody (ANNA-1), which recognizes neuron-specific RNA-binding proteins of the Hu family as an early marker of neuronal phenotype, to study the appearance of enteric neurons in the developing chicken gut. Immunoreactive cells appear first in the gizzard primordium at E3.5 and are found at progressively more caudal locations in the gut as development proceeds. Nascent neurons are present at the yolk stalk at E4.5, at the ileocecal junction at E6.5, and within the rectum at E7.5-8.5. Neurons appear slightly later in the esophagus. Aggregates of cells resembling developing ganglia were first observed at E6.5 in the distal esophagus and at E8.5 in the proximal esophagus. A small number of cells appeared in the vagus nerve trunks at E4.5 and that number increased at E7.5-8.5. Immunoreactive cells were also found in the sympatho-aortic plexus between the mesonephri and in the dorsal mesentery. These cells appeared to coalesce and form the ganglionated Nerve of Remak which contained positive cells at E3.5. This Nerve extended to the yolk stalk at E4.5 and to duodenum at E6.5. We conclude that the appearance of nascent neurons occurs first in the gizzard and proceeds more rapidly in a distal than proximal direction along the gut. Furthermore, cell that appear to be nascent neurons are found in the vagus and in the dorsal mesentery.

Animals↗
PubMed1988

Morphological differentiation of embryonic rat sympathetic neurons in tissue culture. I. Conditions under which neurons form axons but not dendrites.

We have examined the morphology of fetal rat sympathetic neurons grown in serum-free medium in the absence of nonneuronal cells. Because cell density can affect phenotypic expression in vitro, the morphological analysis was subdivided into the study of isolated neurons (neurons whose somata were at least 150 micron from their nearest neighbor) and of more highly aggregated neurons. When isolated neurons were injected with intracellular markers, it was found that most (79%) had a single process emanating from their somata and that this unipolar state persisted for at least 8 weeks in vitro. The processes of unipolar sympathetic neurons had the appearance of axons in that they were thin and long, had a constant diameter, and were relatively unbranched. Cytochemical methods revealed that such processes had other axonal characteristics: (1) they were more reactive with a monoclonal antibody against phosphorylated forms of the M and H neurofilament subunits than with an antibody to nonphosphorylated forms of these proteins; (2) they also reacted with antibodies to the tau microtubule-associated protein and to the phosphorylated forms of the H neurofilament subunit; and (3) they contained only small amounts of RNA as determined by [3H]uridine autoradiography. These data indicate that neurons which normally form dendrites in vivo need not express this capacity in vitro and that axonal and dendritic growth can be dissociated under some conditions in culture. While most isolated neurons were unipolar, neurons in regions of high neuronal cell density were usually multipolar. In addition to axons, multipolar neurons had processes with some of the characteristics expected of rudimentary dendrites: they ended locally (usually within 100 micron), were often highly branched, and reacted with an antibody to nonphosphorylated forms of the M and H neurofilament subunits. The effects of density were most prominent when neurons were within aggregates in which the somata were in close apposition. Density-dependent changes in morphology were less frequently observed when neuronal somata were separated by greater distances (30-100 micron). These data indicate that the morphology of sympathetic neurons is subject to environmental regulation and that neuron-neuron interactions can promote the extension of rudimentary dendrites in vitro.

Animals↗
PubMed2005

Dlgh1 coordinates actin polymerization, synaptic T cell receptor and lipid raft aggregation, and effector function in T cells.

Lipid raft membrane compartmentalization and membrane-associated guanylate kinase (MAGUK) family molecular scaffolds function in establishing cell polarity and organizing signal transducers within epithelial cell junctions and neuronal synapses. Here, we elucidate a role for the MAGUK protein, Dlgh1, in polarized T cell synapse assembly and T cell function. We find that Dlgh1 translocates to the immune synapse and lipid rafts in response to T cell receptor (TCR)/CD28 engagement and that LckSH3-mediated interactions with Dlgh1 control its membrane targeting. TCR/CD28 engagement induces the formation of endogenous Lck-Dlgh1-Zap70-Wiskott-Aldrich syndrome protein (WASp) complexes in which Dlgh1 acts to facilitate interactions of Lck with Zap70 and WASp. Using small interfering RNA and overexpression approaches, we show that Dlgh1 promotes antigen-induced actin polymerization, synaptic raft and TCR clustering, nuclear factor of activated T cell activity, and cytokine production. We propose that Dlgh1 coordinates TCR/CD28-induced actin-driven T cell synapse assembly, signal transduction, and effector function. These findings highlight common molecular strategies used to regulate cell polarity, synapse assembly, and transducer organization in diverse cellular systems.

Actins↗
PubMed1966

Ultrastructural features of Beta leaves infected with beet yellows virus.

A cytochemical and electron microscope study has been made of leaves of sugar beet infected with beet yellows virus. Inclusions of particles, which agree in size with beet yellows virus particles isolated by other investigators, have been localized in the ground cytoplasm, in the chloroplasts, and in the nuclei. These particles are circa 100 A in diameter and have an electron-transparent core of 30 to 40 A. Use of acridine orange, azure B, and pyronine Y has revealed that the cytoplasmic inclusion bodies, which consist wholly of the elongate particles, have a strong RNA reaction removable by RNase pretreatment. Particles observed in the chloroplasts may or may not be associated with lipid spheres. If they are, the particles are confined to the periphery of the spheres. In this position the particles are arranged tangentially and are further arranged parallel into groups which lie at various angles to one another. Within the groups the particles are regularly spaced in a three dimensional lattice. Particles located free in the stromal regions are often arranged regularly in curved rows which lie parallel to one another so that a three dimensional lattice is formed. The dispersed and compact forms of virus inclusions are described and related to the condition of the associated cytoplasm. The ground cytoplasm of cells associated with the sieve elements contains numerous ribosomes. A decrease in the number of ribosomes is concomitant with the increase in size of virus aggregations in a cell. Vesiculation of some component of the cytoplasm occurs during the period of virus replication. The vesicles are approximately 100 mmicro in diameter and could be derived from the dictyosomes. At later stages of infection these vesicles collapse and convoluted membranous material appears.

Cell Nucleus↗
PubMed1995

Recent advances in amelogenin biochemistry.

This paper reviews advances in amelogenin biochemistry in three areas; (i) amelogenin expression; (ii) amelogenin post-translational and post-secretory processing, and (iii) amelogenin structure and function. Recent studies of amelogenin expression have demonstrated that alternative-splicing of mouse amelogenin RNA generates seven distinct mRNAs, coding for amelogenin proteins from 194 to 44 amino acid residues in length. A polyclonal antibody to a sequence of the 194-residue murine amelogenin identified this protein in vivo. While several studies have reported that amelogenins are post-translationally phosphorylated, it has proved difficult to confirm this view. Mass spectrometry studies of bovine and porcine TRAP and LRAP amelogenins have established a phosphoserine residue at position-16 as originally reported by Takagi et al. for a 180-residue bovine amelogenin. Also, we discovered that the detailed mechanism(s) of carboxy-terminal amelogenin proteolytic processing appear different than previously reported. In terms of amelogenin structure, it is well known that amelogenins form aggregated structures. Studies employing a recombinant amelogenin and dynamic light-scattering instrumentation demonstrated aggregate structures of 15-20 nm in radius, corresponding to a mass of 2-3 million daltons. Imaging these aggregates by transmission electron and atomic force microscopy suggested that these structures are equivalent to the "stippled" or "granular" material seen in electron photomicrographs of developing enamel. Collectively, these advances in amelogenin biochemistry lead to a new view of amelogenin structure, processing and functions in enamel biomineralization.

Alternative Splicing↗
PubMedDate not supplied

E. coli minichromosome replication: regulation of initiation at oriC.

The initiation of Escherichia coli DNA replication is a highly regulated event with many parameters exerting positive and negative effects. The activity of the dnaA protein (the initiator protein) is profoundly influenced by the tight binding of the adenine nucleotides ATP and ADP. Further regulation of dnaA protein activity may occur through dnaA protein-cell membrane associations. A replicatively inactive form of dnaA protein is found aggregated with phospholipids; enzymatic treatment of the aggregates with phospholipase A2 or dnaK protein liberates dnaA protein with restored replication activity. Proper DNA structure is essential for replication. The energy stored in the DNA's supercoiling is crucial for dnaA protein's ability to initiate replication. Under conditions where strand-opening by dnaA protein is inhibited, such as low free superhelicity, an R-loop formed by RNA polymerase activates the origin at a distance by aiding strand-opening. A novel protein has been identified as a specific inhibitor of the initiation of DNA replication. This 33-kDa protein binds to the AT rich region of oriC and inhibits strand-opening by dnaA protein.

Bacterial Proteins↗
PubMed1972

Intracellular synthesis, transport, and packaging of proteinaceous yolk in oocytes of Orconectes immunis.

The incorporation of leucine-(3)H into either ovarian or oocyte proteins occurs throughout vitellogenesis, but is at a maximum during early phases of this process. The labeling of ovarian and oocyte proteins is inhibited with cycloheximide. Oocytes are permeable to actinomycin D, and this drug does not affect the incorporation of amino acids into oocyte proteins but does block oocyte RNA synthesis. By means of both light microscope and high resolution radioautography, it has been demonstrated that the initial incorporation of leucine-(3)H under both in vitro and in vivo conditions occurs in elements of the rough-surfaced endoplasmic reticulum in the oocyte. Under pulse-chase conditions, the label subsequently becomes associated with intracisternal (precursor yolk) granules now aggregated within the cisternae of the connected smooth-surfaced endoplasmic reticulum. By 7 days, mature yolk globules are extensively labeled. The results of experiments designed to assess the possible contribution of maternal blood proteins to yolk deposition indicate that such a contribution is minimal. It is concluded that the crayfish oocyte is programmed for and capable of synthesizing the massive store of proteinaceous yolk present in the egg at the end of oogenesis.

Animals↗
PubMed1996

Vaccinia virus A17L open reading frame encodes an essential component of nascent viral membranes that is required to initiate morphogenesis.

We generated an antiserum to the predicted C-terminal peptide of the A17L open reading frame (ORF), which encodes a 23-kDa polypeptide with hydrophobic regions characteristic of membrane proteins. Immuno-electron microscopy of infected cells indicated that the A17L protein is intimately associated with the earliest characteristic viral membranes, even those formed in the presence of the drug rifampin. To study the role of the A17L protein in morphogenesis, we constructed recombinant vaccinia viruses in which the endogenous A17L ORF was deleted and a copy of the ORF under the control of the bacteriophage T7 RNA polymerase and the Escherichia coli lac repressor was inserted into an alternative site in the vaccinia virus genome. Growth of these recombinant viruses was entirely dependent on the induction of A17L expression by isopropyl-beta-D-thiogalactopyranoside. Electron microscopic examination of cells infected in the absence of inducer revealed the accumulation of large, well-demarcated electron-dense aggregates but no characteristic membrane-associated viral structures. Viral late protein synthesis occurred under these conditions, although the maturational proteolytic processing of structural proteins was inhibited. We conclude that the product of the A17L gene is an essential component of the immature viral membrane and has an early function in viral morphogenesis.

Animals↗
PubMed2005

RNA interference of VCP/p97 increases Mallory body formation.

In the present report, valosin-containing protein (VCP) was present in Mallory bodies (MBs). To determine if VCP plays a role in MB formation, primary cultured hepatocytes from drug-primed mice that spontaneously form MBs in vitro were studied. The results were compared with control normal hepatocytes. Gene-specific FITC-labeled gripNA (gVCP) was added to the medium of the primary cultures to inhibit the expression of VCP. gVCP increased MB formation by 230% in drug-primed mouse hepatocytes compared with primed liver cells where no VCP oligos were added. Blocking VCP expression induced both multiple small ubiquitin (Ub) and cytokeratin (CK) aggregates to form within the cytoplasm in normal mouse hepatocytes. Inhibition of VCP expression in both drug-primed and control hepatocytes caused a decrease in proteasome chymotrypsin-like (ChT-L) activity. Overexpression of VCP was achieved by transfecting the hepatocytes with a plasmid containing green fluorescent protein (GFP)-fused VCP (pVCP-GFP). Overexpressed VCP was located in both the cytoplasm and nucleus of pVCP-GFP overexpressing drug-primed hepatocytes. VCP was also concentrated within MBs. MB formation was not decreased by the overexpression of VCP in the cells. These results indicate that VCP plays an important role in inducing MB formation, probably through its molecular chaperone function in the ubiquitin-proteasome system (UPS).

Adenosine Triphosphatases↗
PubMed1997

Production of IL-18 (IFN-gamma-inducing factor) messenger RNA and functional protein by murine keratinocytes.

Recently, the novel cytokine IL-18 (IFN-gamma-inducing factor) has been described as a growth and differentiation factor for Th1 cells. Epidermal keratinocytes (KC) are known to direct T cell education by production of cytokines. Therefore, expression of IL-18 was sought in KC. Epidermal RNA was analyzed following stimulation with contact sensitizers or controls for IL-18 mRNA expression by semiquantitative reverse transcription-PCR. Constitutive expression of IL-18 mRNA was low in untreated epidermal cells (EC), but early up-regulation of IL-18 mRNA signals was detected following application of a contact allergen in vivo. The peak strength of IL-18 signals was observed within 4 to 6 h following stimulation with an allergen. Application of an irritant (benzalconiumchloride) or solvents did not result in increased signal strength. To determine the cellular origin of IL-18 mRNA in EC, depletion experiments were performed. IL-18 signals were not affected by depletion with anti-CD3 (T cells) or anti-MHC class II mAb-coupled beads identifying KC as a major source of IL-18. These results were confirmed by analysis of mRNA derived from the KC cell line PAM 212. Strong IL-18 signals could be detected by reverse transcription-PCR. To delineate whether IL-18 protein was produced by EC/KC, a sandwich ELISA was used to assay for IL-18 production. Supernatants from allergen-stimulated EC and KC showed production of IL-18 protein. To confirm that IL-18 protein was functional, EC or KC supernatants were tested for their ability to induce IFN-gamma production. Significant amounts of IFN-gamma were detected in supernatants of allergen-treated cells. In aggregate, our data indicate that murine KC are a source of both IL-18 mRNA and functional protein.

Animals↗
PubMed2001

Dispersion of cyclin B mRNA aggregation is coupled with translational activation of the mRNA during zebrafish oocyte maturation.

Cyclin B mRNA stored in immature zebrafish oocytes is translationally activated upon the stimulation of 17alpha,20beta-dihydroxy-4-pregnen-3-one (17alpha,20beta-DP), an event prerequisite for initiating oocyte maturation in this species. We investigated localization of cyclin B mRNA in zebrafish oocytes. Cyclin B mRNA was found to be exclusively localized as an aggregation along the cytoplasm at the animal pole of full-grown immature oocytes. When oocytes were treated with 17alpha,20beta-DP, a meshwork of microfilaments in the oocyte cortex disappeared and the aggregation of cyclin B mRNA dispersed just prior to the initiation of cyclin B synthesis and germinal vesicle breakdown (GVBD). Cytochalasin B, but not nocodazole or taxol, deformed the aggregation of cyclin B mRNA, indicating the involvement of microfilaments in organizing this form. Like 17alpha,20beta-DP, cytochalasin B (10 microg/ml) induced both complete dispersion of the aggregation and translational activation of cyclin B mRNA, forcing the oocytes to undergo GVBD without 17alpha,20beta-DP. Conversely, disturbance of the aggregation of cyclin B mRNA with a low concentration (1 microg/ml) of cytochalasin B inhibited 17alpha,20beta-DP-induced GVBD. These results suggest that the direct change in cyclin B mRNA from the aggregated form to the dispersed form is responsible for translational activation of the mRNA during zebrafish oocyte maturation.

Animals↗
PubMed1989

A Dictyostelium mutant deficient in severin, an F-actin fragmenting protein, shows normal motility and chemotaxis.

A severin deficient mutant of Dictyostelium discoideum has been isolated by the use of colony immunoblotting after chemical mutagenesis. In homogenates of wild-type cells, severin is easily detected as a very active F-actin fragmenting protein. Tests for severin in the mutant, HG1132, included viscometry for the assay of F-actin fragmentation in fractions from DEAE-cellulose columns, labeling of blots with monoclonal and polyclonal antibodies, and immunofluorescent-labeling of cryosections. Severin could not be detected in the mutant using these methods. The mutation in HG1132 is recessive and has been mapped to linkage group VII. The mutant failed to produce the normal severin mRNA, but small amounts of a transcript that was approximately 100 bases larger than the wild-type mRNA were detected in the mutant throughout all stages of development. On the DNA level a new Mbo II restriction site was found in the mutant within the coding region of the severin gene. The severin deficient mutant cells grew at an approximately normal rate, aggregated and formed fruiting bodies with viable spores. By the use of an image processing system, speed of cell movement, turning rates, and precision of chemotactic orientation in a stable gradient of cyclic AMP were quantitated, and no significant differences between wild-type and mutant cells were found. Thus, under the culture conditions used, severin proved to be neither essential for growth of D. discoideum nor for any cell function that is important for aggregation or later development.

Actins↗
PubMed2005

Deletions in the polyAlanine-containing transcription factor FOXL2 lead to intranuclear aggregation.

Mutations of FOXL2, a gene encoding a forkhead transcription factor, have been shown to cause the blepharophimosis-ptosis-epicanthus inversus syndrome. This genetic disorder is characterized by eyelid and craniofacial abnormalities associated or not with premature ovarian failure. We have previously shown that mutant FOXL2 with an expanded polyAlanine (polyAla) tract forms large aggregates both in the nucleus and in the cytoplasm of transfected cells, whereas the wild-type protein localizes in the nucleus in a rather diffuse manner. Premature stop codons in FOXL2 have been considered so far as null alleles. However, we demonstrate here that such nonsense mutations may lead to the production of N-terminally truncated proteins by re-initiation of translation downstream of the stop codon. Surprisingly, the truncated proteins strongly aggregate in the nucleus, partially localize in the cytoplasm and retain a fraction of the wild-type protein. We also show that a complete deletion of the polyAla tract of FOXL2 induces a significant intranuclear aggregation. Our results enlarge the spectrum of mutations inducing FOXL2 aggregation.

Amino Acid Sequence↗
PubMed2002

P2Y-receptors mediating an inhibition of the evoked entry of calcium through N-type calcium channels at neuronal processes.

In the search for P2-receptors modulating the stimulation-evoked entry of calcium at processes of PC12 cells differentiated in the presence of nerve growth factor and neurotrophin-3, electrically evoked increases in free calcium were assessed by fura-2 microfluorimetry. Omission of calcium and addition of cadmium (100 microM) or the N-type calcium channel blocker omega-conotoxin GVIA (0.5 microM) abolished or markedly reduced the evoked responses. The P2Y-receptor agonists 2-methylthio adenosine 5'-diphosphate (2-methylthio-ADP), ADP, and adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS) inhibited the electrically evoked entry of calcium without any changes in basal calcium concentrations. 2-Methylthio-ADP was the most potent agonist. Adenosine, P(1),P(4)-di(adenosine-5')-tetraphosphate (Ap4A), UDP, and UTP (30 microM each) had no effect. The effect of ADPbetaS (30 microM) was abolished by the P2-antagonists reactive blue 2 (3 microM), suramin (100 microM), 2-methylthio-AMP (10 microM), p-chloromercuriphenyl sulfonic acid (1 microM), and AR-C 69931MX [N(6)-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-beta,gamma-dichloromethylene adenosine 5'-triphosphate] (300 nM). In contrast, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (10 microM), the selective P2Y1-receptor antagonist MRS 2179 (N(6)-methyl-2'-deoxyadenosine 3',5'-bisphosphate; 10 microM), as well as the adenosine A(1)-receptor antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine; 100 nM), caused no change. Pretreatment with pertussis toxin abolished the effect of ADPbetaS. Reverse transcriptase-polymerase chain reaction revealed the presence of mRNA for P2Y12-receptors in nondifferentiated and differentiated PC12 cells. The results indicate that processes of differentiated PC12 cells possess P2Y12-receptors coupling to pertussis toxin-sensitive G-proteins and mediating an inhibition of the stimulation-evoked entry of calcium through omega-conotoxin GVIA-sensitive calcium channels. This suggests a role of P2Y12-receptors in neuromodulation in addition to their involvement in platelet aggregation.

Adenosine↗
PubMed1991

Proteins isolated from regenerating sciatic nerves of rats form aggregates following posttranslational amino acid modification.

Soluble proteins of regenerating sciatic nerves of rats can be posttranslationally, covalently modified by a variety of radioactive amino acids. The present study shows that once modified by a mixture of 15 amino acids, many of those proteins form aggregates that are unable to pass through a 0.45-micron filter and pellet following 20,000g centrifugation (suggesting a size of greater than 2 x 10(6) Da). Aggregation of proteins also occurs following modification by Arg or Lys alone, but does not occur following protein modification in nonregenerating nerves or in brain. Aggregates are not disrupted by treatment with 100 mM beta mercaptoethanol or by exposure to 1.0 M NaCl, but aggregates are solubilized by treatment with urea and by boiling in 1.5% SDS. Amino acid analysis of proteins modified by a mixture of [3H]amino acids shows a similar proportion of posttranslationally incorporated Ser, Pro, Val, Ala, Leu, Phe, Lys, and Arg in the soluble and pelletable fractions. Two-dimensional PAGE profiles of soluble and pelletable modified proteins show that the modified proteins in both fractions are in similar pI and molecular weight ranges, except that the soluble modified proteins include a high-molecular-weight component that is absent in the pelleted modified proteins. Kinetic studies show that while half-maximal levels of protein modification occur within 30 seconds of incubation, the appearance of the pelletable modified protein fraction is delayed significantly. These results indicate that amino acid modification of soluble proteins in regenerating sciatic nerves of rats results in physical changes in those proteins so that they form high-molecular-weight aggregates.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1999

CXCR4 mRNA expression in colon, esophageal and gastric cancers and hepatitis C infected liver.

We have recently demonstrated by Northern blot and RT-PCR that the mRNA expression of the alpha-chemokine hIRH/SDF-1alpha is reduced in hepatocellular carcinoma (HCC), several digestive tract cancers and premalignant colon adenomas, and that its receptor CXCR4 mRNA expression is reduced in HCC. Here we investigate the expression of CXCR4 mRNA expression in several digestive tract cancers and hepatitis C viral (HCV) infected liver, a premalignant condition. There was no difference in the CXCR4 mRNA expression in colon, esophageal or gastric cancers compared to non-cancerous tissues. This is significantly different from the reduced expression we have seen with hepatocellular carcinoma (p<0.05). To better refine regional tumor or hepatic cytokine mRNA analysis within a biopsy sample we describe a micro-isolation technique for RNA extraction from portal and triad areas of liver biopsies or other small malignant or non-malignant biopsy samples suitable for use in RT-PCR and differential display reactions. In HCV liver biopsies, the expression of hIRH and its receptor CXCR4 mRNA, corrected for G3PDH, was not significantly different from that of control non-HCV (steatosis) biopsies. CXCR4 is expressed on leukocytes and its expression was predicted to correlate with hepatic inflammation. CXCR4 receptor mRNA expression did correlate significantly with that of its ligand hIRH/SDF-1alpha (p=0.001), and with the severity of fibrosis (p<0.05), but not with portal inflammation (p<0.10), piecemeal necrosis (p<0.10), lobular inflammation (p>0.10), the presence of lymphoid aggregates (p>0.10), or the total histological activity index (p=0.07). There was no difference in expression of hIRH or CXCR4 between responders and non-responders to interferon (IFN) treatment, while as a control, the responder group of patients did show a higher expression of IFNalpha receptor than the non-responder group (p=0.05).

Biopsy↗
PubMed1974

Inhibition of translation in liver polyribosomes by a new substituted thiopseudourea with antitumour action.

A new thiopseudourea, S-(10-undecen-1-yl)isothiouronium iodide (compound AHR-1911), was tested for antitumour action and shown to inhibit considerably the growth of the Walker carcinoma in rats. The mechanism of its effect on protein and nucleic acid synthesis was then studied with systems in vitro from rat liver. In incubations of liver slices, 1.4mm-compound AHR-1911 decreased by 96% the incorporation of [(14)C]leucine into microsomal proteins, and mitochondrial protein synthesis measured in the presence of cycloheximide was decreased by 44%. At lower doses, translation, as well as the incorporation of [(3)H]uridine into RNA, was also considerably impaired, compound AHR-1911 being the most active of all the thiopseudoureas tested whereas undecylenic acid and thiourea by themselves showed practically no inhibition. Protein synthesis by cytoplasmic ribosomes (microsomes and C-polyribosomes) was inhibited by compound AHR-1911 at different concentrations (72% at 0.42mm), and again the other related compounds were much less effective, with the exception of one antileukaemic thiopseudourea. The same occurred with the poly(U)-stimulated incorporation of phenylalanine. The puromycin reaction with pulse-labelled C-ribosomes was strongly inhibited, particularly when preincubation with compound AHR-1911 preceded the addition of puromycin, with no release of nascent chains by the thiopseudourea alone. In the presence of GTP and pH5 fraction, to induce translocation and transform all the ribosomes to the donor state, the percentage inhibition remained the same. The ribosomes incubated with the drug are aggregated, as shown by the polyribosome profile, but, when excess of inhibitor was removed, the activity in protein synthesis and the puromycin reaction was restored, indicating that the inhibition is not due to the polyribosomal aggregation. These results suggest that the effect on translation with both 55S and 80S ribosomes is derived from inhibition of chain elongation at the level of transpeptidation and not translocation, probably together with the interference with transcription playing a role in the antitumour activity.

Animals↗