Outcomes of emergent embolisation of ruptured hepatocellular carcinoma in a western population.

AIM: To evaluate the authors' experience with interventional radiological management of tumour rupture in hepatocellular carcinoma (HCC) in a Western population. MATERIALS AND METHODS: A retrospective review was performed of all consecutive patients treated at a single institution with transcatheter embolisation for ruptured HCC between 2000 and 2013. Patient age, sex, aetiology of liver disease, degree of underlying liver dysfunction, and clinical presentation were assessed. Embolisation was performed in a selective fashion when possible. Success, complications, and survival were assessed. RESULTS: Twenty-three patients were treated with embolisation for ruptured HCC. Of these patients, nine, nine, and five patients were Child-Pugh Class A, B, and C respectively. Embolisation was successful in 22 patients; one patient remained haemodynamically unstable and transfusion dependent despite embolisation. No major complications occurred. Median survival time was 260 days and the 30 day and 1 year survival rates were 83% and 45%, respectively. Child-Pugh class B or C (p = 0.04), Model for End-Stage Liver Disease score greater than 10 (p = 0.04), lobar embolisation (p = 0.04), and presence of portal vein thrombosis (p = 0.01) were significantly associated with worse prognosis. CONCLUSION: Transcatheter embolisation is effective at controlling haemorrhage in patients with ruptured HCC. Although major procedural complications are low, embolisation should proceed with an understanding of poor prognosis in patients with decompensated liver disease. Superselective embolisation is associated with improved prognosis and should be performed when feasible.

Molecular characterization of monoamine oxidases A and B.

Monoamine oxidase A and B (MAO A and B) are the major neurotransmitter-degrading enzymes in the central nervous system and in peripheral tissues. MAO A and B cDNAs from human, rat, and bovine species have been cloned and their deduced amino acid sequences compared. Comparison of A and B forms of the enzyme shows approximately 70% sequence identity, whereas comparison of the A or B forms across species reveals a higher sequence identity of 87%. Within these sequences, several functional regions have been identified that contain crucial amino acid residues participating in flavin adenine dinucleotide (FAD) or substrate binding. These include a dinucleotide-binding site, a second FAD-binding site, a fingerprint site, the FAD covalent-binding site, an active site, and the membrane-anchoring site. The specific residues that play a role in FAD or substrate binding were identified by comparing sequences in wild-type and variants of MAO with those in soluble flavoproteins of known structures. The genes that encode MAO A and B are closely aligned on the X chromosome (Xp11.23), and have identical exon-intron organization. Immunocytochemical localization studies of MAO A and B in primate brain showed distribution in distinct neurons with diverse physiological functions. A defective MAO A gene has been reported to associate with abnormal aggressive behavior. A deleterious role played by MAO B is the activation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a proneurotoxin that can cause a parkinsonian syndrome in mammals. Deprenyl, an inhibitor of MAO B, has been used for the treatment of early-stage Parkinson's disease and provides protection of neurons from age-related decay.

Immunochemical characterization of fibrinogen induction in rat liver.

The synthesis of fibrinogen in rat hepatocytes can be induced two to three times its normal level following an injection of turpentine. Detection of the increased synthesis of this protein was accomplished by quantitative immunoprecipitation analysis. The time sequence for maximal intracellular synthesis occurs more rapidly than the concomitant rise in plasma fibrinogen, implying that the secretion of fibrinogen is somewhat slower than was anticipated. Binding studies with 125I-labeled anti-fibrinogen to the nascent chains on the ribosomes showed no detectable changes in the radioactivity distribution before or after stimulation as analyzed by zone sedimentation. It was estimated that only 4.4% of the total polysomes were involved in fibrinogen synthesis prior to induction and this was increased to 15% following turpentine stimulation. The results suggests that the rise in fibrinogen synthesis during the acute-phase response was related to an increase in functional messenger RNA transcripts rather than in an increase in the rate of protein synthesis.

Haptoglobin biosynthesis in rats. Immunological identification of polysomes synthesizing haptoglobin and quantitation of haptoglobin in the cytoplasm of liver cells.

A quantitative enzyme-linked immunosorbent assay was developed and utilized to study the stimulation of haptoglobin biosynthesis during an acute inflammatory challenge. A 10-fold increase in intracellular haptoglobin was measured at the peak of the inflammatory response. The increase in serum haptoglobin levels was concomitant with the intracellular levels, demonstrating the secretory output is also elevated during the inflammatory period. A monospecific antihaptoglobin was produced and used to detect the specific polysomes involved in haptoglobin synthesis. The amount of radioactively labeled antibody bound to the nascent haptoglobin chain was increased approx. 3-fold during the inflammatory response, indicating that new haptoglobin was being synthesized and suggesting an increase in functional haptoglobin mRNA resulting from the inflammatory signal.

Cellular expression of mRNAs encoding monoamine oxidases A and B in the rat central nervous system.

Monoamine oxidases A and B (MAO-A and MAO-B) oxidatively deaminate neurotransmitter and xenobiotic amines. The cellular localization of these isoenzymes in the central nervous system (CNS) differs markedly and only partly reflects the distribution of their presumed natural substrates. In the present study, by using in situ hybridization with 35S-labelled oligonucleotide probes, we examined the distribution of mRNAs encoding MAO-A and MAO-B in the rat CNS. Probes for tyrosine hydroxylase, histidine decarboxylase, and tryptophan hydroxylase mRNAs were used to demonstrate the catecholaminergic, histaminergic, or serotoninergic nature of some cell populations in adjacent sections. The radioligands [3H]-Ro 41-1049 and [3H]lazabemide (reversible and selective inhibitors of MAO-A and MAO-B, respectively) were used to reveal the protein distribution by enzyme radioautography. The distribution and abundance of transcripts for both isoenzymes in the tissues investigated differed markedly but, in general, correlated with the protein distribution. MAO-A mRNA and protein were most abundant in noradrenergic neurons. However, moderate levels of transcript expression and protein were also detected in the serotoninergic neurons, and low but significant levels were detected in the dopaminergic neurons. An unexpectedly remarkable degree of hybridization signal was apparent in nonaminergic cell populations, e.g., in the cerebral cortices, the hippocampal formation (CA1-3, dentate gyrus), the cerebellar granule cell layer, and the spinal cord motoneurons. In contrast, MAO-B mRNA and protein were most abundant in serotoninergic and histaminergic neurons, Bergmann glial cells, and circumventricular organs, including the ependyma. MAO-B transcripts were also weakly expressed in nonaminergic cells, e.g., in the hippocampal formation (CA1-2). A further nonneuronal localization of MAO-B transcripts was also resolved, e.g., in the glia limitans, the olfactory nerve layer, and the cerebellar peduncle. These findings reveal further the potential of various cell populations to synthesize the isoenzymes, and homologous (aminergic) and heterologous (nonaminergic) patterns of expression as well as coexpression of MAO mRNAs are described.

Improved models for pharmacological null experiments: calculation of drug efficacy at recombinant D1A dopamine receptors stably expressed in clonal cell lines.

Modern drug discovery demands accurate knowledge of the drug properties of affinity and efficacy at specific receptor proteins. Furthermore, drugs with well defined properties make better tools with which to explore and understand receptor regulation. The use of clonal cell lines stably expressing a given recombinant receptor may provide a highly useful model in which drug effects may be studied on one receptor subtype at a time. The present report was designed to evaluate the utility of a general method in which a clonal cell line stably expressing a recombinant D1A dopamine receptor was used as a model system for studying drug actions by null models. The null model for receptor occlusion (to calculate agonist Ka) and the null model for relative efficacy (to calculate test agonist affinity and epsilon r) were evaluated in these studies. To initiate these studies, rat C6 glioma cells that do not normally express DA receptors have been modified by stable transfection with the primate D1A DA receptor [Machida et al., 1992 (Molec. Pharmacol. 41: 652-659)] to a density of approximately equal to fmol/mg protein. The recombinant receptors show robust stimulation of cAMP in the stably transfected C6 cells. Calculation of agonist Ka from dose-response data requires that a portion of the cell's receptors be occluded in the absence of changes in post-receptor events leading to the response. Receptor reserve is typically reduced by alkylation, thereby lowering maximal response. Unfortunately, most of the currently available alkylating agents are not selective either for a particular receptor or for receptors vs other proteins within a signaling pathway. Short-term agonist treatment offers a possible complement to the use of non-selective or poorly characterized alkylating drugs for reducing maximum response in appropriate cell systems. The null method of receptor occlusion was used to determine the Ka for dopamine when maximum response was decreased by alkylation vs short-term agonist treatment. Direct non-linear curve fitting was used to analyze the data. In addition to DA, two other compounds were used to reduce receptor reserve to validate the method: fenoldopam (relatively high efficacy) and SKF38393 (low efficacy). Analyses indicated that the affinity of DA was similar whether calculated by alkylation (1.1 +/- 0.58 microM), 75 min DA treatment (0.57 +/- 0.16 microM) or 45 min treatment with DA (0.86 +/- 0.11 microM). Short-term agonist treatment experiments using multiple concentrations of DA, fenoldopam, or SKF38393 to decrease receptor reserve provided additional support for the validity of the Ka determinations using this procedure. Other experiments were conducted according to the null model for relative efficacy in which the affinity for DA is calculated by comparing the DA response before and after receptor occlusion, and the affinity and relative intrinsic efficacy of the test agonist are determined as a function of its actions relative to DA. We used the following four test drugs: + Br-APB, a novel agent with potential dopamine agonist properties, and three high-affinity DA agonists, fenoldopam, R-(-)-apomorphine (APO), and SKF38393. Intrinsic efficacy values relative to that of DA (1.0) were as follows: fenoldopam, 0.46 +/- 0.11; APO, 0.19 +/- 0.13; SKF38393, 0.07 +/- 0.01; and +Br-APB, 0.26 +/- 0.40. The agonist affinities (Ka) were: fenoldopam, 0.018 +/- 0.008 microM; APO, 0.80 +/- 0.18 microM; SKF38393, 0.16 +/- 0.04 microM; BR-APB, 0.43 +/- 0.29 microM; and DA, 0.58 +/- 0.17 microM. EC50/Ka ratios were consistent with relative intrinsic efficacies and Ka values were similar to KL values reported for membrane binding studies. Finally, Monte Carlo simulations were conducted to determine the precision of the parameter estimates...

Molecular properties of monoamine oxidases A and B.

Monoamine oxidases A and B (MAO-A and B) catalyze the oxidative catabolism of biogenic amines and xenobiotics. Investigation of these mitochondrial membrane proteins shows that they differ in substrate preference, inhibitor specificity, tissue and neuronal cell distribution, immunological properties, and nucleotide and deduced amino acid sequences. Comparisons of MAO-A and B from the human, bovine, and rat species show strikingly high similarity (85-88%) in the amino acid sequences of each enzyme. Furthermore, three regions in MAO-A and B have sequence identities across species of 78, 88, and 86%. These regions correspond to a nucleotide-binding site near the N-terminal end that is found in the vast majority of enzymes that require flavin adenine dinucleotide (FAD), a region of unknown function, and the FAD-binding site toward the C-terminal end. Genomic clones of MAO-B which span almost the entire gene (greater than 40 kb) have been isolated, restriction mapped, and partially sequenced. Likewise, genomic clones of MAO-A that correspond to the 3'-flanking region have also been investigated. Current studies which focus on identification of the promoter and regulatory sequences should help to establish why MAO-A and B are localized in different subsets of neurons in brain.

A simplified 14CO2-trapping microassay for tyrosine hydroxylase activity.

This 14CO2-trapping microassay for tyrosine hydroxylase activity uses microtest tubes (1.5 or 2.0 ml) with pierceable caps for injecting the reaction mixture. A folded filter paper strip (1 X 4 cm) impregnated with Protosol is placed directly inside the top of the tube prior to capping in order to trap liberated 14CO2. The effects of several variables and components involved in the assay have been systematically studied. The tyrosine hydroxylation reaction may be optimized by incubating 300 micrograms protein with 150 microM L-Tyr, 0.8 mM 6MPH4, 1 mM FeSO4, and 0.12 M Tris-acetate buffer (pH 5.8) for 10 min at 37 degrees C. The DOPA decarboxylation reaction may be optimized by continual incubation of the tyrosine hydroxylation medium with 175 micrograms hog kidney aromatic-L-amino acid decarboxylase, 6.25 mM 3-iodotyrosine, and 0.125 M potassium phosphate buffer (pH 8.0) for 30 min at 37 degrees C. Under these conditions, the radioactivity of 14CO2 recovered after 1 h at 37 degrees C may reach 14,000 dpm, whereas the blank only has 300 dpm (less than 3% of test value). This microassay is fast (less than 2 h to complete all reactions) and convenient for performing a large number of determinations.

Intracellular distribution of monoamine oxidase A in selected regions of rat and monkey brain and spinal cord.

Monoamine oxidase A and B (MAO A and B; EC 1.4.3.4) are integral proteins of the outer mitochondrial membrane that degrade monoamines including the neurotransmitters norepinephrine, dopamine, and serotonin. In this study, monoclonal antibodies that recognize rat or monkey MAO A were used in immunocytochemical studies to visualize the subcellular localization of this enzyme within neurons in the central nervous system of these species. The regions examined included the locus coeruleus, substantia nigra, spinal cord, and pallidostriatum, which are known to contain MAO A-positive structures. Ultrastructural studies revealed that most MAO A staining was associated with the outer membrane of mitochondria, within the cell bodies, dendrites, axons and terminals. However, some immunoreactive staining for MAO A was also observed in the rough endoplasmic reticulum in the cell bodies. Staining for mitochondrial MAO A in dendrites was observed in terminal fields of the monoamine system, including the spinal cord and the pallidostriatum. The intensity of staining also increased in the subsynaptic density. MAO A was also found associated with mitochondria in ependymal cells lining the fourth ventricle adjacent to the locus coeruleus and in the endothelial cells lining the blood vessels. Localization of MAO A in noradrenergic neurons, ependymal cells, and subsynaptic regions of dendrites in monoamine terminal fields supports the concept that this neurotransmitter-degrading enzyme may play a protective role in the central nervous system.

Nonlinear analysis of partial dopamine agonist effects on cAMP in C6 glioma cells.

Most drugs have some efficacy so that improved methods to determine the relative intrinsic efficacy of partial agonists should be of benefit to preclinical and clinical investigators. We examined the effects of partial D(1) or partial D(2) dopamine agonists using a partial agonist interaction model. The dependent variable was the modulation of the dopamine-receptor-mediated cAMP response in C6 glioma cells selectively and stably expressing either D(1) or D(2) recombinant dopamine receptors. The dissociation constant (K(B)) and relative intrinsic efficacy (E(r)) for each partial agonist were calculated using a partial agonist interaction null model in which the effects of fixed concentrations of each partial agonist on the dopamine dose-response curve were evaluated. This model is an extension of the competitive antagonist null model to drugs with efficacy and assumes only that the log-dose--response curve is monotonic. Generally, the partial agonist interaction model fit the data, as well as fits of the independent logistic curves. Furthermore, the partial agonist K(B) values could be shared across partial agonist concentrations without worsening the model fit (by increasing the residual variance). K(B) values were also similar to drug affinities reported in the literature. The model was validated in three ways. First, we assumed a common tissue stimulus parameter (beta) and calculated the E(r) values. This provided a qualitative check on the interaction model results. Second, we calculated new relative efficacy values, E(r)(beta), using the beta estimate. Third, we calculated relative efficacy using relative maxima times midpoint shift ratios (J. Theor. Biol. 198 (1999) 347.). All three methods indicated that the present model yielded reasonable estimates of affinity and relative efficacy for the set of compounds studied. Our results provide a quick and convenient method of quantification of partial agonist efficacy. Special applications and limitations of the model are discussed. In addition, the present results are the first report of the relative intrinsic efficacy values for this set of D(2) ligands.

Test-retest reliability of the rat N40 auditory evoked response: preliminary data.

The N40 auditory evoked potential (EP) in rats has been used to develop an animal model for the study of sensory gating mechanisms. In an examination of the test-retest reliability of sensory gating indices, derived from the rat N40 EP, six rats were tested four times daily for 5 consecutive days. A paired-click paradigm (S1-S2) was used, and both the S2/S1 amplitude ratio and the S1-S2 amplitude difference measures of sensory gating capacity were calculated. Based on the means of four daily trials, reliability coefficients were as follows; r = 0.69 for S1, and r = 0.66 for S2 amplitudes. A reliability coefficient of 0.59 was found for the S1-S2 difference measure, and one of 0.47 for the S2/S1 ratio measure. These results suggest that the N40 can be used to study sensory gating, but caution has to be exercised in interpreting the data particularly in a repeated measures design.

An immunohistochemical study of retinoblastoma gene product in normal, premalignant and malignant tissues of the uterine cervix.

The retinoblastoma gene was the first tumour suppressor gene identified that was altered not only in retinoblastomas but has been described in a wide variety of human neoplasms. The retinoblastoma gene encodes a nuclear phosphoprotein that in its hypophosphorylated state plays an important role in regulating the cell cycle, thus preventing from tumour formation. Expression of retinoblastoma gene protein product (pRB) was investigated in 118 formalin-fixed, paraffin-embedded cervical tissues by immunohistochemistry using commercially available antibody directed against RB protein. Ten normal ectocervical epithelium, 16 cervical intraepithelial neoplasia (CIN) I, 13 CIN II, 14 CIN III, 53 invasive squamous cell carcinoma, 11 adenocarcinoma and 1 small cell carcinoma were selected for this study. The proportions of pRB-positive cells as well as the extent of pRB expression in ectocervical squamous epithelium were assessed and compared among the lesions. The pRB expression was observed in 100% of normal ectocervical epithelium (n=10), 100% of CIN lesions (n=43) and 98.5% of invasive carcinoma of the uterine cervix (n=65) and were statistically significant when CIN or CIN/invasive were compared to normal cases (P < 0.01, P < 0.05 respectively). While in invasive squamous cell carcinoma (SCC), 81.8% (9/11) pRB-positive cells were found in much higher percentages in well differentiated SCC compared to 64.3% (18/28) of moderately differentiated cases and only 7.1% (1/14) of poorly differentiated SCC (P < 0.01, respectively). The results of this study suggest that loss of RB protein expression is rare in carcinoma of the uterine cervix and this protein may be important in the pathogenesis of cervical carcinoma.

cDNA cloning and sequencing of rat monoamine oxidase A: comparison with the human and bovine enzymes.

1. The nucleotide and deduced amino acid sequences of rat liver MAO A were determined, and sequence identities among MAO A and B from rat, human and bovine were compared. 2. MAO A from rat exhibited greater than 85% sequence identity with bovine and human MAO A, and 70% identity with rat MAO B. 3. Rat adrenal cDNAs were restriction mapped, partially sequenced and found to be identical to rat liver MAO A, suggesting that these two tissues express the same polypeptide.

A particle associated with the polyadenylate segment in mammalian messenger RNA.

A structure consisting of poly(A) complexed with other components is released from polysomes by ribonuclease treatment. The poly(A) complex has a sedimentation value of 12-15, while the corresponding sedimentation value for free poly(A) is 4. The complex does not appear to represent an artifact formed by interaction of free poly(A) with either cytoplasmic or polysomal proteins. The polynucleotide released from the complex by treatment with sodium dodecyl sulfate shows the same electrophoretic mobility as that of poly(A) isolated from deproteinized polysomal RNA. The poly(A) in the complex is partially protected from digestion by T(2) ribonuclease. At least part of the poly(A) is available for base pairing with poly(U). The components associated with the poly(A) cause it to bind to Millipore filters at low ionic strength. These components are removed from the complex by Pronase digestion. The findings indicate that the poly(A) segment in messenger RNA serves as a binding site for a particle. This particle appears to consist of proteins.

An enzyme immunoassay for the quantitation of dihydropteridine reductase.

A competitive, enzyme-linked immunosorbent assay for the quantitative determination of dihydropteridine reductase (DHPR) is described. This highly sensitive method can determine the content of DHPR protein in tissue preparations independently of the enzymatic activity of the protein molecule. The method involves initial incubation of samples containing soluble enzyme in microtiter plates coated with purified goat antibodies to rat DHPR and further incubation with DHPR conjugated to alkaline phosphatase. The assay is used to study the ontogeny of DHPR in rat liver.

Radioimmunological identification of polysomes synthesizing fibrinogen polypeptide chains.

Hepatocytes of rats stimulated by turpentine into a hyperfibrinogenemic state produce sufficient quantities of fibrinogen to permit unequivocal identification of specific polysomal complexes involved in the synthesis of this molecule. Monospecific antibodies directed against intact fibrinogen and one of its subunits, the gamma-chain, have shown two size classes of polysomes. Furthermore, it seems possible that polypeptide chain assembly may occur by having completed nascent chains bind to partially completed chains that are still attached to the polysome.

Arginine-42 and threonine-45 are required for FAD incorporation and catalytic activity in human monoamine oxidase B.

Monoamine oxidase B (MAO B) is an integral protein of the outer mitochondrial membrane that is involved in the deamination of vasoactive and neuroactive amines. The oxidation of these amine substrates requires the cofactor FAD, which is covalently bound to Cys-397 of human MAO B. Previously, Glu-34 and Tyr-44 of MAO B have been identified as residues which engage in noncovalent interactions with FAD that are required for subsequent covalent FAD binding and generation of catalytic activity. In this study, we have identified two additional residues, Arg-42 and Thr-45, which form noncovalent contacts with FAD that are prerequisite steps to the covalent attachment of FAD. Arg-42 and Thr-45, along with Tyr-44, comprise part of a highly conserved flavin binding sequence, RXY(T,S), that is found in other flavoproteins, several of which have well-defined X-ray crystal structures. We tested the roles of Arg-42 and Thr-45 in MAO B by constructing mutant MAO B cDNAs which encode amino acid substitutions at these residues and expressed the variant proteins in COS-7 cells. Substitution of Arg-42 or Thr-45 with alanine resulted in complete loss of MAO B activity and FAD incorporation. However, conservative substitutions of Arg-42 with lysine or Thr-45 with serine resulted in MAO B variants that retain both partial activity and partial FAD incorporation. These results indicate that Arg-42 and Thr-45 form critical noncovalent interactions with FAD that are required for the subsequent activation of MAO B by covalent coupling of FAD.

Diversity and nature of ribosomal pools in hepatoma 7800 and host liver.

1. The ribosomal components in the postmitochondrial supernatant of a rat hepatoma (hepatoma 7800) and the corresponding host liver were examined for diversity and functional competence. 2. The ;free' and ;membrane-bound' polyribosomes of both tissues were equally active in vivo and had equilibrated with newly synthesized ribosomes 4hr. after administration of [6-(14)C]orotic acid. 3. The inactive monomer-dimer pool in hepatoma 7800 was unattached to membranes and a larger fraction of the polyribosomes was free in hepatoma than in liver. 4. By using sensitivity to puromycin as a criterion, evidence was obtained that most of the polyribosomes in hepatoma 7800 were active in vivo. 5. Actinomycin, azaguanine and carbon tetrachloride caused marked conversion of polyribosomes into inactive monomers and dimers in the host liver and moderate conversion in the hepatoma. 6. Significant accumulation of ferritin and shifts in the mean polyribosome size to the lighter species occurred in the host liver of rats bearing large hepatomas.

Molecular mechanisms of extinction of liver-specific functions in mouse hepatoma rat fibroblast hybrids: extinction of alpha-fetoprotein gene.

Somatic cell hybrids formed by the fusion of mouse hepatoma (BWTG3) and rat fibroblast (JF1) cells exhibit the extinction of mouse AFP gene expression. Analysis of HindIII digests clearly shows that both mouse and rat AFP genes are present in the hybrids. DNA-RNA hybridization data show that AFP mRNA is virtually absent in the somatic cell hybrids. Our studies indicate, therefore, that extinction of mouse AFP gene expression in somatic cell hybrids (BJ01, BJ50, and BJ140) occurs in the presence of the rat genes and that the mechanism of extinction involves processes that result in the loss of AFP mRNA. We propose that the mechanism may involve either the inhibition of transcription or an inhibition of the processing of the mRNA transcript.