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PubMedDate not supplied

Development and use of a rat albumin cDNA clone to evaluate the effect of chronic ethanol administration on hepatic protein synthesis.

A rat albumin cDNA probe (pBR alb 149) was developed in order to investigate the molecular mechanisms responsible for changes in hepatic protein synthesis after chronic administration of ethanol to rats. Rats fed a diet for up to 1 year in which 36% of calories were from ethanol, developed fatty livers but not cirrhosis. Cell-free protein synthesis with liver membrane-bound polysomes of ethanol-fed rats was increased as compared to control membrane-bound polysomes, whereas protein synthesis with free polysomes was unchanged. Total RNA extracted from liver membrane-bound polysomes and translated in a rabbit reticulocyte mRNA-dependent system showed a marked increase in albumin synthesis in the ethanol-fed group. Analysis of RNA molecules separated according to molecular weight by gel electrophoresis and hybridized with recombinant-cloned albumin cDNA demonstrated an increase in full-sized albumin mRNA species in ethanol-fed animals. Therefore, chronic ethanol administration appears to increase albumin synthesis by increasing the steady-state level of biologically active albumin mRNA in liver membrane-bound polysomes. Despite development of fatty liver, the protein synthesis machinery functions normally.

Albumins↗
PubMedDate not supplied

Regulation of low density lipoprotein receptor activity in primary cultures of human hepatocytes by serum lipoproteins.

The low density lipoprotein receptor activity was measured in primary cultures of human hepatocytes. The receptor-mediated association and degradation of low density lipoprotein increased gradually up to 140 and 190%, respectively, upon incubation of the cells with increasing amounts of whole serum (up to 100%). Preincubation of the cells with low density lipoprotein resulted in a weak downregulation of the receptor-mediated association of low density lipoprotein (only 35% reduction at 100 micrograms low density lipoprotein per ml). However, preincubation with high density lipoproteins with density between 1.16 and 1.20 gm per ml (heavy high density lipoprotein) resulted in a more than 2-fold stimulation of the receptor-mediated association of low density lipoprotein. This heavy high density lipoprotein-mediated stimulation could not be antagonized by a simultaneous addition of low density lipoprotein during that preincubation. We conclude that, in primary cultures of human hepatocytes, the downregulation of the low density lipoprotein receptor activity by low density lipoprotein is weak and completely overruled by heavy high density lipoprotein. If these results for human hepatocytes in vitro hold true for hepatocytes in vivo, our results might explain why in vivo liver cells still display low density lipoprotein receptor activity notwithstanding the exposure of these cells to physiological concentrations of low density lipoprotein.

Cells, Cultured↗
PubMed1990

Binding of the major and large HBsAg to human hepatocytes and liver plasma membranes: putative external and internal receptors for infection and secretion of hepatitis B virus.

A likely mechanism of the strong hepatotrophism of the hepatitis B virus is the presence of specific receptors for the surface antigen of hepatitis B virus on hepatocyte membranes. To examine this hypothesis, we have performed binding studies using recombinant large (preS1 + preS2 + S) and major (S) proteins with adult human hepatocytes, rat hepatocytes, human fibroblasts, human peripheral blood mononuclear cells and plasma membranes derived from these cell types. We found that major HBsAg was able to bind specifically to human hepatocytes, human fibroblasts and human blood mononuclear cells. This binding could be inhibited by recombinant middle (preS2 + S) protein but not by the recombinant large protein. No binding could be demonstrated between large HBsAg and human hepatocytes. However, large protein bound specifically to plasma membranes derived from human liver tissue, human fibroblasts and HepG2A16 cells. This binding could be partially inhibited by the major protein and by a synthetic preS1 peptide but not by a synthetic preS2 peptide. These results support the assumption that hepatitis B virus absorption and penetration into human hepatocytes is mediated by specific receptors recognizing an amino acid sequence in the S-region. This recognition site is not displayed by the recombinant large protein. However, the large protein is recognized by its preS1 region and by a second binding site in the S-region by a receptor molecule, located on the inner surface of the plasma membranes or intracellular membranes of human hepatocytes and of some other cell types derived from human tissue.

Animals↗
PubMed1993

Regulation of glycogen phosphorylase activity in isolated human hepatocytes.

Hepatocytes were isolated from human liver tissue by a two-step perfusion technique. They were treated with vasopressin, angiotensin, ATP and phenylephrine, which are known to be Ca(2+)-mediated glycogenolytic agents in rat liver tissue, and as a control, they were treated with the cyclic AMP-mediated hormones glucagon and isoproterenol. All agonists induce a time-dependent activation of glycogen phosphorylase. Glucagon and isoproterenol induce a somewhat higher degree of phosphorylase activation compared with vasopressin, angiotensin, ATP and phenylephrine, which all increase inositol tris-phosphate levels and have no effect on the cyclic AMP levels. The total activity of glycogen phosphorylase (a + b), amounting to 30 to 35 mU/mg protein, is found to be much lower than that found in rat liver tissue. Because only minor differences could be found, we conclude that the regulation of glycogen phosphorylase in human liver tissue is basically the same as that found in rat liver tissue.

Adenosine Triphosphate↗
PubMed1996

Modulation of basal hepatic glycogenolysis by nitric oxide.

We perfused livers from fed rats with a balanced salt solution containing 1 mmol/L glucose. Under these conditions a low steady rate of glycogenolysis was observed (approximately 1.7 micromol glucose equivalents/g/min; 20% of the maximal glycogenolytic activity). Nitric oxide (NO) transiently stimulated hepatic glucose production. A maximal response (on average doubling basal glucose output) was observed with 34 micromol/L NO. The same concentration of nitrite (NO2-) was ineffective. Half-maximal effects were seen at 8 to 10 micromol/L NO, irrespective of the flow direction (portocaval or retrograde). This glycogenolytic response to NO corresponded to a partial activation of phosphorylase. The NO effect was not additive to maximal stimulation of glycogenolysis (7.7 +/- 0.2 micromol hexose equivalents/g/min; n = 4) by 100 micromol/L dibutyryl cyclic adenosine monophosphate (Bt2cAMP). The requirement for activation of phosphorylase was also evidenced by the ineffectiveness of NO in phosphorylase-kinase-deficient livers of gsd/gsd rats. The NO effect was blocked by co-administration of cyclooxygenase inhibitors (50 micromol/L ibuprofen, 50 micromol/L indomethacin, or 2 mmol/L aspirin), suggesting a mediatory role of prostanoids from nonparenchymal cells. This conclusion was confirmed by the fact that NO did not activate phosphorylase in isolated hepatocytes. Moreover, NO was no longer glycogenolytic in livers perfused with Ca2+-free medium, in agreement with the known mediatory role of Ca2+ in prostanoid-mediated responses. Surprisingly, in Ca2+-free medium NO inhibited the basal glucose production. This coincided with an increased elution of cyclic guanosine monophosphate (cGMP). Inhibition of glycogenolysis by NO under these conditions was blocked by 1 mmol/L theophylline, suggestive for involvement of cGMP-stimulated cAMP phosphodiesterase. However, we could not confirm that an increase in cGMP resulted in a drop in cAMP. In conclusion, NO recruits opposing mechanisms with respect to modulation of basal hepatic glycogenolysis. In the presence of Ca2+, activation of phosphorylase with stimulation of glycogenolysis dominates. Cyclooxygenase inhibitors abolish this effect. Activation by NO of the cyclooxygenase in nonparenchymal cells is a distinct possibility. In the absence of Ca2+, inhibition of basal glycogenolysis becomes observable. It remains to be established whether this results from cGMP-mediated stimulation of hydrolysis of cAMP.

Animals↗
PubMed1999

Transfection of a rat hepatoma cell line with a construct expressing human liver annexin V confers susceptibility to hepatitis B virus infection.

Previously, we have found that human liver annexin V (hA-V; in earlier reports referred as Endonexin II) is a specific hepatitis B surface antigen (HBsAg) binding protein. In this study, we demonstrate that transfection of rat hepatoma FTO 2B cells, a cell line that is not infectable by hepatitis B virus (HBV) and does not express hA-V, with a construct containing the hA-V gene, resulted in hA-V expressing cells susceptible to HBV infection. After in vitro infection, transfected FTO cells (assigned as FTO 9.1 cells) expressing hA-V in cultures were shown to contain HBV-precore/core, X mRNAs, and covalently closed circular (ccc) DNA as detected by polymerase chain reaction (PCR). The presence of HBV ccc and replicative intermediate DNA was also demonstrated by Southern blot hybridization assay. HBV DNA secreted in the culture medium was also evident as determined by quantitative branched DNA (bDNA) assay. HBsAg and hepatitis B core antigen (HBcAg) could also be detected by an immunocytochemical method in 10% to 15% of the cells at day 3 and day 5 after infection. Infectivity of in vitro-propagated HBV was demonstrated by infection of the naive FTO 9.1 cells with the culture supernatant from HBV-carrier cultures. In contrast to primary cultures of human hepatocytes and FTO 9.1 cells, primary rat and mouse hepatocytes, as well as rat hepatoma cell lines that do not express hA-V, are not susceptible to HBV infection. These findings suggest that hA-V plays a key role in the initial step of HBV infection and that the species-specific susceptibility to HBV infection and replication in hepatocytes is associated with the expression of hA-V.

Animals↗
PubMed1999

A phase I/II study of recombinant human interleukin-12 in patients with chronic hepatitis C.

Interleukin-12 (IL-12) plays a central role in mounting an effective cellular immune response directed towards elimination of intracellular pathogens. The present open-label, multicenter, dose-escalation phase I/II study was designed to assess tolerability, pharmacokinetics, pharmacodynamics, and efficacy of subcutaneously administered recombinant human interleukin-12 (rHuIL-12) in the treatment of chronic hepatitis C. Sixty patients (42 men, 18 women, aged 24-60) were treated with 0.03 microgram/kg (n = 16), 0.1 microgram/kg (n = 14), 0.25 microgram/kg (n = 15), or 0.5 microgram/kg rHuIL-12 (n = 15) for 10 consecutive weeks. rHuIL-12 was generally well tolerated, with 2 patients (3.3%) being withdrawn from treatment for adverse events. Treatment was associated with temporary decreases in neutrophils and lymphocyte counts and with elevations in serum transaminases and bilirubin. Serum IL-12 levels observed were higher at 0.5 microgram/kg compared with 0.25 microgram/kg doses, suggesting a dose-related increase in systemic exposure of IL-12. Measurable levels of interferon gamma (IFN-gamma) were also observed at the highest dose of 0.5 microgram/kg. At the end of treatment hepatitis C virus (HCV) RNA was detectable in all patients. A more than 50% decrease in pretreatment HCV RNA levels was observed in 3 of 16 patients of the 0.03-microgram/kg dose group, in 3 of 14 of the 0.10-microgram/kg dose group, in 6 of 15 of the 0.25-microgram/kg dose group, and in 8 of 15 patients of the 0.5-microgram/kg dose group. Although in several cases serum alanine transaminase (ALT) levels decreased either during or after treatment, ALT normalization was observed in only 4 patients at the end of treatment and in 5 patients at the end of follow-up. Significant anti-rHuIL-12 antibody titers were not detectable in any patient. In conclusion, antiviral activity of rHuIL-12 in patients with chronic hepatitis C does not appear advantageous in comparison with other currently available treatments.

Adolescent↗
PubMed1999

Expression of hepatitis C virus NS5B protein: characterization of its RNA polymerase activity and RNA binding.

The nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) is considered to possess RNA-dependent RNA polymerase (RdRp) activity and to play an essential role for the viral replication. In this study, we expressed the NS5B protein of 65 kd by a recombinant baculovirus. With the highly purified NS5B protein, we established an in vitro system for RdRp activity by using poly(A) as a template and a 15-mer oligo(U) (oligo(U)15) as a primer. Optimal conditions of temperature and pH for primer-dependent polymerase activity of the NS5B were 32 degrees C and pH 8.0. The addition of 10 mmol of Mg2+ increased the activity. The importance of three motifs conserved in RdRp among other positive-strand RNA viruses was confirmed by introduction of an Ala residue to every amino acid of the motifs by site-directed mutagenesis. All mutants lost RdRp activity, but retained the RNA binding activity, except one mutant at Thr287/Asn291. Deletion mutant analysis indicated that the N-terminal region of NS5B protein was critical for the RNA binding. Inhibition of RdRp activity by (-)beta-L-2', 3'-dideoxy-3'-thiacytidine 5'-triphosphate (3TC; lamivudine triphosphate) and phosphonoacetic acid (PAA) was observed after screening of nucleoside analogs and known polymerase inhibitors. These data provide us not only important clues for understanding the mechanism of HCV replication, but also a new target of antiviral therapy.

Animals↗
PubMed2007

Diversity of cystathionine beta-synthase haplotypes bearing the most common homocystinuria mutation c.833T>C: a possible role for gene conversion.

Homozygosity or compound heterozygosity for the c.833T>C transition (p.I278 T) in the cystathionine beta-synthase (CBS) gene represents the most common cause of pyridoxine-responsive homocystinuria in Western Eurasians. However, the frequency of the pathogenic c.833C allele, as observed in healthy newborns from several European countries (q(c.833C) approximately equals 3.3 x 10(-3)), is approximately 20-fold higher than expected on the basis of the observed number of symptomatic homocystinuria patients carrying this mutation (q(c.833C) approximately equals 0.18 x 10(-3)), implying clinical underascertainment. Intriguingly, the c.833C mutation is also present in combination with a 68-bp insertion, c.[833C; 844_845ins68], in a substantial proportion of chromosomes from nonhomocystinuric individuals worldwide. We have sought to study the relationship between the pathogenic and nonpathogenic c.833C-bearing chromosomes and to determine whether the pathogenic c.[833C; -] chromosomes are identical-by-descent or instead arose by recurrent mutation. Initial haplotype analysis of 780 randomly selected Czech and sub-Saharan African wild-type chromosomes, employing 12 intragenic markers, revealed 29 distinct CBS haplotypes, of which 10 carried the c.[833C; 844_845ins68] combination; none carried an isolated c.833C or c.844_845ins68 mutation. Subsequent examination of 69 pathogenic c.[833C; -] chromosomes, derived from homocystinuria patients of predominantly European origin, disclosed three unrelated haplotypes that differed from their wild-type counterparts by virtue of the presence of c.833C, thereby indicating that c.833T>C transition has occurred repeatedly and independently in the past. Since c.833T does not reside within an obvious mutational hotspot, we surmise that the three pathogenic and comparatively prevalent c.[833C; -] chromosomes may have originated by recurrent gene conversion employing the common nonpathogenic c.[833C; 844_845ins68] chromosomes as templates.

Africa↗
PubMedDate not supplied

Clinical pharmacology of intramuscularly administered L-asparaginase.

Clinical pharmacology of L-asparaginase was compared by intramuscular and intravenous injections in 12 patients with metastatic cancer or leukemia. Following a single intramuscular injection at the gluteal site of L-asparaginase (10,000 IU/m2), the enzyme appeared in plasma as measured initially at 1 hour, but plateau levels were not reached in plasma until 14 to 24 hours after drug administration. The peak plasma level was 1.12 IU/ml, only one fourth of that seen when L-asparaginase was given intravenously at equal doses. However, the enzyme level decrease in the plasma after intramuscular injections was slower, with a half-life ot 46 hours, compared to 7 to 28 hours for intravenously administered drug. The apparent volume of distribution indicated that the intravenously injected enzyme was mostly distributed in plasma, whereas the intramuscularly injected enzyme yielded a much larger volume of distribution (63 versus 245 ml/kg). In addition, only 19 per cent of the intramuscularly injected dose was in plasma, and the area under the curve (C X t) was only 1/24 that by intravenous route (20 versus 487 IU/ml.hr). No enzyme was measurable in patients' urine samples collected for three days after intramuscular injections of the enzyme, and only a trace (less than 1 per cent) of the enzyme was detected in urine after intravenous administration.

Asparaginase↗
PubMedDate not supplied

Comparison of different test models for the assessment of cytotoxicity of composite resins.

This study compared the use of different test models to assess the cytotoxicity of a dental composite. The cytotoxicity of a composite polymerized using two halogen-based light-curing units (LCUs) (Max LC and Astralis) and two light-emitting diode LCUs (E-light and Freelight) served as the basis of comparison. Disk-shaped specimens (7 mm diameter, 2 mm high) were fabricated using the four different light sources. The specimens were used in several cytotoxicity test models: direct and indirect contact tests as well as an extract test with an established cell line L-929. The cells were stained with neutral red after cell-material contact for 48 h. Neutral red-stained areas (in mm2, for direct and indirect tests) and absorbance readings (for extract tests) were analysed statistically using ANOVA and the Tukey post hoc test, with P < 0.05 considered to be significantly different. Good correlation between direct and indirect contact tests (r = 0.903) was found. The extract test was the least correlated among the three tests. It was found that the E-light + Freelight-cured composite elicited cytotoxicity from the correlated studies. Uncured specimens were most detrimental to the cells in all tests. Our data demonstrated that composite cured with light-emitting diode LCUs were cytotoxic to L-929 cells. Different test models were found to give rise to different findings. Thus, a good cell-material contact method would replicate more closely the physiological situation in vivo. This in turn would give more clinically relevant results.

Animals↗
PubMed2004

Flexural strength of dental composite restoratives: comparison of biaxial and three-point bending test.

This study compared two test methods used to evaluate the flexural strength of resin-based dental composites. The two test methods evaluated were the three-point bending test4 and the biaxial flexural test. Materials used in this investigation were from the same manufacturer (3M ESPE) and included microfill (A110), minifill (Z100 and Filtek Z250), polyacid modified (F2000), and flowable [Filtek Flowable (FF)] composites. Flexural strength was determined with the use of both test methods after 1 week of conditioning in water at 37 degrees C. Data were analyzed with the use of an ANOVA/Scheffe test and an independent-samples t test at significance level 0.05. Mean flexural strength (n = 7) ranged from 66.61 to 147.21 and 67.27 to 182.81 MPa for three-point bending and ball-on-three-ball biaxial test methods, respectively. In both test methods, Z100 was significantly stronger than all other composites evaluated. In the three-point bending test, flexural strength of Z250 was significantly higher than A110, F2000 and FF, and FF was significantly stronger than A110 and F2000. The biaxial test method arrived at the same conclusions except that there was no significant difference between Z250 and FF. Pearson's correlation revealed a significantly (p < 0.01) positive and good correlation (R2 = 0.72) in flexural strength between the two test methods. Although the biaxial test has the advantage of utilizing small specimens, the low reproducibility of this test method does not support the proposition that it is a more reliable test method when compared to the ISO three-point bending test.

Algorithms↗
PubMed2007

Environmental degradation of glass-ionomer cements: a depth-sensing microindentation study.

This study investigated the effects of various environmental conditions on the hardness and elastic modulus of restorative glass-ionomer cements (GICs). Two resin-modified GICs (RMGICs) (Fuji II LC [FL]; Photac-Fil Quick [PQ]) and three highly viscous GICs (HVGICs) (Fuji IX Fast [FN]; KetacMolar [KM]; KetacMolar Quick [KQ]) were evaluated in this study. Specimens were fabricated according to the manufacturers' instructions and stored under a variety of conditions (n = 7): 100% humidity, distilled water, pH 5 demineralization solution, and pH 7 remineralization solution. The hardness and elastic modulus were measured using a depth-sensing microindentation test after 4 weeks. The results were analyzed using the independent samples T-test and ANOVA/Scheffe's post hoc test (p < 0.05). HVGICs showed significantly higher hardness and elastic modulus than RMGICs under all storage conditions. Storage in distilled water significantly increased the hardness and elastic modulus of FN, but decreased that of PQ. All HVGICs and RMGICs stored in remineralization solution had hardness values and elastic moduli comparable to those stored in water. Compared to remineralization solution, demineralization solution had no significant effects on the modified GICs with the exception of KQ. The results suggest that the mechanical properties of glass-ionomer restoratives are material-type and storage condition dependent. Therefore, the clinical selection of a glass-ionomer material should be based on the oral environment to which it will be subjected.

Biodegradation, Environmental↗
PubMed1990

Sonographic screening for renal tract anomalies associated with congenital heart disease.

The incidence of structural renal tract abnormalities in 109 children with documented congenital heart disease (CHD) was studied using real-time sonography. In these children, 11.9% had associated urologic anomalies, which included hydronephrosis, duplication, ectopia, agenesis, and dysplasia of kidneys. Children with associated extracardiac anomalies had a significantly higher incidence of renal tract anomalies (39.1%) compared to those with isolated CHD (4.7%). Therefore, sonographic screening of the renal tract should be performed routinely in patients with CHD, especially in those with multiple congenital defects.

Abnormalities, Multiple↗
PubMed2006

Transplantation of bone marrow genetically engineered to express proinsulin II protects against autoimmune insulitis in NOD mice.

BACKGROUND: Type 1 diabetes (T1D) is a T-cell-dependent autoimmune disease resulting from destructive inflammation (insulitis) of the insulin-producing pancreatic beta-cells. Transgenic expression of proinsulin II by a MHC class II promoter or transfer of bone marrow from these transgenic mice protects NOD mice from insulitis and diabetes. We assessed the feasibility of gene therapy in the NOD mouse as an approach to treat T1D by ex vivo genetic manipulation of normal hematopoietic stem cells (HSCs) with proinsulin II followed by transfer to recipient mice. METHODS: HSCs were isolated from 6-8-week-old NOD female mice and transduced in vitro with retrovirus encoding enhanced green fluorescent protein (EGFP) and either proinsulin II or control autoantigen. Additional control groups included mice transferred with non-manipulated bone marrow and mice which did not receive bone marrow transfer. EGFP-sorted or non-sorted HSCs were transferred into pre-conditioned 3-4-week-old female NOD mice and insulitis was assessed 8 weeks post-transfer. RESULTS: Chimerism was established in all major lymphoid tissues, ranging from 5-15% in non-sorted bone marrow transplants to 20-45% in EGFP-sorted bone marrow transplants. The incidence and degree of insulitis was significantly reduced in mice receiving proinsulin II bone marrow compared to controls. However, the incidence of sialitis in mice receiving proinsulin II bone marrow and control mice was not altered, indicating protection from insulitis was antigen specific. CONCLUSIONS: We show for the first time that ex vivo genetic manipulation of HSCs to express proinsulin II followed by transplantation to NOD mice can establish molecular chimerism and protect from destructive insulitis in an antigen-specific manner.

Animals↗
PubMed1992

Visual demonstration of the limb-forming zone in the chick embryo lateral plate.

The present study was undertaken to determine whether a visible Wolffian ridge, distinct from the lateral fold, can be identified in chick embryos. Ectoderm thickness was measured in stage 11-17 chick embryos. There was a general trend, from thin ectoderm in the midline, to an ectodermal thickening over the somites, intermediate mesoderm, and lateral plate. Other embryos were cut from the yolk, pinned out, and photographed. The lateral fold was then eliminated, and the embryo was rephotographed. The photographs reveal a definite opaic zone, distinct from the lateral fold, in stage 11-18 chick embryos. Furthermore, there is a direct correlation between the opacity of this cellular band and the limb-forming potential of grafted wing, flank, and leg regions (see Stephens et al., '89). At stages 11-14, the wing, flank, and leg exhibit a uniform opacity, and a uniform capacity for limb formation when grafted to a host celom. From stage 15 to stage 18, the opacity in the flank diminishes, and its limb-forming capability disappears. This study demonstrates the presence of an opaic zone, which we have called the limb-forming zone (LFZ) along the lateral side of early chick embryos, which is independent of the lateral fold, is not as extensive as the lateral plate, and is not simply associated with ectodermal thickening, but which is directly correlated with limb-forming potential in the lateral plate.

Animals↗