Collagen biosynthesis enzymes in serum and hepatic tissue in liver disease

Abstract. Serum immunoreactive prolyl hydroxylase protein was measured in sixty-five patients with liver disease, and liver prolyl hydroxylase activity, immunoreactive prolyl hydroxylase protein and collagen hydroxyproline in forty of these patients. Serum immunoreactive prolyl hydroxylase protein was above the 95% confidence limit of the controls in most patients with primary biliary cirrhosis, portal cirrhosis, acute hepatitis and cancer with liver metastases, but below this in most patients with fatty liver, chronic active hepatitis, extrahepatic cholestasis, cholangitis, cancer without liver metastases and other malignant diseases. Elevated serum immunoreactive prolyl hydroxylase protein decreased rapidly with time in acute hepatitis but not in primary biliary cirrhosis. Liver prolyl hydroxylase activity and immunoreactive prolyl hydroxylase protein were elevated in the same diseases as serum immunoreactive prolyl hydroxylase protein, and correlated significantly with the latter whereas no correlation was found between serum immunoreactive prolyl hydroxylase protein and collagen hydroxyproline. Serum immunoreactive prolyl hydroxylase protein correlated highly significantly with serum alkaline phosphatase and weakly with serum aspartate aminotransferase in primary biliary cirrhosis, but not in any other disease. No correlation was found between serum immunoreactive prolyl hydroxylase protein and other tests of liver function. The results suggest that changes in serum immunoreactive prolyl hydroxylase protein in liver disease primarily reflect changes in this enzyme in the hepatic tissue, and that assays of serum immunoreactive prolyl hydroxylase in liver disease may give useful information on the actual hepatic collagen synthesis.     

SR proteins ASF/SF2 and SRp55 participate in tissue factor biosynthesis in human monocytic cells

Summary.  Background:  Human monocytes express two naturally occurring forms of circulating tissue factor (TF) – full-length TF, a membrane-spanning protein, and alternatively spliced TF, a soluble molecule. Presence of the variable exon 5 in TF mRNA determines whether the encoded TF protein is transmembrane, or soluble. Recently, an essential SR protein ASF/SF2 was implicated in TF pre-mRNA processing in human platelets. Objective:  To examine molecular mechanisms governing regulated processing of TF pre-mRNA in human monocytic cells. Methods and results: In silico analysis of the human TF exon 5, present only in full-length TF mRNA, revealed putative binding motifs termed exonic splicing enhancers (ESE) for the SR proteins ASF/SF2 and SRp55, which were found to be abundantly expressed in monocytic cell lines THP-1 and SC, as well as monocyte-enriched peripheral blood mononuclear cells (PBMC). Using a splice competent mini-gene reporter system transiently expressed in monocytic cells, it was determined that weakening of either five closely positioned ASF/SF2 ESE (bases 87–117) or a single conserved SRp55 ESE (base 39) results in severe skipping of exon 5. ASF/SF2 and SRp55 were found to physically associate with the identified ESE. Conclusions:  SR proteins ASF/SF2 and SRp55 appear to interact with the variable TF exon 5 through ESE at bases 39 and 87–117. Weakening of the above ESE modulates splicing of TF exon 5. This study is the first to identify and experimentally characterize cis -acting splicing elements involved in regulated biosynthesis of human TF.     

Studies on the determination of extracellular galactosyltransferase in human intestinal tissue

The determination of extracellular galactosyl transferase (EC 2.4.1.38) activity in human intestinal tissue by assessment of the incorporation of label after incubation with UDP[3H]galactose was evaluated. Intestinal biopsy specimens were incubated with membrane-permeable L-[1-14C]fucose and non-permeable UDP-D-[6-3H]galactose (UDP[3H]Gal). Comparison of the amounts of 3H- and 14C-label incorporated into subcellular fractions showed uptake and incorporation of galactose formed by the hydrolysis of UDP[3H]Gal by brush-border enzymes. The results indicate that incorporation of galactose after incubation of the tissue with UDP[3H]Gal is not exclusively attributable to extracellular galactosyl transferase.     

Synthesis of albumin and acute-phase proteins in perfused liver after burn injury in rats.

The acute-phase response that follows injury and sepsis is characterized by increased hepatic synthesis of specific secreted proteins while production of albumin is decreased. The effect of burn injury on specific synthesis rates of secreted hepatic proteins has not been reported. In this study, Sprague-Dawley rats received either a 30% flame burn (n = 12) or a sham burn (n = 12) and were allowed to recover for 11 days. Burned animals showed slower weight gains and a 25% to 30% higher resting energy expenditures compared with controls. On postburn day 11, synthesis of secreted hepatic proteins was measured by incorporation of leucine during a 2-hour isolated liver perfusion. Synthesis of total secreted proteins, the seromucoid fraction, and complement component C3 was significantly increased in burned animals, whereas synthesis of albumin was unaltered. In spite of unchanged albumin synthesis, plasma albumin concentrations were 50% lower in burned animals than in control animals throughout the postburn period. These findings suggest that decreased albumin synthesis is not the only mechanism responsible for persistent hypoalbuminemia that follows burn injury.     

Studies on the coumarin anticoagulant drugs: interaction of human plasma albumin and warfarin sodium

In studies by continuous flow electrophoresis the coumarin anticoagulant drug warfarin sodium was found to be bound solely to the albumin fraction of the plasma proteins. The interaction was studied in detail by equilibrium dialysis of solutions of crystalline human plasma albumin and warfarin sodium. Analysis of the data showed that albumin possesses a single strong binding site for warfarin with an association constant of 154,000 at 3 degrees C and secondary classes of several sites with a much lower affinity. The free energy of binding for the first anion determined at 3 degrees and 37 degrees C was -6.54 and -7.01 kcal per mole, respectively. The standard enthalpy change for the interaction was -3.48 kcal per mole, and the entropy change was +11.2 U.The negative enthalpy change was surprisingly large and the positive entropy change small for an anion-albumin interaction, suggesting significant nonionic binding. The inability to saturate the albumin binding sites, even when high concentrations of warfarin were used, is consistent with a reversible configurational alteration of the albumin molecule during the binding process. The thermodynamic data indicate that the albumin binding sites for warfarin sodium are formed during the process of binding, rather than being performed as in antigen-antibody reactions. The strength of the binding process suggests that many of the pharmacodynamic characteristics of warfarin sodium in man are determined by its strong interaction with plasma albumin. Such correlations of the physicochemical interactions and biologic effects of the coumarin anticoagulant drugs should lead to a better understanding of their mechanisms of action.     

人血白蛋白输注速度对肝硬化门静脉高压患者血流动力学的影响

目的：探讨肝硬化门静脉高压患者输入人血白蛋白的适宜速度。方法选择门静脉高压需输入人血白蛋白的患者58例,均输入20％人血白蛋白50 ml,采用抛硬币发随机将患者分为对照组（n＝30）和实验组（n＝28）。对照组患者输注速度为25 gtt/min,输入时间为45 min;实验组输注速度为33 gtt/min,输入时间为30 min。采用彩色多普勒超声诊断仪测量不同时间点门静脉内径（ Dpv）、平均血流速度（Vpv）、血流量（Qpv）及门静脉压力,比较两组差异。结果对照组输入白蛋白结束时,Dpv为（13．03±2．01）mm,Vpv为（9．40±2．06）cm/s,Qpv为（782．29±313．02）ml/min,门静脉压力为（2．747±0．348）kPa,与实验组比较差异均无统计学意义（t 值分别为-0．04,0．81,0．46,0．55;P ＞0．05）。结论在30 min内给肝硬化门静脉高压患者输入20％人血白蛋白50ml是可行的。     

The retention of S35-labelled bovine serum albumin in normal and immunized rabbit liver tissue

The S³⁵-label of S³⁵-BSA was detected in the liver tissue of rabbits to the extent of 0.02 per cent (10 µg or 10¹⁴ molecules) of the injected material at 140 days after injection. The rate of loss of antigen at the termination of the experiment was of such an order that significant amounts would be expected to persist for at least several years. Data are reported which extend the retention data previously reported on S³⁵-labelled hemocyanin. They indicate that amounts of the order of 0.05 per cent (25 µg.) of antigen material persist at 330 days after injection. All of the radioactivity of material retained in the liver tissue 6 weeks after injection was immunologically related to the original S³⁵-BSA antigen. Preliminary studies are reported which indicate that the retained antigen is bound to ribonucleic acid. A new method is described for the isolation of p-azophenylsulfonate bovine serum albumin from tissue extracts by means of a Dowex 2 adsorbent.     

Simultaneous expression of type I procollagen mRNA and albumin in cirrhotic human liver.

The gene expression of human type I procollagen was investigated in cirrhotic human liver by using in situ hybridization with nonradioactive DNA probes. Using in situ hybridization can provide direct evidence for the cell type capable for type I collagen synthesis in tissues. T-T dimerized DNA probes were used and DNAs hybridized in situ were detected immunohistochemically using specific antibodies against T-T dimer. The data demonstrated that type I collagen is synthesized in hepatocytes and stellate cells in pseudolobules and in fibroblasts in Glissons capsules in cirrhotic human livers. We indicated hepatocytes morphologically and functionally by using immunohistochemical localization of albumin, which was used as a marker of hepatocyte, since albumin is synthesized exclusively by hepatocytes.     

Comparison of the properties of ribonucleases in human liver tissue and serum.

Two ribonucleases (RNases) with acidic pH optima were partially purified, one from normal human liver tissue and the other from serum. The properties of the two enzymes were studied and compared. Liver RNase was partially purified about 700-fold by acid fractionation, phosphocellulose column chromatography, Sephadex G-75 gel filtration, and polyguanylate affinity column chromatography. Serum RNase was purified about 1200-fold by phosphocellulose column chromatography and Sephadex G-75 gel filtration. The two RNases showed a similar optimal pH and molecular mass, and similar behaviour towards metal ions, but they differed in their substrate specificity. Liver RNase displayed a higher activity towards polyuridylate (poly(U)) than towards polycytidylate (poly(C)), while serum RNase hydrolysed poly(C) more rapidly than poly(U). These findings suggest that liver RNase is not the primary source of the serum RNase with an acidic pH optimum.     

Dipyridamole binding to proteins in human plasma and tissue culture media

Dipyridamole (Persantin), a commonly used coronary vasodilator and antithrombotic drug, has been intensively studied for its potential use in combination chemotherapy for cancer, and, recently, for acquired immunodeficiency syndrome. However, the strong binding of dipyridamole to proteins in human plasma complicates quantitative extrapolations from in vitro data to the clinic. To aid in such extrapolations, we incubated dipyridamole in human plasma and in tissue culture media containing 10% fetal calf serum (FCS), and determined the equilibrium levels of free drug. In human plasma, after addition of 2 to 10 mumol/L dipyridamole (i.e., in the therapeutically relevant concentration range), the fraction of free drug averaged between 1.9% and 3.5%. In 10% FCS, after addition of 0.08 to 5 mumol/L dipyridamole (i.e., the experimentally relevant concentration range), mean free fractions were in the 75% to 100% range. Relating various total dipyridamole levels in the therapeutically relevant range in human plasma to those in 10% FCS that provided identical fractions of free drug gave ratios in the 24 to 55 range. Thus, a multiplication factor in the above range is suggested for the interconversion of in vitro and in vivo dipyridamole concentrations that provide equivalent levels of free drug.     

Multiple forms of alkaline phosphatases in human liver tissue

Alkaline phosphatases (AP) extracted in the presence of n-butanol from human liver are separated by affinity chromatography on phenylsepharose Cl-4B into two fractions named APII and APIIII. By repeated chromatography, APII was purified to a single enzyme entity with a specific activity of 1,684 kU/g protein. APIIII was purified to a specific activity of 535 kU/g protein. It consisted of only APIIII enzyme activity, but still contained gamma-glutamyltransferase activity. These two forms of AP are different in chromatographic and electrophoretic behaviour, APIIII being a larger molecule than APII. APII and APIIII are very similar in enzyme kinetic behavior, such as substrate activity, thermolability and sensitivity to different inhibitors. It is concluded from these experiments that multiple forms of AP in liver bear identical active centres, the difference is due to a modification of protein residue. It is possible that both are modified forms of one enzyme. Both are different from the AP isoenzyme that appears in serum in cholestatic patients.