104, 120,383, 131,173,197,194RNase is one of several enzymes elaborated by the pancreas. The properties and structure of bovine pancreatic RNase (ribonuclease I; ribonucleate 3'-pyrimidino-oligonucleotidohydrolase; EC 3.1.4.22) have been extensively studied (2). In contrast, information on human pancreatic RNase is meager. Recent studies in this laboratory revealed striking similarities between RNase of hum-an pancreas and that present in human serum (3). Both enzymes have their pH optimum at 6.5 with poly(C) as substrate. Their action on poly(C) is inhibited by poly(A) and poly(G). They are absolutely dependent on phosphate or citrate for their activity. They are thermostable at pH 4.2 and thermolabile at pH 8.5. They are highly specific to secondary phosphate esters of cytidine 3'-phosphates.These biochemical similarities imply that human pancreas is the source of human serum RNase. This conclusion is at variance with earlier studies (4, 5) which suggested that leukocytes are the source of serum RNase. In our studies, the RNase present in leukocytes is highly specific to secondary phosphate esters of uridine 3'-phosphates and has very little activity toward secondary phosphate esters of cytidine 3'-phosphates (6). Hence, white cells could not be the source of serum RNase.The results presented in this paper show abnormally elevated RNase in the sera of patients with pancreatic cancer. This 2308 further supports the proposition that serum RNase is of pancreatic origin. The abnormal activity of serum RNase serves as a biochemical indicator of pancreatic cancer.MATERIALS AND METHODS Reagents. Poly(C) was purchased from Schwarz/Mann, Orangeburg, N.Y. All other reagents used in this investigation were of reagent grade.Patients. The diagnosis of cancer of patients included in this study was proven by biopsy or by autopsy.Serum. Venous blood, drawn either from volunteer laboratory workers or patients, was allowed to clot at room temperature for 1 hr and centrifuged at 750 X g for 15 min at room temperature. The serum was removed with a capillary pipette and assayed immediately or stored at -20°.RNase Assay. Because human serum RNase is highly specific to secondary phosphate esters of cytidine 3'-phosphates, its assay with poly(C) as a substrate is more sensitive than with RNA, which is hydrolyzed only partially. Cleavage of poly(C) by serum RNase was followed by the formation of mono-and oligonucleotides, which were separated from partially digested poly(C) fragments by acid precipitation. The concentration of acid-soluble products was measured at 278 rum (3). Detailed assay procedure is given in the legend to Table 1. One RNase unit is defined as that amount which renders 1Atmol of poly(C) acid-soluble in 1 min at 370 and at pli 6.5 (U374) RESULTS The normal level of serum RNase was 103 4 24 units/ml of serum. Serum RNase activity was stable, and about 12% of its original activity was lost when stored at room temperature for 72 hr. The time of venipuncture or eating had no effect on serum RNase activity. Table 1 Re...
Actinobacillus actinomycetemcomitans is a Gram-negative bacterium implicated in the pathology of localized juvenile periodontitis, a condition involving rapid destruction of alveolar bone. We have established that gentle extraction of this bacterium in saline releases a proteinaceous fraction (which we have termed surface-associated material [SAM]) which has potent osteolytic activity in the murine calvarial bone resorption assay. Fractionation of the SAM has now revealed that activity is associated with a 62-kD protein. This bone-resorbing activity can be blocked by a monoclonal antibody (raised to the whole bacterium) that is claimed to recognize a protein homologous to the Escherichia coil molecular chaperone GroEL. Purification of this bone-resorbing protein to homogeneity has been achieved by a combination of anion exchange, gel filtration, and ATP-affinity chromatography and the NH2-terminal sequence shows > 95% homology to E.
Staphylococcus aureus infections are associated with rapid bone destruction in conditions such as osteomyelitis, bacterial arthritis, and infected orthopedic implant failure. How this bacterium induces bone destruction has not been defined. In studies of the role of oral Gram-negative bacteria in periodontal pathology, we have established that cell surface-associated proteins (SAPs) are potent stimulators of bone resorption. The surface-associated components from S. aureus have now been isolated and demonstrated to be extremely potent stimulators of bone resorption in the murine calvarial bone resorption assay. Bone resorption appears to be due to proteins, is not the result of contamination with lipoteichoic acid or muramyl dipeptide, and is potently inhibited by indomethacin and can be completely blocked by high concentrations of interleukin-1 receptor antagonist or TN3-19.12, a neutralizing monoclonal antibody to murine TNF. The SAP fraction can stimulate fibroblasts or monocytes to release osteolytic cytokines, but only at high concentrations. Fractionation of the SAPs by high performance liquid chromatography demonstrated that a number of fractions were osteolytically active. The most active contained a heterodimeric protein of molecular weight 32-36 kD. The presence of this osteolytically active surface-associated fraction may account for the bone resorption associated with local infection with S. aureus.
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