Elevation of an acid ribonuclease in serum of patients with chronic granulocytic leukaemia.

Ribonuclease (Ribonucleate nucleotide 2'-transferase E.C. 2.7.7.17) activity in serum of patients with chronic granulocytic leukaemia measured at pH 4.5-6.0 amounts to more than three times of that in serum of healthy subjects. At pH 6.0-8.0 the elevation of ribonuclease activity in serum of patients with chronic granulocytic leukaemia is less pronounced and amounts to about two times of that in normal ones. Using chromatography on CM Sephadex C-50 column, serum ribonuclease of both normal and chronic granulocytic leukaemia patients was separated into five distinct fractions. In serum of healthy subjects ribonuclease fractions denoted I-V contribute to 10; 21; 29; 22, and 18 percent of the total ribonuclease activity. In the serum of patients with chronic granulocytic leukaemia a decrease in ribonuclease fraction III to merely 17 percent and an increase in contribution of fraction IV to 32 percent of total ribonuclease activity could be observed. The comparison of each individual concentration of fraction in normal and leukaemia patients serum reveals, that ribonuclease fraction IV will increase about 3 times. A less pronounced increase could also be found for fractions I, II and V. However, ribonuclease fraction IV may be supposed to carry more than 50 percent of the whole extra load of ribonuclease present in the serum of chronic granulocytic leukaemia patients.     

Pyrogenic Principle of the Ribonucleic Acid from the Yeast Candida utilis

Experiments were performed to characterize the pyrogenic principle of ribonucleic acid (RNA) from the yeast Candida utilis. It was shown that ribonuclease hydrolysis of the RNA does not lead to inactivation of the pyrogenicity. Pyrogenicity was, however, destroyed by treatment with sodium deoxycholate. On column chromatography with Biogel under sterile and pyrogen-free conditions, the pyrogenic principle of yeast RNA was eluted together with the RNA. After treatment of the RNA with ribonuclease, it was possible to separate the pyrogenic activity from the RNA (hydrolysis products) to a great extent. Column chromatography of Escherichia coli endotoxin showed that the endotoxin was eluted in the same fractions as the pyrogenic activity of yeast RNA. On the basis of the behavior of the pyrogen, it may very well be that the fever reaction is produced not by the nucleic acid but by pyrogenic contaminants of the RNA preparation.     

Detoxification and immunogenic properties of endotoxin-containing precipitate of Brucella abortus

Badakhsh, Fred F. (University of Georgia, Athens), and John W. Foster. Detoxification and immunogenic properties of endotoxin-containing precipitate of Brucella abortus. J. Bacteriol. 91:494-498. 1966.-Endotoxin-containing precipitates (ECP) were prepared from Brucella abortus strain 19A by aqueous ether extraction followed by ethyl alcohol precipitation. Lysozyme was the most effective of several enzymes tried for detoxification of endotoxin present in the precipitate. Trypsin was shown to reduce mouse lethal toxicity but not rabbit dermal toxicity. Immunological studies of ECP and enzyme-treated ECP demonstrated that lysozyme did not harm the immunogenic property of ECP, whereas heat, ribonuclease, lipase, and proteolytic enzymes had an adverse effect. Serological reactivity of ECP was increased after lysozyme treatment, whereas ribonuclease reduced serological activity.     

Purification and characterization of a ribonuclease from human liver

The major ribonuclease of human liver has been isolated in a four-step procedure. The protein appears homogeneous by several criteria. The amino acid composition and the amino-terminal sequence of the enzyme indicate that the protein is related to human pancreatic ribonuclease and to angiogenin, and that it may be identical with an eosinophil-derived neurotoxin and to a ribonuclease that has been isolated from urine. The catalytic activity of the liver ribonuclease and its sensitivity to iodoacetic acid inactivation also relate the enzyme to the pancreatic RNases, but the liver protein is clearly differentiated by immunological measurements. Antibodies to the liver ribonuclease inhibit its activity, but not that of the human pancreatic enzyme; cross-reactivity in a radioimmunological assay is small but measurable. Immunochemical measurements have been used to examine the distribution of the liver-type protein in other tissues. Inhibition of enzyme activity by anti-liver ribonuclease shows that a cross-reactive enzyme is predominant in extracts of spleen and is a significant component in kidney preparations, while the liver-type protein is almost absent in brain or pancreas homogenates. Cross-reactive ribonuclease is present in serum, but levels are not correlated with any of the disease states examined.     

Effect of α-Tocopherol on Endotoxicosis

The effect of α-tocopherol in endotoxicosis was studied. The α-tocopherol level significantly decreased in mouse liver 18 hr after endotoxin administration, thereafter tending to increase to approach the normal range. In endotoxin-tolerant mouse liver, the lipid peroxide level was reduced to less than half of that in nontolerant animals following endotoxin challenge. The liver lipid peroxide level and serum lactate dehydrogenase or acid phosphatase leakage were studied in mice fed a vitamin E-deficient (ED) diet and a vitamin E-supplemented (ES) diet for 40 days. ED mouse liver exhibited a higher formation of lipid peroxide after endotoxin was given while there was a markedly lower level in ES mouse liver. There was significantly more serum lactate dehydrogenase or acid phosphatase leakage in ED mice than in ES mice after endotoxin administration. There was about a 25% decrease in liver superoxide dismutase (SOD) activity in endotoxin-poisoned mice fed both the normal and the ED diets, while the activity was at a higher level in ES-fed mice. These results suggest that α-tocopherol may be helpful in preventing membrane instability in endotoxin poisoning.     

Protection against hyperoxia by serum from endotoxin treated rats: absence of superoxide dismutase induction

Endotoxin greatly reduces lung injury and pleural effusions in adult rats exposed to normobaric hyperoxia (greater than 98% oxygen for 60 hours). This study reports that serum from endotoxin treated donor rats protects serum recipients against hyperoxic lung injury without altering lung superoxide dismutase (SOD) activity. Rats pretreated with endotoxin alone were protected and exhibited an increase in lung SOD activity as previously reported by others. Protection by serum was not due to the transfer of residual endotoxin or SOD. These results show that protection from oxygen toxicity can occur in rats without an increase in lung SOD and suggest that a serum factor may be involved.     

High serum IL-6 level reflects susceptible status of the host to endotoxin and IL-1/tumor necrosis factor.

Patients with high level of serum endotoxin did not necessarily develop into lethal shock, whereas some patients died of septic shock even when their serum endotoxin levels were low. These results indicate that limiting factor which determines the host to be endotoxin shock principally depends on the host susceptibility to endotoxin instead of serum endotoxin level. To understand this susceptible status of the host to endotoxin, we used Propionibacterium acnes primed mouse endotoxin shock model. We found that P. acnes-primed mice responded to low dose of LPS by enhanced production of IL-1 and TNF. And such mice were highly susceptible to the lethal shock inducing effect of IL-1 and/or TNF, which also induced high level of serum IL-6 in these mice. Therefore, measurement of serum IL-6 level provides us with the information of the preceding exposure of the host to either LPS or IL-1 and/or TNF and the highly susceptible status of the host to these stimuli. Based on these results obtained from animal model, we investigated the relationship between serum IL-6 levels and serum endotoxin levels in the patients with malignant hematologic disorders. We found that these patients fell into two groups; an endotoxin susceptible group, equivalent to P. acnes-primed mice, showing high level of serum IL-6 with low level of serum endotoxin, and a nonendotoxin susceptible group, equivalent to P. acnes-nonprimed mice, showing low or undetectable level of serum IL-6 with high level of serum endotoxin. We propose that the measurement of serum IL-6 level in the patients positive for endotoxin is a useful tool in evaluating diagnosis and prognosis of endotoxin shock.     

Inhibition of ascorbic acid uptake by endotoxin: evidence of mediation by serum factor(s)

The effect of bacterial endotoxin on the ascorbic acid uptake by 3T6 fibroblasts was studied. Endotoxin inhibited ascorbic acid uptake by fibroblasts in a dose dependent manner. The inhibition by endotoxin takes place only in the presence of unheated serum; decomplementing serum by heat inactivation at 56 degrees C for 30 minutes eliminates endotoxin's inhibitory effect on ascorbic acid uptake. The effect of endotoxin appears to be instantaneous since the inhibition seen in the cells without any preexposure was similar to the cells preexposed to endotoxin for up to 6 hours. Polymyxin B sulfate which is known to bind the lipid A portion of endotoxin did not reverse the inhibition of ascorbic acid uptake caused by endotoxin.     

Calcium behavior in endotoxin-poisoned mice: especially calcium accumulation in mitochondria

A possible role of intracellular Ca2+ and participation of calmodulin in cellular metabolism in endotoxin-poisoned mice were investigated. The levels of calcium in liver cytosol and liver mitochondria fractions in poisoned mice were markedly higher 18-48 hr after endotoxin injection than in the control mice. On the other hand, the levels of serum calcium in the poisoned mice were about 20% lower at 18 hr than in the controls. The serum calcium levels in mice injected with 50 and 100 micrograms of endotoxin showed no dose-response effect, but a dose-response effect was observed at a dose of 200-400 micrograms. The serum Ca2+ levels in endotoxin-tolerant mice were similar to those in the control mice. The levels in mice injected with glucocorticoid-antagonizing factor mice were about 14% lower at 3 hr than in the controls. The mice fed a vitamin D3- and calcium-free diet showed a higher mortality rate in the early stage (12-18 hr) of endotoxication than that of the mice fed a normal diet. The lipid peroxide levels and Ca2+-ATPase activity in the liver mitochondria fraction in endotoxin-poisoned mice showed a higher level than those of the control mice. There was little or no difference in the levels of serum glucose between the mice injected with calmodulin antagonist (trifluoperazine, TFP) plus endotoxin and those given endotoxin alone. However, the liver glycogen levels in TFP plus endotoxin-treated mice were markedly higher than that in mice given endotoxin alone. Furthermore, calcium antagonist (verapamil) plus endotoxin-treated mice had about a 40% higher survival rate after 72 hr than those given endotoxin alone. The findings suggest that there is a possibility of participation of the Ca2+-calmodulin system in carbohydrate metabolic disorders during endotoxemia and that the changes in intracellular Ca2+ may result in various metabolic disorders.     

Purification of rat liver particulate neutral ribonuclease and comparison of properties with pancreas and serum ribonucleases.

Rat liver particulate neutral ribonuclease (EC 3.1.4.22) was extensively purified (up to 40000-fold). It is shown to be an endonuclease, specific for pyrimidine bases, hydrolysing 5'-phosphate ester bonds. The enzyme specificity, Km, pH optimum, stability in acid medium and thermal stability at high temperature are the same as those of rat pancreatic and serum ribonucleases. Like pancreatic and serum neutral ribonucleases, the hepatic enzyme is sensitive to the liver natural inhibitor. This inhibitor was purified 8000-fold; its association with ribonuclease follows zero-order kinetics. These identical properties for ribonuclease of rat liver, pancreas and serum support the hypothesis [Bartholeyns, Peeters-Joris & Baudhuin (1975) Eur. J. Biochem. 60, 385-393] of an extrahepatic origin for the liver enzyme, the plasma ribonuclease of pancreatic origin being taken up by endocytosis in the liver. Neutral ribonuclease activity was detected in all rat organs investigated; its distribution among tissues is different from the distribution of the natural ribonuclear inhibitor.     

The inhibition of ribonuclease activity and the isolation of polysomes from leaves of the french bean, Phaseolus vulgaris

The effect of inhibitors on the ribonuclease activity of soluble and microsomal fractions of bean leaves has been examined. The soluble ribonuclease activity could be completely inhibited by Zn²⁺, Cu²⁺, bentonite, and diethylpyrocarbonate, although these inhibitors had little effect on the microsomal ribonuclease activity. Ribonuclease activity in the soluble fraction was completely inhibited by guanosine 2′(3′)-monophosphate, which was the first nucleotide to accumulate on degradation of yeast RNA. Adenosine 2′(3′)-monophosphate, the first nucleotide to accumulate on degradation of yeast RNA by the microsomal preparations, completely inhibited the ribonuclease activity of the microsomal fraction.The ribonuclease activity of both enzyme preparations was completely inhibited by an analog of the transition state of the ribonuclease reaction, a complex of guanosine and vanadyl sulfate. Inclusion of this complex in homogenization media markedly increased the proportion of polysomes isolated from bean leaves.     

Endotoxin inactivating activity of rat serum

The ability of rat serum to inactivate endotoxin (LPS) was assessed with the aid of the limulus amebocyte lysate assay. Following the addition of various amounts of endotoxin to normal serum the mixture was incubated for 1 hr at 37 degrees C and the residual endotoxin activity determined. One milliliter of rat serum inactivated between 5 and 10 micrograms Escherichia coli LPS per hour. Heating serum for 45 min at 56 degrees C resulted in loss of 80-90% of the LPS inhibitor (LPSI) activity. Serum from cobra venom factor (CVF)-treated rats inactivated between 0.5 and 2.5 micrograms LPS/ml serum. Serum from tolerant rats, even after heating for 45 min at 56 degrees C, inactivates between 10 and 15 micrograms LPS/ml serum/hr; decomplemented tolerant rat serum neutralizes between 5 and 10 micrograms LPS/ml serum/hr. Clearly, the tolerant rat has large quantities of LPSI activity, which does not appear to be complement. The inhibitor found in tolerant rat serum is not species specific since it inactivates Salmonella minnesota and Salmonella typhimurium endotoxins to the same degree and in the same amount as E. coli endotoxin, the agent used to induce tolerance. Both heating serum (56 degrees C) and lead acetate reduce LPSI activity.     

The effect of chemical modification of ribonuclease A by serum albumin on its catalytic properties

The effect of chemical modification of ribonuclease A on its catalytic properties was studied. The kinetic parameters were calculated by using the Eisental and Cornish-Bowden direct linear plot. The Km value found for ribonuclease A conjugates with human serum albumin are comparable with those for the native enzyme whereas the Vmax value of the modified enzyme exceeds the maximum velocity of the enzymatic reaction of native ribonuclease A.     

Bac-to-Bac/BmNPV杆状病毒在家蚕幼虫中表达重组鲎C因子

鲎C因子是鲎血细胞中一种对内毒素具有高亲和力的丝氨酸蛋白酶,被内毒素激活后可水解人工合成的三肽底物,因而能替代传统的鲎试剂用于内毒素检测。通过RT-PCR从东方鲎血细胞中扩增出鲎C因子基因(factor C)序列,利用Bac-to-Bac/BmNPV杆状病毒表达系统在家蚕幼虫中表达该蛋白,并对其进行活性测定。结果显示:被感染的家蚕血清稀释后能检测到Factor C活性,稀释500倍的血清可以用于内毒素检测,且其灵敏度能达到0.2 EU/mL,有望开发新型的、低成本的内毒素检测试剂。     

The influence of hormones and inflammatory agents on C-reactive protein, cortisol and alanine aminotransferase in the plaice (Pleuronectes platessa L.)

Endotoxin stimulates production of both C-reactive protein (CRP) and cortisol in the plaice within 24 hr. Cortisol alone (optimum dose i.p. 500 micrograms/300 g wt fish) also stimulates CRP production and the possibility that endotoxin acts through cortisol was examined. Dexamethasone suppresses cortisol production but elevates CRP. Cortisol levels are restored to normal within 24 hr of endotoxin injection. Turpentine and ACTH which stimulate cortisol do not affect CRP. Endotoxin and cortisol have no significant effect on alanine aminotransferase activity in the serum and liver although it is elevated in the serum within 24 hr of the administration of adrenalin or turpentine.     

The ribonuclease activity of nucleolar protein B23.

Protein B23 is an abundant nucleolar protein and putative ribosome assembly factor. The protein was analyzed for ribonuclease activity using RNA-embedded gels and perchloric acid precipitation assays. Three purified bacterially expressed forms of the protein, B23.1, B23.2 and an N-terminal polyhistidine tagged B23.1 as well as the natural protein were found to have ribonuclease activity. However, the specific activity of recombinant B23.1 was approximately 5-fold greater than that of recombinant B23.2. The activity was insensitive to human placental ribonuclease inhibitor, but was inhibited by calf thymus DNA in a dose dependent manner. The enzyme exhibited activity over a broad range of pH with an apparent optimum at pH 7.5. The activity was stimulated by but not dependent on the presence of low concentrations of Ca2+, Mg2+ or NaCl. The Ca2+ effect was saturable and only stimulatory in nature. In contrast, Mg2+ and NaCl exhibited optimal concentrations for stimulation and both inhibited the ribonuclease at concentrations above these optima. These data suggest that protein B23 has intrinsic ribonuclease activity. The location of protein B23 in subcompartments of the nucleolus that contain preribosomal RNA suggests that its ribonuclease activity plays a role in the processing of preribosomal RNA.     

Endotoxin stimulates endothelin-release in vivo and in vitro as determined by radioimmunoassay

A marked increase in immunoreactive endothelin was observed in rat serum collected within 10-15 min after infusion of endotoxin. Endothelin level was 117 +/- 11.5 pg/ml (mean +/- S.E., N = 4) in rats exposed to endotoxin as compared with undetectable levels (less than 2 pg/ml, N = 4) in controls. We have also observed a significant stimulation of endothelin-release by endotoxin from cultured bovine transformed thoractic aortic endothelial cells at concentrations of endotoxin ranging between 0.1 and 10.0 micrograms/ml. Serum was indispensable for the stimulating effect of endotoxin, although serum itself did not show any effect at the concentration used (1%). These results suggest that endothelin plays an important role in mediation of pathophysiological responses caused by endotoxin. The levels of endothelin were measured by radioimmunoassay with high sensitivity.     

The effect of 12-O-tetradecanoyl-phorbol 13-acetate on the ribonuclease activity of circulating human lymphocytes.

12-O-Tetradecanoyl-phorbol 13-acetate is a very effective tumor promotor and inflammatory agent and can act as a mitogen for a subset of T lymphocytes. We report here that even short exposure of lymphocytes to 12-O-tetradecanoyl-phorbol 13-acetate changes the balance between the levels of neutral ribonuclease and ribonuclease inhibitor. The most dramatic change occurs in a B-lymphocyte-enriched population. We find that most, if not all, of the neutral ribonuclease activity in circulating lymphocytes is associated with this population and that this activity is lost with exposure to 12-O-tetradecanoyl-phorbol 13-acetate. Both 12-O-tetradecanoyl-phorbol 13-acetate and phytohaemagglutinin increase the level of ribonuclease inhibitor in T cells. However, phytohaemagglutinin has no effect on the ribonuclease or inhibitor level of the B-cell-enriched population.     

Effect of some proteins on the yeast cell membrane

Yeast cells, Candida utilis, in water suspension and in the absence of electrolytes were found to be very sensitive to several proteins of moderate size, including ribonuclease, protamine, lysozyme, bovine serum albumin, cytochrome c, and myoglobin. Viability ceases rapidly, and ultraviolet-absorbing compounds (260 mmu) and the amino acid pool are released into the medium. The ultraviolet-absorbing material appears to be the nucleotide and coenzyme fraction usually extracted by 0.2 n perchloric acid at low temperature. The ribonucleic acid fraction remains in the cell ghosts and can be released by ribonuclease. The enzymatic properties of some of these proteins have no relation to their damaging effect on the cell membrane. Poly-l-lysine shows the same activity.     

Ribonucleases expressed by human pancreatic adenocarcinoma cell lines.

Human ribonucleases have been considered as a possible tumor marker for pancreatic cancer, and elevated serum levels of ribonuclease activity in patients with pancreatic cancer have been reported by many authors. The reason for this elevation is unknown. In this study, we demonstrate that human pancreatic adenocarcinoma cell lines synthesize and secrete different ribonucleases. We isolated and characterized human pancreatic, or secretory, ribonuclease (RNase 1) from the conditioned media of the human pancreatic adenocarcinoma cell lines Capan-1, MDAPanc-3, IBF-CP3 and Panc-1, and the ampullary adenocarcinoma cell line MDAAmp-7, which represent a wide range of differentiation stages. Only one of these cell lines, Panc-1, produces significant amounts of nonsecretory ribonuclease. We then established a purification procedure for both secretory and nonsecretory ribonucleases, consisting of concentration of the supernatant by tangential filtration, anion-exchange and cation-exchange liquid chromatography and C4 RP-HPLC. Ribonuclease activity fractions were monitored using both the spectrophotometric and negative-staining zymogram techniques. The results of N-terminal sequence analysis, kinetic analysis and endoglycosidase digestion studies indicate that the main ribonuclease secreted by all the cell lines is the secretory-type ribonuclease and that it is composed of several differently N-glycosylated forms. Northern blot analyses confirm that some of the cell lines express secretory ribonuclease mRNA. The mRNA levels produced by Panc-1 and MDAPanc-28 are too low to be detected. Similar levels of expression of nonsecretory ribonuclease are found by Northern blot analysis in all the cell lines except Panc-1, which expresses higher levels. Here, we describe, for the first time, that several human pancreatic cancer cell lines with different degrees of differentiation express and secrete ribonucleases. This fact indicates that one origin of the elevated serum RNase levels in patients with pancreatic cancer are tumor cells. Analysis of the oligosaccharide moiety of the RNase 1 secreted by Capan-1 shows that it is highly glycosylated and its N-glycan chains are significantly different from that of the RNase 1 produced by normal pancreas. These results renew the possibility of using human serum RNase 1 determination as a tumor marker.