Tandemization endows bovine pancreatic ribonuclease with cytotoxic activity

Due to their ability to degrade RNA, selected members of the bovine pancreatic ribonuclease A (RNase A) superfamily are potent cytotoxins. These cytotoxic ribonucleases enter the cytosol of target cells, where they degrade cellular RNA and cause cell death. The cytotoxic activity of most RNases, however, is abolished by the cytosolic ribonuclease inhibitor (RI). Consequently, the development of RNase derivatives with the ability to evade RI binding is a desirable goal. In this study, tandem enzymes consisting of two RNase A units that are bound covalently via a peptide linker were generated by gene duplication. As deduced from the crystal structure of the RNase A.RI complex, one RNase A unit of the tandem enzyme can still be bound by RI. The other unit, however, should remain unbound because of steric hindrance. This free RNase A unit is expected to maintain its activity and to act as a cytotoxic agent. The study of the influence of the linker sequence on the conformation and stability of these constructs revealed that tandemization has only minor effects on the activity and stability of the constructs in comparison to monomeric RNase A. Relative activity was decreased by 10-50% and the melting temperature was decreased by less than 2.5 K. Furthermore, the cytotoxic potency of the RNase A tandem enzymes was investigated. Despite an in vitro inhibition by RI, tandemization was found to endow RNase A with remarkable cytotoxic activity. While monomeric RNase A is not cytotoxic, IC(50) values of the RNase A tandem variants decreased to 70.3-12.9 microM. These findings might establish the development of a new class of chemotherapeutic agents based on pancreatic ribonucleases.     

Interaction of sodium dodecyl sulfate with tyrosyl chromophores in ribonuclease A and model compounds

Ultraviolet difference spectra, solvent perturbation difference spectra, and temperature perturbation difference spectra indicate that tyrosyl residues of model compounds are affected by sodium dodecyl sulfate. This effect is dependent on the nature of the model compound, being enhanced by positive charges, and is attributed to partial masking of the tyrosyl chromophores by sodium dodecyl sulfate. With reduced carboxymethylated ribonuclease as a model, all three difference spectral methods can be interpreted as indicating nearly complete externalization of tyrosyl chromophores in ribonuclease in the detergent. With small tyrosyl model compounds the calculated number of external tyrosyl residues depends on the nature of the model compound. Using net positively charged tyrosyl compounds as models, nearly 6 external tyrosyl residues are calculated for RNase. N-Acetyltyrosine amide or N-acetyltyrosine esters appear to be inadequate models for tyrosine in proteindetergent solutions because of their weak interactions with detergents.     

Changes in colonic mucosal permeability in mouse colitis induced with dextran sulfate sodium.

In this study we examined changes in colonic mucosal permeability induced by dextran sulfate sodium (DSS) during the acute phase of mouse colitis. To induce colitis, the mice were given drinking water containing 5% (w/v) DSS (MW = 40,000) ad libitum. Colonic mucosal permeability was evaluated by the permeation of Evans blue (EB) from the lumen into the wall of the colon on 1, 2, 3 and 7 days postadministration of DSS. Mucosal changes were also histologically examined daily for 7 days postadministration. The permeation of EB increased significantly by days 3 and 7 postadministration. Histological analysis showed that crypt loss was the initial change, with no inflammatory process and the surface mucosal epithelial cells remained morphologically intact. These histological changes developed on 2 to 3 days postadministration. Erosion was first recognized at 5 days postadministration. These findings indicated that the increase in colonic mucosal permeability may have occurred in 3 days postadministration, and the increase in mucosal permeability occurred before the appearance of the inflammatory process. This suggests that an increase in colonic mucosal permeability, leading to the destruction of mucosal barrier function, may play an important role in the induction of DSS-induced murine colitis.     

Fluorescent labeling of proteins in sodium dodecyl sulfate complexes with fluorescamine

The effects of sodium dodecyl sulfate on the fluorescent labeling of proteins were studied. Of 57 primary amine groups in bovine serum albumin, no more than 7 are titrable by fluorescamine. Fluorescamine labeling does not cause appreciable conformational changes of proteins. The extent of labeling of proteins decreases as the concentrations of sodium dodecyl sulfate increases. The fluorescence properties of labeled primary amine are only slightly affected by the polarities of the solvents. The inhibitory effects of sodium dodecyl sulfate upon labelings are interpreted as the low permeability of fluorescamine toward the highly charged envelopes of sodium dodecyl sulfate-protein micelles.     

Inhibition by heparin and dextran sulfate of stimulated rat pancreatic adenylate cyclase

Heparin inhibited the adenylate cyclase activity of semipurified rat pancreatic plasma membranes stimulated by hormones and by Gpp(NH)p but not by fluoride or when in the persistently active state. When observed, the inhibition was rapid and sustained. It was of a noncompetitive type and never exceeded 20% for secretin. The inhibition of Gpp(NH)p-stimulated activity was more pronounced (48% inhibition at a heparin concentration of 50 μg/ml). For the C-terminal octapeptide of pancreozymin (CCK-8)-stimulated adenylate cyclase, the inhibition amounted to 93% at 50 μg/ml. This inhibition was competitive at low heparin concentration and of a mixed type above 10 μg/ml. Besides, heparin inhibited (I₅₀ = 6 μg/ml) the binding of peptides of the CCK family to their specific receptors without affecting the apparent K_d value of binding. Taken together, these relatively specific effects of heparin gave evidence in favor of the existence of CCK spare receptors. Dextran sulfate was more potent than heparin as an inhibitor of adenylate cyclase activation while chondroitin-4-sulfate and chondroitin-6-sulfate were ineffective. Dansylated pancreatic plasma membranes exhibited characteristics of adenylate cyclase activation by CCK-8 which were similar to those found for untreated membranes exposed to heparin.     

Oral in vivo bactofection in dextran sulfate sodium treated female Wistar rats

Salmonella typhimurium SL7207 carrying Cu-Zn superoxide dismutase and an N-terminal deletion mutant of monocyte chemoattractant protein-1 genes was applied to dextran sodium sulfate treated female Wistar rats. Stool quality, food and water intake were monitored. Markers of oxidative stress, interleukin 1, interleukin 6 and tumor necrosis factor alpha were quantified. No differences were found in body weights, markers of oxidative stress in plasma and inflammatory markers in colon homogenates. Plasma concentrations of I11, I16 were lower in the treatment groups than in the dextran sodium sulfate group. However, dextran sodium sulfate induced inflammation could not be confirmed by plasma levels of I11, I16 and TNFalpha. Although some parameters showed a tendency to improve, the inflammation caused by administration of 4% dextran sodium sulfate during 7 days was low and contradictory to other studies. Results showed the potential synergic effect of combined bacteria-mediated antioxidative and anti-inflammatory gene therapy.     

Gender dependent importance of IRAK-1 in dextran sulfate sodium induced colitis

Toll-like receptor (TLR) signaling is important for the induction of pro-inflammatory cytokines and interferon (IFN)-inducible genes in response to bacterial and viral challenge. Interleukin-1 receptor-associated kinase-1 (IRAK-1) is a signaling kinase situated downstream of the adapter protein myeloid differentiation factor 88 (MyD88) in the TLR intracellular signaling cascade and is required for normal signal transduction through this pathway. We investigated the importance of IRAK-1 in intestinal inflammation by using the dextran sulfate sodium (DSS)-colitis model. We show that IRAK-1 deficient mice are protected against systemic signs of inflammation, i.e., weight loss and spleen enlargement compared to wild-type controls irrespective of gender. However, IRAK-1^−/y males but not IRAK-1^−/− females display significant protection against colitis and thymic atrophy compared to wild-type mice.Our results indicate a gender specific effect of IRAK-1 in the DSS-induced colitis, an interesting finding since the Irak-1 gene is located on the X-chromosome and several inflammatory diseases have a gender dependent incidence.     

Novel enzymatic properties of DNA-Pt complexes

DNA-Pt complexes have shown novel enzymatic activity as a peroxidase similar to that of horseradish peroxidase in the colorimetric reaction with its substrate. The enzymatic activity of these complexes increased with increasing reaction time and pH in reaction solutions of DNA and K2[PtCl4]. This enhanced enzymatic activity was attributed to the increase in Pt conjugated to DNA in the complex. The enzymatic activity per unit mole of the DNA-Pt complex was significantly higher for complexes prepared with high molecular weight DNA because the enzymatic activity of the complex per repeat unit of DNA was almost constant for these complexes prepared under the same reaction conditions. All the DNA-Pt complexes in this study prepared with different DNA sequences (i.e., [A]20, [G]20, [C]20, [T]20, and [AG]10) exhibited peroxidase enzymatic activity. These complexes showed good thermal stability as compared to native horseradish peroxidase.     

Dose-dependent promoting effect of dextran sodium sulfate on mouse colon carcinogenesis initiated with azoxymethane

We previously reported a powerful tumor-promoting ability of dextran sodium sulfate (DSS) in a novel mouse model for colitis-related colon carcinogenesis initiated with azoxymethane (AOM). To determine the dose-dependent influence of DSS in our animal model, male ICR mice were given a single intraperitoneal injection of AOM (10 mg/kg body weight), followed by DSS at dose levels of 2, 1, 0.5, 0.25, and 0.1% (w/v) in drinking water for 1 week. All animals were sacrificed at week 14 and histological alterations in their colon and nitrotyrosine immunohistochemistry were examined to evaluate the nitrosative stress. In the mice which received AOM and 2% DSS, the incidences (multiplicity) of colonic tubular adenoma and adenocarcinoma were 75% (1.25+/-1.26/mouse) and 100% (2.75+/-2.22/mouse), respectively. Mice given AOM and 1% DSS had 80% incidence of adenoma (1.00+/-0.71/mouse) and 60% incidence of adenocarcinoma (1.40+/-2.07/mouse) in the colon. In a mouse treated with AOM and 0.5% DSS, only one colonic adenoma (20% incidence with 0.20+/-0.45 multiplicity) developed. Higher frequency of high-grade colonic dysplasia was noted in mice given AOM and 2% or 1% DSS when compared with mice treated with AOM and lower doses of DSS. Also, scoring of inflammation and nitrotyrosine immunoreactivity suggested that severe inflammation and nitrosation stress caused by high-doses (2% and 1%) of DSS contribute its tumor-promoting effects in mouse colon carcinogenesis initiated with a low dose of AOM. Thus, our findings indicate that a tumor-promoting effect of DSS was dose-dependent (1% or more) and the effect might occur under the condition of inflammation and nitrosation stress.     

L-arginine supplementation improves responses to injury and inflammation in dextran sulfate sodium colitis

Inflammatory bowel disease (IBD), consisting of Crohn's disease and ulcerative colitis (UC), results in substantial morbidity and is difficult to treat. New strategies for adjunct therapies are needed. One candidate is the semi-essential amino acid, L-arginine (L-Arg), a complementary medicine purported to be an enhancer of immunity and vitality in the lay media. Using dextran sulfate sodium (DSS) as a murine colonic injury and repair model with similarities to human UC, we assessed the effect of L-Arg, as DSS induced increases in colonic expression of the y(+) cationic amino acid transporter 2 (CAT2) and L-Arg uptake. L-Arg supplementation improved the clinical parameters of survival, body weight loss, and colon weight, and reduced colonic permeability and the number of myeloperoxidase-positive neutrophils in DSS colitis. Luminex-based multi-analyte profiling demonstrated that there was a marked reduction in proinflammatory cytokine and chemokine expression with L-Arg treatment. Genomic analysis by microarray demonstrated that DSS-treated mice supplemented with L-Arg clustered more closely with mice not exposed to DSS than to those receiving DSS alone, and revealed that multiple genes that were upregulated or downregulated with DSS alone exhibited normalization of expression with L-Arg supplementation. Additionally, L-Arg treatment of mice with DSS colitis resulted in increased ex vivo migration of colonic epithelial cells, suggestive of increased capacity for wound repair. Because CAT2 induction was sustained during L-Arg treatment and inducible nitric oxide (NO) synthase (iNOS) requires uptake of L-Arg for generation of NO, we tested the effect of L-Arg in iNOS(-/-) mice and found that its benefits in DSS colitis were eliminated. These preclinical studies indicate that L-Arg supplementation could be a potential therapy for IBD, and that one mechanism of action may be functional enhancement of iNOS activity.     

Expression of catalase in Lactobacillus fermentum and evaluation of its anti-oxidative properties in a dextran sodium sulfate induced mouse colitis model

Lactic acid bacteria are generally sensitive to hydrogen peroxide (H₂O₂). Lactobacillus plantarum ATCC14431 is one of the few lactic acid bacteria able to degrade H₂O₂ through the action of a manganese-dependent catalase (containing the katA gene). However, it is not a natural inhabitant of the intestinal tract and its bio-efficacy and survival in the gastrointestinal tract have never been tested. In this study, we successfully expressed the katA gene from L. plantarum ATCC14431 in L. fermentum I5007 and the recombinant L. fermentum exhibited almost 20-fold higher catalase activity than the empty vector control. The anti-oxidative properties of this catalase-producing L. fermentum were evaluated using a dextran sodium sulphate (DSS) induced colitis mice model. Compared with the control, mice receiving DSS alone had increased diarrhea and mucosa histological scores (P < 0.05), as well as lipid peroxidation (P < 0.05), myeloperoxidase (P < 0.05), and active NF-κB in colonic tissue (P < 0.05). Similar to vitamin E, treatment with recombinant L. fermentum mitigate these effects accompanied by a improvement in mucosa histological scores in the proximal colon (P < 0.05) and decreased lipid peroxidation (P < 0.05), myeloperoxidase (P < 0.05) and active NF-κB in colonic tissue (P < 0.05). In conclusion, the expression of catalase in L. fermentum increased its ability to survive when exposed to aerated environment in vitro and conferred the anti-oxidative and anti-inflammatory effects in the DSS induced colitis model.     

Conformation of sequential and random copolypeptides of lysine and alanine in sodium dodecyl sulfate solution

A series of sequential polypeptides, (Lys_i-Ala_j)_n, and random copolypeptides, (Lys^x, Ala^y)_n, were synthesized. The competitive effect of Ala, a helix former, and Lys, whose homopolymer has a β-form in neutral NaDodSO₄ solution, was determined by CD and absorption spectroscopy. All the polypeptides studied were unordered in neutral solution without the surfactant. Of the six sequential polypeptides only (Lys-Ala)_n adopted a stable β-form in NaDodSO₄ solution. Most striking is the difference between this polypeptide, (Lys₂-Ala₂)_n and (Lys^x, Ala^y)_n, even though they all have equimolar Lys and Ala. (Lys₂-Ala₂)_n was partially helical in 2.5–5 mM NaDodSO₄ but approached the unordered form in 50 mM NaDodSO₄, whereas (Lys⁵⁰, Ala⁵⁰)_n was completely helical in all NaDodSO₄ concentrations. Even Lysrich (Lys₂-Ala)_n and (Lys₃-Ala)_n formed a partial helix and a trace of the β-form, respectively, in low NaDodSO₄ concentrations; both reverted to the unordered form in high NaDodSO₄ concentrations. These results can be explained by Pauling-Corey's model for β-pleated sheets. Only (Lys-Ala)_n has all DodSO-bound Lys⁺ residues on one side and Ala residues on the other side of the polypeptide chain. They can nestle quiet efficiently in a β-sheet and between neighboring β-sheets. Our results further imply that random copolypeptides are not completely random; they comprise varying segments of (Lys_k-Ala_m), where k and m could vary from zero to a small integer.     

Bovine pancreatic ribonuclease: fifty years of the first enzymatic reaction mechanism

Fifty years ago, the group of Tony Mathias and Bob Rabin at University College London deduced the first mechanism for catalysis by an enzyme, ribonuclease [Findlay, D., Herries, D. G., Mathias, A. P., Rabin, B. R., and Ross, C. A. (1961) Nature 190, 781-784]. Here, we celebrate this historic accomplishment by surveying knowledge of enzymology and protein science at that time, facts that led to the formulation of the mechanism, criticisms and alternative mechanisms, data that supported the proposed mechanism, and some of the refinements that have since provided a more precise picture of catalysis of RNA cleavage by ribonucleases. The Mathias and Rabin mechanism has appeared in numerous textbooks, monographs, and reviews and continues to have a profound impact on biochemistry.     

Expression of soluble bovine pancreatic ribonuclease A in Pichia pastoris and its purification and characterization

A Pichia pastoris expression system for bovine pancreatic RNase A was constructed: the RNase A sequence was fused to the PHO1 signal and the AOX1 promoter was used for efficient secretion. Approximately 5 mg of soluble enzymes were secreted per liter of the culture, but one half of them were glycosylated. After a series of purifications by cation-exchange chromatography, the glycosylated enzyme was removed and the pure recombinant soluble unglycosylated RNase A was obtained in the final yield of 1 mg per liter of the culture. N-Terminal sequence, molecular weight, secondary structure, thermal stability, and activity were completely identical with those of commercial RNase A. Glycosylated RNase A had a decreased kcat, 60-70% of the activity of wildtype RNase A, as in the case of RNase B. Its carbohydrate moiety seemed to destabilize the enzyme differently from RNase B since Tm of the glycosylated RNase A was decreased by 6 degrees C. The carbohydrate moiety of the glycosylated enzyme contained no GlcNAc. The N34A mutant RNase A, in which the only potential N-glycosylation site, Asn34, is mutated to alanine, was also glycosylated, implying that glycosylation is not N-linked but O-linked.     

A nuclear localization sequence endows human pancreatic ribonuclease with cytotoxic activity

Some members of the ribonuclease superfamily, such as Onconase, are cytotoxic to cancer cells. This is not the case for human pancreatic ribonuclease. This lack of cytotoxicity is probably a result of the inhibition exerted by the cytosolic ribonuclease inhibitor once the protein has reached the cytosol. Until now, all cytotoxic human pancreatic ribonuclease variants have been described as being resistant to the inhibitor. Here, we report on the characterization of a cytotoxic variant of human pancreatic ribonuclease which has an Arg triplet introduced onto one of its surface-exposed loops. Despite its sensitivity to the inhibitor, this variant, called PE5, was only 5-15 times less cytotoxic than Onconase. When it was taken up by cells, it was only observed within late compartments of the endocytic pathway, probably because the number of molecules transported to the cytosol was too small to allow their visualization. Nuclear import assays showed that the Arg triplet endows PE5 with a nuclear localization signal. In these experiments, PE5 was efficiently transported to the nucleus where it was initially localized in the nucleolus. Although the Arg introduction modified the net charge of the protein and somehow impaired recognition by the cytosolic inhibitor, control variants, which had the same number of charges or were not recognized by the inhibitor, were not toxic. We concluded that targeting a ribonuclease to the nucleus results in cytotoxicity. This effect is probably due to ribonuclease interference with rRNA processing and ribosome assembly within the nucleolus.