Comparative studies between in vitro and in vivo effects of human beta-interferon on natural killer activity and its relevance to immunochemotherapy

Summary A good correlation was found between in vivo and in vitro responses of peripheral MNC from breast cancer patients and the NK boosting effect of human IFN. In vitro immunochemotherapy studies showed that marked antitumor effects were obtained against cultured cancer cells when a widely used chemotherapeutic agent such as 5-FU was combined with nonsensitized spontaneously cytolytic MNC, preactivated in vitro with IFN. These results suggest that the in vitro susceptibility assay of MNC to IFNs could be used for predicting favorable responses to immunochemotherapy regimens employing IFNs as immunomodulating agents. Abbreviations used: IFN, beta-interferon; CM, complete medium; CTX, cyclophosphamide; E:T ratio, effector target ratio; EDTA, ethylenediaminetetraacetic acid; FCS, fetal calf serum; 5-FU, 5-fluorouracil; IFN, gamma-interferon; Hepes, N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid IFNs, interferons; KC, killed cells; LDIF, low doses beta-interferon; LU, lytic unit; MAT macrotest; MIT, microtest; MNC, mononuclear cells; MNC/IFN, IFN-pretreated MNC; MTX, methotrexate; NB, no boosting; NK, natural killer; NKA, NK activity; NR, natural resistance; PM, positive modulation; SE, standard error; WM, washing medium     

Specific immobilization of laccase onp-benzoquinone-activated supports

Summary A specific immobilization of laccase (EC 1.10.3.2) onto a ready-to-usep-benzoquinone-activated agarose support is described. The single-step procedure leads to a laccase protein coupling of I8% and an enzyme activity immobilization yield of 27%, while the retained specific activity of the immobilized enzyme was 150% of the specific activity of the free laccase. This peculiar result is thought to be related to the fact that during the process of support activation byp-benzoquinone, a significant amount of the hydroquinone by-product of the activation process is coupled to the support. These coupled derivatives constitute substrate (hydroquinone) analogues for which laccase exhibits a high affinity. Therefore, simultaneous affinity retention on the hydroquinone groups and covalent coupling on the p-benzoquinone groups allow the binding of the enzyme in an advantageous conformation which can generate this increase specific activity by immobilization. The entire process can be considered as an affinity immobilization. The immobilized enzyme is much more stable to the inhibitory action of chloride and azide ions, with a recovery of 100% of the activity, than the free laccase, with a recovery of 67% and 32%, respectively, after removal of the inhibitors by dialysis. The stability was 95% after storage for 14 months at 4° C.Abbreviations HQ hydroquinone - p-BQ p-benzoquinone - U enzyme units Part of the work was presented at the Satellite FEBS 1989 Symposium onBiochemical and biophysical approaches to the study of copper proteins, Camerino, Italy.     

The role of copper in bovine serum amine oxidase

Summary The role of copper in bovine serum amine oxidase was investigated by studying the effect of copper-binding inhibitors on the reactions of the pyrroloquinoline quinone carbonyl and on the reaction with oxygen. Hydrazines and hydrazides were used as carbonyl reagents and one of the hydrazines, benzylhydrazine, which was found to behave as a pseudo-substrate, was used to probe the reaction with oxygen. The presence ofN,N-diethyldithiocarbamate, a chelator that binds copper irreversibly, did not prevent the reactions at the carbonyl, but slowed down their rate and modified the conformation of the adducts. The same happened to the reaction with oxygen, which was slowed down but not abolished. Copper, which was never seen in the reduced state, thus appears to control all reactions without being directly involved in the binding of either hydrazines or oxygen. The enzyme functionality was in fact preserved upon substitution of copper with cobalt. The specific activity of the cobalt-substituted enzyme was only reduced to about 40% the native amine oxidase value. This is the first case so far in which the role of copper can be performed by a different metal ion.Abbreviations BSAO bovine serum amine oxidase - DDC N,N-diethyldithiocarbamate - PQQ pyrroloquinoline quinone     

Spectrophotometric Assay of Lipase Activity: A New 4-nitrophenyl Ester of a Dialkylglycerol Suitable as a Chromogenic Substrate of Pseudomonas cepacia Lipase

Evaluation of Pseudomonas cepacia lipase (PCL) activity by a titrimetric method with triacylglycerols (TAG) and synthetic dialkylglycerol esters (DAGE) established the chain length selectivity of the enzyme and this information has been used to design a new chromogenic substrate [1,2-di-O-octyl-sn-glycerol-3-O-(4-nitrophenyl) glutarate] for the determination of the lipolytic activity of PCL.     

Dietary supplementation with essential amino acids boosts the beneficial effects of rosuvastatin on mouse kidney

The effects of high-potency statins on renal function are controversial. To address the impact of statins on renal morpho-functional aspects, normotensive young mice were treated with rosuvastatin (Rvs). Moreover, because statins may impair mitochondrial function, mice received either dietary supplementation with an amino acid mixture enriched in essential amino acids (EAAm), which we previously demonstrated to increase mitochondrial biogenesis in muscle or an unsupplemented control diet for 1 month. Mitochondrial biogenesis and function, apoptosis, and insulin signaling pathway events were studied, primarily in cortical proximal tubules. By electron microscopy analysis, mitochondria were more abundant and more heterogeneous in size, with dense granules in the inner matrix, in Rvs- and Rvs plus EAAm-treated animals. Rvs administration increased protein kinase B and endothelial nitric oxide synthase phosphorylation, but the mammalian target of rapamycin signaling pathway was not affected. Rvs increased the expression of sirtuin 1, peroxisome proliferator-activated receptor γ coactivator-1α, cytochrome oxidase type IV, cytochrome c, and mitochondrial biogenesis markers. Levels of glucose-regulated protein 75 (Grp75), B-cell lymphoma 2, and cyclin-dependent kinase inhibitor 1 were increased in cortical proximal tubules, and expression of the endoplasmic reticulum–mitochondrial chaperone Grp78 was decreased. EAAm supplementation maintained or enhanced these changes. Rvs promotes mitochondrial biogenesis, with a probable anti-apoptotic effect. EAAm boosts these processes and may contribute to the efficient control of cellular energetics and survival in the mouse kidney. This suggests that appropriate nutritional interventions may enhance the beneficial actions of Rvs, and could potentially prevent chronic renal side effects.     

Oxidative 4-dechlorination of 2,4,6-trichlorophenol catalyzed by horseradish peroxidase

 The well-known and easily available horseradish peroxidase (HRP) catalyzes the H₂O₂-dependent oxidative 4-dechlorination of the pollutant 2,4,6-trichlorophenol, which is recalcitrant to many organisms except those producing ligninases. UV-visible spectroscopy and gas chromatography-mass spectrometry identified the oxidized reaction product as 2,6-dichloro-1,4-benzoquinone. NMR and IR spectroscopic data further supported the above characterization. Experimental evidence for the elimination of HCl from the substrate was acquired by detecting the decrease in pH of the reaction mixture, and by observing the presence of the β-chlorocyclopentadienone cation fragment in the mass spectrum of 2,6-dichloro-1,4-benzoquinone. Consequently, nucleophilic attack by water on the 2,4,6-trichlorocyclohexadienone cation was proposed to give the final product. Our results indicate an oxidative dechlorination pathway catalyzed by HRP for 2,4,6-trichlorophenol, similar to that by extracellular lignin peroxidases. The relative catalytic efficiency of HRP seems higher than that of lignin peroxidases. The HRP-H₂O₂ catalytic system could be utilized in the degradation of polychlorinated phenols for industrial and biotechnological purposes. Received: 20 November 1998 / Accepted: 29 January 1999     

Autoantibody signature in human ductal pancreatic adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy characterized by rapid progression, invasiveness, and resistance to treatment. It is the fourth leading cause of cancer death with a 2% 5-year survival rate. Biomarkers for its early detection are lacking. This study was designed to use a proteomics-based approach as a means of identifying antigens that elicit a humoral response in PDAC patients. Antibodies against PDAC-associated antigens are useful for early cancer diagnosis and therapy. Proteins from PDAC cell lines were separated by 2-DE, and the serum IgG reactivity of 70 PDAC patients, 40 healthy subjects (HS), 30 non-PDAC tumor patients, and 15 chronic pancreatitis (CP) patients was tested by Western blot analysis. Spots specifically recognized by PDAC sera and revealed by mass spectrometry corresponded to metabolic enzymes or cytoskeletal proteins. Most were up-regulated in PDAC tissues. Thus, it seems that metabolic enzymes and cytoskeletal proteins are specific targets of the humoral response during PDAC. The results of further studies of these serological-defined antigens could be of diagnostic and therapeutic significance in PDAC.     

Fat facets deubiquitylation of Medea/Smad4 modulates interpretation of a Dpp morphogen gradient

The ability of secreted Transforming Growth Factor β (TGFβ) proteins to act as morphogens dictates that their influence be strictly regulated. Here, we report that maternally contributed fat facets (faf; a homolog of USP9X/FAM) is essential for proper interpretation of the zygotic Decapentaplegic (Dpp) morphogen gradient that patterns the embryonic dorsal-ventral axis. The data suggest that the loss of faf reduces the activity of Medea (a homolog of Smad4) below the minimum necessary for adequate Dpp signaling and that this is likely due to excessive ubiquitylation on a specific lysine. This study supports the hypothesis that the control of cellular responsiveness to TGFβ signals at the level of Smad4 ubiquitylation is a conserved mechanism required for proper implementation of a morphogen gradient.