Preparation and use of media for protease-producing bacterial strains based on by-products from Cuttlefish (Sepia officinalis) and wastewaters from marine-products processing factories

Cuttlefish powder (CFP) from Sepia officinalis by-products was prepared and tested as a fermentation substrate for microbial growth and protease production by several species of bacteria: Bacillus licheniformis, Bacillus subtilis, Pseudomonas aeruginosa, Bacillus cereus BG1, and Vibrio parahaemolyticus. All microorganisms studied grew well and produced protease activity when cultivated in medium containing only CFP indicating that the strains can obtain their carbon and nitrogen source requirements directly from whole by-product proteins. Moreover, it was found that the addition to the cuttlefish medium of diluted fishery wastewaters (FWW), generated by marine-products processing factories, enhanced the production of protease. Maximum activity was obtained when cells were grown in cuttlefish media containing 5-times or 10-times diluted FWW. Five-times diluted FWW enhanced protease production by B. cereus BG1 and B. subtilis by 467% and 75% more than control media, respectively. The enhancement could have been due to the high organic content or high salts in FWW.As a result, cuttlefish by-products powder enriched with diluted FWW was found to be a suitable growth media for protease-producing strains. This new process, which converts underutilized wastes (liquid and solid) into more marketable and acceptable forms, coupled with protease production, can be an alternative way to the biological treatment of solid and liquid wastes generated by the cuttlefish processing industry.     

Antisense raf oligodeoxyribonucleotide is a radiosensitizer in vivo.

Raf-1, a cytosolic protein serine/threonine kinase, plays important roles in cell growth, proliferation, transformation, and cell survival. The aim of the present study was to evaluate the radiotherapeutic efficacy of a fully phosphorothioated and well-characterized antisense raf oligodeoxyribonucleotide (ODN) corresponding to the 3'-untranslated region of human c-raf-1 mRNA (ISIS 5132/5132). Using our recently developed liposome encapsulation of ODN approach, we first compared the pharmacokinetic parameters of a liposomal formulation of 5132 (LE-5132) and 5132. The peak plasma concentrations 5 minutes after ODN administrations (30 mg/kg i.v.) were 28.5 microg/ml and 13.5 microg/ml for LE-5132 and 5132, respectively. The decrease in plasma concentration of LE-5132 and 5132 followed a biexponential pattern, with initial distribution half-lives (t1/2alpha) of 34.8 minutes and 21.6 minutes, respectively. The terminal half-lives (t1/2beta) with LE-5132 and 5132 were 14.5 hours and 4.3 hours, respectively. The area under the plasma concentration-time curve (AUC) was 5.8 times higher with LE-5132 than with 5132. Significantly higher intact ODN levels could be measured in most organs within 48 hours of administration of LE-5132 compared with 5132 (liver 18.4-fold, spleen, 31-fold, heart 3-fold, lungs 1.5-fold). In kidneys, the level was lower with LE-5132 (0.77-fold). LE-5132 composition, unlike 5132, did not affect clotting time in vitro. Significant decline in the level of Raf-1 protein was observed in vitro in relatively radioresistant human laryngeal squamous cell carcinoma cells (SQ-20B) treated with LE-5132 compared with SQ-20B cells treated with equimolar concentration of 5132 or liposome-encapsulated mismatched 5132 (0.5 microM LE-5132, 71.3%+/-22.5%; 1.0 microM LE-5132, 79.6%+/-16.7%). In addition, LE-5132 appeared to be a more potent antitumor compound than 5132 (p < 0.001). These data established the suitability of LE-5132 for in vivo radiotherapeutic efficacy studies. Intravenous administration of LE-5132 into SQ-20B tumor-bearing athymic mice inhibited Raf-1 expression in tumor tissue compared with blank liposome-treated or untreated control groups. LE-5132 or ionizing radiation (IR) treatment alone caused significant but transient inhibition of SQ-20B tumor growth but not tumor regression. Remarkably, a combination of LE-5132 and IR treatments led to significant and sustained tumor regression for at least 27 days after the last treatment (< 0.001). Histopathologic examination of tumor samples revealed a significant proportion of cells containing fragmented chromatin in the LE-5132 + IR treatment group as compared with single agent and untreated control groups. These in vivo data support the notion that Raf-1 has proliferative and survival functions and advance the scientific and technologic bases for the use of antisense raf ODN in the management of radioresistant malignancies.     

Mitochondrial DNA Sequence Variation Suggests the Lack of Genetic Heterogeneity in the Adriatic and Ionian Stocks of Sardina pilchardus

A genetic stock structure analysis of 11 sardine samples from the Adriatic Sea and Ionian neighboring area was carried out through sequence variation analysis of a 307-bp cytochrome b gene fragment in order to identify self-recruiting units in the Adriatic Sardina pilchardus stock. The overall lack of genetic subdivision among samples detected by analysis of molecular variance, pairwise Φ_st values, and the exact test of population differentiation indicates this sardine stock is part of a larger self-recruiting population whose boundaries are larger than the investigated area. This conclusion is in agreement with preliminary allozymic and mitochondrial DNA restriction fragment length polymorphism data, but contradicts the previous identification of 2 subpopulations of sardines in the Adriatic Sea argued on morphologic differences, which could be rather attributed to different hydrographic or ecologic conditions occurring in different areas of the Adriatic Sea. The reduced gene flow observed between Adriatic-Ionian and Spanish sardine geographic samples (P < 0.001) suggests that reproductively isolated populations of sardines may occur in the Mediterranean Sea.     

Nitric oxide regulates immune cell bioenergetic: a mechanism to understand immunomodulatory functions of nitric oxide-releasing anti-inflammatory drugs

The 2-(acetyloxy)benzoic acid 3-(nitrooxymethyl)phenyl ester (NCX-4016) is a NO-releasing derivative of aspirin. In this study, we provide evidence that NCX-4016 delivered to PMBC-derived T lymphocytes and monocytes causes a transitory inhibition of cell respiration and approximately 50% reduction of cellular ATP, which translates in a time-reversible inhibition of cell proliferation and IL-2, IL-4, IL-5, and IFN-gamma secretion. Exposure of lymphocytes and monocytes to aspirin, 2-(acetyloxy)benzoic acid 3-(hydroxymethyl)phenyl ester (NCX-4017), a non-NO-releasing analog of NCX-4016, and cyclooxygenase inhibitors, reduced PG formation, but has no effect on cytokine/chemokine release. In contrast, delivering NO with (z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino] diazen-1-ium-1,2 diolate (DETA-NO) reproduced most of the metabolic and anti-cytokine activities of NCX-4016. Scavenging NO with hemoglobin or adding selective substrates of complex II, III, and IV of the mitochondrial respiratory chain reverses NCX-4016' inhibitory activities. Exposure to DETA-NO and NCX-4016 enhances glucose uptake, glycolytic rate, and lactate generation in CD3/CD28-costimulated lymphocytes, while reduced citric acid cycle intermediates. These effects were not reproduced by selective and nonselective cyclooxygenase 2 inhibitors. In summary, we demonstrated that exposure of lymphocytes to NCX-4016 causes a metabolic hypoxia that inhibits lymphocyte reactivity to costimulatory molecules, providing a potential counteregulatory mechanism to control activated immune system.     

Early preconditioning prevents the loss of endothelial nitric oxide synthase and enhances its activity in the ischemic/reperfused rat heart

Cardiac ischemia may be responsible for either the loss of endothelial nitric oxide synthase (eNOS) or changes in its activity, both conditions leading to coronary dysfunction. We investigated whether early ischemic preconditioning was able to preserve eNOS protein expression and function in the ischemic/reperfused myocardium. Langendorff-perfused rat hearts were subjected to 20 min global ischemia, followed by 30 min reperfusion (I/R). A second group of hearts was treated as I/R, but preconditioned with three cycles of 5 min-ischemia/5 min-reperfusion (IP). Cardiac contractility markedly decreased in I/R, consistently with the rise of creatine kinase (CK) activity in the coronary effluent, whilst ischemic preconditioning significantly improved all functional parameters and reduced the release of CK. Western blot analysis revealed that the amount of eNOS protein decreased by 54.2% in I/R with respect to control (p < 0.01). On the other hand, NOS activity was not significantly reduced in I/R, as well as cGMP tissue levels, suggesting that a parallel compensatory stimulation of this enzymatic activity occurred during ischemia/reperfusion. Ischemic preconditioning completely prevented the loss of eNOS. Moreover, both NOS activity and cGMP tissue level were significantly higher (p < 0.05) in IP (12.7 +/- 0.93 pmol/min/mg prot and 58.1 +/- 12.2 fmol/mg prot, respectively) than I/R (7.34 +/- 2.01 pmol/min/mg prot and 21.4 +/- 4.13 fmol/mg prot, respectively). This suggest that early ischemic preconditioning may be useful to accelerate the complete recovery of endothelial function by preserving the level of cardiac eNOS and stimulating the basal production of nitric oxide.     

Cholesterol Conjugated Uniform and Gapmer Phosphorothioate Oligonucleotides Targeted Against PKC-α and C-raf Gene Expression

Abstract

In vitra and in vivo antitumor activity of phosphorothioate antisense oligonucleotides targeted against two protein kinases within the mitogen-activated protein (MAP) kinase signaling cascade has been well documented by ISIS 3521/CGP 6412XA (targeted against PKC-α protein) and ISIS 5132KGP69846A (targeted against C-rwfl kinase). For both of these compounds, cationic lipid formulations are necessary to observe any pharmacological activity in cell culture. In contrast, in vivo functional delivery of phosphorothioate oligonucleotides to cells in tissues does not appear to be a prohlem. These oligonucleotides have demonstrated reduction in either PKC-α or C-raf gene expression in tissues or human tumor xenografts following systemic administration.