Production and Properties of Lipase from a Newly Isolated Chromobacterium

Out of some 750 strains of microorganisms, a potent bacterium for lipase production was isolated from soil and was identified as Chromobacterium viscosum.

The bacterium accumulates lipase in culture fluid when grown aerobically at 26°C for 3 days in a medium composed of soluble starch, soy bean meal, lard and inorganic salts.

Chromobacterium lipase had an optimum pH of 7.0 for activity at 37°C, and an optimal temperature of 65°C at pH 7.0. The enzyme retained 80% of the activity when heated for 10 min at 70°C. This lipase was capable of hydrolyzing a variety of natural fats and oils, and it was more active on lard and butter than on olive oil. The activity was stimulated by Ca²⁺, Mg²⁺, Mn²⁺ and inhibited by Cu²⁺, Hg²⁺ and Sn²⁺. It was not diminished but rather stimulated by a high concentration of bile-salts.     

One rotary mechanism for F1-ATPase over ATP concentrations from millimolar down to nanomolar

F₁-ATPase is a rotary molecular motor in which the central γ-subunit rotates inside a cylinder made of α₃β₃-subunits. The rotation is driven by ATP hydrolysis in three catalytic sites on the β-subunits. How many of the three catalytic sites are filled with a nucleotide during the course of rotation is an important yet unsettled question. Here we inquire whether F₁ rotates at extremely low ATP concentrations where the site occupancy is expected to be low. We observed under an optical microscope rotation of individual F₁ molecules that carried a bead duplex on the γ-subunit. Time-averaged rotation rate was proportional to the ATP concentration down to 200 pM, giving an apparent rate constant for ATP binding of 2 × 10⁷ M⁻¹s⁻¹. A similar rate constant characterized bulk ATP hydrolysis in solution, which obeyed a simple Michaelis-Menten scheme between 6 mM and 60 nM ATP. F₁ produced the same torque of ~40 pN·nm at 2 mM, 60 nM, and 2 nM ATP. These results point to one rotary mechanism governing the entire range of nanomolar to millimolar ATP, although a switchover between two mechanisms cannot be dismissed. Below 1 nM ATP, we observed less regular rotations, indicative of the appearance of another reaction scheme.     

Enhanced tolerance to ozone and drought stresses in transgenic tobacco overexpressing dehydroascorbate reductase in cytosol

Ascorbate (vitamin C) is a potent antioxidant protecting plants against oxidative damage imposed by environmental stresses such as ozone and drought. Dehydroascorbate reductase (DHAR; EC 1.8.5.1) is one of the two important enzymes functioning in the regeneration of ascorbate (AsA). To examine the protective role of DHAR against oxidative stress, we developed transgenic tobacco plants overexpressing cytosolic DHAR gene from Arabidopsis thaliana . Incorporation of the transgene in the genome of tobacco plants was confirmed by polymerase chain reaction and Southern blot analysis, and its expression was confirmed by Northern and Western blot analyses. These transgenic plants exhibited 2.3–3.1 folds higher DHAR activity and 1.9–2.1 folds higher level of reduced AsA compared with non-transformed control plants. The transgenic plants showed maintained redox status of AsA and exhibited an enhanced tolerance to ozone, drought, salt, and polyethylene glycol stresses in terms of higher net photosynthesis. In this study, we report for the first time that the elevation of AsA level by targeting DHAR overexpression in cytosol properly provides a significantly enhanced oxidative stress tolerance imposed by drought and salt.     

Near-full-length REV3L appears to be a scarce maternal factor in Xenopus laevis eggs that changes qualitatively in early embryonic development

REV3 is the catalytic subunit of DNA polymerase ζ (pol ζ), which is responsible for the damage-induced mutagenesis that arises during error-prone translesion synthesis in eukaryotes. The related REV3L genes in human and mouse encode proteins of approximately 350 kDa, twice as large as yeast REV3, but full-length REV3L has not been identified in any vertebrate cell. We report that Xenopus laevis REV3L encodes a 352-kDa protein that has high overall amino acid sequence similarity to its mammalian counterparts, and, for the first time in a vertebrate species, we have detected putative REV3L polypeptides of 300 and 340 kDa in X. laevis oocytes. Only the 300-kDa form is stored in eggs, where its concentration of about 65 pM is much lower than those of other replication and repair proteins including the accessory pol ζ subunit REV7. In fertilized eggs, the levels of this polypeptide did not change until neurula; the larger 340-kDa form first appeared at stages after gastrula, suggesting a pattern of regulation during development. These observations indicate the existence of REV3L as a scarce protein, of approximately the full predicted size, whose level may impose severe constraints on the assembly of pol ζ in X. laevis.     

Characterization of exo-(1,4)-alpha glucan lyase from red alga Gracilaria chorda. Activation, inactivation and the kinetic properties of the enzyme

Exo-(1,4)-alpha glucan lyase (GLase) was purified from a red alga Gracilaria chorda. The enzyme was activated 1.3-fold in the presence of Ca(2+) and Cl(-) ions. The ions also stabilized the enzyme increasing the temperature of its maximum activity from 45 degrees C to 50 degrees C. GLase was inactivated by chemical modification with carbodiimide and a carboxyl group of the enzyme was shown essential to the lyase activity. A tryptophanyl residue(s) was also shown to be important for the activity and was probably involved in substrate binding. K(m) values of the enzyme were 2.3 mM for maltose, 0.4 mM for maltotriose and 0.1 mM for maltooligosaccharides of degree of polymerization (dp) 4-7, and the k(0) values for the oligosaccharides were similar (42-53 s(-1)). The analysis of these kinetic parameters showed that the enzyme has four subsites to accommodate oligosaccharides. The subsite map of GLase was unique, since subsite 1 and subsite 2 have large positive and small negative affinities, respectively. The subsite map of this type has not been found in other enzymes with exo-action on alpha-1,4-glucan. The K(m) and k(0) values for the polysaccharides were lower (0.03 mM) and higher (60-100 s(-1)), respectively, suggesting the presence of another affinity site specific to the polysaccharides.     

Edaravone inhibits acute renal injury and cyst formation in cisplatin-treated rat kidney

Background: Although cis-diamminedichloroplatinum (II) (cisplatin) is an effective anticancer agent, its clinical use is highly limited predominantly due to its adverse effects on renal functions. The present work examined the therapeutic potential of edaravone, a free radical scavenger, for inhibiting cisplatin-induced renal injury.

Methods: Edaravone, 3-methyl-1-phenyl-pyrazolin-5-one, was administrated intravenously at a dose of 30 mg/kg of body weight to male Wistar rats (200-220 g). After 30 min, cisplatin was injected intraperitoneally at a dose of 5 mg/kg of body weight. At the indicated times after the treatment, functions and histological changes of the kidney were analyzed. To test the therapeutic potential of edaravone in chemotherapy, its effect on the anticancer action of cisplatin was examined in ascites cancer-bearing rats.

Results: We found that cisplatin rapidly impaired the respiratory function and DNA of mitochondria in renal proximal tubules, thereby inducing apoptosis of tubular epithelial cells within a few days and chronic renal dysfunction associated with multiple cysts one-year after the administration. Administration of edaravone inhibited the cisplatin-induced acute injury of mitochondria and their DNA and renal epithelial cell apoptosis as well as the occurrence of chronic renal dysfunction and multiple cyst formation. The anticancer effect of cisplatin remained unaffected by intravenous administrating of edaravone.

Conclusions: These results indicate that edaravone may have therapeutic potential for inhibiting the acute and chronic injury of the kidney induced by cisplatin.