Comparison of the dynamics of the primary events of bacteriorhodopsin in its trimeric and monomeric states

In this paper, femtosecond pump-probe spectroscopy in the visible region of the spectrum has been used to examine the ultrafast dynamics of the retinal excited state in both the native trimeric state and the monomeric state of bacteriorhodopsin (bR). It is found that the excited state lifetime (probed at 490 nm) increases only slightly upon the monomerization of bR. No significant kinetic difference is observed in the recovery process of the bR ground state probed at 570 nm nor in the fluorescent state observed at 850 nm. However, an increase in the relative amplitude of the slow component of bR excited state decay is observed in the monomer, which is due to the increase in the concentration of the 13-cis retinal isomer in the ground state of the light-adapted bR monomer. Our data indicate that when the protein packing around the retinal is changed upon bR monomerization, there is only a subtle change in the retinal potential surface, which is dependent on the charge distribution and the dipoles within the retinal-binding cavity. In addition, our results show that 40% of the excited state bR molecules return to the ground state on three different time scales: one-half-picosecond component during the relaxation of the excited state and the formation of the J intermediate, a 3-ps component as the J changes to the K intermediate where retinal photoisomerization occurs, and a subnanosecond component during the photocycle.     

Purification and characterization of trypsin from an entomopathogen,Nomuraea rileyi NRRL 13755

Nomuraea rileyi isolate NRRL-13755 produced a large amount of trypsin enzyme when cultured on basal salt medium containing 1% (w/v) gelatin. The trypsin was purified nearly 60-fold, with a recovery of about 13% of the initial activity from the culture supernatant. This protease exhibited a remarkably high specific activity of nearly 370,000 IU/mg protein. The native molecular weight was estimated by gel permeation chromatography to be 30 kDa, and the subunit molecular weight was determined to be about 30 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The pH and temperature optima were determined to be 8.5 and 35°C, respectively. With a relative trypsin activity of 100%, this purified preparation showed about 10% chymoelastase and nearly 50% chymotrypsin activity. Metal-chelating agents such as EDTA and EGTA at 2mm inhibited the enzyme activity by 40%, whereas N-carbobenzoxy-glycyl-l-phenylalaninamide (CBZ-gly-phe-NH₂) (2mm) and DTT (2mm) had no effect on activity. Trypsin inhibitor from turkey egg-white at 100 g/ml strongly inhibited the enzyme activity.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

Production of dextransucrase and dextran by Leuconostoc mesenteroides immobilized in calcium-alginate beads: II. Semicontinuous fed-batch fermentations

Cells of Leuconostoc mesenteroides immobilized in calcium alginate beads were used to produce dextransucrase (DS) in three sequential cycles of semicontinuous fed-batch fermentations. Each cycle consisted of a fed-batch DS production period of 24 h followed by a batch dextran production period for another 24 h. Free, suspended cells were used in only one cycle of fed-batch DS production followed by a dextran production period. It was impractically tedious to separate and reuse free cells. Increasing sucrose feed rate from 5 to 10 g/L h led to increases of the total enzymatic activity by about 88% with immobilized cells and by about 100% with free cells. In DS fed-batch semicontinuous fermentation, total enzymatic activity produced by immobilized cells was 1.35 and 1.56 times greater than that produced by free cells with respective sucrose feeding rates of 10 and 5 g/L h. These increases in enzyme productivity with immobilized cells, however, required total overall operating times three times longer (three cycles) than with free cells (one cycle). Growing the microorganism at optimum conditions for DS production also increased the dextran yield and shortened the time of conversion of sucrose to dextran, regardless of whether the cells were free or immobilized. Moreover, during three cycles of semicontinuous operation (144 h) immobilized cells produced more than three times as much dextran as free cells during one cycle (24 h).     

Effect of beta-cyclodextrin on production of L-phenylacetyl carbinol by immobilized cells of Saccharomyces cerevisiae

The rate and extent of microbial transformation of higher concentrations of benzaldehyde substrate to L-phenylacetyl carbinol (L-PAC) by immobilized cells of Saccharomyces cerevisiae ATCC 834 was markedly stimulated by addition of different concentrations of beta-cyclodextrin (BCD) to the fermentation medium. With 0.5, 1.0, and 1.5% BCD in the fermentation medium and cumulative doses of benzaldehyde of 12 and 14 g/L, significantly higher yields of L-PAC were obtained, about one- to twofold that of the yields of the control experiments. The favorable effects of BCD were evident in spite of its presence in stoichiometric concentrations significantly lower than those of benzaldehyde. The presence of BCD also appeared to stimulate microbial growth slightly. Enhanced cellular activity was reflected by faster D-glucose consumption and faster benzaldehyde utilization in the presence of BCD.     

Comparative study of production of dextransucrase and dextran by cells of Leuconostoc mesenteroides immobilized on Celite and in calcium alginate beads

In fed-batch fermentation, cells of L. mesenteroides immobilized on three types of Celite were used to produce dextransucrase (DS) followed by production of dextran. A layer of calcium alginate on the porous Celite R630 particles improved their mechanical stability, increased the amount of soluble DS produced and decreased the cell leakage from the highly porous support. Enzyme production with the immobilized cell cultures was significantly affected by both pore and particle size. Immobilized cultures using Celite R648 (average particle radius of 200 mum and pore size of 0.14 mum) produced the highest total enzymatic activity, followed by Celite R633, alginate-coated Celite R630, Celite R630, and then calcium alginate beads. Culture of free cells produced about 18% more total enzymatic activity than immobilized cells in calcium alginate beads, but about 64% less than immobilized cells on Celite R630. It is expected that larger amounts of enzymatic activity than measured are immobilized inside the alginate-coated Celite R630 and calcium alginate beads due to the mass transfer limitation conferred by the dextran product formed therein. The dextran yield from conversion of sucrose to dextran and fructose with all such enzyme-enriched, immobilized-cell cultures was higher than that obtained from free-cell culture under similar conditions.     

Biochemical and histological studies on H2-receptor antagonist ranitidine-induced hepatotoxicity in rats

This study was designed to investigate the hepatotoxicity of ranitidine treatment in dose levels of 10, 30, and 50 mg/kg b.wt. for 3 weeks period in male rats. The results showed some adverse changes in rats treated with either 10 or 30 mg/kg. Treatment with dose of 50 mg/kg produced marked increase in the activity of both acid phosphatase in liver and aspartate aminotransferase in serum and liver, with a tendency for increase in serum alanine aminotransferase activity. Also, a significant decrease in the serum activity of both amylase and alkaline phosphatase was noted. Microscopic examination of livers of the same animals revealed absence of some hepatic cells, pyknotic nuclei, dilatation of blood sinusoids, binucleated cells, and infiltration of lymphocytes. These biochemical and histological changes indicate that ranitidine when given chronically in high dose could produce hepatotoxicity in rats.     

The protonation-deprotonation kinetics of the protonated Schiff base in bicelle bacteriorhodopsin crystals

In the recently published x-ray crystal structure of the "bicelle" bacteriorhodopsin (bbR) crystal, the protein has quite a different structure from the native and the in cubo bacteriorhodopsin (cbR) crystal. Instead of packing in parallel trimers as do the native membrane and the cbR crystals, in the bbR crystal the protein packs as antiparallel monomers. To date, no functional studies have been performed, to our knowledge, to investigate if the photocycle is observed in this novel protein packing structure. In this study, both Raman and time-resolved transient absorption spectroscopy are used to both confirm the presence of the photocycle and investigate the deprotonation-reprotonation kinetics of the Schiff base proton in the bbR crystal. The observed rates of deprotonation and reprotonation processes of its Schiff base have been compared to those observed for native bR under the same conditions. Unlike the previously observed similarity of the rates of these processes for cbR crystals and those for native bacteriorhodopsin (bR), in bbR crystals the rate of deprotonation has increased by 300%, and the rate of reprotonation has decreased by nearly 700%. These results are discussed in light of the changes observed when native bR is delipidated or monomerized by detergents. Both the change of the hydrophobicity of the environment around the protonated Schiff base and Asp85 and Asp96 (which could change the pKa values of proton donor-acceptor pairs) and the water structure in the bbR crystal are offered as possible explanations for the different observations.     

The effect of protein conformation change from alpha(II) to alpha(I) on the bacteriorhodopsin photocycle

The bacteriorhodopsin (bR) photocycle was followed by use of time-resolved Fourier-transform infrared (FTIR) spectroscopy as a function of temperature (15-85 degrees C) as the alpha(II) --> alpha(I) conformational transition occurs. The photocycle rate increases with increasing temperature, but its efficiency is found to be drastically reduced as the transition takes place. A large shift is observed in the all-trans left arrow over right arrow 13-cis equilibrium due to the increased stability of the 13-cis isomer in alpha(I) form. This, together with the increase in the rate of dark adaptation as the temperature increases, leads to a large increase in the 13-cis isomer concentration in bR in the alpha(I) form. The fact that 13-cis retinal has a much-reduced absorption cross-section and its inability to pump protons leads to an observed large reduction in the concentration of the observed photocycle intermediates, as well as the proton gradient at a given light intensity. These results suggest that nature might have selected the alpha(II) rather than the alpha(I) form as the helical conformation in bR to stabilize the all-trans retinal isomer that is a better light absorber and is capable of pumping protons.     

Production of thaxtomin a by two species ofStreptomyces causing potato scab

A total of nine isolates of streptomycetes were isolated from scab lesions on potato tubers. Five of nine isolates were pathogenic on potato minitubers. Four of the pathogenic isolates produced thaxtomin A (ThxA) in infected tuber tissues. The lesion surface areas inducing ThxA were highest in treatment of the minitubers with an extract of OMB inoculated with S-66 and S-67, intermediate with that inoculated with S-64 and lowest with S-63. The pathogenic isolates were identified by gray aerial mycelia, melanin pigment productivity, the type of spore chain morphology and carbon utilization asS. scabies strains S-63, S-64 and S-68, andS. acidiscabies strains S-66 and S-67. Strains S-63 and S-64 produced 0.65 and 1.60 mg ThxA per L of OMB, respectively, strains S-66 and S-67 producing similar amounts,viz. 2.36 and 2.10 mg/L, respectively. The optimal temperature for production (by both species) was 28 °C. Production of ThxA byS. scabies strain S-64 andS. acidiscabies strain S-66 was suppressed at least 50-fold at 0.5 and 0.3 % of glucose, respectively. Fructose enhanced the production by both species.     

Chemopreventive activity of selenocysteine prodrugs against tobacco-derived nitrosamine (NNK) induced lung tumors in the A/J mouse

Prodrugs of L-selenocysteine have potential utility in cancer chemoprevention. This study reports the efficacy of three selenazolidine-4(R)-carboxylic acids, (2-unsubstituted, 2-oxo, and 2-methyl derivatives; SCA, OSCA, and MSCA, respectively) against tobacco-related lung tumorigenesis in a mouse model. Seven days after initiation of an AIN-76A diet supplemented with sodium selenite (5 ppm Se), L-selenomethionine (3.75 ppm Se), Se-methyl-L-selenocysteine (3 ppm Se), L-selenocystine (15 ppm Se), SCA (15 ppm Se), OSCA (15 ppm Se), or MSCA (15 ppm Se), mice received 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK; 10 micromol, i.p.). After an additional 16 weeks on the diets, two compounds, OSCA and selenocystine, significantly reduced lung adenoma multiplicity from 7.2 tumors per mouse in the NNK group to 4.5 and 4.6 tumors per mouse, respectively. Neither selenium concentration nor glutathione peroxidase activity in either RBCs or liver served as surrogate indicators of tumor reduction. Hepatic selenium levels were significantly elevated by all selenium-containing compounds except Se-methyl-L-selenocysteine and SCA; RBC selenium levels by all except sodium selenite and MSCA. With the exception of L-selenomethionine, RBC glutathione peroxidase activity was increased along with the elevated selenium levels. Hepatic glutathione peroxidase activity was elevated by all Se-compounds except SCA. The two compounds showing significant tumor reduction (OSCA and selenocystine) were the only two compounds that showed ubiquity of changes, elevating both selenium levels and GPx activity in both liver and RBC.