Biochemical characterization and immunocytochemical localization of EM66, a novel peptide derived from secretogranin II, in the rat pituitary and adrenal glands.

Characterization of secretogranin II (SgII) mRNA in various vertebrates has revealed selective conservation of the amino acid sequences of two regions of the protein, i.e., the bioactive peptide secretoneurin and a flanking novel peptide that we named EM66. To help elucidate the possible role of EM66, we examined the occurrence as well as the cellular and subcellular distribution of EM66 in rat pituitary and adrenal glands by using a polyclonal antibody raised against the recombinant human EM66 peptide. High-performance liquid chromatography (HPLC) analysis of rat pituitary and adrenal extracts combined with a radioimmunoassay resolved EM66-immunoreactive material exhibiting the same retention time as recombinant EM66. In the rat pituitary, double-labeling immunohistochemical (IHC) studies showed that EM66 immunoreactivity (IR) was present in gonadotrophs, lactotrophs, thyrotrophs, and melanotrophs, whereas corticotrophs were devoid of labeling. EM66-IR was also observed in nerve endings in the neural lobe. Immunocytochemical staining at the electron microscopic level revealed that EM66-IR is sequestered in the secretory granules within gonadotrophs and lactotrophs. In the adrenal medulla, double IHC labeling showed that EM66-IR occurs exclusively in epinephrine-synthesizing cells. At the ultrastructural level, EM66-IR was seen in chromaffin vesicles of adrenomedullary cells. These results demonstrate that post-translational processing of SgII generates a novel peptide that exhibits a cell-specific distribution in the rat pituitary and adrenal glands where it is stored in secretory granules, supporting the notion that EM66 may play a role in the endocrine system.     

New action pattern of a maltose-forming alpha-amylase from Streptomyces sp. and its possible application in bakery

An a-Amylase (EC 3.2.1.1) was purified that catalyses the production of a high level of maltose from starch without the attendant production of glucose. The enzyme was produced extracellularly by thermophilic Streptomyces sp. that was isolated from Thailand's soil. Purification was achieved by alcohol precipitation, DEAE-Cellulose, and Gel filtration chromatographies. The purified enzyme exhibited maximum activity at pH 6-7 and 60 degrees C. It had a relative molecular mass of 45 kDa, as determined by SDS-PAGE. The hydrolysis products from starch had alpha-anomeric forms, as determined by 1H-NMR. This maltose-forming alpha-Amylase completely hydrolyzed the soluble starch to produce a high level of maltose, representing up to 90%. It hydrolyzed maltotetrose and maltotriose to primarily produce maltose (82% and 62% respectively) without the attendant production of glucose. The high maltose level as a final end-product from starch and maltooligosaccharides, and the unique action pattern of this enzyme, indicate an unusual maltose-forming system. After the addition of the enzyme in the bread-baking process, the bread's volume increased and kept its softness longer than when the bread had no enzyme.     

Zinc inhibition of bacterial cytochrome bc(1) reveals the role of cytochrome b E295 in proton release at the Q(o) site

The cytochrome (cyt) bc(1) complex (cyt bc(1)) plays a major role in the electrogenic extrusion of protons across the membrane responsible for the proton motive force to produce ATP. Proton-coupled electron transfer underlying the catalysis of cyt bc(1) is generally accepted, but the molecular basis of coupling and associated proton efflux pathway(s) remains unclear. Herein we studied Zn(2+)-induced inhibition of Rhodobacter capsulatus cyt bc(1) using enzyme kinetics, isothermal titration calorimetry (ITC), and electrochemically induced Fourier transform infrared (FTIR) difference spectroscopy with the purpose of understanding the Zn(2+) binding mechanism and its inhibitory effect on cyt bc(1) function. Analogous studies were conducted with a mutant of cyt b, E295, a residue previously proposed to bind Zn(2+) on the basis of extended X-ray absorption fine-structure spectroscopy. ITC analysis indicated that mutation of E295 to valine, a noncoordinating residue, results in a decrease in Zn(2+) binding affinity. The kinetic study showed that wild-type cyt bc(1) and its E295V mutant have similar levels of apparent K(m) values for decylbenzohydroquinone as a substrate (4.9 ± 0.2 and 3.1 ± 0.4 μM, respectively), whereas their K(I) values for Zn(2+) are 8.3 and 38.5 μM, respectively. The calorimetry-based K(D) values for the high-affinity site of cyt bc(1) are on the same order of magnitude as the K(I) values derived from the kinetic analysis. Furthermore, the FTIR signal of protonated acidic residues was perturbed in the presence of Zn(2+), whereas the E295V mutant exhibited no significant change in electrochemically induced FTIR difference spectra measured in the presence and absence of Zn(2+). Our overall results indicate that the proton-active E295 residue near the Q(o) site of cyt bc(1) can bind directly to Zn(2+), resulting in a decrease in the electron transferring activity without changing drastically the redox potentials of the cofactors of the enzyme. We conclude that E295 is involved in proton efflux coupled to electron transfer at the Q(o) site of cyt bc(1).     

Investigation of Design Space for Freeze-Drying: Use of Modeling for Primary Drying Segment of a Freeze-Drying Cycle

In this work, we explore the idea of using mathematical models to build design space for the primary drying portion of freeze-drying process. We start by defining design space for freeze-drying, followed by defining critical quality attributes and critical process parameters. Then using mathematical model, we build an insilico design space. Input parameters to the model (heat transfer coefficient and mass transfer resistance) were obtained from separate experimental runs. Two lyophilization runs are conducted to verify the model predictions. This confirmation of the model predictions with experimental results added to the confidence in the insilico design space. This simple step-by-step approach allowed us to minimize the number of experimental runs (preliminary runs to calculate heat transfer coefficient and mass transfer resistance plus two additional experimental runs to verify model predictions) required to define the design space. The established design space can then be used to understand the influence of critical process parameters on the critical quality attributes for all future cycles.     

In situ biosurfactant production by Bacillus strains injected into a limestone petroleum reservoir

Biosurfactant-mediated oil recovery may be an economic approach for recovery of significant amounts of oil entrapped in reservoirs, but evidence that biosurfactants can be produced in situ at concentrations needed to mobilize oil is lacking. We tested whether two Bacillus strains that produce lipopeptide biosurfactants can metabolize and produce their biosurfactants in an oil reservoir. Five wells that produce from the same Viola limestone formation were used. Two wells received an inoculum (a mixture of Bacillus strain RS-1 and Bacillus subtilis subsp. spizizenii NRRL B-23049) and nutrients (glucose, sodium nitrate, and trace metals), two wells received just nutrients, and one well received only formation water. Results showed in situ metabolism and biosurfactant production. The average concentration of lipopeptide biosurfactant in the produced fluids of the inoculated wells was about 90 mg/liter. This concentration is approximately nine times the minimum concentration required to mobilize entrapped oil from sandstone cores. Carbon dioxide, acetate, lactate, ethanol, and 2,3-butanediol were detected in the produced fluids of the inoculated wells. Only CO(2) and ethanol were detected in the produced fluids of the nutrient-only-treated wells. Microbiological and molecular data showed that the microorganisms injected into the formation were retrieved in the produced fluids of the inoculated wells. We provide essential data for modeling microbial oil recovery processes in situ, including growth rates (0.06 +/- 0.01 h(-1)), carbon balances (107% +/- 34%), biosurfactant production rates (0.02 +/- 0.001 h(-1)), and biosurfactant yields (0.015 +/- 0.001 mol biosurfactant/mol glucose). The data demonstrate the technical feasibility of microbial processes for oil recovery.     

Structure of trichamide, a cyclic peptide from the bloom-forming cyanobacterium Trichodesmium erythraeum, predicted from the genome sequence

A gene cluster for the biosynthesis of a new small cyclic peptide, dubbed trichamide, was discovered in the genome of the global, bloom-forming marine cyanobacterium Trichodesmium erythraeum ISM101 because of striking similarities to the previously characterized patellamide biosynthesis cluster. The tri cluster consists of a precursor peptide gene containing the amino acid sequence for mature trichamide, a putative heterocyclization gene, an oxidase, two proteases, and hypothetical genes. Based upon detailed sequence analysis, a structure was predicted for trichamide and confirmed by Fourier transform mass spectrometry. Trichamide consists of 11 amino acids, including two cysteine-derived thiazole groups, and is cyclized by an N C terminal amide bond. As the first natural product reported from T. erythraeum, trichamide shows the power of genome mining in the prediction and discovery of new natural products.     

Acid ceramidase but not acid sphingomyelinase is required for tumor necrosis factor-{alpha}-induced PGE2 production

Sphingolipids are well established effectors of signal transduction downstream of the tumor necrosis factor (TNF) receptor. In a previous study, we showed that the sphingosine kinase/sphingosine 1-phosphate (S1P) pathway couples TNF receptor to induction of the cyclooxygenase 2 gene and prostaglandin synthesis (Pettus, B. J., Bielawski, J., Porcelli, A. M., Reames, D. L., Johnson, K. R., Morrow, J., Chalfant, C. E., Obeid, L. M., and Hannun, Y. A. (2003) FASEB J. 17, 1411-1421). In this study, the requirement for acid sphingomyelinase and sphingomyelin metabolites in the TNFalpha/prostaglandin E(2) (PGE(2)) pathway was investigated. The amphiphilic compound desipramine, a frequently employed inhibitor of acid sphingomyelinase (ASMase), blocked PGE(2) production. However, the action of desipramine was independent of its action on ASMase, since neither genetic loss of ASMase (Niemann-Pick fibroblasts) nor knockdown of ASMase using RNA interference affected TNFalpha-induced PGE(2) synthesis. Further investigations revealed that desipramine down-regulated acid ceramidase (AC), but not sphingosine kinase, at the protein level. This resulted in a time-dependent drop in sphingosine and S1P levels. Moreover, exogenous administration of either sphingosine or S1P rescued PGE(2) biosynthesis after desipramine treatment. Interestingly, knockdown of endogenous AC by RNA interference attenuated cyclooxygenase 2 induction by TNFalpha and subsequent PGE(2) biosynthesis. Taken together, these results define a novel role for AC in the TNFalpha/PGE(2) pathway. In addition, the results of this study warrant careful reconsideration of desipramine as a specific inhibitor for ASMase.     

Synthetic adjuvants for vaccine formulations: evaluation of new phytol derivatives in induction and persistence of specific immune response

Terpenoids are ubiquitous natural compounds that have been shown to improve vaccine efficacy as adjuvants. To gain an understanding of the structural features important for adjuvanticity, we studied compounds derived from a diterpene phytol and assessed their efficacy. In a previous report, we showed that phytol and one of its derivatives, PHIS-01 (a phytol-derived immunostimulant, phytanol), are excellent adjuvants. To determine the effects of varying the polar terminus of PHIS-01, we designed amine and mannose-terminated phytol derivatives (PHIS-02 and PHIS-03, respectively). We studied their relative efficacy as emulsions with soluble proteins, ovalbumin, and a hapten-protein conjugate phthalate-KLH. Immunological parameters evaluated consisted of specific antibody responses in terms of titers, specificities and isotype profiles, T cell involvement and cytokine production. Our results indicate that these new isoprenoids were safe adjuvants with the ability to significantly augment immunogen-specific IgG1 and IgG2a antibody responses. Moreover, there was no adverse phthalate cross-reactive anti-DNA response. Interestingly, PHIS-01 and PHIS-03 influenced differentially T-helper polarization. We also observed that these compounds modulated the immune response through apoptotic/necrotic effects on target tumor cells using murine lymphomas. Finally, unlike squalene and several other terpenoids reported to date, these phytol derivatives did not appear arthritogenic in murine models.     

Cytotoxicity and acid ceramidase inhibitory activity of 2-substituted aminoethanol amides

The acid ceramidase (AC) inhibitory activity of octanoylamides, p-tert-butylbenzamides and pivaloylamides of several 2-substituted aminoethanols is reported. All the aminoethanol amides bearing a hexadecyl substituent (C16), as well as (S)-N-(1-(hexadecylthio)-3-hydroxypropan-2-yl)pivaloylamide (SC16-tb) were inhibitory in cell lysates overexpressing AC, while all other compounds were not inhibitors. Kinetic experiments with (R,E)-N-(1-hydroxyoctadec-3-en-2-yl)pivaloylamide (E-tb) and SC16-tb showed that inhibition was competitive, with K(i) values of 34 and 94.0 microM, respectively. None of the compounds inhibited neutral ceramidase. Compounds E-tb and E-c7 (the octanoylamide of the unsaturated base E), which elicited a dose-response inhibition with IC(50) values around 15 microM, were the only AC inhibitors in intact cells. Both compounds were toxic to A549 cells with LD(50) values nearly 40 microM. Flow cytometry studies with E-tb evidenced that this compound induced a concentration-dependent cell cycle arrest at G(1) and a 20-25% apoptosis/late apoptosis/necrosis after a 24-h incubation at 50 microM. In agreement with its activity as acidic ceramidase inhibitor, this effect was accompanied with an increase in the amounts of C14, C16 and C18 ceramides (LC-MS analyses), which suggested that these lipids may be responsible for the cytotoxic activity of E-tb.