Backbone amide linker (BAL) strategy for Nalpha-9-fluorenylmethoxycarbonyl (Fmoc) solid-phase synthesis of peptide aldehydes.

A rapid and efficient strategy has been developed for the general synthesis of complex peptide aldehydes. N(alpha)-Benzyloxycarbonylamino acids were converted to protected aldehyde building blocks for solid-phase synthesis in four steps and moderate overall yields. The aldehydes were protected as 1,3-dioxolanes except for one case where a dimethyl acetal was used. These protected amino aldehyde monomers were then incorporated onto 5-[(2 or 4)-formyl-3,5-dimethoxyphenoxy]butyryl-resin (BAL-PEG-PS) by reductive amination, following which the penultimate residue was introduced by HATU-mediated acylation. The resultant resin-bound dipeptide unit, anchored by a backbone amide linkage (BAL), was extended further by routine Fmoc chemistry procedures. Several model peptide aldehydes were prepared in good yields and purities. Some epimerization of the C-terminal residue occurred (10% to 25%), due to the intrinsic stereolability conferred by the aldehyde functional group, rather than any drawbacks to the synthesis procedure.     

Monitoring growth phase-related changes in phosphatidylcholine-specific phospholipase C production,adhesion properties and physiology of <Emphasis Type="Italic">Bacillus cereus</Emphasis> vegetative cells

The physiological status and metabolic heterogeneity of Bacillus cereus cells within a culture during an 8-h batch fermentation process was measured using flow cytometry (FCM). Concurrently, production of the toxin, PC-PLC, and the extent of cell adhesion of live and dead cells were monitored using novel fluorescent assays. Flow cytometry analysis detected growth phase-related changes in the physiological profiles of cells over the course of the fermentation, with variation in the percentage of cells displaying membrane damage and intracellular esterase and redox activities. As the exponential phase proceeded, populations became more uniform in terms of protein content as measured using FCM in tandem with a cell tracking dye, with the majority of cells becoming membrane intact, esterase positive and redox active. PC-PLC activity appeared strongly related to cell density. Permeabilisation of cells was accompanied by a loss in adherent properties, while 25–100% of cells with intracellular esterase activity possessed adhesion properties. Cells in late exponential phase appeared to have reduced adherence properties compared to cells in early exponential or lag phase. As well as demonstrating the utility of FCM for measuring heterogeneity in terms of cell physiological status throughout the course of batch cultures, the methods utilised in this study could be used to relate processes such as toxin production or cell adhesion to cell physiological state.     

A SXRF method for determining the relative concentration of trace elements in plasma protein affected by cisplatin

This article presents an analytical method for the determination of the relative concentrations of trace elements in plasma protein by gel chromatography combined with SXRF (synchrotron radiation X-ray fluorescence). The fraction of plasma protein of male Kunming mice (body weight 24.2±0.3 g), treated with a cisplatin ip injection at a dose of 10 mg/kg, was obtained by the separation of a Sephadex G-50 column (buffered with ammonium acetate, pH 5.7). The SXRF experiments were performed at the Beijing Electron Positron Collider synchrotron radiation facility. The elements (Pt, S, Ca, Fe, Ni, Cu, Zn, Se, Br, and Sr) in the fraction of the plasma proteins (>22 kDa) were assayed using highly sensitive SXRF. The relative concentrations of elements were calculated by a normalization of Compton scattering intensity around 22 keV, after the normalization for the collection time of the X-ray spectrum and the counting of the ion chamber, and subtracting the contribution of the polycarbonate film for the supporting sample. The determination could prove that the element Pt in plasma was bound with macromolecular protein. Cu and S were present in the fraction of the protein in mice treated with cisplatin increase, and their ratios of treated/control were 1.66±0.06 and 1.78±0.33, respectively; Zn decreased to a ratio of 0.78±0.09. Our results are in agreement with others that cisplatin exposure leads to a marked loss of kidney copper and a moderate rise in kidney zinc. However, this article primarily describes one of the analytical methods used; it does not emphasize the results of the effect of cisplatin on trace elements in plasma protein.     

RNA-binding protein Csx1 is phosphorylated by LAMMER kinase, Lkh1, in response to oxidative stress in Schizosaccharomyces pombe

Recent studies have shown that global gene expression during oxidative stress in Schizosaccharomyces pombe is regulated by stress-induced activation and binding of Csx1 to atf1(+) mRNA. However, the kinase responsible for the activation of Csx1 has not been identified. Here, we describe, for the first time, that Csx1 is phosphorylated by S. pombe LAMMER kinase, Lkh1, under oxidative conditions and that the stress-activated binding of the Csx1 to the atf1(+) mRNA was also affected by Lkh1 and Spc1. These data indicate that concerted actions of Spc1 and Lkh1 are required for the activation of Csx1 during oxidative condition in the fission yeast S. pombe.     

Alkylglucosides with isoprenoid-type hydrophobic chains-effects of hydrophobic chain size on the aqueous phase behavior

Aqueous phase diagrams were constructed for two new alkylglucosides with isoprenoid-type hydrophobic chains, viz. 1-O-beta-(3,7-dimethyloctyl)-D-glucopyranoside, beta-Glc(Ger), and 1-O-beta-(3,7,11,15-tetramethylhexadecyl)-D-glucopyranoside, beta-Glc(Phyt). In a low concentration regime, from 0.17 to 34 wt.% beta-Glc(Ger), the beta-Glc(Ger)/water system exhibits two phase, a dilute (L1dil) and a concentrated isotropic phase (L1con), coexistence region. Above about 62 wt.% beta-Glc(Ger), an Lalpha phase is formed. The extent of the L1dil + L1conc two-phase region decreases as temperature increases and totally disappears above 130 degrees C, exhibiting an upper critical temperature. The beta-Glc(Phyt)/water system exhibits an Lalpha phase above 78 wt.% surfactant below which, an Lalpha + water two-phase region appears. One notable feature of these compounds is their low values of Krafft-eutectic temperature, TK, e.g. the value of TK for beta-Glc(Phyt) is below 0 degrees C although the total number of carbon atoms in the hydrophobic chain is as large as 20.     

Evaluation of mono or mixed cultures of lactic acid bacteria in type II sourdough system

The aim of this study was to evaluate the use of mono and mixed lactic acid bacteria (LAB) cultures to determine suitable LAB combinations for a type II sourdough system. In this context, previously isolated sourdough LAB strains with antimicrobial activity, which included Lactobacillus plantarum PFC22, Lactobacillus brevis PFC31, Pediococcus acidilactici PFC38, and Lactobacillus sanfranciscensis PFC80, were used as mono or mixed culture combinations in a fermentation system to produce type II sourdough, and subsequently in bread dough production. Compared to the monoculture fermentation of dough, the use of mixed cultures shortened the adaptation period by half. In addition, the use of mixed cultures ensured higher microbial viability, and enhanced the fruity flavor during bread dough production. It was determined that the combination of L. plantarum PFC22 + P. acidilactici PFC38 + L. sanfranciscensis PFC80 is a promising culture mixture that can be used in the production of type II sourdough systems, and that may also contribute to an increase in metabolic activity during bread production process.     

A detailed one-dimensional model of combustion of a woody biomass particle

A detailed one-dimensional model for combustion of a single biomass particle is presented. It accounts for particle heating up, pyrolysis, char gasification and oxidation and gas phase reactions within and in the vicinity of the particle. The biomass pyrolysis is assumed to take place through three competing reactions yielding char, light gas and tar. The model is validated using different sets of experiments reported in the literature. Special emphasis is placed on examination of the effects of pyrolysis kinetic constants and gas phase reactions on the combustion process which have not been thoroughly discussed in previous works. It is shown that depending on the process condition and reactor temperature, correct selection of the pyrolysis kinetic data is a necessary step for simulation of biomass particle conversion. The computer program developed for the purpose of this study enables one to get a deeper insight into the biomass particle combustion process.     

Isolation and Characterization of the Putative Photoreceptor for Phototaxis in Amoebae of the Cellular Slime Mold,Dictyostelium discoideum

Amoebae of the cellular slime mold Dictyostelium discoideum (strain AX2) produce a pigment with an absorption spectrum that closely resembles the action spectrum for phototaxis. The protein-pigment complex was isolated and purified by sucrose gradient centrifugation, fast protein liquid chromatography (FPLC) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). It is tightly membrane-bound and the bulk of it is located in the mitochondrial membrane fraction, while a small part is located in the cytoplasmic membrane fraction, as indicated by marker enzyme tests (succinate dehydrogenase for mitochondria and alkaline phosphatase for the cytoplasmic membrane). It is speculated that the pigment bound to the cytoplasmic membrane acts as photoreceptor and that bound to the mitochondria operates as a shading pigment in the light direction perception mechanism of Dictyostelium amoebae.     

Relationship between excision repair and the cytotoxic and mutagenic effect of the ‘anti’ 7,8-diol-9,10-epoxide of benzo[a]pyrene in human cells

The cytotoxic and mutagenic effect of (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti BPDE) in normally excision diploid human cells treated just prior to onset of S was compared with that of cells allowed ～ 16 h for excision repair before onset of S and with that observed in excision-deficient serodema pigmentosum (SP12BE) cells. The cells were synchronized by release from density inhibition of cell replication. DNA synthesis began ～ 22 h after the cells were plated at lower density (i.e., 1.4 × 10⁴ cells/cm²). The frequency of thioguanine-resistant mutants induced in normal cells treated just prior to onset of S was ～ 12- to 16-fold higher than that observed in cells treated in early G₁ or treated in G₀ (confluence) and then plated at lower density. The frequency approximated that expected for XP12BE cells from extrapolation of data obtained at lower doses. The frequency of mutants measured in normal cells treated in exponential growth was also much higher than that in the cells treated in early G₁ or in G₀, No such difference could be seen in XP12BE cells treated in exponential growth or in G₀. In contrast to the mutagenicity data in the normal cells, there was no significant difference in the slope of the survival curve of normal cells treated at various times prior to S phase at low densities. However, normal cells treated even at the onset of S exhibited survival equal to XP12BE cells give a 4- to 5-fold lower dose. The data support the hypothesis that DNA synthesis is the cellular event which converts unexcised DNA lesions into mutations. However, they indicate that S is not the event primarily responsible for translating DNA damage into cell death. Accompanying studies on the rate of excision of anti BPDE adducts from the normal cells during the period priot to S support the conclusions.