Microsomal and cytosolic cytochrome P450 mediated benzo(a)pyrene hydroxylation in Pleurotus pulmonarius

Summary Microsomal and soluble fractions of Pleurotus pulmonarius exhibited a reduced carbon monoxide difference spectrum with P450 maxima at 448nm and 450–452nm respectively. Substrate induced Type I spectra were observed on addition of benzo(a)pyrene to both fractions. Benzo(a)pyrene hydroxylation was measured using the aryl hydrocarbon hydroxylase assay and was observed to be P450 dependent as indicated by carbon monoxide inhibition together with the substrate binding characteristics. The activity of the fractions were observed to give K_m of 200mM and 660mM and V_max of 1.25 nmol/min/nmol P450 and 0.57 nmol/min/nmol P450 for the microsomal and cytosolic fractions respectively.     

Lambda transducing phages derived from a FinO- R100::lambda cointegrate plasmid: proteins encoded by the R100 replication/incompatibility region and the antibiotic resistance determinant.

Summary Three transducing phages have been isolated from pEDR20, an R100:: cointegrate plasmid in which the insertion inactivated the R100 finO gene. Physical analysis of the three phages showed that the is inserted at kilobase coordinate 81.3 of R100. All three phages carry different amounts of R100 DNA in the left arm of . Each phage contains IS1b, the mer genes and the region between coordinate 81.3 and 88.6; thus, all contain the genes necessary for R100 replication. One phage, VA73, contains the entire r-determinant of R100 in addition to the above DNA. Five proteins coded by the region between 81.3 and 88.6 were detected. These had subunit molecular weights of 10,400; 12,200; 16,200; 19,600; and 38,300. The first was made constitutively and the other four only from a promoter. Other constitutive proteins were one from the cml fus region with a molecular weight of 22,400 (cml) and two from the str sul region with molecular weights of 31,500 (str?) and 30,100 (sul?). Mercuric ion induced synthesis of at least 10 proteins. Six of these were known from earlier work. The total size of the proteins which appear to derive from the mer genes exceeds by a factor of 1.5, the coding capacity of this region without overlapping genes. Some, or all of these extra proteins may be chromosomal in origin, possibly derepressed in response to mercury gene products.     

A rapid and effecient method for the isolation of proteins from polyacrylamide cells.

A procedure is described for the rapid and efficient electrophoretic elution of protein from polyacrylamide gels which is then collected in a dialysis bag tied to the end of a tube containing the gel slices. To illustrate the method a heterogeneous preparation of alkaline phosphatase was used from which a single homogeneous component was isolated in six hours with a recovery of 86%. The eluted protein is collected in a volume which can easily be kept below 1.5 ml, thus eliminating the need for subsequent concentration. The method has also been used successfully in two other systems in which a human lung tumor-associated antigen and glycogen synthetase from yeast were isolated. Since the method utilizes a standard analytical gel electrophoresis apparatus with no modifications or accessories, it should be immediately applicable for the isolation of many different proteins from polyacrylamide gels.     

Methylated DNA/RNA in Body Fluids as Biomarkers for Lung Cancer

DNA/RNA methylation plays an important role in lung cancer initiation and progression. Liquid biopsy makes use of cells, nucleotides and proteins released from tumor cells into body fluids to help with cancer diagnosis and prognosis. Methylation of circulating tumor DNA (ctDNA) has gained increasing attention as biomarkers for lung cancer. Here we briefly introduce the biological basis and detection method of ctDNA methylation, and review various applications of methylated DNA in body fluids in lung cancer screening, diagnosis, prognosis, monitoring and treatment prediction. We also discuss the emerging role of RNA methylation as biomarkers for cancer.     

Flow cytometric analysis of DNA in cells obtained from deparaffinized formalin-fixed lymphoid tissues

Flow cytometric analysis of DNA content was performed on nuclear suspensions prepared from fresh and from paraffin-embedded, formalin-fixed lymphoid tissues. We confirmed previous reports that it is possible to obtain nuclear suspensions from deparaffinized, formalin-fixed tissues, suitable for DNA analysis by flow cytometry. We observed a tendency for a larger coefficient of variation (CV) of the DNA measurements in the fixed tissues than in the unfixed material causing abnormalities in 2 of 19 lymphomas to become undetectable. Furthermore, samples from different paraffin blocks of a single tumor with an extra G1 (hyperdiploid) peak showed marked differences in the CV of the hyperdiploid peak while the CV of the diploid peak was similar in all samples. In both benign and malignant lymphoid tissues, the S-phase fraction was higher in paraffin-embedded tissues than in unfixed cells. This difference could be attributed to 4', 6'-diamidino-2-phenylindole dihydrochloride (DAPI), a DNA-binding dye commonly used in this technique. Nevertheless, intermediate and high grade lymphomas from paraffin-embedded tissues generally showed a greater S-fraction than low grade lymphomas, a similar observation as with unfixed tissues. Therefore, DNA content analysis of nuclei extracted from paraffin sections may be inadequate to resolve slight aneuploidy, but the measurement of S-fraction size may remain diagnostically or prognostically valuable. Large retrospective studies will be necessary to determine the clinical impact of this technique in the analysis of lymphomas.     

DISTRIBUTION OF INTERTIDAL DIATOMS ASSOCIATED WITH SEDIMENTS IN YAQUINA ESTUARY,OREGON1,2

Sediment samples and environmental data were collected from November 1973 to August 1974 to analyze the distribution of sediment-associated diatom assemblages relative to vertical, horizontal, and seasonal gradients in Yaquina Estuary, Oregon. The distribution of diatoms was regulated primarily by mean salinity and characteristics of the sediment, i.e., mean sediment size and percentage of organic carbon. Prominent taxa in the river above Yaquina Bay exhibited overlapping distributions along the salinity gradient to a location in brackish water where the mean salinity was approximately 5%_o. At this salinity, a relatively sharp discontinuity in the diatom flora existed which appeared to represent the biochemical and biophysical mechanisms involved in the osmotic regulation of fresh- and brackish-water diatoms. Relatively large disparities m the structure of diatom assemblages were found within relatively small areas of Yaquina Bay. These differences were attributed to properties of the sediment. Differences in diatom assemblages relative to variations in light intensity, water temperature and exposure to intertidal emergence were not apparent from the analysis of field data.     

A scaleable manufacturing process for pro-EP-B2, a cysteine protease from barley indicated for celiac sprue

Celiac Sprue is an inflammatory disease of the small intestine triggered by ingestion of dietary gluten, a family of glutamine and proline rich proteins found in common foodgrains such as wheat, rye, and barley. One potential therapy for this lifelong disease anticipates using an oral protease to detoxify gluten in vivo. Recent studies have shown that EP-B2 (endoprotease B, isoform 2) from barley is a promising example of such a glutenase, thus warranting its large-scale production for animal safety and human clinical studies. Here we describe a scaleable fermentation, refolding and purification process for the production of gram to kilogram quantities of pro-EP-B2 (zymogen form of EP-B2) in a lyophilized form. A fed-batch E. coli fermentation system was developed that yields 0.3-0.5 g purified recombinant protein per liter culture volume. Intracellular degradation of pro-EP-B2 during the fermentation was overcome by manipulating the fermentation temperature and duration of protein expression. A simple refolding protocol was developed using a fast dilution method to refold the enzyme at concentrations greater than 0.5 mg/mL. Kinetic analysis showed that pro-EP-B2 refolding is a first-order reaction with an estimated rate constant of 0.15 h(-1). A lyophilization procedure was developed that yielded protein with 85% recoverable activity after 7 weeks of storage at room temperature. The process was successfully scaled up to 100 L with comparable purity and recovery.     

NetLogoR: a package to build and run spatially explicit agent‐based models in R

NetLogoR is an R package to build and run spatially explicit agent‐based models (SE‐ABMs) using the R language. SE‐ABMs are models that simulate the fate of entities at the individual level within a spatial context and where patterns emerge at the population level. NetLogoR follows the same framework as the NetLogo software (Wilensky 1999). Rather than a call function to use the NetLogo software, NetLogoR is a translation into the R language of the structure and functions of NetLogo. Models built with NetLogoR are written in R language and are run on the R platform; no other software or language has to be involved. NetLogoR provides new R classes to define model agent objects and functions to implement spatially explicit agent‐based models in the R environment. Users of this package benefit from the fast and easy coding provided by the highly developed NetLogo framework, coupled with the versatility, power and massive resources of the R language.     

Effects of noncovalent and covalent FAD binding on the redox and catalytic properties of p-cresol methylhydroxylase

Each flavoprotein subunit (alpha or PchF) of the alpha(2)beta(2) flavocytochrome p-cresol methylhydroxylase (PCMH) from Pseudomonas putida contains FAD covalently attached to Tyr384. PCMH oxidizes p-cresol to 4-hydroxybenzyl alcohol, which is oxidized subsequently by PCMH to 4-hydroxybenzaldehyde. The Y384F mutant form of PchF (apo-PchF[Y384F]) displayed stoichiometric noncovalent FAD binding. PchF[Y384F]FAD associated with the cytochrome subunit (beta or PchC) (producing PCMH[Y384F]), although not as avidly as with wild-type PchF containing covalently bound FAD (PchF(C)). Dramatic increases in the two-electron E(m,7) (NHE) values for FAD were observed when it bound noncovalently to either apo-PchF or apo-PchF[Y384F], and the two-electron E(m,7) value for FAD was increased further by about 75 mV upon covalent binding to PchF, i.e., PchF(C). The E(m,7) values increased by approximately 20 and 45 mV, respectively, when PchF(C) and PchF[Y384F]FAD associated with PchC. The two-electron E(m,7) for covalently bound FAD in PCMH is 84 mV, the highest measured for a flavoprotein. The values for the one-electron redox potentials (E(m,7), NHE) for FAD were measured also for various forms of PchF. Under anaerobiosis, the reduction of PchF[Y384F]FAD by substrates was similar to that observed previously for PchF containing noncovalently bound FAD. Stopped-flow kinetic studies indicated a rapid substrate reduction of the FAD and heme in PCMH[Y384F] which produced PchF[Y384F]FAD(rad) x PchC, the mutant enzyme containing the flavin radical and reduced heme. These experiments also revealed a slow reduction of unassociated PchC(ox) by PchF[Y384F]FAD(rad) x PchC. Steady-state kinetic studies of the reaction of PCMH[Y384F] with p-cresol indicated that the K(m) for this substrate was unchanged relative to that of PCMH, but that the k(cat) was diminished by an order of magnitude. The data indicate that the covalent attachment of FAD to PchF assists catalysis by raising the E(m,7) of the flavin. Contributions to this effect likely result from conformational changes.     

The G protein beta subunit is a determinant in the coupling of Gs to the beta 1-adrenergic and A2a adenosine receptors

The signaling specificity of five purified G protein betagamma dimers, beta(1)gamma(2), beta(2)gamma(2), beta(3)gamma(2), beta(4)gamma(2), and beta(5)gamma(2), was explored by reconstituting them with G(s) alpha and receptors or effectors in the adenylyl cyclase cascade. The ability of the five betagamma dimers to support receptor-alpha-betagamma interactions was examined using membranes expressing the beta(1)-adrenergic or A2a adenosine receptors. These receptors discriminated among the defined heterotrimers based solely on the beta isoform. The beta(4)gamma(2) dimer demonstrated the highest coupling efficiency to either receptor. The beta(5)gamma(2) dimer coupled poorly to each receptor, with EC(50) values 40-200-fold higher than those observed with beta(4)gamma(2). Strikingly, whereas the EC(50) of the beta(1)gamma(2) dimer at the beta(1)-adrenergic receptor was similar to beta(4)gamma(2), its EC(50) was 20-fold higher at the A2a adenosine receptor. Inhibition of adenylyl cyclase type I (AC1) and stimulation of type II (AC2) by the betagamma dimers were measured. betagamma dimers containing Gbeta(1-4) were able to stimulate AC2 similarly, and beta(5)gamma(2) was much less potent. beta(1)gamma(2), beta(2)gamma(2), and beta(4)gamma(2) inhibited AC1 equally; beta(3)gamma(2) was 10-fold less effective, and beta(5)gamma(2) had no effect. These data argue that the beta isoform in the betagamma dimer can determine the specificity of signaling at both receptors and effectors.