Phenotypic expression in Escherichia coli and nucleotide sequence of two Chinese hamster lung cell cDNAs encoding different dihydrofolate reductases.

Nucleotide sequence analysis of two cDNA clones, one shown to direct the synthesis in Escherichia coli of the pI 6.7 form of the 20,000-molecular-weight class of Chinese hamster lung cell dihydrofolate reductase, and the other shown to direct the synthesis of the pI 6.5 form of the 21,000-molecular-weight class of the enzyme, has revealed the following: (i) the differences in physical and enzymatic properties displayed by these two proteins are due to two variations in their respective amino acid sequences with the conversion of Leu to Phe at position 22 probably responsible for the differential sensitivity of these two enzymes to methotrexate and methasquin; (ii) the multiple mRNAs responsible for the synthesis of each of these proteins differ in size due, at least in part, to a length heterogeneity at their 3' ends; (iii) these two proteins are encoded by different genes; and (iv) the sequence AAATATA appears to be a major polyadenylation signal in one Chinese hamster lung cell dihydrofolate reductase gene and a minor signal in another.     

The "Alzheimer's disease signature": potential perspectives for novel biomarkers

Alzheimer's disease is a progressive and neurodegenerative disorder which involves multiple molecular mechanisms. Intense research during the last years has accumulated a large body of data and the search for sensitive and specific biomarkers has undergone a rapid evolution. However, the diagnosis remains problematic and the current tests do not accurately detect the process leading to neurodegeneration. Biomarkers discovery and validation are considered the key aspects to support clinical diagnosis and provide discriminatory power between different stages of the disorder. A considerable challenge is to integrate different types of data from new potent approach to reach a common interpretation and replicate the findings across studies and populations. Furthermore, long-term clinical follow-up and combined analysis of several biomarkers are among the most promising perspectives to diagnose and manage the disease. The present review will focus on the recent published data providing an updated overview of the main achievements in the genetic and biochemical research of the Alzheimer's disease. We also discuss the latest and most significant results that will help to define a specific disease signature whose validity might be clinically relevant for future AD diagnosis.     

A High-Content Small Molecule Screen Identifies Sensitivity of Glioblastoma Stem Cells to Inhibition of Polo-Like Kinase 1

Glioblastoma multiforme (GBM) is the most common primary brain cancer in adults and there are few effective treatments. GBMs contain cells with molecular and cellular characteristics of neural stem cells that drive tumour growth. Here we compare responses of human glioblastoma-derived neural stem (GNS) cells and genetically normal neural stem (NS) cells to a panel of 160 small molecule kinase inhibitors. We used live-cell imaging and high content image analysis tools and identified JNJ-10198409 (J101) as an agent that induces mitotic arrest at prometaphase in GNS cells but not NS cells. Antibody microarrays and kinase profiling suggested that J101 responses are triggered by suppression of the active phosphorylated form of polo-like kinase 1 (Plk1) (phospho T210), with resultant spindle defects and arrest at prometaphase. We found that potent and specific Plk1 inhibitors already in clinical development (BI 2536, BI 6727 and GSK 461364) phenocopied J101 and were selective against GNS cells. Using a porcine brain endothelial cell blood-brain barrier model we also observed that these compounds exhibited greater blood-brain barrier permeability in vitro than J101. Our analysis of mouse mutant NS cells (INK4a/ARF^−/−, or p53^−/−), as well as the acute genetic deletion of p53 from a conditional p53 floxed NS cell line, suggests that the sensitivity of GNS cells to BI 2536 or J101 may be explained by the lack of a p53-mediated compensatory pathway. Together these data indicate that GBM stem cells are acutely susceptible to proliferative disruption by Plk1 inhibitors and that such agents may have immediate therapeutic value.     

Species of the genus Quercus in Italy: Characterization by means of leaf surface observation

Abstract

We propose a study of the main species belonging to the genus Quercus in Italy, characterized and identified by means of leaf surface observation, with special attention devoted to waxes, trichomes and stomata. Comparing our results with the classification proposed by SCHWARZ (1984), we find that species belonging to Schwarz's subgenus Quercus are recognizable because their waxes are structured in vertical scales; the two other subgenera (Sclerophyllodrys and Cerris) present smooth wax structures, their distinctive feature being the shape of the stomatal rima, which is roundish in Sclerophyllodrys and elliptical in Cerris. The study characterizes Quercus pubescens Willd. and Quercus petraea Liebl. by analyzing some morphometric traits; but the authors feel that further research is needed on these critical taxonomic entities. Lastly, the study examines forms of was degeneration correlated to the phenomenon known as oak decline.     

Influence of ova within rat oviducts on spontaneous motility and on prostaglandin production

The present study was performed in order to explore the influence of ova present within rat oviducts on: a) tubal spontaneous motility and b) oviduct prostaglandin production. It was found that the isometric developed tension (IDT) of tubes isolated from proestrous rats (preovulatory oviducts) was significantly higher (P less than 0.01) than the IDT of tubes from rats at estrus and at metestrus (postovulatory oviducts). After flushing the oviducts with KRB solution (i.e., after removing existing ova) the IDT of the oviducts obtained from estrous rats increased significantly (P less than 0.01), whereas the IDT of tubes isolated from proestrous rats (i.e., preparations without ova) was not modified. On the other hand, isolated tubes containing their corresponding ova released into the suspending solution significantly more PGE1 than PGE2 or PGF2 alpha (P less than 0.005). It was particularly interesting to find that after flushing the oviducts, tissue production of PGE1, PGE2 and PGF2 alpha was similar. Finally, when dose response curves for PGE1 and for PGE2 on the spontaneous contractions of oviducts isolated from rats at proestrus, estrus and metestrus were constructed, both PGs evoked an inhibitory inotropic action. The ED50 for PGE1 in tubes from estrous rats was significantly smaller (P less than 0.01) than that for metestrous animals but significantly greater (P less than 0.01) than that observed in oviducts from proestrous rats. The ED50 for PGE2 did not change in the different tested periods of the sex cycle. Results reported herein suggest the possibility that the ova present within rat oviducts, may influence their own transport along the tubes by modifying the amount of prostaglandins produced by the oviducts or via their own prostaglandin synthesis.     

Functional and spectral characterization of the respiratory chain of Chloroflexus aurantiacus grown in the dark under oxygen-saturated conditions

In this paper we attempt a functional and spectral characterization of the membrane-bound cytochromes involved in respiratory electron transport by membranes from cells of Chloroflexus aurantiacus grown in the dark under oxygen saturated conditions. We conclude that the NADH-dependent respiration is carried out by a branched respiratory chain leading to two oxidases which differ in sensitivity to CN^- and CO. The two routes also show a different sensitivity to the ubiquinone analogue, HQNO, the pathway through the cytochrome c oxidase being fully blocked by 5 M HQNO, whereas the alternative one is insensitive to this inhibitor. The cytochrome c oxidase containing branch is composed by at least two c-type haems with E _m 7.0 of +130 and +270 mV ( bands at 550/553 nm and 549 nm, respectively), plus a b-type cytochrome with E _m 7.0 of +50 mV ( band at 561 nm). From this, and previous work, we conclude that respiratory and photosynthetic electron transport components are assembled together and function on a single undifferentiated plasma membrane.Abbreviations HQNO heptylhydroxy-quinoline-N-oxide - UHDBT undecyl-hydroxydioxobenthiazole - Q/b-c ubiquinol/cytochrome c oxidoreductase complex - BChl bacteriochlorophyll     

The electron transport system of Alcaligenes eutrophus H16

In a previous work (Kömen et al. 1991) it has been concluded that membrane fragments isolated from autotrophically grown Alcaligenes eutrophus H16 contain several iron-sulphur centres along with haems of a-, b-, c-, and d-type. These redox components have been proposed to be part of a branched respiratory chain leading to multiple membrane bound oxidases. Here, some of the respiratory activities catalyzed by membrane fragments from wild type cells of A. eutrophus (H16) and, for comparison, Paracoccus denitrificans, have been investigated through the use of electron transport inhibitors. Cyanide (CN^-) titration curves indicated that in A. eutrophus H16 oxidation of succinate and H₂ preferentially proceeds via the cytochrome c oxidase(s) branch (I ₅₀=2 · 10^-5 M) whereas the NADH dependent respiration started being inhibited at higher CN^- concentrations (I ₅₀=5 · 10^-4 M). In membranes isolated from both, cells harvested at late growth-phase (OD 12) and from a mutant deficient in cytochrome c oxidase activity (A. eutrophus RK1), respiration was insensitive to low CN^- concentrations (< 10^-4 M), and it was sustained by the high catalytic activities of two quinol oxidases. These alternative oxidases of b- (formally o-) and d-type showed different sensitivities to KCN (I ₅₀=10^-3 M and 10^-2 M, respectively). Interestingly, the cytochrome c oxidase(s) dependent respiration of H16 membranes was insensitive to antimycin A but largely inhibited by myxothiazol (10^-6 M). This, and previous work (Kömen et al. 1991), suggest that although the respiratory chain of A. eutrophus is endowed with a putative bc ₁ complex, its biochemical nature and role in respiration of this organism are apparently different from those of P. denitrificans. The peculiarity of the respiratory chain of A. eutrophus is confirmed by the rotenone insensitivity of the NADH oxidation in both protoplasts and membrane fragments from wild type and soluble hydrogenase deficient cells (HF14 and HF160). A tentative model of the respiratory chain of autotrophically grown A. eutrophus is presented.     

Effect of oxygen limitation on the formation of the electron transport system of the phytopathogenic fluorescent bacteriumPseudomonas cichorii

The composition of the membrane-bound electron transport system of the phytopathogenic bacteriumPseudomonas cichorii underwent modification in response to oxygen supply. Growth adaptation to low oxygen concentrations was characterized by repression of cytochromes involved in ubiquinol-cyt.c oxidoreductase and cyt.c oxidase activities. By contrast, cyto.o, i.e., the alternative cyanide-insensitive oxidase ofP. cichorii, was unaffected by low oxygen tension. Noa-type cytochromes could be detected at any stage of growth.