The metalloproteinase ADAM‐12 regulates bronchial epithelial cell proliferation and apoptosis

Abstract. Objectives: The ADAMs (a disintegrin and metalloproteinase) enzymes compose a family of membrane‐bound proteins characterized by their multi‐domain structure and ADAM‐12 expression is elevated in human non‐small cell lung cancers. The aim of this study was to investigate the roles played by ADAM‐12 in critical steps of bronchial cell transformation during carcinogenesis. Materials and methods: To assess the role of ADAM‐12 in tumorigenicity, BEAS‐2B cells were transfected with a plasmid encoding human full‐length ADAM‐12 cDNA, and then the effects of ADAM‐12 overexpression on cell behaviour were explored. Treatment of clones with heparin‐binding epidermal growth factor (EGF)‐like growth factor (HB‐EGF) neutralizing antibodies as well as an EGFR inhibitor allowed the dissection of mechanisms regulating cell proliferation and apoptosis. Results: Overexpression of ADAM‐12 in BEAS‐2B cells promoted cell proliferation. ADAM‐12 overexpressing clones produced higher quantities of HB‐EGF in their culture medium which may rely on membrane‐bound HB‐EGF shedding by ADAM‐12. Targeting HB‐EGF activity with a neutralizing antibody abrogated enhanced cell proliferation in the ADAM‐12 overexpressing clones. In sharp contrast, targeting of amphiregulin, EGF or transforming growth factor‐α failed to influence cell proliferation; moreover, ADAM‐12 transfectants were resistant to etoposide‐induced apoptosis and the use of a neutralizing antibody against HB‐EGF activity restored rates of apoptosis to be similar to controls.Conclusions: ADAM‐12 contributes to enhancing HB‐EGF shedding from plasma membranes leading to increased cell proliferation and reduced apoptosis in this bronchial epithelial cell line.     

The Distribution of Coumarins and Furanocoumarins in Citrus Species Closely Matches Citrus Phylogeny and Reflects the Organization of Biosynthetic Pathways

Citrus plants are able to produce defense compounds such as coumarins and furanocoumarins to cope with herbivorous insects and pathogens. In humans, these chemical compounds are strong photosensitizers and can interact with medications, leading to the “grapefruit juice effect”. Removing coumarins and furanocoumarins from food and cosmetics imply additional costs and might alter product quality. Thus, the selection of Citrus cultivars displaying low coumarin and furanocoumarin contents constitutes a valuable alternative. In this study, we performed ultra-performance liquid chromatography coupled with mass spectrometry analyses to determine the contents of these compounds within the peel and the pulp of 61 Citrus species representative of the genetic diversity all Citrus. Generally, Citrus peel contains larger diversity and higher concentrations of coumarin/furanocoumarin than the pulp of the same fruits. According to the chemotypes found in the peel, Citrus species can be separated into 4 groups that correspond to the 4 ancestral taxa (pummelos, mandarins, citrons and papedas) and extended with their respective secondary species descendants. Three of the 4 ancestral taxa (pummelos, citrons and papedas) synthesize high amounts of these compounds, whereas mandarins appear practically devoid of them. Additionally, all ancestral taxa and their hybrids are logically organized according to the coumarin and furanocoumarin pathways described in the literature. This organization allows hypotheses to be drawn regarding the biosynthetic origin of compounds for which the biogenesis remains unresolved. Determining coumarin and furanocoumarin contents is also helpful for hypothesizing the origin of Citrus species for which the phylogeny is presently not firmly established. Finally, this work also notes favorable hybridization schemes that will lead to low coumarin and furanocoumarin contents, and we propose to select mandarins and Ichang papeda as Citrus varieties for use in creating species devoid of these toxic compounds in future breeding programs.     

Are There Circadian Variations of Polymorphonuclear Phagocytosis in Man?

Eleven male subjects were investigated to detect a possible circadian rhythm of the polymorphonuclear phagocytosis. Both cell activity and serum opsonins were studied for numerical detection of granulocytes having ingested at least one particle and for the mean number of ingested particles per cell. No significant temporal differences (ANOVA and cosinor) were found.     

A Three-Tiered Study of Differences in Murine Intrahost Immune Response to Multiple Pneumococcal Strains

We apply a previously developed 4-variable ordinary differential equation model of in-host immune response to pneumococcal pneumonia to study the variability of the immune response of MF1 mice and to explore bacteria-driven differences in disease progression and outcome. In particular, we study the immune response to D39 strain of bacteria missing portions of the pneumolysin protein controlling either the hemolytic activity or complement-activating activity, the response to D39 bacteria deficient in either neuraminidase A or B, and the differences in the response to D39 (serotype 2), 0100993 (serotype 3), and TIGR4 (serotype 4) bacteria. The model accurately reproduces infection kinetics in all cases and provides information about which mechanisms in the immune response have the greatest effect in each case. Results suggest that differences in the ability of bacteria to defeat immune response are primarily due to the ability of the bacteria to elude nonspecific clearance in the lung tissue as well as the ability to create damage to the lung epithelium.     

Chromosomal initiation in Bacillus subtilis may involve two closely linked origins

Summary When the dnaB37 initiation mutant of Bacillus subtilis is returned to a permissive temperature following a period at 45° C, a synchronous round of DNA replication immediately ensues. Using this system we have been able to analyse the first fragments to be replicated while avoiding the use of thymine starvation or inhibitors of DNA replication. Such treatments are necessary to achieve even modest synchrony in germinating spores. Our results showed that the first fragment to be replicated was a 4kb BamHI-SalI restriction fragment, BS6. In contrast, when the analysis was performed out in the presence of novobiocin, an inhibitor of DNA gyrase, replication from BS6 was inhibited and the first fragment to be replicated was BS5, a 5.6 kb fragment located 1.7 kb to the right of BS 6. Replication from both putative origins was suppressed by rifamycin and was dependent upon dnaB. The results are discussed in relation to previous attempts to identify the first replicating fragment in germinating spores. We also discuss the possibility that B. subtilis contains two origins and suggest that either can act as the primary origin under certain conditions, or alternatively that both origins may act in concert in normal bidirectional replication, each site being required for the leading strand in each direction.     

Structural and catalytic characteristics of Escherichia coli adenylate kinase

The adk gene encoding adenylate kinase in Escherichia coli was cloned in pBR322. Adenylate kinase represented about 4% of total proteins in extracts of cells containing the pBR322:adk plasmid. This allowed preparation of more than 90% pure enzyme in a single-step purification procedure. Amino acid analysis, high performance liquid chromatography separation of trypsin digests, sequence analysis of most peptides, and determination of the N-terminal sequence of the whole protein confirmed the primary structure of E. coli adenylate kinase predicted from the nucleotide sequence of the adk gene (Brune, M., Schumann, R., and Wittinghofer, F. (1985) Nucleic Acids Res. 13, 7139-7151). 2-Nitro-5-thiocyanatobenzoic acid reacted with the single cysteine residue of E. coli adenylate kinase. The cyanylated protein was cleaved upon exposure to alkaline pH, yielding two peptides corresponding to residues 1-76 and 77-214, respectively. A mixture of purified peptides tended to reassociate, recovering both catalytic activity and binding properties for adenine nucleotides. E. coli adenylate kinase has a broader specificity for nucleoside monophosphates than does the mammalian enzyme. In addition to 2'-dAMP, other nucleoside monophosphates such as 3'-dAMP, adenine-9-beta-D-arabinofuranoside 5'-monophosphate, and 7-deazaadenosine (tubercidine) 5'-monophosphate were able to replace AMP as substrate.