A plant type 2 metallothionein (MT) from cork tissue responds to oxidative stress

Expression of plant metallothionein genes has been reported in a variety of senescing tissues, such as leaves and stems, ripening fruits, and wounded tissues, and has been proposed to function in both metal chaperoning and scavenging of reactive oxygen species. In this work, it is shown that MT is also associated with suberization, after identifying a gene actively transcribed in Quercus suber cork cells as a novel MT. This cDNA, isolated from a phellem cDNA library, encodes a MT that belongs to type 2 plant MTs (QsMT). Expression of the QsMT cDNA in E. coli grown in media supplemented with Zn, Cd, or Cu has yielded recombinant QsMT. Characterization of the respective metal aggregates agrees well with a copper-related biological role, consistent with the capacity of QsMT to restore copper tolerance to a MT-deficient, copper-sensitive yeast mutant. Furthermore, in situ hybridization results demonstrate that RNA expression of QsMT is mainly observed under conditions related to oxidative stress, either endogenous, as found in cork or in actively proliferating tissues, or exogenous, for example, in response to H(2)O(2) or paraquat treatments. The putative role of QsMT in oxidative stress, both as a free radical scavenger via its sulphydryl groups or as a copper chelator is discussed.     

Deformation of helix C in the low temperature L-intermediate of bacteriorhodopsin

X-ray and electron diffraction studies of specific reaction intermediates, or reaction intermediate analogues, have produced a consistent picture of the structural mechanism of light-driven proton pumping by bacteriorhodopsin. Of central importance within this picture is the structure of the L-intermediate, which follows the retinal all-trans to 13-cis photoisomerization step of the K-intermediate and sets the stage for the primary proton transfer event from the positively charged Schiff base to the negatively charged Asp-85. Here we report the structural changes in bacteriorhodopsin following red light illumination at 150 K. Single crystal microspectrophotometry showed that only the L-intermediate is populated in three-dimensional crystals under these conditions. The experimental difference Fourier electron density map and refined crystallographic structure were consistent with those previously presented (Royant, A., Edman, K., Ursby, T., Pebay-Peyroula, E., Landau, E. M., and Neutze, R. (2000) Nature 406, 645-648; Royant, A., Edman, K., Ursby, T., Pebay-Peyroula, E., Landau, E. M., and Neutze, R. (2001) Photochem. Photobiol. 74, 794-804). Based on the refined crystallographic structures, molecular dynamic simulations were used to examine the influence of the conformational change of the protein that is associated with the K-to-L transition on retinal dynamics. Implications regarding the structural mechanism for proton pumping by bacteriorhodopsin are discussed.     

Effect of substratum surface chemistry and surface energy on attachment of marine bacteria and algal spores

Two series of self-assembled monolayers (SAMs) of omega-substituted alkanethiolates on gold were used to systematically examine the effects of varying substratum surface chemistry and energy on the attachment of two model organisms of interest to the study of marine biofouling, the bacterium Cobetia marina (formerly Halomonas marina) and zoospores of the alga Ulva linza (formerly Enteromorpha linza). SAMs were formed on gold-coated glass slides from solutions containing mixtures of methyl- and carboxylic acid-terminated alkanethiols and mixtures of methyl- and hydroxyl-terminated alkanethiols. C. marina attached in increasing numbers to SAMs with decreasing advancing water contact angles (theta(AW)), in accordance with equation-of-state models of colloidal attachment. Previous studies of Ulva zoospore attachment to a series of mixed methyl- and hydroxyl-terminated SAMs showed a similar correlation between substratum theta(AW) and zoospore attachment. When the hydrophilic component of the SAMs was changed to carboxylate, however, the profile of attachment of Ulva was significantly different, suggesting that a more complex model of interfacial energetics is required.     

Development and senescence of <Emphasis Type="Italic">Grevillea</Emphasis> ‘Sylvia’ inflorescences,flowers and flower parts

To characterise the physiology of development and senescence for Grevillea Sylvia floral organs, respiration, ethylene production and ACC concentrations in harvested flowers and flower parts were measured. The respiration rate of harvested inflorescences decreased over time during senescence. In contrast, both ethylene production and ACC concentration increased. Individual flowers, either detached from cut inflorescences held in vases at 20 °C or detached from in planta inflorescences at various stages of development, had similar patterns of change in ACC concentration and rates of respiration and ethylene production as whole inflorescences. The correlation between ACC concentration and ethylene production by individual flowers detached from cut inflorescences held in vases was poor (r² = 0.03). The isolated complete gynoecium (inclusive of the pedicel) produced increasing amounts of ethylene during development. Further sub-division of flower parts and measurement of their ethylene production at various stages of development revealed that the distal part of the gynoecium (inclusive of the stigma) had the highest rate of ethylene production. In turn, anthers had higher rates of ethylene production and also higher ACC concentrations than the proximal part of the gynoecium (inclusive of the ovary). Rates of ethylene production and ACC concentrations for tepal abscission zone tissue and adjacent central tepal zone tissue were similar. ACC concentration in pollen was similar to that in senescing perianth tissue. Overall, respiration, ethylene and ACC content measurements suggest that senescence of G. Sylvia is non-climacteric in character. Nonetheless, the phytohormone ethylene is produced and evidently mediates normal flower development and non-climacteric senescence processes.     

Myogenesis during holothurian intestinal regeneration

Echinoderms are well known as being able to regenerate body parts and thus provide excellent models for studying regenerative processes in adult organisms. We are interested in intestinal regeneration in the sea cucumber, Holothuria glaberrima, and focus here on the regeneration of intestinal muscle components. We have used immunohistochemical techniques to describe the formation of the intestinal muscle layers. Myoblasts are first observed within the regenerating structure, adjacent to the coelomic epithelia. Within a few days, these cells acquire muscle markers and form a single cell layer that underlies the epithelia. Animals injected with BrdU at various regeneration stages have been subsequently analyzed for the presence of muscle differentiation markers. BrdU-labeled muscle nuclei are observed in myocytes of 3-week regenerates, showing that these cells originate from proliferating precursors. The peak in muscle precursor proliferation appears to occur during the second week of regeneration. Therefore, new muscle cells in the regenerating intestine originate from precursors that have undergone cell division. Our results suggest that the precursor cells arise from the coelomic epithelia. We also provide a comparative view of muscle regeneration in an echinoderm, a topic of interest in view of the many recent studies of muscle regeneration in vertebrate species. This work was supported by NSF (IBN-0110692) and NIH-MBRS (S06GM08102). We also acknowledge partial support from NIH-RCMI (RRO-3641-01) and the University of Puerto Rico     

Colon cancer specific nuclear matrix protein alterations in human colonic adenomatous polyps

Most colon cancers arise within preexisting adenomatous polyps or adenomas. The slow evolution from the non-invasive premalignant lesion, the adenomatous polyp, to invasive cancer supports a strategy of early detection. Recently, we identified unique nuclear matrix proteins (NMPs) specific for colon cancer (CC2, CC3, CC4, CC5). Most of the NMPs identified are common to all cell types, but several identified NMPs are tissue and cell line specific. The objective of this study is to describe and characterize the NMP profile of premalignant adenomatous colon polyps. Specifically when in the adenoma-carcinoma sequence four specific colon cancer NMPs, previously described, appear. Using two-dimensional (2-D) gel analysis 20 colon polyps (one juvenile polyp, six tubular adenoma (TA), seven tubulovillous adenoma (TVA), six TVA with focal high-grade dysplasia (HGD), were analyzed for the presence of four (CC2, CC3, CC4, CC5) specific NMPs. CC2 was not seen in any of the premalignant polyps. CC5 was present in only two premalignant TVA with HGD and in one TA. CC3 and CC4 were present in most adenomas. None of the NMPs were seen in the juvenile polyp, which is not considered to be a precursor of colon cancer. CC2 and CC5 are NMPs expressed at the junction of an advanced adenoma and invasive colorectal cancer. CC3 and CC4 are expressed earlier in the evolution of adenomatous polyps. Development of an assay to these proteins may serve as a new method for early detection of colorectal cancer.     

Fatty acid transfer from intestinal fatty acid binding protein to membranes: electrostatic and hydrophobic interactions

Intestinal fatty acid binding protein (IFABP) is thought to participate in the intracellular transport of fatty acids (FAs). Fatty acid transfer from IFABP to phospholipid membranes is proposed to occur during protein-membrane collisional interactions. In this study, we analyzed the participation of electrostatic and hydrophobic interactions in the collisional mechanism of FA transfer from IFABP to membranes. Using a fluorescence resonance energy transfer assay, we examined the rate and mechanism of transfer of anthroyloxy-fatty acid analogs a) from IFABP to phospholipid membranes of different composition; b) from chemically modified IFABPs, in which the acetylation of surface lysine residues eliminated positive surface charges; and c) as a function of ionic strength. The results show clearly that negative charges on the membrane surface and positive charges on the protein surface are important for establishing the "collisional complex", during which fatty acid transfer occurs. In addition, changes in the hydrophobicity of the protein surface, as well as the hydrophobic volume of the acceptor vesicles, also influenced the rate of fatty acid transfer. Thus, ionic interactions between IFABP and membranes appear to play a primary role in the process of fatty acid transfer to membranes, and hydrophobic interactions can also modulate the rates of ligand transfer.     

FAPP2 is involved in the transport of apical cargo in polarized MDCK cells

Phosphatidylinositol-4-phosphate (PI(4)P) is the main phosphoinositide in the Golgi complex and has been reported to play a pleiotropic role in transport of cargo from the trans-Golgi network to the plasma membrane (PM) in polarized Madin-Darby canine kidney (MDCK) cells. Overexpression of the chimeric fluorescent protein encoding the pleckstrin homology domain, which is specific for PI(4)P, inhibited both apical and basolateral transport pathways. The transport of apical cargo from the Golgi was shown to be specifically decreased by adenovirus-mediated RNA interference directed against PI(4)P adaptor protein (FAPP) 2. FAPP1 depletion had no effect on transport. On the other hand, FAPP2 was not involved in the Golgi-to-PM transport of cargo that was targeted to the basolateral membrane domain. Thus, we conclude that FAPP2 plays a specific role in apical transport in MDCK cells.     

Genetic analysis of the species cytoplasm specific gene (scs d) derived from durum wheat

The action of species cytoplasm specific (scs) gene(s) can be observed when a durum (Triticum turgidum L.) nucleus is placed in the Aegilops longissimum S. & M. cytoplasm. This alloplasmic combination, (lo) durum, results in nonviable progeny. A scs gene derived from T. timopheevii Zhuk. (scs(ti)) produced compatibility with the (lo) cytoplasm. The resulting hemizygous (lo) scs(ti)- durum line was male sterile and when crossed to normal durum produced a 1:1 ratio of plump, viable (PV) seeds with scs(ti) and shriveled inviable (SIV) seeds without scs(ti). In a systematic characterization of durum lines an unusual line was identified that when crossed to (lo) scs(ti)- produced all PV seeds. When planted these PV seeds segregated at a 1:1 ratio of normal vigor plants (NVPs) and low vigor plants (LVPs). The LVP senescence before full maturity. The NVPs were male sterile and when crossed to common durum lines resulted in all plump seeds that again segregated at a 1:1 ratio of NVPs to LVPs. The crosses of these NVPs to common durum lines resulted in a 1:1 ratio of PV to SIV seeds. This study was extended to 317 individuals segregating for scs(ti) and the new locus, derived from durum wheat (scs(d)), establishing the allelic relationship of these two genes.