Relation of changes in amount and type of dietary fat to fecapentaenes in premenopausal women

Correlation studies suggest that fecal mutagenicity is increased in groups eating high-fat diets, the same groups who are often found to have high colorectal cancer incidence and mortality. The fecapentaenes are the best characterized class of fecal mutagens, but the relationship of dietary fat intake to the excretion of these potent genotoxins is unknown. We studied the effect of changes in amount and type of dietary fat on fecapentaene levels in 31 premenopausal women 20-40 years of age who participated in a controlled feeding study. After a pre-diet free-living period lasting 1 menstrual cycle, women were placed on a high-fat (40% energy from fat) diet for 4 menstrual cycles and then switched to a low-fat (20% energy from fat) diet for an additional 4 menstrual cycles. One-half the subjects were maintained throughout the study at a ratio of polyunsaturated-to-saturated fatty acids (P/S ratio) of 1.0, the other half at 0.3; body weight was constant. All meals during the controlled diet periods were prepared at the Human Study Facility of the Beltsville Human Nutrition Research Center. Fecapentaene and fecapentaene precursor levels were measured in acetone extracts from 3-day pooled stool samples collected during the study. No differences in fecapentaene or precursor levels were observed between the high- and low-fat diets at either P/S ratio. Fecapentaene and precursor levels were higher while on controlled diets than during the pre-diet free-living period, and levels declined again in the post-diet free-living period. We conclude that dietary fat has no significant effect on fecapentaene or precursor levels in acetone extracts of stool in premenopausal women. The effect of other dietary or non-dietary factors on fecapentaenes remains unknown.     

Effects of climate change and horticultural use on the spread of naturalized alien garden plants in Europe

Climate warming is supposed to enlarge the area climatically suitable to the naturalization of alien garden plants in temperate regions. However, the effects of a changing climate on the spread of naturalized ornamentals have not been evaluated by spatially and temporarily explicit range modelling at larger scales so far. Here, we assess how climate change and the frequency of cultivation interactively determine the spread of 15 ornamental plants over the 21st century in Europe. We coupled species distribution modelling with simulations of demography and dispersal to predict range dynamics of these species in annual steps across a 250 × 250 m raster of the study area. Models were run under four scenarios of climate warming and six levels of cultivation intensity. Cultivation frequency was implemented as size of the area used for planting a species. Although the area climatically suitable to the 15 species increases, on average, the area predicted to be occupied by them in 2090 shrinks under two of the three climate change scenarios. This contradiction obviously arises from dispersal limitations that were pronounced although we assumed that cultivation is spatially adapting to the changing climate. Cultivation frequency had a much stronger effect on species spread than climate change, and this effect was non‐linear. The area occupied increased sharply from low to moderate levels of cultivation intensity, but levelled off afterwards. Our simulations suggest that climate warming will not necessarily foster the spread of alien garden plants in Europe over the next decades. However, climatically suitable areas do increase and hence an invasion debt is likely accumulating. Restricting cultivation of species can be effective in preventing species spread, irrespective of how the climate develops. However, for being successful, they depend on high levels of compliance to keep propagule pressure at a low level.     

Phosphatidyl inositol 3-kinase-like serine/threonine protein kinases (PIKKs) are required for DNA damage-induced phosphorylation of the 32 kDa subunit of replication protein A at threonine 21

Replication protein A (RPA) is a single-stranded DNA (ssDNA) binding protein involved in various processes, including nucleotide excision repair and DNA replication. The 32 kDa subunit of RPA (RPA32) is phosphorylated in response to various DNA-damaging agents, and two protein kinases, ataxia-telangiectasia mutated (ATM) and the DNA-dependent protein kinase (DNA-PK) have been implicated in DNA damage-induced phosphorylation of RPA32. However, the relative roles of ATM and DNA-PK in the site-specific DNA damage-induced phosphorylation of RPA32 have not been reported. Here we generated a phosphospecific antibody that recognizes Thr²¹-phosphorylated RPA32. We show that both DNA-PK and ATM phosphorylate RPA32 on Thr²¹ in vitro. Ionizing radiation (IR)-induced phosphorylation of RPA32 on Thr²¹ was defective in ATM-deficient cells, while camptothecin (CPT)-induced phosphorylation of RPA32 on Thr²¹ was defective in cells lacking functional DNA-PK. Neither ATM nor DNA-PK was required for etoposide (ETOP)-induced RPA32 Thr²¹ phosphorylation. However, two inhibitors of the ATM- and Rad3-related (ATR) protein kinase activity prevented ETOP-induced Thr²¹ phosphorylation. Inhibition of DNA replication prevented both the IR- and CPT-induced phosphorylation of Thr²¹, whereas ETOP-induced Thr²¹ phosphorylation did not require active DNA replication. Thus, the regulation of RPA32 Thr²¹ phosphorylation by multiple DNA damage response protein kinases suggests that Thr²¹ phosphorylation of RPA32 is a crucial step within the DNA damage response.     

Biochemical and ultrastructural characterization of the 1,4-dihydropyridine receptor from rabbit skeletal muscle. Evidence for a 52,000 Da subunit

The 1,4-dihydropyridine receptor purified from rabbit skeletal muscle contains four polypeptide components of 175,000 Da (nonreduced)/150,000 Da (reduced), 170,000, 52,000, and 32,000 Da (Leung, A. T., Imagawa, T., and Campbell, K. P. (1987) J. Biol. Chem. 262, 7943-7946). A monoclonal antibody specific to the 52,000-Da polypeptide component of the dihydropyridine receptor has been produced and used in immunoprecipitation and immunoblotting experiments to demonstrate that the 52,000-Da polypeptide is an integral subunit of the purified dihydropyridine receptor. Peptide mapping experiments with 32P-labeled dihydropyridine receptor have also demonstrated that the 52,000-Da polypeptide is distinct from and not a proteolytic fragment of the 170,000-Da subunit. Densitometric scanning of Coomassie Blue-stained sodium dodecyl sulfate-polyacrylamide gels of the purified dihydropyridine receptor has demonstrated that the 52,000-Da polypeptide exists in a 1:1 stoichiometric ratio with the 170,000-, 175,000/150,000-, and 32,000-Da subunits of the dihydropyridine receptor. Electron microscopy of the freeze-dried, rotary-shadowed dihydropyridine receptor has shown that the preparation contains a homogeneous population of 16 x 22-nm ovoidal particles large enough to contain all four polypeptides of the dihydropyridine receptor. The particles have two distinct components of similar size which may represent the location in the molecule of the two larger subunits.     

Identification and Purification of a Derepressible Alkaline Phosphatase from Anacystis nidulans R2

We have examined the increase in alkaline phosphatase activity in the cyanobacterium Anacystis nidulans R2 upon phosphate deprivation. Much of the activity is released into the medium when A. nidulans is osmotically shocked, indicating that the enzyme is located either in the periplasmic space or is loosely bound to the cell wall. The polypeptide associated with phosphatase activity has been identified as a single species of M_r 160,000. Several lines of evidence demonstrate that this polypeptide is responsible for alkaline phosphatase activity: (a) It is absent when cells are grown in the presence of phosphate and specifically accumulates during phosphate deprivation. (b) It is the major periplasmic polypeptide extracted by osmotic shock. (c) It represents over 90% of the protein in a fraction of periplasmic polypeptides enriched for phosphatase activity. (d) Antibodies raised against the purified species of M_r 160,000 inhibit phosphatase activity by approximately 70%.     

Pollen morphology of the Pavetteae (Rubiaceae,Ixoroideae) and its taxonomic significance

Pollen grains of the tribe Pavetteae (Rubiaceae, subfamily Ixoroideae) are examined using LM and SEM. Grains are 3‐ or 4‐colporate and (semi‐) tectate (in one Versteegia species atectate). Sexine patterns vary between perforate, microreticulate, reticulate, rugulate and striato‐reticulate. Supratectal elements are sometimes present. The variation in pollen morphology in the Pavetteae allows to recognize seven pollen types, the distribution of which is useful to evaluate generic delimitations and relationships within the tribe. Pollen characters corroborate the close relationships between the genera Coleactina, Dictyandra and Leptactina and between Homollea, Homolliella and Paracephaelis. All the genera of the tribe proved to be stenopalynous (the species examined possess the same pollen type), except Pavetta, Rutidea, Versteegia and Tarenna which are eurypalynous. In the huge genus Pavetta the existing infrageneric classification is supported pollen morphologically. Pollen morphology further indicates that the genus Tarenna is badly delimited and strongly in need of a revision. The small genus Versteegia is in need of further taxonomic and palynological study to understand the pollen morphological variation encountered here. At a higher rank, pollen morphology also does not contradict the recent division of the Pavetteae in the Ixoreae (a stenopalynous tribe with presumably primitive pollen) and the Pavetteae sensu stricto (eurypalynous).     

Expression of foreign genes in regenerated plants and in their progeny

Chimeric genes comprised of the nopaline synthase promoter and bacterial coding sequences specifying resistance to kanamycin, chloramphenicol or methotrexate, were inserted into the non-oncogenic Ti plasmid vector pGV3850 by recombination (through homologous pBR322 sequences present in the chimeric gene constructs and pGV3850). These co-integrates in Agrobacterium were used to infect single plant protoplasts of Nicotiana by co-cultivation. The resistance traits allowed the selection of transformed calli in tissue culture in the presence of the appropriate antibiotic. Furthermore, as a non-oncogenic Ti plasmid was used for the protoplast transformation, phenotypically normal and fertile plants could be regenerated from the resistant calli. We have shown that these fully differentiated plant tissues exhibit functional expression of resistance traits (Km^R and Cm^R). All plants carrying the chimeric genes developed normally, flowered, and set seeds. The inheritance of several of these resistance traits was analyzed and shown to be Mendelian. These results are model experiments to demonstrate that genes of interest can be systematically transferred to the genome of plants using non-oncogenic Ti plasmid derivatives; and that transformed plants are capable of normal growth and differentiation, thus providing a natural environment for the study of gene expression and development of plant cells.     

In vitro and in vivo evaluation of 6-aminopyrazolyl-pyridine-3-carbonitriles as JAK2 kinase inhibitors

Synthesis and biological evaluation of a series of 6-aminopyrazolyl-pyridine-3-carbonitriles as JAK2 kinase inhibitors was reported. Biochemical screening, followed by profile optimization, resulted in JAK2 inhibitors exhibiting good kinase selectivity, pharmacokinetic properties, physical properties and pharmacodynamic effects.     

Hepatocyte growth factor induces epithelial cell motility through transactivation of the epidermal growth factor receptor

Hepatocyte growth factor (HGF) is a potent inducer of motility in epithelial cells. Since we have previously found that activation of the epidermal growth factor receptor (EGFR) is an absolute prerequisite for induction of motility of corneal epithelial cells after wounding, we investigated whether induction of motility in response to HGF is also dependent on activation of the EGFR. We now report that HGF induces transactivation of the EGFR in an immortalized line of corneal epithelial cells, in human skin keratinocytes, and in Madin-Darby canine kidney cells. EGFR activation is unconditionally required for induction of motility in corneal epithelial cells, and for induction of a fully motile phenotype in Madin-Darby canine kidney cells. Activation of the EGFR occurs through amphiregulin and heparin-binding epidermal growth factor-like growth factor. Early after HGF stimulation, blocking EGFR activation does not inhibit extracellular-signal regulated kinase 1/2 (ERK1/2) activation by HGF, but the converse is seen after approximately 1 h, indicating the existence of EGFR-dependent and -independent routes of ERK1/2 activation. In summary, HGF induces transactivation of the EGFR in epithelial cells, and this is a prerequisite for induction of full motility.     

Conformation, localization, and integrin binding of talin depend on its interaction with phosphoinositides

Talin is a structural component of focal adhesion sites and is thought to be engaged in multiple protein interactions at the cytoplasmic face of cell/matrix contacts. Talin is a major link between integrin and the actin cytoskeleton and was shown to play an important role in focal adhesion assembly. Consistent with the view that talin must be activated at these sites, we found that phosphatidylinositol 4-monophosphate and phosphatidylinositol 4,5-bisphosphate (PI4,5P(2)) bound to talin in cells in suspension or at early stages of adhesion, respectively. When phosphoinositides were associated with phospholipid bilayer, talin/phosphoinositide association was restricted to PI4,5P(2). This association led to a conformational change of the protein. Moreover, the interaction between integrin and talin was greatly enhanced by PI4,5P(2)-induced talin activation. Finally, sequestration of PI4,5P(2) by a specific pleckstrin homology domain confirms that PI4,5P(2) is necessary for proper membrane localization of talin and that this localization is essential for the maintenance of focal adhesions. Our results support a model in which PI4,5P(2) exposes the integrin-binding site on talin. We propose that PI4,5P(2)-dependent signaling modulates assembly of focal adhesions by regulating integrin-talin complexes. These results demonstrate that activation of the integrin-binding activity of talin requires not only integrin engagement to the extracellular matrix but also the binding of PI4,5P(2) to talin, suggesting a possible role of lipid metabolism in organizing the sequential assembly of focal adhesion components.