Lectin microarray profiling of metastatic breast cancers

Altered protein glycosylation compared with the disease-free state is a universal feature of cancer cells. It has long been established that distinct glycan structures are associated with specific forms of cancer, but far less is known about the complete array of glycans associated with certain tumors. The cancer glycome has great potential as a source of biomarkers, but progress in this field has been hindered by a lack of available techniques for the elucidation of disease-associated glycosylation. In the present study, lectin microarrays consisting of 45 lectins with different binding preferences covering N- and O-linked glycans were coupled with evanescent-field activated fluorescent detection in the glycomic analysis of primary breast tumors and the serum and urine of patients with metastatic breast cancer. A single 50 μm section of a primary breast tumor or <1 μL of breast cancer patient serum or urine was sufficient to detect glycosylation alterations associated with metastatic breast cancer, as inferred from lectin-binding patterns. The high-throughput, sensitive and relatively simple nature of the simultaneous analysis of N- and O-linked glycosylation following minimal sample preparation and without the need for protein deglycosylation makes the lectin microarray analysis described a valuable tool for discovery phase glycomic profiling.     

Diversity in and evidence for selection on the mitochondrial genome of Phytophthora infestans

Two extant nomenclature systems were reconciled to relate six mitochondrial DNA (mtDNA) haplotypes of Phytophthora infestans, the oomycete pathogen causing late blight disease on potato and tomato. Carter's haplotypes I-a and I-b were included in Goodwin's haplotype A, while Carter's haplotypes II-a and II-b were included in Goodwin's haplotype B. In addition, haplotypes E and F were included in Carter's haplotype I-b. The mutational differences separating the various haplotypes were determined, and we propose that either haplotype I-b(A) or haplotype I-a(A) is the putative ancestral mtDNA of P. infestans, because either can center all the other haplotypes in a logical stepwise network of mutational changes. The occurrence of the six haplotypes in 548 isolates worldwide was determined. Haplotypes I-a and II-a were associated with diverse genotypes worldwide. As previously suggested, haplotype I-b was found only in the US-1 clonal lineage and its variants (n = 99 isolates from 16 countries on 5 continents), and haplotype II-b was limited to the US-6 clonal lineage and its derivatives (n = 36). In a confirmation of a previous suggestion, the randomly mating population in the Toluca Valley of central Mexico (n = 78) was monomorphic for mtDNA haplotype I-a(A). We hypothesize that selection there may be driving the dominance of that single mtDNA haplotype.     

Placental Cadmium Levels Are Associated with Increased Preeclampsia Risk

Environmental exposure to heavy metals is a potentially modifiable risk factor for preeclampsia (PE). Toxicologically, there are known interactions between the toxic metal cadmium (Cd) and essential metals such as selenium (Se) and zinc (Zn), as these metals can protect against the toxicity of Cd. As they relate to preeclampsia, the interaction between Cd and these essential metals is unknown. The aims of the present study were to measure placental levels of Cd, Se, and Zn in a cohort of 172 pregnant women from across the southeast US and to examine associations of metals levels with the odds of PE in a nested case-control design. Logistic regressions were performed to assess odds ratios (OR) for PE with exposure to Cd controlling for confounders, as well as interactive models with Se or Zn. The mean placental Cd level was 3.6 ng/g, ranging from 0.52 to 14.5 ng/g. There was an increased odds ratio for PE in relationship to placental levels of Cd (OR = 1.5; 95% CI: 1.1–2.2). The Cd-associated OR for PE increased when analyzed in relationship to lower placental Se levels (OR = 2.0; 95% CI: 1.1–3.5) and decreased with higher placental Se levels (OR = 0.98; 95% CI: 0.5–1.9). Similarly, under conditions of lower placental Zn, the Cd-associated OR for PE was elevated (OR = 1.8; 95% CI: 0.8–3.9), whereas with higher placental Zn it was reduced (OR = 1.3; 95% CI: 0.8–2.0). Data from this pilot study suggest that essential metals may play an important role in reducing the odds of Cd-associated preeclampsia and that replication in a larger cohort is warranted.     

Inhibition of auxin-stimulated growth of pea stem segments by a specific nonasaccharide of xyloglucan

Hemicellulose extracted from cell walls of suspension-cultured rose (Rosa Paul's Scarlet) cells was digested with cellulase from Trichoderma viride. The quantitatively major oligosaccharide products, a nonasaccharide and a heptasaccharide derived from xyloglucan, were purified by gel permeation chromatography. The nonasaccharide was found to inhibit the 2,4-dichlorophenoxy-acetic-acid-induced elongation of etiolated pea (Pisum sativum) stem segments. This confirms an earlier report (York et al., 1984, Plant Physiol. 75, 295–297). The inhibition of elongation by the nonasaccharide showed a maximum at around 10^-9M with higher and lower concentrations being less effective. The heptasaccharide did not significantly inhibit elongation at 10^-7–10^-10M and also did not affect the inhibition caused by the nonasaccharide when co-incubated with the latter.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - XG xyloglucan - XG7 xyloglucan heptasaccharide (Glc₄·Xyl₃) - XG9 xyloglucan nonasaccharide (Glc₄·Xyl₃·Gal·Fuc)     

Activated phosphoinositide 3-kinase associates with membrane skeleton in thrombin-exposed platelets.

Human platelets undergo a rapid, major reorganization of the cytoskeletal matrix upon exposure to thrombin, and accumulate 3-phosphorylated phosphoinositides in a protein kinase C (PKC)-dependent manner. These phosphoinositides have been suggested to be involved in actin polymerization/depolymerization. We reasoned that, if newly generated 3-phosphorylated phosphoinositide modulates cytoskeletal reorganization, a prerequisite for such action would be generation near cytoskeletal proteins. We have found that, after platelet activation, phosphatidylinositol 3-kinase and phosphatidylinositol(4)P 3-kinase activities, antibody-detectable phosphoinositide 3-kinase, and PKC become markedly and specifically enriched in a Triton X-100-insoluble cytoskeletal fraction that contains GPIIb/IIIa (integrin) and pp60c-src. The cytoskeletal fraction then accounts for up to 70% of total phosphoinositide 3-kinase activity, a function of recruited activated enzyme. These proteins are not occluded or directly associated with newly polymerized actin, since blockage by cytochalasin D of actin polymerization, and consequent inhibition of accumulation of about 40% of incremental protein and actin in this fraction, has no effect on its content of phosphoinositide 3-kinase, GPIIb/IIIa, pp60c-src, or PKC. Depolymerization of actin with DNase I, or inhibition of ligand binding to GPIIb/IIIa by RGDS, however, in combination with cytochalasin D, further depletes actin and significantly decreases sedimentability of GPIIb/IIIa as well as phosphoinositide 3-kinase, pp60c-src, and PKC, without inhibiting total 3-kinase activity. Our results suggest that, as a function of platelet activation, enzymes that regulate the synthesis of 3-phosphorylated phosphoinositides rapidly associate with the membrane skeleton and that skeletally associated phosphoinositide 3-kinase is more active than the Triton-soluble form.     

Production and harvesting of ionically wall-bound extensin from living cell suspension cultures

Cell-suspension cultures ofSpinacia andRosa accumulated a cell wall protein, extensin, in a form that was amenable to leaching from the surface of the living cells by a brief treatment with non-toxic salts. Cultures ofLycopersicon, Capsicum, Acer andFestuca did not accumulate this class of extensin. InSpinacia andRosa, optimum yields of leachable extensin were achieved from young cultures, in media at relatively low pH, by leaching with 0.1 M CaCl₂. Older cultures, pH values >6.5, and LaCl₃ or higher concentrations of CaCl₂ were less effective.Abbreviation TCA trichloroacetic acid     

Isotope effects associated with the anaerobic oxidation of sulfite and thiosulfate by the photosynthetic bacterium, Chromatium vinosum

Abstract The purple photosynthetic bacterium Chromatium vinosum , strain D, catalyzes several oxidations of reduced sulfur compounds under anaerobic conditions in the light: e.g., sulfide → sulfur → sulfate, sulfite → sulfate, and thiosulfate → sulfur + sulfate. Here it is shown that no sulfur isotope effect is associated with the last of these processes; isotopic compositions of the sulfur and sulfate produced can differ, however, if the sulfane and sulfonate positions within the thiosulfate have different isotopic compositions. In the second process, an observed change from an inverse to a normal isotope effect during oxidation of sulfite may indicate the operation of 2 enzymatic pathways. In contrast to heterotrophic anaerobic reduction of oxidized sulfur compounds, anaerobic oxidations of inorganic sulfur compounds by photosynthetic bacteria are characterized by relatively small isotope effects.