The pattern of clinical breast cancer metastasis correlates with a single nucleotide polymorphism in the C1qA component of complement

Complement is one of primary defense mechanisms against intravascular microorganisms and could play a role in the immune response to malignancy and hence its clinical behavior. We evaluated if the sole coding polymorphism of C1qA associates with outcome in patients with breast carcinoma. Genotyping for C1qA_[276A/G] was performed in 63 breast cancer subjects with localized tumor and compared with that in 38 breast cancer subjects with metastasis. Established risk factors for clinical outcome were considered and evaluated in multivariable analysis. Breast cancer subjects with heterozygous or homozygous C1qA_[276G] genotype had a higher rate of metastasis than subjects with the homozygous C1qA_[276A] genotype [hazard ratio (HR) 2.4, 95% confidence interval (CI) 1.1–4.1]. This association was stronger when only metastatic sites associated with hematogenous spread, i.e., to the bone, liver, and brain, were considered (HR 3.5, 95% CI 1.4–5.6) and remained statistically significant after adjustment for the number of positive lymph nodes, estrogen receptor status, and progesterone receptor status. There was no statistical difference in the C1qA_[276A/G] allelic distribution between all subjects with breast cancer and controls. These results suggest there could be an association of a single nucleotide polymorphism at position 276 of the C1qA component of complement with breast cancer metastasis to sites linked to hematogenous spread of disease. The C1qA polymorphism associated with decreased distant metastasis has also been correlated with an increased incidence of subcutaneous systemic lupus and C1q deficiencies, suggesting that an altered immune response may play a role in the observed association. Supported in part by National Institute of Health grant R21-CA90822, Friends You Can Count On! grant 1-87093-00, and the Woody and Louise White Cancer Research Fund.     

Crystal structure of thioltransferase at 2.2 A resolution.

We report here the first three-dimensional structure of a mammalian thioltransferase as determined by single crystal X-ray crystallography at 2.2 A resolution. The protein is known for its thiol-redox properties and dehydroascorbate reductase activity. Recombinant pig liver thioltransferase expressed in Escherichia coli was crystallized in its oxidized form by vapor diffusion technique. The structure was determined by multiple isomorphous replacement method using four heavy-atom derivatives. The protein folds into an alpha/beta structure with a four-stranded mixed beta-sheet in the core, flanked on either side by helices. The fold is similar to that found in other thiol-redox proteins, viz. E. coli thioredoxin and bacteriophage T4 glutaredoxin, and thus seems to be conserved in these functionally related proteins. The active site disulfide (Cys 22-Cys 25) is located on a protrusion on the molecular surface. Cys 22, which is known to have an abnormally low pKa of 3.8, is accessible from the exterior of the molecule. Pro 70, which is in close proximity to the disulfide bridge, assumes a conserved cis-peptide configuration. Mutational data available on the protein are in agreement with the three-dimensional structure.     

Prediction of the profiles of the elution bands of proteins in preparative liquid chromatography

A semiequilibrium model of chromatography has been developed to account for the elution profile of high concentration bands. The use of this model requires the prior determination of the equilibrium isotherms of the studied compounds in the chromatographic phase system. The model is applied here to a study of the isocratic elution bands of chicken albumin and conalbumin on a weak anion exchanger in aqueous buffered solutions. Satisfactory agreement is observed between the experimental elution profiles of large-sample-size bands of chicken albumin and conalbumin.     

6‐O‐(3″, 4″‐di‐O‐trans‐cinnamoyl)‐α‐l‐rhamnopyranosylcatalpol and verbascoside: Cytotoxicity,cell cycle kinetics,apoptosis, and ROS production evaluation in tumor cells

The aim of this study was to evaluate the impact that 6‐O‐(3″, 4″‐di‐O‐trans‐cinnamoyl)‐α‐ l ‐rhamnopyranosylcatalpol (Dicinn) and verbascoside (Verb), two compounds simultaneously reported in Verbascum ovalifolium, have on tumor cell viability, apoptosis, cell cycle kinetics, and intracellular reactive oxygen species (ROS) level. At 100 µg/mL and 48 hours incubation time, Dicinn and Verb produced good cytotoxic effects in A549, HT‐29, and MCF‐7 cells. Dicinn induced cell‐cycle arrest at the G₀/G₁ phase and apoptosis, whereas Verb increased the population of subG₁ cells and cell apoptosis rates. Furthermore, the two compounds exhibited time‐dependent ROS generating effects in tumor cells (1‐24 hours). Importantly, no cytotoxic effects were induced in nontumor MCF‐10A cells by the two compounds up to 100 µg/mL. Overall, the effects exhibited by Verb in tumor cells were more potent, which can be correlated with its structural features, such as the presence of phenolic hydroxyl groups.     

The formation of syncytia within the visceral musculature of the Drosophila midgut is dependent on duf, sns and mbc.

The visceral musculature of the Drosophila midgut consists of an inner layer of circular and an outer layer of longitudinal muscles. Here, we show that the circular muscles are organised as binucleate syncytia that persist through metamorphosis. At stage 11, prior to the onset of the fusion processes, we detected two classes of myoblasts within the visceral trunk mesoderm. One class expresses the founder-cell marker rP298-LacZ in a one- to two-cells-wide strip along the ventralmost part of the visceral mesoderm, whereas the adjacent two to three cell rows are characterised by the expression of Sticks-and-stones (SNS). During the process of cell fusion at stage 12 SNS expression decreases within the newly formed syncytia that spread out dorsally over the midgut. At both margins of the visceral band several cells remain unfused and continue to express SNS. Additional rP298-LacZ-expressing cells arise from the posterior tip of the mesoderm, migrate anteriorly and eventually fuse with the remaining SNS-expressing cells, generating the longitudinal muscles. Thus, although previous studies proposed a separate primordium for the longitudinal musculature located at the posteriormost part of the mesoderm anlage, our cell lineage analyses as well as our morphological observations reveal that a second population of cells originates from the trunk mesoderm. Mutations of genes that are involved in somatic myoblast fusion, such as sns, dumbfounded (duf) or myoblast city (mbc), also cause severe defects within the visceral musculature. The circular muscles are highly unorganised while the longitudinal muscles are almost absent. Thus the fusion process seems to be essential for a proper visceral myogenesis. Our results provide strong evidence that the founder-cell hypothesis also applies to visceral myogenesis, employing the same genetic components as are used in the somatic myoblast fusion processes.     

Genetic analysis of a contact zone between two lineages of the ocellated lizard (Lacerta lepida Daudin 1802) in south‐eastern Iberia reveal a steep and narrow hybrid zone

Measuring the diffusion of genes between diverging taxa through zones of secondary contact is an essential step to understand the extent and nature of the reproductive isolation that has been achieved. Previous studies have shown that the ocellated lizard (Lacerta lepida Daudin, 1802) has endured repeated range fragmentation associated with the climatic oscillations of the Plio‐Pleistocene that promoted diversification of many different evolutionary units within the species. However, the oldest divergence within the group is estimated to have occurred much earlier, during the Miocene, around 9 Ma and corresponds to the split between the subspecies Lacerta lepida nevadensis Buchholz (1963) and Lacerta lepida lepida Daudin (1802). Although these two evolutionary units have documented genetic and morphological differentiation, most probably accumulated during periods of allopatry, little is known about patterns of gene flow between them. In this study, we performed a population genetic analysis of a putative area of secondary contact between these two taxa, using mtDNA and microsatellite data. We assessed levels of gene flow across the contact zone to clarify to what extent gene flow may be occurring. Hybridization between the subspecies was observed by the presence of genetically introgressed individuals. However, the overall coincidence of mitochondrial and multilocus nuclear clines and generally steep clines support the idea that this contact zone is acting as a barrier to gene flow. Taken together, these results suggest that L. l. lepida and L. l. nevadensis are in independent evolutionary trajectories and should be considered as two different species.     

The cytoplasmic domain of neuropilin-1 regulates focal adhesion turnover

Though the vascular endothelial growth factor coreceptor neuropilin-1 (Nrp1) plays a critical role in vascular development, its precise function is not fully understood. We identified a group of novel binding partners of the cytoplasmic domain of Nrp1 that includes the focal adhesion regulator, Filamin A (FlnA). Endothelial cells (ECs) expressing a Nrp1 mutant devoid of the cytoplasmic domain (nrp1^cytoΔ/Δ) migrated significantly slower in response to VEGF relative to the cells expressing wild-type Nrp1 (nrp1^+/+ cells). The rate of FA turnover in VEGF-treated nrp1^cytoΔ/Δ ECs was an order of magnitude lower in comparison to nrp1^+/+ ECs, thus accounting for the slower migration rate of the nrp1^cytoΔ/Δ ECs.     

The Delta fbpA mutant derived from Mycobacterium tuberculosis H37Rv has an enhanced susceptibility to intracellular antimicrobial oxidative mechanisms, undergoes limited phagosome maturation and activates macrophages and dendritic cells

Mycobacterium tuberculosis H37Rv (Mtb) excludes phagocyte oxidase (phox) and inducible nitric oxide synthase (iNOS) while preventing lysosomal fusion in macrophages (MPhis). The antigen 85A deficient (Delta fbpA) mutant of Mtb was vaccinogenic in mice and the mechanisms of attenuation were compared with MPhis infected with H37Rv and BCG. Delta fbpA contained reduced amounts of trehalose 6, 6, dimycolate and induced minimal levels of SOCS-1 in MPhis. Blockade of oxidants enhanced the growth of Delta fbpA in MPhis that correlated with increased colocalization with phox and iNOS. Green fluorescent protein-expressing strains within MPhis or purified phagosomes were analysed for endosomal traffick with immunofluorescence and Western blot. Delta fbpA phagosomes were enriched for rab5, rab11, LAMP-1 and Hck suggesting enhanced fusion with early, recycling and late endosomes in MPhis compared with BCG or H37Rv. Delta fbpA phagosomes were thus more mature than H37Rv or BCG although, they failed to acquire rab7 and CD63 preventing lysosomal fusion. Finally, Delta fbpA infected MPhis and dendritic cells (DCs) showed an enhanced MHC-II and CD1d expression and primed immune T cells to release more IFN-gamma compared with those infected with BCG and H37Rv. Delta fbpA was thus more immunogenic in MPhis and DCs because of an enhanced susceptibility to oxidants and increased maturation.