Combined DNA flow cytometry and sorting with k-ras2 mutation spectrum analysis and the prognosis of human sporadic colorectal cancer

BACKGROUND: Activation of the k-ras2 pathways and chromosomal instability leading to aneuploidy in human sporadic colorectal cancer (sCRC) is essential to the tumor cell ability to survive, grow, and metastatize. METHODS: The study included 135 patients with sCRC who were followed up for a median of 72 months. Multiple fresh-frozen fragments obtained from superficial and invasive areas of the tumors were mixed and used to detect the degree of DNA aneuploidy (DNA index [DI]) and S-phase fraction by two scatter signals and 4,6-diamidino-2-phenylindole-2-hydrocloride (DAPI) fluorescence flow cytometry (FCM). PCR amplification and k-ras2 mutation spectrum analysis were performed using enriched epithelial nuclei after sorting DNA aneuploid nuclei and DNA diploid nuclei from which tissue-infiltrating lymphocytes were absent. RESULTS: DNA aneuploidy was detected in 98 (73%) and k-ras2 mutations in 54 cases (40%). Univariate analyses of overall survival with both Dukes' A to D or B to C series of cases showed that DNA multiple aneuploidy, k-ras2 mutations, older age, and distal site, but not increased S-phase fraction, were predictive of worse outcome. Multivariate Cox models strongly indicated that k-ras2 mutations, but neither single nor multiple DNA aneuploidy, were an independent prognostic factor in both series of patients. In particular, with B and C Dukes' stage patients (n = 110), the relative risk (RR) of death was above 2.5 with k-ras2 mutations and above 3 with the G-->C/T transversions. CONCLUSION: Combined FCM and k-ras2 analysis may be used to predict long-term increased risk of death in sCRC patients.     

The evolution of polar fish hemoglobin: a phylogenetic analysis of the ancestral amino acid residues linked to the root effect

Originating from a benthic ancestor, the suborder Notothenioidei (the dominant fish fauna component of the Antarctic sea) underwent a remarkable radiation, which led notothenioids to fill several niches. The ecological importance of notothenioids in Antarctica and their biochemical adaptations have prompted great efforts to study their physiology and phylogeny, with special attention to the evolutionary adaptation of the oxygen-transport system. We herewith report the evolutionary history of alpha- and beta-globins under the assumption of the molecular clock hypothesis as a basis for reconstructing the phylogenetic relationships among species. These studies have been extended to fish species of other latitudes, including the Arctic region. The northern and southern polar oceans have very different characteristics; indeed, in many respects the Antarctic and Arctic ichthyofaunas are more dissimilar than similar. Our results show that the inferred phylogeny of Arctic and Antarctic globins is different. Taking advantage of the wealth of information collected on structure and function of hemoglobins, we have attempted to investigate the evolutionary history of an important physiological feature in fish, the Root effect. The results suggest that the amino acid residues reported to play a key role in the Root effect may be regarded as ancestor characters, but the lack of this effect in extant species can hardly be associated with the presence of synapomorphies.     

Relationship between adenylate cyclase sensitivity to follitropin and FSH receptor mRNA expression in the ovary of the lizard Podarcis sicula

In mammals, gonadal functions are regulated by two pituitary gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), that interact with gonadal membrane receptors to activate adenylate cyclase. In comparison to mammalian systems, in squamate reptiles a reduced amount of information exists on gonadotropins and their related receptors. This study is aimed at clarifying if, in the lizard Podarcis sicula, the ovarian sensitivity to FSH is correlated to the reproductive cycle and to the expression of membrane receptors involved in the hormone recognition. The results demonstrate that the ovarian adenylate cyclase responsiveness to FSH parallels ovarian functions, being maximal during the ovulatory period. The ovarian sensitivity to FSH is also related to oocyte growth and vitellogenesis. Northern blot analyses reveal that the FSH receptor mRNA is maximally expressed in vitellogenic oocytes during the reproductive period. These results suggest that, in lizard ovary, hormone activation of adenylate cyclase is mediated by de novo synthesis of receptors specifically involved in FSH recognition. In lizards treated in vivo with FSH during the pre-ovulatory period, adenylate cyclase becomes refractory to further FSH stimulation 2 hr after treatment, but sensitivity to the hormone is restored after 2 weeks. Nevertheless, while the restored level of activity never exceeds that observed during the nonreproductive period, the expression level of FSH receptor mRNAs is significantly enhanced in these animals. These results suggest that in lizard the processes that regulate ovarian growth, vitellogenesis, and ovulation are controlled by a complex network of signals including gonadotropin, FSH receptor expression, and adenylate cyclase.     

Respiratory syndrome very similar to extrinsic allergic alveolitis due to Penicillium verrucosum in workers in a cheese factory

A respiratory syndrome very similar to extrinsic allergic alveolitis due to Penicillium verrucosum was recognized in 4 workers employed in a Gorgonzola cheese factory. A mycogen allergy to P. verrucosum, used as starter in the production, was demonstrated by positive sputum culture and detection of specific antibodies in the blood. Intense and prolonged exposure to inhalation of fungal spores could have lead to the development of this allergic response. The fact that 2 of the subjects are siblings seems to indicate host susceptibility or immunological constitution in the pathogenesis of the respiratory allergy. This revised version was published online in June 2006 with corrections to the Cover Date.     

Pathway engineering for healthy phytochemicals leading to the production of novel flavonoids in tomato fruit

Flavonoids are a large family of plant polyphenolic secondary metabolites. Although they are widespread throughout the plant kingdom, some flavonoid classes are specific for only a few plant species. Due to their presumed health benefits there is growing interest in the development of food crops with tailor-made levels and composition of flavonoids, designed to exert an optimal biological effect. In order to explore the possibilities of flavonoid engineering in tomato fruits, we have targeted this pathway towards classes of potentially healthy flavonoids which are novel for tomato. Using structural flavonoid genes (encoding stilbene synthase, chalcone synthase, chalcone reductase, chalcone isomerase and flavone synthase) from different plant sources, we were able to produce transgenic tomatoes accumulating new phytochemicals. Biochemical analysis showed that the fruit peel contained high levels of stilbenes (resveratrol and piceid), deoxychalcones (butein and isoliquiritigenin), flavones (luteolin-7-glucoside and luteolin aglycon) and flavonols (quercetin glycosides and kaempferol glycosides). Using an online high-performance liquid chromatography (HPLC) antioxidant detection system, we demonstrated that, due to the presence of the novel flavonoids, the transgenic tomato fruits displayed altered antioxidant profiles. In addition, total antioxidant capacity of tomato fruit peel with high levels of flavones and flavonols increased more than threefold. These results on genetic engineering of flavonoids in tomato fruit demonstrate the possibilities to change the levels and composition of health-related polyphenols in a crop plant and provide more insight in the genetic and biochemical regulation of the flavonoid pathway within this worldwide important vegetable.     

IGF-1, IGFBP-1, and IGFBP-3 polymorphisms predict circulating IGF levels but not breast cancer risk: findings from the Breast and Prostate Cancer Cohort Consortium (BPC3)

IGF-1 has been shown to promote proliferation of normal epithelial breast cells, and the IGF pathway has also been linked to mammary carcinogenesis in animal models. We comprehensively examined the association between common genetic variation in the IGF1, IGFBP1, and IGFBP3 genes in relation to circulating IGF-I and IGFBP-3 levels and breast cancer risk within the NCI Breast and Prostate Cancer Cohort Consortium (BPC3). This analysis included 6,912 breast cancer cases and 8,891 matched controls (n = 6,410 for circulating IGF-I and 6,275 for circulating IGFBP-3 analyses) comprised primarily of Caucasian women drawn from six large cohorts. Linkage disequilibrium and haplotype patterns were characterized in the regions surrounding IGF1 and the genes coding for two of its binding proteins, IGFBP1 and IGFBP3. In total, thirty haplotype-tagging single nucleotide polymorphisms (htSNP) were selected to provide high coverage of common haplotypes; the haplotype structure was defined across four haplotype blocks for IGF1 and three for IGFBP1 and IGFBP3. Specific IGF1 SNPs individually accounted for up to 5% change in circulating IGF-I levels and individual IGFBP3 SNPs were associated up to 12% change in circulating IGFBP-3 levels, but no associations were observed between these polymorphisms and breast cancer risk. Logistic regression analyses found no associations between breast cancer and any htSNPs or haplotypes in IGF1, IGFBP1, or IGFBP3. No effect modification was observed in analyses stratified by menopausal status, family history of breast cancer, body mass index, or postmenopausal hormone therapy, or for analyses stratified by stage at diagnosis or hormone receptor status. In summary, the impact of genetic variation in IGF1 and IGFBP3 on circulating IGF levels does not appear to substantially influence breast cancer risk substantially among primarily Caucasian postmenopausal women.     

Antibody Constant Region Peptides Can Display Immunomodulatory Activity through Activation of the Dectin-1 Signalling Pathway

We previously reported that a synthetic peptide with sequence identical to a CDR of a mouse monoclonal antibody specific for difucosyl human blood group A exerted an immunomodulatory activity on murine macrophages. It was therapeutic against systemic candidiasis without possessing direct candidacidal properties. Here we demonstrate that a selected peptide, N10K, putatively deriving from the enzymatic cleavage of the constant region (Fc) of human IgG(1), is able to induce IL-6 secretion and pIkB-α activation. More importantly, it causes an up-regulation of Dectin-1 expression. This leads to an increased activation of β-glucan-induced pSyk, CARD9 and pIkB-α, and an increase in the production of pro-inflammatory cytokines such as IL-6, IL-12, IL-1β and TNF-α. The increased activation of this pathway coincides with an augmented phagocytosis of non opsonized Candida albicans cells by monocytes. The findings suggest that some Fc-peptides, potentially deriving from the proteolysis of immunoglobulins, may cause an unexpected immunoregulation in a way reminiscent of innate immunity molecules.     

The transmembrane prolines of the mitochondrial ADP/ATP carrier are involved in nucleotide binding and transport and its biogenesis

The mitochondrial ADP/ATP carrier (Ancp) is a paradigm of the mitochondrial carrier family, which allows cross-talk between mitochondria, where cell energy is mainly produced, and cytosol, where cell energy is mainly consumed. The members of this family share numerous structural and functional characteristics. Resolution of the atomic structure of the bovine Ancp, in a complex with one of its specific inhibitors, revealed interesting features and suggested the involvement of some particular residues in the movements of the protein to perform translocation of nucleotides from one side of the membrane to the other. They correspond to three prolines located in the odd-numbered transmembrane helices (TMH), Pro-27, Pro-132, and Pro-229. The corresponding residues of the yeast Ancp (Pro-43, Ser-147, and Pro-247) were mutated into alanine or leucine, one at a time and analysis of the various mutants evidenced a crucial role of Pro-43 and Pro-247 during nucleotide transport. Beside, replacement of Ser-147 with proline does not inactivate Ancp and this is discussed in view of the conservation of the three prolines at equivalent positions in the Ancp sequences. These prolines belong to the signature sequences of the mitochondrial carriers and we propose they play a dual role in the mitochondrial ADP/ATP carrier function and biogenesis. Unexpectedly their mutations cause more general effects on mitochondrial biogenesis and morphology, as evidenced by measurements of respiratory rates, cytochrome contents, and also clearly highlighted by fluorescence microscopy.