Nucleon to Delta Weak Excitation Amplitudes in the Non-relativistic Quark Model

We investigate the nucleon to Delta(1232) vector and axial vector amplitudes in the non-relativistic quark model of the Isgur-Karl variety. A particular interest is to investigate the SU(6) symmetry breaking, due to color hyperfine interaction. We compare the theoretical estimates to recent experimental investigation of the Adler amplitudes by neutrino scattering.Comment: \documentstyle[aps]{revtex}, 21pages; 11 postscript figures. Accepted for publication by Phys. Rev.

Alteration of AKT Activity Increases Chemotherapeutic Drug and Hormonal Resistance in Breast Cancer yet Confers an Achilles Heel by Sensitization to Targeted Therapy

The PI3K/PTEN/Akt/mTOR pathway plays critical roles in the regulation of cell growth. The effects of this pathway on drug resistance and cellular senescence of breast cancer cells has been a focus of our laboratory. Introduction of activated Akt or mutant PTEN constructs which lack lipid phosphatase [PTEN(G129E)] or lipid and protein phosphatase [PTEN(C124S)] activity increased the resistance of the cells to the chemotherapeutic drug doxorubicin, and the hormonal drug tamoxifen. Activated Akt and PTEN genes also inhibited the induction of senescence after doxorubicin treatment; a phenomenon associated with unrestrained proliferation and tumorigenesis. Interference with the lipid phosphatase domain of PTEN was sufficient to activate Akt/mTOR/p70S6K as MCF-7 cells transfected with the mutant PTEN gene lacking the lipid phosphatase activity [PTEN(G129E)] displayed elevated levels of activated Akt and p70S6K compared to empty vector transfected cells. Cells transfected with mutant PTEN or Akt constructs were hypersensitive to mTOR inhibitors when compared with the parental or empty vector transfected cells. Akt-transfected cells were cultured for over two months in tamoxifen from which tamoxifen and doxorubicin resistant cells were isolated that were >10-fold more resistant to tamoxifen and doxorubicin than the original Akt-transfected cells. These cells had a decreased induction of both activated p53 and total p21Cip1 upon doxorubicin treatment. Furthermore, these cells had an increased inactivation of GSK-3β and decreased expression of the estrogen receptor-α. In these drug resistant cells, there was an increased activation of ERK which is associated with proliferation. These drug resistant cells were hypersensitive to mTOR inhibitors and also sensitive to MEK inhibitors, indicating that the enhanced p70S6K and ERK expression was relevant to their drug and hormonal resistance. Given that Akt is overexpressed in greater than 50% of breast cancers, our results point to potential therapeutic targets, mTOR and MEK. These studies indicate that activation of the Akt kinase or disruption of the normal activity of the PTEN phosphatase can have dramatic effects on activity of p70S6K and other downstream substrates and thereby altering the therapeutic sensitivity of breast cancer cells. The effects of doxorubicin and tamoxifen on induction of the Raf/MEK/ERK and PI3K/Akt survival pathways were examined in unmodified MCF-7 breast cells. Doxorubicin was a potent inducer of activated ERK and to a lesser extent Akt. Tamoxifen also induced ERK. Thus a consequence of doxorubicin and tamoxifen therapy of breast cancer is the induction of a pro-survival pathway which may contribute to the development of drug resistance. Unmodified MCF-7 cells were also sensitive to MEK and mTOR inhibitors which synergized with both tamoxifen and doxorubicin to induce death. In summary, our results point to the key interactions between the PI3K/PTEN/Akt/mTOR and Raf/ MEK/ERK pathways in regulating chemotherapeutic drug resistance/sensitivity in breast cancer and indicate that targeting these pathways may prevent drug and hormonal resistance. Orignally published Advances in Enzyme Regulation, Vol. 48, No. 1, 2008

Breed and adaptive response modulate bovine peripheral blood cells’ transcriptome

Background: Adaptive response includes a variety of physiological modifications to face changes in external or internal conditions and adapt to a new situation. The acute phase proteins (APPs) are reactants synthesized against environmental stimuli like stress, infection, inflammation. Methods: To delineate the differences in molecular constituents of adaptive response to the environment we performed the whole-blood transcriptome analysis in Italian Holstein (IH) and Italian Simmental (IS) breeds. For this, 663 IH and IS cows from six commercial farms were clustered according to the blood level of APPs. Ten extreme individuals (five APP+ and APP- variants) from each farm were selected for the RNA-seq using the Illumina sequencing technology. Differentially expressed (DE) genes were analyzed using dynamic impact approach (DIA) and DAVID annotation clustering. Milk production data were statistically elaborated to assess the association of APP+ and APP- gene expression patterns with variations in milk parameters. Results: The overall de novo assembly of cDNA sequence data generated 13,665 genes expressed in bovine blood cells. Comparative genomic analysis revealed 1,152 DE genes in the comparison of all APP+ vs. all APP- variants; 531 and 217 DE genes specific for IH and IS comparison respectively. In all comparisons overexpressed genes were more represented than underexpressed ones. DAVID analysis revealed 369 DE genes across breeds, 173 and 73 DE genes in IH and IS comparison respectively. Among the most impacted pathways for both breeds were vitamin B6 metabolism, folate biosynthesis, nitrogen metabolism and linoleic acid metabolism. Conclusions: Both DIA and DAVID approaches produced a high number of significantly impacted genes and pathways with a narrow connection to adaptive response in cows with high level of blood APPs. A similar variation in gene expression and impacted pathways between APP+ and APP- variants was found between two studied breeds. Such similarity was also confirmed by annotation clustering of the DE genes. However, IH breed showed higher and more differentiated impacts compared to IS breed and such particular features in the IH adaptive response could be explained by its higher metabolic activity. Variations of milk production data were significantly associated with APP+ and APP- gene expression patterns

A Mouse Model of Acrodermatitis Enteropathica: Loss of Intestine Zinc Transporter ZIP4 (Slc39a4) Disrupts the Stem Cell Niche and Intestine Integrity

Loss-of-function of the zinc transporter ZIP4 in the mouse intestine mimics the lethal human disease acrodermatitis enteropathica. This is a rare disease in humans that is not well understood. Our studies demonstrate the paramount importance of ZIP4 in the intestine in this disease and reveal that a root cause of lethality is disruption of the intestine stem cell niche and impaired function of the small intestine. This, in turn, leads to dramatic weight loss and death unless treated with exogenous zinc

Comparison of the functional and structural characteristics of rare TSC2 variants with clinical and genetic findings

The TSC1 and TSC2 gene products interact to form the tuberous sclerosis complex (TSC), an important negative regulator of the mechanistic target of rapamycin complex 1 (TORC1). Inactivating mutations in TSC1 or TSC2 cause TSC, and the identification of a pathogenic TSC1 or TSC2 variant helps establish a diagnosis of TSC. However, it is not always clear whether TSC1 and TSC2 variants are inactivating. To determine whether TSC1 and TSC2 variants of uncertain clinical significance affect TSC complex function and cause TSC, in vitro assays of TORC1 activity can be employed. Here we combine genetic, functional, and structural approaches to try and classify a series of 15 TSC2 VUS. We investigated the effects of the variants on the formation of the TSC complex, on TORC1 activity and on TSC2 pre-mRNA splicing. In 13 cases (87%), the functional data supported the hypothesis that the identified TSC2 variant caused TSC. Our results illustrate the benefits and limitations of functional testing for TSC

ZIP8 Zinc Transporter: Indispensable Role for Both Multiple-Organ Organogenesis and Hematopoiesis In Utero

Previously this laboratory characterized Slc39a8-encoded ZIP8 as a Zn2+/(HCO3–)2 symporter; yet, the overall physiological importance of ZIP8 at the whole-organism level remains unclear. Herein we describe the phenotype of the hypomorphic Slc39a8(neo/neo) mouse which has retained the neomycin-resistance gene in intron 3, hence causing significantly decreased ZIP8 mRNA and protein levels in embryo, fetus, placenta, yolk sac, and several tissues of neonates. The Slc39a8(neo) allele is associated with diminished zinc and iron uptake in mouse fetal fibroblast and liver-derived cultures; consequently, Slc39a8(neo/neo) newborns exhibit diminished zinc and iron levels in several tissues. Slc39a8(neo/neo) homozygotes from gestational day(GD)-11.5 onward are pale, growth-stunted, and die between GD18.5 and 48 h postnatally. Defects include: severely hypoplastic spleen; hypoplasia of liver, kidney, lung, and lower limbs. Histologically, Slc39a8(neo/neo) neonates show decreased numbers of hematopoietic islands in yolk sac and liver. Low hemoglobin, hematocrit, red cell count, serum iron, and total iron-binding capacity confirmed severe anemia. Flow cytometry of fetal liver cells revealed the erythroid series strikingly affected in the hypomorph. Zinc-dependent 5-aminolevulinic acid dehydratase, required for heme synthesis, was not different between Slc39a8(+/+) and Slc39a8(neo/neo) offspring. To demonstrate further that the mouse phenotype is due to ZIP8 deficiency, we bred Slc39a8(+/neo) with BAC-transgenic BTZIP8-3 line (carrying three extra copies of the Slc39a8 allele); this cross generated viable Slc39a8(neo/neo)_BTZIP8-3(+/+) pups showing none of the above-mentioned congenital defects–proving Slc39a8(neo/neo) causes the described phenotype. Our study demonstrates that ZIP8-mediated zinc transport plays an unappreciated critical role during in utero and neonatal growth, organ morphogenesis, and hematopoiesis

Targeted Genomic Sequencing of TSC1 and TSC2 Reveals Causal Variants in Individuals for Whom Previous Genetic Testing for Tuberous Sclerosis Complex Was Normal

Tuberous sclerosis complex (TSC) is caused by inactivating variants in TSC1 and TSC2. Somatic mosaicism, as well as the size and complexity of the TSC1 and TSC2 loci, makes variant identification challenging. Indeed, in some individuals with a clinical diagnosis of TSC, diagnostic testing fails to identify an inactivating variant. To improve TSC1 and TSC2 variant detection, we screened the TSC1 and TSC2 genomic regions using targeted HaloPlex custom capture and next-generation sequencing (NGS) in genomic DNA isolated from peripheral blood of individuals with definite, possible or suspected TSC in whom no disease-associated variant had been identified by previous diagnostic genetic testing. We obtained &gt;95% target region coverage at a read depth of 20 and &gt;50% coverage at a read depth of 300 and identified inactivating TSC1 or TSC2 variants in 83/155 individuals (54%); 65/113 (58%) with clinically definite TSC and 18/42 (43%) with possible or suspected TSC. These included 19 individuals with deep intronic variants and 54 likely cases of mosaicism (variant allele frequency 1-28%; median 7%). In 13 cases (8%), we identified a variant of uncertain significance (VUS). Targeted genomic NGS of TSC1 and TSC2 increases the yield of inactivating variants found in individuals with suspected TSC.</p

The Death Effector Domains of Caspase-8 Induce Terminal Differentiation

The differentiation and senescence programs of metazoans play key roles in regulating normal development and preventing aberrant cell proliferation, such as cancer. These programs are intimately associated with both the mitotic and apoptotic pathways. Caspase-8 is an apical apoptotic initiator that has recently been appreciated to coordinate non-apoptotic roles in the cell. Most of these functions are attributed to the catalytic domain, however, the amino-terminal death effector domains (DED)s, which belong to the death domain superfamily of proteins, can also play key roles during development. Here we describe a novel role for caspase-8 DEDs in regulating cell differentiation and senescence. Caspase-8 DEDs accumulate during terminal differentiation and senescence of epithelial, endothelial and myeloid cells; genetic deletion or shRNA suppression of caspase-8 disrupts cell differentiation, while re-expression of DEDs rescues this phenotype. Among caspase-8 deficient neuroblastoma cells, DED expression attenuated tumor growth in vivo and proliferation in vitro via disruption of mitosis and cytokinesis, resulting in upregulation of p53 and induction of differentiation markers. These events occur independent of caspase-8 catalytic activity, but require a critical lysine (K156) in a microtubule-binding motif in the second DED domain. The results demonstrate a new function for the DEDs of caspase-8, and describe an unexpected mechanism that contributes to cell differentiation and senescence

Mechanisms of Intragastric pH Sensing

Luminal amino acids and lack of luminal acidity as a result of acid neutralization by intragastric foodstuffs are powerful signals for acid secretion. Although the hormonal and neural pathways underlying this regulatory mechanism are well understood, the nature of the gastric luminal pH sensor has been enigmatic. In clinical studies, high pH, tryptic peptides, and luminal divalent metals (Ca2+ and Mg2+) increase gastrin release and acid production. The calcium-sensing receptor (CaSR), first described in the parathyroid gland but expressed on gastric G cells, is a logical candidate for the gastric acid sensor. Because CaSR ligands include amino acids and divalent metals, and because extracellular pH affects ligand binding in the pH range of the gastric content, its pH, metal, and nutrient-sensing functions are consistent with physiologic observations. The CaSR is thus an attractive candidate for the gastric luminal sensor that is part of the neuroendocrine negative regulatory loop for acid secretion