Tyrosyl-DNA Phosphodiesterase I Catalytic Mutants Reveal an Alternative Nucleophile That Can Catalyze Substrate Cleavage

Tyrosyl-DNA phosphodiesterase I (Tdp1) catalyzes the repair of 3′-DNA adducts, such as the 3′-phosphotyrosyl linkage of DNA topoisomerase I to DNA. Tdp1 contains two conserved catalytic histidines: a nucleophilic His (His^nuc) that attacks DNA adducts to form a covalent 3′-phosphohistidyl intermediate and a general acid/base His (His^gab), which resolves the Tdp1-DNA linkage. A His^nuc to Ala mutant protein is reportedly inactive, whereas the autosomal recessive neurodegenerative disease SCAN1 has been attributed to the enhanced stability of the Tdp1-DNA intermediate induced by mutation of His^gab to Arg. However, here we report that expression of the yeast His^nucAla (H182A) mutant actually induced topoisomerase I-dependent cytotoxicity and further enhanced the cytotoxicity of Tdp1 His^gab mutants, including H432N and the SCAN1-related H432R. Moreover, the His^nucAla mutant was catalytically active in vitro, albeit at levels 85-fold less than that observed with wild type Tdp1. In contrast, the His^nucPhe mutant was catalytically inactive and suppressed His^gab mutant-induced toxicity. These data suggest that the activity of another nucleophile when His^nuc is replaced with residues containing a small side chain (Ala, Asn, and Gln), but not with a bulky side chain. Indeed, genetic, biochemical, and mass spectrometry analyses show that a highly conserved His, immediately N-terminal to His^nuc, can act as a nucleophile to catalyze the formation of a covalent Tdp1-DNA intermediate. These findings suggest that the flexibility of Tdp1 active site residues may impair the resolution of mutant Tdp1 covalent phosphohistidyl intermediates and provide the rationale for developing chemotherapeutics that stabilize the covalent Tdp1-DNA intermediate.     

Targeting reverse tetracycline-dependent transactivator to murine mammary epithelial cells that express the progesterone receptor

Through an established gene-targeting strategy, reverse tetracycline-dependent transactivator (rtTA) was targeted downstream of the murine progesterone receptor (PR) promoter. Mice were generated in which one (PR(+/rtTA)) or both (PR(rtTA/rtTA)) PR alleles harbor the rtTA insertion. The PR(+/rtTA) and PR(rtTA/rtTA) knockins exhibit phenotypes identical to the normal and the progesterone receptor knockout mouse, respectively. Crossed with the TZA reporter, which carries the TetO-LacZ responder transgene, the PR(+/rtTA)/TZA and PR(rtTA/rtTA)/TZA bigenics exhibit doxycycline-induced beta-galactosidase activity specifically in progesterone responsive target tissues such as the mammary gland, uterus, ovary, and pituitary gland. In the case of the PR(+/rtTA)/TZA mammary epithelium, dual immunofluorescence demonstrated that PR expression and doxycycline-induced beta-galactosidase activity colocalized; beta-galactosidase was not detected in the absence of doxycycline. Although both the PR(+/rtTA) and PR(rtTA/rtTA) knockins represent innovative animal models with which to further query progesterone's mechanism of action in vivo, the PR(rtTA/rtTA) mouse in particular promises to provide unique insight into the paracrine mechanism of action, which underpins progesterone's involvement in mammary morphogenesis with obvious implications for extending our understanding of this steroid's role in breast cancer progression.     

Cambial activity of Pinus elliottii var. densa reveals influence of seasonal insolation on growth dynamics in the Florida Keys

We determined the temporal and seasonal dynamics of intra-annual cell formation of south Florida slash pine (Pinus elliottii Engelm. var. densa Little & Dor.), the southernmost native pine in the United States and the foundation species of globally endangered pine rockland ecosystems. To assess intra-annual cambial activity and identify possible relationships between cell production and climatic factors, wood micro-cores were extracted monthly from six trees during the period March 2010 to March 2011. The results confirmed annual growth ring formation in P. elliottii var. densa and indicated that its growing season extends from February to December, with a short period of dormancy that varied little between individuals. Within the growing season, earlywood cells were produced from February to July, latewood cells produced from July to December, and intra-annual density fluctuations (IADFs) occurred in the growth rings of four of six trees between July and August. A principal component analysis indicated a homogeneous response of cambial activity among trees to site-specific climatic factors. The first principal component axis explained 71?% of the total variance in cell production during the study period. Our results indicated that the dynamics of seasonal cambial activity of P. elliottii var. densa are controlled by solar radiation (r?=?0.51, p?<?0.10) in the Florida Keys. The nature of our data allow us to only speculate on the ecophysiological processes responsible for IADFs in P. elliottii var. densa, and additional research is needed to better understand the relationship between their formation and the environment in the Lower Florida Keys.     

Genetic distinction of four haplochromine cichlid fish species in a satellite lake of Lake Victoria,East Africa

Lake Victoria is famous for its in evolutionary terms young but species‐rich assemblage of cichlid fishes. This ‘superflock’ also includes additional species from adjacent water systems. Lake Victoria is surrounded by several smaller lakes that are connected to the main water body of Lake Victoria only through swampy areas. Lake Kanyaboli is one such lake, harbouring a much poorer species diversity, mostly comprised of Lake Victoria endemics, some of which are now considered extirpated from the main lake. The focus of this study was on the modern haplochromine component of the cichlid fauna, represented by Lipochromis maxillaris, Astatotilapia nubila, Xystichromis phytophagus and Astatotilapia sp. ‘Bigeye’, as well as a number of morphologically distinct haplochromine specimens that could not be assigned to any of the recognized species. We used five microsatellite markers to distinguish these five taxa. Genetically, L. maxillaris was clearly differentiated from all other taxa, and A. sp. ‘Bigeye’ was moderately differentiated from the remaining three. Astatotilapia nubila, X. phytophagus and the unidentified specimens constituted a partially overlapping cluster. As each of the clusters had several (5–14) private alleles, extremely recent divergence is suggested. As all taxa except for A. sp. ‘Bigeye’ and the unidentified specimens also occur or at least occurred in Lake Victoria, it is likely that they evolved as part of the Lake Victoria superflock, while A. sp. ‘Bigeye’ and the unidentified specimens may have currently evolved in situ. The observation of slightly distinct albeit overlapping body shapes and the extremely close genetic relationship between three of the five taxa are fully compatible and in support of the hybrid swarm theory of adaptive radiation.     

Enterocyte TLR4 mediates phagocytosis and translocation of bacteria across the intestinal barrier

Translocation of bacteria across the intestinal barrier is important in the pathogenesis of systemic sepsis, although the mechanisms by which bacterial translocation occurs remain largely unknown. We hypothesized that bacterial translocation across the intact barrier occurs after internalization of the bacteria by enterocytes in a process resembling phagocytosis and that TLR4 is required for this process. We now show that FcgammaRIIa-transfected enterocytes can internalize IgG-opsonized erythrocytes into actin-rich cups, confirming that these enterocytes have the molecular machinery required for phagocytosis. We further show that enterocytes can internalize Escherichia coli into phagosomes, that the bacteria remain viable intracellularly, and that TLR4 is required for this process to occur. TLR4 signaling was found to be necessary and sufficient for phagocytosis by epithelial cells, because IEC-6 intestinal epithelial cells were able to internalize LPS-coated, but not uncoated, latex particles and because MD2/TLR4-transfected human endothelial kidney (HEK)-293 cells acquired the capacity to internalize E. coli, whereas nontransfected HEK-293 cells and HEK-293 cells transfected with dominant-negative TLR4 bearing a P712H mutation did not. LPS did not induce membrane ruffling or macropinocytosis in enterocytes, excluding their role in bacterial internalization. Strikingly, the internalization of Gram-negative bacteria into enterocytes in vivo and the translocation of bacteria across the intestinal epithelium to mesenteric lymph nodes were significantly greater in wild-type mice as compared with mice having mutations in TLR4. These data suggest a novel mechanism by which bacterial translocation occurs and suggest a critical role for TLR4 in the phagocytosis of bacteria by enterocytes in this process.     

Lipid metabolism response to a single, prolonged bout of endurance exercise in healthy young men

To discover the alterations in lipid metabolism linked to postexercise hypotriglyceridemia, we measured lipid kinetics, lipoprotein subclass distribution and lipid transfer enzymes in seven healthy, lean, young men the day after 2 h of cycling and rest. Compared with rest, exercise increased fatty acid rate of appearance and whole body fatty acid oxidation by approximately 65 and 40%, respectively (P < 0.05); exercise had no effect on VLDL-triglyceride (TG) secretion rate, increased VLDL-TG plasma clearance rate by 40 +/- 8%, and reduced VLDL-TG mean residence time by approximately 40 min and VLDL-apolipoprotein B-100 (apoB-100) secretion rate by 24 +/- 8% (all P < 0.05). Exercise also reduced the number of VLDL but almost doubled the number of IDL particles in plasma (P < 0.05). Muscle lipoprotein lipase content was not different after exercise and rest, but plasma lipoprotein lipase concentration increased by approximately 20% after exercise (P < 0.05). Plasma hepatic lipase and lecithin:cholesterol acyltransferase concentrations were not affected by exercise, whereas cholesterol ester transfer protein concentration was approximately 10% lower after exercise than after rest (P = 0.052). We conclude that 1) greater fatty acid availability after exercise does not stimulate VLDL-TG secretion, probably because of the increase in fatty acid oxidation and possibly also fatty acid use for restoration of tissue TG stores; 2) reduced secretion of VLDL-apoB-100 lowers plasma VLDL particle concentration; and 3) increased VLDL-TG plasma clearance maintains low plasma TG concentration but is not accompanied by similar increases in subsequent steps of the delipidation cascade. Acutely, therefore, the cardioprotective lowering of plasma TG and VLDL concentrations by exercise is counteracted by a proatherogenic increase in IDL concentration.     

Apolipoprotein B gene variants are involved in the determination of blood glucose and lipid levels in patients with non-insulin dependent diabetes mellitus

We have examined the frequency of the EcoRI, XbaI and MspI RFLPs of the apolipoprotein B (apo B) gene in 110 type 2 diabetic patients and 91 healthy control subjects in order to ascertain whether variation in this gene may influence the development of non-insulin dependent diabetes mellitus (type 2 diabetes). Serum lipids including total-cholesterol (T-Chol), triacylglycerol (TAG), apolipoprotein E (apo E), apolipoprotein AI (apo AI), apolipoprotein B and lipoprotein (a) (Lp(a)) were analysed. Genomic DNA was extracted and the apo B polymorphic regions amplified by the polymerase chain reaction. Regions carrying EcoRI, XbaI, and MspI restriction sites present in the apo B gene were amplified and digested separately by the respective enzymes. No significant difference for genotypic frequencies was observed for the EcoRI, XbaI and MspI restriction sites in type 2 diabetic patients as compared to controls. Type 2 diabetic patients and controls with EcoRI +/+ and XbaI +/+ genotypes had higher apo E levels. The MspI +/+ genotype is more frequent in the patient and control groups with elevated T-Chol. Furthermore, the EcoRI -/-, XbaI -/-, and MspI +/+ genotypes were found to be significantly more frequent in type 2 diabetic patients with higher blood glucose levels. This study identifies the apo B gene polymorphisms in modulating plasma lipid/lipoprotein and glucose levels in patients with type 2 diabetes.