Frequency of sex chromosomal mosaicism in bovine embryos and its effects on sexing using a single blastomere by PCR

Assessment of nuclear status is important when a biopsied single blastomere is used for embryo sexing. In this study we investigated the nuclear status of blastomeres derived from 8- to 16-cell stage in vitro fertilised bovine embryos to determine the representativeness of a single blastomere for embryo sexing. In 24 embryos analysed, the agreement in sex determination between a biopsied single blastomere and a matched blastocyst by polymerase chain reaction (PCR) was 83.3%. To clarify the discrepancies, karyotypes of blastomeres in 8- to 16-cell stage bovine embryos were analysed. We applied vinblastine sulfate at various concentrations and for different exposure times for metaphase plate induction in 8- to 16-cell stage bovine embryos. The 1.0 mg/ml vinblastine sulfate treatment for 15 h was selected as the most effective condition for induction of a metaphase plate (> 45%). Among 22 embryos under these conditions, only 8 of 10 that had a normal diploid chromosome complement showed a sex chromosomal composition of XX or XY (36.4%) and 2 diploid embryos showed mosaicism of the opposite sex of XX and XY in blastomeres of the embryo (9.1%). One haploid embryo contained only one X-chromosome (4.5%). Four of another 11 embryos with a mixoploid chromosomal complement contained a haploid blastomere with a wrong sex chromosome (18.2%). In conclusion, assessment of nuclear status of 8- to 16-cell stage bovine embryos revealed that morphologically normal embryos had a considerable proportion of mixoploid blastomeres and sex chromosomal mosaicism; these could be the cause of discrepancies in the sex between biopsied single blastomeres and matched blastocysts by PCR.     

Mapping the binding interface of the cytochrome b5-cytochrome c complex by nuclear magnetic resonance

The interaction between bovine cytochrome b(5) (cyt b(5)) and horse heart cytochrome c (cyt c) is investigated by NMR spectroscopy. Chemical shifts of cyt b(5) backbone resonances and side chain methyl resonances were monitored as a function of cyt c concentration. The shifts are small but saturatable and indicate that the binding of cyt b(5) with cyt c is in fast exchange. An equilibrium association constant of (6 +/- 3) x 10(4) M(-1) was obtained with a lower limit of 180 s(-1) for the dissociation rate of the complex. To resolve considerable ambiguities in the interpretation of the chemical shift mapping, (15)N relaxation experiments and cross-saturation experiments were used as alternative methods to map the cyt b(5)-cyt c binding interface. Results from the three experiments combined demonstrate that the conserved negatively charged region of cyt b(5) surrounding the solvent-exposed heme edge is involved in the interaction with cyt c. These data support the models proposed by Salemme and Mauk [(1976) J. Mol. Biol. 102, 563-568; (1993) Biochemistry 32, 6613-6623].     

Interference of ferric ions with ferrous iron quantification using the ferrozine assay

The ferrozine assay is a widely used colorimetric method for determining soluble iron concentrations. We provide evidence for a heretofore unrecognized interference of ferric ions (Fe^3 +) on ferrous iron (Fe^2 +) measurements performed in the dark. Fe^3 + concentrations affected the absorbance measurements, which linearly increased with incubation time.     

Overexpression of A-kinase anchoring protein 12A activates sterol regulatory element binding protein-2 and enhances cholesterol efflux in hepatic cells

A-kinase anchoring protein 12 (AKAP12) is known to function as a scaffold protein and as a putative tumor suppressor. However, little is known about the biological role of AKAP12 in hepatic cells. In this study, we performed micro-array analysis to identify the downstream pathway of AKAP12A, and found that AKAP12A overexpression up-regulates the expressions of several cholesterol-associated genes including HMG-CoA reductase and LDL receptor, which have been reported to be controlled by sterol regulatory element binding protein-2 (SREBP-2). It was found that AKAP12A activates SREBP-2 in hepatic cells, as demonstrated by the presence of its cleavage product, whereas the activation of sterol regulatory element binding protein-1 was not remarkably changed. Moreover, AKAP12A-induced SREBP-2 activation was found to depend on SREBP cleavage-activating protein (SCAP), as inhibition of SCAP by RNAi or sterols blocked SREBP-2 activation in response to AKAP12A overexpression. Interestingly, the hydrophobic amine U18666A caused dramatic movement of AKAP12A from the plasma membrane to cytosol and lysosomal membranes. Moreover, cholesterol depletion from the plasma membrane (using methyl-beta-cyclodextrin) caused a shift of AKAP12A from the plasma membrane to the cytoplasm. Cholesterol binding assay revealed that the N-terminal region of AKAP12A binds directly to cholesterol in vitro. Furthermore, AKAP12A overexpression enhanced [3H]-cholesterol efflux to extracellular acceptors, suggesting that AKAP12A may activate SREBP-2 by increasing cholesterol efflux. In conclusion, the present study suggests that AKAP12A is a novel regulator of cellular cholesterol metabolism.     

A Repulsive Electrostatic Mechanism for Protein Export through the Type III Secretion Apparatus

Many Gram-negative bacteria initiate infections by injecting effector proteins into host cells through the type III secretion apparatus, which is comprised of a basal body, a needle, and a tip. The needle channel is formed by the assembly of a single needle protein. To explore the export mechanisms of MxiH needle protein through the needle of Shigella flexneri, an essential step during needle assembly, we have performed steered molecular dynamics simulations in implicit solvent. The trajectories reveal a screwlike rotation motion during the export of nativelike helix-turn-helix conformations. Interestingly, the channel interior with excessive electronegative potential creates an energy barrier for MxiH to enter the channel, whereas the same may facilitate the ejection of the effectors into host cells. Structurally known basal regions and ATPase underneath the basal region also have electronegative interiors. Effector proteins also have considerable electronegative potential patches on their surfaces. From these observations, we propose a repulsive electrostatic mechanism for protein translocation through the type III secretion apparatus. Based on this mechanism, the ATPase activity and/or proton motive force could be used to energize the protein translocation through these nanomachines. A similar mechanism may be applicable to macromolecular channels in other secretion systems or viruses through which proteins or nucleic acids are transported.     

Mutation at Intronic Repeats of the Ataxia-Telangiectasia Mutated (ATM) Gene and ATM Protein Loss in Primary Gastric Cancer with Microsatellite Instability

Ataxia-telangiectasia mutated (ATM) is a Ser/Thr protein kinase that plays a critical role in DNA damage-induced signaling and initiation of cell cycle checkpoint signaling in response to DNA-damaging agents such as ionizing radiation. We have previously reported the ATM protein loss by immunohistochemistry (IHC) in 16% of human gastric cancer (GC) tissue. We hypothesized that ATM gene intron mutations targeted by microsatellite instability (MSI) cause ATM protein loss in a subset of GC. We studied mononucleotide mutations at the intron of ATM gene, ATM IHC and MSI in GC. Ten human gastric cancer cell lines were studied for the ATM gene mutation at introns, RT-PCR, direct sequencing, and immunohistochemistry. GC tissues of 839 patients were analyzed for MSI and ATM IHC. Among them, 604 cases were analyzed for the ATM mutations at introns preceding exon 6, exon 10 and exon 20. Two human GC cell lines (SNU-1 and -638) showed ATM intron mutations, deletion in RT-PCR and direct sequencing, and ATM protein loss by IHC. The frequencies of ATM mutation, MSI, and ATM protein loss were 12.9% (78/604), 9.2% (81/882) and 15.2% (134/839), respectively. Analysis of associations among MSI, ATM gene mutation, and ATM protein loss revealed highly co-existing ATM gene alterations and MSI. ATM intron mutation and ATM protein loss were detected in 69.3% (52/75) and 53.3% (40/75) of MSI positive GC. MSI positivity and ATM protein loss were present in 68.4% (52/76) and 48.7% (37/76) of GC with ATM intron mutation. ATM mutation and ATM protein loss had characteristics of old age, distal location of tumor, large tumor size, and histologic intestinal type. Our study might be interpreted as that ATM gene mutation at intron might be targeted by MSI and lead to ATM protein loss in a selected group of GC.     

Decline of dark coniferous stands in Baikal Region

The reasons for the decline in Siberian pine and fir in the Baikal Region (Khamar-Daban) were analyzed using remote sensing techniques, dendrochronology and GIS-technology methods, and in situ observations. It is found that a decrease in the value of the growth index (R ² = 0.69) and an decrease in the SPEI drought index (Standardized Precipitation–Evapotranspiration Index) (R ² = 0.72) has been observed since the 1980s. In the mid-2000s, the increase in aridity led to the division of Siberian pine trees into two cohorts: “survivors” and “decliners.” The spatial distribution of these cohorts is different: dead and declining stands are localized mainly on relief elements with increased risk of water stress (steep and convex slopes of southwestern exposure). The growth index of the trees is closely related to the dryness index in June (r ² = 0.55). Along with water stress, declining trees were also exposed to stem pests and plant pathogens. The primary cause of Siberian pine decline is water stress due to the increasing climate aridity. The weakened waterstressed trees were sensitized to pathogens. The synergism of climatic and biotic effects led to the decline of Siberian pine stands. On the whole, heavily damaged and declining stands (over 50% of dead and declining trees) within the Khamar-Daban ridge are 8–10% of the total area of dark coniferous forests.     

Association of CD38 with nonmuscle myosin heavy chain IIA and Lck is essential for the internalization and activation of CD38

Activation of CD38 in lymphokine-activated killer (LAK) cells involves interleukin-8 (IL8)-mediated protein kinase G (PKG) activation and results in an increase in the sustained intracellular Ca(2+) concentration ([Ca(2+)](i)), cADP-ribose, and LAK cell migration. However, direct phosphorylation or activation of CD38 by PKG has not been observed in vitro. In this study, we examined the molecular mechanism of PKG-mediated activation of CD38. Nonmuscle myosin heavy chain IIA (MHCIIA) was identified as a CD38-associated protein upon IL8 stimulation. The IL8-induced association of MHCIIA with CD38 was dependent on PKG-mediated phosphorylation of MHCIIA. Supporting these observations, IL8- or cell-permeable cGMP analog-induced formation of cADP-ribose, increase in [Ca(2+)](i), and migration of LAK cells were inhibited by treatment with the MHCIIA inhibitor blebbistatin. Binding studies using purified proteins revealed that the association of MHCIIA with CD38 occurred through Lck, a tyrosine kinase. Moreover, these three molecules co-immunoprecipitated upon IL8 stimulation of LAK cells. IL8 treatment of LAK cells resulted in internalization of CD38, which co-localized with MHCIIA and Lck, and blebbistatin blocked internalization of CD38. These findings demonstrate that the association of phospho-MHCIIA with Lck and CD38 is a critical step in the internalization and activation of CD38.