DNA replication initiates non-randomly at multiple sites near the c-myc gene in HeLa cells.

The origin of replication of the c-myc gene in HeLa cells was previously identified at low resolution within 3.5 kb 5' to the P1 promoter, based on replication fork polarity and the location of DNA nascent strands. To define the initiation events in the c-myc origin at higher resolution the template bias of nascent DNAs in a 12 kb c-myc domain has been analyzed by hybridization to strand specific probes. Strong switches in the asymmetry of nascent strand template preference confirm that replication initiates non-randomly at multiple sites within 2.4 kb 5' to the c-myc P1 promoter, and at other sites over a region of 12 kb or more. The strongest template biases occur in the 2.4 kb region 5' of the P1 promoter, shown earlier to contain sequences which allow the autonomous semiconservative replication of c-myc plasmids. An asymmetric pyrimidine heptanucleotide consensus sequence has been identified which occurs 12 times in the c-myc origin zone, and whose polarity exactly correlates with the polarity of nascent strand synthesis.     

Oxide-Based Solid-State Batteries: A Perspective on Composite Cathode Architecture

The garnet-type phase Li₇La₃Zr₂O₁₂ (LLZO) attracts significant attention as an oxide solid electrolyte to enable safe and robust solid-state batteries (SSBs) with potentially high energy density. However, while significant progress has been made in demonstrating compatibility with Li metal, integrating LLZO into composite cathodes remains a challenge. The current perspective focuses on the critical issues that need to be addressed to achieve the ultimate goal of an all-solid-state LLZO-based battery that delivers safety, durability, and pack-level performance characteristics that are unobtainable with state-of-the-art Li-ion batteries. This perspective complements existing reviews of solid/solid interfaces with more emphasis on understanding numerous homo- and heteroionic interfaces in a pure oxide-based SSB and the various phenomena that accompany the evolution of the chemical, electrochemical, structural, morphological, and mechanical properties of those interfaces during processing and operation. Finally, the insights gained from a comprehensive literature survey of LLZO–cathode interfaces are used to guide efforts for the development of LLZO-based SSBs.     

Structural and systematic study of an unusual tracheid type in cacti

Wide-band tracheids are a specialized tracheid type in which an annular or helical secondary wall projects deeply into the cell lumen. They are short, wide and spindle-shaped, and their bandlike secondary walls cover little of the primary wall, leaving most of it available for water diffusion. Wide-band tracheids appear to store and conduct water while preventing the spread of embolisms. They may be the most abundant tracheary element in the xylem, but they are always accompanied by at least a few vessels. Typically, fibers are absent wherever wide-band tracheids are present. Wide-band tracheids occur in the primary and secondary xylem of succulent stems, leaves and roots in genera of all three subfamilies of Cactaceae but were not found in the relictual genusPereskia, which lacks succulent tissues. In the large subfamily Cactoideae, wide-band tracheids occur only in derived members, and wide-band tracheids of North American Cactoideae are narrower and are aligned in a more orderly radial pattern than those of South American Cactoideae. Wide-band tracheids probably arose at least three times in Cactaceae.     

Methylation and expression of a metallothionein promoter ovine growth hormone fusion gene (MToGH1) in transgenic mice

We have examined transgene methylation in the DNA from the livers of a pedigree of mice carrying three copies of an integrated MToGH1 transgene. Utilizing the methylation-sensitive isoschizomersMsp I andHpa II, Southern blot analysis revealed that all second generation animals derived from a transgenic female had hypermethylated DNA, whereas first generation animals sired by a transgenic male displayed a range of methylation phenotypes ranging from no methylation to hypermethylation of the transgene sequences. Of the mice that exhibited hypermethylation of the transgene in CpG dinucleotides (CmCGG), a minority of these animals also exhibited apparent CpC methylation (i.e. inhibition ofMsp I cutting, presumably blocked by methylation of the outer C of CCGG). Methylation was also examined in the inner C of CC(A/T)GG sequences in the MToGH1 transgene using the isoschizomer pairBstN I andEcoR II. A minority of MToGH1 animals in the F₁ generation showed clear evidence of methylation in these sites as well as in the inner and outer Cs of CCGG sites. An examination of MToGH1 expression in terms of oGH levels in serum revealed that there was a high degree of variation in the levels of circulating oGH between animals of this pedigree. There was a weak inverse relationship between the serum level of oGH and the extent of methylation of the transgene. In particular, mice exhibiting CpC together with CpG methylation were found to have very low levels of circulating oGH. Our results highlight the nature and complexity of epigenetic factors associated with transgene sequences which may ultimately influence expression of introduced genes in the mammalian genome.     

PURIFICATION AND PROPERTIES OF CHOLINE ACETYLTRANSFERASE FROM TORPEDO CALIFORNICA

Abstract— Choline acetyltransferase (ChAT), the enzyme responsible for the biosynthesis of acetylcholine in nervous tissue, has been purified to apparent homogeneity from the electric organ of the electric fish Torpedo californica using ion-exchange, gel filtration, and hydroxyapatite chromatography. The final preparation had been purified 8570-fold to a specific activity of 30μmol ACh formed/min/mg protein. The purified protein has a pH optimum of 6.8 (phosphate buffer), is activated by low concentrations (ca. 10 m m ) of ammonium or alkylammonium ions, and is strongly inhibited by a sulfhydryl blocking reagent (DTNB). ChAT has a mol. wt. of 63000 when measured by SDS-polyacrylamide gel electrophoresis or gel filtration.
A new method for the rapid assay of ChAT activity is described in which unreacted substrate ([³H]acetyl-CoA) is removed from reaction mixtures by adsorption to charcoal: some advantages of this technique are discussed.     

Glucocorticoids inhibit estradiol-mediated uterine growth: possible role of the uterine estradiol receptor

Stress-related activation of the hypothalamic-pituitary-adrenal axis (HPA) is associated with suppression of the reproductive axis. This effect has been explained by findings indicating that corticotropin-releasing hormone suppresses hypothalamic gonadotropin-releasing hormone (GnRH) secretion via an opioid peptide-mediated mechanism, and that glucocorticoids suppress both GnRH and gonadotropin secretion and inhibit testosterone and estradiol production by the testis and ovary, respectively. To evaluate whether glucocorticoids suppress the effects of estradiol on its target tissues, we examined the ability of dexamethasone to inhibit estradiol-stimulated uterine and thymic growth in ovariectomized rats. Estradiol alone, given daily for 5 days, caused dose-dependent uterine and thymic growth. Dexamethasone alone, given daily for 5 days, caused a dose-dependent decrease in body weight gain and in thymic growth. When estradiol and dexamethasone were administered simultaneously, however, body weight gain and thymic growth were also inhibited (p less than 0.05). Dexamethasone decreased estradiol-induced uterine cytosolic and nuclear estrogen receptor concentrations (E2 R0, p less than 0.05; E2nR0, respectively), but had no effect on estradiol-induced progesterone receptor concentrations (P4R0, p greater than 0.05). Levels of uterine glucocorticoid receptors were not affected by estrogen and/or dexamethasone treatment. These findings suggest that stress levels of glucocorticoids, administered over a 5-day interval, block the estradiol-stimulated growth of female sex hormone target tissues. This effect may be partially mediated by a glucocorticoid-induced decrease of the estradiol receptor concentration. Thus, another mechanism by which the HPA may influence reproductive function during stress is by a direct effect of glucocorticoids on the target tissues of sex steroids.     

Purification of human C3b inactivator by monoclonal-antibody affinity chromatography

Monoclonal antibody has been obtained to the human complement control protein C3b inactivator after immunization of mice with the enzyme prepared by conventional methods. Antibody from ascitic fluid was purified and coupled to Sepharose-CL-4B to give a specific affinity column, which was used to isolate C3b inactivator from human serum in 70% yield. The product was characterized by size, chain structure, amino acid analysis and proteolytic activity.