Genetic analysis of a mutation affecting ribosomal protein S1 in Escherichia coli

Molecular Genetics and Genomics - Ribosomal protein S1 from a newly isolated Escherichia coli mutant has a molecular weight of about 54,000 which is smaller than the wild type S1 (M.W. 65,000). The...     

Re-examination ofPyrenomonas andRhodomonas (class cryptophyceae) through ultrastructural survey of red pigmented cryptomonads

Several taxa of cryptomonads, including species of marineChroomonas, Cryptomonas and freshwaterRhodomonas were examined using transmission electron microscopy. They have cellular structures fundamentally in common: a single bilobed chlorplast, a single pyrenoid between the chloroplast lobes, and a nuclemorph embedded within a cleft of the pyrenoidal matrix. These features are in accordance with the taxonomic characteristics of the recently established genusPyrenomonas. The algae also have similar pigmentation to that ofRhodomonas andPyrenomonas which is red or reddish-brown. On the basis of these observations, the genusRhodomonas Karsten (1898) is redescribed in this paper and the genusPyrenomonas Santore is considered to be synonymous withRhodomonas.     

Pneumocystis carinii infection in corticosteroid-treated cats

Corticosteroids were administered to produce Pneumocystis carinii infection in cats. Six of 10 cats, injected intramuscularly for 97-141 days with 2 mg/cat twice weekly of betamethasone sodium phosphate, developed a light infection with P. carinii. Six of 7 cats, injected intramuscularly for 11-168 days with 10-25 mg/cat weekly of prednisolone acetate, also developed a light infection with P. carinii. There was no significant difference in the infection rate between the sexes and ages of the cats. Using Giemsa staining and Gomori's methenamine silver nitrate stain, P. carinii organisms were indistinguishable morphologically from human and rat P. carinii. The cysts and trophozoites were usually present singly or in small groups, and they always were adhering to the periphery of alveoli. The inflammatory changes were inconspicuous except for the fact that alveolar macrophages often were seen. Corticosteroid-treated cats should be useful in the study of experimental P. carinii infection. This is the first reported case of experimentally induced P. carinii infection in cats.     

Development of the fetal mouse palate in suspension organ culture

Explanted palates of day 12 and day 13 mouse fetuses were cultured in a chemically defined serumless medium for 48-72 h by a suspension culture technique. The palate of day 12 fetuses closed successfully within 72 h and that of day 13 fetuses within 48 h. Both macroscopically and histologically, the in vitro fusion of palatal shelves simulated the palatogenetic process in vivo. This novel technique for culturing the fetal mouse palate may be of potential use for the study of palatogenesis and in developmental toxicology.     

The oxidative cleavage of folates. A critical study

Alkaline permanganate oxidation has been used to determine the chain length of naturally occurring pteroylpolyglutamates on the assumption that all forms of folates cleave at the C⁹N¹⁰ bond to produce the corresponding p-aminobenzoyl-polyglutamates. The chain length of the latter could be determined by cochromatography with synthetic markers. The products of alkalinc (ammonium bicarbonate buffer, pH 9.0) permanganate oxidation of a number of reduced and oxidized, one-carbon-substituted and unsubstituted folic acid derivatives have been identified, and their yields and stability to the oxidative treatment have been determined. Unsubstituted, oxidized and reduced folic acid and N⁵-formyl-tetrahydrofolic acid are cleaved at the C⁹N¹⁰ bond to produce p-aminobenzoylglutamic acid. N⁵, N¹⁰-methenyl-tetrahydrofolic acid, N⁵,N¹⁰-methylene-tetrahydrofolic acid, and N¹⁰-formyl-tetrahydrofolic acid are not cleaved but are oxidized to N¹⁰-formyl-folic acid which is completely stable to the oxidative treatment employed. N⁵-methyl-tetrahydrofolic acid is not cleaved either but is oxidized to N⁵-methyl-dihydrofolic acid which upon continued oxidation decomposes slowly to unidentified products. The γ-glutamyl peptide linkage is completely stable to oxidation. Using p-amino-[3,5-³H]benzoylglutamic acid, it is also shown that this product, previously thought to be stable to the oxidative treatment is decomposed by it. The significance of these findings in terms of the errors that may have been introduced in prior estimations of the chain length and pool sizes of the naturally occurring pteroylpolyglutamates is discussed. The possibility of developing a method for the chain length determination of noncleavable pools of one-carbon-substituted folates using [2-¹⁴C]folic acid to label the folates in vivo is presented.     

1H NMR studies of deuterated ribonuclease HI selectively labeled with protonated amino acids

Summary Two-dimensional (2D)¹H NMR experiments using deuterium labeling have been carried out to investigate the solution structure of ribonuclease HI (RNase HI) fromEscherichia coli (E. coli), which consists of 155 amino acids. To simplify the¹H NMR spectra, two fully deuterated enzymes bearing several prototed amino acids were prepared from an RNase HI overproducing strain ofE. coli grown in an almost fully deuterated medium. One enzyme was selectively labeled by protonated His, He. Val. and Leu. The other was labeled by only protonated His and Ile. The 2D¹H NMR spectra of these deuterated R Nase H1 proteins, selectively labeled with protonated amino acids, were much more simple than those of the normally protonated enzyme. The simplified spectra allowed unambiguous assignments of the resonance peaks and connectivities in COSY and NOESY for the side-chain protons. The spin-lattice relaxation times of the side-chain protons of the buried His residue of the deuterated enzyme became remarkably longer than that of the protonated enzyme. In contrast, the relaxation times of the side-chain protons of exposed His residues remained essentially unchanged.     

Sonic hedgehog is involved in formation of the ventral optic cup by limiting Bmp4 expression to the dorsal domain

Accumulating evidence suggests that Sonic hedgehog (Shh) signaling plays a crucial role in eye vesicle patterning in vertebrates. Shh promotes expression of Pax2 in the optic stalk and represses expression of Pax6 in the optic cup. Shh signaling contributes to establishment of both proximal–distal and dorsal–ventral axes by activating Vax1, Vax2, and Pax2. In the dorsal part of the developing retina, Bmp4 is expressed and antagonizes the ventralizing effects of Shh signaling through the activation of Tbx5 expression in chick and Xenopus. To examine the roles of Shh signaling in optic cup formation and optic stalk development, we utilized the Smoothened (Smo) conditional knockout (CKO) mouse line. Smo is a membrane protein which mediates Shh signaling into inside of cells. Cre expression was driven by Fgf15 enhancer. The ventral evagination of the optic cup deteriorated from E10 in the Smo-CKO, whereas the dorsal optic cup and optic stalk develop normally until E11. We analyzed expression of various genes such as Pax family (Pax2/Pax6), Vax family (Vax1/Vax2) and Bmp4. Bmp4 expression was greatly upregulated in the optic vesicle by the 21-somite stage. Then Vax1/2 expression was decreased at the 20- to 24-somite stages. Pax2/6 expression was affected at the 27- to 32-somite stages. Our data suggest that the effects of the absence of Shh signaling on Vax1/Vax2 are mediated through increased Bmp4 expression throughout the optic cup. Also unchanged patterns of Raldh2 and Raldh3 suggest that retinoic acid is not the downstream to Shh signaling to control the ventral optic cup morphology.     

DNA methylation profiles provide a viable index for porcine pluripotent stem cells

Porcine induced pluripotent stem cells (iPSCs) provide useful information for translational research. The quality of iPSCs can be assessed by their ability to differentiate into various cell types after chimera formation. However, analysis of chimera formation in pigs is a labor‐intensive and costly process, necessitating a simple evaluation method for porcine iPSCs. Our previous study identified mouse embryonic stem cell (ESC)‐specific hypomethylated loci (EShypo‐T‐DMRs), and, in this study, 36 genes selected from these were used to evaluate porcine iPSC lines. Based on the methylation profiles of the 36 genes, the iPSC line, Porco Rosso‐4, was found closest to mouse pluripotent stem cells among 5 porcine iPSCs. Moreover, Porco Rosso‐4 more efficiently contributed to the inner cell mass (ICM) of blastocysts than the iPSC line showing the lowest reprogramming of the 36 genes (Porco Rosso‐622‐14), indicating that the DNA methylation profile correlates with efficiency of ICM contribution. Furthermore, factors known to enhance iPSC quality (serum‐free medium with PD0325901 and CHIR99021) improved the methylation status at the 36 genes. Thus, the DNA methylation profile of these 36 genes is a viable index for evaluation of porcine iPSCs. genesis 51:763–776. © 2013 Wiley Periodicals, Inc.