Cholinesterases and cell proliferation in “nonstratified” and “stratified” cell aggregates from chicken retina and tectum

Summary Dissociated single cells from chicken retina or tectum kept in rotation-mediated cell culture aggregate, proliferate and establish a certain degree of histotypical cellto-cell relationships (sorting out), but these systems never form highly laminated aggregates (nonstratified R- and T-aggregates). In contrast, a mixture of retinal plus pigment epithelial cells forms highly stratified aggregates (RPE-aggregates, see Vollmer et al. 1984). The present comparative study of stratified and nonstratified aggregates enables us to investigate the process of cell proliferation uncoupled from that of tissue stratification. Here we try to relate these two basic neurogenetic processes with patterns of expression of cholinesterases (AChE, BChE) during formation of both types of aggregates.During early aggregate formation, in both stratified and nonstratified aggregates an increased butyrylcholinesterase activity is observed close to mitotically active cells. Quantitatively both phenomena show their maxima after 2–3 days in culture. In contrast, AChE-expression in all systems increases with incubation time. In nonproliferative areas, in the center of RPE-aggregates, the formation of plexiform layers is characterized initially by weak BChE and then strong AChE-activity. These areas correspond with the inner (IPL) and outer (OPL) plexiform layers of the retina in vivo. Although by sucrose gradient centrifugation we find that the 6S- and the fiber-associated 11S-molecules of AChE are present in all types of aggregates, during the culture period the ratio of 11S/6S-forms increases only in RPE-aggregates, which again indicates the advanced degree of differentiation within these aggregates.It is thus demonstrated that cholinesterases first correlate with neuronal cell proliferation and later with stratification, which indicates functions of both enzymes during both developmental periods.Abbreviations AChE acetylcholinesterase - BChE butyrylcholinesterase - iso-OMPA specific inhibitor of BChE - BW 284C51 specific inhibitor of AChE - IPL inner plexiform layer - OPL outer plexiform layer     

Adenine nucleotide contents and energy charge of Azotobacter vinelandii grown at low phosphate concentration

The effect of a low phosphate concentration on intracellular adenine nucleotide content, oxygen consumption and poly--hydroxybutyrate deposition was investigated with N-free and NH ₄ ⁺ batch cultures of Azotobacter vinelandii. When the microorganisms were cultured under low-phosphate concentrations the cells contained much larger amounts of poly--hydroxybutyrate, but displayed lower oxygen consumption activities and energy charge values than did control cells. Also, the ratio ATP to ADP was much higher in control cells and the intracellular levels of ATP were lower in low-phosphate cells.     

Cloning of a Vero toxin (VT2) gene from a VT2-converting phage isolated from Escherichia coli 0157: H7

Abstract A Vero toxin (VT2 or Shiga-like toxin II)-converting phage was isolated from Escherichia coli 0157: H7 strain J-2. Nontoxigenic E. coli C600 produced VT2 when lysogenized with the toxin-converting phage. Eco RI fragments of the phage DNA were ligated with Eco RI-digested pBR322 or pUC118 and were transformed into E. coli MC1061 or MV1184. Transformants exhibiting VT2 production commonly contained a 4.6 kb Eco RI fragment. It was found that a 2.3 kb Kpn I- Sph I fragment coded VT2 production and that this fragment hybridized weakly with the 2.1 kb fragment encoding VT1.     

Genetic deletions between directly repeated sequences in bacteriophage T7

Summary DNA sequence analysis of genetic deletions in bacteriophage T7 has shown that these chromosomal rearrangements frequently occur between directly repeated DNA sequences. To study this type of spontaneous deletion in more quantitative detail synthetic fragments of DNA, made by hybridizing two complementary oligonucleotides, were introduced into the non-essential T7 gene 1.3 which codes for T7 DNA ligase. This insert blocked synthesis of functional ligase and made the phage that carried an insert unable to form plaques on a host strain deficient in bacterial ligase. The sequence of the insert was designed so that after it is put into the T7 genome the insert is bracketed by direct repeats. Perfect deletion of the insert between the directly repeated sequences results in a wild-type phage. It was found that these deletion events are highly sensitive to the length of the direct repeats at their ends. In the case of 5 bp direct repeats excision from the genome occurred at a frequency of less than 10⁻¹⁰, while this value for an almost identical insert bracketed by 10 bp direct repeats was approximately 10⁻⁶. The deletion events were independent of a hostrecA mutation.     

The organization of repetitive sequences in the albumin and alpha-fetoprotein gene loci in the rat

Summary The distribution of middle repetitive sequences in the genic and extragenic regions of the rat albumin and -fetoprotein genes was analyzed. Their presence was determined by probing Southern blots of restriction fragments of albumin and -fetoprotein genomic subclones with ³²P-labeled total rat DNA. Repetitive sequences were detected in both genes. They were classified as weak, moderate and intense hybridizing elements according to the intensity of hybridization. Weak repetitive sequences were characterized as dG·dT repeats by using ³²P-labeled poly-(dG·dT)(dC·dA) oligomer probe. They occurred in 5 and 3 extragenic regions of the two genes and in introns 4 and 5 of the albumin gene. The moderate repetitive sequence present in intron 6 of the albumin gene was identified as the rat SINES element, 4D12. The intense repetitive sequence, localized in the 3 non-coding region of the albumin gene, corresponded to the terminal segment of a rat high repeat long interspersed DNA family, L1Rn. 4D12 and L1Rn sequences were also scattered throughout the -fetoprotein locus as moderate and intense repetitive elements, respectively, but their distribution was different from that of the albumin genomic region. These results indicate that repetitive sequences invaded the two loci in a non-conservative manner.     

Specificity towards oligomannoside and hybrid type glycans of the endo-β-N-acetylglucosaminidase B from the basidiomy ceteSporotrichum dimorphosporum

We have previously shown that an endo--N-acetylglucosaminidase (EC 3.2.1.96) named Endo B, isolated from culture filtrates of the basidiomyceteSporotrichum dimorphosporum cleaves asialo-, and to some extent, monosialylated bi-antennary glycans of theN-acetyllactosamine type linked to the asparagine residue of peptide or protein moieties [Bouquelet S, Strecker G, Montreuil J, Spik G (1980) Biochimie 62:43–49]. In the present paper, the substrate specificity of the enzyme towards oligomannoside and hybrid type glycans has been analyzed. The results obtained indicate that ovalbumin glycopeptides containing four to seven mannose residues and bovine lactotransferrin glycopeptides containing four to nine mannose residues were completely hydrolyzed by the enzyme. The degree of cleavage was variable among hybrid type structures, since glycopeptides containing the following glycans: (Gal)₁(GlcNAc)₃(Man)₅(GlcNAc)₂; (GlcNAc)₃(Man)₅(GlcNAc)₂; (GlcNAc)₃(Man)₄(GlcNAc)₂ were not hydrolyzed by the enzyme while the percentage of hydrolysis of a glycopeptide containing (GlcNAc)₂(Man)₅(GlcNAc)₂ glycan reached 90%. The bovine lactotransferrin was partially deglycosylated (40%) in the absence of non-ionic detergent while native ovalbumin glycoprotein was not hydrolyzed by the enzyme.The oligomannoside-and theN-acetyllactosamine-type degrading activities present in the culture filtrates were not separated at any step of the purification procedure. Both activities were eluted as a single component with an apparent molecular mass of 89 kDa suggesting that they are located on the same enzyme molecule.Endo B represents a powerful tool for removing oligomannoside-andN-acetyllactosamine-type glycans fromN-glycopeptides andN-glycoproteins. Moreover, advantages in the use of Endo B in a soluble form as well as in an immobilized form result in its high activity and in its stability to heat denaturation and storage.Abbreviations Gal d-galactose - Man d-mannose - GlcNAc N-acetyl-d-glucosamine - Con A concanavalin A - Asn asparagine - GLC gas liquid chromatography - TLC thin layer chromatography - Endo endo--N-acetylglucosaminidase - Endo B endo--N-acetylglucosaminidase isolated fromSporotrichum dimorphosporum - PBE polybuffer exchanger - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis     

Binding of the transition metal ion [(H20)(NH3)5Ru(II)]2+ to nucleosomal core and internucleosomal DNA

The goal of this study is to establish the nature of pentammineruthenium(III) binding to DNA in intact mouse liver nuclei. Also, we wish to determine whether the nucleosomal organization of mouse chromatin has a substantial effect on the relative Ru(III) binding levels of internucleosomal and nucleosomal core DNA. These questions are important because ammineruthenium compounds share chemical and biological properties with the cis-dichlorodiammineplatinum(II) or cisplatin chemotherapeutic agent. Therefore, they represent a potential class of new chemotherapeutic agents. We find that in intact nuclei the predominant DNA binding site for pentammineruthenium(II), followed by air oxidation to pentammineruthenium(III), is N-7 guanine, as is the case with cisplatin. Also, the Ru(III) distribution between internucleosomal and nucleosomal core DNA was found to be nearly identical as probed with three non-specific deoxyribonucleases.     

Gibberellin involvement in dormancy-break and germination of seeds of celery (Apium graveolens L.)

The temperature-dependent, primary dormancy of cv. Florida 683 celery seeds in darkness was partially broken by a 30 min light exposure on the third day of incubation at 20–22°C, resulting in c 50 percent germination after 20 days. This light stimulation was negated by including different inhibitors of gibberellin biosynthesis in the incubation medium. Subsequent addition of a solution of the gibberellins A₄ and A₇ or of the gibberellin-active compound (1-3-chlorophthalimido)-cyclohexane carboxamide (AC94,377) overcame the inhibitory effects on germination of these GA-biosynthesis inhibitors. It is suggested that light stimulates the biosynthesis of gibberellins which are essential for dormancy-break in celery seeds and that this biosynthesis is either directly or indirectly controlled through phytochrome.Abbreviations AC94,377 1-(3-chlorophthalimido)-cyclohexane carboxamide; ancymidol, -cyclopropyl--(4-methoxyphenyl)-5-pyrimidinemethanol - AMO1618 N,N,N-2-tetramethyl-5-(1-methyl ethyl)-4-(1-piperidinylcarbonyl)oxy-benzenaminium chloride - BTS44584 S-2,5-dimethyl-4-pentamethylenecarbamoyloxyphenyl-SS-dimethyl sulphonium - P toluenesulphonate; chlormequat chloride, 2-chloroethyltrimethylammonium chloride; daminozide - N dimethylaminoscuccinamic acid; paclobutrazol, (2RS, 3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1H-1,2,4-triazol-1-yl pentan-3-ol)