Characterization of Caedibacter endonucleobionts from the macronucleus of Paramecium caudatum and the identification of a mutant with blocked R-body synthesis

Cytology, DNA and host-symbiont relationships of x-like endosymbionts from Paramecium caudatum are described. The symbionts (Caedibacter caryophila, sp. nov.) live in the macronuclei of their hosts. They confer the killer trait upon their hosts and appear well adapted to their endonucleobiotic way of life. R bodies (proteinaceous ribbons associated with killing) are produced, but differ significantly from any of the four R-body classes previously described. C. caryophila and their R bodies were isolated. DNA was extracted from purified symbionts and used to demonstrate that one P. caudatum line harbors a natural mutant which is deficient in R-body production. Melting studies indicate a GC content of 34.6%. No sequence homology between the C. caryophila DNA and the coding sequence for type 51 R-body production was observed. C. caryophila is parasitic, causing the death of its hosts in starving cultures.     

Immortalized differentiated hepatocyte lines derived from transgenic mice harboring SV40 T-antigen genes

Hepatocytes of transgenic mouse fetuses harboring SV40 virus transforming gene sequences in the SV delta e-MGH fusion gene construct 202 driven by the mouse metallothionein (MT-I) enhancer [R. D. Palmiter, H. Y. Chen, A. Messing, and R. L. Brinster (1985) Nature (London) 316, 457-460] were cultured at Day 19 of gestation and established as a differentiated line expressing albumin and alpha-fetoprotein (AFP) mRNAs. Hepatocyte line FMH-202 contains integrated SV40 sequences, expresses SV40 T-antigen genes, and exhibits unlimited growth potential because it has been cultured 18 months without apparent decrease in cell viability or in growth rate that could suggest the occurrence of a crisis period. Immortalized cells multiply in chemically defined medium deficient in arginine with transferrin plus insulin, whereas EGF, insulin, and transferrin are obligatory requirements for fetal or newborn mouse hepatocyte multiplication in primary cultures. Cells did not grow in agar and were not tumorigenic in nude mice. Their immortalized, nonmalignant phenotype was further documented by low saturation densities of confluent monolayers showing no overgrowth, and by growth arrest in the absence of insulin with subsequent induction of DNA synthesis and resumption of cell growth in response to insulin. Thus, it appears that immortalized SV40 T-antigen-expressing hepatocytes are present in the liver of the transgenic mice. However, at later points in liver development the transforming activity of T-antigen becomes apparent and leads to hepatocellular carcinoma formation in vivo.     

Hepatocyte heterogeneity in response to icosanoids. The perivenous scavenger cell hypothesis

1. The metabolic and hemodynamic effects of prostaglandin F2 alpha, leukotriene C4 and the thromboxane A2 analogue U-46619 were studied during physiologically antegrade (portal to hepatic vein) and retrograde (hepatic to portal vein) perfusion and in a system of two rat livers perfused in sequence. 2. The stimulatory effects of prostaglandin F2 alpha (3 microM) on hepatic glucose release, perfusion pressure and net Ca2+ release were diminished by 77%, 95% and 64%, respectively, during retrograde perfusion when compared to the antegrade direction, whereas the stimulation of 14CO2 production from [1-14C]glutamate by prostaglandin F2 alpha (which largely reflects the metabolism of perivenous hepatocytes) was lowered by only 20%. Ca2+ mobilization and glucose release from the liver comparable to that seen during antegrade perfusion could also be observed in retrograde perfusions; however, higher concentrations of the prostaglandin were required. 3. The glucose, Ca2+ and pressure response to leukotriene C4 (20 nM) or the thromboxane A2 analogue U-46619 (200 nM) of livers perfused in the antegrade direction were diminished by about 90% during retrograde perfusion. Sodium nitroprusside (20 microM) decreased the pressure response to leukotriene C4 (20 nM) and U-46619 (200 nM) by about 40% and 20% in antegrade perfusions, respectively, but did not affect the maximal increase of glucose output. 4. When two livers were perfused antegradely in series, such that the perfusate leaving the first liver (liver I) entered a second liver (liver II), infusion of U-46619 at concentrations below 200 nM to the influent perfusate of liver I increased the portal pressure of liver I, but not of liver II. At higher concentrations of U-46619 there was also an increase of the portal pressure of liver II and with concentrations above 800 nM the pressure responses of both livers were near-maximal [19.6 +/- 0.8 (n = 7) cm H2O and 16.5 +/- 1.1 (n = 8) cm H2O for livers I and II, respectively]. There was a similar behaviour of glucose release from livers I and II in response to U-46619 infusion. When liver I was perfused in the retrograde direction, a significant pressure or glucose response of liver II (antegrade perfusion) could not be observed even with U-46619 concentrations up to 1000 nM. 5. Similarly, the perfusion pressure increase and glucose release induced by leukotriene C4 (10 nM) observed with liver II was only about 20% of that seen with liver I.(ABSTRACT TRUNCATED AT 250 WORDS)     

The tertiary structure of Aspergillus saitoi minor ribonuclease (Ms) predicted from the structure of RNase T1

Ribonuclease Ms from Aspergillus saitoi is a small acidic protein (11 714 Da) containing 106 amino acids of known sequence. Unlike other enzymes belonging to the RNase T₁ family this ribonuclease is base-unspecific. Using interactive computer graphics and energy minimisation we predicted the structure of RNase Ms on the basis of sequence homology to RNase T₁ of known structure. In this report the predicted structure of this protein is presented and characterised.     

Complete amino acid sequence of human intestinal aminopeptidase N as deduced from cloned cDNA

The complete primary structure (967 amino acids) of an intestinal human aminopeptidase N (EC 3.4.11.2) was deduced from the sequence of a cDNA clone. Aminopeptidase N is anchored to the microvillar membrane via an uncleaved signal for membrane insertion. A domain constituting amino acid 250-555 positioned within the catalytic domain shows very clear homology to E. coli aminopeptidase N and contains Zn2+ ligands. Therefore these residues are part of the active site. However, no homology of the anchor/junctional peptide domain is found suggesting that the juxta- and intra-membraneous parts of the molecule have been added/preserved during development. It is speculated that this part carries the apical address.     

Mutations of plasmid pBS286 blocking the initial stages of naphthalene oxidation induced by Tn5

A collection of Tn5 transposon Nah- mutants of the plasmid pBS286 was obtained. The insertion sites were localized and orientation of Tn5 determined. The mutants obtained were biochemically analyzed, the nah-region map of the plasmid being elaborated. Structural genes of the nah operon were shown to be organized similarly to those of the nah1 operon of the NAH7 plasmid discussed in the literature. The data obtained are in favour of the previously published information on the presence of elements operating the pBS286 plasmid. The results are given indicating a possibility of regulating the expression of catechol splitting meta-pathway genes with participation of products on early stages of naphthalene oxidation.     

Recapitulation in the development of the components of the counterflow system in the vertebrate metanephros

The investigation has been performed on 107 renal preparations obtained from persons of various age (from 5-month-old fetuses up to 45 years of age), certain representatives of other classes of the Vertebrata are also included: fish, amphibia, reptile and mammalia at various stages of pre- and postnatal periods of ontogenesis by means of preparing graphic and plastic reconstructive models, histological investigation and microdissection. The complexity of the intrarenal branching of derivatives of the mesonephric duct diverticulum, development and structure of the canalicular part in nephrons directly depend on the phylogenetic position of the animal. Complexity of the nephron architectonics occurs along the progressive line of taxonomic groups of higher Vertebrata. The nephron loop becomes longer, thin segment of the nephron canalicular part increases in its length and, at last, in mammalia a cone-shaped fasciculus appears as a structural-functional unit of the osmoregulating apparatus of the constant kidney. In the comparative anatomical and comparative embryological aspects recapitulation is observed concerning certain morphological signs of derivatives of the metanephric duct and nephron.     

Phenolsulphonphthalein conjugation and biliary excretion in the rat: influence of phenobarbital and 3-methylcholanthrene

The effect of phenobarbital and 3-methylcholanthrene pretreatment on the biliary excretion of phenolsulphonphthalein (PSP) was investigated in male Wistar rats. The dye was injected at a single dose of 200 mumol/kg body wt. About 20% of the compound was excreted as a glucuronide in the controls, the liver UDP-glucuronyltransferase activity toward PSP being 0.064 +/- 0.005 nmol.min-1.mg protein-1. Treatment for two weeks with phenobarbital (354 mumol.kg body wt-1.day-1) caused a transient increase in conjugated and unconjugated PSP excretion, but glucuronyltransferase activity was not modified. 3-Methylcholanthrene pretreatment for 4 days (75 mumol.kg body wt-1.day-1) also enhanced biliary excretion of the dye, but the increase corresponded only to the glucuronide and glucuronyltransferase activity was significantly enhanced by 20%. Our data indicate that not only the rate of biotransformation but also other factors could be responsible for increased PSP biliary excretion following administration of microsomal enzyme inducers.     

Molecular and catalytic properties of a butyrylesterase from human red cells and brain

A butyrylesterase from human red cells was prepared to homogeneity using DEAE-cellulose, Ultrogel ACA-34, DEAE-Sephacel, and precipitation with 1.5 M (NH4)2SO4. The yield was 25-35% relative to the enzyme activity of the hemolysate. Because of its preference for butyric acid esters the enzyme was designated a butyrylesterase. With alpha-naphthyl butyrate the Km was 7.6 microM and the kcat, 48 s-1. The molecular weight was 340,000 and the subunit weight 85,000, indicating a tetrameric structure. The isoelectric pH was 4.0. The enzyme preparation did not contain cystine. Sialic acid or other carbohydrate components could not be detected. The enzyme was irreversibly inhibited by organophosphate esters and the second-order rate constant was 192 M-1 s-1 for diethyl p-nitrophenyl phosphate. For the brain enzyme the constant was 206 M-1 s-1. The enzyme was irreversibly inhibited by sulfhydryl reagents, indicating that the enzyme is a sulfhydryl-dependent serine esterase. The enzyme was identical to the butyrylesterase from human brain, and the two enzymes were immunochemically identical. An amino acid ester has been shown to be split at a higher rate than butyric acid esters; however, the specificity constant (kcat/Km) was lower for the amino acid ester than for the butyric acid ester. The enzyme did not exhibit amidase activity.