Expression patterns of mRNAs for α-fetoprotein and albumin in the developing rat: the ontogenesis of hepatocyte heterogeneity

Summary In developing and normal adult rat liver the expression patterns of the mRNAs for -fetoprotein (AFP) and albumin (ALB) were analysed byin situ hybridization using specific³⁵S-labelled complementary DNA probes. In the developing liver AFP and ALB mRNA are found from embryonic day (ED) 11 and 12, respectively, onward. At ED 20 the first signs of a zonal distribution of these mRNAs across the liver lobule can be observed, AFP mRNA concentration being higher in the pericentral area and ALB mRNA concentration higher in the periportal area. This distribution pattern of reciprocal, overlapping gradients of mRNA can be clearly recognized in the neonatal period. In the adult liver AFP mRNA can no longer be detected and similar to the neonatal situation, ALB mRNA is expressed across the entire porto-central distance decreasing in concentration going from the portal to the central area.Transient extra-hepatic expression of AFP mRNA is found in the embryonic heart and in the epithelial lining of intestine and lung furthermore, AFP and ALB mRNA are found to be transiently expressed in the developing renal tubules. Similar expression patterns have been observed for other liver-characteristic mRNAs (Moormanet al., 1990), suggesting that common regulatory factors are operative during development.     

Functional analysis of Arg-308 mutants of Flp recombinase. Possible role of Arg-308 in coupling substrate binding to catalysis

The arginine residue at position 308 in the Flp recombinase corresponds to the only invariant arginine within the Int family of recombinases. Alterations of this residue result in Flp variants that retain substrate recognition, but form weaker protein-DNA complexes than wild type Flp. Furthermore, their DNA cleavage activity is significantly diminished. A conservative change of R308K results in a functional Flp variant; however, this protein has a lowered temperature optimum for recombination. The Arg-308 mutants can be stabilized on the DNA substrate through cooperativity with a partner Flp mutant that is tight binding. Thus, interactions between Flp monomers must be a relevant feature of the normal recombination reaction.     

Cysteine-108 is an acylation site in myelin proteolipid protein.

Myelin proteolipid protein (PLP) contains covalently bound long-chain fatty acids. A large proportion of these acyl moieties are bound in thioester linkages, as demonstrated by alkylation of newly formed SH groups upon deacylation. To identify the Cys residue(s) involved in the thioester linkage(s), reduced and carboxyamidomethylated proteolipid protein was labeled with [14C]iodoacetamide upon deacylation with neutral hydroxylamine. The labeled protein was digested with trypsin or pepsin, and peptides analyzed by RP-HPLC. Identification of the isolated radioactive peptides by amino acid analysis, peptide sequencing and/or fast-atom bombardment-mass spectrometry revealed that Cys108 in the bovine PLP sequence is an acylated site. The sequence surrounding the palmitoylation site in the myelin PLP is strikingly similar to that found in rhodopsin. Furthermore, as in rhodopsin and other members of the G protein-coupled receptor family, this Cys residue is located within a hydrophilic, basic, and possibly cytoplasmic, domain.     

Cation radius effects on the helix-coil transition of DNA. Cryptates and other large cations.

Most polyelectrolyte theories of the effect of ions on the thermal melting of DNA assume that the predominant influence of the cations comes through their charge. Ion size and structure are treated, for analytic convenience, as negligible variables. We have examined the validity of this assumption by measuring the melting temperature of calf thymus DNA as a function of salt concentration with four univalent cations of different hydrated radii. These are K+ (3.3 A), (n-Pr)4N+ (4.5 A), (EtOH)4N+ (4.5 A), and C222-K+ (5 A). C222-K+ is a complex of cryptand C222 with K+. With K+ as the sole cation, Tm varies linearly with the log of ionic strength over the range 0.001-0.1 M. With all the K+ sequestered by an equimolar amount of C222, Tm is depressed by 10-20 degrees C and the slope of Tm vs. ionic strength is lower. At low ionic strength, an even greater reduction in Tm is achieved with (n-Pr)4N+; but the similar-sized (EtOH)4N+ gives a curve more similar to K+. Theoretical modeling, taking into account cation size through the Poisson-Boltzmann equation for cylindrical polyelectrolytes, predicts that larger cations should be less effective in stabilizing the double helix; but the calculated effect is less than observed experimentally. These results show that valence, cation size, and specific solvation effects are all important in determining the stability of the double-helical form of DNA.     

The identification and characterization of an epidermal growth factor-stimulated phosphorylation of a specific low molecular weight GTP-binding protein in a reconstituted phospholipid vesicle system

The abilities of different GTP-binding proteins to serve as phosphosubstrates for the epidermal growth factor (EGF) receptor/tyrosine kinase have been examined in reconstituted phospholipid vesicle systems. During the course of these studies we discovered that a low molecular mass, high affinity GTP-binding protein from bovine brain (designated as the 22-kDa protein) served as an excellent phosphosubstrate for the tyrosine-agarose-purified human placental EGF receptor. The EGF-stimulated phosphorylation of the purified 22-kDa protein occurs on tyrosine residues, with stoichiometries approaching 2 mol of 32Pi incorporated/mol of [35S]guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)-binding sites. The EGF-stimulated phosphorylation of the brain 22-kDa protein requires its reconstitution into phospholipid vesicles. No phosphorylation of this GTP-binding protein is detected if it is simply mixed with the purified EGF receptor in detergent solution or if detergent is added back to lipid vesicles containing the EGF receptor and the 22-kDa protein. The EGF-stimulated phosphorylation of this GTP-binding protein is also markedly attenuated by guanine nucleotides, i.e. GTP, GTP gamma S, or GDP, suggesting that maximal phosphorylation occurs when the GTP-binding protein is in a guanine nucleotide-depleted state. Purified preparations of the 22-kDa phosphosubstrate do not cross-react with antibodies against the ras proteins. However, they do cross-react against two different peptide antibodies generated against specific sequences of the human platelet (and placental) GTP-binding protein originally designated Gp (Evans, T., Brown, M. L., Fraser, E. D., and Northrup, J. K. (1986) J. Biol. Chem. 261, 7052-7059) and more recently named G25K (Polakis, P. G., Synderman, R., and Evans, T. (1989) Biochem. Biophys. Res. Commun. 160, 25-32). When highly purified preparations of the human platelet Gp (G25K) protein are reconstituted with the purified EGF receptor into phospholipid vesicles, an EGF-stimulated phosphorylation of the platelet GTP-binding protein occurs with a stoichiometry approaching 2 mol of 32Pi incorporated/mol of [35S]GTP gamma S-binding sites. As is the case for the brain 22-kDa protein, the EGF-stimulated phosphorylation of the platelet GTP-binding protein is attenuated by guanine nucleotides. Overall, these results suggest that the brain 22-kDa phosphosubstrate for the EGF receptor is very similar, if not identical, to the Gp (G25K) protein. Although guanine nucleotide binding to the brain 22-kDa protein or to the platelet. GTP-binding protein inhibits phosphorylation, the phosphorylated GTP-binding proteins appear to bind [35S]GTP gamma S slightly better than their nonphosphorylated counterparts.     

Stereoselective fungal metabolism of methylated anthracenes.

The metabolism of 9-methylanthracene (9-MA), 9-hydroxymethylanthracene (9-OHMA), and 9,10-dimethylanthracene (9,10-DMA) by the fungus Cunninghamella elegans ATCC 36112 is described. The metabolites were isolated by high-performance liquid chromatography and characterized by UV-visible, mass, and 1H nuclear magnetic resonance spectral techniques. The compounds 9-MA and 9,10-DMA were metabolized by two pathways, one involving initial hydroxylation of the methyl group(s) and the other involving epoxidation of the 1,2- and 3,4- aromatic double bond positions, followed by enzymatic hydration to form hydroxymethyl trans-dihydrodiols. For 9-MA metabolism, the major metabolites identified were trans-1,2-dihydro-1,2-dihydroxy and trans-3,4-dihydro-3,4-dihydroxy derivatives of 9-MA and 9-OHMA. 9-OHMA was also metabolized to trans-1,2- and 3,4-dihydrodiol derivatives. The absolute configuration and optical purity were determined for each of the trans-dihydrodiols formed by fungal metabolism and compared with previously published circular dichroism spectral data obtained from rat liver microsomal metabolism of 9-MA, 9-OHMA, and 9,10-DMA. Circular dichroism spectral analysis revealed that the major enantiomer for each dihydrodiol was predominantly in the S,S configuration, in contrast to the predominantly R,R configuration of the trans-dihydrodiol formed by mammalian enzyme systems. These results indicate that C. elegans metabolizes methylated anthracenes in a highly stereoselective manner that is different from that reported for rat liver microsomes.     

Sulphate uptake in the unicellular marine red algaRhodella maculata

Sulphate uptake by the unicellular marine red algaRhodella maculata conforms to Michaelis-Menten kinetics. Two uptake systems have been found: a low affinity system with an apparentK _m of 22 mM, and a high affinity system with an apparentK _m of 63.4 M. Transition from the low to the high affinity system can occur within 2.5 min, in response to a decrease in the ambient sulphate concentration to below 10 mM. Assimilation rates in the dark are about 20% those in the light, although enhancement by light is independent of the quanlity of light supplied above 27 mol m^-2 s^-1. Use of metabolic inhibitors indicates that photophosphorylation provides the main source of energy for sulphate assimilation, through both cyclic and non-cyclic electron flow.Abbreviations used APS-kinase ATP:adenylyl-sulphate 3-phosphotransferase (E.C. 2.7.1.25) - ATP-sulphurylase ATP:sulphate adenylyltransferase (E.C.2.7.74) - DCMU [3-(3,4-dichlorophenyl)]-1,1 dimethylurea - 2,4 DNP 2,4-dinitrophenol - DBMIB Dibromothymoquinone (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone)     

Homozygous Robertsonian translocations in a fetus with 44 chromosomes

Summary We report the unique finding of a human fetus with 44 chromosomes with homozygous 14;21 translocations. This fetus appeared phenotypically normal but the long-term neurodevelopmental outcome had this pregnancy continued could not be predicted. We speculate one 14;21 translocation was inherited from her father and one arose de novo being maternal in origin. A previous sibling with psychomotor retardation has an abnormal chromosome complement of 45,XX,dup(7)(q21pter), t(14;21)(p11;q11). The mother's underlying disease, systemic lupus erythematosis (SLE), and her prior chemotherapy may have contributed to the appearance of these chromosome aberrations. It is interesting that although 14;21 translocations are among the commonest structural chromosome rearrangements in man, there are no previous reports in newborn surveys of a child with 44 chromosomes resulting from the mating of two identical Robertsonian translocation carrier parents.     

The effect of inhibition of hexosemonophosphate shunt on the metabolism of glucose and function in rat brain in vivo

Rats treated 4 hr previously with 6-aminonicotinamide showed a twenty-four fold increase of [¹⁴C]phosphogluconate in the adult brain at 30 min after injection of [U-¹⁴C]glucose indicating a blockade of the hexosemonophosphate shunt. There was a significant increase in the¹⁴C-content of glucose and glucose 6-phosphate, and a decrease in that of amino acids. [¹⁴C]Phosphoglycerate content showed no consistent change after 6-aminonicotinamide treatment. The concentration of glucose and glucose 6-phosphate increased significantly without a significant change in the lactate pool in the brain of 6-aminonicotinamide treated rats. The rate of utilization of glucose in the brain of control rats was 0.73 mol/min per g of brain. It decreased by 16% in rats treated with 6-aminonicotinamide; the results suggested that both glycolysis and pyruvate oxidation were affected. The amount of glucose utilized in the brain by the hexosemonophosphate shunt was approximately 0.0093 mol/min per g of brain, i.e. 1.3% of the total rate of utilization of glucose. The observed changes were not due to hypothermia. The rate of glucose utilization was higher in animals exposed to higher ambient temperature and to stress caused by handling. The results were explained by postulating a role for the hexosemonophosphate shunt in providing neurotransmitter amino acids glutamate and -aminobutyrate, and interdependence of brain function and glucose utilization.This paper is dedicated to Dr. Derek Richter on his seventy-fifth birthday.     

Uterine prostaglandin concentrations following fetal death in sheep

Concentrations of prostaglandin E (PGE), PGF and 6-oxo-PGF_1α (the hydrolytic product of PGI₂) were measured by radioimmunoassay (RIA) in myometrium, endometrium, cotyledons, amnion and chorioallantois taken from different uterine areas from chronically catheterized sheep bearing fetuses which had died 12–26 h previously (n=4) or 34–72 h previously (n=4). These two groups of animals were designated fetuses dead <30 h and >30 h respectively. The time of fetal death was assessed on the basis of fetal heart rate and blood gases. At the time of the tissue collection the ewes were between 123 and 130 days after mating. For comparative purposes, tissues also were collected from four sheep bearing live chronically catheterized fetuses at 130 days of gestation.For myometrium, concentration of PGF, PGE and 6-oxo-PGF_1α were significantly higher in sheep bearing dead fetuses, compared to those bearing live fetuses. Analysis of variance also showed a significant effect of uterine area on myometrial PGE concentrations, concentrations being higher in tubal areas than elsewhere. Concentrations of PGE, PGF and 6-oxo-PGF_1α were higher in endometrium taken from uteri containing dead fetuses. In cotyledons, concentrations of PGF and 6-oxo-PGF_1α but not PGE, were significant elevated following fetal death. Concentrations of 6-oxo-PGF_1α, but not PGE or PGF, were elevated in both chorioallantois and amnion of sheep bearing dead fetuses, compared to those bearing live fetuses. In association with elevated PG concentrations, there was a progressive increase in the frequency and maximum amplitude of uterine contractions. These results show that PG concetrations are elevated following fetal death in sheep, and suggest an association between elevated PG concentrations and delivery of the dead fetus.