Genetic Analysis of Murine Arylsulfatase C and Steroid Sulfatase

SWR/J mice possess two- to threefold higher 4-methylumbelliferyl sulfate (4MUS), dehydroepiandrosterone sulfate (DHEAS) and estrone sulfate (E1S) sulfatase activities in liver and kidney extracts than do A/J mice. These interstrain activity differences are maintained throughout the 6- to 45-day postnatal period. Characteristics of the hepatic activities of SWR/J mice suggest that all three activities reside in the same enzyme. Biochemical properties of the SWR/J and A/J enzyme were not significantly different. Expression of hepatic enzyme activity is subject to regulation by an autosomal locus possessing two alleles with additive effects. Postnuclear E1S- and DHEAS-sulfatase activities are primarily microsomal. Although postnuclear hepatic 4MUS-sulfatase activity is predominantly microsomal, renal activity is primarily nonmicrosomal. Only that portion of 4MUS-sulfatase occurring in cell membranes appears capable of hydrolyzing E1S and DHEAS. The hepatic- and renal-specific subcellular distributions of 4MUS-sulfatase activity may reflect tissue differences in enzyme processing. Renal 4MUS-sulfatase activity is also controlled by an autosomal gene with two alleles having additive effects. Positive correlation between hepatic and renal 4MUS-sulfatase activities indicates that both activities are most likely influenced by the same gene.     

Characterization of the Blood-Brain Barrier: Protein Composition of the Capillary Endothelial Cell Membrane

Microvessels were isolated from canine cerebral cortex, and the composition of the endothelial cell membrane was investigated. Endothelial cell membranes were separated from the surrounding basement membrane, solubilized, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 12% gels. Staining with Coomassie Blue revealed a characteristic banding pattern of at least 12 major proteins with apparent molecular weights between 14,000 and 250,000. When proteins from red blood cell ghosts were run simultaneously, no similarities were observed, except for proteins at apparent molecular weights of 43,000 (band 3) and 35,000 (band 4). These two proteins migrated exactly to the positions of the erythrocyte proteins actin and glyceraldehyde 3-phosphate dehydrogenase, respectively. Membrane glycoproteins in gels were also examined by the use of fluorescent lectins. Of the fluorescein isothiocyanate-conjugated (FITC) lectins tested, only FITC-concanavalin A had an affinity for any membrane components. Diazotized [125I]iodosulfanilic acid, a membrane-impermeable reagent, was used to label the internal (lumen) cell surface and the external (antilumen) cell surface. Autoradiography and determination of radioactivity levels in gel slices showed that several proteins were specifically labeled, and that major differences in radioactivity of proteins existed in internal and external labeling experiments. It is concluded that the protein composition of the luminal membrane is different from that of the antiluminal membrane.     

The in vitro synthesis and characteristics of ribosomal RNA in imaginal discs of Drosophila melanogaster

Summary Late third instar imaginal discs of Drosophila melanogaster cultured in vitro in Robb's tissue culture medium synthesize 38S, 28S and 18S ribosomal RNAs which are qualitatively indistinguishable from their in vivo synthesized counterparts (Fig. 1). As found in other insect systems, the 38S molecule appears to be the precursor for both the 28S and 18S rRNAs (Figs. 2, 3 and 4). The 28S rRNA and a portion of the 38S pre-rRNA shift in sedimentation value upon exposure to heat or dimethylsulfoxide (Figs. 5 and 8). Studies of the thermal denaturations of these molecules (Figs. 6, 7 and 9) indicate the existence of a single class of 28S rRNA, but three classes of 38S pre-rRNAs. The addition of -ecdysone to the in vitro culture medium stimulates the net amount of rRNA synthesized, increases the rate of processing of the 38S precursor and increases the relative amount of 18S material produced (Figs. 10 and 12).This work was supported in part by grants from the National Science Foundation (GB-8176) and from the Atomic Energy Commission (AT-04-3-34).Predoctoral Trainees, PHS Training Grant No. 2-Tl-GM367 from Research Training Grants Branch, National Institute of General Medical Sciences.1 For purposes of simplification we shall refer to the rRNA molecules of D. melanogaster as being 38S, 30S, 28S and 18S; however, it should be noted that these values are approximate (see Hastings and Kirby, 1966; Greenberg, 1969; Tartof and Perry, 1970).     

Inhibition of enzymes by metal ion-chelating reagents. Theory and new graphical methods of study

1. The mechanism of inhibition of enzymes by metal ion-chelating reagents is discussed and equations derived. 2. Two distinct mechanisms are postulated and graphical methods are given for differentiating between them. 3. Where the metal ion is actually removed from the enzyme to form a co-ordination complex in solution, a procedure is described for obtaining the stability constant for metal-enzyme interaction, the number of metal ions involved and the stoicheiometry of metal ion-ligand interaction.     

Purification of human placental alkaline phosphatase. Salt effects in affinity chromatography

Human placental alkaline phosphatase was chromatographed on Sepharose derivatives of d- and l-phenylalanine, l-leucine, glycine, aniline and p-aminobenzoic acid in high concentrations of (NH(4))(2)SO(4). Retention on these columns was greatest at the highest concentrations of (NH(4))(2)SO(4). By using decreasing concentrations and changing the types of salts, elution was effected from each of the columns. The (NH(4))(2)SO(4)-mediated retention appeared to be related to the hydrophobic character of the substituted Sepharose, rather than to any specific binding site of the enzyme. It is suggested that this provides a way of controlling hydrophobic affinity chromatography. By use of chromatography on l-phenylalanine-Sepharose and of DEAE-Sephadex chromatography in the presence of Triton X-100 detergent, a preparation of highly purified (1000-fold) human placental alkaline phosphatase was obtained in 22% yield.     

Inherited and/or conditioned changes in host-plant preference in Pieris

The ability to survive of a phytophagous insect, as illustrated by the genus Pieris, is a quantitative phenomenon, not an all or none reaction. This has been shown by various tests involving Pieris rapae and P. protodice on each of six different larval food plants. There are significant genetic differences between these species with regard to survival, growth rate and size differences. Strains of P. rapae have been induced to prefer one or another host-plant by prior conditioning on that plant. Prior conditioning is effective during the life of the larva (1 st to 5th instars), or the individual (larva to adult) or subsequent generations. Hybridization between conditioned strains gives (1) the effect of hybrid vigor and (2) multiple factor differentiation of F₁ and F₂ progeny. Testing of conditioned strains indicates that the larvae select not only different plants but also different concentrations of mustard oil. The F₁ larvae select a concentration intermediate between parental strains. The F₂ select a wider range. Similar tests on oviposition selection by adult females derived from conditioned strains indicate that the females follow the larval conditioning pattern, with similar results on inheritance. Conditioned strains have been shown to be alterable; that is, a strain conditioned to one plant can be reconditioned to another and vice versa. The process is constantly reversable as long as there are individuals which survive the changes.

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Zusammenfassung Wie an der Gattung Pieris gezeigt wird, ist die Fähigkeit eines phytophagen Insekts, zu überleben, ein quantitatives Phänomen, keine Alles-oder-Nichts-Reaktion. Dies hat sich bei verschiedenen Untersuchungen mit Pieris rapae und P. protodice an jeder von 6 verschiedenen Larven-Futterpflanzen gezeigt. Im Hinblick auf Überlebensdauer, Wachstumsrate und Größenunterschiede bestehen zwischen diesen Arten signifikante genetische Unterschiede. Stämme von P. rapae können durch vorangehende Gewöhnung (conditioning) an eine oder die andere Wirtspflanze zur Bevorzugung der betreffenden Fraßpflanze gebracht werden. Anpassung durch vorangehende Gewöhnung ist während des Larvenlebens (vom 1. bis 5. Stadium) oder während der einzelnen Entwicklung (Larve bis Imago) oder während aufeinanderfolgender Generationen wirksam. Kreuzungen zwischen so angepaßten Stämmen ergaben 1. den Effekt der Hybridsteigerung und 2. die Vielfach-Faktoren-Trennung in der F₁-und F₂-Nachkommenschaft. Prüfung der angepaßten Stämme zeigten, daß die Larven nicht nur verschiedene Pflanzen, sondern auch verschiedene Konzentrationen von Senföl wählen. Die F₁-Larven wählen eine Konzentration, die intermediär zu den von den Ausgangsstämmen bevorzugten liegt. Die F₂ wählt einen weiteren Bereich. Entsprechende Untersuchungen über die Wahl der Eiablage-Pflanzen durch reife Weibchen aus angepaßten Stämmen erwiesen, daß die Weibchen den larvalen Gewöhnungsmustern folgen, mit entsprechenden Konsequenzen für die Vererbung. Es ließ sich zeigen, daß angepaßte Stämme abgeändert werden können, d.h. daß ein an eine Wirtspflanzenart gewöhnter Stamm auf eine andere umgestimmt werden kann und umgekehrt. Dieser Vorgang ist konstant umkehrbar, solange nur einzelne Individuen die Veränderungen überleben.

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Aided by the National Science Foundation, Washington, D.C., the California Arboretum Foundation, Inc. and the California State College, Los Angeles.