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S D Black

Publications and source records attributed to S D Black.

At least 19 recordsLinked to original sources
PubMed1992

Development of hydrophobicity parameters for prenylated proteins.

We have determined hydrophobicity parameters for the side-chains of the prenyl thioether protein modifications, farnesyl-cysteine and geranylgeranyl-cysteine. Farnesyl-Cys is somewhat more hydrophobic than palmitoyl-Cys, but geranylgeranyl-Cys is more than two log(P) units more non-polar. These post-translational modifications represent the most hydrophobic residues yet described quantitatively. Furthermore, such modifications occur at the COOH-terminus which is generally methyl esterified. Loss of the terminal negative charge and formation of the ester proceeds with the gain of an additional 2.343 log(P) units of hydrophobicity. Clearly, COOH-terminal prenylation and esterification impart sufficient potential to render the terminus membrane bound. Thus, hydrophobicity parameters presented here for the prenylated amino acyl residues extend our understanding of these important physiological derivatives and enable computational analysis of proteins thus modified.

Amino Acid Sequence↗
PubMed1992

Deployment of extracellular matrix proteins in sea urchin embryogenesis.

The apical extracellular matrix of the sea urchin embryo, known as the hyaline layer (HL), is a multi-laminate organelle composed of at least 10 polypeptides. Although integrated into one ECM, HL proteins exhibit individual temporal and spatial dynamics throughout development. These molecules are stockpiled in the oocyte during vitellogenesis in at least four distinct vesicle populations. They are released onto the cell surface at fertilization in a specific order, and interact differentially with embryonic cells as development proceeds. Many experiments have suggested that the HL is vital for embryogenesis, but relatively little is known about the functions and interactions of its constituent molecules. The purpose of the present review has been to gather information on the basic characteristics of the known HL proteins together with data on their expression in the embryo, and where possible, their biological activities. Compiled, these observations may provide some insight into the workings of a uniquely embryonic organelle.

Animals↗
PubMed1992

Comparative metabolism in vitro of a novel carcinogenic polycyclic aromatic hydrocarbon, 1,2,3,4-tetrahydro-7,12-dimethylbenz[a]anthracene, and its two regioisomeric B-ring fluoro analogues.

A novel biotransformation pathway likely exists for carcinogenic 1,2,3,4-tetrahydro-7,12-dimethylbenz[a]anthracene (THDMBA), since this A-ring-reduced polycyclic aromatic hydrocarbon does not have an aromatic bay-region. The comparative metabolism of THDMBA, a non-carcinogenic 5F analogue, and a more carcinogenic 6F-THDMBA species was examined to determine potential DNA-bonding metabolites. Rat liver microsomes from phenobarbital-treated animals were incubated in the presence of THDMBA (or fluoro-THDMBA), NADPH, and O2. Metabolic products and the parent compound were extracted into organic solvent and analyzed/purified using reversed-phase high-performance liquid chromatography. Structure identification of metabolites using proton nuclear magnetic resonance, mass spectroscopy, and ultraviolet/visible spectroscopy indicated that hydroxylations at benzylic C1 and at the C7- and C12-CH3 functions are major oxidation products of THDMBA. Major metabolites for the noncarcinogenic 5F-THDMBA are the C4-hydroxy, C7-hydroxymethyl, and C12-hydroxymethyl derivatives. However, the potent carcinogen 6F-THDMBA only yielded major hydroxylation products at C1 and C12-CH3. These results together with a consideration of the electronic and steric effects of fluorine and the biological activities of these polycyclic aromatic hydrocarbons suggest that hydroxylation at the hindered benzylic C1 position or the C12-CH3 group of THDMBA is important for the biotransformation of such polycyclic aromatic hydrocarbons to DNA-bonding species.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1992

Membrane topology of the mammalian P450 cytochromes.

The membrane topology of the mammalian P450 cytochromes has been studied intensively by computational approaches, proteolysis, chemical modification, genetic engineering, and immunochemistry. Initial results for the cytochromes of the endoplasmic reticulum appeared to indicate a polytopic, four to eight transmembrane anchor model with an active site buried in the membrane. However, recent findings show that the microsomal P450s are bound to the endoplasmic reticulum by only one or two transmembrane peptides located at the NH2-terminal end, and that the active site is part of a large cytoplasmic domain that may have one or two additional peripheral membrane contacts. The membrane-bound state is viewed as rather rigid, and the plane of the heme lies between perpendicular and parallel to the plane of the endoplasmic reticulum. The mitochondrial P450 cytochromes lack a hydrophobic NH2 terminus in the mature form, and thus differ from the microsomal isozymes in this significant way. However, although the exact topology of cytochrome P450 in the inner mitochondrial membrane remains to be elucidated, certain features are clearly comparable to those of microsomal P450. Therefore, the membrane topology of the P450 gene superfamily may follow a similar pattern.

Animals↗
PubMed1992

Modulation of carcinogenicity in 1,2,3,4-tetrahydro-7,12- dimethylbenz[a]anthracene (THDMBA) by fluorine substitution: crystal structures of the 5- and 6-fluoro regioisomers.

1,2,3,4-Tetrahydro-7,12-dimethylbenz[a]anthracene (THDMBA) is an animal carcinogen which lacks an aromatic bay-region and shows promise as a model to investigate non-classical mechanisms of carcinogenesis. The fluorine-substituted derivatives at positions 5 and 6 on the B-ring exhibit a striking range of oncogenic potential wherein the 6F-THDMBA is twice as potent as the parent carcinogen, but the 5F-THDMBA is virtually inactive. To study structure-reactivity relationships for these fluorine regioisomers, we have determined the three-dimensional structures of the compounds by single-crystal X-ray diffraction. These crystal structures are the first such to be reported for any monofluoro anthracene (or pyrene) derivative. The partially-reduced A-ring exists in both enantiomeric half-chair conformers in the crystalline state, and the compounds have quasi-planar anthracene ring systems which exhibit a right-handed twist in the 'beta'-conformer, with the expected opposite twist in the other. A complete analysis of bond lengths, bond angles and torsion angles is presented. Preliminary electrostatic potentials have been derived from the X-ray data sets, and the results indicate significant differences in potential between 5F- and 6F-THDMBA at positions near the partially reduced bay region. Such results are likely to be of importance in the understanding of metabolic activation to reactive intermediates capable of bonding covalently to DNA.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1992

Commitment along the dorsoventral axis of the sea urchin embryo is altered in response to NiCl2.

Few treatments are known that perturb the dorsoventral axis of the sea urchin embryo. We report here that the dorsoventral polarity of the sea urchin embryo can be disrupted by treatment of embryos with NiCl2. Lytechinus variegatus embryos treated with 0.5 mM NiCl2 from fertilization until the early gastrula stage appear morphologically normal until the midgastrula stage, when they fail to acquire the overt dorsoventral polarity characteristic of untreated siblings. The ectoderm of normal embryos possesses two ventrolateral thickenings just above the vegetal plate region. In nickel-treated embryos, these become expanded as a circumferential belt around the vegetal plate. The ectoderm just ventral to the animal pole normally invaginates to form a stomodeum, which then fuses with the tip of the archenteron to produce the mouth. In nickel-treated embryos, the stomodeal invagination is expanded to become a circumferential constriction, and it eventually pinches off as the tip of the archenteron fuses with it to produce a mouth. Primary mesenchyme cells form a ring in the lateral ectoderm, but as many as a dozen spicule rudiments can form in a radial pattern. Dorsoventral differentiation of ectodermal tissues is profoundly perturbed: nickel-treated embryos underexpress transcripts of the dorsal (aboral) gene LvS1, they overexpress the ventral (oral) ectodermal gene product, EctoV, and the ciliated band is shifted to the vegetal margin of the embryo. Although some dorsoventral abnormalities are observed, animal-vegetal differentiation of the archenteron and associated structures seems largely normal, based on the localization of region-specific gene products. Gross differentiation of primary mesenchyme cells seems unaffected, since nickel-treated embryos possess the normal number of these cells. Furthermore, when all primary mesenchyme cells are removed from nickel-treated embryos, some secondary mesenchyme cells undergo the process of "conversion" (Ettensohn, C. A. and McClay, D. R. (1988) Dev. Biol. 125, 396-409), migrating to sites where the larval skeleton would ordinarily form and subsequently producing spicule rudiments. However, the skeletal pattern formed by the converted cells is completely radialized. Our data suggest that a major effect of NiCl2 is to alter commitment of ectodermal cells along the dorsoventral axis. Among the consequences appears to be a disruption of pattern formation by mesenchyme cells.

Animals↗
PubMed1992

Two monoclonal antibodies recognizing different epitopes on rat cytochrome IIB1 react with human IIE1.

To identify human cytochromes P450 (P450) in the CYP2B subfamily, 14 human liver microsomal samples were screened by immunoblots developed with monoclonal antibodies that recognized seven distinct epitopes on rat IIB1. Two of these antibodies recognized a protein in all of the samples. This protein was termed P450BE. Using video-imaging densitometry, the levels of P450BE were determined and compared with levels of other P450s. An excellent correlation was seen (r = 0.87) between P450BE and human IIE1. However, rat IIE1 did not react in immunoblot and enzyme-linked immunosorbant assays with the two anti-rat IIB1 monoclonal antibodies. As previously observed, the levels of IIE1 in the samples correlated well (r = 0.88) with the ability of these human liver microsomes to N-demethylate N-nitrosodimethylamine. The levels of P450BE also correlated well (r = 0.91) with the ability of the microsomes to N-demethylate N-nitrosodimethylamine. In addition, excellent correlations were obtained when the levels of P450BE and IIE1 were compared with the ability of the microsomes to O-deethylate ethoxycoumarin (r = 0.87 and r = 0.85, respectively). To identify the protein recognized by the anti-rat IIB1 antibodies, P450BE was purified from microsomes prepared from human liver D. Amino-terminal amino acid sequence analyses of P450BE revealed that the 18-amino acid sequence obtained matched the corresponding sequence of human IIE1. In addition, purified human IIE1 and P450BE migrated with the same apparent molecular weight in polyacrylamide gels. Furthermore, proteolytic maps of P450BE and IIE1, generated with two proteases, were found to be identical. Sequence alignments and antigenicity calculations identified three regions of rat IIB1 as likely candidates for the epitopes shared in common with human IIE1. In conclusion, this study indicates that caution must be taken when interpreting the results of immunochemical assays when species lines are crossed.

Adolescent↗
PubMed1991

Development of hydrophobicity parameters to analyze proteins which bear post- or cotranslational modifications.

We have determined hydrophobicity parameters for the side chains of 22 common post- or cotranslationally modified amino acyl residues and for the standard unmodified amino acids as well. This "comprehensive" parameter set is the first such reported. Parameters determined for the side chains of the standard 20 amino acids correlate well with those of widely accepted sets. Our parameters have also been evaluated by hydrophobicity profiles and by transverse hydrophobic moment calculations on cytosolic, secreted, and membranous model proteins, with favorable results. Many of the hydrophobicity parameters for the post- or cotranslationally modified derivatives are of remarkable magnitude, especially those for oligosaccharide-bound Asn and fatty-acylated Cys or amino terminus. Thus, the comprehensive parameter set determined here greatly extends our ability to analyze homology, membrane directedness, and folding potential of proteins.

Amino Acids↗
PubMed1989

Membrane topology of mammalian cytochromes P-450 from liver endoplasmic reticulum. Determination by trypsinolysis of phenobarbital-treated microsomes.

We have studied the membrane topology of liver microsomal cytochromes P-450 derived from phenobarbital-treated rabbits via trypsinolysis of intact microsomes, recovery of solubilized peptide fragments by ultracentrifugation and liquid chromatography, primary structure determination by Edman microsequence analysis, and database searching to match isolated fragments with parent sequences. Relative to the primary structure of isozyme 2, the major phenobarbital-inducible form, fragments were isolated beginning at residues Glu86, Ile101, Arg126, Cys152, Leu198, Ser211, Asn237, Glu327, Asn385, Thr407, Phe408, Phe413, and Thr444. Such results show that this family of structurally related cytochromes is bound to the endoplasmic reticulum membrane by only one or two transmembrane segments, located at the NH2-terminal end of the polypeptide chain. The remainder of the protein, from residue approximately 50 to the COOH terminus must exist as a catalytic, heme-containing domain exposed on the cytosolic side of the membrane. Furthermore, our results indicate that the catalytic domain must be peripherally associated with the membrane surface. This would imply that substrates might have access to the active site of the cytochrome P-450 either by diffusion from the cytosol or from within the lipid bilayer.

Amino Acid Sequence↗
PubMed1989

Experimental reversal of the normal dorsal-ventral timing of blastopore formation does not reverse axis polarity in Xenopus laevis embryos.

During gastrulation in Xenopus laevis, the dorsal lip of the blastopore normally appears before the ventral lip. Metabolic gradient models propose that the dorsal lip develops from the region of highest metabolic activity and somehow dominates other regions to prevent them from becoming dorsal. To test these ideas, I applied a temperature gradient of 12 degrees C across the embryo. Localized heating of the prospective ventral vegetal region from early in the first cleavage period until gastrulation causes the blastopore lip to form first by 2 hr at the prospective ventral meridian rather than at the prospective dorsal meridian. Despite this reversal of the timing of blastopore formation, gastrulation is completed, and the neural plate forms at its usual position on the prospective dorsal meridian. This demonstrates that the earliest gastrulating regions of the blastopore do not necessarily become dorsal, nor do they inhibit dorsal development by other regions. It is unlikely that axis polarity is based on regional differences in energy metabolism.

Animals↗
PubMed1988

The first cleavage plane and the embryonic axis are determined by separate mechanisms in Xenopus laevis. I. Independence in undisturbed embryos.

We examined the spatial relationships between the meridian of sperm entry the plane of first cleavage, and the embryonic axis (defined by the neural groove) in eggs of Xenopus laevis. Direct measurement of the angular separations between these embryonic structures in gelatin-embedded eggs confirmed the classical conclusion that the sperm entry point and neural groove tend to form on opposite sides of the egg, and also revealed that the first cleavage plane has a nearly random orientation with respect to the neural groove. We next examined the distortion of the first cleavage plane that results from the normal processes of convergence and extension during gastrulation and neurulation. We permanently marked the first cleavage plane by injecting one blastomere of the two-cell embryo with a fluorescent lineage marker. At the start of gastrulation, the interface between the labeled and unlabeled regions was almost randomly oriented relative to the dorsal blastopore lip, confirming our first set of observations. In embryos with the interface less than 60 degrees to the plane passing through the midline of the dorsal lip, convergent movements of cells produced a confrontation of labeled and unlabeled cells along much of the dorsal midline. Thus, although the first cleavage plane and the bilateral plane were frequently not congruent, the morphogenetic movements of gastrulation and neurulation brought about an apparent congruence in many half-labeled embryos.

Animals↗
PubMed1988

The first cleavage plane and the embryonic axis are determined by separate mechanisms in Xenopus laevis. II. Experimental dissociation by lateral compression of the egg.

In eggs of Xenopus laevis, the meridian of sperm entry (SEP meridian), the direction of subcortical rotation, and the first cleavage furrow have been used to predict, with varying degrees of accuracy, the position of the plane of bilateral symmetry of the embryo. We show here that altering the shape of the uncleaved egg by lateral compression disrupts some of these topographical relationships in a reproducible way. The neural groove, which identifies the embryonic dorsal midline, usually forms at either of the two narrow ends of the compressed egg, regardless of the position of the SEP meridian, whereas the first cleavage furrow divides the compressed egg across its shorter dimension, regardless of the position of the SEP meridian. Thus the positions of the SEP meridian, the cleavage plane, and the embryonic bilateral plane can be completely uncoupled from each other. In contrast, the direction of subcortical rotation is usually parallel to the plane of compression and predicts the position of the neural groove in all cases. Since the direction of subcortical rotation and the plane of bilateral symmetry still correlate under conditions of compression, we conclude that subcortical rotation is the crucial early step in the process of axis specification.

Animals↗
PubMed1986

High-frequency twinning of Xenopus laevis embryos from eggs centrifuged before first cleavage.

In embryos of the frog Xenopus laevis, the dorsal structures normally develop from regions of the egg opposite the side of sperm entry. Gravity and centrifugal force, applied at an angle of 90 degrees to the animal-vegetal axis of the egg, can override this topographic relationship and can cause the dorsal structures to be positioned according to the force vector (S. Black and J. Gerhart, 1985, Dev. Biol. 108, 310-324). We report here that at time 0.40 (40% of the first cleavage interval), an average of 60% of eggs centrifuged at 30g for 4 min in this orientation form conjoined twins with one body axis arising from the centripetal side of the egg and one arising from the centrifugal side of the egg. This positioning is observed regardless of the orientation of the side of sperm entry in the centrifugal field. If, after the 0.40 centrifugation, the eggs are inclined with the centripetal side up, they do not make twins; instead, they make only a single axis at the centripetal side. This indicates that the second axis in twins is caused to form by postcentrifugation gravity-driven internal rearrangements of materials that were displaced by the centrifugation. Twins also form at high frequency in eggs centrifuged twice, first at an inclination of 90 degrees, and then at an inclination of 0 degrees. The second centrifugation yields secondary axes even when it is begun midway in the second cell cycle, well after the time of grey crescent formation. Double centrifugation also causes twinning ("double rescue") of uv-irradiated eggs which otherwise would not develop axial structures. This suggests that the internal displacements caused by the centrifugations can substitute for a step in the normal axis specification process that is impaired in irradiated eggs.

Ambystoma mexicanum↗
PubMed1985

Analysis of plasma cyanate as 2-nitro-5-thiocarbamylbenzoic acid by high-performance liquid chromatography.

A method for the determination of cyanate concentration in blood plasma over the range 1 to 1000 microM is presented. Cyanate present in the dried residue of acetone-deproteinized plasma is converted to a chromophoric thiocarbamyl derivative by addition of pH 3.0-buffered thionitrobenzoic acid. The derivative is then analyzed by reversed-phase high-performance liquid chromatography with detection at 313 nm, near the absorption maximum. Carbamyl thionitrobenzoic acid peak height is quantified by comparison to a standard curve made by analysis of plasma samples to which known quantities of cyanate have been added. This technique is sensitive and linear with respect to cyanate concentration, and is faster than other reported methods; sample analysis and column regeneration are accomplished within 20 min.

Chromatography, High Pressure Liquid↗
PubMed1985

Studies on the identity of the heme-binding cysteinyl residue in rabbit liver microsomal cytochrome P-450 isozyme 2.

The reaction of purified rabbit liver microsomal P-450 isozyme 2 with 4,4'-dithiobis(2-nitrobenzoate) (DTNB) exhibits first order kinetics and results in the modification of a single thiol, but causes no net loss of the native ferrous-carbonyl spectrum. Inclusion of both phospholipid and a tight-binding nitrogenous ligand, 1-benzylimidazole, in the reaction medium produces a burst-phase of DTNB modification, but the stoichiometry remains one thiol modified per polypeptide chain. The site of isozyme 2 rapidly labeled by DTNB and by monobromobimane, a fluorescent reagent for thiol groups, was shown to be Cys152. Results obtained strongly suggest that Cys152 does not provide the proximal thiolate ligand to the heme iron atom. Since Cys152 represents one of the two highly conserved cysteine-containing regions in the P-450 cytochromes, it appears likely that the other region, containing Cys436 in this rabbit cytochrome (corresponding to Cys355 in bacterial P-450 cam, Cys436 in rat P-450 b or e, Cys461 in rat P-450 c, Cys456 in rat P-450 d or mouse isozyme 3, and Cys458 in mouse isozyme 1) is the source of the thiolate ligand to the heme.

Animals↗
PubMed1985

Experimental control of the site of embryonic axis formation in Xenopus laevis eggs centrifuged before first cleavage.

In Xenopus laevis, the dorsal structures normally develop from regions of the egg opposite the side of sperm entry. Gravity is known to affect this topographic relationship in eggs inclined obliquely from their normal vertical orientation in the period before first cleavage. This effect has been explored in detail, making use of low-speed centrifugation (10-50 g) for short durations (4 min). Eggs were immobilized in gelatin and oriented with their animal-vegetal axes 90 degrees to the force vector, with the sperm entry point (SEP) side of the egg either toward or away from the center of the rotor. It has been found that the egg shows three distinct periods of response to centrifugal force in the interval from fertilization to first cleavage: Prior to 0.4 (40% of the first cleavage interval), the egg is very sensitive to centrifugal force and develops dorsal structures from its centrifugal side, regardless of the position of the SEP in the centrifugal field. Thus, the dorsal structures of the embryo are reversed from normal in eggs centrifuged with the SEP away from the center of the rotor. In the period 0.4 to 0.7, the egg is still very sensitive to centrifugal force and develops dorsal structures from its centripetal side, regardless of the position of the SEP in the centrifugal field. Thus, the dorsal structures of the embryo are reversed from normal in eggs centrifuged with the SEP toward the center of the rotor. In the period 0.7-1.0, the egg becomes increasingly resistant to centrifugal force and forms dorsal structures at the normal position opposite the SEP side. This resistance can be overcome in some egg clutches by 50 g centrifugation followed by prolonged 90 degrees off-axis inclination at 1g. Midway in the second cell cycle, there is a brief period of sensitivity to centrifugal force. These These results are discussed in terms of the types of cytoplasmic rearrangements occurring in the egg at different times of the cell cycle, and in terms of the process of cytoplasmic localization of determinants of dorsal axial development.

Animals↗