bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of functional domains of human erythrocyte spectrin.
Authors: Morrow J S , Speicher D W , Knowles W J , Hsu C J , Marchesi V T
Published: May 2006

Isolated human erythrocyte spectrin is a dimer of two unique polypeptide chains. The dimer (alpha beta) undergoes reversible salt- and temperature-dependent association to form (alpha beta)2 tetramers. Spectrin also binds with high affinity to a protein receptor on the cytoplasmic surface of erythrocyte membrane vesicles. By cleavage of spectrin at its cysteine residues with 2-nitro-5-thiocyanobenzoic acid, a 50,000-dalton peptide fragment has been isolated which inhibits the binding of spectrin to erythrocyte membrane vesicles. This peptide arises from a terminal region of the beta chain. An 80,000-dalton peptide generated by restricted trypsin digestion binds preferentially to dimeric spectrin. This peptide arises from a terminal portion of the alpha chain. Multiple peptides involved in noncovalent associations between the chains have also been identified. These associations indicate that the two subunits of spectrin are aligned parallel to one another and that the tetramer formation site and the high-affinity membrane binding site are in close proximity to one another.
Prostaglandin I2 modifies both prolactin binding capacity and fluidity of mouse liver membranes.
Authors: Dave J R , Knazek R A
Published: May 2006

The objective of this study was to determine if prostaglandins alter the fluidity of hepatic membranes and if this, in turn, modifies their ability to bind prolactin. Liver homogenates of adult C3H female mice incubated with 1-1000 nM prostaglandin A1, A2, B2, F1 alpha, 6-keto-F1 alpha, E1, E2, I2, or thromboxane B2 provided the 100,000 X g membrane pellets for subsequent ovine prolactin binding and membrane fluidity studies. Only membrane preparations treated with prostaglandin I2 showed an increase in specific binding of ovine prolactin. The effect (40-50%) was maximal at 100 nM prostaglandin I2 after 30 min of incubation and was due to an increase in the number of receptor sites. Under the same conditions, prostaglandin I2 induced a 17% decrease in membrane microviscosity. These data suggest that specific prostaglandins may modulate the number of prolactin receptors in vivo by modifying the fluidity of the lipid bilayer and the subsequent ease with which receptors can assume active configurations within the matrix.
Monoclonal antibodies to sucrase/isomaltase: probes for the study of postnatal development and biogenesis of the intestinal microvillus membrane.
Authors: Hauri H P , Quaroni A , Isselbacher K J
Published: May 2006

Two monoclonal antibodies, designated BB 3/34/12 and BB 5/8/40/90, have been produced to rat intestinal sucrase/isomaltase (SI) by the hybridoma technique using microvillus membranes as antigen. The BB 3/34/12 antibody was shown to be specific for the sucrase subunit. These antibodies provided new information regarding the biosynthesis and postnatal development of SI. In rat intestinal fetal transplants, SI was found exclusively in the form of an enzymatically active high molecular weight precursor, confirming our previous observations concerning the role of luminal proteases for the processing of SI in the microvillus membrane. The SI precursor, purified by affinity chromatography using the BB 3/34/12 antibody, had both sucrase and isomaltase activities, suggesting that a single precursor protein generates both sucrase and isomaltase subunits by proteolytic cleavage. The initial appearance of SI during normal postnatal development in the rat intestine was found to be confined to the cells present at the base of the villi. The same localization was observed after precocious induction of SI by cortisone acetate. In both cases, no immunofluorescence was observed in the crypts, suggesting that only the differentiated enterocyte is capable of synthesizing this enzyme. Even at the earliest times of appearance, newly synthesized SI was found almost completely split into its subunits, suggesting that the protease(s) responsible for the processing of the precursor in the microvillus membrane develop(s) in parallel with SI or earlier.
Receptor-mediated internalization of fluorescent gonadotropin-releasing hormone by pituitary gonadotropes.
Authors: Hazum E , Cuatrecasas P , Marian J , Conn P M
Published: May 2006

A bioactive, fluorescent derivative of gonadotropin-releasing hormone, < Glu-His-Trp-Ser-Tyr-D-Lys(N epsilon-tetramethylrhodamine)-Leu-Arg-Pro-Gly-NH2, was prepared. This peptide retained high-affinity binding (apparent dissociation constant, 3 nM) to the receptor for gonadotropin-releasing hormone and was utilized for microscopic visualization and localization of gonadotropin-releasing hormone receptors in cultured rat pituitary cells. The fluorescently labeled receptors were initially distributed uniformly on the cell surface and formed patches, which subsequently internalized (at 37 degrees C) into endocytic vesicles. These processes were dependent on specific binding sites for the rhodamine-labeled peptide to gonadotrope cells. Cluster formation and internalization were markedly reduced in the absence of Ca2+, which is required for gonadotropin secretion. It is possible that cluster formation, microaggregation, and internalization of gonadotropin-releasing hormone receptors may be important in eliciting biological effects or for the observed loss of tissue responsiveness after desensitization due to exposure to the homologous hormone.
Separation of antibody helper and antibody suppressor human T cells by using soybean agglutinin.
Authors: Reisner Y , Pahwa S , Chiao J W , Sharon N , Evans R L , Good R A
Published: May 2006

Soybean agglutinin (SBA) binds specifically to mouse B cells and has been used in the past to separate mouse B and T spleen cells by differential agglutination of the B cells. In the present study it was found that a major T-cell subpopulation of human peripheral blood mononuclear cells is agglutinated by SBA along with the B cells and monocytes. Tests of such cell surface markers as Fc receptors for IgG and IgM, as well as functional assays of antibody production by B cells, revealed that the SBA-agglutinated cell fraction contains the antibody helper T cells whereas the unagglutinated fraction is enriched with antibody suppressor T cells. Similar observations were made in tests of the proliferative response to mumps antigen. A recently prepared monoclonal antibody, anti-Leu 2a, which recognized the same thymus-dependent antigen previously defined by a heterologous anti-human T cell serum (alpha TH2), was found to define by indirect immunofluorescence a subpopulation of SBA- cells of intermediate staining intensity which was not detectable in the SBA+ population.
Assembly of subcomponents C1r and C1s of first component of complement: electron microscopic and ultracentrifugal studies.
Authors: Tschopp J , Villiger W , Fuchs H , Kilchherr E , Engel J
Published: May 2006

Monomeric C1s (Mr, 85,000; s20,w, 4.3S), a subcomponent of first component of complement (C1), the dimer (Mr, 170,000; s20,w, 6.7 S) of C1r, another subcomponent, and the tetrameric complex (C1r,C1s)2 (Mr, 340,000; s20,w, 8.7 S) are elongated molecules. Hydrodynamic equivalents of cylindrical shape have a diameter of 3.3 nm and lengths of 20 nm for C1s, 36 nm for (C1r)2, and 64 nm for (C1r,C1s)2. In electron micrographs the C1r,C1s complex appears as a chain composed of six to eight globular domains with a contour length of 51 nm. A structure is proposed in which (C1r)2 forms a core to which C1s protomers are associated at both ends. The C1 complex (s20,w, 16.3 S) reconstituted from C1q, C1r, and C1s dissociates under the conditions used for electron microscopy. Some features of the C1 complex are revealed in the dissociation products.
Characterization of a protease apparently involved in processing of pre-ornithine transcarbamylase of rat liver.
Authors: Mori M , Miura S , Tatibana M , Cohen P P
Published: May 2006

The precursor of rat liver ornithine transcarbamylase (ornithine carbamoyltransferase; carbamoylphosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3) (pre-ornithine transcarbamylase), which was synthesized in a reticulocyte lysate cell-free system, was converted to an apparently mature form of the enzyme by isolated rat liver mitochondria. The proteolytic processing involved two steps: (i) conversion of pre-ornithine transcarbamylase (39,400 daltons) to a product of about 37,000 daltons and (ii) further conversion to the apparently mature form of the enzyme (36,00 daltons). When mitochondria were subfractionated by digitonin treatment followed by sonication of a mitoplast fraction, the proteolytic activity catalyzing the first step was recovered mainly in a matrix fraction. Some activity was found in an intermembrane space fraction. The enzyme activity in the matrix fraction has an optimal pH at about 7.5. The activity was inhibited almost completely by 2 mM leupeptin and partly by 2 mM antipain but not significantly by other microbial protease inhibitors or serine protease inhibitors. It was inhibited strongly by 2 mM EDTA, 2 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetate, 2 mM p-chloromercuriphenylsulfonate, and 2 mM Hg(CH3COO)2 but not by N-ethylmaleimide or iodoacetamide. These results suggest that pre-ornithine transcarbamylase is first transported into the mitochondrial matrix and converted there to the mature form of the enzyme by a novel neutral protease(s).
Phosphorylated oligosaccharides in lysosomal enzymes: identification of alpha-N-acetylglucosamine(1)phospho(6)mannose diester groups.
Authors: Hasilik A , Klein U , Waheed A , Strecker G , von Figura K
Published: May 2006

In human fibroblasts, the recognition of lysosomal enzymes by cell surface receptors is mediated by mannose 6-phosphate residues located on oligosaccharides that can be cleaved by endo-beta-N-acetylglucosaminidase H. About half of these oligosaccharides, as isolated from beta-hexosaminidase and cathepsin D secreted by human skin fibroblasts, are anionic. Most of these are resistant to alkaline phosphatase. The resistance is due to alpha-N-acetylglucosamine residues linked to mannose 6-phosphate by a phosphodiester bond. The major phosphorylated oligosaccharides contain one and two and possibly three phosphate groups blocked by N-acetylglucosamine. Besides the blocked phosphate groups these oligosaccharides contain a common inner core consisting of Man alpha 1,6-(Man alpha 1,3)Man alpha 1,6(Man alpha 1,3)Man beta GlcNAc and either one or two alpha 1,2-linked mannose residues.
Interaction between the intermediary electron acceptor (pheophytin) and a possible plastoquinone-iron complex in photosystem II reaction centers
Authors: Klimov V. V. , Dolan Ed , Shaw E. R. , Ke Bacon
Published: May 2006

Photoreduction of the intermediary electron acceptor, pheophytin (Pheo), in photosystem II reaction centers of spinach chloroplasts or subchloroplast particles (TSF-II and TSF-IIa) at 220 K and redox potential
E
h
= -450 mV produces an EPR doublet centered at
g
= 2.00 with a splitting of 52 G at 7 K in addition to a narrow signal attributed to Pheo
[unk]
(
g
= 2.0033, Δ
H
≈ 13 G). The doublet is eliminated after extraction of lyophilized TSF-II with hexane containing 0.13-0.16% methanol but is restored by reconstitution with plastoquinone A (alone or with β-carotene) although not with vitamin K
1
. TSF-II and TSF-IIa are found to contain ≈2 nonheme Fe atoms per reaction center. Incubation with 0.55 M LiClO
4
plus 2.5 mM
o
-phenanthroline (but not with 0.55 M LiClO
4
alone) decreases this value to ≈0.6 and completely eliminates the EPR doublet, but photoreduction of Pheo is not significantly affected. Partial restoration of the doublet (about 25%) was achieved by subsequent incubation with 0.2 mM Fe
2+
, but not with either Mn
2+
or Mg
2+
. The Fe removal results in the development of a photoinduced EPR signal (
g
= 2.0044 ± 0.0003, Δ
H
= 9.2 ± 0.5 G) at
E
h
= 50 mV, which is not observed after extraction with 0.16% methanol in hexane. It is ascribed to plastosemiquinone no longer coupled to Fe in photosystem II reaction centers. The results show that a complex of plastoquinone and Fe can act as the stable “primary” electron acceptor in photosystem II reaction centers and that the interaction of its singly reduced form with the reduced intermediary acceptor, Pheo
[unk]
, is responsible for the EPR doublet.
Noninvasive, nondestructive approaches to cell bioenergetics.
Authors: Chance B , Eleff S , Leigh J S
Published: May 2006

To demonstrate the feasibility of using NMR spectra of human limbs and larger animals for continuous, noninvasive, nondestructive evaluation of cell bioenergetics, we have constructed a relatively simple and inexpensive 31P NMR apparatus. This apparatus consists of an 18-cm (7-in.) bore superconducting magnet and appropriate transmit-receive components for Fourier transform NMR. The principal signals observed by this instrument in the tissues are due to phosphocreatine and inorganic phosphate. The apparatus can be used to detect tissue normoxia and hypoxia. The large phosphocreatine/phosphate ratio (greater than 10:1), and the low phosphate signal from normoxic tissue (approximately 10% of the phosphocreatine signal from brain and human skeletal tissue) make an increased phosphate peak a very sensitive indicator of tissue hypoxia. Direct experiments on the human forearm and leg and the brains of dog and rabbit suggest the applicability of 31P NMR to humans and animals. This method and optical methods can both be used for quantitative determination of oxygen delivery to tissue, function of mitochondria, and the coupling of bioenergetic processes to functional activity in skeletal tissue and brain.
Quantitative genetic variation of enzyme activities in natural populations of
Drosophila melanogaster
Authors: Laurie-Ahlberg C. C. , Maroni G. , Bewley G. C. , Lucchesi J. C. , Weir B. S.
Published: May 2006

The genetic component of variation of enzyme activity in natural populations of
Drosophila melanogaster
was investigated by using two sets of chromosome substitution lines. The constitution of a line of each type is:
i
1
/
i
1
;+
2
/ +
2
;
i
3
/
i
3
and
i
1
/
i
1
;
i
2
/
i
2
;+
3
/+
3
, where
i
refers to a chromosome from a highly inbred line and + refers to a chromosome from a natural population. The + but not the
i
chromosomes vary within a set of lines. By use of a randomized block design to test and estimate components of variance, 50 of the second- and 50 of the third- chromosome substitution lines have been screened for variation in the activity levels of seven enzymes. Six of the seven enzymes show a significant genetic component in at least one set of lines, and five of the seven enzymes show activity variations attributable to factors that are not linked to the structural gene. These unlinked activity modifiers identify possible regulatory elements. Analyses of covariance show that most of the genetic variation of enzyme activities cannot be accounted for by genetic variation of live weight or protein content. These results and the lack of strong correlations between the genetic effects on the activities of different enzymes indicate that the effects are mainly specific for individual enzymes.
Role of minimization of chemical distances between amino acids in the evolution of the genetic code.
Authors: Wong J T
Published: May 2006

The allocation of codons in the genetic code makes possible a moderate minimization of the chemical distances between pairs of neighboring amino acids in the code. However, the code is neither a global nor a local optimum with respect to distance minimization. These findings do not support the physicochemical postulate that distance minimization was a major factor shaping the evolution of the genetic code. They agree with the coevolution theory, which proposes that genetic code evolution was predominantly determined by the concession of codons from precursor to product amino acids in an expansion of the code to accommodate new varieties of amino acids, with distance minimization playing a subsidiary role in deciding the choice of codons to be acquired by the product amino acids from the codon domains of the precursor amino acids.
Self-association of human immunoglobulin kappa I light chains: role of the third hypervariable region.
Authors: Stevens F J , Westholm F A , Solomon A , Schiffer M
Published: May 2006

Gel electrophoresis and molecular sieve chromatography were used to compare 17 different human kappa I type Bence Jones proteins including 5 for which the amino acid sequence is known. Although electrophoresis in the presence of NaDodSO4 showed uniformity of covalent dimer and monomer molecular weights, Sephadex chromatography under nondissociating conditions showed that monomers eluted with different apparent molecular weights. These differences were attributed to heterogeneity in light chain self-association; dimerization constants of the 17 proteins, calculated from a computer simulation of their behavior upon gel filtration, ranged from less than 10(3) to greater than 10(6) M-1. The variable region, more specifically the third hypervariable region, appears to be responsible for the variation in the dimerization constant. Association properties of light chains of known sequence suggest that the presence of an aromatic or hydrophobic residue at position 96 enhances dimer formation whereas a charged residue at that position results in light chains remaining stable monomers. The location of hypervariable residue 96 within the amino-terminal portion of the joining segment of the variable region suggests that the joining region may account for the variability of self-association of light chains and, moreover, that it has a function in determining the selective association of immunoglobulin polypeptide chains.
Membrane-active drugs potentiate the killing of tumor cells by D-glucosamine
Authors: Friedman Susan J. , Skehan Philip
Published: May 2006

D-Glucosamine is toxic to several malignant cell lines and
in vivo
tumors at concentrations that have little effect upon normal host tissues. Evidence is presented to support the hypothesis that cellular membranes may be the primary targets of glucosamine's tumoricidal activity. Treatment of rat C6 glioma cells with a cytotoxic concentration of glucosamine (20 mM) caused fragmentation of rough endoplasmic reticulum, proliferation of Golgi complexes, evagination of outer nuclear and mitochondrial membranes, and the accumulation of membranous vacuoles and lipid droplets in the cytoplasm. These changes were detected within the first 3 hr after treatment of cultures with glucosamine and became increasingly severe until cell lysis occurred between 24 and 48 hr of treatment. The cytotoxicity of glucosamine was potentiated by the local anesthetic lidocaine, and by other membrane-active drugs, at concentrations that were growth inhibitory but nonlytic. Most of these drugs possessed local anesthetic activity and inhibited glioma sterol synthesis. Within the same period of time required for ultrastructural changes in cellular membranes, glucosamine inhibited the incorporation of [2-
14
C]acetate into sterols and into an unidentified 400-dalton lipid that migrated close to sterols on thin-layer chromatograms. This inhibition was potentiated by lidocaine and increased over the same range of D-glucosamine concentrations that led to increased cell toxicity after a 48-hr treatment. These findings suggest that the effects of glucosamine upon cellular membranes may be central to its tumoricidal activity and that glucosamine, in combination with membrane-active drugs, may be useful in the treatment of certain types of tumors, particularly those of the central nervous system.
Agonist-promoted coupling of the beta-adrenergic receptor with the guanine nucleotide regulatory protein of the adenylate cyclase system.
Authors: Limbird L E , Gill D M , Lefkowitz R J
Published: May 2006

Binding of the beta-adrenergic agonist [3H]hydroxybenzylisoproterenol to the beta-adrenergic receptor of rat reticulocyte membranes results in the coupling of the receptor to the guanine nucleotide regulatory protein associated with the adenylate cyclase system. This regulatory component, referred to as the G-protein, was identified by its specific [32P]-ADP-ribosylation catalyzed by cholera toxin. Incubation of [32P]ADP-ribosylated rat reticulocyte membranes with the [3H]hydroxybenzylisoproterenol agonist prior to membrane solubilization and gel exclusion chromatography resulted in the coelution of the 42,000 Mr [32P]ADP-ribosylated G-proteins with the agonist-occupied beta-adrenergic receptors. The receptor-G-protein complex was not formed when receptors were unoccupied or occupied with antagonists at the time of solubilization. Incubation of rat reticulocyte membranes with [3H]hydroxybenzylisoproterenol in the presence of guanine nucleotides reversed or prevented the formation of this receptor-G-protein complex. These data provide direct evidence for the molecular interactions promoted by agonist occupancy of beta-adrenergic receptors. It is probable that the formation of a receptor-G-protein complex is crucial for catecholamine stimulation of the adenylate cyclase enzyme and, hence, transmembrane information transfer.
Mechanisms of membrane assembly: effects of energy poisons on the conversion of soluble M13 coliphage procoat to membrane-bound coat protein.
Authors: Date T , Zwizinski C , Ludmerer S , Wickner W
Published: May 2006

The coat protein (gene 8 product) of coliphage M13 spans the host cell plasma membrane prior to its assembly into extruding virions. It is made as a soluble precursor, termed procoat, with an extra 23 NH2-terminal amino acid residues. We have examined the effect of metabolic poisons on the assembly of procoat into the plasma membrane and its proteolytic conversion to coat protein. Protein synthesis and proline uptake were measured to assess the effect of each poison on cellular high-energy phosphate and on the transmembrane protonmotive force, respectively. Arsenate, which abolished protein synthesis but did not affect proline uptake, had no measurable effect on the conversion of procoat to coat protein. In contrast, the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) blocked conversion of procoat to coat protein. Neither CCCP nor arsenate inhibited the ability of a detergent-solubilized and highly purified preparation of leader peptidase to convert procoat to coat protein in the presence of detergents. The procoat that accumulated in the presence of CCCP was membrane bound. A spontaneous mutant that grows in the presence of CCCP showed (i) CCCP-resistant proline uptake in whole cells, (ii) CCCP-resistant uptake in inner membrane vesicles, and (iii) CCCP-resistant conversion of procoat protein to coat protein. These data suggest that an electrochemical gradient is at least indirectly necessary for the proper assembly of procoat into the cellular membrane.
Chemical mechanisms for cytochrome P-450 hydroxylation: evidence for acylation of heme-bound dioxygen.
Authors: Sligar S G , Kennedy K A , Pearson D C
Published: May 2006

Using isotopic tracer methods, we have shown that dihydrolipoic acid (2,3-thioctic acid) acylates the distal oxygen of ferrous oxygenated Pseudomonas cytochrome P-450, forming a transient acyl peroxide intermediate that facilitates oxygen-oxygen bond cleavage. Single-turnover studies with 18O2 indicate one oxygen-18 atom incorporated into the carboxylate group of lipoic acid for each oxygen-18 inserted into the substrate, camphor, forming the product, exo-5-hydroxycamphor. Such a branching ratio for label indicates that water is initially released from an unlageled position and illustrates that the general P-450 mixed-function oxidase stoichiometry generates H218O from 18O2 only after multiple-turnover equilibration with the acylating carboxylate oxygen. Formation of an acyl peroxide state is a natural intermediate in peracid, "oxene", or radical mechanisms for methylene carbone oxygenation.
Site-specific phosphorylation of the α subunit of eukaryotic initiation factor eIF-2 by the heme-regulated and double-stranded RNA-activated eIF-2α kinases from rabbit reticulocyte lysates
Authors: Ernst Vivian , Levin Daniel H. , Leroux Alena , London Irving M.
Published: May 2006

The site specificity of phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF-2α) by the heme-regulated and double-stranded RNA-activated eIF-2α kinases were compared by phosphopeptide mapping. eIF-2α was maximally phosphorylated
in vitro
with [γ-
32
P]ATP and either crude or partially purified preparations of the kinases.
32
P-Labeled eIF-2α was isolated by electrophoresis in sodium dodecyl sulfate/polyacrylamide gels. The fixed, stained, and dried polypeptide band was excised and then exhaustively digested directly in the gel slice with one of several proteases (trypsin, chymotrypsin, subtilisin, or thermolysin); the resultant [
32
P]phosphopeptides were analyzed by one-dimensional chromatography or by two-dimensional chromatography and high-voltage electrophoresis. In addition, limited proteolysis of [
32
P]eIF-2α contained in fixed, dried, and stained gel slices was achieved with
Staphylococcus aureus
protease V8, chymotrypsin, or subtilisin, and the partial
32
P-labeled cleavage products were analyzed by gel electrophoresis. Each protease produced distinct and reproducible [
32
P]phosphopeptide profiles after partial or exhaustive proteolysis of [
32
P]eIF-2α. With a given protease, identical [
32
P]phosphopeptide patterns were obtained whether eIF-2α was phosphorylated by the heme-regulated or the double-stranded RNA-activated kinase. These data indicate that,
in vitro
, the kinases phosphorylate sites on eIF-2α that are identical or proximally located in the primary sequence. In this report we also provide preliminary evidence that the two eIF-2α kinases activated in lysates by heme deficiency or double-stranded RNA phosphorylate site(s) of endogenous eIF-2α that are similar, if not identical, to the sites phosphorylated
in vitro
with partially purified eIF-2α kinase(s) and eIF-2.
Supercoils in prokaryotic DNA restrained in vivo.
Authors: Pettijohn D E , Pfenninger O
Published: May 2006

Cells of Escherichia coli containing the plasmid F were gamma-irradiated with various doses to introduce determined numbers of single-strand breaks in the F DNA. The cells were then incubated to permit repair of the breaks while DNA gyrase was inhibited with coumermycin to limit restoration of any relaxed supercoil. Repaired, covalently continuous F DNA was isolated and its superhelical density was measured by two different methods. Both indicated that a major part (50-60%) of the negative superhelical turns were maintained in the repaired molecules, suggesting that the supercoils are restrained in vivo.
Ubiquitous, interspersed repeated sequences in mammalian genomes.
Authors: Jelinek W R , Toomey T P , Leinwand L , Duncan C H , Biro P A , Choudary P V , Weissman S M , Rubin C M , Houck C M , Deininger P L , Schmid C W
Published: May 2006

DNA base sequence comparisons demonstrate that the principal family of 300-nucleotide interspersed human DNA sequences, the repetitive double-strand regions of HeLa cell heterogeneous nuclear RNA, and specific RNA polymerase III in vitro transcripts of cloned human DNA sequences are all representatives of a closely related family of sequences. A segment of approximately 30 residues of these sequences is highly conserved in mammalian evolution because it is also present in the interspersed repeated DNA sequences of Chinese hamsters. Further DNA sequence comparisons demonstrate that a portion of this highly conserved segment of repetitive mamalian DNA sequence is similar to a sequence found within a low molecular weight RNA that hydrogen-bonds to poly(A)-terminated RNA molecules of Chinese hamsters and a sequence that forms half of a perfect inverted repeat near the origin of DNA replication in papovaviruses.
Clonal anergy: Persistence in tolerant mice of antigen-binding B lymphocytes incapable of responding to antigen or mitogen
Authors: Nossal G. J. V. , Pike Beverley L.
Published: May 2006

The purpose of these experiments was to determine the degree of reduction in the number of antigen-binding B lymphocytes in the spleens of mice that had been rendered tolerant in the perinatal period. Newborn or pregnant mice were injected with fluorescein (Flu) coupled onto human gamma globulin, and the spleen cells of the neonatally injected mice, or of the offspring of the pregnant mice, were analyzed 1-6 weeks later. Tolerogen doses were chosen so as to achieve either a two-thirds reduction (low dose) in the number of anti-Flu B cells capable of yielding anti-hapten plaque-forming cell clones after
in vitro
stimulation, or as representing a supra-optimal tolerogenic stimulus (high dose). Antigen-binding B cells were studied by a two-cycle procedure, namely an initial cycle of binding to Flu-gelatin thin layers, followed by analysis of the binding cells in the fluorescence-activated cell sorter (FACS) after suitable staining with Flu-protein conjugates. With the high dose of tolerogen, a modest diminution in Flu-binding cell numbers down to 56-71% of control values could be induced. When these residual Flu-specific B cells were analyzed in the FACS to quantitate their spectrum of Flu-binding avidities, profiles identical to those of controls were obtained. The reduction proved transient in nature, binding cell numbers having returned to 80% of normal by 2 weeks and to normal by 6 weeks. Nevertheless, the Flu-specific B cells were incapable of responding to antigen or mitogen by antibody formation. With the low dose of tolerogen, despite the desired degree of functional silencing of Flu-specific B cells, the numbers and avidity spectra of antigen-binding cells were entirely normal in both the neonatally injected and
in utero
-injected groups. The results indicate that tolerance induced amongst immature B lymphocytes is not due to a physical elimination of the relevant B cell clones or to a modulation or blockade of their surface Ig receptors. Rather, it is due to the recognition and storage of negative signals amongst cells that continue to display a normal complement of receptors. We therefore propose that the term “clonal anergy” is a more accurate description than either “clonal deletion” or “clonal abortion.”
Autoantibody to centromere (kinetochore) in scleroderma sera.
Authors: Moroi Y , Peebles C , Fritzler M J , Steigerwald J , Tan E M
Published: May 2006

Sera from patients with scleroderma contained several autoantibodies to nuclear antigens which were distinguished by different patterns of nuclear immunofluorescence staining. One of these autoantibodies reacted with centromeric regions of chromosomes. In chromosome spreads, the staining appeared as two small spheres at the centromere, resembling kinetochores. The antigenic determinant appeared to be a protein or polypeptide tightly bound to DNA. The autoantibody was reactive with centromeres of cells derived from humans, mice, and Chinese hamsters. The autoantibody was present in high frequency in the calcinosis/Raynaud's phenomenon/esophageal dysmotility/sclerodactyly/telangiectasia variant (CREST) of scleroderma.
A mechanism for RNA splicing.
Authors: Rogers J , Wall R
Published: May 2006

The most abundant of the stable small nuclear RNAs of eukaryotic cells, U-1 small nuclear RNA, is exactly complementary to the consensus sequences at RNA splice sites. We propose that this RNA is the recognition component of the nuclear RNA splicing enzyme and forms base pairs with both ends of an intron so as to align them for cutting and splicing.
Rate theory calculation of gramicidin single-channel currents using NMR-derived rate constants.
Authors: Urry D W , Venkatachalam C M , Spisni A , Läuger P , Khaled M A
Published: May 2006

By means of 23Na NMR, two ion binding sites were observed in phospholipid-packaged gramicidin channels and the four associated rate constants were approximated. Limits also were placed on a fifth rate constant for an intrachannel ion translocation. By using Eyring rate theory to introduce voltage dependence, these rate constants were used in steady-state-current equations for calculation of gramicidin single-channel currents for two- and three-site models. Calculated single-channel currents are compared with previously published experimental single-channel currents obtained by electrical measurements on Na+ transport across gramicidin-doped planar lipid bilayers. The calculated results for the two- and three-site models compare favorably with the experimental results. Accordingly, it is demonstrated that NMR-derived rate constants can be coupled with Eyring rate theory to calculate currents through a transmembrane channel and to do so within levels of variation that compare with the differences obtained on planar lipid bilayers formed with different lipids.
Isolation of genes by complementation in yeast: molecular cloning of a cell-cycle gene.
Authors: Nasmyth K A , Reed S I
Published: May 2006

cdc28, one of several genes required for cell division in the yeast Saccharomyces cerevisiae, has been isolated on recombinant plasmids. A recombinant plasmid pool containing the entire yeast genome was constructed by partial digestion of yeast DNA with the four-base recognition restriction endonuclease Sau3A to give the equivalent of random fragments, size selection on sucrose gradients, and introduction of the fragments into the yeast vector YRp7 by use of the homology of Sau3A ends with those generated in the vector by cleavage with BamHI. Recombinant plasmids capable of complementing cdc28 mutations were isolated by transformation of a cdc28ts strain and selection for clones capable of growth at the restrictive temperature. Plasmids responsible for complementing the cdc28ts phenotype were shown to recombine specifically with the chromosomal cdc28 locus, confirming the identity of the cloned sequences. In addition, one of the recombinant plasmids was capable of complementing a mutation in tyr1, a gene genetically linked to cdc28. This method of gene isolation and identification should be applicable to all yeast genes for which there are readily scorable mutants.
DNA sequence of baboon highly repeated DNA: evidence for evolution by nonrandom unequal crossovers.
Authors: Donehower L , Furlong C , Gillespie D , Kurnit D
Published: May 2006

A highly repeated DNA was isolated from the West African baboon (Papio papio) as a 343-base-pair fragment after digestion of total baboon DNA with the restriction endonuclease BamHI. The DNA sequence of this fragment was obtained by chemical cleavage methods and is compared with the DNA sequence of related highly repeated primate DNAs from African green monkey (Cercopithecus aethiops) and man. The 343-base-pair baboon repeat consists of two related but nonidentical wings of 172 and 171 base pairs, respectively. The baboon 172-base-pair wing shares more homology with the African green monkey 172-base-pair repeat than with the baboon 171-base-pair wing. Comparison with the previously published monkey and human DNA sequences indicates that: (i) All the DNA sequences apparently arose from a common ancestral sequence. (ii) Evolution of the primate DNA sequences can be explained by a model involving unequal crossovers at specific points within the repeated DNA, possibly mediated by the sequence 5'-AAGG-3' 3'-TTCC-5' or its invert 5'-GGAA-3' 3'-CCTT-5'. (iii) There are alternating domains of conserved and divergent DNA sequences within each greater than 170-base-pair wing sequence. Taken together, the DNA sequences of these primates suggest a model whereby highly repeated DNAs are established and evolve as a consequence of unequal nonrandom exchanges of DNA duplexes. These exchanges may be mediated by short repeated nucleotide sequences and involve exchanges within and between the greater than 170-base-pair wings.
Identification of a cell surface protein, p97, in human melanomas and certain other neoplasms.
Authors: Woodbury R G , Brown J P , Yeh M Y , Hellström I , Hellström K E
Published: May 2006

BALB/c mice were immunized with a human melanoma cell line, SK-MEL 28, and their spleen cells were fused with mouse NS-1 myeloma cells. Hybrid cells were tested in an indirect 125I-labeled protein A assay for production of antibodies that bound to surface antigens of SK-MEL 28 melanoma cells but not to autologous skin fibroblasts. One hybridoma, designated 4.1, had the required specificity. It was cloned and grown in mice as an ascites tumor. The monoclonal IgG1 antibody produced by the hybridoma was purified from the ascites fluid and labeled with 125I. The labeled antibody bound, at significant levels, to approximately 90% of the melanomas tested and to approximately 55% of other tumor cells, but not to three B-lymphoblastoid cell lines or to cultivated fibroblasts from 15 donors. Immunoprecipitation and sodium dodecyl sulfate gel electrophoresis were used to detect the target antigen in 125I-labeled cell membranes of both cultivated cells and tumor biopsy samples. A protein with a molecular weight of 97,000 was identified. This protein, designated p97, was present in both cultured cells and biopsy material from melanomas and certain other tumors, but it was not detected in eight different samples of normal adult epithelial or mesenchymal tissues obtained from five donors.
Lipid-protein interaction in the glycophorin-dipalmitoylphosphatidylcholine system: Raman spectroscopic investigation
Authors: Taraschi Theodore , Mendelsohn Richard
Published: May 2006

Raman spectra have been recorded as a function of temperature for lipid-protein complexes of glycophorin isolated from erythrocyte membranes reconstituted with dipalmitoylphosphatidylcholine (DPPC) and its chain perdeuterated analogue ([
2
H
62
]DPPC). The conformation of the phospholipid hydrocarbon chains in the vicinity of protein is drastically altered from that in pure lipid dispersions. Analysis of the chain C-
2
H stretching vibrations for complexes of [
2
H
62
]-DPPC-glycophorin shows that at lipid:protein mole ratios of 125:1, a broad melting event occurs that is not observable by calorimetric techniques. The midpoint occurs at temperatures about 15°C below that of the gel/liquid crystal phase transition for [
2
H
62
]DPPC in multilamellar dispersions. The same number of
gauche
rotamers form in the phospholipid hydrocarbon chains during the melting process as in the phase transition of the unperturbed molecule. Analysis of the C-H stretching region of the Raman spectrum in DPPC-glycophorin complexes indicates that lateral interactions between phospholipid chains in the complex are reduced so that interchain vibrational coupling is minimized. The observed differences between the Raman melting curves and the calorimetric endothermic transitions arise because different populations of phospholipid molecules are sampled in the two experiments. The advantages of Raman spectroscopy for the study of lipid-protein interaction are demonstrated in the current work. Implications for the structure of the lipid in the immediate vicinity of membrane protein are discussed.
A suggestion for naming faces of ring compounds
Authors: Rose Irwin A. , Hanson Kenneth R. , Wilkinson Keith D. , Wimmer Mary J.
Published: May 2006

Discussion of the topology of interaction of ring compounds with macromolecules and receptors requires a system for naming the faces of the cyclic compound. An α/β-face nomenclature is suggested that is based on the clockwise/counterclockwise direction of ascending numbering in the ring with the lowest numbered unshared ring atom. This system is applicable to a very broad range of compounds: sugars, cyclic bases, steroids, cyclitols, porphyrins, etc., and molecules with a single ring, fused rings, encompassing rings, and rings formed by head-to-tail polymerization.
Measurement of the internal pH of yeast spores by 31P nuclear magnetic resonance.
Authors: Barton J K , den Hollander J A , Lee T M , MacLaughlin A , Shulman R G
Published: May 2006

The asci and single spores of the yeast Pichia pastoris were examined by using 31P NMR. The pH within the spores was determined from the pH-dependent chemical shifts of the internal orthophosphate (Pi) peaks. In spectra of asci, two internal Pi peaks corresponding to two compartments were observed. Only small variations in the internal pH values were found upon incubation of the asci in buffers ranging in pH from 3 to 10. For this range in external pH, the internal pH values calculated from the Pi chemical shifts were 5.5-6.3 and 5.1-5.9. The two internal Pi peaks, which have line widths of approximately 60 Hz, have been assigned to spore and epiplasmic compartments, respectively. Spectra of single spores revealed only one Pi peak, and, upon germination of either the single spores or asci, only this Pi peak disappeared. Peaks corresponding to ATP were not found in the dormant spores and asci but did appear upon germination. These NMR studies show the yeast spore to have a mobile and relatively acidic interior.
Electron transport to nitrogenase in Klebsiella pneumoniae.
Authors: Nieva-Gómez D , Roberts G P , Klevickis S , Brill W J
Published: May 2006

Cell-free extracts of nifF and nifJ mutants of Klebsiella pneumoniae are unable to couple acetylene reduction (N2 fixation) by nitrogenase to the oxidation of organic metabolites. However, nifF and nifJ mutants can complement each other in vitro to establish the coupling. This indicates that the products of the nifF and nifJ genes constitute essential elements of the physiological electron pathway to nitrogenase. The electron-transfer-active product of the nifF gene, a flavoprotein, has been purified.
Conformation of the helical nucleocapsids of paramyxoviruses and vesicular stomatitis virus: reversible coiling and uncoiling induced by changes in salt concentration.
Authors: Heggeness M H , Scheid A , Choppin P W
Published: May 2006

The conformations of the helical nucleocapsids of the paramyxoviruses Sendai virus and simian virus 5, and of a rhabdovirus, vesicular stomatitis virus, have been found to vary extensively with changes in salt concentration. In 10 mM sodium phosphate buffer at pH 7.2, the nucleocapsids are loosely coiled or almost completely extended; with increasing concentrations of NaCl they become more tightly coiled and less flexible. Under isotonic conditions (150 mM) the Sendai virus nucleocapsid is moderately tightly coiled but still curved and apparently flexible, whereas at 400 mM or higher it is very tightly coiled, with the appearance of a rigid rod. These salt-dependent changes in conformation were also found with nucleocapsids composed of proteolytically cleaved protein subunits. Because of the effect of salt concentration, and the fact that it may change during the preparation of negatively stained samples of electron microscopy, it was necessary to fix that nucleocapsids before negative staining to preserve their original conformation. The striking changes in nucleocapsid conformation in response to the ionic milieu indicate the plasticity of its helical structure and suggest that changes in the microenvironment of the nucleocapsid could influence its conformation during viral RNA transcription and replication or during virus assembly by budding, processes in which changes in the coiling of the nucleocapsid or its flexibility could be important.
Proteins encoded by
Agrobacterium tumefaciens
Ti plasmid DNA (T-DNA) in crown gall tumors
Authors: McPherson Joan C. , Nester Eugene W. , Gordon Milton P.
Published: May 2006

In order to detect proteins that may be produced in crown gall tumors as a result of expression of incorporated
Agrobacterium tumefaciens
Ti plasmid DNA (T-DNA), we have isolated mRNA complementary to T-DNA and translated this in a protein-synthesizing system derived from wheat germ. mRNA prepared from cultured E1 tumor from
Nicotiana tabacum
hybridized with
Hin
dIII fragment 1 sequences of T-DNA immobilized on cellulose nitrate filters. Two proteins of 30,000 and 16,500
M
r
were produced when this selected RNA was released and translated. Other tumor lines from
N. tabacum
were investigated, and a protein of slightly less than 30,000
M
r
was encoded by
Hin
dIII fragment 1 sequences of 15955/01 tumor. No products were observed for 15955/1 tumor line, which differs from E1/B6-806 and 15955/01 in that it does not produce octopine. mRNA species of each of the tumor lines hybridized to
Bst
I fragment 8 sequences of T-DNA and produced a common protein of 15,000
M
r
. Because this protein is derived from the region of the T-DNA that is conserved in octopine- and nopaline-type crown gall tumors, it may play a role in oncogenicity.
Anion channel blockers inhibit lysosomal enzyme secretion from human neutrophils without affecting generation of superoxide anion.
Authors: Korchak H M , Eisenstat B A , Hoffstein S T , Dunham P B , Weissmann G
Published: May 2006

The role of permeant anions in lysosomal enzyme secretion from human neutrophils was investigated by means of anion-channel-blocking agents: 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), and pyridoxal phosphate. Lysosomal enzyme release from cytochalasin B-treated human neutrophils stimulated by immune complexes (bovine serum albumin and IgG anti-bovine serum albumin) was inhibited by DIDS, SITS, and pyridoxal phosphate at concentrations that inhibited sulfate fluxes. Enzyme secretion triggered by calcium ionophore A23187 was also inhibited by DIDS and SITS; these agents acted on secretory events subsequent to Ca2+ influx. Neither the species of permeant anion(s) nor the role of anion fluxes in degranulation was identified, although influxes of chloride, hydroxide, or phosphate ions were not critical. In contrast to degranulation, generation of superoxide anions (O2.-) stimulated by immune complex or A23187 was not inhibited by these agents. Ultrastructural cytochemical studies demonstrated that, although lysosomal contents were not discharged from stimulated cells, vacuole formation and lysosome-lysosome fusion were unaffected by SITS or DIDS. Data suggest that anion channel blockers specifically inhibit fusion of lysosomes with the plasma membrane or its invaginations.
Establishment of a fetal rat liver cell line that retains differentiated liver functions.
Authors: Schlegel-Haueter S E , Schlegel W , Chou J Y
Published: May 2006

Fetal rat liver cells were transformed with a temperature-sensitive A mutant (tsA255) of simian virus 40. A CLONAL CELL LINE, RLA255-4, which was temperature sensitive in the maintenance of the transformed phenotype, was isolated. This cell line expressed the transformed phenotype (rapid growth, high cell density, overgrowth of normal cells, and cloning in soft agar) at the permissive temperature (33 degrees C) and the nontransformed phenotype (slower growth, lower saturation density, decreased efficiency of overgrowth of normal cells, and lower cloning efficiency in soft agar) at the restrictive temperature (40 degrees C). The tsA255-transformed cells expressed differentiated liver functions under controllable conditions. At the permissive temperature, they produced low levels of albumin and transferrin, whereas at the restrictive temperature the transformed phenotype was lost and the production of these hepatic proteins was greatly enhanced. RLA255-4 cells contained functional receptors for glucagon, as shown by the stimulation of intracellular cyclic AMP accumulation by glucagon. The response to glucagon was dose dependent (Kact = 5 x 10(-8) M) and could be demonstrated in cells grown at both permissive and restrictive temperatures after 7 days in culture (i.e., at a cell density of approximately 4 x 10(5) cells per cm2 or higher). Addition of cortisol to the culture medium enhanced the glucagon response selectively at the restrictive temperature.