bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of HLA-DR polymorphism by two-dimensional peptide mapping.
Authors: Corte G , Damiani G , Calabi F , Fabbi M , Bargellesi A
Published: May 2006

Two-dimensional peptide mapping was used to study the polymorphism of DR antigens, membrane glycoproteins composed of two chains, alpha and beta, and encoded by the human major histocompatibility complex (MHC). Four DR antigens were purified by immunoabsorption from four human lymphoblastoid cell lines homozygous at the DR locus. After labeling with 125I, alpha and beta chains were separated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and digested with pepsin. Comparison of the peptide maps showed a marked degree of polymorphism among beta chains: only 43% of peptides were common to all four chains and 15-21% of the spots were unique to a given chain. By contrast, only a limited variability was observed among alpha chains. Homology was 75% for the four chains and the percentage of unique peptides was very low. DR7 did not possess even a single unique peptide. The limited variability among alpha chains and the lack of "private" peptides in one of them point to the conclusion that the beta chain is the unique carrier of the alloantigenic specificities. Higher homology within the known crossreactive groups was not observed, suggesting that the determinants responsible for crossreactivity are on different molecules. From a genetic point of view, because beta chains show allele-associated polymorphism, they are likely to be MHC encoded, whereas the minor differences among alpha chains do not allow a similar conclusion. The available data point to an analogy between these DR antigens and the mouse I-E/C antigens.
Complete amino acid sequence of murine beta 2-microglobulin: structural evidence for strain-related polymorphism.
Authors: Gates F T , Coligan J E , Kindt T J
Published: May 2006

Primary structural analyses of beta 3-microglobulin isolated from the tumor cell lines EL4.BU (derived from a C57BL/6 mouse) and C14 (derived from a BALB/c mouse) have revealed the presence of an amino acid difference at position 85 of this molecule. beta 2-Microglobulin isolated from histocompatibility antigens of EL4.BU has alanine at this position, whereas that from C14 has aspartic acid. Determination of the sequence of these molecules has employed radiochemical methodology that was developed in studies of murine histocompatibility antigens. The sequence obtained in this study is: Ile - Gln - Lys - Thr - Pro - Gln - Ile - Gln - Val - Tyr - Ser - Arg - His - Pro - Pro - Glu - Asn - Gly - Lys - Pro - Asn - Ile - Leu - Asn - Cys - Tyr - Val - Thr - Gln - Phe - His - Pro - Pro - His - Ile - Glu - Ile - Gln - Met - Leu - Lys - Asn - Gly - Lys - Lys - Ile Pro - Lys - Val - Glu - Met - Ser - Asp - Met - Ser - Phe - Ser - Lys - Asp - Trp - Ser - Phe - Tyr - Ile - Leu - Ala - His - Thr - Glu - Phe - Thr - Pro - Thr - Glu - Thr - Asp - Thr - Tyr - Ala - Cys - Arg - Val - Lys - His - Ala/Asp - Ser - Met - Ala - Glu - Pro - Lys - Thr - Val - Tyr - Trp - Asp - Arg - Asp - Met. Comparison of the sequence of murine beta 2-microglobulin to the sequences reported for the homologues from man, rabbit, and guinea pig indicate identities of 68%, 66%, and 61%, respectively.
Autoradiographic localization of the sites of uptake, cellular transport, and catabolism of low density lipoproteins in the liver of normal and estrogen-treated rats
Authors: Chao Yu-Sheng , Jones Albert L. , Hradek Gary T. , Windler Eberhard E. T. , Havel Richard J.
Published: May 2006

The hepatic uptake and catabolism of low density lipoproteins are stimulated severalfold in rats treated with large amounts of 17α-ethinylestradiol. To determine the sites within the liver at which these processes occur,
125
I-labeled human low density lipoproteins were injected intravenously into intact control and estradiol-treated rats or added to perfusates of their isolated livers. The livers were fixed by perfusion and processed for light and electron microscopic autoradiography. Distribution of autoradiographic silver grains was estimated qualitatively in light micrographs and quantitatively in electron micrographs. Many more silver grains were seen in livers from estradiol-treated than from control rats, but the processing of labeled low density lipoprotein was indistinguishable. Three minutes after intravenous injection or perfusion of livers, the grains were concentrated over the microvillous surface of parenchymal cells bordering the space of Disse. Many of these grains were within two half-distances from endocytic pits. Only 5-15% of the grains were seen over endothelial and Kupffer cells. Silver grains were also observed over vesicles beneath the plasma membrane whose size and shape suggested that they were derived from fusion of endocytic vesicles. By 15 min, grains were predominantly located in structures like multivesicular bodies in the region of the GERL (Golgi complex-endoplasmic reticulum-lysosomes) near the bile canaliculi. These bodies were packed with small vesicle-like structures and a few larger vesicles, the latter possessing a unit membrane. Between 15 and 30 min, when proteolysis of low density lipoproteins is known to begin, the initially clear matrix of the multivesicular body-like structures became dark and the structures frequently had a dense tail-like appendage. At the same time, silver grains began to appear over secondary lysosomes. These and other results indicate that the hepatic uptake of low density lipoproteins that is stimulated in rats given large amounts of estradiol follows a pathway that closely resembles that of the well-defined “LDL receptor” in cultured cells. In the liver these lipoproteins appear to be transported in endocytic vesicles; the vesicles fuse to form multivesicular body-like structures that acquire lysosomal enzymes and are converted to secondary lysosomes as the lipoproteins are degraded.
General quantitative treatment for the binding of divalent antibodies to antigens immobilized on a solid phase
Authors: Pincus Matthew R. , Rendell Marc
Published: May 2006

A general expression for the binding isotherm of divalent antibodies to antigens bound to solid lattices with excluded volume effects is derived by using a statistical mechanical approach. The method is based on matrix generation of the partition function for the system, from which the degree of saturation of the antigen lattice may be computed directly. The saturation function has a simple form that under appropriate conditions reduces to forms derived previously for simpler cases (such as monovalent binding with and without excluded volume effects). Solution by numerical techniques of a simple secular equation for each concentration of antibody allows computation of the saturation function. Thus, the binding expression derived is easily fit to any set of binding data obtained and results in the determination of such parameters as intrinsic affinity constants, length of excluded space (and hence molecular size), and number of sites. It predicts that under conditions wherein divalent binding is favored over univalent binding, biphasic adsorption isotherms will be obtained.
Synthesis, intracellular transport, and processing of the precursors for mitochondrial ornithine transcarbamylase and carbamoyl-phosphate synthetase I in isolated hepatocytes.
Authors: Mori M , Morita T , Ikeda F , Amaya Y , Tatibana M , Cohen P P
Published: May 2006

The synthesis and intracellular transport of the mitochondrial matrix enzymes ornithine transcarbamylase (carbamoylphosphate: L-ornithine carbamoyltransferase, EC 2.1.3.3.) and carbamoyl-phosphate synthetase (ammonia) I [carbon-dioxide:ammonia ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.4.16] were studied in isolated rat hepatocytes. In pulse experiments at 37 degrees C, the larger precursors of the two enzymes appeared in the cytosol of the liver cells, where radioactivity levels of the precursors reached a plateau in 10-20 min after the pulse. The pulse-labeled mature enzymes appeared in the particulate fraction (containing mitochondria) after a time lag and increased almost linearly with time up to 40 min. The specific radioactivities of the precursors in the cytosol were much higher than those of the mature enzymes in the particulate fraction. In pulse--chase experiments, the labeled precursors disappeared from the cytosol with estimated half-lives of about 1-2 min. These results indicate that ornithine transcarbamylase and carbamoyl-phosphate synthetase I are initially synthesized as larger precursors and exist in a cytosolic pool from which they are transported into mitochondria and processed there to the mature enzymes concomitantly with or immediately after transport. Although the rates of synthesis, transport, and processing were decreased about 3-fold at 25 degrees C (as compared to incubation at 37 degrees C), the pool size of the precursors in the cytosol were somewhat larger at this temperature.
Heteroduplex formation by recA protein: polarity of strand exchanges.
Authors: West S C , Cassuto E , Howard-Flanders P
Published: May 2006

Purified recA protein promotes strand exchanges between linear duplex DNA and homologous circular single-stranded phage phi X174 DNA that carries a short hybridized fragment [West, S. C., Cassuto, E. & Howard-Flanders, P. (1981) Proc. Natl. Acad. Sci. USA 78, 2100-2104]. In this paper we investigate the mechanism of this strand exchange reaction. We show that recA protein initiates strand exchanges by pairing the free end of the duplex fragment with the single-stranded DNA. In addition, we find that strand exchanges are polar, stable heteroduplex molecules being formed by the directional transfer transfer of the (-) strands starting at 3' termini.
Molecular complexes involved in the regulation of adenylate cyclase.
Authors: Sahyoun N E , LeVine H , Davis J , Hebdon G M , Cuatrecasas P
Published: May 2006

Selective extraction of the adenylate cyclase regulatory protein (N-protein) from pigeon erythrocyte plasma membranes provided evidence for its cytoskeletal association. Cholate, but not Triton X-100 or digitonin, was effective in solubilizing the ADP-ribosylated N-protein. The labeled protein complex or components thereof that were associated with the Triton-insoluble cytoskeleton (shells) could be partly released by 0.1 mM EDTA; 1 M KCl in the presence of Triton X-100 achieved complete solubilization. 5'-Guanylyl imidodiphosphate (p[NH]ppG) and NaF, activators of adenylate cyclase, promoted the release of the regulatory protein from the cytoskeleton but MnCl2, an "uncoupler" of the adenylate cyclase system, had the opposite effect. The solubilized, labeled N-protein was able to bind specifically to rat erythrocyte inside-out vesicles in the presence of divalent cations. A proteolytic product of inside-out vesicles inhibited the binding of the N-protein to fresh vesicles. Three molecular species which contained the Mr 45,000 polypeptide component of the N-protein were identified by gel permeation chromatography and by sucrose density gradient velocity sedimentation. p[NH]ppG appeared to convert the two larger molecular complexes to a smaller molecular entity. Such a molecular dissociation might be relevant to the effects of guanyl nucleotides on the activity of adenylate cyclase and on the affinity of hormone receptors.
Regulation of protein synthesis: translational control by procollagen-derived fragments.
Authors: Hörlein D , McPherson J , Goh S H , Bornstein P
Published: May 2006

Previous studies have shown that a type I procollagen-derived peptide, called pN alpha 1(I)-Col 1, selectively inhibits collagen synthesis by fibroblasts in culture and the translation of procollagen mRNA in a rabbit reticulocyte lysate system. We prepared the 10,700-dalton peptide from dermatosparactic calf skin, which contained high levels of incompletely processed type I procollagen, by collagenase digestion. Time-course and dose-response studies showed that the peptide specifically inhibited the translation of procollagen mRNA in preparations of human fibroblast RNA while not affecting the translation of globin mRNA or of other messenger RNAs in fibroblast RNA. After reduction and alkylation, the peptide lost its specificity but became a nonspecific inhibitor of translation. Enzymatic cleavage enabled us to localize the nonspecific activity to a short sequence -Pro-Thr-Asp-Glu, an assignment confirmed by peptide synthesis. Using pactamycin, a specific inhibitor of translational initiation, we showed that the intact peptide acts on procollagen mRNA translation by inhibition of polypeptide chain elongation or termination, or both, whereas the nonspecific inhibitory activity of the unfolded peptide and its derivatives can be attributed to an inhibition of chain initiation. Although the native peptide may function in feedback regulation of collagen synthesis, the physiological role of the lower molecular weight fragments, if any, is uncertain.
Successive generations of mice produced from an established culture line of euploid teratocarcinoma cells
Authors: Stewart Timothy A. , Mintz Beatrice
Published: May 2006

The possibility of utilizing mouse teratocarcinoma stem cells as intermediaries for production of new strains of mice with preselected mutant or foreign genes requires that, after propagation in culture (to allow for genetic manipulation and selection), the cells be capable of normalization and orderly development in carrier embryos and, ultimately, of germ-cell formation. Heretofore, no
in vitro
cell line has fulfilled all these requirements. A karyotypically normal teratocarcinoma culture line was recently established in this laboratory and now has been investigated as a candidate. The line, designated METT-1, is chromosomally female (X/X) and was obtained from the 129 (agouti-colored) inbred strain [Mintz, B. & Cronmiller, C. (1981)
Somat Cell Genet
7, 489-505]. The developmental potential of these cells was tested, after prolonged culture and freezing and thawing, by microinjecting them into early (blastocyst stage) embryos of the C57BL/6 (black) strain. Among 312 experimental animals examined at 1 week of age, there were 41 mice (21 females and 20 males) that displayed the coat colors of both strains. This frequency (13%), as well as the extent of the coat areas derived from the cell line, greatly surpasses the contributions observed in all previous experiments, whether with other
in vitro
teratocarcinoma cell lines or with
in vivo
transplant lines. The developmental totipotency of METT-1 cells became evident from the presence of substantial amounts of 129-strain cells (bearing an isozyme marker) in all internal tissues of an individual whose coat was largely agouti. The culture-cell lineage also proved to be capable of giving rise to reproductively functional oocytes. Of nine mosaic-coat females testmated to C57BL/6 males, one produced progeny of the diagnostic agouti color in two litters; these heterozygous F
1
offspring in turn transmitted their marker genes to F
2
homozygous segregants. Thus, the METT-1 teratocarcinoma line bridges the gap between
in vitro
cell propagation and
in vivo
development and between the soma and the germ line. This creates the option of producing new mouse strains with predetermined genetic changes designed as probes of developmental regulation or as models of human genetic diseases.
Abnormal action potentials associated with the
Shaker
complex locus of
Drosophila
Authors: Tanouye Mark A. , Ferrus Alberto , Fujita Shinobu C.
Published: May 2006

Intracellular recordings of action potentials were made from the cervical giant axon in
Shaker
(
Sh
) mutants and normal
Drosophila
. The mutants showed abnormally long delays in repolarization. The defect is not due to abnormal Ca
2+
channels, because it persists in the presence of Co
2+
, a Ca
2+
-channel blocker. On the other hand, the K
+
-channel blocker 4-aminopyridine causes a similar effect in normal animals, suggesting that the
Sh
mutant may have abnormal K
+
conductance. Gene-dosage analysis of
Sh
shows that the defect is not due to underproduction of an otherwise normal molecule; it may be due to an abnormal molecule produced by the mutated gene. Gel electrophoresis failed to detect an abnormal protein, suggesting that, if
Sh
codes for a nervous system protein, it is rare. Genetic analysis of the
Sh
locus indicates three regions. Mutations or chromosome breaks in the two flanking regions cause
Sh
mutant physiology; the central region shows a “haplolethal effect”—i.e., heterozygous females are lethal.
Radial distribution function of liquid argon according to significant structure theory
Authors: Yoon Byoung Jip , Jhon Mu Shik , Eyring Henry
Published: May 2006

The significant structure theory of liquids is tested by evaluating the radial distribution function. The calculations are based on face-centered cubic quasi-lattice structure, and the fluctuation in volume is used to describe the thermal displacements of molecules. It is shown that the local structure of liquids has inhomogeneous distributions. The correlation at long ranges becomes negligible because of the fluidized vacancies in our model. The calculations are performed for liquid argon, and the results are in good agreement with experiments.
Tyrosylprotein kinase and phosphatase activities in membrane vesicles from normal and Rous sarcoma virus-transformed rat cells.
Authors: Gallis B , Bornstein P , Brautigan D L
Published: May 2006

Membrane vesicles; isolated from normal and Rous sarcoma virus-transformed rat cells, have an associated cyclic-AMP independent kinase that phosphorylates a Mr 37,000 protein in vesicles from normal cells and proteins of Mr 37,000, 50,000, and 67,000 in vesicles from transformed cells. Proteins in vesicles from normal and transformed cells contain 9% and 77%, respectively, of their labeled phospho amino acids as phosphotyrosine. Thus, isolation of vesicles and subsequent incubation with [gamma-32P]ATP enriches the proportion of labeled phosphotyrosine in proteins (relative to other phospho amino acids) by two orders of magnitude over that found in intact cells. The in vitro phosphorylation of each of these proteins is enhanced in the presence of 10 microM Zn2+, a phosphotyrosylprotein phosphatase inhibitor. From these studies it appears that membrane vesicles may be a valuable system for examination of transformation-specific phosphorylation of proteins.
Laser flash photolysis studies of electron transfer between semiquinone and fully reduced free flavins and horse heart cytochrome c.
Authors: Ahmad I , Cusanovich M A , Tollin G
Published: May 2006

Laser flash photolysis has been used to determine the second-order rate constants for the reduction of horse heart cytochrome c by the semiquinone and fully reduced forms of various flavin analogs. We find that substitution in the dimethylbenzene ring of the flavin causes appreciable changes in the rate constants, whereas substitutions at the N-10 position do not. Placing a charged phosphate group in the N-10 ribityl side chain leads to only small ionic strength effects on the rate constants, whereas a charged group attached to the dimethylbenzene ring produces a large ionic strength effect. These results can be accounted for by assuming that a productive collision between flavin and cytochrome involves an orientation that positions the aromatic ring--N-5 region of the flavin toward the heme crevice and the N-10--pyrimidine ring region away from it. Our observations have implications for mechanistic understanding of biological electron transfer reactions and are discussed in this context.
Differential transcription
in vivo
and
in vitro
of two adjacent maize chloroplast genes: The large subunit of ribulosebisphosphate carboxylase and the 2.2-kilobase gene
Authors: Jolly Setsuko O. , McIntosh Lee , Link Gerhard , Bogorad Lawrence
Published: May 2006

The transcription of cloned maize plastid DNA sequences
in vitro
by maize plastid DNA-dependent RNA polymerase has been studied to expose the roles of the enzyme, polypeptide cofactors, and DNA sequences in the regulation of gene expression. The 4.35-kilobase pair
Bam
HI fragment 9 carries the maize plastid gene for the large subunit of ribulosebisphosphate carboxylase and part of the gene for a 2.2-kilobase RNA. These two genes are separated by ≈330 base pairs and are transcribed divergently. Transcripts of the gene for the large subunit of ribulosebisphosphate carboxylase are abundant in bundle sheath cells of maize leaves and we show here that transcripts of the 2.2-kilobase RNA gene are present in both mesophyll cells and the adjacent bundle sheath cells.
In vitro
, in the presence of the S factor, maize chloroplast DNA-dependent RNA polymerase produces a transcript of the gene for the large subunit of ribulose-bisphosphate carboxylase with a 5′ terminus like that of the corresponding mRNA isolated from plastids, transcribes chloroplast DNA sequences of
Bam
fragment 9 in a chimeric plasmid in preference to the vehicle RSF 1030 and, in a ratio of 3:1, preferentially transcribes the gene for the large subunit of ribulosebisphosphate carboxylase over the 2.2-kilobase RNA gene from supercoiled chimeric plasmid DNA.
Morphology of a novel cyanobacterium and characterization of light-harvesting complexes from it: Implications for phycobiliprotein evolution
Authors: Kursar Thomas A. , Swift Hewson , Alberte Randall S.
Published: May 2006

The morphology of the marine cyanobacterium DC-2 and two light-harvesting complexes from it have been characterized. DC-2 has an outer cell wall sheath not previously observed, the purified phycoerythrin shows many unusual properties that distinguish it from all phycoerythrins characterized to date, and isolated phycobilisomes have a single absorption band at 640 nm in the phycocyanin-allophycocyanin region of the spectrum. On the basis of these observations we suggest that DC-2, rather than being a member of the
Synechococcus
group, should be placed in its own taxonomic group. In addition, the particular properties of the isolated phycoerythrin suggest that it may be representative of an early stage in the evolution of the phycoerythrins. These observations are of special interest in light of the contribution DC-2 and related cyanobacteria may make to global primary productivity.
Phosphorylation of the same specific protein during amylase release evoked by beta-adrenergic or cholinergic agonists in rat and mouse parotid glands.
Authors: Jahn R , Söling H D
Published: May 2006

Stimulation of amylase secretion from the rat parotid gland by beta-adrenergic agonists is associated with a specific phosphorylation of three membrane-bound proteins designated as proteins I, II, and III [Jahn, R., Unger, C. & Söling, H. D. (1980) Eur. J. Biochem. 112, 345-352]. In contrast, stimuliation by carbachol induced significant phosphorylation of only protein I. This phosphorylation was low compared to isoproterenol-induced phosphorylation but corresponded to the smaller enhancement of amylase secretion. The mouse organ, however, is almost equally sensitive to beta-adrenergic and to cholinergic agonists. Incubation of mouse parotid gland slices with either 20 microM isoproterenol or 10 microM carbachol resulted in strong and comparable releases of amylase, which were accompanied by comparable phosphorylations of protein I. Proteins II and III were phosphorylated only in the presence of isoproterenol. Removal of external calcium by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetate abolished the carbachol-induced release of amylase but not the phosphorylation of protein I. Isoproterenol-induced secretion of amylase and phosphorylation of proteins I, II, and III were not inhibited under these conditions. Amylase release stimulated by the ionophore A-23187 was accompanied by the phosphorylation of protein I. Two-dimensional electrophoresis revealed that the radioactive spot corresponding to protein I was located at the same position after cholinergic and after beta-adrenergic stimulation, indicating that both stimuli led to the phosphorylation of the same membrane-associated protein. These findings strongly support the view that the phosphorylation of protein I is an important step in the sequence of events leading from receptor activation to exocytosis.
In vitro translation of hypoxanthine/guanine phosphoribosyltransferase mRNA: characterization of a mouse neuroblastoma cell line that has elevated levels of hypoxanthine/guanine phosphoribosyltransferase protein.
Authors: Melton D W , Konecki D S , Ledbetter D H , Hejtmancik J F , Caskey C T
Published: May 2006

Antibody specific for the native form of Chinese hamster hypoxanthine/guanine phosphoribosyltransferase (HPRT; IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) was used to detect the synthesis of HPRT protein in a rabbit reticulocyte lysate translation system primed with mRNA from Chinese hamster tissues and cultured cells. Electrophoretic analysis of the immunopurified products from the translation of mRNA from wild-type and a series of mutant Chinese hamster cells indicated that HPRT synthesis in vitro qualitatively and quantitatively corresponded to synthesis in vivo. The translation system was used to identify two mRNA sources producing high levels of HPRT protein: Chinese hamster brain and a mouse neuroblastoma HPRT revertant cell line, NBR4. Translation of NBR4 mRNA generated 25-50 times more HPRT protein than mRNA from wild-type cells. The basis for HPRT overproduction is considered in view of an X chromosome alteration found in NBR4 cells.
Developmental onset of mixed-function oxidase activity in preimplantation mouse embryos.
Authors: Filler R , Lew K J
Published: May 2006

Two-cell embryos, obtained from the C57BL/6N and DBA/2N strains, were cultured in media that supported in vitro differentiation and that contained [3H]benzo[a]pyrene. High-pressure liquid chromatography of the activated intermediates formed during in vitro early embryonic development indicated that the onset of polynuclear aromatic hydrocarbon activation coincided with blastocyst formation. Comparison of individual oxygenated intermediates metabolically formed from embryos genetically "responsive" or "nonresponsive" to aromatic hydrocarbons revealed significant quantitative differences in the production of dihydrodiol, quinone, and phenolic derivatives. In addition to exhibiting basal mixed-function oxidase activity, blastocysts were also responsive to enzymatic induction when exposed to 2,-3,7,8-tetrachlorodibenzo-p-dioxin. The presence of operative metabolite-detoxifying pathways was also assayed. Enzymatic treatment of water-soluble metabolites with beta-glucuronidase or arylsulfatase revealed that neither glucuronic acid conjugates nor sulfate ester derivatives were present. These data, therefore, provide direct evidence that late preimplantation mouse embryos (day 3 1/2 of gestation) are similar to later developmental stages in having the enzymatic capability for xenobiotic activation and enzyme induction but are dissimilar with respect to their detoxification mechanism(s). Moreover, the ability of preimplantation embryos to activate directly polynuclear aromatic hydrocarbon to bioreactive intermediates may be of importance in assessing the ontological susceptibility of the developing embryo to carcinogenic or teratogenic chemicals.
Cloning of chromosomal genes in Streptococcus pneumoniae.
Authors: Stassi D L , Lopez P , Espinosa M , Lacks S A
Published: May 2006

A system for molecular cloning in Streptococcus pneumoniae was developed. The multicopy plasmids pMV158 (5.4 kilobases) and pLS1 (4.3 kilobases), which confer tetracycline resistance, were used as vectors to clone chromosomal genes of S. pneumoniae in host cells of this species. A 3.3-kilobase restriction fragment containing the malM gene, which codes for amylomaltase, was cloned in a deletion mutant lacking chromosomal homology with the fragment. The recombinant plasmid pLS70, could transform over 50% of a recipient population to maltose utilization. Amylomaltase constituted up to 10% of the protein of cells containing pLS70. A derivative with a deletion, pLS69, appeared to gain a selective advantage by producing less enzyme. A 10-kilobase restriction fragment containing the sul-d gene for sulfonamide resistance was cloned in the presence of the homologous chromosomal gene. De novo establishment of a recombinant plasmid was just as frequent as transformation in an endogenous plasmid. Despite the processing of DNA during uptake in the transformation of S. pneumoniae, recombinant plasmids can be introduced. Models for the reconstruction of recombinant DNA in cells of S. pneumoniae and Bacillus subtilis are considered and compared.
Superoxide dismutase in Drosophila melanogaster: biochemical and structural characterization of allozyme variants.
Authors: Lee Y M , Misra H P , Ayala F J
Published: May 2006

Superoxide dismutase (SOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1) is known to be polymorphic in many organisms; in Drosophila, the degree of polymorphism has a wide range of variation from locality to locality within a given species. We have thoroughly purified from D. melanogaster the two common electromorphs, SODS and SODF. These differ in properties such as isoelectric point, specific activity, rate constant, thermostability, and amino acid composition. The specific activity is three times greater in SODS than in SODF, but the latter is more thermostable. In strains from California, SODS differs from SODF by at least one amino acid substitution: lysine in SODS is replaced by either aspartic acid or asparagine in SODF. This difference is consistent with the electrophoretic mobility and isoelectric points of the two electromorphs. In strains from Africa, SODS and SODF differ by two amino acid substitutions (histidine and proline in SODS vs. serine and either glutamic acid or glutamine in SODF) in addition to the one distinguishing the California strains. Thus the SODF electromorphs from California and from Tunisia, in spite of their identical electrophoretic mobility, differ by at least two amino acid substitutions.
Structural comparisons of TL antigens derived from normal and leukemia cells of Tl+ and TL- strains and relationship to genetically linked H-2 major histocompatibility complex products.
Authors: Yokoyama K , Stockert E , Old L J , Nathenson S G
Published: May 2006

Comparative tryptic peptide analysis was used to probe the structure of the TL products coded for by normally expressed TL alleles (Tlaa and Tlac haplotypes) and the structure of the TL product on ERLD, a TL+ leukemia occurring in a strain that does not normally express TL antigens (Tlab haplotype). In mice that have the Tlaa haplotype, the peptide maps of TL glycoproteins from normal thymocytes and from TL+ leukemias were identical, a finding that is consistent with the indistinguishable serologically defined TL phenotype of these cells. Analysis of the TL product on ERLD leukemia cells (TL phenotype, TL.1,2,4) indicates a single TL glycoprotein species with the TL.4 determinant (restricted to leukemias of Tlab and Tlac haplotypes) coexisting on the same glycoprotein as the Tl.1 determinant (normally expressed by thymocytes of Tlaa haplotype). Comparison of the peptides of the TL product of ERLD and the products of the normally expressed Tlaa and Tlac loci showed a high degree of structural similarity (i.e., 70-80%) of shared peptides. In contrast, the products of the Tlaa, Tlab, and Tlac loci shared relatively few peptides with the products of the K, D, and L loci of the closely linked major histocompatibility complex (15-35%). The TL family, therefore, consists of alleles that are highly homologous. This contrasts with the marked diversity and polymorphism among the alleles of the K, D, and L loci.
Uptake of aspartic and glutamic acid by photoreceptors in goldfish retina.
Authors: Marc R E , Lam D M
Published: May 2006

The uptake of acidic amino acids by goldfish photoreceptors was investigated by light microscope autoradiography. Isolated retinas were incubated in media containing micromolar amounts of L-[3H]aspartate, L-[3H]glutamate, and D-[3H]aspartate. We have four major observations. (i) Rods accumulate L-[3H]glutamate with a high-affinity transport system; they exhibit a glutamate-to-aspartate selectivity ratio of 30:1. When incubated in 1-10 microM L-[3H]glutamate, rods label more densely than cones. A unit area of rod membrane transports glutamate 30 times better than a unit area of cone membrane. (ii) Red-sensitive and green-sensitive cones show accumulation of L-[3H]aspartate, D-[3H]aspartate, and L-[3H]glutamate, apparently with high affinity, but with little selectivity. Because rods have poor aspartate uptake, red-sensitive and green-sensitive cones may be preferentially labeled with L-[3H]aspartate or D-[3H]aspartate, (iii) Blue-sensitive cones show no uptake of L-[3H]aspartate, D-[3H]aspartate, or L-[3H]glutamate other than that attributable to low-affinity transport. (iv) Various cell types in the goldfish retina can clearly discriminate between glutamate and aspartate, unlike acidic amino acid transport systems described in mammalian brain.
Pattern recognition applied to presidential elections in the United States, 1860-1980: Role of integral social, economic, and political traits
Authors: Lichtman A. J. , Keilis-Borok V. I.
Published: May 2006

The outcome of American presidential elections in 1860-1980 follows certain regular patterns which can be described phenomenologically by simple integral parameters of “common sense” type.
Kinematic model for B-DNA.
Authors: Dickerson R E , Drew H R
Published: May 2006

X-ray structure analysis of B-DNA double helix with sequence C-G-C-G-A-A-T-T-C-G-C-G has revealed several sequence-dependent structural features. Four of these are shown in this paper to be related to one another by simple structural or kinematic principles: (i) the correlation between glycosyl torsion and chi and main chain C4'--C3' torsion angle delta, (ii) the observations that purines prefer larger phi and delta angles than do pyrimidines, (iii) the anticorrelation of phi or of delta angles between sugars associated with one base pair, and (iv) the observation that successive base planes in purine-pyrimidine steps open up the angle between them toward the major groove, whereas pyrimidine-purine steps open toward the minor groove. These features offer the beginning of an understanding of the way in which specific base sequences can perturb the structure of a B-DNA double helix so as to be "read" by intercalating drugs, repressors, and other recognition proteins.
Plasmid-directed synthesis of enzymes required for D-mannitol transport and utilization in Escherichia coli.
Authors: Lee C A , Jacobson G R , Saier M H
Published: May 2006

A transformant Escherichia coli colony bank [Clarke, L. & Carbon, J. (1976) Cell 9, 91-99] has been screened for hybrid ColE1 plasmids carrying the genes for D-mannitol utilization. Two of the plasmids, pLC11-7 and pLC15-48, were shown to contain the mannitol operon, which includes the structural genes for the mannitol-specific enzyme II of the phosphotransferase system and mannitol-1-phosphate dehydrogenase. One E. coli strain harboring plasmid pLC15-48 overproduced mannitol-1-phosphate dehydrogenase activity 4- to 5-fold. However, there was no corresponding increase in mannitol enzyme II activity. Plasmid pLC15-48 was shown to direct the synthesis of two polypeptides in E. coli minicells in the presence of cyclic AMP and mannitol. The larger (Mr = 60,000) was membrane bound and was specifically precipitated by antibody directed against purified mannitol-specific enzyme II. The smaller (Mr = 40,000) was soluble and had an electrophoretic mobility indistinguishable from that of the major component in a partially purified mannitol-1-phosphate dehydrogenase preparation. These data are consistent with previous genetic studies of the mannitol locus and confirm an independent conclusion [Jacobson, G. R., Lee, C. A. & Saier, M. H., Jr. (1979) J. Biol. Chem. 254, 249-252] that mannitol enzyme II consists of a single type of polypeptide chain that has a Mr of 60,000. The plasmid pLC15-48 DNA was characterized by mapping of restriction endonuclease cleavage sites.
Sequence determination of the thiolester site of the fourth component of human complement.
Authors: Harrison R A , Thomas M L , Tack B F
Published: May 2006

The fourth component of complement (C4) is inactivated by treatment with methylamine. This property is shared wit the third component (C3) and with alpha 2-macroglobulin. In each instance, the reaction with methylamine is stoichiometric, covalent, and accompanied by the appearance of a thiol group. These data are consistent with the presence of an internal thiolester bond. Incubation of C4 with [14C] methylamine in the presence of activated thiol-Sepharose resulted in immobilization of the protein via its active-site thiol. Analysis of bound C4 indicated incorporation of 1.12 mol of [14C]methylamine per mol of protein. Digestion of the immobilized protein with porcine elastase resulted in the release of C4 beta- and gamma-chains and lower molecular weight fragments. The 14C label, however, was retained on the Sepharose beads. Subsequent release of bound material with L-cysteine indicated that the radiolabel was associated with two polypeptides of Mr 25,000 [C4d(ela25)]. The released material was dialyzed and the active-site thiol was radioalkylated with iodo[2-3H]acetic acid. C4d(ela25) was further purified by chromatography on Sephadex G-100 and, after reduction and alkylation, on Sepharose CL-6B in 0.2% NaDodSO4. The C4d(ela25) pool, containing 0.83 mol of [14C]methylamine per mol of iodo[2-3H]acetic acid, was subjected to automated sequence analysis. S-carboxy-[3H]methylcysteine was released at step 21 and gamma-glutamyl-[14C]methylamide was released at step 24. The recovery of radiolabel at positions 21 and 24 confirmed the originally calculated 14C/3H incorporation ratio and further indicated that the radiolabels were present at single sites in the C4 molecule. Comparison of the derived primary structure for the thiolester site in C4 with those for the corresponding regions in C3 and alpha 2-macroglobulin has shown sequence identity. Further comparisons among these three proteins have indicated additional homologies on both the NH2- and COOH-terminal sides of the thiolester site.
Glutathione export by human lymphoid cells: depletion of glutathione by inhibition of its synthesis decreases export and increases sensitivity to irradiation.
Authors: Dethmers J K , Meister A
Published: May 2006

Glutathione (in the form of GSH) is transported out of cultured human lymphoid cells at rates proportional to the intracellular glutathione levels. Inhibition of glutathione synthesis by buthionine sulfoximine, a potent selective inhibitor of gamma-glutamylcysteine synthetase, leads to exponential decrease in intracellular glutathione, a large fraction of which appears extracellularly, indicating that glutathione turnover is associated with its export. Although cells with 0.09 mM glutathione (4% of controls) were 85% viable, further decrease was associated with marked loss of viability. Cells with 4-5% of control glutathione levels were much more sensitive than control cells to the effects of gamma radiation and of 5,5'-dithiobis(2-nitrobenzoate). Depletion of glutathione by use of buthionine sulfoximine has advantages over other reagents (such as diamide, other oxidizing agents, and diethylmaleate, which affect other cellular components and may increase glutathione disulfide levels) and therefore has potential usefulness in sensitizing cells to the effects of radiation and to therapeutic agents that are detoxified by reactions involving glutathione.
Mouse macrophages synthesize and secrete a protein resembling apolipoprotein E.
Authors: Basu S K , Brown M S , Ho Y K , Havel R J , Goldstein J L
Published: May 2006

Monolayers of mouse peritoneal macrophages were shown to synthesize and secrete a protein that resembles apoprotein E (apoE), a normal constituent of plasma lipoproteins. Synthesis and secretion were studied by incubation of macrophages with L-[35S]methionine and analysis of the 35S-labeled proteins secreted into the culture medium. The 35S-labeled protein resembling apoE showed the following properties: (i) it floated in the ultracentrifuge at a density less than 1.215 g/ml, indicating that it was associated with lipid; (ii) by NaDodSO4/polyacrylamide gel electrophoresis, its Mr of 35,000 was identical to that of authentic apoE obtained from mouse plasma very low density lipoprotein; (iii) its isoelectric point of 5.4 was the same as that of authentic mouse apoE; (iv) it comigrated with authentic mouse apoE after two-dimensional isoelectric focusing/NaDodSO4/polyacrylamide gel electrophoresis; and (v) it was quantitatively precipitated by a monospecific antibody directed against rat apoE. Synthesis and secretion of the apoE-like protein was stimulated 3- to 8-fold when the macrophages were loaded with cholesterol by incubation with either acetylated low density lipoprotein (acetyl-LDL) or beta-migrating very low density lipoprotein from cholesterol-fed rabbits. When the cells were incubated with acetyl-LDL, the apoE-like protein composed approximately 2% of the total 35S-labeled protein synthesized by the cells and approximately 10% of the total 35S-labeled protein secreted into the medium. The current findings suggest a role for apoE in the plasma transport of cholesterol excreted from cholesterol-loaded macrophages.
Successful treatment of murine muscular dystrophy with the proteinase inhibitor leupeptin.
Authors: Sher J H , Stracher A , Shafiq S A , Hardy-Stashin J
Published: May 2006

Mice with genetic muscular dystrophy were treated with intraperitoneal injections of the proteinase inhibitor leupeptin, beginning before the onset of weakness. A significant number of the treated animals failed to develop histological evidence of dystrophy, compared with controls. Leupeptin treatment prevented (or delayed) the onset of muscular dystrophy in this experiment.
Hamster activity and estrous cycles: control by a single versus multiple circadian oscillator(s).
Authors: Carmichael M S , Nelson R J , Zucker I
Published: May 2006

Running activity onset and estrous onset were recorded for hamsters exposed to progressively shorter daily light/dark (T) cycles. The period of the estrous cycle was a quadruple multiple of the period of the activity rhythm during entrainment to T cycles of 23.5-21.5 hr. There was no evidence of desynchronization of the activity and estrus rhythms. The very short estrous periods shown during exposure to short T cycles indicate that an intrinsic 96-hr interval for ovarian follicular maturation does not determine the period of the estrous cycle. Dissociation of estrous and running activity onsets occurred for all hamsters: estrous onset generally preceded running activity onset for T greater than or equal to 23.0 hr; for shorter T cycles, estrous onset generally lagged behind running activity onset. Wheel-running activity was intermittently split into entrained and free-running components for one female: at T = 22.0 hr, estrous onset was coupled first with one and then with the other of the split activity components. These findings suggest that two or more separate circadian oscillators may control timing of the activity and estrous rhythms.
Saccharin and cyclamate inhibit binding of epidermal growth factor.
Authors: Lee L S
Published: May 2006

The binding of 125I-labeled mouse epidermal growth factor (EGF) to 18 cell lines, including HeLa (human carcinoma), MDCK (dog kidney cells), HTC (rat hepatoma), K22 (rat liver), HF (human foreskin), GM17 (human skin fibroblasts), XP (human xeroderma pigmentosum fibroblasts), and 3T3-L1 (mouse fibroblasts), was inhibited by saccharin and cyclamate. The human cells were more sensitive to inhibition by these sweeteners than mouse or rat cells. EGF at doses far above the physiological levels reversed the inhibition in rodent cells but not in HeLa cells. In HeLa cells, the doses of saccharin and cyclamate needed for 50% inhibition were 3.5 and 9.3 mg/ml, respectively. Glucose, 2-deoxyglucose, sucrose, and xylitol did not inhibit EGF binding. Previous studies have shown that phorbol esters, strongly potent tumor promoters, also inhibit EGF binding to tissue culture cells. To explain the EGF binding inhibition by such greatly dissimilar molecules as phorbol esters, saccharin, and cyclamate, it is suggested that they operate through the activation of a hormone response control unit.
Engrailed: a gene controlling compartment and segment formation in Drosophila.
Authors: Kornberg T
Published: May 2006

A total of 58 mutations at the engrailed locus were isolated. Analysis suggests that this genetic locus is necessary for survival but required only in the cells of the posterior compartments. Inactivation of the engrailed locus renders the animal incapable of maintaining the separation between the groups of cells that constitute either the compartments that subdivide each segment or the individual segments themselves.
Isolation and characterization of polyoma host range mutants that replicate in nullipotential embryonal carcinoma cells.
Authors: Sekikawa K , Levine A J
Published: May 2006

Polyoma wild-type virus replicates in most murine differentiated cells but fails to produce virus in murine embryonal carcinoma cells. Polyoma host range mutants have been isolated that replicate in several nullipotential embryonal carcinoma cell lines but fail to replicate in a pluripotential cell line. The final virus yield of these host range mutants is dependent upon the multiplicity of infection in both differentiated cells and nullipotent embryonal carcinoma cells. Two independently derived host range mutants contain the same single base pair change (A.T to G.C) and a 33- and 67-base pair duplication of those viral DNA sequences containing this point mutation. This duplication of viral DNA is located at 69 map units on the polyoma genome on the late gene side of the origin of viral DNA replication (70.5 map units). This type of mutation suggests several models to explain the polyoma host range restriction in embryonal carcinoma cells.
Evidence that ribosomal protein S10 participates in control of transcription termination.
Authors: Friedman D I , Schauer A T , Baumann M R , Baron L S , Adhya S L
Published: May 2006

We report the isolation of an Escherichia coli K-12 strain with a mutation, nusE71, that results in a change in ribosomal protein S10. Phage lambda fails to grow in hosts carrying the nusE71 mutation because the lambda N gene product is not active. The N product regulates phage gene expression by altering transcription complexes so that they can overcome termination barriers. This suggests that a ribosomal protein is involved in antitermination of transcription.
Effects of chaotropic and antichaotropic agents on elution of poliovirus adsorbed on membrane filters.
Authors: Farrah S R , Shah D O , Ingram L O
Published: May 2006

The association of poliovirus with membrane filters results from both electrostatic and hydrophobic interactions. At low pH, electrostatic interactions appear to dominate. However, at high pH, hydrophobic interactions appear to dominate with both Millipore and Zeta plus filters. With both filters, viral elution was prevented at high pH by the presence of antichaotropic salts, which strengthen hydrophobic associations. This effect was antagonized by detergents and by chaotropic salts, which weaken hydrophobic associations. Excellent correlation was observed between viral elution, the solubilization of adenine, and the micelle-formation process in the presence of chaotropic and antichaotropic agents. Such simple measurements of the relative ability of solvents to accommodate hydrophobic groups may be of predictive value in designing methods for concentration and purification of viruses. The hypothesis that hydrophobic interactions are the primary force involved in the stabilization of virus attachment at high pH is consistent with observations that cannot be explained by consideration of electrostatic interactions as the major stabilizing factor.