bluuebunny/crossref_metadata_2025 huggingface dataset:
Prediction of protein antigenic determinants from amino acid sequences.
Authors: Hopp T P , Woods K R
Published: May 2006

A method is presented for locating protein antigenic determinants by analyzing amino acid sequences in order to find the point of greatest local hydrophilicity. This is accomplished by assigning each amino acid a numerical value (hydrophilicity value) and then repetitively averaging these values along the peptide chain. The point of highest local average hydrophilicity is invariably located in, or immediately adjacent to, an antigenic determinant. It was found that the prediction success rate depended on averaging group length, with hexapeptide averages yielding optimal results. The method was developed using 12 proteins for which extensive immunochemical analysis has been carried out and subsequently was used to predict antigenic determinants for the following proteins: hepatitis B surface antigen, influenza hemagglutinins, fowl plague virus hemagglutinin, human histocompatibility antigen HLA-B7, human interferons, Escherichia coli and cholera enterotoxins, ragweed allergens Ra3 and Ra5, and streptococcal M protein. The hepatitis B surface antigen sequence was synthesized by chemical means and was shown to have antigenic activity by radioimmunoassay.
Conditioning of leg position in normal and mutant Drosophila.
Authors: Booker R , Quinn W G
Published: May 2006

Individual Drosophila melanogaster, with or without heads, can be trained to lift their legs to avoid electric shock. This behavior is similar to the operant conditioning previously demonstrated in intact and headless cockroaches. More than 90% of headless wild-type flies learned to our criterion. In contrast, three mutants (dunce, cabbage, and turnip), originally selected for failure to learn in an olfactory discrimination paradigm, tended to perform poorly in this new learning situation. The difference in learning behavior between normal and mutant flies is distinguishable in individuals and may be useful for mosaic analysis.
Calcium-binding sites on sensory processes in vertebrate hair cells.
Authors: Moran D T , Rowley J C , Asher D L
Published: May 2006

Vertebrate lateral line and vestibular systems center their function on highly mechanosensitive hair cells. Each hair cell is equipped with one kinocilium (which resembles a motile cilium) and 50-100 actin-containing stereocilia (which resemble microvilli) at the site of stimulus reception. This report describes electron-microscopic localization of calcium-binding sites on the sensory processes of vertebrate hair cells. Using the Oschman-Wall technique for calcium localization [Oschman, J. L. & Wall, B. J. (1972) J. Cell Biol. 55, 58-73] together with electron-probe x-ray microanalysis of thin sections, we observed: (i) calcium- and iron-containing deposits in the region of the ciliary necklace in goldfish lateral line hair cells, (ii) calcium deposits upon the surface of stereocilia of hair cells of the bullfrog inner ear, and (iii) calcium deposits upon stereocilia of hair cells of the guinea pig vestibular system.
Regulation of glycoprotein biosynthesis by formation of specific glycosyltransferase complexes.
Authors: Ivatt R J
Published: May 2006

Eukaryotic surfaces simultaneously express a complex array of protein-linked oligosaccharides and, during differentiation and on neoplastic transformation, these arrays change. The oligosaccharides linked to asparagine residues of proteins are a complex family of structures that are derived from a common precursor oligosaccharide by a network of competing biosynthetic pathways. The glycosylation reactions that make up these biosynthetic pathways often share common intermediates, requiring the competition between rival glycosyltransferases to be regulated at the supramolecular level. Investigation of two sequential glycosyltransferases that together add N-acetyllactosamine to glycoproteins has revealed their ability to form specific complexes. The functional consequences of complex formation were assessed by investigating the coupled reaction carried out by these sequential enzymes. The membrane enzymes are readily adsorbed by preformed liposomes, and their ability to interact after liposome adsorption has allowed direct investigation of the action carried out by these sequential enzymes when they are coadsorbed to the same liposome or adsorbed to separate liposomes shows the preferential use of endogenously generated intermediate over exogenously added glycoproteins. This facilitated passage of the intermediate glycosylated glycoprotein within the complex results in a tight coupling of the sequential enzymes. The formation of such complexes by sequential glycosyltransferases is proposed as a mechanism for controlling the competition between potentially rival glycosylation sequences during glycoprotein synthesis. The formation of different specific complexes under different conditions provides a flexible mechanism for regulating the synthesis of cell surface glycoproteins during development.
On the attribution and additivity of binding energies
Authors: Jencks William P.
Published: May 2006

It can be useful to describe the Gibbs free energy changes for the binding to a protein of a molecule, A—B, and of its component parts, A and B, in terms of the “intrinsic binding energies” of A and B, Δ
G
A
i
and Δ
G
B
i
, and a “connection Gibbs energy,” Δ
G
s
that is derived largely from changes in translational and rotational entropy. This empirical approach avoids the difficult or insoluble problem of interpreting observed ΔH and
T
Δ
S
values for aqueous solutions. The Δ
G
i
and Δ
G
s
terms can be large for binding to enzymes and other proteins.
Sequences of mRNAs derived from genome RNA segment 7 of influenza virus: colinear and interrupted mRNAs code for overlapping proteins.
Authors: Lamb R A , Lai C J , Choppin P W
Published: May 2006

RNA segment 7 of the influenza A virus genome codes for at least two proteins, M1 and M2, which are synthesized from separate mRNA species. Sequence analysis of the M2 mRNA has shown that it contains an interrupted sequence of 689 nucleotides. The approximately 51 virus-specific nucleotides comprising the 5'-end leader sequence of the M2 mRNA are the same as those found at the 5' end of the colinear M1 mRNA. Following the leader sequence of the M2 mRNA, where is a 271-nucleotide body region that is 3' coterminal with the M1 mRNA. Another small potential mRNA (mRNA3) related to RNA segment 7 has been found. mRNA3 has a leader sequence of approximately 11 virus-specific nucleotides that are the same as the 5' end of the M1 and M2 mRNAs, followed by an interrupted sequence of 729 nucleotides, and then a body region of approximately 271 nucleotides that is the same as that of the M2 mRNA. The nucleotide sequences found at the junctions of the interrupted sequences in M2 mRNA and mRNA3 are similar to those found at the splicing points of intervening sequences in eukaryotic mRNAs. In addition, both mRNAs contain 10-15 heterogeneous nonviral nucleotides at their 5' ends that appear to be derived from cellular RNAs used for priming the transcription of viral RNAs. Because the 5'-end sequences of the M1 mRNA and the M2 mRNA are the same and share the 5'-proximal initiation codon for protein synthesis, the first nine amino acids would be the same in the M1 and M2 protein and then the sequences would diverge. The approximately 271-nucleotide body region of the M2 mRNA can be translated in the +1 reading frame, and the sequence indicates that M1 and M2 overlap by 14 amino acids. The coding potential of the mRNA3 is for only nine amino acids, and these would be identical to the COOH-terminal region of the membrane protein (M1).
N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, a calmodulin antagonist, inhibits cell proliferation.
Authors: Hidaka H , Sasaki Y , Tanaka T , Endo T , Ohno S , Fujii Y , Nagata T
Published: May 2006

N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and its derivatives are putative calmodulin antagonists that bind to calmodulin and inhibit Ca2+/calmodulin-regulated enzyme activities. Autoradiographic studies using tritiated W-7 showed that this compound penetrates the cell membrane, is distributed mainly in the cytoplasm, and inhibits proliferation of Chinese hamster ovary K1 (CHO-K1) cells. Cytoplasmic [3H]W-7 was excluded completely within 6 hr after removal of [3H]W-7 from the culture medium. N-(6-aminohexyl)-1-naphthalenesulfonamide, an analogue of W-7 that interacts only weakly with calmodulin, proved to be a much weaker inhibitor of cell proliferation. CHO-K1 cells were synchronized by shaking during mitosis and then released into the cell cycle in the presence of 25 microM W-7 or 2.5 mM thymidine for 12 hr. Cell division was observed approximately 6 hr later. The results suggest that the effect of W-7 on cell proliferation might be through selective inhibition of the G1/S boundary phase, which is similar to the effect of excess thymidine. This pharmacological demonstration that cytoplasmic calmodulin is involved in cell proliferation is significant; W-7 and its derivatives may be useful tools for research on calmodulin and cell biology-related studies.
Platelet-derived growth factor-modulated proteins: constitutive synthesis by a transformed cell line.
Authors: Pledger W J , Hart C A , Locatell K L , Scher C D
Published: May 2006

Platelet-derived growth factor (PDGF) initiates replication of density-arrested BALB/c-3T3 mouse cells by rendering them "competent" to respond to factors contained in plasma. Treatment of quiescent cells with PDGF rapidly stimulates the preferential synthesis of several cytoplasmic proteins (molecular weights 29,000 to 70,000). Four of these proteins were noted within 1.5 hr of PDGF addition and one (pI) within 40 min. Inhibitors of RNA synthesis prevented the synthesis of these proteins. Both the synthesis of pI and the stimulation of DNA synthesis displayed a similar dose response to PDGF concentration. Pituitary fibroblast growth factor, which also induces competence, stimulated pI and pII synthesis. Plasma, epidermal growth factor, or insulin, which do not induce competence, did not stimulate selective synthesis of these proteins. A transformed variant of BALB/c-3T3 cells, which has retained the growth requirement for plasma factors but lost the requirement for PDGF, synthesizes these PDGF-modulated proteins constitutively.
Cyclic AMP: a mitogenic signal for Swiss 3T3 cells.
Authors: Rozengurt E , Legg A , Strang G , Courtenay-Luck N
Published: May 2006

Addition of cholera toxin (100 ng/ml) to quiescent cultures of Swiss 3T3 cells acts synergistically with serum (2-4%), insulin, phorbol esters, epidermal growth factor, and fibroblast-derived growth factor to stimulate DNA synthesis. In the presence of insulin, cholera toxin caused a dose-dependent increase in cumulative [3H]thymidine incorporation into acid-insoluble material and in the intracellular cyclic AMP (cAMP) level. The dose--response curves for the two processes were similar. Furthermore, addition of 1-methyl-3-isobutylxanthine (15--500 microM) or of 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (5--100 microM), both of which are potent inhibitors of cyclic nucleotide phosphodiesterase which are potent inhibitors of cyclic nucleotide phosphodiesterase activity, stimulated DNA synthesis and increased cAMP levels in Swiss 3T3 cells. These compounds strikingly potentiated the effect of cholera toxin on DNA synthesis and on cAMP levels. When quiescent Swiss 3T3 cells were exposed to cholera toxin (100 ng/ml) and insulin at 10 micrograms/ml (4- to 7-fold increase in cAMP level) or to these agents and 1-methyl-3-isobutyl xanthine at 50 microM (35-fold increase in cAMP level), DNA synthesis began after a lag of 16 hr. These results indicate that cAMP acts as a mitogenic signal for Swiss 3T3 cells and differ from the widely held view that cyclic AMP inhibits the proliferation of fibroblast cells.
31P NMR reveals increased intracellular pH after fertilization in Xenopus eggs.
Authors: Nuccitelli R , Webb D J , Lagier S T , Matson G B
Published: May 2006

31P NMR spectra of mature eggs of the frog (Xenopus laevis) were taken prior to and after both fertilization and activation by a Ca2+/H+ ionophore (A23187). The eggs were constantly perfused with fresh well-buffered solution during the experiments, and the intracellular pH (pHi) was determined from the pH-dependent chemical shift of the internal Pi peak. The detection of this Pi peak in the presence of overlapping yolk phosphoprotein signals was accomplished by a T2 experiment which discriminated against the broader yolk phosphoprotein peak. The average pHi of the unfertilized, fertilized, and activated eggs was 7.42, 7.66, and 7.64, respectively. Thus, a cytoplasmic alkalinization of 0.24 pH unit occurs within 90 min 90 min after fertilization. These values are practically identical to pHi measurements made in this laboratory on Xenopus eggs by using pH-sensitive glass microelectrodes. These 31P NMR studies also indicate that extracellular pH changes as large as 3 pH units had no effect on pHi. We also found that phosphocreatine levels are very sensitive to metabolic perturbations such as oxygen depletion or metabolic inhibitor application. These treatments resulted in a rapid decrease in the phosphocreatine concentration; the ATP concentration declined only slowly after the phosphocreatine peak had disappeared.
Sequence homology among different size classes of plant mtDNAs
Authors: Dale Roderic M. K.
Published: May 2006

Supercoiled mtDNAs were isolated from tissue culture cells of tobacco, bean, and corn, and the smallest size classes were used to study the relationships among the different size classes of each species through restriction digests and hybridizations. Three of the smallest tobacco mtDNAs [10.1, 20.2, and 28.8 kilobases (kb)], the two smallest bean mtDNAs (1.9 and 3.8 kb), and the two smallest corn mtDNAs (1.5 and 1.8 kb) were extracted from the gels and nick translated. The 10.1-kb tobacco mtDNA hybridizes to all the other tobacco mtDNA size classes and a large percentage of the tobacco mtRNAs. Restriction digests indicate that the 20.2-kb size class is a dimer of the 10.1-kb size class. The 1.9-kb bean mtDNA hybridizes to all but three of the bean mtDNA size classes and hybridizes to two mtRNAs. Restriction digests indicate that the 3.8-kb size class is a dimer of the 1.9-kb size class. The 1.5- and 1.8-kb corn mtDNAs, which do not have any
Hha
I restriction fragments in common, both hybridize to many of the same size classes of the corn mtDNA profile and, in addition, each hybridizes to a few size classes not recognized by the other. The 1.5- and 1.8-kb size classes both hybridize to two RNAs, one of which they appear to have in common. However, with both the 1.9-kb bean mtDNA and the two corn mtDNAs, the molecular sizes of the two RNAs exceed those of the respective DNAs. The possible role and origin of the many size classes are discussed.
Lymphocyte function-associated antigen 1 (LFA-1): a surface antigen distinct from Lyt-2,3 that participates in T lymphocyte-mediated killing.
Authors: Davignon D , Martz E , Reynolds T , Kürzinger K , Springer T A
Published: May 2006

Monoclonal antibodies (MAb) have been used to probe the relationship of cytolytic T lymphocyte (CTL) surface molecules to CTL function. Rat MAb to mouse CTL were generated. Twelve MAb so obtained gave preferential binding to T cells as compared to B cells, and three of these recognized previously undescribed surface polypeptides. These Mab and more broadly reactive and previously obtained MAb were tested for their ability to block CTL-mediated killing in the absence of complement. To ensure that any observed blocking was due to binding of MAb to the effector cell rather than the target cell, a xenogeneic mouse CTL anti-rat BN lymphoma target cell system was utilized (MAb and target cells both of rat origin). Of 24 MAb tested here, 21 had little or no effect on CTL function, including those to H-2, Thy-1, Lyt-1, Ly 5, Ly 6, Lgp 100, and at least six other defined antigens. We confirmed inhibition of killing with two MAb to Lyt-2,3. Another MAb, M7/14, gave profound and consistent blockade of CTL function. It was confirmed that M7/14 MAb blocks killing by binding to the mouse CTL and does not bind to the rat lymphoma target cells used for the CTL assay. The findings suggest that the antigen defined by M7/14, termed a lymphocyte function-associated antigen, LFA-1, participates in or is closely associated with the mechanism of CTL-mediated killing. LFA-1 contains two polypeptide chains of 180,000 and 95,000 Mr and is distinct from other described lymphocyte glycoproteins. LFA-1 thus represents both a previously undescribed lymphocyte surface antigen and molecular site for blockade of CTL-mediated killing.
Contractile activities of structural analogs of leukotrienes C and D: role of the polar substituents.
Authors: Lewis R A , Drazen J M , Austen K F , Toda M , Brion F , Marfat A , Corey E J
Published: May 2006

Twenty-three structural analogs of the leukotriene components of slow reacting substance of anaphylaxis (SRS-A), in which the polar regions of the leukotriene were systematically modified, were tested for their contractile activities on guinea pig pulmonary parenchymal strips and guinea pig ileum. The structural modifications allowed evaluation of the separate contributions of the four polar units in the C-1 to C-6 region of the SRS-A leukotrienes to smooth muscle spasmogenic activity. The free NH2-terminal amino group of the S-linked peptide was necessary for full activity, and its deletion or substitution reduced activity by more than one but less than two orders of magnitude. A similar level of importance was apparent for the free glycine carboxyl group. In contrast, a free eicosanoid carboxyl at C-1 is not required for full activity on the airway and for substantial activity on the ileum. A role for the C-5 hydroxyl is indicated by the inactivity of the one available 5-desoxy analog. Nucleophilic, divalent sulfur is not critical to leukotriene D (LTD) activity, in that one sulfoxide had substantial function. The conformational relationship between the eicosanoid and peptide moieties of LTD is of considerable importance in that epimers at the C-5 or C-6 position were less active than LTD by more than two orders of magnitude. Several lines of evidence suggest that the relative geometrical arrangement of the C20 chain and the peptide unit is important to activity, consistent with the existence of a true receptor for LTD.
Purification of the saxitoxin receptor of the sodium channel from rat brain.
Authors: Hartshorne R P , Catterall W A
Published: May 2006

The saxitoxin (STX) receptor has been purified 740-fold from rat brain by a combination of ion exchange chromatography, wheat germ agglutinin chromatography, and sedimentation on sucrose gradients to a specific activity of 1488 pmol/mg of protein. The best fractions were estimated to be 47% pure from their specific activity or 66% pure on the basis of NaDodSO4 gel electrophoresis. Two polypeptides, alpha (Mr approximately equal to 270,000 +/- 10,000) and beta (Mr approximately equal to 38,300 +/- 2000) (mean +/- SD) copurify with STX binding activity. Two polypeptides of the same apparent Mr are specifically covalently labeled by photoreactive derivatives of 125I-labeled scorpion toxin in rat brain synaptosomes and are likely to be identical to alpha and beta. The solubilized STX receptor has a Mr of 316,000 +/- 63,000, limiting its composition to one alpha polypeptide and one or more beta polypeptides per soluble receptor. Our results suggest that the alpha and beta polypeptides contain both the STX binding site and the scorpion toxin binding site of the mammalian sodium channel.
Structure of the body-centered cubic phase of lipid systems does not consist of indefinitely long straight rods
Authors: Luzzati Vittorio , Tardieu Annette , Gulik-Krzywicki Tadeusz
Published: May 2006

The observed intensities of the reflections from the body-centered cubic phase of lipid systems are shown to be incompatible with a recently reported model consisting of straight, indefinitely long rods.
Streptococcal M protein: alpha-helical coiled-coil structure and arrangement on the cell surface.
Authors: Phillips G N , Flicker P F , Cohen C , Manjula B N , Fischetti V A
Published: May 2006

The conformation and molecular dimensions of purified type 6 streptococcal M proteins establish the close structural relationship of these molecules to tropomyosin. Ultracentrifuge studies reveal that the M molecules exist as stable dimers; circular dichroism spectra indicate that the molecules contain about 70% alpha helix; and fiber x-ray diffraction diagrams show the characteristic reflections of the alpha-helical pattern. Electron microscopic images of M protein shadowed with platinum reveal rod-shaped molecules having the same width as tropomyosin. However, the lengths of the M molecules are about 30% shorter than lengths predicted by assuming a completely alpha-helical molecule. These findings indicate that the structure of the M6 protein is primarily alpha-helical coiled coil. Comparison of the lengths of the fibers on the surface of the streptococcus and the isolated M proteins suggests that each fiber on the cell wall consists of a single M-protein molecule approximately 500 A long. The structure determined for these fimbriae is the first alpha-helical coiled-coil conformation to be demonstrated for bacterial surface projections.
A mitochondrial locus is necessary for the synthesis of mitochondrial tRNA in the yeast Saccharomyces cerevisiae.
Authors: Martin N C , Underbrink-Lyon K
Published: May 2006

We have used a cloned yeast mitochondrial tRNAUCNSer gene as a probe to detect RNA species that are transcripts from this gene in wild-type Saccharomyces cerevisiae and in petite deletion mutants. In RNA from wild-type cells, the tRNA is the most prominent transcript of the gene. In RNA from deletion mutants that retain this gene but have lost other regions of mtDNA, high molecular weight transcripts containing the tRNAUCNSer sequences accumulate but tRNAUCNSer is not made. tRNAUCNSer synthesis can be restored in these mutants when they are mated to other deletion mutants that retain a different portion of the mitochondrial genome. Protein synthesis is not necessary for the restoration, and the restoration is not due to a nuclear effect or to an effect of mating alone, because strains without mtDNA are not able to restore tRNA synthesis. These results definitively demonstrate the existence of a yeast mitochondrial locus that is necessary for tRNA synthesis and, because the restoration of tRNAUCNSer synthesis appears to result from intergenic complementation, not recombination, indicate that this locus acts in trans.
Induction of prothrombin synthesis by prothrombin fragments.
Authors: Graves C B , Munns T W , Carlisle T L , Grant G A , Strauss A W
Published: May 2006

The mechanisms by which blood levels of prothrombin (PT) are regulated in the vitamin K-sufficient state are unknown. We have studied PT synthesis by Reuber H-35 rat hepatoma cells exposed to vitamin K and [3H]leucine in serum-free cultures. Administration to the culture system of exogenous bovine PT and rat PT was characterized by increases in endogenous PT synthesis and secretion of 2- and 3-fold, respectively. This induction required endogenous proteolytic degradation of PT. Studies conducted with bovine PT fragment 1 (residues 1-156) demonstrated up to 5-fold increases in PT synthesis. This induction was dose dependent and saturable. Addition of bovine PT chymotryptic fragments to the cells indicated that the NH2-terminal peptide of prothrombin (residues 1-42) contained the requisite structural elements for the induction. Peptide-bound gamma-carboxyglutamate residues were required for the observed stimulation of PT synthesis. These results suggest that PT synthesis might be regulated physiologically by the products formed during its normal turnover and consumption during blood coagulation.
Direct demonstration of glycosylation of insulin receptor subunits by biosynthetic and external labeling: evidence for heterogeneity.
Authors: Hedo J A , Kasuga M , Van Obberghen E , Roth J , Kahn C R
Published: May 2006

Insulin receptors of human lymphocytes (IM-9 line) were biosynthetically labeled with [3H]glucosamine, [3H]galactose, [3H]fucose, or [3H]mannose. After solubilization in Triton X-100, cell extracts were immunoprecipitated with serum from a patient containing autoantibodies to the insulin receptor. Na-DodSO4/polyacrylamide gel electrophoresis of the immunoprecipitates under reducing conditions showed the presence of major labeled subunits of apparent Mr 134,000 and 98,000 and a minor component of Mr 206,000. The ratio of activity in the 134,000 versus 98,000 Mr bands varied from 2:1 for mannose to 1.2:1 for galactose. In addition, the receptor subunits could be demonstrated when the cell surface of intact lymphocytes was labeled with NaB3H4 by using either the galactose oxidase (acts on nonreducing terminal galactose and N-acetylgalactosamine) technique or the periodate (oxidizes sialic acid) technique. With the periodate treatment, NaB3H4 labeled preferentially the Mr 98,000 band. With the galactose oxidase procedure, on the other hand, NaB3H4 labeled only the Mr 134,000 band; prior treatment with neuraminidase increased the labeling of this band and also revealed the Mr 98,000 subunit. These data demonstrate that the major subunits of the insulin receptor are complex glycoproteins that have differences in the nonreducing ends of the carbohydrate chains. In the Mr 134,000 subunit, there appear to be more exposed galactosyl or N-acetylgalactosaminyl (or both) residues, whereas the Mr 98,000 subunit appears to have a higher degree of sialylation. These labeling techniques provide new tools to examine the role of the carbohydrate moiety in insulin receptor function and turnover.
Expression of the J chain gene during B cell differentiation is inversely correlated with DNA methylation.
Authors: Yagi M , Koshland M E
Published: May 2006

During B cell differentiation, transcription of the J chain gene is initiated. To determine the regulatory mechanism involved, we have analyzed the structure of the J chain gene in cell lines representing successive stages in B cell development. Comparison of restriction sites showed that the J chain gene does not require a rearrangement of DNA for expression; cleavage sites present in embryonic J chain DNA were preserved through all the subsequent differentiative steps. However, comparison of 5-methylcytosine contents showed that J chain gene expression correlates with a loss of methyl groups. The J chain gene was heavily methylated in cell types not expressing J chain (i.e., embryo and lymphomas representative of immature and mature B cells) and significantly less methylated in cell lines representative of antigen-stimulated lymphocytes synthesizing J chain. These changes in J chain gene methylation represent a specific differentiation-induced response. Analyses of the heavy chain C mu and C gamma 2b genes, which are expressed at earlier and later stages than the J chain gene, showed that the C mu sequences were undermethylated in all cell types examined whereas the C gamma 2b sequences were undermethylated only in cell lines expressing the IgG2b protein. The primary encounter of a B cell with antigen must therefore trigger events that effect J chain gene transcription through a mechanism involving loss of methyl groups from cytosine.
Diphtheria toxin fragment forms large pores in phospholipid bilayer membranes.
Authors: Kagan B L , Finkelstein A , Colombini M
Published: May 2006

The cytotoxic effect of diphtheria toxin requires the entry of its enzymatic A fragment (Mr approximately 21,000) into the cytosol of sensitive cells. We show that the B45 fragment (Mr approximately 24,000) forms, in lipid bilayers, pores that are large enough (diameter greater than or equal to 18 A) to allow the passage of extended fragment A. Pore formation is maximal when the B45-containing side is at low pH (4.7) and the opposite side is at high pH (7.4). These conditions resemble the pH gradient existing across lysosomal membranes. We suggest that fragment A passes through these pores from acidic endocytotic vesicles (lysosomes?) to the cytosol.
Adrenoleukodystrophy: evidence for X linkage, inactivation, and selection favoring the mutant allele in heterozygous cells.
Authors: Migeon B R , Moser H W , Moser A B , Axelman J , Sillence D , Norum R A
Published: May 2006

Skin fibroblasts of human males affected with adrenoleukodystrophy (ALD) have previously been shown to be abnormal with respect to C26 fatty acid content. Skin fibroblast clones from heterozygotes in three families segregating this mutation have been analyzed and are of two types: clones with normal ratios of C26 to C22 fatty acids and clones with an excess of C26 fatty acids similar to that found in cells of affected males. This indicates not only that the locus is X linked but also that it is subject to inactivation. In most of the heterozygotes there were significantly more clones of abnormal type than those expressing the normal allele, indicating a proliferative advantage in vitro for skin fibroblasts of mutant type. The increased levels of fatty acids in plasma in most heterozygotes and the phenotype of blood cells of women heterozygous for both ALD and glucose-6-phosphate dehydrogenase (G6PD) in one family are evidence that selection favoring the mutant allele may occur in vivo as well as in vitro and may explain why many heterozygotes manifest clinical symptoms of the disease. These studies have also revealed the close linkage between ALD and G6PD loci, because there are no recombinants among 18 informative offspring of doubly heterozygous mothers. Therefore, the ALD locus can be mapped on the human X chromosome near the G6PD locus at Xq28.
Chemical composition of high-excitation planetaries
Authors: Aller L. H. , Czyzak S. J.
Published: May 2006

Nebular spectral line intensities measured mostly in the optical region, but also in the IR and satellite UV (where possible), are used to derive plasma diagnostics and ionic concentrations
n
(X
i
)/
n
(H
+
). Then, we use theoretical nebular models to represent as closely as possible certain excitation-sensitive line ratios of HeII/HeI, [OIII]/[OII], and [NeIII]/[NeV]. Also, we try to reproduce the line intensities themselves. These models are used as devices to allow for unobserved ionization stages. Although He, C, and N show significant variations among different nebulae, heavier elements such as O, and probably Ne, S, Cl, and Ar are more nearly constant, suggesting that progenitor stars underwent nuclear transformations in their interiors that affected C and N but not heavier elements.
New class of transforming growth factors potentiated by epidermal growth factor: isolation from non-neoplastic tissues.
Authors: Roberts A B , Anzano M A , Lamb L C , Smith J M , Sporn M B
Published: May 2006

Proteins potentiated by epidermal growth factor (EGF) to induce a transformed phenotype in non-neoplastic rat kidney fibroblasts in cell culture have been isolated from many non-neoplastic tissues of the adult mouse, including submaxillary gland, kidney, liver, muscle, heart, and brain. They resemble previously described transforming growth factors (TGFs) isolated from neoplastic cells as follows: they are extractable by acid/ethanol and are acid-stable, low molecular weight (6000-10,000) polypeptides requiring disulfide bonds for activity; and they cause anchorage-independent growth of non-neoplastic indicator cells that will not grow in soft agar in their absence. Partial purification of these TGFs from submaxillary glands of male mice shows that they are distinct from EGF. Unlike previously described extracellular TGFs, but like certain cellular TGFs from neoplastic cells, they are potentiated by EGF in their ability to promote anchorage-independent growth. The isoelectric point of the submaxillary gland TGF protein is near neutrality. Chromatography on Bio-Gel P-30 followed by high-pressure liquid chromatography has resulted in a 22,000-fold overall purification. The most purified protein is active in inducing growth in soft agar at 1 ng/ml when assayed in the presence of EGF. The data add further evidence to the concept that neoplasia may result from a quantitative, rather than qualitative, alteration in non-neoplastic biochemical processes.
Detection and partial characterization of proenkephalin mRNA.
Authors: Gubler U , Kilpatrick D L , Seeburg P H , Gage L P , Udenfriend S
Published: May 2006

We have used an oligodeoxynucleotide of defined sequence to detect and quantitate proenkephalin mRNA in the poly(A)-containing fraction of RNA from bovine adrenal medullas. The decahexamer 5'-d(G-G-T-A-G-T-C-C-A-T-C-C-A-C-C-A)-3' was synthesized to be complementary to the codons specifying the amino acid sequence NH2-Trp-Trp-Met-Asp-Tyr-Gln-COOH. This stretch of amino acids occurs in peptide I, one of the intermediates in the biosynthetic pathway of the enkephalins in bovine adrenal medulla. This pathway starts with a precursor (proenkephalin) of about 45 kilodaltons [Stern, A. S., Jones, B. N., Shively, J. E., Stein, S. & Udenfriend, S. (1981) Proc. Natl. Acad. Sci. USA 78, 1962-1966]. The decahexamer hybridized to adrenal poly(A)+RNA and was extended into cDNA with reverse transcriptase (RNA-dependent DNA nucleotidyltransferase). Five main discrete products ranging in size from 115 to 168 nucleotides were observed. The sequences of these extensions were found to be identical over the approximately 70 nucleotides sequenced from their 5' termini and corresponded exactly to the sequence expected from the amino acid sequence of peptide I. These cDNAs and the decahexamer itself hybridized to an adrenal medullary poly(A)+RNA species of about 1500 nucleotides, sufficient in size to code for the proposed proenkephalin. At saturation, approximately 2 fmol of the decahexamer were bound per microgram of mRNA; thus, the proenkephalin mRNA represents about 0.1% of the total poly(A)+RNA population in the tissue.
Phosphorylation of ornithine decarboxylase by a polyamine-dependent protein kinase.
Authors: Atmar V J , Kuehn G D
Published: May 2006

This paper presents evidence that a polyamine-dependent protein kinase (EC 2.7.1.37) purified from nuclei of the slime mold Physarum polycephalum catalyzes phosphorylation of ornithine decarboxylase (OrnDCase; L-ornithine carboxy-lyase, EC 4.1.1.17). The protein kinase had properties similar to OrnDCase antizyme. Phosphocellulose chromatography of nuclear preparations from P. polycephalum yielded the polyamine-dependent protein kinase of subunit Mr 26,000 that was resolved from a second fraction in which the protein kinase copurified with a phosphate-acceptor protein of subunit Mr 70,000. At Na+ concentrations less than approximately 150 mM, a complex formed between the protein kinase and the phosphate-acceptor protein. The complex did not demonstrate protein kinase or OrnDCase activity. The complex was dissociated by greater than 150 mM Na+ into its constituent proteins. The dissociated complex catalyzed phosphorylation of the Mr 70,000 component in the presence of spermidine and spermine, and it also demonstrated OrnDCase activity. The purified Mr 70,000 component from the complex and authentic OrnDCase, purified by procedures previously reported, were virtually identical with respect to OrnDCase activity, capacity to be phosphorylated by the polyamine-dependent protein kinase, amino acid composition, and immunological crossreactivity. Phosphorylation of OrnDCase by the polyamine-dependent protein kinase sharply inhibited OrnDCase activity. Thus, this is an example of posttranslational covalent modification of OrnDCase with concurrent alteration of its catalytic function. It is also an unusual example of control of the first enzyme in a biosynthetic pathway by a protein kinase that is, in turn, modulated by the immediate end products of the pathway.
Reaction kinetics in living systems.
Authors: Eyring H , Ma S M , Ueda I
Published: May 2006

In this report we treat reaction rates, equilibrium theory, and irreversible thermodynamics as different aspects of a single discipline. In biological reactions the rate is ultimately controlled by enzymes and other proteins of complex structure and high molecular weight. The needed formalism can be placed in one-to-one correspondence with appropriate electrical and mechanical networks. An enzyme molecule has zwitter ions anchored in the polypeptide chain, which enable it to distort the substrate by electrostatic polarization. Water weakens the induced or existing polar bonds and so speeds reaction. Several biological processes, such as luminescence, catalysis, nerve excitation, and anesthesia, in which enzymatic reactions play a major part are discussed from this point of view. We also have discussed the energy consumption and coupling effect in living systems. It is likely that a small fraction of bonds can become energy rich through the process of quenching and that unsymmetrical barriers in biological systems act like transistors in making the driving forces more efficient by a valve effect.
Electrostatic control of the efficiency of light-induced electron transfer across membranes
Authors: Warshel Arieh , Schlosser Donald W.
Published: May 2006

The energetics and efficiency of light-induced electron transfer across membranes is examined on a molecular level. It is found that the activation energies that control the efficiency are determined by the solvation energies of the charge-transfer states, the redox potentials of the donors and acceptors, and the dielectric relaxation of the system. The formalism developed allows one to assess the efficiency of any artificial photosynthetic system in terms of its molecular components and their local environment. It is pointed out that the key problem in designing an efficient photosynthetic system is the transfer of a charge through a low dielectric environment and that this problem cannot be overcome by choosing the position of the primary donor and acceptor in the membrane. It is predicted that artificial photosynthetic systems can be optimized by placing the acceptors in polar sites that provide a large effective dielectric constant and low dielectric relaxation and by arranging the acceptors in order of increasing redox potentials. The implication regarding bacterial photosynthesis is discussed.
Isolation and characterization of human muscle cells.
Authors: Blau H M , Webster C
Published: May 2006

We have developed an in vitro system for the study of postnatal human muscle under standardized conditions. The technique utilizes cloning to isolate pure populations of muscle cells. By manipulating culture conditions we can maximize either proliferation or differentiation of individual clones or of clones pooled to yield mass cultures of muscle cells. The muscle phenotype is stable; cells can be stored in liquid nitrogen for long-term use without loss of proliferative or differentiative potential. We have determined proliferative capacity of muscle cells from an analysis of clonal growth kinetics; we determined differentiative capacity from morphological evidence (cell fusion, striations, contractions, and the appearance of acetylcholine receptors) and biochemical analysis of muscle protein synthesis (creatine kinase, alpha-actin, tropomyosin, and myosin light chains). Our approach eliminates the variability in cellular composition that has complicated studies of primary muscle to date. We can now study in a controlled fashion the interactions and contributions of different cell types to the development of normal and genetically dystrophic human muscle.
Requirement for an accessory factor for binding of [3H]estradiol to protein in the cytosol fraction of rat pancreas.
Authors: Boctor A M , Band P , Grossman A
Published: May 2006

Supernatant fractions from rat pancreas bind approximately 300 fmol of [3H]estradiol per mg of protein when incubated with 5 nM [3H]estradiol for 1 hr at room temperature. Passage through gel filtration columns reduces binding in the eluate to approximately 1% of its initial activity. Extracts of the supernatant contain a factor that reactivates binding in gel filtrates. Addition of accessory factor to fractional eluates gives one sharp peak of activity. Since fractions that cannot be reactivated contain as much or more protein as fractions that can be reactivated, it is concluded that interaction of accessory factor and [3H]estradiol-binding protein is specific. Peptides such as antipain [(1-carboxy-2-phenylethyl)carbamoyl-L-arginyl-L-valyl-L-argininal] and, especially, N-benzoyl-L-phenylalanyl-L-valyl-L-arginine-p-nitroanilide also enhanced binding of [3H]estradiol. Accessory factor is water soluble, dialyzable, and heat stable. Although as currently purified, it contains several substances, the data suggest that at least one component of accessory factor is an oligopeptide.
Glucocorticoid-mediated inhibition of ornithine decarboxylyase activity in S49 lymphoma cells.
Authors: Insel P A , Honeysett J M
Published: May 2006

Incubation of wild-type S49 lymphoma cells with glucocorticoids, such as dexamethasone and hydrocortisone, inhibits the activity of ornithine decarboxylase (L-ornithine carboxylyase, EC 4.1.1.17), the rate-limiting enzyme in the pathway of polyamine biosynthesis. The kinetics of this inhibition are more rapid than the glucocorticoid-mediated growth arrest in the G1 phase of the cell cycle or in glucocorticoid-mediated cytolysis of these cells. The inhibition of ornithine decarboxylase activity by corticosteroids is specific for steroids of the glucocorticoid class. Results obtained with variant S49 cells having lesions in the pathways of glucocorticoid or cyclic AMP action indicate that cytoplasmic glucocorticoid receptors, as well as nuclear transfer of steroid--receptor complexes, are required for the inhibition of ornithine decarboxylase activity but that this inhibition does not require hormonal activation of adenylate cyclase or cyclic AMP-dependent protein kinase. Because glucocorticoid-mediated inhibition of ornithine decarboxylase occurs when cellular protein synthesis has decreased less than 20%, this inhibition may represent a specific glucocorticoid-mediated deinduction of ornithine decarboxylase in S49 cells. Inhibition of ornithine decarboxylase activity may offer a useful marker for suppression of growth and cell cycle progression in these and other lymphoma cells.
Inducibility of a gene product required for UV and chemical mutagenesis in Escherichia coli.
Authors: Bagg A , Kenyon C J , Walker G C
Published: May 2006

The product of the umuC gene is required for UV and chemical mutagenesis in Escherichia coli. By the use of the Mud(Ap, lac) bacteriophage, we have obtained an operon fusion of the lac structural genes to the promoter/regulatory region of the umuC gene. The strain containing the umuC::Mud(Ap, lac) fusion was identified on the basis of its UV nonmutability. Strains containing this putative null allele of umuC were (i) nonmutable by UV and other agents, (ii) slightly UV sensitive, and (iii) deficient in their ability to carry out Weigle reactivation of UV-irradiation bacteriophage lambda. The UV nonmutability of the strain could be suppressed by a derivative of the mutagenesis-enhancing plasmid pKM101. beta-Galactosidase synthesis in umuC::Mud(Ap, lac) fusion strains was inducible by UV and other DNA-damaging agents. Genetic analysis of the regulation of beta-galactosidase in umuC::Mud(Ap, lac) strains suggests that the lexA protein is the direct repressor of the umuC gene and that a function of the recA protein, probably its protease activity, is required for the removal of the lexA repressor at the time of umuC induction.
Homozygosity and patch structure in plant populations as a result of nearest-neighbor pollination
Authors: Turner Monte E. , Stephens J. Claiborne , Anderson Wyatt W.
Published: May 2006

The population genetic consequences of nearest-neighbor pollination in an outcrossing plant species were investigated through computer simulations. The genetic system consisted of two alleles at a single locus in a self-incompatible plant that mates by random pollen transfer from a neighboring individual. Beginning with a random distribution of genotypes, restricted pollen and seed dispersal were applied each generation to 10,000 individuals spaced uniformly on a square grid. This restricted gene flow caused inbreeding, a rapid increase in homozygosity, and striking microgeographic differentiation of the populations. Patches of homozygotes bordered by heterozygotes formed quickly and persisted for many generations. Thus, high levels of inbreeding, homozygosity, and patchiness in the spatial distribution of genotypes are expected in plant populations with breeding systems based on nearest-neighbor pollination, and such observations require no explanation by natural selection or other deterministic forces.
DNA sequences affecting specific initiation of transcription in vitro from the EIII promoter of adenovirus 2.
Authors: Lee D C , Roeder R G , Wold W S
Published: May 2006

We have identified those sequences affecting the level of specific initiation of transcription in vitro from the EIII promoter of adenovirus 2. Mutants containing deletions in and around the initiation sites were constructed in cloned viral DNA fragments and assayed for their ability to initiate transcription in vitro. Three classes of mutants were studied with deletions in the following regions: -38 to -268, -21 to -71 (which includes the T-A-T-A-A box), and -29 through the cap sites (+1 and +3). Deletions that remove some or all of the area from -28 to several nucleotides downstream from the cap sites essentially abolished specific transcription. Small deletions in the region -30 to -41 reduced transcription to approximately 60% of wild type; larger deletions in the region -35 to -268 reduced transcription to 30-40% of wild type. Deletions beginning from approximately +10 to +25 and extending further downstream reduced transcription to 20-40% of wild type, whereas a deletion beginning at +31 had little or no effect. Our results suggest that the region including the T-A-T-A-A box and extending to the area immediately beyond the cap sites is essential for specific transcription in vitro from the EIII promoter. However, sequences upstream from the T-A-T-A-A box and those downstream from the cap sites appear to significantly modulate the levels of transcription.
Transposon-specified site-specific recombination.
Authors: Kitts P , Symington L , Burke M , Reed R , Sherratt D
Published: May 2006

Cointegrate DNA molecules containing two copies of a transposable element appear to be intermediates in the transposition process. These structures are resolved by site-specific recombination to yield the normal end products of transposition. The transposable element gamma delta (Tn1000) synthesizes a product interchangeable with the Tn1/3tnpR protein in promoting Tn1/3 site-specific recombination. These data support the hypothesis that cointegrates containing directly repeated copies of Tn1/3 are obligatory intermediates in interreplicon transposition of Tn1/3. In addition, we show here that the reaction is independent of the element-encoded tnpA gene product. Tn501, which specifies mercury resistance, also produces cointegrates as intermediates in interreplicon transposition. The appearance of Tn501-specified recombination activity that can act on these cointegrates requires growth of cells in the presence of Hg2+.
Sizes and map positions of several plasmid-DNA-encoded transcripts in octopine-type crown gall tumors.
Authors: Gelvin S B , Thomashow M F , McPherson J C , Gordon M P , Nester E W
Published: May 2006

The sizes and relative map positions of RNA molecules encoded by the plasmid DNA of several octopine-type tobacco crown gall tumors were determined by blot hybridization. At least six discrete polyadenylylated RNAs (sizes 0.73-1.75 kilobases) were detected in octopine-producing tumors (lines A6S/2, E9, and 15955/01). In one tumor (line 15955/1) in which octopine could not be detected, one of the RNA species was missing and another was diminished in abundance. Evidence is presented suggesting that the transcripts are encoded entirely by the plasmid DNA and do not originate from the surrounding plant DNA sequences.