bluuebunny/crossref_metadata_2025 huggingface dataset:
Serological characterization of B-tropic viruses of C57BL mice: possible origin by recombination of endogenous N-tropic and xenotropic viruses.
Authors: Benade L E , Ihle J N , Declève A
Published: May 2006

The serological properties of the gag gene products p15 and p12 of N- and B-tropic viruses of C57BL mice have been examined. Although these viruses were serologically identical by competition assays for proteins gp71 and p30, they were readily distinguishable in competition assays for proteins p15 and p12. Two isolates of N-tropic viruses had p12s serologically indistinguishable from AKR murine leukemia virus p12, while two B-tropic isolates had distinctly different p12s. The latter p12s were serologically indistinguishable from the p12 purified from the B-tropic radiation leukemia virus (RadLV)/VL-3. Moreover, this p12 was indistinguishable from the p12 of the endogenous C57BL/Ka xenotropic virus. Similarly, the p15s of the B-tropic viruses were serologically distinct from the AKR murine leukemia virus type of p15, as was the p15 of one C57BL N-tropic virus, whilc another N-tropic isolate had a p15 identical to the AKR murine leukemia virus p15. These results are interprered to suggest that the endogenous N-tropic virus of C57BL mice undergoes recombination with the endogenous, xenotropic virus and that this mechanism is involved in the generation of B-tropic viruses in C57BL mice.
RNA synthesis in isolated nuclei: in vitro initiation of adenovirus 2 major late mRNA precursor.
Authors: Manley J L , Sharp P A , Gefter M L
Published: May 2006

We have analyzed the RNA produced in vitro by incubating nuclei from HeLa cells infected with adenovirus serotype 2. Our results show that adenovirus-specific RNA is produced at a linear rate for up to 2.5 hr. Hybridization analysis of RNA produced in nuclei isolated 18 hr after infection indicated that transcription begins at the "late promoter" at map position 16.5. Sequence analysis of the 5' termini of the in vitro transcripts showed that this system initiates RNA chains de novo at the correct promoter and that the 5' terminus is capped.
Activation of high levels of endogenous phospholipase A2 in cultured cells.
Authors: Shier W T
Published: May 2006

Activatable cellular phospholipase A2 (PLase; phosphatide 2-acyl-hydrolase, EC 3.1.1.4) has been proposed to constitute the first and rate-limiting step in prostaglandin synthesis and to regulate membrane function by altering the levels in the membrane of the detergent lipids lysolecithin and free fatty acids. We have observed that a wide variety of cells in culture contain high levels of endogenous PLase that can be activated by polypeptide toxins, such as melittin purified from bee venom and direct lytic factor purified from the venom of African Ringhals cobra (Hemachatus hemachatus). Activation of PLase by sublytic concentrations of these agents results in the synthesis and release of prostaglandins. Melittin concentrations greater than or equal to 10 microgram/ml activate sufficient PLase in 3T3-4a mouse fibroblasts to hydrolyze 10% of the cellular lecithin in less than 5 min and virtually all of it within 30 min, demonstrating the existence of sufficient activatable PLase to provide the basis for the proposed mechanism of regulation of membrane function by alteration of membrane lipid composition. Lipases, phospholipases B and C, and sphingomyelinases are not activated by melittin. The PLase activated in 3T3-4a cells exhibits little, if any, specificity for individual phosphoglycerides. The PLase activated by direct lytic factor exhibits a Ca2+ dependence characteristic of lysosomal PLase, wherease the Ca2+ dependence of PLase activated by melittin is consistent with the activation of a cell-surface enzyme. The extent of cell death correlates with percent of maximal PLase activation.
Kinetics of initiation of bacterial protein synthesis.
Authors: Blumberg B M , Nakamoto T , Kézdy F J
Published: May 2006

The 30S initiation complex, formed with the 30S ribosomal subunit, mRNA, and fMet-tRNA, has been shown by kinetic analysis with limiting concentrations of Escherichia coli ribosomes to be an obligatory intermediate in the formation of the 70S initiation complex. The formation of the 70S initiation complex began with an induction period and was proportional to the concentration of the 30S complex, which rapidly rose to a peak. The entire time course of the sequential pseudo-first-order, second-order reaction was reproduced accurately by the overall rate expression, in which we used rate constants that were determined by carrying out 30S and 70S complex formation separately. By using limiting concentrations of mRNA, we showed that phage MS2 RNA contained no specific signal that enhanced its rate of 30S complex formation with E. coli ribosomes and initiation factors; the pseudo-first-order rate constants obtained with poly(A3C9G1U1), poly(C15G1U4), and poly(G1U3) were 12-45 times higher than that with MS2 RNA. The observation that the rate constants for binding of fMet-tRNA and AcPhe-tRNA with a given synthetic RNA were comparable indicated that the initiator codon is recognized only indirectly through the initiator tRNA.
Quaternary enhancement in binding of oxygen by human hemoglobin
Authors: Mills Frederick C. , Ackers Gary K.
Published: May 2006

From an experimental study on the linkage between dimer-tetramer association and oxygen binding in human hemoglobin it is found that triliganded tetramers α
2
β
2
(O
2
)
3
have an affinity for oxygen that is significantly higher than that of α
1
β
1
dimers (superscripts denote intersubunit contacts). This conclusion is based upon a newly determined series of accurate oxygen binding isotherms, which were analyzed in conjunction with independently determined values of the dimer-tetramer equilibrium constants in the unliganded and fully oxygenated states [Ip, S. H. C. & Ackers, G. K. (1977)
J. Biol. Chem
. 252, 82-87]. The results imply that in the molecule α
2
β
2
(O
2
)
3
the interactions at the α
1
β
2
intersubunit contacts are propagated to the unliganded heme in a manner that
increases
its affinity for oxygen. This effect contrasts sharply with the well-known reduction in oxygen affinity arising from these same contacts when unliganded dimers are assembled to form unliganded α
2
β
2
tetramers. The magnitude of the enhancement in affinity at the unliganded site in triliganded tetramers (0.81 kcal, 3.39 kJ) is approximately one-fourth as great as the reduction in affinity on each heme site that arises from subunit assembly of the unliganded tetramer. The terms “quaternary constraint” and “quaternary enhancement” are employed to describe these oppositely directed effects of intersubunit interaction upon heme-site affinity. Experimental results also suggest that dimers bind oxygen with a higher affinity than monomeric α and β chains do under the same temperature and buffer conditions (21.5°C, pH 7.40, 0.1 M Tris·Hcl/0.1 M NaCl/1 mM Na
2
EDTA). Thus quaternary enhancement may be manifested at the α
1
β
1
contacts. Implications of these results for models of the cooperative mechanism are discussed.
Import of proteins into mitochondria: precursor forms of the extramitochondrially made F1-ATPase subunits in yeast.
Authors: Maccecchini M L , Rudin Y , Blobel G , Schatz G
Published: May 2006

The mitochondrial F1-ATPase consists of five nonidentical subunits that are synthesized outside the mitochondria and imported across both mitochondrial membranes to the matrix side of the inner membrane. In order to study the mechanism of this import, we synthesized the F1-ATPase subunits of yeast either in vitro (in a reticulocyte lysate programmed with yeast RNA) or in vivo (in pulsed and pulsed-chased yeast spheroplasts). Both in vitro and in vivo, each of the three largest ATPase subunits was synthesized as a larger precursor. When the precursors that had been synthesized in vitro were incubated with isolated yeast mitochondria, they were converted to "mature" subunits that were no longer susceptible to externally added proteases. The uptake of the subunit into the mitochondria was thus accompanied by conversion of the precursor. Since uptake of precursors into mitochondria was independent of protein synthesis and since the precursors could also be detected in vivo, the transfer of proteins from the cytosol across both mitochondrial membranes does not occur by vectorial translation. Instead, the proteins destined for import are first made outside the mitochondria as precursors and only subsequently transported into the mitochondria. This step is accompanied by proteolytic conversion of the mature subunit.
Evolutionary change in 5S RNA secondary structure and a phylogenic tree of 54 5S RNA species.
Authors: Hori H , Osawa S
Published: May 2006

Secondary structure models of 54 5S RNA species are constructed based on the comparative analyses of their primary structure. All 5S RNAs examined have essentially the same secondary structure. However, there are revealing characteristic differences between eukaryotic and prokaryotic types. The prokaryotic 5S RNAs may be further classified into two types, one having 120 nucleotides (120-N type) and another having 116 (116-N type). A possible mechanism for the conversion of the prokaryotic 116-N type to the 120-N type 5S RNAs (or vice versa) is discussed on the basis of their nucleotide alignments. Finally, by comparing the nucleotide alignments, we propose a phylogenic tree of the 54 5S RNA species.
Phase variation in Salmonella: genetic analysis of a recombinational switch.
Authors: Silverman M , Zieg J , Hilmen M , Simon M
Published: May 2006

The alternative expression of Salmonella genes H1 and H2, which specify different flagellar antigens, results in the oscillation of phenotype known as phase variation. This alternation is controlled by the inversion of an 800-base-pair sequence of DNA adjacent to, or including part of, the H2 gene. The invertable region was presumed to regulate the function of a promoter and to include specific sites at which a recombinational event, resulting in the inversion, could occur. Here we report genetic manipulations of hybrid lambda phage carrying the H2 gene that were used to define the H2 promoter region and the recombinational sites. The H2 gene fragment was inserted on a hybrid lambda phage next to the cheW gene, which lacked a promoter element. In the resulting fusion, cheW gene activity was restored, the expression of the H2 and cheW genes was controlled coordinately by the inversion, and the polarity of transcription and location of the H2 gene could be determined. Evidence from this type of gene fusion suggested that the H2 gene promoter is included in the inversion region. Hybrid H2 phage were constructed that contained substitutions for regions of the H2 gene. In contrast to hybrid lambda containing the H2 gene, which alternate between "on" and "off" states, several substituted lambdaH2 were fixed in the "on" state. A site necessary for the recombinational event must have been removed in these fixed lambdaH2.
In vitro suppression of UAG and UGA mutants in the thymidine kinase gene of herpes simplex virus.
Authors: Cremer K J , Bodemer M , Summers W P , Summers W C , Gesteland R F
Published: May 2006

We have studied the in vitro translation, in nuclease-treated reticulocyte lysates, of mRNA from cells infected with several thymidine kinase-deficient mutants of herpes simplex virus type I. The addition of suppressor tRNAs from yeast resulted in suppression of the mutant property in the case of two mutants. Synthesis of enzymatically active viral thymidine kinase was restored by serine-inserting amber suppressor tRNA in the case of HSV TK4- and synthesis of the intact, but inactive, thymidine kinase protein was restored by serine- and leucine-inserting UGA suppressor tRNAs in the case of HSV TK43-. Read-through of the normal termination at the end of the thymidine kinase gene was promoted by UGA suppressor tRNAs. We conclude that HSV-TK4- is an amber (UAG) mutant and that HSV-TK43- is an opal (UGA) mutant.
Self-avoiding random walks at finite concentrations: The bulk phase limit
Authors: Wall Frederick T. , Seitz William A.
Published: May 2006

In infinitely dilute solutions, macromolecules exhibit non-Gaussian distributions for their end-to-end separations. This occurs under such circumstances because intramolecular interactions are more important than intermolecular forces. On the other hand, when a macromolecular solution becomes so concentrated that it approaches its bulk phase, then the end-to-end length distribution becomes substantially Gaussian. A theoretical explanation for the observed behavior is obtained by taking cognizance of a balance between inter-and intramolecular forces acting on self-avoiding random chains. Such a balance causes the chains to behave very much like random walks of order 2—that is to say, walks for which the only restriction against double occupancy is that identified with immediate return steps. This is demonstrated by taking Monte Carlo data for chains of various concentrations and analyzing the distributions of a component of length by using expansions involving orthogonal vectors. Although Gaussian behavior is more or less achieved for bulk polymers, slight deviations from that behavior still persist at the origin.
Crystallographic and kinetic investigations of the covalent complex formed by a specific tetrapeptide aldehyde and the serine protease from Streptomyces griseus.
Authors: Brayer G D , Delbaere L T , James M N , Bauer C A , Thompson R C
Published: May 2006

X-ray crystallographic data show that a specific tetrapeptide aldehyde inhibitor (N-acetylprolylalanylprolylphenylalaninal) forms a stable, covalent, tetrahedral addition complex with the serine protease, SGPA, from Streptomyces griseus. Earlier proposals, based on kinetic measurements, for the covalent nature of such linkages are confirmed, and the difference electron density map of this aldehyde inhibitor indicates that a major conformational change of the histidyl-57 side chain occurs on inhibitor binding.
Sequential adsorption-electrophoresis: combined procedure for purification of calcium-dependent cyclic nucleotide phosphodiesterase.
Authors: Kincaid R L , Vaughan M
Published: May 2006

A procedure for combined sequential affinity adsorption-electrophoresis has been devised. Its use for the rapid purification of a calcium-dependent cyclic nucleotide phosphodiesterase from bovine brain in high yield is described. In this procedure, proteins bound to a solid phase of calcium-dependent regulatory protein (CDR) linked to Sepharose 4B were electrophoretically eluted, concentrated, and separated, thus avoiding the large losses in activity incurred during attempts to purify further the phosphodiesterase eluted by conventional means. The highly purified phosphodiesterase prepared by this method was stable for months at -60 degrees C in the presence of glycerol. It has a higher affinity for cyclic GMP than for cyclic AMP, and hydrolysis of both substrates is stimulated 5- to 6-fold by calcium plus CDR. Factors that influence adsorption of the enzyme to CDR-Sepharose and selection of optimal conditions for electrophoresis were investigated. Sequential adsorption-electrophoresis should be generally useful in the purification of macromolecules for which affinity adsorbents are available. The procedures described here could be directly applicable to the purification of proteins that, like the phosphodiesterase, interact with CDR.
Control features within the rplJL-rpoBC transcription unit of Escherichia coli.
Authors: Barry G , Squires C L , Squires C
Published: May 2006

Gene fusions constructed in vitro have been used to examine transcription regulatory signals from the operon which encodes ribosomal proteins L10 and L7/12 and the RNA polymerase beta and beta' subunits (the rplJL-rpoBC operon). Portions of this operon, which were obtained by in vitro deletions, have been placed between the ara promoter and the lacZ gene in the gene-fusion plasmid pMC81 developed by M. Casadaban and S. Cohen. The effect of the inserted DNA segment on the expression of the lacZ gene (in the presence and absence of arabinose) permits the localization of regulatory signals to discrete regions of the rplJL-rpoBC operon. An element that reduces the level of distal gene expression to one-sixth is located on a fragment which spans the rplL-rpoB intercistronic region. This strongly supports the idea that there is an attenuator in this region. The terminator for the operon is located on a fragment which spans the 3' end of the rpoC gene. The major promoter for the operon precedes the rplJ gene [Yamamoto, M. & Nomura, M. (1978) Proc. Natl. Acad. Sci. USA 75, 3891-3895 and Linn, T. & Scaife, J. (1978) Nature (London) 276, 33-37] and was not examined in this study. However, a weak promoter is observed on the fragment that spans the rplJ-rplL intercistronic region. Other regions of the operon may also contain weak promoters. The contribution of these elements to the regulation of this complex operon is discussed.
Interaction site of Escherichia coli cyclic AMP receptor protein on DNA of galactose operon promoters.
Authors: Taniguchi T , O'Neill M , de Crombrugghe B
Published: May 2006

Cyclic AMP (cAMP) and its receptor protein (CRP) have a dual role in the regulation of the two promoters that control the galactose (gal) operon of Escherichia coli. One promoter, P1, requires cAMP-CRP for activity; the other, P2, is inhibited by these factors. We have examined the interactions site of cAMP-CRP on gal DNA by using two types of protection experiments, involving DNase digestion and methylation by dimethyl sulfate. Our results indicate that cAMP-CRP binds to gal DNA in a segment located between 50 and 24 base pairs preceding the P1 start point for transcription. Although the location of the cAMP-CRP interaction site is clearly different in gal and lac DNA, comparison of the DNA sequences suggests a similar recognition sequence. The location of the cAMP . CRP-binding site in gal further suggests that protein-protein interactions between RNA polymerase and cAMP . CRP play an important role in transcription initiation at the gal and possibly other cAMP-dependent promoters.
Electrophoretically pure mouse interferon exerts multiple biologic effects.
Authors: Gresser I , De Maeyer-Guignard J , Tovey M G , De Maeyer E
Published: May 2006

Electrophoretically pure mouse interferon was examined for a number of biologic effects previously ascribed to crude or partially purified interferon preparations. These effects include: inhibition of the growth of a transplantable tumor in mice; inhibition of cell multiplication of mouse tumor cells in vitro; enhancement of the expression of histocompatibility antigens on mouse tumor cells in vitro; inhibition of antibody formation in vitro; inhibition of sensitization to sheep erythrocytes and the expression of delayed type hypersensitivity in mice; enhancement of natural killer cell activity in vivo and in vitro; enhancement of cell sensitivity to the toxicity of poly(I)-poly(C); and enhanced production ("priming") of interferon production in vitro. Our results establish that the molecules responsible for the antiviral action of interferon are also responsible for these varied biologic effects.
Human epidermal cell cultures: growth and differentiation in the absence of differentiation in the absence of dermal components or medium supplements.
Authors: Eisinger M , Lee J S , Hefton J M , Darzynkiewicz Z , Chiao J W , de Harven E
Published: May 2006

Human epidermal cells grew and differentiated in vitro, provided that the pH of the culture medium was at 5.6-5.8, the seeding density was optimal (approximately 2.5 x 10(5) cells per cm2), and the incubation temperature was maintained at 35-37 degrees C. Under these conditions, epidermal cells from many different skin locations grew to confluency within 15-20 days and formed multi-layered sheets whose differentiated structure resembled that of the full depth of skin epidermis. Cell proliferation and differentiation did not require a feeder layer, a collagen substrate, a high concentration of fetal bovine serum, or added hormones. The sheets of differentiated epidermal cells could be dissociated from the plastic surfaces of the tissue culture flasks. The use of such cultured cells for wound dressing is proposed.
Evidence for hydrophobic region within heavy chains of mouse B lymphocyte membrane-bound IgM
Authors: Vassalli Pierre , Tedghi Rachel , Lisowska-Bernstein Barbara , Tartakoff Alan , Jaton Jean-Claude
Published: May 2006

The gel filtration behavior, in the presence of detergents, of membrane-bound IgM from normal mouse spleen B lymphocytes was compared to that of secretory IgM from mouse plasma cells. The proteins were labeled either by surface radioiodination or biosynthetically with radioactive amino acids. Cell lysates were fractionated on calibrated Sepharose 6B columns in the presence of the detergents Nonidet P-40 or deoxycholate. Eluted fractions were immunoprecipitated and the reduced or unreduced precipitates were analyzed by sodium dodecyl sulfate gel electrophoresis followed by radioautography. Surface
125
I-labeled 8S IgM exhibited a gel filtration pattern in Nonidet P-40 corresponding to much higher apparent molecular weight than that of secretory 8S IgM, a difference that almost disappeared when gel filtration was performed in the presence of deoxycholate, which forms much smaller micelles than does Nonidet P-40. Biosynthetically labeled lymphocytes contain two types of IgM molecules differing in their gel filtration behavior and fate: one identical to secretory 8S IgM of plasma cells and secreted in the medium during chase periods, and the other identical to surface
125
I-labeled IgM and remaining cell-associated. Because the surface-bound 8S IgM was not found to be associated with other labeled molecules, it is likely that the detergent-binding behavior of surface IgM is due to a hydrophobic segment carried by these Ig molecules. That lymphocytes synthesize two types of μ chains was also shown by the use of tunicamycin, an inhibitor of glycosylation. In its presence, two unglycosylated μ chains were observed: one identical in size to that made by tunicamycin-treated plasma cells, and the second slightly larger. Gel filtration in Nonidet P-40 of the cell lysates of tunicamycin-treated lymphocytes showed that the nonsecretory 8S IgM contains this second type of μ chains, whereas the IgM molecules of the secretory type contain plasma cell-like μ chains. It is suggested that membrane IgM μ chains contain a hydrophobic segment which is responsible for its association to the membrane.
Termination of transcription in nucleoli isolated from Tetrahymena.
Authors: Leer J C , Tiryaki D , Westergaard O
Published: May 2006

Correct termination of transcription of the rRNA gene in nucleoli isolated from Tetrahymena is dependent on a protein factor with a molecular weight between 50,000 and 100,000. At low ionic strength the endogenous RNA polymerase synthesizes a transcript identical in size to the precursor rRNA extracted from cells. In the presence of ammonium sulfate, however, the RNA polymerase reads through the normal termination point as demonstrated by size and hybridization studies of the transcript. After ammonium sulfate treatment, rDNA associated with chromosomal proteins (r-chromatin) can be separated from the termination factor by differential centrifugation. The endogenous RNA polymerase on the salt-treated r-chromatin is no longer able to recognize the normal terminator even at low ionic strength. Normal termination properties can be reconstituted by complementation with intact nucleoli or with a protein factor extracted from nucleoli.
Evidence for a dissociable protein subunit required for calmodulin stimulation of brain adenylate cyclase.
Authors: Toscano W A , Westcott K R , LaPorte D C , Storm D R
Published: May 2006

An adenylate cyclase [ATP pyrophosphatelyase (cyclizing), EC 4.6.1.1] preparation that is not stimulated by NaF,5'-guanylyl imidodiphosphate, or Ca2+.calmodulin has been isolated from bovine cerebral cortex by Affi-Gel Blue chromatography and calmodulin-Sepharose chromatography. Sensitivity to these effectors was restored by incubation of the adenylate cyclase preparation with detergent-solubilized protein from bovine cerebral cortex. Reconstitution of of Ca2+.calmodulin activation required the presence of 5'-guanylyl imidodiphosphate. The factor required for restoration of Ca2+.calmodulin stimulation was sensitive to heat, trypsin digestion, and N-ethylmaleimide. These observations suggest that this adenylate cyclase activity requires the presence of one or more guanyl nucleotide binding subunits for calmodulin sensitivity.
Evolution of eusociality in termites
Authors: Bartz Stephen H.
Published: May 2006

A model is presented that demonstrates that asymmetries in relatedness such that individuals are more closely related to siblings than to offspring develop in diploid pedigrees under conditions of inbreeding. Given also certain incestuous conditions, the model predicts that eusocial-type helping behavior can be favored by natural selection. Three cases of the model are discussed along with implications for termite eusociality. Several aspects of termite biology, particularly the existence of replacement reproductives, are interpreted as evidence that termite eusociality may have arisen under the special conditions specified in the models.
Effect of interchain disulfide bond on hapten binding properties of light chain dimer of protein 315.
Authors: Zidovetski R , Licht A , Pecht I
Published: May 2006

The hapten binding characteristics of the covalent light chain dimer, derived from the murine IgA secreted by plasmacytoma MOPC-315, to two nitroaromatic compounds, epsilon-N-(2,4-dinitrophenyl)-L-lysine and 4-(alpha-N-alanine)-m-nitrobenz-2-oxa-1,3-diazole, were investigated by differential spectroscopic titrations. The binding curves for both haptens were found to display sigmoidity similar to that reported earlier for the reduced and alkylated dimer held together by noncovalent bonds only. However, the presence of the interchain disulfide bond in the covalent dimer was found to cause marked changes in its binding properties. The data, like those obtained for the noncovalent dimer, fit the allosteric model of Monod, Wyman, and Changeux in which binding of the first hapten to the dimer causes a conversion of both sites of the protein molecule from a lower to a higher affinity conformation. However, the binding parameters show that both the affinity and the positive cooperativity in the interaction between haptens and the covalent dimer are significantly enhanced. The differences in the parameters of the binding and of the allosteric transition caused by the presence of the interchain disulfide bond demonstrate the existence of longitudinal interactions in immunoglobulin derivatives. These properties of the light chain dimer make it a potential model for the receptors present on thymus-derived lymphocytes.
Antigen-induced strain-specific autoantiidiotypic antibodies modulate the immune response to dextran B 512.
Authors: Fernandez C , Möller G
Published: May 2006

Sera from CBA and C57BL mice immunized against dextran B 512 eight or more days earlier contain mainly IgG antibodies that specifically suppress the development of plaque-forming cells against dextran in vitro. Suppression was not caused by antibodies against the alpha 1-6 epitope of dextran to any major extent. Sera from CBA mice (IgCH allotype j) suppressed plaque-forming cells from CBA mice, whereas sera from C57BL mice (allotype b) inhibited plaques from C57BL and CBA/Ig-1b mice. Sera from dextran-tolerant mice or from mice that are genetic unresponders to dextran did not suppress plaque-forming cells. Both thymus-independent and thymus-dependent forms of dextran induced the appearance of these autoantiidiotypic antibodies that can distinguish between anti-alpha 1-6 antibodies produced in mouse strains having different IgCH-locus-determined allotypes.
Social gliding is correlated with the presence of pili in Myxococcus xanthus.
Authors: Kaiser D
Published: May 2006

Myxococcus xanthus, an organism whose motility involves cell interactions, normally bears pili. Myxococcal pili are found only at cell poles, are less than 10 nm in diameter, and may be longer than a cell. Myxococcus has two basic patterns of cell movement, adventurous (A-motility) and social (S-motility). Pili are found to be completely correlated with the presence of S-motility. (The S-motility pattern has many groups of cells, almost no single cells, and is governed by a set of genes called system S.) On the other hand, A-motility is in dependent of piliation. (The A-motility pattern has many single, isolated cells and it is governed by a second set of genes called system A.) Electron microscopic examination of more than 40 genetically different strains shows that all A+S+ (wild-type) and A-S+ strains have pili, but A+S- and A-S- strains lack them. Mutations in four different loci belonging to system S were tested and were found to stop productions of pili: the loci sg1A, sg1B, sg1G, and tg1. When brought into contact with tg1+ cells, cells of a tg1- strain, which lack pili, become phenotypically S+, produce pili, and become S-motile. Both motility and the production of pili are transient when initiated in this way. Thus it appears that pili permit cells that are close to one another to move.
Structure of carboxymyoglobin in crystals and in solution.
Authors: Makinen M W , Houtchens R A , Caughey W S
Published: May 2006

The configuration of the heme-carbonyl group upon binding of carbon monoxide to sperm whale myoglobin (Mb) in crystals is evaluated on the basis of infrared spectroscopic methods. Multiplets of the totally symmetric C-O stretching mode are observed for the heme-bound ligand near 1933, 1944, and 1967 cm-1, corresponding to three different heme-carbonyl conformers. Variations in the relative proportions of these conformers can be induced by incorporation of small fractions of metMb or deoxyMb into MbCO crystals. The configuration of the iron-carbonyl with respect to the immediate coordination environment of the heme iron is assigned for each v(CO) stretching frequency on the basis of a detailed comparison of the three-dimensional structures of the heme environments of MbCO, metMb, and deoxyMb defined by crystallographic methods. The structures of the three heme-carbonyl conformers account for the v(CO) infrared absorption bands that can be observed for MbCO in solution.
Small circular DNA of Drosophila melanogaster: chromosomal homology and kinetic complexity.
Authors: Stanfield S W , Lengyel J A
Published: May 2006

Nucleic acid reassociation techniques were used to determine the kinetic complexity of small circular DNA in cultured cells of Drosophila melanogaster. Two kinetic components are present. One of these constitutes 82% of the mass of the circular DNA and has a complexity of 1.8 x 10(4) nucleotide pairs; the other constitutes 18% of the mass and the a significantly higher but undefined sequence complexity. We have demonstrated that these circular molecules hybridize to middle repetitive chromosomal sequences by hybridization of in vitro-labeled circular DNA tracer with a vast excess of Drosophila chromosomal DNA. Thermal stability measurements indicate that base-pair mismatch between small circular DNA and middle repetitive chromosomal DNA does not exceed 2%. We discuss possible functions of these small circular DNAs in light of the above findings.
Role of proline isomerization in folding of ribonuclease A at low temperatures
Authors: Cook Kem H. , Schmid Franz X. , Baldwin Robert L.
Published: May 2006

In unfolded RNase A there is an interconversion between slow-folding and fast-folding forms (U
S
⇌ U
F
) that is known to show properties characteristic of proline isomerization in model peptides. Here, we accept the evidence that U
S
molecules contain nonnative proline isomers and we ask about the isomerization of these proline residues during folding. The U
S
⇌ U
F
reaction in unfolded RNase A is used both to provide data on the kinetics of proline isomerization in the unfolded protein and as the basis of an assay for measuring proline isomerization during folding.

The tyrosine-detected folding kinetics at low temperatures have been compared to those of proline isomerization in unfolded RNase A. The comparison is based on the recent observation that the U
S
⇌ U
F
kinetics are independent of guanidinium chloride concentration, so that they can be extrapolated to low guanidinium chloride concentrations, at which folding takes place. At 0°C the tyrosine-detected folding reaction is 100-fold faster than the conversion of U
S
to U
F
in unfolded RNase A. Consequently, the folding reaction is not rate-limited by proline isomerization as it occurs in unfolded RNase A.

An assay is given for proline isomerization during folding. The principle is that native RNase A yields U
F
on unfolding, whereas protein molecules that still contain nonnative proline isomers yield U
S
. Unfolding takes place at 0°C, at which proline isomerization is slow compared to unfolding. This assay yields two important results: (
i
) The kinetics of proline isomerization during folding are substantially faster than in unfolded RNase A—e.g., 40-fold at 0°C. The mechanism of the rate enhancement is unknown. (
ii
) At low temperatures (0-10°C), and also in the presence of (NH
4
)
2
SO
4
, the tyrosine-detected folding reaction occurs before proline isomerization and yields a folded intermediate I
N
that is able to bind the specific inhibitor 2′-CMP. The results demonstrate that a folding intermediate is spectrally detectable when folding occurs at low temperatures. They suggest that low temperatures provide suitable conditions for determining the kinetic pathway of folding by characterizing folding intermediates.
Characterization of cyanobacterial phycobilisomes in zwitterionic detergents
Authors: Glazer Alexander N. , Williams Robley C. , Yamanaka Gregory , Schachman H. K.
Published: May 2006

Properties of cyanobacterial phycobilisomes (from
Synechococcus
spp. 6301 and 6312 and
Synechocystis
sp. 6701) prepared in the presence of two different zwitterionic detergents were compared to those of phycobilisomes detached from membranes with the nonionic detergent Triton X-100 and then freed from Triton by sedimentation through high-salt sucrose density gradients. The absorption spectra, polypeptide composition, and ultrastructure of phycobilisomes were independent of the detergent used during the preparation. Phycobilisomes from certain cyanobacteria aggregated in the absence of detergent. Such aggregation was not seen in preparations containing zwitterionic detergents. Aggregation of phycobilisomes led to a partial quenching of their fluorescence. Electron microscopy showed the three types of phycobilisomes to have a hemi-disc appearance, although their detailed structures were quite different.
Uroporphyrinogen I synthase from human erythrocytes: separation, purification, and properties of isoenzymes.
Authors: Miyagi K , Kaneshima M , Kawakami J , Nakada F , Petryka Z J , Watson C J
Published: May 2006

Uroporphyrinogen I synthase [porphobilinogen ammonia-lyase (polymerizing), EC 4.3.1.8] from human erythrocytes was separated into two active protein peaks (A and B on DEAE-cellulose, by ammonium sulfate fractionation, on Sephadex G-100, and on DEAE-Sephadex A-50 with a NaCl gradient. The final purification was 613 and 743 times for A and B, respectively. The corresponding yields were 2.2 and 3.4% Fraction A was separated further into two (A1 and A2) active protein bands and fraction B into three (B1, B2, and B3) on analytical polyacrylamide disc gel electrophoresis. Bands A1 and A2 were identical with B1 and B2; B3 represented a third isoenzyme. Molecular weights (mean +/- SEM), measured by gel filtration and sodium dodecyl sulfate/polyacrylamide gel electrophoresis, were 38,000 +/- 1000 for B1 and 40,000 +/- 1000 for B2 and B3. Isoelectric focusing on 4% polyacrylamide gel separated both fractions A and B into three active protein bands. Maximal activity of the enzyme was found in gel cuts (5-mm) at pH 5.6 for both fractions A and B.
Removal of an adenine-like molecule during activation of dinitrogenase reductase from Rhodospirillum rubrum.
Authors: Ludden P W , Burris R H
Published: May 2006

During the activation of the inactive dinitrogenase reductase from Rhodospirillum rubrum, an adenine-like molecules is lost and phosphate is found on both active and inactive forms of the protein. ATP and divalent metals are required for activation of the reduced protein, but ATP is not required for activation of phenazine methosulfate-oxidized dinitrogenase reductase. Snake venom diesterase and spleen diesterase have no effect on the inactive protein; alkaline phosphatase removes phosphate from the activated protein but not from the inactive protein. ATP binds to both active and inactive forms of the protein.
Changes in activity of fructose-1,6-bisphosphate aldolase in livers of fasted rabbits and accumulation of crossreacting immune material.
Authors: Pontremoli S , Melloni E , Salamino F , Sparatore B , Michetti M , Horecker B L
Published: May 2006

The activity of fructose-1,6-bisphosphate aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) in livers of fasted rabbits decreases to less than one-half the value found in livers of fed rabbits. However, the concentration of aldolase protein in the liver extracts, measured with a specific antibody, remains unchanged. More than twice as much antibody is required to neutralize the aldolase activity in liver extracts from fasted compared with fed rabbits. The results suggest that modification of liver aldolase occurs during fasting, resulting in loss of catalytic activity without loss of immunoreactivity.
Organization of spacer DNA in chromatin.
Authors: Lohr D , Van Holde K E
Published: May 2006

Detailed analysis of the DNA fragment patterns produced by DNase I digestion of yeast, HeLa, and chicken erythrocyte nuclei reveals surprising features of nucleosome phasing. First, the spacer regions in phased yeast chromatin must be of lengths (10m + 5) base pairs, where m = 0, 1, 2,....This feature is not seen in parallel studies of chicken erythrocyte chromatin. The 5-base pair increment in the yeast spacer imposes interesting restraints on the higher order structure of yeast chromatin. Second, we have been able to simulate the DNase I cutting patterns and get good agreement with the observed yeast patterns. Third, three different chromatins show a long range periodicity in the DNase I digest pattern, with a period half that of the staphylococcal nuclease repeat. These results suggest that the amount of chromatin observed in discrete extended-ladder bands is a minimum estimate of phasing and in fact phasing may be a more general feature.
Mechanism of acetylcholine release: possible involvement of presynaptic muscarinic receptors in regulation of acetylcholine release and protein phosphorylation.
Authors: Michaelson D M , Avissar S , Kloog Y , Sokolovsky M
Published: May 2006

Acetylcholine (AcCho) release from purely cholinergic Torpedo synaptosomes was evoked by K+ depolarization in the presence of Ca2+. Activation of muscarinic receptors, present in the synaptosomal fraction, by the agonist oxotremorine resulted in the inhibition of AcCho liberation. This inhibition was abolished by the muscarinic antagonist atropine, which by itself has no effect. These findings suggest that the muscarinic receptor, present in the electric organ of Torpedo is presynaptic and that its physiological function is to regulate AcCho release by negative feedback. The mechanism of presynaptic muscarinic inhibition was investigated by examining the effect of muscarinic ligands on synaptosomal 45Ca2+ uptake and on the level of phosphorylation of specific synaptosomal proteins. Ca2+-dependent K+ depolarization-induced synaptosomal AcCho release was accompanied by 45Ca2+ uptake and by a marked increase in the phosphorylation of a specific synaptosomal protein (band alpha) of approximately 100,000 daltons. Activation of the muscarinic receptor by the agonist oxotremorine had no detectable effect on synaptosomal 45Ca2+ uptake but resulted in the concomitant inhibition of AcCho release and of phosphorylation of band alpha. The muscarinic antagonist atropine abolished the inhibitory effect of oxotremorine both on AcCho liberation and on phosphorylation of band alpha. These findings suggest that phosphorylation of band alpha may be involved in regulation of the presynaptic processes that underly AcCho release and that activation of the muscarinic receptor by agonists may inhibit AcCho release by blocking the phosphorylation of band alpha.
Functional reconstitution of photosynthetic reaction centers in planar lipid bilayers
Authors: Schönfeld M. , Montal M. , Feher G.
Published: May 2006

Planar lipid bilayers containing reaction centers from
Rhodopseudomonas sphaeroides
R-26 were formed by apposing two reaction center—lipid monolayers formed from a reaction center—lipid complex in hexane. Secondary donors (cytochrome
c
) and acceptors (ubiquinone-0) were added on opposite sides of the membrane. Upon illumination, this system generated transient and steady-state voltages and currents. The wavelength dependence of the photoresponse matched the absorption spectrum of reaction centers. A simple model based on the transfer of charges across the membrane that explains the salient features of the photoresponse is presented.
Photosynthetic carbon reduction pathway is absent in chloroplasts of
Vicia faba
guard cells
Authors: Outlaw William H. , Manchester Jill , DiCamelli Cynthia A. , Randall Douglas D. , Rapp Barbara , Veith George M.
Published: May 2006

Four cell types from
Vicia faba
Linnaeus “Long Pod” leaflets were assayed for three enzymes unique to the photosynthetic carbon reduction pathway. The enzymes were ribulosebisphosphate carboxylase [3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39], phosphoribulokinase (ATP:D-ribulose-5-phosphate 1-phosphotransferase, EC 2.7.1.19), and glyceraldehyde-phosphate dehydrogenase (NADP
+
) (phosphorylating) [D-glyceraldehyde-3-phosphate:NADP
+
oxidoreductase (phosphorylating), EC 1.2.1.13]. On a dry weight basis, these enzyme activities were about twice as high in palisade as in spongy parenchyma. Two of the enzymes were not detected in epidermal cells and the other was present in only a trace amount. In guard cells, these enzyme activities were absent or present at les than 1% of the amount in palisade cells. Immunoelectrophoresis showed that ribulosebisphosphate carboxylase was absent in extracts of guard cell protoplasts. Microscopy confirmed the abundance of typical guard cell chloroplasts. These results demonstrate the absence of the photosynthetic carbon reduction pathway in guard cell chloroplasts. This is the only chloroplast type known to be deficient in this pathway in plants whose primary CO
2
acceptor is ribulose bisphosphate. Possible reasons for the absence of this pathway in guard cells are discussed.
Uncouplers of oxidative phosphorylation promote derepression of the hexose transport system in cultures of hamster cells.
Authors: Kalckar H M , Christopher C W , Ullrey D
Published: May 2006

Long-term (18-24 hr) preincubation of NIL hamster cell cultures with D-glucose or D-glucosamine (both of which repress the hexose transport system) gave rise to a striking loss of the hexose transport system ("super-repression") when cycloheximide was also present in the culture medium. However, if 0.2 mM 2,4-dinitrophenol (DNP) was also present, the cycloheximide-mediated super-repression was prevented. Moreover, the presence of DNP at this low concentration contributed to an increase in hexose uptake such that it was substantially higher than that permitted by either of the two repressive sugars alone. When the cultures were maintained in medium containing D-fructose in place of glucose, a marked increase in uptake occurred, and this increase (derepression) was not affected by DNP. The derepression due to glucose deprivation and the increases caused by DNP treatment were also observed when 3-O-methylglucose was used to measure hexose transport. Although cultures maintained in the presence of glucosamine exhibited a repressed hexose transport rate, they did not generate significant amounts of lactic acid. DNP, and other uncouplers of oxidative phosphorylation, promoted a derepressed state of hexose transport but did not stimulate the generation of lactate from glucosamine. These data suggest that the metabolic repression phenomena of hexose transport do not depend on glycolysis but rather on the "energized" state of the cell. The energized state of the cell may also be required for the super-repression of hexose transport that is especially apparent when protein synthesis is blocked by cycloheximide.
Structural studies of human IgD: Isolation by a two-step purification procedure and characterization by chemical and enzymatic fragmentation
Authors: Lin Lien-Ching , Putnam Frank W.
Published: May 2006

A myeloma IgD immunoglobulin (designated WAH) that was present in high concentration in plasma (≈3.5 g/dl) was purified in >90% yield by a two-step procedure of ammonium sulfate precipitation plus AcA 34 gel filtration. Although the plasma had been stored for 2 years without the addition of a proteolytic inhibitor, no “spontaneous” degradation was apparent and the isolated IgD remained structurally intact. However, the purified IgD showed extreme susceptibility
in vitro
to various proteolytic enzymes; e.g., Fab
δ
(
M
r
≈ 47,000) and Fc
δ
(
M
r
≈ 80,000) fragments were generated quantitatively after only 10 min of incubation with papain in the absence of cysteine. By combining limited enzymatic digestion, reductive cleavage, and cyanogen bromide fragmentation, several series of well defined fragments corresponding to the different regions and domains of the IgD molecule were generated. These fragments are useful for physical, chemical, and immunological studies, as well as for the sequence determination of the IgD δ chain. A model of the IgD molecule was derived from such studies and from overlapping of the series of fragments. The possible existence of an extra constant domain in the δ chain appears unlikely in view of our finding of an extended hinge region of about 50 residues which can be cleaved off the amino terminus of the papain Fc
δ
by brief treatment with trypsin. In addition to a distinct stretch of carbohydrate attachment sites, the δ-chain hinge region contains a segment unusually rich in electrical charge. This charged segment is responsible for the lability of IgD to spontaneous degradation and may be related to its biological role as a B lymphocyte receptor.
Cellular site of gastric acid secretion.
Authors: DiBona D R , Ito S , Berglindh T , Sachs G
Published: May 2006

Isolated gastric glands of the rabbit were examined both with differential interference-contrast microscopy and with electron microscopy to describe the morphologic correlates of acid secretion. Stimulation of the glands with histamine resulted in the development of intracellular spaces within the parietal cells. A similar transformation was produced by addition of 1 mM aminopyrine, whether the weak base was added in the presence of normal-K+ (5.4 mM) or high-K+ (108 mM) solutions. The intracellular space was compatible with the expanded canaliculus described in stimulated parietal cells. Confirmation that the space produced by histamine is the site of acid secretion was gained by combining fluorescence and interference-contrast methods in the presence of the dye acridine orange, which displays a pH-dependent metachromasia in its emission spectrum. Human gastrin I resulted in an observable discharge of peptic granules.