bluuebunny/crossref_metadata_2025 huggingface dataset:
Collagen reduces glycosaminoglycan degradation by cultured mammary epithelial cells: possible mechanism for basal lamina formation.
Authors: David G , Bernfield M R
Published: May 2006

Collagenous substrates are reported to promote the accumulation of extracellular matrix materials by epithelia in culture. Glycosaminoglycan (GAG) metabolism is compared in secondary cultures of mouse mammary epithelial cells maintained on plastic or type I collagen gel substrates. The incorporation of 35SO42- into GAG during brief labeling indicates no difference between substrates in the rate of GAG synthesis. During prolonged labeling, however, accumulation of [35S]GAG in cultures on colllagen exceeds that of cultures on plastic. This increased accumulation is due to a markedly reduced rate of GAG degradation. GAG degradation does not occur in the medium, indicating that degradation is localized to the cells. The cultures on collagen contain a slowly degrading cell-associated [35S]GAG pool and a ruthenium red-stained basal lamina, neither of which is present in cultures on plastic. The cell-associated [35S]GAG in cultures on collagen is, in part, localized to the site of the ultrastructurally identified basal lamina. Formation of the basal lamina, therefore, may result from collagen-mediated reduction in the degradation of GAG-containing molecules.
Coupling between oxidation state and hydrogen bond conformation in heme proteins
Authors: Valentine Joan S. , Sheridan Robert P. , Allen Leland C. , Kahn Peter C.
Published: May 2006

In all heme proteins for which crystal structures are available, the N
ε
of a histidyl residue is bonded to the heme iron and N
δ
is hydrogen bonded to a carbonyl oxygen of the peptide backbone. We investigate here the possibility that a change in oxidation state of the iron or a change in the geometry of this hydrogen bond might change the hydrogen bond strength in a functionally significant way. Dimerization energies obtained from
ab initio
molecular orbital calculations on the hydrogen-bonded dimer of imidazole and planar formamide are used to represent the strength of this hydrogen bond in heme proteins. The effect of a change in iron oxidation state is modeled by varying the positive charge on imidazole. The effect of a change in hydrogen bond geometry is studied by employing x-ray coordinates for reduced and oxidized cytochrome
c
, deoxy- and metmyoglobin, and deoxy- and methemoglobin. Our conclusions are that the strength of this hydrogen bond in heme proteins is sensitive to both the oxidation state of the iron atom and to geometry changes on the order of those obtained from the x-ray coordinates. We speculate that the changes in oxidation state may be functionally coupled with changes in hydrogen bond geometry and that this hydrogen bond represents a feasible pathway to link protein conformation with redox potential or reactivity of the iron atom.
Mutations reducing the activity of c17, a promoter of phage lambda formed by a tandem duplication.
Authors: Mozola M A , Friedman D I , Crawford C L , Wulff D L , Shimatake H , Rosenberg M
Published: May 2006

We report the isolation of four independently selected mutations (scs) in the c17 promoter of phage lambda that reduce or eliminate the promoter activity. The c17 promoter is not normally present in lambda, and has been shown to be generated by a tandem duplication which creates a "Pribnow Box," a heptamer sequence implicated in promoter activity. This sequence is located upstream from the site of transcription initiation and is present, with some variation, in all promoters whose sequences have been determined. Analysis of the c17 duplications carrying the scs mutations reveals that three of these mutants carry single base-pair changes in the most highly conserved base pairs of the Pribnow Box and that the other mutation is a reversion to the wild type sequence in this region (i.e., a loss of the duplicated base pairs).
Formation of 5-hydroxykynurenine and 5-hydroxykynurenamine from 5-hydroxytryptophan in rabbit small intestine
Authors: Fujiwara Motokazu , Shibata Masakatsu , Nomiyama Yoshitsugu , Sugimoto Tetsuo , Hirata Fusao , Tokuyama Takashi , Senoh Siro , Hayaishi Osamu
Published: May 2006

In order to clarify the role of indoleamine 2,3-dioxygenase [indole:oxygen 2,3-oxidoreductase (decyclizing), EC 1.13.11.17] in the metabolism of serotonin, DL-5-hydroxy[
methylene
-
14
C]tryptophan, a precursor of serotonin, was incubated with slices of rabbit ileum. Resulting metabolites were separated by DEAE-cellulose column and polyamide column chromatography and identified by various chromatographic techniques and enzymatic analysis. Metabolites obtained in significant amounts were serotonin, 5-hydroxyindoleacetic acid, 5-hydroxytryptophol, 5-hydroxykynurenine, 5-hydroxykynurenamine, and 4,6-dihydroxyquinoline, representing 13.2, 15.8, 7.0, 21.9, 1.3, and 2.6% of the total metabolites, respectively. The first three compounds were previously reported to be major metabolites produced from 5-hydroxytryptophan by the action of aromatic L-amino acid decarboxylase and monoamine oxidase, whereas the last three are formed by the cleavage of the indole ring by the action of indoleamine 2,3-dioxygenase. In the presence of pargyline, a monoamine oxidase inhibitor, the major metabolites obtained were serotonin, 5-hydroxykynurenine, and 5-hydroxykynurenamine, representing 29.6, 26.6, and 5.4% of the total metabolites, respectively. In the presence of RO4-4602, an aromatic amino acid decarboxylase inhibitor, 5-hydroxykynurenine was the sole major product. These results strongly suggest that the newly discovered metabolic pathway involving the cleavage of the indole ring of 5-hydroxytryptophan operates
in vivo
to a significant extent and that indoleamine 2,3-dioxygenase plays an important role in the regulation of serotonin levels in the small intestine of the rabbit.
Nucleotide sequence of the region of an origin of replication of the antibiotic resistance plasmid R6K.
Authors: Stalker D M , Kolter R , Helinski D R
Published: May 2006

A 2.1-kilobase segment of the antibiotic resistance plasmid R6K carries sufficient information to replicate as a plasmid in Escherichia coli. This segment contains a functional origin of replication and a structural gene for a protein, designated pi, that is required for the initiation of R6K replication. The nucleotide sequence of a 520-base-pair portion of this 2.1-kilobase segment that includes the functional origin of replication and the region adjacent to the start of the pi structural gene was determined. A striking feature of the sequence is the presence of seven 22-base-pair direct repeats joined in tandem in the region adjacent to the start of the pi gene. A possible role of the tandem repeats in the regulation of expression of the pi protein and the control of initiation of replication of the plasmid R6K is discussed.
Vasopressin stimulation of mouse 3T3 cell growth.
Authors: Rozengurt E , Legg A , Pettican P
Published: May 2006

Vasopressin is shown to be a potent mitogen for Swiss 3T3 cells. The hormone (1--10 ng/ml) causes a striking shift of the dose--response curve for the effect of serum on thymidine incorporation by cultures of 3T3 cells arrested in the G1/G0 phase of the cell cycle. In the absence of added serum, the effect of vasopressin on DNA synthesis is greatly potentiated by insulin, epidermal growth factor, and a factor isolated from medium conditioned by simian virus 40-infected baby hamster kidney cells. The mitogenic effect of vasopressin is dependent on time and hormone concentration. In the presence of insulin, the half-maximal effect elicited by the peptide is obtained at 0.6 ng/ml. [Arg]Vasopressin and [Lys]vasopressin are equally potent. The vasopressins are 10(3)-fold more potent than oxytocin. In the presence of a low (2.5%) concentration of serum, vasopressins stimulate cell proliferation.
Antigenic drift in type A influenza virus: Peptide mapping and antigenic analysis of A/PR/8/34 (HON1) variants selected with monoclonal antibodies
Authors: Laver W. G. , Gerhard W. , Webster R. G. , Frankel M. E. , Air G. M.
Published: May 2006

Variants of A/PR/8/34 (HON1) influenza virus, having hemagglutinin molecules with probably a single altered antigenic determinant, were isolated by growing the virus in the presence of the monoclonal hybridoma antibody PEG-1. The variants were analyzed by peptide mapping and characterized antigenically by using PEG-1 and four other monoclonal hybridoma antibodies to PR8 hemagglutinin. Peptide maps of the large hemagglutinin polypeptide, HA1, from 8 out of 10 variants showed a single changed peptide. This peptide from two of the variants was analyzed, and in each case a serine residue in the wild-type hemagglutinin was replaced by leucine in the variant. Although these eight variants showed identical peptide maps, one could be discriminated antigenically from the others with one of the hybridomas. (The peptide maps represented about one-third of the HA1 molecule.) Of the other two variants, one gave the same HA1 map as the wild type, but could be distinguished antigenically from wild-type virus by two of the hybridomas. The other was unique, and could be distinguished, both antigenically and by peptide mapping, from the other variants.

Since a large number of the variants selected with PEG-1 showed the same peptide change, it is likely that this alteration in amino acid sequence (serine to leucine) was responsible for the inability of the variants to bind PEG-1 monoclonal antibody. We do not know, however, whether the changed amino acids were located within the antigenic sites or whether the change occurred somewhere else in the hemagglutinin molecule and altered the determinants through conformational changes.
Neurotoxicity of human eosinophils
Authors: Durack David T. , Sumi S. Mark , Klebanoff Seymour J.
Published: May 2006

Eosinophils contain a substance that is neurotoxic when injected intracerebrally or intrathecally into laboratory animals—an effect known as the “Gordon phenomenon.” We found neurotoxic activity in eosinophils from three patients with eosinophilic syndromes by injecting cell preparations into rabbits and guinea pigs. These animals developed a syndrome of muscular rigidity and ataxia, progressing to severe paralysis. No neurotoxic activity was found in preparations of polymorphonuclear or mononuclear leukocytes from normal donors. Examination of the brains of affected animals confirmed widespread loss of Purkinje cells, as described by earlier investigators. A new finding was severe spongy change occurring in the white matter of the cerebellum, brainstem, and spinal cord. Electron microscopic examination showed that vacuoles formed within the myelin sheaths of axons by separation of lamellae. Associated axonal degeneration was common and was also seen occasionally in peripheral nerves. Gray matter in the cerebral hemispheres and spinal cord was normal. This eosinophil-derived neurotoxin was partially purified by ultracentrifugation of sonicated eosinophils and fractionation of the supernate by gel filtration. Fractions with neurotoxic activity eluted at a position consistent with a molecular weight of approximately 15,000. The neurotoxic activity of this material withstood lyophilization and dialysis but was destroyed by heating to 90°C. Injection of eosinophil-derived neurotoxin into laboratory animals may provide a useful short-term experimental model for study of mechanisms of damage to myelinated nerve fibers. The clinical significance of the Gordon phenomenon has yet to be established.
On integral representations for the Neumann operator
Authors: Phong D. H.
Published: May 2006

This paper is devoted to the derivation of an approximate formula for the kernel of the Neumann operator
N
on strongly pseudo-convex domains. In particular, a new class of singular integral operators arises that are infinitely smoothing in the interior and preserve singular supports at the boundary and whose kernels are products of isotropic kernels with parabolic ones.
Choleragen (cholera toxin): a bacterial lectin.
Authors: Richards R L , Moss J , Alving C R , Fishman P H , Brady R O
Published: May 2006

Choleragen (cholera toxin) agglutinated erythrocytes and liposomes containing the toxin receptor, galactosyl-N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosylglucosylceramide (ganglioside GM1). Cells that had been exposed to GM1 were agglutinated, but agglutination was not observed when cells had been exposed to other gangliosides (GM2, GM3, GD1a, GD1b). Choleragen-dependent agglutination of liposomes was slightly less specific, because liposomes containing either GM1 or GD1b, but neither GM2, GD1a, nor GM3 were agglutinated. The oligosaccharide isolated from GM1 inhibited both the agglutination of cells and liposomes containing GM1 and the binding of choleragen to liposomes containing GM1. Galactose and sialic acid were less effective inhibitors of liposomal agglutination and did not inhibit cellular agglutination or binding of choleragen to liposomes. Liposomal agglutination was dependent on choleragen concentration and occurred with the B but not the A protomer of choleragen. These results suggest that choleragen, through its binding to the oligosaccharide portion of a glycolipid, exhibits lectinlike activity, which results in agglutination of liposomes and erythrocytes.
Regulation of the threonine operon: tandem threonine and isoleucine codons in the control region and translational control of transcription termination.
Authors: Gardner J F
Published: May 2006

The DNA sequence of 178 base pairs preceding the first structural gene of the threonine operon of Escherichia coli has been determined. A region of perfect 2-fold rotational symmetry, involving 28 base pairs, precedes the first structural gene. The structural similarity of this sequence to known RNA polymerase termination sites suggests that this region is the termination site of the threonine operon leader RNA. Moreover a mutation (thr 79-20), which confers a depressed, constitutive phenotype, was sequenced and found to be a G.C insertion in the putative terminator. A potential coding region for a 21-amino acid leader peptide ends approximately 18 base pairs before the terminator. This peptide contains eight threonine and four isoleucine codons. Eleven of these codons are in tandem. A model for threonine operon regulation, involving alternative secondary RNA structures and translation of leader RNA, is discussed.
Secretion and cell-surface growth are blocked in a temperature-sensitive mutant of
Saccharomyces cerevisiae
Authors: Novick Peter , Schekman Randy
Published: May 2006

Saccharomyces cerevisiae
cells contain a small internal pool of the secretory enzymes invertase and acid phosphatase. This pool increases up to 8-fold at 37°C in a temperature-sensitive, secretion-defective mutant strain (
sec
1-1). Cell division and incorporation of a sulfate permease activity stop abruptly at the restrictive temperature, while protein synthesis continues for several hours. Electron microscopy of mutant cells incubated at 37°C reveals a large increase in the number of intracellular membrane-bound vesicles, which are shown by histochemical staining to contain the accumulated acid phosphatase. The vesicles are removed and the accumulated enzymes are secreted when cells are returned to a permissive temperature in the presence or absence of cycloheximide. These results are consistent with a vesicle intermediate in the yeast secretory pathway and suggest that exocytosis may contribute to cell-surface growth.
Methods for identifying proteins by using partial sequences
Authors: Dayhoff M. O. , Orcutt B. C.
Published: May 2006

Methods for the identification of a protein segment by using the information from partial sequence analyses are described. If the protein sequence is known, a segment can usually be identified with confidence through comparison with the data file of all known sequences when the identity and position of only seven amino acid residues (not necessarily contiguous) are known. Partial sequences are obtained from extremely sensitive microsequencing procedures. Tissue is incubated with amino acids, one or more of which are distinctively radiolabeled. Proteins of interest are isolated and a sequenator experiment performed to locate the positions of radioactivity in an NH
2
-terminal segment of approximately 30 residues. We derive and investigate an equation for the probability of finding a unique match to any pattern of radioactivity. From this we suggest a new strategy. In one incubation, several amino acids are labeled with each kind of isotope. The most information is contained in patterns in which approximately equal numbers of positions are occupied by residues distinguished by different labels (including no label). The amino acid composition of the segment will typically not be known in advance. Labeling residues expected to occupy 36% of the positions suffices for a 98% chance of success in uniquely characterizing any human segment. Such a strategy will permit the identification of most proteins from a single tissue incubation. The mathematical discussion is general and applies to any segment from a sequence and to sequences obtained by any method. Improved identification procedures should expedite the accumulation of information on the expression and function of proteins.
Cloning in Escherichia coli and physical structure of hepatitis B virion DNA.
Authors: Charnay P , Pourcel C , Louise A , Fritsch A , Tiollais P
Published: May 2006

A restriction map of hepatitis B virion DNA was established after cloning of the whole viral genome in Escherichia coli. By use of EcoRI, Xho I, Bgl II, Xba I, BamHI, HincII, and Hae III endonucleases, a total of 28 restriction sites were mapped. The single-stranded region was localized on the restriction map and 5' end of the short strand was mapped at a fixed position.
Rate and equilibrium constants for binding of apo-E HDL
c
(a cholesterol-induced lipoprotein) and low density lipoproteins to human fibroblasts: Evidence for multiple receptor binding of apo-E HDL
c
Authors: Pitas R. E. , Innerarity T. L. , Arnold K. S. , Mahley R. W.
Published: May 2006

Competitive binding assays have demonstrated that a cholesterol-induced canine lipoprotein containing only the E apoprotein (apo-E HDL
c
) binds to the same cell surface receptors of human fibroblasts as human low density lipoproteins (LDL). However, the apo-E HDL
c
have a much greater binding activity than LDL. Equilibrium and kinetic binding studies were conducted at 4°C to determine the mechanism for this enhanced receptor binding activity. Based on the data, the binding of both LDL and apo-E HDL
c
appears to be a simple bimolecular receptor interaction, and no heterogeneity of binding sites or cooperative effects among the receptor sites were observed. Equilibrium dissociation constants determined by Scatchard analysis of the equilibrium binding data for apo-E HDL
c
(
K
d
= 0.12 × 10
-9
M) and LDL (
K
d
= 2.8 × 10
-9
M) revealed a 23-fold greater affinity of HDL
c
for the receptors. Association and dissociation rate constants for the lipoprotein-receptor complex were determined from the time course of binding at various lipoprotein concentrations. The equilibrium dissociation constants calculated from these kinetic data confirmed that apo-E HDL
c
had a much higher affinity for the receptor than LDL. Furthermore, the kinetic studies indicated that apo-E HDL
c
bound more rapidly than LDL with rates of association of 18.0 × 10
4
and 5.5 × 10
4
M
-1
sec
-1
, respectively. The rate of dissociation of the apo-E HDL
c
-receptor complex (1.7 × 10
-5
sec
-1
) was slower than that of the LDL receptor complex (6.3 × 10
-5
sec
-1
). An additional important difference between the binding of apo-E HDL
c
and LDL was that 4 times (3.6 ± 0.4) as many LDL particles as HDL
c
particles were required for saturation of the receptors at maximal binding. These data indicate that each HDL
c
particle binds to multiple cell surface receptors at a ratio of 4:1 for LDL receptor binding.
Villin: The major microfilament-associated protein of the intestinal microvillus
Authors: Bretscher Anthony , Weber Klaus
Published: May 2006

The major protein associated with actin in the microfilament core of intestinal microvilli has been purified. This protein, for which we propose the name villin, has a polypeptide molecular weight of approximately 95,000. Two arguments suggest that villin may be the microvillus crossfilament protein that links the microfilament core laterally down its length to the cytoplasmic side of the plasma membrane. First, electron microscopy shows that crossfilaments stay attached to isolated membrane-free microvillus cores. Calculation of the expected abundance of the crossfilament protein shows that only villin is present in sufficient quantity to account for these structures. Second, decoration of microvillus cores by antibodies to either actin or villin, followed by ferritin-labeled second antibody in a sandwich procedure, results in specific labeling of the cores in both cases. The antivillin decoration, however, gives rise to a greater increase in diameter, in agreement with a model in which villin projects from the F-actin microfilament core. Villin is distinct from α-actinin, a protein suggested to be involved in membrane anchorage of microfilaments in nonmuscle cells. The two proteins differ in molecular weight. Specific antibodies against villin and α-actinin show no immunological crossreactivity. Immunofluorescence microscopy reveals that villin is located in the microvilli of the brush border whereas α-actinin is absent from the microvilli but is found in the terminal web. In addition, villin is not found in microfilament bundles of tissue culture cells, which are rich in α-actinin. Thus, villin and α-actinin appear to be immunologically and functionally different proteins.
Induction of prostaglandin E synthesis in normal and neoplastic macrophages: role for colony-stimulating factor(s) distinct from effects on myeloid progenitor cell proliferation.
Authors: Kurland J I , Pelus L M , Ralph P , Bockman R S , Moore M A
Published: May 2006

The biosynthesis of prostaglandin E (PGE) by normal and neoplastic macrophages is intrinsically linked to their synthesis of, and exposure to, myeloid colony-stimulating factors (CS-factors). The defect in responsiveness to endotoxin lipopolysaccharide (LPS) by macrophages from C3H/HeJ mice extends equally to the synthesis of CS-factor and PGE. However, C3H/HeJ macrophages can be stimulated to synthesize PGE by treatment with agents other than LPS [zymosan, tuberculin purified protein derivative, concanavalin A, poly(I).poly(C)], which also stimulate CS-factor production, or by the addition of various preparations of soluble CS-factor. In peritoneal wash preparations, constitutive PGE synthesis occurred in rapidly sedimenting macrophage cells, whereas constitutive CS-factor production and inducible PGE synthesis occurred in slower sedimenting adherent cells. A similar functional heterogeneity in CS-factor and PGE production was found in neoplastic macrophagae cell lines. The association of elevated CS-factor levels and PGE synthesis by macrophages suggests a role for CS-factor in many of the physiological responses heretofore associated with elevated tissue levels of the E type prostaglandins.
Direct demonstration of binding of a lysosomal enzyme, alpha-L-iduronidase, to receptors on cultured fibroblasts.
Authors: Rome L H , Weissmann B , Neufeld E F
Published: May 2006

Receptor-binding of "high-uptake" forms of lysosomal enzymes to human diploid skin fibroblasts had been predicted from the Michaelis--Menten kinetics of uptake of these enzymes [e.g., Sando, G.N. & Neufeld, E.F. (1977) Cell 12, 619--627]. We have now demonstrated such binding directly by using a sensitive assay for the bound enzyme. Cells deficient in alpha-L-iduronidase were detached from plastic dishes by mild trypsinization, allowed to recover, and used in suspension. They were incubated with urinary alpha-L-iduronidase at 0 degrees C for 90 minutes and then washed by centrifugation through concentrated bovine serum albumin; the activity of the cell-associated enzyme was measured with 4-methylumbelliferyl alpha-L-iduronide as substrate. A Scatchard analysis showed 14,000 binding sites per cell and a Kd of 1 x 10(-9) M for high-uptake alpha-L-iduronidase; binding of the low-uptake form was barely detectable. Mannose 6-phosphate, a known competitive inhibitor of uptake, inhibited the binding competitively, with Ki = 1 x 10(-4) M. Unexpectedly, mannose 6-phosphate greatly accelerated the dissociation of bound enzyme. During uptake of alpha-L-iduronidase at 35 degrees C, the receptors were regenerated every few minutes, even in the absence of protein synthesis.
Bedload transport associated with high stream power, Jordan River, Israel
Authors: Inbar Moshe , Schick Asher P.
Published: May 2006

During a flood of a magnitude that recurs once in 100 years, boulders up to 1700 mm in size were transported in the Jordan and Meshushim Rivers, northern Israel. Bedload discharge rates were estimated for periods of 3-72 hr of peak flow by a combination of hydrologic and geomorphic methods. Bedload transport rate is proportional to unit stream power in excess of that necessary for initial motion, raised to the power 3/2, as has been shown for data on other rivers.
Inhibition of myosin ATPase by vanadate ion.
Authors: Goodno C C
Published: May 2006

Inhibition of the myosin ATPase by vanadate ion (Vi) has been studied in 90 mM NaCl/5 mM MgCl2/20 mM Tris-HCl, pH 8.5, at 25 degrees C. Although the onset of inhibition during the assay is slow and dependent upon Vi concentration (kapp approximately 0.3 M-1 s-1), the final level of inhibition approaches 100%, provided the Vi concentration is in slight excess over the concentration of ATPase sites. Inhibition is not reversible by dialysis or the addition of reducing agents. The source of this irreversible inhibition consists of the formation of a stable, inactive complex with the composition M . ADP . Vi (where M represents a single myosin active site). The complex has been isolated, and its mechanism of formation from M, ADP, and Vi has been studied. Omission of ATP increases the rate of formation by about 35-fold (kapp approximately 11 M-1 s-1), yet this rate is still low in comparison with the rates of simple protein-ligand association reactions. This slowness is interpreted in terms of a rate-limited isomerization step that follows the association of M+, ADP, and Vi: M+ . ADP . Vi leads to M+. ADP . Vi (+ indicates the inactive product of the isomerization). The properties of M.ADP.Vi are compared with those of the ATPase intermediate M**.ADP . Pi, and the possible role of Vi as an analog of Pi is discussed.
lac repressor changes conformation upon binding to poly[dA-T)]
Authors: Kelsey D E , Rounds T C , York S S
Published: May 2006

N-(Iodoacetylaminoethyl)-1-naphthylamine-5-sulfonate reacts with Escherichia coli lac repressor to selectively label cysteine-140 with the fluorescent N-(acetylaminoethyl)-1-naphthylamine-5-sulfonate group. The fluorescence intensity of this label decreases by 20% when labeled repressor associates with poly[d(A-T)]. Fifteen base pairs of poly[d(A-T)] per repressor tetramer are required to complete this decrease. Stopped-flow experiments have shown that the repressor undergoes at least two conformational changes as it binds to poly[d(A-T)], with half-lives of 5.0 +/- 1.2 msec and 3.5 +/- 1.0 sex. Quite likely, these conformational changes serve to strengthen the interaction of repressor with DNA.
Transcription of Ti plasmid-derived sequences in three octopine-type crown gall tumor lines.
Authors: Gurley W B , Kemp J D , Albert M J , Sutton D W , Callis J
Published: May 2006

Total RNA isolated from three octopine-type crown gall lines contains sequences homologous to specific regions of the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens strain 15955. A comparison of transcripts in these three tumor lines suggests that tumor cells transcribe various sequences within a sector of plasmid DNA of 13 x 10(6) daltons and that transcription may not be uniform across the plasmid derived sequences (T-DNA). Transcription of T-DNA by octopine-type tumors occurs at four major sites. The levels of transcription occurring at three of these sites appear to vary considerably among the three tumor lines investigated. Part of this variability may reflect differences in the organization and copy number of T-DNA. One of the transcription sites maps within a region of DNA with common sequence homology with all Ti plasmids. Varying amounts of transcript homologous to this region of T-DNA are present in all three tumor lines. It is suggested that transcription of these conserved sequences in the plant may have significance regarding the mechanism of tumorigenesis.
Physical map of chromosomal nitrogen fixation (
nif
) genes of
Klebsiella pneumoniae
Authors: Riedel Gerard E. , Ausubel Frederick M. , Cannon Frank C.
Published: May 2006

We describe a method for the rapid determination of the physical location of mutations caused by insertion of transposable elements. We used this method to construct a detailed physical map of the nitrogen fixation (
nif
) gene cluster of
Klebsiella pneumoniae
and to correlate it with the genetic map. Total cellular DNA was isolated from individual strains, each carrying an insertion in 1 of 15 different
nif
genes. The DNA was digested with a restriction endonuclease, fractionated by agarose gel electrophoresis, denatured, and blotted onto nitrocellulose filter paper. The DNA on the filters was hybridized with
32
P-labeled DNA fragments derived from amplifiable plasmids carrying cloned
nif
DNA fragments from
K. pneumoniae
. Altered hybridization patterns caused by insertions into
nif
genes allowed us to map
nif
mutations with respect to the previously mapped cleavage sites for various restriction endonucleases. We have used the same method to map the end points of
nif
deletions. Using this procedure, we assigned physical locations on the
K. pneumoniae
chromosome to 86
nif
insertion mutations and 13
nif
deletion end points. This mapping procedure provides a convenient alternative to deletion mapping as a definitive method for mapping insertion mutations within a gene or for ordering genes within a gene cluster. This procedure will be especially useful for mapping mutations conferring phenotypes that are difficult to monitor and for mapping mutations in bacterial species in which techniques for conducting deletion mapping have not been devised.
Amino acid sequence of a mouse immunoglobulin mu chain.
Authors: Kehry M , Sibley C , Fuhrman J , Schilling J , Hood L E
Published: May 2006

The complete amino acid sequence of the mouse mu chain from the BALB/c myeloma tumor MOPC 104E is reported. The C mu region contains four consecutive homology regions of approximately 110 residues and a COOH-terminal region of 19 residues. A comparison of this mu chain from mouse with a complete mu sequence from human (Ou) and a partial mu chain sequence from dog (Moo) reveals a striking gradient of increasing homology from the NH2-terminal to the COOH-terminal portion of these mu chains, with the former being the least and the latter the most highly conserved. Four of the five sites of carbohydrate attachment appear to be at identical residue positions when the constant regions of the mouse and human mu chains are compared. The mu chain of MOPC 104E has a carbohydrate moiety attached in the second hypervariable region. This is particularly interesting in view of the fact that MOPC 104E binds alpha-(1 leads to 3)-dextran, a simple carbohydrate. The structural and functional constraints imposed by these comparative sequence analyses are discussed.
Virolysis of mouse mammary tumor virus by sera from breast cancer patients.
Authors: Witkin S S , Egeli R A , Sarkar N H , Good R A , Day N K
Published: May 2006

All type C retroviruses are lysed by human serum in apparently antibody-independent, complement-mediated reactions. In contrast, we have now determined that the mouse mammary tumor virus (MMTV), a type B retrovirus, is not disrupted by normal human serum. MMTV was lysed, however, when rabbit antibody to whole MMTV was added to the serum. By taking advantage of this dependence of MMTV lysis on specific antibody, a virolytic assay was developed, based on the measurement of reverse transcriptase released from disrupted virions, to search for evidence of antibodies to MMTV in human sera. Significantly greater virolytic activity was detected in the sera of patients with breast cancer than in sera of patients with benign disease (P less than 0.001) or colorectal cancer (P less than 0.001) or in sera from apparently healthy individuals (P less than 0.002). This assay thus appears to be able to detect a unique attribute, possibly the presence of an antibody crossreacting with MMTV, in serum in patient with breast cancer.
Resolution of the ATP-dependent proteolytic system from reticulocytes: a component that interacts with ATP.
Authors: Hershko A , Ciechanover A , Rose I A
Published: May 2006

The ATP-dependent proteolytic cell-free system from reticulocytes has been resolved into three components, each of which is absolutely required for acid solubilization of 125I-labeled bovine serum albumin radioactivity. In addition to the previously reported heat-stable polypeptide [Ciechanover, A., Hod, Y. & Hershko, A. (1978) Biochem. Biophys. Res Commun. 81, 1100-1105], we now describe a protein of high molecular weight (approximately 450,000) that is labile at 42 degrees C. The extremely heat-labile factors is remarkably stabilized by ATP. GTP and CTP, which do not stimulate protolysis, do not stabilize this factor. Adenylate nucleotides such as ADP or the nonhydrolyzable beta,gamma imido or methylene analogues of ATP cause stabilization although they do not activate proteolysis. A third protein component of the protease system, stable at 42 degrees C, has been separated from the heat-labile species by salt precipitation. All three components are required with ATP for proteolytic activity, but thus far only the heat-labile factor has been shown to interact directly with ATP.
Nucleoside triphosphate regeneration decreases the frequency of translation errors.
Authors: Jelenc P C , Kurland C G
Published: May 2006

The addition of naturally occurring polyamines and inorganic ions to an in vitro protein-synthesizing system improved the extent and fidelity of translation. In such an optimized system, regeneration of the nucleoside triphosphates with phosphoenolpyruvate and pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) reduced further the missense error frequency to the in vivo level as well as enhanced the extent of translation. The effect of nucleoside triphosphate regeneration was shown to be due primarily to the increase in the ratio of adenosine and guanosine triphosphates to their hydrolysis products and only marginally due to the increase in the absolute concentrations of the nucleoside triphosphates.
Diglyceride lipase: a pathway for arachidonate release from human platelets.
Authors: Bell R L , Kennerly D A , Stanford N , Majerus P W
Published: May 2006

We provide evidence that the mechanism for arachidonate release from stimulated human platelets involves two enzymes: a phosphatidylinositol-specific phospholipase C (EC 3.1.4.10) and a diglyceride lipase. After incubation of platelets with thrombin for 15 seconds, 1.2 nmol of 1-stearoyl-2-arachidonoyl diglyceride per 10(9) platelets, was isolated. Arachidonate was released from this substrate by the action of diglyceride lipase located in the particulate fraction of platelets. The enzyme has a pH optimum of 7.0, is stimulated by calcium ions and reduced glutathione, and liberates 31 nmol of fatty acid per min per mg of platelet particulate protein. The diglyceride lipase has sufficient activity to account for the 5-10 nmol of arachidonate released per 10(9) platelets upon thrombin stimulation. That only arachidonate is released upon thrombin stimulation may be explained by the fact that the diglyceride substrate in platelets contains only arachidonate in the 2 position. The lipase activity found in platelet membranes can also hydrolyze the 1-position fatty acid. Stearate is not released when intact platelets are stimulated with thrombin, and the fate of this fatty acid remains to be elucidated.
The zero dispersion limit for the Korteweg-deVries KdV equation
Authors: Lax Peter D. , Levermore C. David
Published: May 2006

We use the inverse scattering method to determine the weak limit of solutions of the Korteweg-deVries equation as dispersion tends to zero. The limit, valid for all time, is characterized in terms of a quadratic programming problem which can be solved with the aid of function theoretic methods. For large
t
, the solutions satisfy Whitham's averaged equations at some times and the equations found by Flaschka
et al.
at other times.
Sequential cleavage of proinsulin by human pancreatic kallikrein and a human pancreatic kininase
Authors: ole-MoiYoi Onesmo , Seldin David C. , Spragg Jocelyn , Pinkus Geraldine S. , Austen K. Frank
Published: May 2006

A pancreatic endopeptidase localized to the β-cells of the pancreas by immunohistochemical techniques has been purified to homogeneity by following its functional and antigenic characteristics as a glandular kallikrein (EC 3.4.21.8). The enzyme gave a single stained band on alkaline disc gel electrophoresis which corresponded in location with the kinin-generating activity eluted from a replicate gel, was of 54,000 molecular weight by gel filtration, was devoid of caseinolytic activity, elicited a monospecific antiserum in a rabbit, and gave a line of complete identity with a single constituent in pancreatic extract, crude urine, and purified urokallikrein when analyzed with monospecific antibody to urokallikrein. The pancreatic glandular kallikrein generated three cleavage products of increasing anodal mobility from bovine and porcine proinsulin, and the presence of pancreatic kininase or bovine carboxypeptidase B increased the quantity of these products. Although the conversion products did not correspond to diarginyl- and monoarginylinsulin, the product of intermediate mobility was also obtained when proinsulin was treated with a low concentration of trypsin in the presence of kininase. The most rapidly migrating product did correspond to desalanylinsulin in the reference standard. Kininase alone had no action on proinsulin, and aprotinin prevented cleavage by kallikrein alone or in combination with kininase. Although the chemical structure of the proinsulin cleavage products has not been established, human pancreatic kallikrein is considered a putative activator of proinsulin because of its location in the β-cell, its preferential action on proinsulin and kininogen as compared to azocasein, and its capacity to generate insulin intermediate products that are further modified by human pancreatic kininase or bovine carboxypeptidase B.
Permeabilization of cells for studies on the biochemistry of bacterial chemotaxis.
Authors: Paoni N F , Koshland D E
Published: May 2006

The cell membranes of Salmonella typhimurium and Escherichia coli have been made permeable in order to introduce S-adenosylmethionine into the cell for study of the course of methylation. A series of protein bands in the Mr 60,000 region were methylated, the specific bands and the extent of methylation depending on the attractant used. The change in levels of methylation was essentially the same as the in vivo responses, indicating that the permeabilization procedure maintains the relative relationships of the cellular proteins. A shift in intensity of the methylated bands occurred over time, indicating that a sequential process is involved in the methylation of these proteins. The permeabilization technique appears to offer major advantages in tracing the biochemical processes of the behavioral system.
Platelet-derived growth factor: purification and partial characterization.
Authors: Heldin C H , Westermark B , Wasteson A
Published: May 2006

A cationic protein that stimulates DNA synthesis in human cultured cells was isolated from human platelets by ion exchange chromatography, hydrophobic chromatography, gel chromatography, and gel electrophoresis in sodium dodecyl sulfate. The electrophoretic behavior of biologically active or radioiodinated and reduced growth factor indicated that the native protein (approximately 30,000 daltons) was composed of two different polypeptides (approximately 13,000-14,000 and 16,000-17,000 daltons, respectively) linked via reduction-susceptible bonds. The stimulatory activity on human glial cells of the purified product at a concentration of approximately 4 ng/ml (0.13 nM) was equal to that of 1% human serum.
Isolation of a condensed, intracellular form of the 2-micrometer DNA plasmid of Saccharomyces cerevisiae.
Authors: Livingston D M , Hahne S
Published: May 2006

We have initiated an investigation of the proteins bound in vivo to the 2-micrometer DNA plasmid found in the yeast Saccharomyces cerevisiae by examining its intracellular form. To isolate 2-micrometer DNA without disturbing proteins bound to the plasmid, an extract was prepared from a strain lacking mitochondrial DNA and the nuclear chromatin was removed from the extract by centrifugation. When the DNA in this extract was sedimented through a sucrose gradient containing 0.15 M NaCl, plasmid DNA had a sedimentation coefficient of approximately 70. This S value is greater than the S value of 25 for naked, superhelical 2-micrometer DNA. Cosedimenting with the DNA were proteins of the same size as the histone proteins associated with yeast nuclear chromatin. Digestion of the plasmid DNA with micrococcal nuclease and electrophoresis of the resulting DNA fragments revealed that segments of discrete sizes are protected from degradation. Examination of the plasmid DNA protein complex by electron microscopy showed nucleosome structures. We conclude that 2-micron DNA exists as a condensed chromosome body within the cell.
Aspirin-like drugs interfere with arachidonate metabolism by inhibition of the 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid peroxidase activity of the lipoxygenase pathway.
Authors: Siegel M I , McConnell R T , Cuatrecasas P
Published: May 2006

Aspirin, indomethacin, and sodium salicylate are anti-inflammatory, analgesic, and antipyretic. Whereas aspirin and indomethacin inhibit prostaglandin synthetase (cyclo-oxygenase; 8,11,14-eicosatrienoate, hydrogen-donor: oxygen oxidoreductase, EC 1.14.99.1), salicylate does not. However, all three drugs affect the metabolism of arachidonate via the lipoxygenase pathway by inhibiting the conversion of 12-hydroperoxy- to 12-hydroxy-5,8,10,14-eicosatetraenoic acid.
Coexistence of desmin and the fibroblastic intermediate filament subunit in muscle and nonmuscle cells: identification and comparative peptide analysis.
Authors: Gard D L , Bell P B , Lazarides E
Published: May 2006

Extraction of chicken embryo fibroblasts (CEF) or baby hamster kidney (BHK) cells with 1% Triton X-100 and 0.6 M KCl leaves an insoluble cytoskeletal residue composed primarily of the 52,000 Mr subunit of intermediate filaments (F-IFP). In addition, CEF cytoskeletons exhibit a minor component with Mr of 50,000, identified as alpha-desmin, one of the two major isoelectric variants of the intermediate filament subunit from smooth muscle. BHK cytoskeletons contain the 50,000 Mr mammalian desmin variant. Cytoskeletons prepared from chicken embryonic myotubes contain F-IFP and both alpha- and beta-desmin. These data suggest that two distinct 10-nm filament subunits coexist in a single cell. One-dimensional peptide analysis of F-IFP and desmin from avian and mammalian cells reveals significant interspecies homology, as well as homology between F-IFP and desmin from the same species. Peptide analyses of 32P-labeled intermediate filament subunits suggest that there is considerable similarity in the phosphorylation sites of these proteins. These results indicate that F-IFP and desmin might be evolutionally related.
Postreplication repair: questions of its definition and possible alteration in xeroderma pigmentosum cell strains.
Authors: Park S D , Cleaver J E
Published: May 2006

DNA synthesis in normal cells and in excision-defective and variant xeroderma pigmentosum cells was investigated after irradiation with ultraviolet light. The sizes of DNA synthesized during brief pulses of [3H]thymidine 1-2 hr after irradiation were decreased, the xeroderma pigmentosum variant showing the smallest molecular weight. Once synthesized, however, labeled DNA increased in size at the same rat as control in all cell strains, and the rate was relatively insensitive to caffeine. After 2-3 hr, labeled DNA in each cell type reached a maximum size that was less than that in control cells, indicating the presence of long-lived blocks to DNA chain growth. This kind of experiment (pulse-chase) has in the past been used to investigate a repair process believed to be associated with the bypass of damaged sites in parental DNA: postreplication repair. We present an alternative model that does not involve a specific postreplication repair mechanism, but involve a specific postreplication repair mechanism, but involves normal chain elongation and termination mechanisms in which we conceive that dimers and other damaged sites act as well-or-nothing blocks to the progress of replication forks. No evidence could be found for any inducible process that enhanced the bypass of damaged sites.
Isolation and characterization of germ line DNA from mouse sperm.
Authors: Shiurba R , Nandi S
Published: May 2006

Mouse germ line DNA was isolated from sperm by a physicochemical procedure that preferentially destroys contaminating somatic cell DNA. The use of reducing conditions and chelating agents in combination with phenol permitted extraction of molecular weight DNA from mature sperm nuclei with approximately 80% efficiency. Less than 0.1% somatic cell DNA contamination remained in sperm DNA prepared by this method. Germ line DNA was characterized by determination of its ultraviolet absorbance spectrum, buoyant density in cesium chloride, and melting profile on a hydroxyapatite column. Contamination by mitochondrial DNA was assessed by cesium chloride/ethidium bromide gradient centrifugation. The significance of the mouse germ line DNA isolation procedure is discussed with respect to the possible genetic transmission of mammary tumor virus and leukemia virus, the origin of antibody diversity, and the origin of testicular teratomas.
Mutation in a structural gene for a beta-tubulin specific to testis in Drosophila melanogaster.
Authors: Kemphues K J , Raff R A , Kaufman T C , Raff E C
Published: May 2006

By two-dimensional gel electrophoresis of tubulins prepared from tissues of Drosophila melanogaster we have identified a beta-tubulin subunit that is present only in the testis. Furthermore, we have isolated, as a male sterile, a third chromosome dominant mutation [ms(3)KKD] in the structural gene for this beta-tubulin. Males heterozygous for this mutation produce no motile spermatozoa. Beginning with meiosis, all processes in spermatogenesis are abnormal to some extent. Many microtubules (including both cytoplasmic microtubules and doublet tubules of the axoneme) show aberrant structure in cross section, and the overall morphology of the developing spermatids is disorganized. Testes from these males were shown, by two-dimensional gel electrophoresis, to contain both the normal testis-specific beta-tubulin and an electrophoretic variant of this tubulin in equal amounts. Both wild-type and mutant testis-specific beta-tubulins were characterized by vinblastine sulfate precipitation, coassembly with purified Drosophila embryo tubulin, and peptide mapping.