bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence that fibronectin is the collagen receptor on platelet membranes.
Authors: Bensusan H B , Koh T L , Henry K G , Murray B A , Culp L A
Published: May 2006

Evidence is presented that fibronectin is present on the platelet cell membrane and that it is a receptor for collagen in the platelet-collagen interaction. First, sodium dodecyl sulfate/acrylamide gel electrophoresis was performed on the proteins remaining attached to the surface of collagen after the removal of the remainder of the platelet by sonication. The material was relatively enriched in a glycoprotein that comigrated with cold-insoluble globulin (CIG), a form of fibronectin, and in other proteins which comigrated with myosin, actin, and tropomyosin. The presumptive presence of contractile proteins is consistent with the presence of microfibrillar proteins. Second, the collagen-attached material was shown to contain a protein that reacted with anti-CIG serum by immunoelectrophoresis. Third, when CIG was preincubated with fibrous collagen, the platelet-collagen interaction was inhibited. Fourth, rabbit anti-human CIG stimulated human platelets to secrete the contents of their dense granules. The stimulation was not due to antibody complexes present in the solution. Fifth, a protein was extracted from well-washed platelets and purified on affinity columns of anti-CIG antibodies. The isolated protein was found to bind to fibrous collagen.
Refolding of a bifunctional enzyme and its monofunctional fragment.
Authors: Dautry-Varsat A , Garel J R
Published: May 2006

The renaturation of the bifunctional enzyme aspartokinase II-homoserine dehydrogenase II has been studied by using the reappearance of its two activities. The same kinetics of renaturation are obtained for the dehydrogenase (EC 1.1.1.3) and the kinase activity (EC 2.7.2.4). The mechanism of refolding of the enzyme apparently involves two steps, a folding step occurring within a monomer and a subsequent dimerization step. The reappearance of the two activities depends on this dimerization step, suggesting that monomeric species are inactive. A proteolytic fragment possessing full dehydrogenase activity is shown to be able to renature, as judged by the recovery of its activity. In this case also, the refolding depends on the formation of dimeric species. However, the refolding of this fragment is much faster than that of the dehydrogenase region in the intact enzyme. These results suggest that, although the dehydrogenase region can refold by itself when isolated as a fragment, refolding of this same region in the whole protein involves interactions with the remainder of the protein.
Cluster of ribosomal protein genes in Escherichia coli containing genes for proteins S6, S18, and L9.
Authors: Isono K , Kitakawa M
Published: May 2006

A mutant of Escherichia coli K-12 isolated for temperature-sensitive growth was found to harbor an alteration in robosomal protein L9. Because the chromosomal location of the structural gene for this protein (rplI) was not known, we mapped the mutation by using various Hfr strains. The fine mapping of this gene by Plkc phage-mediated transductions has revealed that it forms a gene cluster at 94 min on the E. coli genetic map together with the genes coding for two other ribosomal proteins, S6 (rpsF) and S18 (rpsR). Furthermore, the region of the E. coli genetic map containing this cluster was found to be shorter than previously estimated by approximately 2 min.
Idiotypic analysis of antibodies to poly(Glu60Ala30Tyr10): interstrain and interspecies idiotypic crossreactions.
Authors: Ju S T , Benacerraf B , Dorf M E
Published: May 2006

A common idiotype was defined by a guinea pig anti-idiotypic antiserum made against D1.LP antibodies specific to the synthetic terpolymer poly(Glu60Ala30Tyr10), referred to as GAT. This idiotype was found in the anti-GAT antibodies of all individuals of all inbred strains of mice tested. Due to its common (C) occurrence in the anti-GAT antibodies of all mouse strains, it was termed the CGAT idiotype. The presence of CGAT idiotype in mice is independent of the expression of various Ig-1, H-2, or Vg genetic markers. In contrast, antibodies produced in response to the crossreactive poly(Glu60-Ala40), termed GA, bound to the GAT ligand but did not bear the CGAT idiotype. This suggests that GAT, although bearing GA determinants, preferentially stimulates clones of CGAT idiotype. Some of the CGAT idiotypic specificities were also identified in sera of WF inbred rats after immunization with GAT. However, no significant levels of CGAT idiotype were detected in LEW rats, F344 rats, guinea pigs, or rabbits, although measurable quantities of anti-GAT antibody were present.
Expression of Ia-like antigens on cultured human malignant melanoma cell lines.
Authors: Winchester R J , Wang C Y , Gibofsky A , Kunkel H G , Lloyd K O , Old L J
Published: May 2006

Human malignant melanoma cell lines were found to bear Ia-like cell surface determinants demonstrable by hetero- or alloantisera and by direct identification of the characteristic bimolecular glycoprotein complex. Immunoprecipitation confirmed the presence of Ia determinants on the bimolecular complex. The quantity of Ia molecules determined by these methods and by absorption experiments was relatively constant for each cell line. Among different lines, however, the amount of Ia antigens ranged from a level equal to that expressed by B-cell lines to a small fraction of this amount. This variation in level of Ia contrasted with the more uniform amount of beta2-microglobulin detected on the cell surface. The Ia alloantigen specificity DRw2 was the most frequently encountered specificity. Ia determinants were also found on the surface of an epidermoid carcinoma line, but not on various other cell lines of normal or neoplastic origin.
"Dark-active" rat transformed into "light-active" rat by destruction of 24-hr clock: function of 24-hr clock and synchronizers.
Authors: Richter C P
Published: May 2006

In alternating 12-hr periods of light and dark the rat is active mainly in the dark. Its activity in the dark (beginning at 1800) depends exclusively on release of activity by the 24-hr clock. In the light (beginning at 0600) the 24-hr clock inhibits activity; the normal rat becomes totally inactive in the light except for activity resulting from external stimulation. After section of the connections between the optic chiasma and the hypothalamus, some rats become totally and permanently inactive in the dark. This sectioning destroys the 24-hr clock. After destruction of the clock removes inhibition of activity in the light period, the rat becomes active promptly at start of the light period--i.e., becomes a "light-active" animal. In the normal rat, activity becomes synchronized to start of the dark (by the electric clock at 1800), regardless of the amounts of activity. Destruction of the 24-hr clock eliminates the synchronizer at 1800. However, almost at once, activity, eating, and drinking are kept together by a second synchronizer, start of the light (by the electric clock at 0600). This may explain the ability of the rat to survive after destruction of the 24-hr clock.
Inhibition of putrescine synthesis blocks development of the polychete Ophryotrocha labronica at gastrulation.
Authors: Emanuelsson H , Heby O
Published: May 2006

Development eggs of the polychete Ophryotrocha labronica were analyzed for polyamines during the first 6 days after fertilization. The spermine content dominated initially, but gradually decreased. It was surpassed by putrescine, which rapidly increased to a maximum on the 3rd day, i.e., at the inception of grastrulation. The spermidine content was low during the entire period. Treatment of eggs with the putrescine synthesis inhibitor alpha-methylornithine from the onset of development led to developmental arrest at gastrulation and to an abnormally low content of putrescine in the treated embryos. Methylglyoxal bis(guanylhydrazone), an inhibitor of spermine and spermidine synthesis, had no visible effect of development. Our observations strongly suggest that putrescine synthesis is indispensable in early embryonic development of Ophryotrocha.
Translational control: recognition of the methylated 5' end and an internal sequence in eukaryotic mRNA by the initiation factor that binds methionyl-tRNAfMet.
Authors: Kaempfer R , Rosen H , Israeli R
Published: May 2006

Structural analogs of the methylated 5' end (cap) of eukaryotic mRNA, such as 7-methylguanosine 5'-monophosphate, specifically inhibit both GTP-dependent binding of Met-tRNAfMet and binding of globin mRNA to eukaryotic initiation factor 2 (eIF-2). Addition of purified eIF-2 effectively relieves the cap analog-induced inhibition of globin mRNA translation. The analog competitively inhibits the function of eIF-2 and of mRNA in protein synthesis. Binding to eIF-2 of capped mRNA as well as noncapped mRNA, such as Mengo virus RNA, can be inhibited completely by free cap molecules, but much more cap is needed to inhibit binding of Mengo virus RNA. mRNA, whether or not it is capped, competitively inhibits the binding of Met-tRNAfMet to eIF-2. These results provide compelling evidence that eIF-2 recognizes mRNA. It is shown that binding of mRNA to eIF-2 is primarily at an internal sequence, and secondarily through the cap. A model for the function of eIF-2 is presented that can account for all these properties. This model can provide a molecular basis for the differential translation of mRNA species, whether or not they are capped.
Interaction of mycobacterial polymethylpolysaccharides with paranaric acid and palmitoyl-coenzyme A: Structural specificity and monomeric dissociation constants
Authors: Yabusaki Kenichi K. , Ballou Clinton E.
Published: May 2006

The long-chain polyenoic fatty acids α- and β-paranaric acid form complexes with the 6-
O
-methylglucose polysaccharide from
Mycobacterium smegmatis
as demonstrated by an enhanced fluorescence emission of the paranaric acid. This interaction is eliminated by digestion of the methylglucose polysaccharide with α-amylase and glucoamylase, which removes four hexose units from the nonreducing end of the chain. Titration of the methylglucose polysaccharide with either paranaric acid isomer suggests formation of a 1:1 complex with a dissociation constant (
K
d
) of 0.4 μM. The fluorescence emission of this complex is quenched by palmitoyl-CoA, which indicates that the paranaric acid can be displaced by the acoyl-CoA, a conclusion confirmed by gel filtration. The presumed polysaccharide/palmitoyl-CoA complex has a
K
d
of about 0.1 μM. Acoyl-CoA derivatives with shorter fatty acid chains and free palmitic acid complete less effectively, indicating that they form weaker complexes with the polysaccharide. The methylmannose polysaccharides with 12 or 13 sugar units also complex paranaric acid strongly (
K
d
∼ 0.4 μM), whereas the isomer with 11 sugar units complexes weakly.

The methylglucose polysaccharide has been coupled to L-tryptophan methyl ester. The fluorescence emission spectrum of the attached tryptophan group is shifted to a shorter wavelength relative to
N
-acetyl-L-tryptophan methyl ester, and this effect is enhanced in the corresponding derivative made with the amylase-digested polysaccharide. The circular dichroism spectrum of the polysaccharide—tryptophan derivative shows three bands with negative ellipticity, in the 270-300 nm region, not observed in the amylase-digested derivative. These results imply that the tryptophan is in a more structured environment in the former than in the latter derivative. α-Paranaric acid binds to the polysaccharide—tryptophan conjugate and shows an enhanced fluorescence emission with partial quenching of the tryptophan fluorescence emission, suggestive of Förster energy transfer from tryptophan to paranaric acid.
Translation of rat liver fatty acid synthetase mRNA in a cell-free system derived from wheat germ
Authors: Flick Parke K. , Chen Julie , Alberts A. W. , Vagelos P. Roy
Published: May 2006

Total liver polysomes were isolated from rats that had fasted for 48 hr and that then had been re-fed a high-carbohydrate, fat-free diet for 20-24 hr. Indirect immunoprecipitation of the polysomes with purified antibody to rat liver fatty acid synthetase and deproteination on sodium dodecyl sulfate-containing sucrose gradients gave an RNA fraction which, when translated in a cell-free system derived from wheat germ, yielded a major polypeptide of apparent molecular weight 225,000 when the translation products were analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The polypeptide was specifically precipitated with antibody against rat liver fatty acid synthetase and competed with unlabeled fatty acid synthetase for binding to the antibody. It was somewhat smaller than native fatty acid synthetase subunits (molecular weight 240,000). The peptide accounted for approximately 65% of the radioactive, antibody-precipitable product, the remainder being peptides in the molecular weight range 100,000-150,000. Synthesis of the polypeptide was optimized with respect to K
+
, Mg
2+
, and spermine concentrations. The quantity of fatty acid synthetase mRNA obtained by the above procedure and measured by translation was a function of the nutritional state of the animal. The relative activity in fasting rats compared to rats that were re-fed for 12 hr was 1:12. The data suggest that rat liver fatty acid synthetase is synthesized as intact subunits from a large mRNA molecule or molecules.
Ligand-promoted transfer of proteins between phases: spontaneous and electrically helped.
Authors: Mustacich R V , Weber G
Published: May 2006

A model system for the partitioning of peripheral membrane proteins into membranes by ligand binding has been examined experimentally. Both bovine serum albumin and lysozyme partition between water and 1-butanol by the addition of sodium p-toluene sulfonate at pH 2.4. The partitioning is characterized by high orders of reaction: 25 and 10, respectively. Theory indicates that these high orders of reaction need not result from cooperative ligand binding in either phase, but depend primarily upon the number N of protein sites at which the transfer-promoting ligant binds, and on the difference in free energy of formation delta F0s of the protein--ligand complexes in the two phases. From the reaction orders and the experimental values of N, 80 for albumin and 11 for lysozyme, delta F0s was calculated to be --0.5 kcal/mol (--2.1 kJ/mol) and --0.8 kcal/mol (--2.5 kJ/mol) per ligand bound, respectively. Experiments measuring the dependence on ligand concentration of the rate of protein electrophoresis across the water/butanol interface are described. These rates increase by more than two orders of magnitude as the ligand concentration approaches the critical value for partition and are inversely dependent on the number of ligant sites for the two proteins studied.
Messenger RNA is conserved during drying of the drought-tolerant moss
Tortula ruralis
Authors: Dhindsa Rajinder S. , Bewley J. Derek
Published: May 2006

On rehydration after complete drying, the drought-tolerant moss
Tortula ruralis
(Hedw.) Gaertn., Meyer, and Scherb reforms its polyribosomes and can resume protein synthesis without new RNA synthesis. Results obtained with the double-label ratio technique coupled with polyacrylamide disc gel electrophoresis show that (
i
) the proteins synthesized on rehydration are largely similar to those synthesized at the time of dehydration and (
ii
) inhibition of RNA synthesis does not alter the rate or the pattern of protein synthesis during rehydration of slowly dried moss. Poly(A)-rich RNA has been isolated from fresh and dried moss by chromatography on oligo(dT)-cellulose and has been translated
in vitro
in the cell-free wheat germ system. It is concluded that mRNA is conserved during complete drying of
T. ruralis
and supports protein synthesis on subsequent rehydration. This ability to conserve mRNA is a characteristic held in common by vegetative drought-tolerant tissue of
T. ruralis
and air-dried seeds of higher plants.
Cyclic AMP-dependent protein kinase mediates a cyclic AMP-stimulated decrease in ornithine and S-adenosylmethionine decarboxylase activities.
Authors: Insel P A , Fenno J
Published: May 2006

Incubation of S49 lymphoma cells with N6,O2'-dibutyryl cyclic AMP (Bt2cAMP) decreases the activities of ornithine decarboxylase (L-ornithine carboxy-lyase; EC 4.1.1.17) and S-adenosylmethionine decarboxylase (S-adenosyl-L-methionine carboxy-lyase; EC 4.1.1.50), the two principal enzymes in the pathway of polyamine synthesis. This decrease is dose-dependent, commences after a 3-hr delay, virtually abolishes the assayable activities of the two enzymes, and is not associated with a soluble inhibitor of the enzyme activities. Studies in mutant S49 clones that have altered protein kinase indicate that cAMP-dependent protein kinase mediates the decreases in enzyme activities. The dose-response pattern for the cAMP-stimulated decrease in enzyme activities parallels the pattern for the cAMP-stimulated, cell cycle-specific (G1) growth arrest of S49 cells. The activity of ornithine decarboxylase decreases faster than Bt2cAMP arrests wild-type S49 cells and, similarly, release of cells from the cAMP-stimulated arrest in G1 increases the activity of ornithine decarboxylase faster than cells exit from G1. These findings contrast with reports that cAMP induces ornithine decarboxylase in other cell types and further suggest that passage of cells through cell cycle is required for maintaining the activities of ornithine and S-adenosylmethionine decarboxylases.
Expression of human adenosine deaminase after fusion of adenosine deaminase-deficient cells with mouse fibroblasts
Authors: Siciliano Michael J. , Bordelon Mary R. , Kohler Peter O.
Published: May 2006

Two human choriocarcinoma cell lines were shown to be deficient in adenosine deaminase (ADA; adenosine aminohydrolase, EC 3.5.4.4) such that they did not produce bands on starch gels after electrophoresis and histochemical staining. Radiometric assay indicated that their ADA specific activity was approximately 2% that of HeLa (human) cell controls. Subclone analysis of one of the lines indicated that this deficiency was representative of individual cells of the line. After fusion of these cells with mouse fibroblasts having high ADA activity, most independently isolated hybrid clones expressed one of two, or both, additional (to the mouse) bands of ADA activity after electrophoresis. The expression of these extra bands in hybrids was dependent upon actual fusion. The phenomenon was observed in 30 of 45 independently derived hybrid clones from four different fusion experiments involving two different parental lines from each species. The pattern of appearance of the extra bands in independent hybrid clones and the tendency of a hybrid clone to lose one of the extra bands through subsequent passages suggests that the bands were the products of human genetic material. The extra bands electrophoretically comigrated with human ADA 1 and 2 from human
ADA-1-2
heterozygotes and the faster-migrating of the two extra bands comigrated with human ADA 1 from HeLa cells. Therefore, we suggest that the bands appearing in hybrids are the products of the
1
and
2
alleles of the human
ADA
locus. The human cells used for fusion were deficient in ADA activity but contained the genetic information for ADA 1 and 2. Fusion with mouse cells having ADA activity resulted in the activation of both human gene products coded for on separate homologous chromosomes. We conclude that the human
ADA
locus is under manipulatable genetic regulation.
Vesicular stomatitis virus glycoprotein is necessary for H-2-restricted lysis of infected cells by cytotoxic T lymphocytes.
Authors: Hale A H , Witte O N , Baltimore D , Eisen H N
Published: May 2006

Vesicular stomatitis virus (VSV) elicited cytotoxic thymus-derived lymphocytes (CTLs) in mice of the BALB/c and three congenic strains (BALB.b, BALB.k, BALB.HTG). CTL lysis of VSV-infected fibroblasts from the four strains was restricted by the target cells' major histocompatibility complex (H-2). Target cells were also infected with two temperature-sensitive mutants of VSV, tsM and tsG in which, respectively, the viral matrix protein and glycoprotein are not expressed at 39 degrees (restrictive temperature) on the infected cell's surface membrane. At the restrictive temperature, cells infected with wild-type VSV or tsM were lysed by CTLs, but cells infected with tsG were not. The requirement for the glycoprotein on the target cell was also evident from the ability of antisera to the glycoprotein to block completely CTL lysis of VSV-infected cells.
Cytotoxicity of 5-fluoro-2'-deoxyuridine: requirement for reduced folate cofactors and antagonism by methotrexate.
Authors: Ullman B , Lee M , Martin D W , Santi D V
Published: May 2006

Protein in vitro inhibition of thymidylate synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) by 5-fluoro-2'-deoxyuridylate requires 5,10-methylenetetrahydrofolate. The cytoxicity of 5-fluoro-2'-deoxyuridine towards cultured L1210 mouse leukemia cells is reduced when intracellular reduced folates are depleted, either by limiting the source in media or by inhibition of dihydrofolate reductase with methotrexate. Likewise, the intracellular amount of 5-fluoro-2'-deoxyuridylate covalently bound to thymidylate synthase in L1210 cells treated with 5-fluoro-2'-deoxyuridine is greatly diminished when cells are depleted of folate cofactors. The folate requirement for optimal growth of L1210 cells is lower than that required for maximal cytotoxicity of 5-fluoro-2'-deoxyuridine. These findings provide a biochemical rationale that may be useful in designing clinical protocols that use 5-fluorinated uracil analogs.
Chemistry of low-valent molybdenum phosphite complexes: Models of seven-coordinate reaction intermediates
Authors: Muetterties E. L. , Kirner J. F. , Evans W. J. , Watson P. L. , Abdel-Meguid S. , Tavanaiepour I. , Day V. W.
Published: May 2006

A simple synthesis of the zerovalent complex Mo[P(OCH
3
)
3
]
6
has been devised from a potassium reduction of MoCl
4
(tetrahydrofuran)
2
followed by reaction with trimethyl phosphite at 70°. Protonation of this octahedral complex gave only low yields of the expected seven-coordinate hydride, HMo[P(OCH
3
)
3
]
6
+
. The major product was an octahedral nonhydridic cation, Mo[P(OCH
3
)
3
]
5
P(OCH
3
)
2
+
, derived from proton cleavage of the P—O phosphite ester bond. This octahedral cation was stereochemically nonrigid, apparently through facile methoxy group migration. Close packing by methoxy groups in this fluxional cation was established through an x-ray crystallographic study of Mo[P(OCH
3
)
3
]
5
P(OCH
3
)
2
+
-PF
6
-
. Extended reaction of trifluoroactic acid with Mo[P(OCH
3
)
3
]
6
yielded the seven-coordinate hydride, HMo[P(OCH
3
)
3
]
4
(O
2
CCF
3
), which was near pentagonal bipyramidal and was stereochemically nonrigid.
“Pretranscriptional capping” in the biosynthesis of cytoplasmic polyhedrosis virus mRNA
Authors: Furuichi Yasuhiro
Published: May 2006

The
in vitro
synthesis of cytoplasmic polyhedrosis virus (CPV) mRNA was previously shown to be dependent upon the presence of the methyl donor
S
-adenosylmethionine (AdoMet). We now find that the competitive inhibitor of methylation,
S
-adenosylhomocysteine (AdoHcy), also stimulates CPV mRNA synthesis efficiently, resulting in the synthesis of viral mRNAs containing 5′-terminal GpppA and ppA, rather than m
7
GpppAm as observed with Adomet. In addition to AdoHcy, other AdoMet analogues, including
S
-adenosylethionine and adenosine, also stimulate CPV mRNA synthesis but to a smaller extent than does AdoHcy or AdoMet. In order to study the relationship between cap formation and mRNA synthesis, nucleoside triphosphates were replaced in the RNA-synthesizing reaction mixture (containing AdoMet) by the corresponding β,γ-imido analogues, which are resistant to nucleotide phosphohydrolase, an enzyme involved in cap formation. Although mRNA synthesis occurred in the presence of UMP-pNHp or GMP-pNHp, none was observed when AMP-pNHp was substituted for ATP. Because the ATP molecule that becomes the 5′-terminal nucleotide of CPV mRNA must be cleaved at the β-γ position during cap formation, the results suggest that, in this viral transcription system, cap formation is prerequisite to mRNA synthesis—i.e., a “pretranscriptional” event.
The disulfide bond connecting the chains of ricin.
Authors: Lappi D A , Kapmeyer W , Beglau J M , Kaplan N O
Published: May 2006

Studies on the disulfide bond connecting the two polypeptide chains of ricin are reported. Reduction of this bond in the native protein requires approximately 50-fold more mercaptoethanol than the reduction of the bond in the protein denatured by sodium dodecyl sulfate. An improved procedure for the formation of this disulfide bond from recombined chains is reported. A and B chains spontaneously and rapidly reassociate into a stable complex with a sedimentation velocity similar to that of native oxidized ricin before the disulfide bond reforms. The mixture of both chains also behaves on Bio-Gel P-100 like native oxidized ricin. However, the complex formed by the two chains, assayed before the disulfide bond can reform, and reduced ricin, carboxymethylated to prevent reoxidation, shows a significant decrease in toxicity to mice and a decrease in ability to inhibit protein synthesis in HeLa cells in culture.
Altered nucleosome structure containing DNA sequences complementary to 19S and 26S ribosomal RNA in Physarum polycephalum.
Authors: Johnson E M , Allfrey V G , Bradbury E M , Matthews H R
Published: May 2006

The localization of DNA sequences coding for ribosomal RNA was studied by hybridization of purified ribosomal RNA to DNA from chromatin fragments prepared by limited digestion of Physarum nuclei with staphylococcal nuclease. The 32P-labeled 19S and 26S RNA hybridized to DNA from nucleosome monomers, dimers, trimers, and higher oligomers, separated by sucrose gradient centrifugation, although the level of hybridization to DNA from nucleosome fractions was less than the level of hybridization to undigested nuclear DNA. The distribution of 19S and 26S rDNA sequences in the nucleosome fractions differed from the distribution of bulk DNA in that the rDNA sequences were recovered primarily in two fractions containing monomer-sized DNA lengths (140-160 base pairs). The percentage of DNA hybridizing to 19S plus 26S RNA was greater in peak A, the more slowly sedimenting monomer peak, than in any other chromatin fraction at all stages of digestion. Peak A and monomer particles differed in protein content and distribution. The presence of ribosomal cistrons in an altered nucleosome configuration may be related to changes in functional states of rDNA chromatin.
Common ancestor for concanavalin A and lentil lectin?
Authors: Foriers A , de Neve R , Kanarek L , Strosberg A D
Published: May 2006

The primary structure of the alpha subunit from Lens culinaris lectin was determined by analysis of tryptic peptides and was shown to consist of 52 amino acid residues. The molecular weight calculated on the basis of the sequence is 5928. The whole chain is homologous with the region between positions 75 and 121 from concanavalin A. The NH2-terminal sequence of the beta chain, determined by automated Edman degradation, is homologous to another portion of the concanavalin A molecule, between positions 123 and 165. Comparison of the 94 residues from the lentil lectin alpha and beta chains with concanavalin A reveals the existence of 43 identities. Thirty-four other homologies could have arisen, each by a single nucleotide substitution. This extensive homology suggests that the lentil lectin alpha and beta chains may be proteolytic fragments from a single polypeptide chain of the same length as concanavalin A.
Analysis of mRNA populations by cDNA·mRNA hybrid-mediated inhibition of cell-free protein synthesis
Authors: Hastie Nicholas D. , Held William A.
Published: May 2006

Hybridization of mRNA to its corresponding cDNA was found to specifically inhibit translation of the mRNA
in vitro
. Using hybridization of globin cDNA to globin mRNA as a model system, we found that equivalent amounts of cDNA were required both for the saturation of the mRNA hybridization and for complete inhibition of globin synthesis. Also, the rate of inactivation of translation was identical to the rate of hybridization and followed the predicted kinetic form. This assay has been applied to the analysis of a set of abundant mRNAs in mouse liver. Hybridization of liver mRNA with total liver cDNA in slight excess to a low C
0
t value specifically inhibited translation of several major polypeptides. Melting of the hybrids prior to translation restored synthesis of these polypeptides. Moreover, we found that different liver mRNAs are inactivated with different kinetics; the results suggest that the mRNAs for the major urinary polypeptide and for albumin are the most abundant and second most abundant, respectively, in mouse liver. The general applications of this technique are discussed.
The intervening sequence of a mouse beta-globin gene is transcribed within the 15S beta-globin mRNA precursor.
Authors: Tilghman S M , Curtis P J , Tiemeier D C , Leder P , Weissmann C
Published: May 2006

Mouse beta-globin in encoded in a discontinuous structural gene interrupted by a 550-base pair intervening sequence of DNA. Correspondingly, the mature beta-globin mRNA appears to be synthesized via a 15S precursor, the length of which roughly equals the total length of the coding and intervening sequences of the beta-globin gene. Using the electron microscope to visualize hybrid structures formed between this gene and the purified 15S beta-globin mRNA precursor, we show that the intervening sequence is present within the larger precursor molecule. This finding suggests that the precursor mRNA is processed through the removal and rejoining of internal RNA sequences.
Indirect induction of differentiation in myeloid leukemic cells by lipid A
Authors: Weiss Benny , Sachs Leo
Published: May 2006

Normal myeloid and MGI
+
D
+
clones of myeloid leukemic cells can be induced for Fc and complement component 3 rosettes, lysozme, and mature macrophages and granulocytes by a protein with macrophage- and granulocyte-inducing (MGI) activity, whereas MGI
+
D
-
clones can be induced by this protein for rosettes and lysozme but not mature cells. Lipopolysaccharides (LPS) from different bacteria induced the appearance of rosettes, lysozyme, and macrophages in some MGI
+
D
+
clones but did not induce any of these changes in MGI
+
D
-
clones. Lipid A gave the same results as LPS. Incubation of MGI
+
D
+
cells with LPS also induced an MGI activity detectable in the culture medium. This activity behaved like MGI in inducing (
i
) rosettes, lysozyme, and mature cells in MGI
+
D
+
leukemic cells including a clone resistant to LPS, (
ii
) rosettes and lysozyme in MGI
+
D
-
leukemic cells, and (
iii
) differentiation of normal myeloid cells to mature macrophages and granulocytes. This activity was induced in MGI
+
D
+
cells by LPS before the induction of rosettes or lysozyme. The results indicate that the lipid A portion of LPS indirectly induces differentiation of MGI
+
D
+
myeloid leukemic cells by inducing MGI protein. It is suggested that induction of specific regulatory proteins may be a more general mechanism for the induction of differentiation by surface-acting compounds.
Haemophilus influenzae
bacteremia and meningitis resulting from survival of a single organism
Authors: Moxon E. Richard , Murphy Patrick A.
Published: May 2006

Infant rats were infected intranasally with mixtures of streptomycin-sensitive and streptomycin-resistant strains of
Haemophilus influenzae
type b and cultures of nasopharyngeal washings, blood, and cerebrospinal fluid were obtained. If the infecting organisms cooperated with each other during the establishment of infection, nasopharyngeal, blood, and cerebrospinal fluid cultures should have contained mixtures of the variants. If each organism acted independently, then with small infecting inocula all the organisms in nasopharynx, blood, or cerebrospinal fluid should be descended from a single bacterium. Cultures should then contain only one of the variants.

Single variant nasopharyngeal cultures were obtained from 8 out of 19 (42%) rats when the intranasal inoculum was <100 organisms. As the inoculum was increased, single variant cultures were less frequently observed. When the inoculum was ≥10
5
organisms, nasopharyngeal cultures were always mixtures. Single variant blood cultures were obtained in 46 of 67 (68.7%) episodes of bacteremia when rats were inoculated intranasally with 10
8
organisms. Single variants were isolated from the cerebrospinal fluid of 13 of 19 (68.4%) rats with meningitis whose blood contained both streptomycin-sensitive and streptomycin-resistant variants. When the blood contained a single variant, this same variant was cultured from the cerebrospinal fluid on 39 of 40 (97.5%) occasions. These studies demonstrated that invasive.
H. influenzae
b infections of infant rats resulted from independent action, as opposed to cooperative interaction of intransally inoculated organisms. The results also suggested that the meninges were invaded by the hematogenous route.
Unique structures formed by pyrimidine-purine DNAs which may be four-stranded.
Authors: Johnson D , Morgan A R
Published: May 2006

Pyrimidine-purine DNAs with repeating sequences can be made to undergo a reversible transition to possibly a tetra-stranded complex. Physicochemical characterization of the new structures and model building are consistent with, in the case of d(TC)n-d(GA)n, a tetra-stranded complex forming by the addition of d(GA)n to the remaining space in the major groove of the triple-stranded complex d(TC)n-d(GA)n-d(CT)n. A possible role for tetra-stranded complexes in chromosome condensation is suggested by the natural occurrence of repeating sequence pyrimidine-purine DNAs and the properties of condensed chromosomes.
Mapping of adenovirus late promoters with nascent mercurated RNA.
Authors: Weinmann R , Aiello L O
Published: May 2006

Nascent RNA molecules were labeled in vivo and elongated in vitro by incubation of the isolated nuclei in the presence of mercurated nucleotides. The RNA molecules initiated and labeled in vivo and elongated in vitro were then selectively purified on a thiopropyl 6-B Sepharose affinity column. The procedure was shown to be free of artifacts since the addition of mercurated nucleotides and the retention on the affinity column is mediated by the endogenous RNA polymerase II (nucleoside triphosphate:RNA nucleotidyltransferase; EC 2.7.7.6), is sensitive to actinomycin D, and is dependent on the presence of all four ribonucleotide triphosphates. This general procedure was applied to the mapping of viral promoters late after adenovirus 2 infection of HeLa cells. RNA purified as described above was hybridized to restriction enzyme fragments attached to nitrocellulose filters. The 5' ends of the nascent RNA chains are located in coordinates 9.5-17 for a rightward transcript, 0-25 for a leftward transcript, and possibly 60-70 for a second rightward transcript. These locations clearly differ from locations of the early promoters and therefore suggest that the transition from early to late functions is controlled at the transcriptional level.
Attack of sea urchin eggs by dogfish phagocytes: Model of phagocyte-mediated cellular cytotoxicity
Authors: Weissmann Gerald , Finkelstein Morris C. , Csernansky John , Quigley James P. , Quinn Ruth S. , Techner Lee , Troll Walter , Dunham Philip B.
Published: May 2006

To test whether lysosomal degranulation of phagocytes is associated with antibody-dependent cytotoxicity, eggs of
Arbacia punctulata
were used as targets for blood phagocytes of
Mustelus canis
. Eggs were coated with heat-aggregated dogfish IgM and exposed to phagocytes, and cytolysis of eggs was observed by Nomarski optics. Phagocytes adhered, degranulated, and raised fertilization membranes resembling those induced by sperm or ionophore A23187. Lysis was then observed as damage radiating from the point of phagocyte-egg contact. By 4 hr, coated eggs exposed to phagocytes released 8.9, 12.3, and 7.4% of total catalase (EC 1.11.1.6), β-glucuronidase (EC 3.2.1.31), and superoxide dismutase (EC 1.15.1.1) into the medium. Cytotoxic enzyme release significantly exceeded that from uncoated eggs incubated with phagocytes or eggs alone (uncoated or coated). Because activated eggs release a neutral protease, it was considered possible that this enzyme might be responsible for autolysis of eggs. This possibility was excluded because (
i
) lysis of eggs was not inhibited by soybean trypsin inhibitor (SBTI) whereas the egg protease was sensitive to SBTI, and (
ii
) the major trypsin-like activity of phagocytes was not inhibited by SBTI. These experiments demonstrate that Ig-coated cells are first activated, and then killed, when exposed to degranulating phagocytes and suggest that enzymes from attacking phagocytes, and not target cells, are responsible for cell death.
Growth of embryonal carcinoma cells in serum-free medium.
Authors: Rizzino A , Sato G
Published: May 2006

Two mouse embryonal carcinoma cell lines, PCC.4 aza-1 and F9, have been grown in serum-free F-12 medium supplemented with Pedersen fetuin, insulin, transferrin, and 2-mercaptoethanol. This medium supports long-term growth of both cell lines. When these cells are transferred from medium containing serum to this serum-free medium, growth continues without any detectable lag. PCC.4 aza-1 grown in this medium for over 20 generations retains the capacity to differentiate in vivo. This medium appears to be a general serum-free medium for the growth of embryonal carcinoma cells.
Calcium-dependent regulator protein: Localization in mitotic apparatus of eukaryotic cells
Authors: Welsh Michael J. , Dedman John R. , Brinkley B. R. , Means Anthony R.
Published: May 2006

Calcium-dependent regulator protein is a low molecular weight (17,000), thermostable, calcium binding protein which is structurally homologous to skeletal muscle troponin C. This protein is present in all nonmuscle cells and has been shown to decorate stress fibers in interphase cells by indirect immunofluorescence. Using this procedure we have investigated the distribution of the protein during mitosis of eukaryotic cells. As the cells enter prophase, the distinct cytoplasmic localization disappears commensurate with the dissolution of the cytoskeleton. The regulator protein seems to be randomly distributed throughout the prophase cell, including the region around the condensed chromosomes. However, at prometaphase, it is localized in association with the half-spindles of the mitotic apparatus. Through metaphase and most of anaphase, the protein remains localized between the chromosomes and the poles of the spindle. During late anaphase the protein is also found in the interzone region but rapidly condenses into two small regions, one on each side of the midbody that separates the daughter cells. The regulator protein is not localized in the cleavage furrow during telophase, whereas actin is demonstrable in this region. Indeed, placement of the protein during mitosis is distinct from both that of actin and that of tubulin. The localization of calcium-dependent regulator protein during mitosis suggests that it may mediate the calcium effects on the mitotic apparatus and thus play a role in chromosome movement.
Chimeric mice derived from human—mouse hybrid cells
Authors: Illmensee Karl , Hoppe Peter C. , Croce Carlo M.
Published: May 2006

Mouse teratocarcinoma cells from the OTT6050 ascites tumor were established in tissue culture and selected for 5-bromodeoxyuridine (BrdUrd) resistance. The embryonal carcinoma cells grew without a feeder layer, remained deficient for thymidine kinase (EC 2.7.1.75), and differentiated like the original tumor into various tissues after subcutaneous injection into 129 mice. We fused the BrdUrd-resistant mouse teratocarcinoma cells with HT1080-6TG human diploid fibrosarcoma cells deficient in hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) and selected for hybrid cells in hypoxanthine/aminopterin/thymidine medium. The resulting hybrid cells segregated human chromosomes quickly and retained one to three human chromosomes including chromosome 17 that carries the human genes for thymidine kinase and galactokinase (EC 2.7.1.6). Single hybrid cells from five independent clones containing human chromosome 17 were injected into mouse blastocysts bearing several genetic markers that affect the coat color phenotype and strain-specific enzyme variants in order to detect tissue differentiation derived from the injected cells. After the injection of single hybrid cells into a total of 103 experimental blastocysts that had been surgically transferred to pseudopregnant foster mothers, 49 mice were born and 2 of them clearly revealed coat mosaicism. In 2 of 17 mice thus far analyzed, the injected hybrid cells proved to be capable of participating substantially in development of seven different organs. However, human gene products have not yet been detected unequivocally in those tissues and weak human-specific galactokinase activity could be recovered only from two mosaic tissues.

Our results demonstrate that, after
in vitro
culture and selection, at least some of the human-mouse hybrid cells still retain their
in vivo
potential to differentiate and become functionally integrated in the living organism. It now seems feasible to cycle mouse teratocarcinoma cells carrying human genetic material through mice via blastocyst injection to study human gene expression during differentiation.
Hydrolysis of poly (A) to adenine nucleotides by purified poly (A) polymerase.
Authors: Abraham A K , Jacob S T
Published: May 2006

Highly purified poly(A) polymerase (polynucleotide adenylyltransferase, EC 2.7.7.19), which synthesizes poly(A) from ATP substrate, can also catalyze hydrolysis of poly(A). The enzyme, designated as poly(A) hydrolase, requires either Mn2+ or Mg2+ for activity. Although AMP is the predominant product of the reaction, ADP and ATP are also formed. The enzyme is a 3'-exonuclease that does not degrade poly(A) associated with poly(A) poly(U) helical structure. AMP, ADP, and ATP inhibit the hydrolytic reaction. These data suggest that (i) the levels of adenine nucleotides regulate synthesis and degradation of poly(A), (ii) poly(A) itself is a storage form of adenine nucleotides, (iii) the hydrolytic reaction is responsible for poly(A) shortening or turnover observed in vivo, and (iv) the synthetic and hydrolytic activities are functions of the same protein molecule.
General characteristics of protein degradation in diabetes and starvation.
Authors: Dice J F , Walker C D , Byrne B , Cardiel A
Published: May 2006

The enhanced protein degradation associated with diabetes and starvation is fundamentally different from normal protein catabolism. In normal eukaryotic cells large molecular weight proteins tend to be degraded more rapidly than small proteins, acidic proteins tend to be degraded more rapidly than neutral or basic proteins, and glycoproteins tend to be degraded more rapidly than nonglycoproteins. All three of these general correlations are absent or markedly reduced in liver and muscle of diabetic and starved rats. In contrast, the correlations between proteins size and half-life, between protein net charge and half-life, and between protein carbohydrate content and half-life are not affected in brain of diabetic or starved animals. These results suggest that diabetes and starvation alter the general characteristics of intracellular protein degradation in target tissues of insulin. Degradation of serum proteins is also affected in diabetes and starvation. In normal animals a general correlation exists between isoelectric points of serum proteins and their degradative rates. This relationship is abolished in diabetes and starvation, as it is among liver and muscle proteins. The implications of our findings are discussed with regard to possible mechanisms of the enhanced protein breakdown.
Micrococcus luteus DNA gyrase: active components and a model for its supercoiling of DNA.
Authors: Liu L F , Wang J C
Published: May 2006

Two active components alpha and beta of micrococcus luteus DNA gyrase, of peptide weights of 115,000 and 97,000, respectively, have been purified. Each individual component exhibits little DNA gyrase activity; the ATP-dependent negative supercoiling of a covalently closed circular DNA duplex is catalyzed by a combination of the two. Covalent closure by Escherichia coli ligase of a circular DNA containing single-chain scissions, when carried out in the presence of a combination of the DNA gyrase components alpha and beta, gives a positively supercoiled DNA upon removal of the bound protein molecules. ATP was not present during the ligase treatment; therefore the positive supercoiling of DNA observed is a result of the binding of gyrase molecules, presumably as multi-subunit oligomers, during the ligation step. This is in contrast to the negative supercoiling of DNA catalyzed by gyrase in the presence of ATP. A model in which negative supercoiling of DNA is achieved by ATP-modulated repetitive wrapping of the DNA around gyrase is described. The model also suggests a plausible mode of action by which translocation of a DNA along its helix axis can be actively driven by an ATPase.
Nucleotide sequence of the simian virus 40 small-t gene
Authors: Volckaerţ G. , Voorde A. Van De , Fiers W.
Published: May 2006

The nucleotide sequence of the segment of simian virus 40 DNA between standard map positions 0.53 and 0.65, i.e., approximately half of the restriction fragment
Hin
d A, is reported. This segment is located near the beginning of the early region and is transcribed counterclockwise. There is a potential initiating ATG signal at 13 nucleotides from the
Hin
d C-
Hin
d A junction in the strand with the same polarity as the early mRNA. From this signal on, an open reading frame is present which would allow the synthesis of a polypeptide of 174 amino acids until a TAA termination codon is reached at nucleotide 602 (map position 0.547). This polypeptide, revealed by the DNA sequence, corresponds almost certainly to small-t antigen. Correlation of the deduced amino acid sequence with the NH
2
-terminal sequences of small-t and large-T (tumor) antigens of simian virus 40, as established by Paucha
et al.
[Paucha, E., Mellor, A., Harvey, R., Smith, A. E., Hewick, R. M. & Waterfield, M. D. (1978) [
Proc. Natl. Acad. Sci. USA
75, 2165-2169], strongly argues that both proteins are indeed initiated at the ATG triplet. Because the DNA region between 0.547 and 0.534 is blocked for translation in all three reading frames by multiple termination condons, we conclude that the large-T antigen must be coded for by two noncontiguous DNA segments: the segment from 0.65 to around 0.60, which small-t and large-T antigens share, and another segment starting at some point after position 0.534 and continuing counterclockwise until it terminates at map position 0.174. Small-t antigen is methionine-rich and has a remarkably high number of cysteine residues clustered mainly in its COOH-terminal half. It is rich in both basic and acidic residues, the former being slightly in excess.
Natural species-restricted attachment of human and murine T lymphocytes to various cells.
Authors: Galili U , Galili N , Vánky F , Klein E
Published: May 2006

Murine and human T lymphocytes bear on their surface a receptor that confers on them the ability to attach to a variety of target cells from the same species, derived in vivo and in vitro. Thymocytes and activated T cells attached readily to target cells, while blood T lymphocytes were able to do so only after the removal of sialic acid from either their cell membrane or that of the target cell. The natural attachment (NA) receptor and the corresponding site on the target cells are trypsin sensitive and the conjugation between them is temperature dependent. The phenomenon may be a manifestation of self recognition in a broader sense--recognizing the species--which is also reflected in the reactivity of mitogen-activated T cells and specific immune responses against allo- or other antigens expressed on target cell surfaces.