bluuebunny/crossref_metadata_2025 huggingface dataset:
Naturally soluble component(s) that confer(s) guanine nucleotide and fluoride sensitivity to adenylate cyclase.
Authors: Bhat M K , Iyengar R , Abramowitz J , Bordelon-Riser M E , Birnbaumer L
Published: May 2006

Supernatant fractions (300,000 x g, 60 min) from homogenates of rat liver, heart, and skeletal muscle, dog liver, and rabbit liver prepared without detergent in the homogenization medium (referred to as S300) are shown to contain an activity that restores Mg2+-dependent fluoride- and guanine nucleotide-stimulated cyclizing activity to the adenylate cyclase system [ATP pyrophosphate-lyase (cyclizing); EC 4.6.1.1] in cyc- S49 murine lymphoma cell membranes. Approximately 25% of the total cyc- reconstituting activity in the above tissues is present in S300. Reconstituting activity is proportional to S300, is sensitive to trypsin, is protected against heat inactivation by guanine nucleotide, and has a sedimentation coefficient of 5.3 in both H2O and 2H2O linear sucrose density gradients. Treatment with cholera toxin and NAD+ results in reconstitution of cyc- adenylate cyclase with enhanced activity in the presence of GTP. Reconstituion with S300 is stable, as seen in cyc- membranes after washing. All of these properties of S300 are similar to those of membrane-derived cyc- reconstituting activity. It is concluded that cell cytoplasm contains a naturally soluble protein or mixture of proteins having guanine nucleotide regulatory component activity of adenylate cyclase.
Cloning and mapping of BamHi endonuclease fragments of DNA from the transforming B95-8 strain of Epstein-Barr virus.
Authors: Skare J , Strominger J L
Published: May 2006

DNA from the B95-8 strain of Epstein-Barr virus was cleaved into 29 different fragments by BamHI endonuclease (EC 3.1.23.6). All of the fragments except the terminal fragments have been inserted into the pBR322 cloning vector and replicated in Escherichia coli. The location of each cloned DNA fragment in the viral genome has been determined, providing a more detailed physical map of the genome than has been available previously.
Involvement of cyclic GMP in intracellular signaling in the chemotactic response of Escherichia coli.
Authors: Black R A , Hobson A C , Adler J
Published: May 2006

The intracellular signal that produces changes in swimming behavior when bacteria encounter attractants or repellents has not previously been identified. We suggest, based on the following lines of evidence, that cyclic GMP (cGMP) is involved in this signaling process in chemotaxis by Escherichia coli. (i) The addition of attractants to bacteria causes a transient increase in the intracellular level of cGMP, whereas a repellent stimulus decreases the level transiently. These changes do not generally occur in a mutant lacking chemotaxis-specific proteins. (ii) In the absence of chemoeffectors, both addition of cGMP to bacteria and reducing the intracellular cGMP level produce changes in swimming behavior, and a mutant with an abnormal swimming pattern has an altered intracellular cGMP level. (iii) cGMP modulates the demethylation reaction responsible for adaptation to stimuli. (iv) Mutants defective in components of the adaptation system have altered cGMP metabolism.
Initiation of genetic recombination: homologous pairing between duplex DNA molecules promoted by recA protein.
Authors: Cassuto E , West S C , Mursalim J , Conlon S , Howard-Flanders P
Published: May 2006

recA protein has been shown to promote hydrogen bonding between single-stranded DNA fragments and duplex DNA molecules homologous to them. However, genetic and biochemical evidence indicates that genetic exchanges generally take place between duplex molecules. We therefore chose to study the interactions promoted by recA protein between intact duplex DNA molecules and molecules containing gaps that are believed to increase the frequency of genetic exchanges. In the present paper, we show that incubation of intact and gap-containing plasmid DNA in the presence of recA protein leads to homologous pairing between duplex molecules which can be detected by centrifugation analysis and electron microscopy. The reaction is completely dependent on an active recA gene product, on genetic homology between the DNA species involved, and on the presence of ATP; under certain conditions, its efficiency can be increased considerably by the presence of the single-stranded DNA binding protein of Escherichia coli.
Active efflux of tetracycline encoded by four genetically different tetracycline resistance determinants in Escherichia coli.
Authors: McMurry L , Petrucci R E , Levy S B
Published: May 2006

Tetracycline resistance encoded by four genetically different determinants residing on plasmids in Escherichia coli was shown to be associated in each case with an energy-dependent decrease in accumulation of the antibiotic in whole cells in which resistance had been induced. The different class determinants examined were those on plasmids RP1 (class A), R222 (class B), R144 (class C), and RA1 (class D). This decrease in accumulation was attributable to an active efflux, because everted (inside-out) membrane vesicles made from tetracycline-induced E. coli cells containing any one of the four plasmids were shown to concentrate tetracycline by an active influx. This active uptake was not seen in inside-out vesicles from sensitive cells or uninduced R222-containing cells. In vesicles from induced R222-containing cells, the efflux appeared to be carrier-mediated with a Km of about 6 microM. These results demonstrate that active export of tetracycline is a common component of the mechanism for tetracycline resistance encoded by different plasmid-borne determinants in bacteria.
Reversibility of sodium-induced aggregation of sonicated phosphatidylserine vesicles.
Authors: Day E P , Kwok A Y , Hark S K , Ho J T , Vail W J , Bentz J , Nir S
Published: May 2006

The kinetics of sodium-induced aggregation of sonicated phosphatidylserine vesicles has been studied as a function of sodium concentration and temperature. The concentration threshold for aggregation induced by monovalent sodium has been found to be 550 mM sodium by stopped-flow rapid-mixing techniques. This aggregation is completely reversible to changes in sodium ion concentration and to changes in temperature. The aggregation rate decreases with increasing temperature, indicating that the backward reaction rate increases more rapidly with temperature than does the forward rate.
Resource variation and the structure of British bird communities
Authors: Lister Bradford C.
Published: May 2006

Data on the foraging microhabitats of British birds are reanalyzed with the aim of understanding how fluctuations in resource abundance affect niche relationships and community structure. At Marley Wood, overlap in the foraging sites of resident bird species increased during the late spring and summer and decreased during the fall and winter. Among bird species coexisting in the pine forests at Thetford Chase, spatial overlap and spatial niche widths were positively correlated with food abundance over a 4-year period. These results suggest that in variable environments similarity in spatial niches is an inverse function of the intensity of competition for food. As food supplies drop, consumer species must apparently occupy increasingly different foraging areas in order to coexist. In less variable environments, however, resource stability may allow finer partitioning of the available foraging space and greater spatial overlap within a given foraging area. The results of this paper also suggest a reinterpretation of MacArthur's study of resource partitioning among warbler species in the boreal forests of New England. Rather than providing an instance of niche segregation in order to avoid intense competition, MacArthur's warblers may actually represent another example of increased spatial similarity when food resources are abundant and competition is reduced.
A cis-active regulatory gene in the mouse: direct demonstration of cis-active control of the rate of enzyme subunit synthesis.
Authors: Bernstine E G , Koh C
Published: May 2006

Mouse mitochondrial malic enzyme [L-malate:NADP+ oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.40] is a tetrameric protein. Two alleles of the structural gene (Mod-2) are known which code for electrophoretically distinct enzyme subunits: Mod-2a and Mod-2b. A regulatory gene (Mdr-1), closely linked to Mod-2 on chromosome 7, determines the rate of mitochondrial malic enzyme synthesis in brain. Two alleles of Mdr-1 are known: Mdr-1a (high activity) and Mdr-1b (low activitiy). By pulse-labeling with [35S]methionine, immune precipitation, and isoelectric focusing under dissociating conditions, we have measured the relative rates of synthesis of the two types of enzyme subunit in animals of genotypes Mdr-1a Mod-2a/Mdr-1a Mod-2b and Mdr-1S Mod-2a/Mdr-1b Mod-2b. The results show that in the former animals both types of subunit are made at an identical rate, whereas in the latter animals the Mod-2a gene product is synthesized at a rate 2.2 times that of the Mod-2b-coded subunit. Thus we have unambiguously demonstrated that Mdr-1 is cis-active in its control of the expression of the Mod-2 structural gene.
Placental alkaline phosphatase in nonmalignant human cervix.
Authors: Goldstein D J , Blasco L , Harris H
Published: May 2006

At least three loci determine human alkaline phosphatases [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1]: one coding for the placental form of the enzyme; at least one coding for the intestinal forms; and at least one for the liver, bone, and kidney forms. It is generally believed that the locus determining the placental form is, in the normal individual, expressed only in placenta. However, ectopic or aberrant expression of this locus occurs in certain malignancies of other tissues and in cell lines, such as HeLa, derived from malignancies. We have examined by thermostability, inhibition, and immunologic studies the alkaline phosphatases in endocervix, cervical mucus, and endometrium from nonpregnant women with no evidence of malignancy. It was found that on the average about 18% of the alkaline phosphatase activity in endocervix and in cervical mucus is placental in type, the remainder being of the liver/bone/kidney type. The quantity of the placental enzyme is, however, low and amounts to only about 0.5% of the amount in normal term placenta. In endometrium all the alkaline phosphatase activity was of the liver/bone/kidney type. Thus the placental alkaline phosphatase locus is expressed in nonmalignant endocervix. The result is of some significance in connection with the widely held view that the expression of placental alkaline phosphatase in certain malignancies (including cervical malignancy) is due to derepression of a locus for a fetal enzyme protein normally repressed in adult tissues.
Serological survey of normal humans for natural antibody to cell surface antigens of melanoma.
Authors: Houghton A N , Taormina M C , Ikeda H , Watanabe T , Oettgen H F , Old L J
Published: May 2006

Sera of 106 normal adult men were tested for antibodies reacting with cell surface antigens of three established lines of cultured malignant melanoma. Positive reactions with a protein A assay for IgG antibodies were extremely rare (1-2%). The frequency of positive reactions with assays for IgM antibodies was higher: 5-15% in immune adherence assays and 55-82% in anti-C3 mixed hemadsorption assays. After low-titered sera and sera reacting with fetal calf serum components, conventional alloantigens, and widely distributed class 3 antigens were excluded, sera from seven individuals (one with IgG antibody and six with IgM antibodies) were selected for detailed analysis. The serum containing the IgG antibody came from a healthy 65-year-old Caucasian man; titers of antibody in his serum ranged from < 1/10 to 1/40,000 in tests with different melanoma cell lines. This IgG antibody identifies a differentiation antigen of melanocytes, provisionally designated Mel 1, that distinguishes two classes of melanomas: 22 melanoma cell lines typed Mel 1+ and 17 types Mel 1-. Mel 1 is expressed by fetal fibroblasts but not adult fibroblasts and can be found on a proportion of cultured epithelial cancer cell lines (5 out of 23) but not on glioma or B-cell lines. The melanoma antigens detected by the naturally occurring IgM antibodies are serologically unrelated to Mel 1 but, like Mel 1, appear to be differentiation antigens that distinguish subsets of melanoma. These IgM antibodies detect antigens that are identical or closely related to the AH antigen, a melanoma surface antigen that was initially defined by autologous antibody in a patient with melanoma. In view of the immunogenicity of both Mel 1 and the AH antigens in humans and their occurrence on more than 50% of melanomas, it remains to be seen whether antibody to these antigens can be elicited by specific vaccination of seronegative melanoma patients and whether this will have an influence on the clinical course of the disease.
Partial amino acid sequences of the murine fourth component of complement (C4): demonstration of homology with human C4 and identification of the amino-terminal subunit in pro-C4.
Authors: Parker K L , Shreffler D C , Capra J D
Published: May 2006

Radiolabeled murine C4 and C4 precursor (pro-C4) from cultured peritoneal macrophages were purified by immunoprecipitation and preparative gel electrophoresis. The partial NH2-terminal amino acid sequences of the isolated subunits demonstrated that murine C4 alpha, beta-, and gamma-chains all show sequence homology with the corresponding subunits of human C4; of the 10 residues identified in murine C4, all are identical to those in human C4. These data extend to the primary structural level the homology between the murine serum substance (Ss) protein and human C4. Comparisons of the amino acid sequences of murine pro-C4 and the constituent polypeptide chains of secreted C4 indicate that pro-C4 and the C4 beta-chain have an identical amino acid sequence. Both molecules have lysine at position 1, leucine at positions 4, 5, and 6, and phenylalanine at position 7. No sequence homology was found between pro-C4 and either the alpha- or gamma-subunits. These results define the beta-chain as the NH2-terminal subunit in the C4 precursor molecules.
Resistance of an adenosine kinase-deficient human lymphoblastoid cell line to effects of deoxyadenosine on growth, S-adenosylhomocysteine hydrolase inactivation, and dATP accumulation.
Authors: Hershfield M S , Kredich N M
Published: May 2006

Accumulation of dATP derived from 2'-deoxyadenosine (dAdo), causing inhibition of ribonucleotide reductase and depletion of the other deoxynucleotide substrates required for DNA synthesis, has been suggested as the cause of the lymphopenia and immune defect in inheritable deficiency of adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4). dAdo also inactivates the enzyme S-adenosylhomocysteine hydrolase (AdoHcyase; S-adenosyl-L-homocystein hydrolase EC 3.3.1.1) which is involved in the catabolism of S-adenosyl-L-homocysteine (AdoHcy), both a product and a potent inhibitor of S-adenosylmethionine-dependent transmethylation. We have tried to determine whether inactivation of AdoHcyase might also contribute to dAdo toxicity to adenosine deaminase-inhibited cells. dAdo rapidly inactivates intracellular AdoHcyase and causes the accumulation of AdoHcy in WI-L2 human B lymphoblastoid cells. Low concentrations of adenosine (Ado), which block binding of dAdo to purified AdoHcyase, prevented inactivation of intracellular AdoHcyase and also lessened the growth-inhibitory effect of dAdo. A mutant of this cell line which lacks Ado kinase and accumulated endogenously synthesized Ado was resistant to the effects of dAdo on both growth and AdoHcyase activity. The mutant also accumulated far less dATP from dAdo than did its parent and was resistant to the inhibitory effect of dAdo on DNA synthesis, indicating the Ado kinase is involved in dAdo phosphorylation in these cells. Combinations of deoxycytidine, thymidine, and deoxyguanosine that could prevent dATP-mediated depletion of deoxynucleotide pools but not AdoHcyase inactivation were less effective than Ado in preventing dAdo toxicity to normal lymphoblasts. Our results suggest that inactivation of AdoHcyase, as well as dATP accumulation, contributes to dAdo toxicity.
In vitro synthesis of repressible yeast acid phosphatase: identification of multiple mRNAs and products.
Authors: Bostian K A , Lemire J M , Cannon L E , Halvorson H O
Published: May 2006

Antibodies to repressible nonspecific acid phosphatase [APase; orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2] purified from Saccharomyces cerevisiae were used to detect the in vitro products of APase mRNA. Immunoprecipitation of cell-free synthesized protein and of in vivo enzyme from cell extracts has shown that derepression of enzyme synthesis in situ is the result of de novo appearance of functional mRNA followed by de novo protein synthesis. At least three unique APase polypeptides are synthesized in vitro from separate mRNAs and appear to be glycosylated in vivo to form secreted enzyme.
Fungal oxidation of (+/-)-9,10-dihydroxy-9,10-dihydrobenzo[a]pyrene: formation of diastereomeric benzo[a]pyrene 9,10-diol 7,8-epoxides.
Authors: Cerniglia C E , Gibson D T
Published: May 2006

The filamentous fungus Cunninghamella elegans oxidized (+/-) trans-9,10-dihydroxy-9,10-dihydrobenzo[a]-pyrene to a complex mixture of metabolites which were detected by high-pressure liquid chromatography. Two of the metabolites were identified as (+/-)7 beta, 8 alpha, 9 alpha, 10 beta-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene and (+/-)-7 beta,8 alpha,9 beta,10 alpha-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene. A third product gave absorption and mass spectra consistent with a diol-epoxide structure. Hydrolysis of this compound gave (+/-)-7 beta, 8 alpha, 9 beta, 10 alpha-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene as the major identifiable product with a minor unidentified tetraol. Synthetic (+/-)-9 alpha, 10 beta-dihydroxy-7 beta, 8 beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene gave the same hydrolysis products and had the same retention time on high-pressure liquid chromatography as did the fungal metabolite. The trans-9,10-dihydroxy-9,10-dihydrobenzo[a]pyrene recovered at the end of the experiment showed no optical activity, indicating that both enantiomers were metabolized by the fungus. the results suggest that C. elegans oxidizes (+/-)-trans-9,10-dihydroxy-9,10-dihydrobenzo[a]pyrene to diastereomeric benzo[a]pyrene 9,10-diol 7,8-epoxides.
Elongation mechanism and substrate specificity of 2',5'-oligoadenylate synthetase.
Authors: Justesen J , Ferbus D , Thang M N
Published: May 2006

2',5'-Oligoadenylate synthetase has been purified from a rabbit reticulocyte lysate to a high degree of purity. The enzyme contained no detectable interfering activities that could degrade the nucleoside triphosphate substrate or the oligomeric products. Two basic properties of this enzyme have been examined: the elongation mechanism for the synthesis of oligoadenylates and the substrate specificity for nucleotides. Kinetic studies on the formation of different oligomeric intermediates show that the dimer pppA2'p5'A is the first product to accumulate in predominant proportion during the first period of reaction; the trimer and other longer oligomers appear after a lag phase. The amount of the trimer increases at the expense of the dimer. Preformed dimers and trimers added to the incubation mixture were readily incorporated into higher oligomers, suggesting the free access of these dimers and trimers to the active center after the onset of polymerization of ATP. The results indicate clearly that the enzyme catalyzes the de novo synthesis of the oligonucleotide from ATP and that the mechanism of elongation of the 2',5'-oligonucleotides catalyzed by the enzyme is not processive. Polymerization of a mixture of ATP and another nucleoside triphosphate shows that the enzyme is not only an ATP polymerase. The 2',5'-oligoadenylate synthetase is in fact a 2',5'-nucleotidyltransferase that catalyzes the formation of co-oligonucleotides. However, the purified reticulocyte enzyme catalyzed only the addition of one unit of GMP, UMP, CMP, 2'-dAMP, 3'-dAMP, dCMP, dGMP, or TMP to the 2'-OH end of a preformed oligoadenylate. A procedure for the separation of 2',5'-oligonucleotides with or without the 5'triphosphate end also is described.
Fidelity of structure representation in electron micrographs of negatively stained protein molecules.
Authors: Steven A C , Navia M A
Published: May 2006

We have investigated the fidelity of structure representation in electron micrographs of negatively stained proteins by conducting a systematic evaluation of such micrographs in terms of a known molecular structure, solved by x-ray crystallography. Microcrystals of immunoglobulin G Dob were used as specimens in this comparison between micrograph images, optimized by computer image processing, and reference images derived computationally from the crystal structure. To an effective resolution of 2 nm, we observed a remarkably good correlation between the experimental images and their idealized counterparts, which are unaffected by those factors--electron irradiation and dehydration--that are thought to be primarily responsible for perturbation of protein structure during electron microscopy. Separate structural features resolved in these micrographs do not, in general, correspond to specific components of individual molecules but arise instead from complex superpositions involving several overlapping molecules.
Inhibition of Ca2+ accumulation in mitogen-activated lymphocytes: role of membrane-bound plasma lipoproteins.
Authors: Hui D Y , Harmony J A
Published: May 2006

Low density lipoproteins (LDL) in which apoB is the only protein constituent inhibit lymphocyte activation induced by the mitogen phytohemagglutinin. LDL suppress primary induction events, most notably enhanced Ca2+ accumulation, which occur as a result of mitogen-triggered alterations of the lymphocyte plasma membrane. Receptors in the lymphocyte membrane--designated "immunoregulatory receptors"--recognize and bind apoB-LDL. Under conditions of receptor saturation, about 22,500 +/- 4500 LDL particles are bound to the lymphocyte surface at 4 and 37 degrees C. The dissociation constant, Kd, is 1.76 X 10(-7) M and is also independent of temperature. Suppression of phytohemagglutinin-induced 45Ca2+ accumulation by LDL is a receptor-dependent process: inhibition is contingent on attachment of LDL to the immunoregulatory receptors. This conclusion is supported by three lines of evidence. First, heparin restores the susceptibility of the lymphocytes to mitogenic challenge by displacing membrane-bound LDL. Second, the immnoregulatory potency of LDL correlates directly with the amount of LDL bound at the cell surface. Third, LDL-Sepharose complexes, which cannot be internalized by the cells, are as immunosuppressive as soluble LDL. This demonstrates that membrane-bound plasma lipoproteins influence the behavior of intact cells.
Linkage of alpha G-Philadelphia to alpha-thalassemia in African-Americans .
Authors: Surrey S , Ohene-Frempong K , Rappaport E , Atwater J , Schwartz E
Published: May 2006

We have studied the inheritance of the alpha-chain hemoglobin variant Hb G-Philadelphia (alpha 2(68 Asn leads to Lys)Beta 2) in two African-American families. Expression of the alpha-globin loci was monitored by the percentage of Hb G in these individuals. The variant represented approximately 33% of the total adult hemoglobin in some and 50% in others. alpha-Globin gene fragments were analyzed by using restricton endonucleases that cleave outside (EcoRI), within (HindIII), and between (Bgl II) the normal duplicated alpha-globin loci (alpha alpha/alpha alpha). Individuals having 33% variant lack one functioning alpha gene (alpha G/alpha alpha); those with 50% variant lack two genes, one missing on each chromosome (alpha G/alpha). Inheritance of alpha G was therefore linked to that of a chromosome with only one functional alpha-globin gene locus. This locus is probably the result of a nonhomologous crossover. Our results also suggest equal expression of the alpha-globin loci in humans because the percentages of the variant could be explained solely on the basis of the total number of alpha genes present. The percentages of Hb G as well as other hematologic data all were consistent with the number of alpha-globin genes identified by restriction endonuclease mapping. Gene mapping yields a more precise determination of the number of alpha-globin genes than does study of globin synthesis.
Partial sequence of human complement component factor B: novel type of serine protease.
Authors: Christie D L , Gagnon J , Porter R R
Published: May 2006

Factor B (a component of the alternative pathway of complement) is believed to contain the proteolytic site of the complex enzymes C3 convertase (C3bB) and C5 convertase (C3bnB). Conflicting results have been obtained in regard to the inactivation of these enzymes by diisopropyl phosphorofluoridate but it has been suggested that activated Factor B (Factor B) is a serine protease with the active site in Bb, a COOH-terminal fragment of approximately 60,000 molecular weight. Partial amino acid sequence studies of Bb derived from human Factor B have shown that the NH2-terminal 40 residues have no homology with NH2-terminal sequences of other serine proteases. However, positioning of a further 170 residues out of approximately 290 residues in two continuous CNBr fragments from the COOH terminus has shown that there is a strong homology of sequence in this section. The active site residues histidine, aspartic acid, and serine all are present in positions corresponding with those of typical serine proteases. It is suggested that Factor B is a novel type of serine protease with a catalytic chain of molecular weight twice that of proteases previously studied and probably with a different activation mechanism.
Efficient genetically controlled formation of antibody to a synthetic antigen [poly(LTyr, LGlu)-poly(DLAla)- -poly(LLys)] covalently bound to a synthetic adjuvant (N-acetylmuramyl-L-alanyl-D-isoglutamine).
Authors: Mozes E , Sela M , Chedid L
Published: May 2006

The synthetic polypeptide antigen poly(LTyr, LGlu)-poly(DLAl)- -poly(LLys)[T,G)-A- -L] was covalently linked to N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), which is the minimal adjuvant-active structure that can substitute for Mycobacteria in complete Freund's adjuvant. When injected in aqueous solution into mice, the completely synthetic conjugate elicited significant antibody responses specific to (T,G)-A- -L, whereas (T,G,)-A- -L alone administered under the same conditions did not lead to antibody production. The conjugate was much more efficient in eliciting (T,G)-A- -L responses than was a mixture of DMP and (T,G)-A- -L. One hundred micrograms of MDP mixed with 10 micrograms of (T,G)-A- -L resulted in production of (T,g)-A- -L-specific antibodies. However, the titers obtained were much lower than those observed with 10 micrograms of the conjugate, MDP-(T,G)-A- -L, which contained less than 1 microgram of MDP. MDP was enhanced when the mixture was administered in incomplete Freund's adjuvant, the adjuvant did not significantly affect the (T,G)-A- -L-specific antibody responses in mice immunized with MDP-(T,G)-A- -L. The isoelectric focusing pattern of antibodies obtained with MDP-(T,G)-A- -L was similar to that obtained after immunization with (T,G)-A- -L in complete Freund's adjuvant. The pattern of high-responder and low-responder mice to (T,G)-A- -L, the immune response to which is genetically controlled, was retained when MDP-(T,G)-A- -L was used as the immunogen. Conjugation of (T,G)-A- -L was creased the immunogenicity of MDP and affected its biological properties. It is thus possible to obtain efficient immune responses to synthetic polypeptide antigens that produce poor reactions when injected in aqueous solution by conjugating them to small molecular weight synthetic adjuvants.
Active site amino acid sequence of human factor D.
Authors: Davis A E
Published: May 2006

Factor D was isolated from human plasma by chromatography on CM-Sephadex C50, Sephadex G-75, and hydroxylapatite. Digestion of reduced, S-carboxymethylated factor D with cyanogen bromide resulted in three peptides which were isolated by chromatography on Sephadex G-75 (superfine) equilibrated in 20% formic acid. NH2-Terminal sequences were determined by automated Edman degradation with a Beckman 890C sequencer using a 0.1 M Quadrol program. The smallest peptide (CNBr III) consisted of the NH2-terminal 14 amino acids. The other two peptides had molecular weights of 17,000 (CNBr I) and 7000 (CNBr II). Overlap of the NH2-terminal sequence of factor D with the NH2-terminal sequence of CNBr I established the order of the peptides. The NH2-terminal 53 residues of factor D are somewhat more homologous with the group-specific protease of rat intestine than with other serine proteases. The NH2-terminal sequence of CNBr II revealed the active site serine of factor D. The typical serine protease active site sequence (Gly-Asp-Ser-Gly-Gly-Pro was found at residues 12-17. The region surrounding the active site serine does not appear to be more highly homologous with any one of the other serine proteases. The structural data obtained point out the similarities between factor D and the other proteases. However, complete definition of the degree of relationship between factor D and other proteases will require determination of the remainder of the primary structure.
Isolation of BPgp70, a fibroblast receptor for the envelope antigen of Rauscher murine leukemia virus.
Authors: Landen B , Fox C F
Published: May 2006

A protein that avidly binds gp70, the envelope antigen of Rauscher murine leukemia virus (RMuLV), has been purified from the culture medium used for growth of BALB/c 3T3 mouse cells. Gel filtration chromatrography revealed the apparent Mr 10,000 BPgp70 was efficiently labeled when BALB/c 3T3 cells were grown in medium containing [3H]leucine, indicating a cellular origin for BPpg70. Metabolically labeled [3H]BPgp70 was not immunoprecipitated by IgG-anti RMuLV-gp&) alone, but was immunoprecipitated when gp70 was added, an indicaton of BPgp70 x gp70 complex formation. The dissociation constant estimated by immunoprecitipation agreed with the apparent Kd for binding of gp70 to BALB/c 3T3 cells. BPgp70 reversibly inhibited specific binding of 125I-labeled BMuLV-gp70 to BALB/c 3T3 cells when it was incubated with the 125I-labeled gp70 first. These data yielded a dissociation constant similar to that calculated from the immunoprecipitation data. 125I-Labeled BPgp70 also bound specifically to cells infected with RMuLV, but not to uninfected cells. Incubation of BALB/c 3T3 cells with the IgG fraction of an antiserum to BPgp70 inhibited the specific binding of 125I-labeled gp70 to these cells, but preimmune IgG did not. Complete inhibition was achieved at a less than 100:1 ratio of IgG anti-BPgp70 to gp70 binding sites.
An attempt to distinguish between the actions of neuromuscular blocking drugs on the acetylcholine receptor and on its associated ionic channel
Authors: Lambert Jeremy J. , Volle Robert L. , Henderson Edward G.
Published: May 2006

The effects of lobeline and tubocurarine on the voltage-clamped endplates of frog sartorius and cutaneous pectoris muscles were examined at room temperature (20-23°C). Like tubocurarine, lobeline causes nondepolarizing neuromuscular blockade. The half-time of decay (
t
½
) of endplate currents (e.p.c.s) recorded at a holding potential (
V
m
) of -90 mV was significantly shorter in endplates treated with lobeline (50 μM; mean
t
½
± SEM = 0.41 ± 0.02 ms) or tubocurarine (11.4 μM;
t
½
= 0.64 ± 0.04 ms) than in those treated with Mg
2+
(13 mM;
t
½
= 1.39 ± 0.11 ms) or a low concentration of tubocurarine (3 μM;
t
½
= 0.87 ± 0.05 ms). Similarly, lobeline (10 μM) shortened the
t
½
of untreated miniature e.p.c.s by 35%; tubocurarine, however, abolished miniature e.p.c.s at the concentration required to observe its actions on e.p.c. decay kinetics. The
t
½
of e.p.c.s recorded from preparations treated with Mg
2+
(13 mM), tubocurarine at low concentrations (3 μM), or untreated miniature e.p.c.s was logarithmically related to
V
m
, being slower at more hyperpolarized values. By contrast, the
t
½
s of e.p.c.s recorded in either lobeline (50 μM) or tubocurarine (11.4 μM) were independent of voltage in the range -150 to -80 mV. The ability of lobeline to shorten
t
½
and to remove the voltage dependence of
t
½
was partially antagonized by Mg
2+
(13 mM). As expected, when lobeline or tubocurarine was removed from the bath or when acetylcholine release from the motor nerve terminals was increased by 4-aminopyridine (20 μM) and Ca
2+
(10 mM) (in the presence of lobeline or tubocurarine), the amplitude of e.p.c.s increased as a function of time. However, the
t
½
of the decay phase of the e.p.c.s remained shortened (i.e., unaltered from the earlier treatment). These results suggest that both tubocurarine and lobeline have at least two distinct postjunctional actions including: (
i
) a block of the acetylcholine receptor and (
ii
) a block of the ionic channel associated with the acetylcholine receptor.
Relationship between transfer activity coefficients,

S
1

γ

S
2

, of cryptate 2.2.2 complexes and their stability constant in various solvents
Authors: Kolthoff Izaak M. , Chantooni Miran K.
Published: May 2006

Between dipolar aprotic solvents S
1
and S
2
, the transfer activity coefficients,

S
1

γ

S
2

, of complexed sodium, potassium, thallium(I), and silver ions with cryptand 2.2.2 have been found to be equal to that of the cryptand. With reference to methanol (S
1
), this equality does not hold, the cryptates being more or less solvated by hydrogen bonding in this solvent. In water as a solvent, the cryptates are on a considerably higher level of free energy than in the organic solvents. This is attributed to hydrophobic hydration of the cryptates.
Helper virus is not required for
in vitro
erythroid transformation of hematopoietic cells by Friend virus
Authors: Hankins W. David , Krantz S. B.
Published: May 2006

The Friend polycythemia virus complex (FVP), consisting of the replication-defective spleen focus-forming virus (SFFV) and a helper Friend murine leukemia virus (MuLV-F), produces erythroleukemia within 2-3 weeks
in vivo
. We have recently reported
in vitro
transformation of bone marrow cells by FVP, producing clusters of erythroid colonies (erythroid bursts) 4-6 days after infection. In contrast to uninfected bone marrow cells, FVP-treated cells proliferated and differentiated (synthesized hemoglobin) in the absence of added erythropoietin, the physiologic regulator of erythropoiesis. The relative roles of helper murine leukemia virus (MuLV) and SFFV in the
in vitro
erythroid transformation have now been examined. Pseudotype studies and the finding that cloned MuLV-F (free of SFFV) did not induce burst formation indicated that SFFV was essential for this
in vitro
effect of FVP. Because SFFV could not be obtained free of helper MuLV, we assessed the requirement of MuLV in the transformation by kinetic analyses of helper-deficient and helper-excess FVP preparations. Whereas helper-excess FVP gave single-hit kinetics both
in vivo
and
in vitro
, the helper-deficient FVP followed multiple-hit kinetics when titrated for spleen focus formation
in vivo
. Addition of MuLV-F to helper-deficient FVP prior to injection resulted in a marked enhancement of spleen focus formation and a conversion from multiple-hit to single-hit kinetics. In contrast, titration of this same preparation for erythroid burst transformation
in vitro
yielded single-hit kinetics, and the addition of helper MuLV-F had no effect. The time course of burst development was similar with or without added MuLV-F. Unlike burst transformation, SFFV production by these infected cultures followed multiple-hit kinetics. Addition of MuLV-F at the time of infection led to an enhancement of SFFV production and conversion of the titration curve from multiple-hit to single-hit. These data are consistent with the idea that SFFV is competent for erythroid transformation
in vitro
, but requires helper MuLV for its replication.
Messenger RNA prevalence in sea urchin embryos measured with cloned cDNAs.
Authors: Lasky L A , Lev Z , Xin J H , Britten R J , Davidson E H
Published: May 2006

mRNA prevalence during sea urchin development was measured by treating cDNA clone colonies with labeled cDNAs transcribed from unfertilized egg and embryo poly(A)-RNAs. The number of cytoplasmic transcripts per embryo complementary to several clones was determined independently by titration with poly(A)-RNA in solution, and the amount of cDNA bound to these clones in colony hybridizations was shown to be proportional to the concentration of the respective poly(A)-RNAs in the embryo cytoplasm. At the gastrula stage, the most prevalent mRNA species occur in about 10(6) molecules per embryo. If all cells were equivalent, this would be a few hundred molecules per cell. By pluteus stage, the prevalence of some sequences has increased more than 10-fold. Most, though not all, sequences prevalent in later embryos are also present in the maternal RNA of the unfertilized egg. For most poly(A)-RNA sequences, the prevalence levels determined during oogenesis are maintained through the pluteus stage, whereas a minority of sequences display sharp stage-specific changes in representation during development.
Overlapping genes at the cheA locus of Escherichia coli.
Authors: Smith R A , Parkinson J S
Published: May 2006

The cheA locus of Escherichia coli, which is required for chemotactic behavior, encodes two polypeptide products designated p[cheA]L and p[cheA]S. The mode of synthesis of these two proteins was investigated by transferring various missense and nonsense mutations to a lambda transducing phage and observing the mutant cheA products made after infection of ultraviolet-irradiated host cells. Missense mutations had no effect on either the size or the relative amounts of the two cheA polypeptides. Most nonsense mutations caused premature translational termination of both cheA products, indicating that p[cheA]L and p[cheA]S must be translated from the same coding sequence in the same reading frame. Two exceptional nonsense alleles at the promoter-proximal end of cheA made an intact p[cheA]s but no detectable p[cheA]L. These findings show that the cheA locus may contain two different sites for initiation of translation. The synthesis of both proteins can be effected by the same promoter, but it is not yet clear whether both are translated from identical mRNA molecules. Complementation studies of cheA mutants provided evidence for two functional activities, one associated with the amino terminus of p[cheA]L and the other with the common portions of p[cheA]L and p[cheA]S. It is possible that each cheA product has a different function required for chemotaxis. The possible roles of these two products and the functional significance of bacterial genes with overlapping coding sequences are discussed.
Segment-directed mutagenesis: construction in vitro of point mutations limited to a small predetermined region of a circular DNA molecule.
Authors: Shortle D , Koshland D , Weinstock G M , Botstein D
Published: May 2006

A general method for efficiently mutagenizing a predetermined segment of a closed circular duplex DNA molecule was used to construct mutations in two specific regions of the beta-lactamase (bla) gene carried by the small plasmid pBR322. The principle of segment-directed mutagenesis is the use of a single-stranded homologous DNA fragment to direct the nicking of circular duplex DNA within a segment defined by the DNA fragment in a two-step reaction. First, Escherichia coli recA protein is used to catalyze assimilation of the homologous single-stranded DNA, producing a displacement loop ("D-loop") in the circular DNA. Second, a small amount of the single-strand-specific S1 nuclease is used to nick the displaced DNA. The segment-directed nicks are converted to small gaps, which are then mutagenized specifically with sodium bisulfite. A short (128-base pair) restriction endonuclease fragment from the center of the bla gene was used to direct mutagenesis with the result that 7.5% of the recovered plasmids were bla- mutants and 49/51 of these mutants, mapped genetically, were found to lie in a deletion interval whose endpoints approximate those of the restriction fragment. Similar results were obtained when another short fragment covering the beginning of the gene was used; many of these mutations map in the region coding the "signal" sequence thought to be involved in secretion of beta-lactamase.
Syngeneic tumor cells can induce alloreactive T killer cells: a biological role for transplantation antigens.
Authors: Schirrmacher V , Hübsch D , Garrido F
Published: May 2006

A chemically induced sarcoma of BALB/c (H-2d) mice, MCG4, is shown to induce in BALB/c lymphocytes a primary anti-tumor cytolytic T lymphocyte (CTL) reaction in vitro. The anti-tumor CTL showed tumor specificity but reacted also with normal cells expressing distinct H-2 alloantigens. The CTL response could be shown to be induced by and directed against alloantigenic determinants expressed on two different molecules, one H-2Kk-like the other H-2Dk-like. The biological significance of these findings is discussed with regard to (i) possibility of derepression of normally silent H-2 genes in tumor cells and normal cells, (ii) generation of alloreactivity in ontogeny, and (iii) role of alloreactive T cells in eliminating cells expressing wrong gH-2 antigens.
In vitro autoradiography of opiate receptors in rat brain suggests loci of "opiatergic" pathways.
Authors: Herkenham M , Pert C B
Published: May 2006

Slide-mounted sections of unfixed frozen rat brain can be labeled in vitro with [3H]naloxone to show the mu-like ligand selectivity characterized in previous studies. We have developed an autoradiographic technique using hot paraformaldehyde vapors to prevent diffusion of ligands with reversible binding. Resolution at the light level is sufficient to detect concordance between receptor patterns and terminal fields of axonal projections marked by tract-tracing techniques. The opiate receptor distribution suggests the existence of widespread intrinsic and several longer multisynaptic "opiatergic pathways within sensory and limbic circuits. One multisynaptic pathway may link olfactory structures with limbic circuits in the amygdala and habenula. Another may lie in limbic cortical structures. Opiate receptors are numerous also in sensory systems, and within primary sensory nuclei (visual, auditory, olfactory, somatic) they are found superficially in laminated structures. Together, the opiate receptors are well placed to control incoming sensory and subsequent limbic information processing.
Mechanism of mRNA capping by vaccinia virus guanylyltransferase: characterization of an enzyme--guanylate intermediate.
Authors: Shuman S , Hurwitz J
Published: May 2006

Vaccinia virus RNA guanylyltransferase catalyzes the transfer of GMP from GTP to the 5'-triphosphate or diphosphate terminus of RNA to generate the cap structure G(5')ppp(5')N-. The guanylylation reaction consists of a series of at least two partial reactions: (i) GTP + E in equilibrium E-pG + PPi, (ii) E-pG + (p)ppNpNpN- leads to GpppNpNpN- + E. Inthe first of these, GTP reacts with capping enzyme in the absence of an RNA acceptor to form a covalent enzyme-guanylate intermediate. The GMP is linked to the Mr 95,000 subunit of the capping enzyme via a phosphoamide bond, as judged by the acid-labile, alkali-stable nature of the bond and by the susceptibility of the linkage to cleavage by hydroxylamine at pH 4.75. The isolated enzyme-guanylate complex is able to transfer the guanylate moiety to triphosphate-terminated poly(A) to yield the 5' cap structure GpppA or to pyrophosphate to regenerate GTP. Both partial reactions of transguanylylation require a divalent cation.
Membrane potential changes during the first steps of coliphage infection.
Authors: Labedan B , Letellier L
Published: May 2006

Immediately after adsorption, phages T4 and T5 induce a partial depolarization of the host cytoplasmic membrane. Infected bacteria respond to this phage-induced effect by a repolarization that leads to a new steady state of reduced membrane potential. The rate and extent of repolarization are adjusted to the intensity of depolarization, which depends on the number of adsorbed phages. Consequently, the new steady state membrane potential is attained in the same interval of time regardless of the maximum depolarization. These membrane potential changes appear to be independent of phage-specific properties (type of phage, presence of DNA and internal proteins, injection process) and of several membrane-related parameters (temperature, external pH, preinfectious level of membrane potential). We propose that phage adsorption to the outer membrane triggers the emission of a signal that is transmitted to the cytoplasmic membrane. Additivity of independent signals is possible when stimuli (phages) are added at the same time. Additional adsorption of phages has no further depolarizing effect as soon as the repolarization begins. We propose that this refractoriness to secondary depolarization nd the shut-off of the first depolarization are induced by the same chemical modification also initiated by adsorption of the first phage.
Colloidal gold--low density lipoprotein conjugates as membrane receptor probes.
Authors: Handley D A , Arbeeny C M , Witte L D , Chien S
Published: May 2006

We have developed a method for conjugating low density lipoproteins (LDL) with colloidal gold. Conjugation, complete after 1 min, occurs by electrostatic adsorption of the LDL to the negatively charged gold particle. Each conjugate consists of approximately eight biologically active LDL molecules clustered around a central 19-nm gold granule. Acidic (pH 4), alkaline (pH 9), or high ionic (600 milliosmolar NaCl) environments do not dissociate the conjugate. Colloidal gold is an electron-dense, nondegradable marker that is easily identified within the cell and serves as a valuable probe for studying receptor binding and endocytosis. By using a modified method of ruthenium red staining, the LDL molecules of the conjugate can be directly visualized when they are bound to the cell surface receptor. Receptor binding (4 degrees C) of the conjugate by cultured human fibroblasts reveals that the gold granule is positioned 18-21 nm from the coated pit region of the membrane. This distance, similar to the diameter of LDL, suggests concomitant internalization of the receptor during vesicular endocytosis and early lysosomal incorporation (10 min at 37 degrees C). Continued internalization (30-60 min at 37 degrees C) results in the formation of free pools of gold within the lysosome.
Excision of transposon Tn5 is dependent on the inverted repeats but not on the transposase function of Tn5.
Authors: Egner C , Berg D E
Published: May 2006

The excision of Tn5 from sites of insertion in the Escherichia coli genome was studied by examining the reversion of lac::Tn5 insertion mutations to lac+. We find that: (i) the frequency of excision depends on the site of Tn5 insertion, (ii) excision occurs efficiently in recA- cells, (iii) excision does not require a Tn5-encoded transposition function, and (iv) efficient excision requires the inverted repeats of Tn5. We propose that excision of Tn5 is similar to the formation of spontaneous deletions and occurs by slippage during DNA synthesis.