bluuebunny/crossref_metadata_2025 huggingface dataset:
ATP-driven sodium pump in Streptococcus faecalis.
Authors: Heefner D L , Harold F M
Published: May 2006

Sodium extrusion by bacteria is generally attributed to secondary antiport of Na+ for H+ energized by the proton circulation. Streptococcus faecalis is an exception, in that sodium expulsion from intact cells requires the generation of ATP but does not depend on the protonmotive force. Unfortunately, studies with everted membrane vesicles failed to reveal the expected sodium pump; instead, the vesicles contained a conventional secondary Na+/H+ antiporter. We report here that everted membrane vesicles prepared in the presence of protease inhibitors retain an ATP-driven sodium transport system. The evidence includes the findings that (i) accumulation of 22Na+ by these vesicles is resistant to reagents that dissipate the protonmotive force but requires ATP and (ii) the vesicles contain a sodium-stimulated ATPase that is distinct from F1F0 ATPase, and whose presence is correlated with sodium transport activity. Sodium movements appear to be electroneutral and are accompanied by movement of H+ in the opposite direction. When membranes are incubated in the absence of protease inhibitors, a secondary Na+/H+ antiport activity emerges, possibly by degradation of the sodium pump. We suggest that S. faecalis expels Na+ by means of an ATP-driven primary transport system that mediates exchange of Na+ for H+. The Na+/H+ antiporter seen in earlier membrane preparation is an artefact of proteolytic degradation.
Staphylococcal plasmids that replicate and express erythromycin resistance in both Streptococcus pneumoniae and Escherichia coli.
Authors: Barany F , Boeke J D , Tomasz A
Published: May 2006

Plasmid pSA5700 from Staphylococcus aureus coding for erythromycin (EmR) and chloramphenicol (CmR) resistance was transformed into Streptococcus pneumoniae. High-copy-number and EmR constitutive mutants of this plasmid were isolated. Transformation frequencies in S. pneumoniae as high as 70% were obtained with a constitutive plasmid as donor DNA, into a recipient cell containing a resident, inducible, high-copy-number plasmid. With the aid of these high frequencies, the site of constitutive mutations could be mapped via a simple marker rescue technique that uses purified restriction endonuclease-generated fragments. One of the EmR constitutive mutants, pFB9, a plasmid originating from a Gram-positive host, was shown to replicate and express EmR and CmR in a Gram-negative organism, Escherichia coli. Four derivatives of pFB9 containing large (0.6-0.9 megadalton) insertion sequences that arose spontaneously in E. coli demonstrated unusual transforming activity, as well as enhanced EmR, in E. coli. The inserted elements mapped to the region in front of the EmR gene. Three of these inserted elements had the size and restriction patterns of insertion sequence IS1, IS2, and IS5. Plasmid pFB9 and derivatives are useful for isolation of new insertion sequences and for comparison of gene expression and illegitimate recombination between Gram-positive and Gram-negative species.
Immunoglobulin heavy chain gene rearrangement and transcription in murine T cell hybrids and T lymphomas.
Authors: Züñiga M C , D'Eustachio P , Ruddle N H
Published: May 2006

We have examined the arrangement of immunoglobulin heavy chain constant (CH) and joining (JH) region genes in murine T cell hybrid lines and in T lymphomas. CH genes derived from both parental cell types were present in all hybrids for which polymorphism in sequences flanking CH genes permitted us to distinguish parental CH genes. All T lymphomas and T cell hybrids retained the C alpha gene in germ-line configuration and all but one cell line had germ-line C mu genes. Novel DNA fragments reactive with JH probes were observed in six of nine T cell hybrids, as well as in two T lymphomas, WEHI7.1 and YAC-1, but not in the fusion parent, BW5147. No RNA homologous to C gamma 2b, C alpha, or lambda genes was detected in any of the T cell lines. T cell lines contained poly(A)+ RNA homologous to a C mu cDNA probe. More importantly, in several cell lines the C mu RNAs were associated with membrane-bound polyribosomes. These results suggest that both JH rearrangements and C mu RNA production occur in at least some mature, antigen-specific T cells. They may therefore reflect events in normal T cell development and function related to those involved in the generation of the T receptor for antigen.
Developmental regulation of mRNA in mouse heart.
Authors: Ouellette A J , Croall D E , Van Ness J , Ingwall J S
Published: May 2006

The myocardium contains abundant translatable mRNAs that change during development. Maximal cell-free synthesis of [3H]leucine-, [35S]methionine-, and [35S]cysteine-labeled translation products directed by poly(A)-containing mRNAs from 12-, 14-, and 17-day fetal; 5-day-old neonatal; and 30-day-old adult mouse heart was determined by using one- and two-dimensional polyacrylamide gels. Three general developmental patterns of heart-specific mRNA translation products were observed: two translatable mRNAs were most abundant in 12-day fetal heart; five mRNAs were most abundant in 14- and 17-day fetal heart and occurred only at low concentrations in 12-day fetal and adult heart; four mRNAs, including mRNAs coding for actin, tropomyosin, and myosin light chains 1 and 2, were most abundant in the adult heart. Thus, differentiating cardiac muscle is characterized by a complex pattern of mRNA regulation.
Identification and partial characterization of the secreted form of the fourth component of human complement: evidence that it is different from major plasma form.
Authors: Chan A C , Mitchell K R , Munns T W , Karp D R , Atkinson J P
Published: May 2006

Immunoprecipitation of human C4 from plasma followed by NaDodSO4/polyacrylamide gel electrophoresis under reducing conditions revealed the expected alpha, beta, and gamma chains as well as a smaller quantity of a molecule containing an alpha chain (p98) approximately equal to 5,000 daltons heavier than the normal alpha chain. Further studies on p98 indicated that it covalently incorporated methyl-amine, was present at a concentration of approximately equal to 8% of the principal plasma form of the C4 alpha chain, and was found in highly purified C4 preparations. Hep G2, a human hepatoma-derived cell line, was found to secrete a C4 molecule in which the alpha chain had a molecular weight identical to that of the p98 protein found in plasma. The secreted C4 molecule possessed hemolytic activity. The 5,000-dalton difference in the alpha chain was localized to the COOH terminus and was attributed to an additional polypeptide. We propose that p98 is the alpha chain of the secreted form of C4, which is processed extracellularly by proteolytic cleavage to the principal C4 molecule found in plasma.
Competitive advantage of diferric transferrin in delivering iron to reticulocytes.
Authors: Huebers H A , Csiba E , Huebers E , Finch C A
Published: May 2006

Radioiron- and radioiodine-labeled forms of human diferric and monoferric transferrin and apotransferrin, isolated by preparative isoelectric focusing, were used to define transferrin-iron uptake by human reticulocytes. In mixtures of human diferric and monoferric transferrin, the diferric molecule had a constant 7-fold advantage in delivering iron to reticulocytes, as compared with the 2-fold advantage when single solutions of mono- and diferric transferrins were compared. This was shown to be due to competitive interaction in iron delivery, probably at a common membrane-receptor binding site for transferrin. Apotransferrin did not interfere with the iron-donating process and its limited cellular uptake was inhibited in noncompetitive fashion by diferric transferrin.
Chromatographic resolution and kinetic characterization of glucokinase from islets of Langerhans.
Authors: Meglasson M D , Burch P T , Berner D K , Najafi H , Vogin A P , Matschinsky F M
Published: May 2006

Glucokinase (ATP:D-glucose 6-phosphotransferase, EC 2.7.1.2) from rat islets of Langerhans was partially purified by chromatography on DEAE-Cibacron blue F3GA agarose. The enzyme eluted in two separate peaks. Sigmoidal rate dependence was found with respect to glucose (Hill coefficient = 1.5) for both enzyme fractions. Km values for glucose were 5.7 mM for the major fraction and 4.5 mM for the minor fraction. Neither fraction phosphorylated GlcNAc. A GlcNAc kinase (ATP:2-acetamido-2-deoxy-D-glucose 6-phosphotransferase, EC 2.7.1.59)-enriched fraction, prepared by affinity chromatography on Sepharose-N-(6-aminohexanoyl)-GlcNAc, had a Km of 25 microM for GlcNAc. Islet tissue also contained hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) eluting in multiple peaks. The results are consistent with the concept that glucokinase serves as the glucose sensor of pancreatic beta cells.
Diversity and joining segments of mouse immunoglobulin heavy chain genes are closely linked and in the same orientation: implications for the joining mechanism.
Authors: Wood C , Tonegawa S
Published: May 2006

We have mapped 12 diversity (D) gene segments of mouse Ig heavy chains between 80 and 1 kilobases 5' to the four joining (JH) gene segments by molecular analysis of DNA clones isolated from recombinant phage and cosmid libraries. All identified D and JH segments are in the same 5'-3' orientation. The significance of these findings with respect to the joining mechanism for Ig gene segments is discussed.
Structural bases for public idiotypic specificities of monoclonal antibodies directed against poly(Glu60Ala30Tyr10) and poly(Glu60Ala40) random copolymers.
Authors: Ruf J , Tonnelle C , Rocca-Serra J , Moinier D , Pierres M , Ju S T , Dorf M E , Thèze J , Fougereau M
Published: May 2006

NH2-terminal amino acid sequences of heavy and light chains of seven poly(Glu60Ala30Tyr10) (GAT) specific hybridoma products derived from DBA/2 and (DBA/2 X BALB/c)F1 hybrid mice and those of BALB/B polyclonal antibodies have been determined over the first 40 residues. Comparison of these sequences with those of nine other GAT or poly(Glu60Ala40) (GA) specific hybridoma products previously reported allowed the following conclusions. (i) Sequences of hybridoma H and L chains are present in the pool of polyclonal antibodies. (ii) The public CGAT (or pGAT) idiotypic specificities are strictly confined to antibodies exhibiting limited heterogeneity with regard to both the variable heavy (VH) and the variable kappa (V kappa) sequences that may be accounted for by one and two germ-line genes, respectively. (iii) The public idiotypic specificities GA-1, expressed by some anti-GAT and most anti-GA antibodies, make use of the same (or similar) VH germ-line genes as the CGAT or pGAT antibodies but possess a distinctive V kappa sequence. (iv) Antibodies expressing neither of the alternative public specificities mentioned above appear to be more heterogeneous and express VH and V kappa sequences that were found to differ from the basic structures defining the CGAT (pGAT) or GA-1 correlates. It is concluded that CGAT (or pGAT) and GA-1 public idiotypic specificities are germ-line markers of both VH and V kappa regions, an observation in agreement with previously reported serological data.
Immunological evidence for deficiency in an activator protein for sulfatide sulfatase in a variant form of metachromatic leukodystrophy.
Authors: Inui K , Emmett M , Wenger D A
Published: May 2006

Cultured skin fibroblasts from the patient described by Shapiro and co-workers as having a variant form of metachromatic leukodystrophy (MLD) [Shapiro, L.J., Aleck, K. A., Kaback, M.M., Itabashi, H., Desnick, R.J., Brand, N., Stephens, R.L., Fluharty, A.L. & Kihara, H. (1979) Pediatr. Res. 13, 1179-1181] were confirmed to have a partial deficiency (25-40% of controls) of arylsulfatase A activity in vitro and a severe inability to metabolize [14C]stearic acid-labeled sulfatide presented in the medium. When 150 micrograms of purified activator protein for GM1 ganglioside beta-galactosidase and sulfatide sulfatase was added in 4 ml of medium with the 14C-labeled sulfatide, correction of the sulfatide metabolism to the normal range was found. Monospecific antibodies to this activator protein were prepared in rabbits, and they were used to examine cultured cells for the presence of crossreacting material by Ouchterlony double immunodiffusion and rocket immunoelectrophoresis. Cell extracts from controls and from patients with GM1 gangliosidosis and MLD were found to have a single line of identity. By comparison to known concentrations of purified activator protein, cell extracts from controls were found to have 0.76 +/- 0.32 micrograms of activator protein (mean +/- 1 SD, n = 10) per mg of solubilized protein, whereas those from patients with type 1 GM1 gangliosidosis and late infantile MLD had 1.53 and 1.41 micrograms/mg, respectively. Cell extracts from the patient with a variant form of MLD had no visible precipitin line by Ouchterlony double immunodiffusion and only a diffuse nonspecific region of staining by rocket immunoelectrophoresis. These immunologic studies provide evidence for a deficiency in the activator protein required for normal catabolism of sulfatide in the cells from this patient and possibly provide a method for diagnosis of similar patients.
Microheterogeneity ("neurotypy") of neurofilament proteins.
Authors: Goldstein M E , Sternberger L A , Sternberger N H
Published: May 2006

Neurofilaments purified from adult rat brainstem by two methods were electrophoresed on NaDodSO4/polyacrylamide gels to separate the triplet proteins (approximate Mrs of 200,000, 155,000, and 68,000) which, in turn, were electroblotted onto nitrocellulose paper. On Coomassie blue-stained gels that were not electroblotted, the same banding pattern was seen with both methods of preparation. Immunocytochemical staining of the electroblots with each of five monoclonal antibodies revealed that three of the monoclonal antibodies were specific for the Mr 200,000 neurofilament protein and two, for both the Mrs 200,000 and 155,000 neurofilament proteins. None of the antibodies reacted with the Mr 68,000 band. The Mr 200,000 band could be resolved into doublet bands. Individual monoclonal antibodies reacted with either one or both of the Mr 200,000 doublets. The immunocytochemical staining of the neurofilament triplets on electroblots was compared to that of adult rat cerebellar paraffin sections. Each monoclonal antibody had a unique pattern of staining, reacting only with certain subpopulations of neurons or their processes. Correlation of the staining patterns in cerebellar tissue sections with those of neurofilament polypeptides on electroblots suggested that different neurofilament polypeptides can be localized to different structures and subpopulations of neurons and that molecular heterogeneity ("neurotypy") may be revealed within the Mrs 200,000 and 155,000 neurofilament polypeptides.
Catecholamine-induced desensitization of turkey erythrocyte adenylate cyclase is associated with phosphorylation of the beta-adrenergic receptor.
Authors: Stadel J M , Nambi P , Shorr R G , Sawyer D F , Caron M G , Lefkowitz R J
Published: May 2006

Preincubation of turkey erythrocytes with catecholamines desensitizes the beta-adrenergic receptor-adenylate cyclase complex in the plasma membranes of these cells. Photoaffinity labeling of the beta-adrenergic receptors with 125I-labeled p-azidobenzylcarazolol (125I-pABC) and subsequent analysis by NaDodSO4/polyacrylamide gel electrophoresis demonstrates an altered mobility of receptor peptides from desensitized cells compared to controls [Stadel, J.M., Nambi, P., Lavin, T.N., Heald, S.L., Caron, M.G. & Lefkowitz, R.J. (1982) J. Biol. Chem. 257, 9242-9245]. The time course of alteration in beta-adrenergic receptor mobility correlates with that for desensitization of isoproterenol-stimulated adenylate cyclase activity. The altered mobility of the receptor peptides from desensitized cells is also observed if the receptors are first purified and then photoaffinity labeled with 125I-pABC. The cyclic nucleotide analog 8-bromoadenosine 3',5'-cyclic monophosphate partially mimics catecholamines in promoting desensitization of the adenylate cyclase and modification of the receptor. Phosphorylation of the beta-adrenergic receptor in intact turkey erythrocytes was assessed by preincubating the cells with [32P]orthophosphate, desensitizing them with catecholamine, purifying the receptors, and then subjecting them to NaDodSO4/polyacrylamide gel electrophoresis. Desensitization is associated with a 2- to 3-fold increase in 32P incorporation into the receptor, which also demonstrates the characteristic alterations in mobility. These effects are blocked by the beta-adrenergic antagonist propranolol. Purified turkey erythrocyte beta-adrenergic receptors could be phosphorylated by incubation with [gamma-32P]ATP and the catalytic subunit of cAMP-dependent protein kinase. The mobility of the phosphorylated receptor peptides on NaDodSO4/polyacrylamide gel electrophoresis appears to correspond to that of the desensitized receptors. These data show that catecholamine-induced desensitization of adenylate cyclase in turkey erythrocytes correlates with a stable modification of the beta-adrenergic receptor and is associated with agonist-promoted phosphorylation of beta-receptor peptides.
Complete nucleotide sequence of a human c-onc gene: deduced amino acid sequence of the human c-fos protein.
Authors: van Straaten F , Müller R , Curran T , Van Beveren C , Verma I M
Published: May 2006

The complete nucleotide sequence of the c-fos(human) gene, the human cellular homolog of the oncogene (v-fos) of Finkel-Biskis-Jinkins murine osteosarcoma virus, has been determined. The c-fos(human) gene contains four discontinuous regions when compared with the v-fos gene. Three of the discontinuities are flanked by sequences characteristic of introns, while the fourth discontinuity is due to a deletion of 104 base pairs in the v-fos gene. As a consequence of the deletion, the predicted c-fos(human) and v-fos gene products differ at their carboxyl termini. Transcripts of 2.2 kilobases from the c-fos(human) gene have been identified in human cells. The sizes of these transcripts are in close agreement with the size expected from the nucleotide sequence after removal of introns.
Cleavage of chromatin with methidiumpropyl-EDTA . iron(II).
Authors: Cartwright I L , Hertzberg R P , Dervan P B , Elgin S C
Published: May 2006

Methidiumpropyl-EDTA . iron(II) [MPE . Fe (II)] cleaves double-helical DNA with considerably lower sequence specificity than micrococcal nuclease. Moreover, digestions with MPE . Fe(II) can be performed in the presence of certain metal chelators, which will minimize the action of many endogenous nucleases. Because of these properties MPE . Fe(II) would appear to be a superior tool for probing chromatin structure. We have compared the patterns generated from the 1.688 g/cm3 complex satellite, 5S ribosomal RNA, and histone gene sequences of Drosophila melanogaster chromatin and protein-free DNA by MPE . Fe(II) and micrococcal nuclease cleavage. MPE . Fe(II) at low concentrations recognizes the nucleosome array, efficiently introducing a regular series of single-stranded (and some double-stranded) cleavages in chromatin DNA. Subsequent S1 nuclease digestion of the purified DNA produces a typical extended oligonucleosome pattern, with a repeating unit of ca. 190 base pairs. Under suitable conditions, relatively little other nicking is observed. Unlike micrococcal nuclease, which has a noticeable sequence preference in introducing cleavages, MPE . Fe(II) cleaves protein-free tandemly repetitive satellite and 5S DNA sequences in a near-random fashion. The spacing of cleavage sites in chromatin, however, bears a direct relationship to the length of the respective sequence repeats. In the case of the histone gene sequences a faint, but detectable, MPE . Fe(II) cleavage pattern is observed on DNA, in some regions similar to and in some regions different from the strong chromatin-specified pattern. The results indicate that MPE . Fe(II) will be very useful in the analysis of chromatin structure.
Topology of the lac carrier protein in the membrane of Escherichia coli.
Authors: Goldkorn T , Rimon G , Kaback H R
Published: May 2006

Proteolysis of topologically sealed right-side-out and inside-out membrane vesicles from Escherichia coli with chymotrypsin, trypsin, or papain inactivates lac carrier function in a symmetrical manner. Concomitantly, the electrophoretic mobility of lac carrier protein photoaffinity labeled in situ with p-nitro[2-3H]phenyl-alpha-D-galactopyranoside is altered from a relative Mr of 33,000 to 20,000, and the time course of proteolysis is almost identical in vesicles of opposite polarities. In contrast, solubilization of the vesicles in NaDodSO4 followed by proteolysis causes fragmentation of the Mr 33,000 band into material that electrophoreses at the solvent front. Notably, proteolysis has no effect whatsoever on the ability of the lac carrier protein to bind substrate, as judged by photoaffinity-labeling experiments. Furthermore, the electrophoretic patterns of samples proteolyzed prior to photoaffinity labeling are the same as those observed when the procedures are reversed. These results show that the lac carrier protein spans the membrane and indicate that the binding site resides within a segment that is embedded in the bilayer.
Topographical arrangement of basement membrane proteins in lactating rat mammary gland: comparison of the distribution of type IV collagen, laminin, fibronectin, and Thy-1 at the ultrastructural level.
Authors: Monaghan P , Warburton M J , Perusinghe N , Rudland P S
Published: May 2006

The topographical distribution of type IV collagen, laminin, fibronectin, and the thymocyte differentiation antigen Thy-1 in the basement membrane of the lactating rat mammary gland was investigated. Small cubes of tissue, which had not been subjected to prior fixation or freezing, were incubated with monospecific or monoclonal antibodies to these proteins, and the antibodies were located by an indirect immunoperoxidase staining technique and observed in the electron microscope. The lamina densa stained uniformly with antibodies to type IV collagen and laminin. In addition, both laminin and type IV collagen were present in semiperiodic clusters that traversed the lamina lucida from the cell surface to the lamina densa. Fibronectin was present only in the semiperiodic clusters and not elsewhere in the basement membrane. These clusters were irregularly spaced along the cell surface and heterogeneous in size. It remains to be determined if these three proteins are present in the same clusters. Thy-1 was largely present on the lamina densa and not on the lamina lucida. The Thy-1 staining of the lamina densa occurred in discrete maxima and minima. These maxima occurred in regions adjacent to Thy-1-bearing stromal cells. Thus, the topographical distribution of proteins within a basement membrane varies in a nonrandom manner, and local factors can modify this distribution. We suggest that this topographical variability may play a role in cell recognition and signalling processes that occur across the basement membrane.
Hexamethylenebisacetamide-resistant murine erythroleukemia cells have altered patterns of inducer-mediated chromatin changes.
Authors: Sheffery M , Rifkind R A , Marks P A
Published: May 2006

We determined inducer-mediated changes in chromatin structure near the globin genes in a variant line of murine erythroleukemia cells (MELC). The variant cell line, R1, was derived from the inducer-sensitive DS19 cell line by selection for inducer-resistance. R1 cells are resistant to induction of erythroid differentiation by hexamethylenebisacetamide (HMBA) whereas the parental line is HMBA-sensitive. Uninduced MELC (both inducer-sensitive DS19 cells and inducer-resistant R1 cells) have DNase I-sensitive sites in chromatin containing the alpha 1- and beta maj-globin genes. These nuclease-sensitive regions are located within the beta maj-globin second intervening sequence (IVS2) and near the alpha 1-globin gene 5' cap site. Culture with HMBA causes changes in chromatin structure in both parental and variant cell lines. In DS19 cells, the DNase I-sensitive site within the beta maj-globin IVS2 becomes more resistant to nuclease cleavage, and a new DNase I-sensitive region develops near the beta maj-globin cap site. In addition, the nuclease-sensitive region adjacent to the cap site of the alpha 1-globin gene increases, and a novel 5' nuclease-sensitive site is also established. In R1 cells, HMBA-mediated changes in chromatin structure are incomplete. The DNase I-sensitive site within the beta maj-globin IVS2 becomes more resistant to nuclease cleavage, but the nuclease sensitivity near the beta maj-globin cap site does not increase to the extent observed in DS19 cells. The pattern of nuclease sensitivity near the alpha 1-globin gene is essentially unchanged after culture of R1 cells with HMBA. Thus, in R1 cells, resistance to HMBA-induced expression of globin genes is associated with failure to detect inducer-mediated changes in chromatin structure 5' to the cap site of the alpha 1- and beta maj-globin genes. These results also suggest that the increased nuclease resistance of a site in the beta maj-globin IVS2 does not depend on the establishment of a DNase I-sensitive region near the beta maj-globin gene cap site.
Transcription of repetitive sequences on Xenopus lampbrush chromosomes.
Authors: Jamrich M , Warrior R , Steele R , Gall J G
Published: May 2006

We reinvestigated the lampbrush chromosomes of Xenopus laevis and found them well suited for the study or transcription by in situ hybridization to nascent RNA transcripts. Using this technique, we analyzed the transcription of three repetitive sequences that do not show any sequence homology and that differ in their degree of interspersion. We found that they are located on different parts of the chromosomes: two are clustered, one is interspersed. All three of these sequences are transcribed at the lampbrush chromosome stage and transcripts from both strands of each sequence can be detected. The amount of transcription is apparently not proportional to the number of copies of the repetitive sequence at a given chromosomal locus, suggesting that other sequences are involved in the regulation of their transcription.
Role of antigen structure in the regulation of IgE isotype expression.
Authors: Francus T , Siskind G W , Becker C G
Published: May 2006

Tobacco glycoprotein (TGP) is a glycoprotein containing rutin-like polyphenol groups that is purified from cured tobacco leaves and can be detected in condensates of tobacco smoke. One-third of normal humans have been shown to manifest immediate, IgE-mediated, wheal and flare reactions to an intradermal injection of TGP. Rutin-like moieties are also found in a wide variety of vegetable foods. The possible importance of sensitivity to TGP in the pathogenesis of the vascular and pulmonary complications of tobacco smoking has stimulated us to study the immune response of mice to TGP and the role of rutin groups in influencing isotype expression. A series of three intradermal injections of TGP elicits a long-lasting IgE antibody response in mice. However, no hemagglutinating antibodies are produced. Similarly, immunization with a rutin derivative of bovine serum albumin stimulates IgE antibodies to bovine serum albumin but little hemagglutinating antibodies. In contrast, mice injected in the same manner with bovine serum albumin produce both IgE and hemagglutinating antibodies. Thus, the rutin moiety is implicated as exerting a regulatory effect on isotype expression by suppressing the production of serum antibodies of isotypes other than IgE. The immunization procedure employed (which involves an initial injection of 100 micrograms of antigen in phosphate-buffered saline, followed, at monthly intervals, by two intradermal injections of 100 micrograms of antigen precipitated on alum) apparently fails to stimulate the normal "down-regulation" of the IgE response so that a persisting high-titered response is obtained.
Evidence for an interleukin-independent pathway for human lymphocyte activation.
Authors: Koretzky G A , Daniele R P , Greene W C , Nowell P C
Published: May 2006

Though lectin mitogen stimulation of T-cell proliferation is an interleukin 1- (IL 1), interleukin 2- (IL 2) dependent process, the calcium ionophore A23187 may be able to initiate T-lymphocyte proliferation by an additional pathway. That the action of A23187 is IL 1 independent was demonstrated by its ability to stimulate monocyte-depleted cells without the addition of exogenous IL 1. The IL 2 independence of A23187 was indicated by (i) the inability of exogenous IL 2 to augment A23187-induced proliferation and (ii) the inability of the monoclonal antibody anti-Tac (with specificity for the human IL 2 receptor) to inhibit proliferation mediated by A23187. By contrast, in cultures stimulated with the lectin mitogen phytohemagglutinin, additional IL 2 considerably increased proliferation, whereas anti-Tac routinely caused 60-90% inhibition. Although the ionophore caused some IL 2 production and resulted in IL 2 receptor expression, quantitative studies showed that our results could not be explained by excessive amounts of endogenous IL 2 interfering with the blocking action of the antibody. Therefore, these data suggest that the action of A23187 as a human lymphocyte mitogen may be the result of at least two pathways--one dependent on the interleukin-cell interactions and the other independent of these mediators.
Enzymatic deamidation of methyl-accepting chemotaxis proteins in Escherichia coli catalyzed by the cheB gene product.
Authors: Kehry M R , Bond M W , Hunkapiller M W , Dahlquist F W
Published: May 2006

The methyl-accepting chemotaxis proteins (MCPs) of Escherichia coli undergo reversible methylation that has been correlated with adaptation of cells to environmental stimuli. MCPI, the product of the tsr gene, accepts methyl groups at multiple sites that are located on two tryptic peptides, denoted K1 and R1. A second modification of the MCPs, which is not methylation, has been designated the CheB-dependent modification. A CheB-dependent modification occurs on methyl-accepting peptide K1 and allows additional methyl groups to be incorporated into this peptide. We have performed partial amino acid sequence analyses on radiolabeled peptides K1 and R1 derived from MCPI and have identified several methyl-accepting sites. We found that, in the absence of CheB-dependent modification, a site in peptide K1 is unable to accept methyl groups. Correlation of this protein sequence data with the nucleotide sequence of the tsr gene [Boyd, A., Kendall, K. & Simon, M.I. (1983) Nature (London) 301, 623-626] suggests that CheB-dependent modification of MCPI is the enzymatic deamidation of glutamine to methyl-accepting glutamic acid. Possible roles for this deamidation in bacterial chemotaxis are discussed.
Nucleotide sequence analysis of human c-myc locus, chicken homologue, and myelocytomatosis virus MC29 transforming gene reveals a highly conserved gene product.
Authors: Watson D K , Psallidopoulos M C , Samuel K P , Dalla-Favera R , Papas T S
Published: May 2006

We have determined the complete nucleotide sequence of human cellular c-myc, which is homologous to the transforming gene, v-myc, of myelocytomatosis virus MC29. Analysis of the genetic information and alignment with the known sequence of chicken c-myc and v-myc indicates: (i) An intervening sequence can be identified by consensus splice signals. The unique 5' sequence of c-myc and its junction with the v-myc region may be a canonical 3' splice acceptor. (ii) The c-myc locus can generate a mRNA whose termination signals are downstream from the translational termination signal. (iii) The three myc genes share the same reading frame, including translational termination signals. (iv) The homology is conserved only in the coding region. (v) Most changes at the nucleotide level result in no change in the amino acid. (vi) There are two distinct domains--the 5' unique domain, which is different from the viral, and the 3' coding domain, which contains amino acids coded by the two exons whose sequences have been determined here. In the latter domain, the amino acid variation between v-myc and chicken c-myc is less than 2%, whereas that between the chicken v-myc and the human is 27%, with the variation concentrated in the region that flanks the splicing points.
Rapid spontaneous dihydrofolate reductase gene amplification shown by fluorescence-activated cell sorting.
Authors: Johnston R N , Beverley S M , Schimke R T
Published: May 2006

We have determined whether the gene encoding dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) varies spontaneously in gene copy number in cells in vitro. Cells were stained under nonselective conditions with fluoresceinated methotrexate, which binds quantitatively to dihydrofolate reductase. Cells with the highest fluorescence were collected by a fluorescence-activated cell sorter and subsequently grown in the absence of methotrexate. At no time during the experiment were the cells placed under metabolic stress. After 10 successive rounds of growth and sorting, the derived population showed a 50-fold increase in fluorescence intensity, was highly resistant to methotrexate, and was amplified 40-fold in content of dihydrofolate reductase gene. We also found that cells already having amplified genes can undergo increases or decreases in their fluorescence and in gene copy number even more rapidly (at rates as high as 3 X 10(-2) amplification events per cell division) than do parental cells (ca. 10(-3) events per division). We therefore conclude that gene amplification can occur spontaneously in cells and that the rate of its occurrence varies with gene copy number.
Expression of human myeloid-associated surface antigens in human-mouse myeloid cell hybrids.
Authors: Geurts van Kessel A H , Tetteroo P A , von dem Borne A E , Hagemeijer A , Bootsma D
Published: May 2006

Hybrid cell lines were obtained after fusion of mouse myeloid cells (WEHI-TG) with leukocytes from two patients with chronic myeloid leukemia. A third fusion was carried out with leukocytes from a patient with acute lymphocytic leukemia. All three patients carried the Philadelphia chromosome (Ph1) in the leukemia cell population. Cytochemical analysis confirmed the myelo-monocytic nature of the hybrid cell lines. The presence of Ph1 translocation products could be established in most hybrids derived from the two chronic myeloid leukemic patients, which confirms that indeed human myeloid cells were fused. Several of these hybrid lines showed reactivity with monoclonal antibodies known to be specific for human myeloid cells, whereas interlineage Chinese hamster fibroblast-human chronic myeloid leukemia hybrids failed to react with these antibodies. Five independently obtained monoclonal antibodies--MI/NI, UJ-308, VIM-D5, FMC-10, and B4.3--showed very similar reactivity patterns when tested on the hybrid clones. This result substantiates the evidence obtained from other studies, that these five antibodies are directed against the same myeloid-associated antigen. The gene(s) for expression of the latter antigen could be assigned to human chromosome 11.
Definition of two pathways for generation of suppressor T-cell activity.
Authors: Hu S K , Eardley D D , Cantor H , Gershon R K
Published: May 2006

Antigen-stimulated Ly1 cells induce T cells from nonimmune donors to develop potent feedback suppressive activity. Suppression is mediated by Ly23 suppressor T (Ts) cells, which are generated from either Ly23 or Ly123 precursors. Ts activity generated from Ly23 precursors requires a strong inducer signal and is rapidly expressed but short lived. In contrast, Ts activity from Ly123 precursors is relatively long lived and is efficiently generated by relatively low levels of inducer signals. Induction of both Ly123 and Ly23 precursors to become Ts cells requires that both cells share genes linked to the Ig-H locus.
Properties of the major outer membrane protein from Neisseria gonorrhoeae incorporated into model lipid membranes.
Authors: Young J D , Blake M , Mauro A , Cohn Z A
Published: May 2006

The major outer membrane protein from Neisseria gonorrhoeae was incorporated into artificial planar bilayer membranes by a detergent-dilution procedure. The integrated protein forms voltage-dependent aqueous pores with a minimal pore diameter estimated to be 11 A. A pore of this size suggests a role for this protein in macromolecular sieving at the level of the outer membrane. This protein self-associates preferentially in triplets of three equal unit conductance steps of 130 pS (in 0.1 M NaCl) each. The two-state model may be applied to explain the voltage-dependent conductance. The average lifetime of the open state of single channels is strongly dependent on the applied voltage, the channels shifting to the closed state at higher voltages. The pore is anion selective, differing from porins of other Gram-negative bacteria studied so far but resembling the voltage-dependent anion-selective channel of the outer membrane of mitochondria.
Regulation of microtubule cold stability by calmodulin-dependent and -independent phosphorylation.
Authors: Job D , Rauch C T , Fischer E H , Margolis R L
Published: May 2006

Cold-labile microtubule protein can be rendered cold-stable by addition of a fraction containing a small number of polypeptides that are derived from cold-stable microtubules. These polypeptides can be obtained from purified cold-stable microtubules by passage through a DEAE-cellulose (DE-52) ion exchange column from which they emerge in the first eluate fraction. The stabilizing activity of these proteins is abolished by phosphorylation catalyzed by two types of protein kinases, one dependent on calmodulin and the other independent of that regulatory protein. The calmodulin-dependent reaction appears to phosphorylate mainly two polypeptides, 56 and 72 kilodaltons; the reaction is blocked by trifluoperazine. The calmodulin-independent reaction appears to phosphorylate different cold-stable microtubule-associated proteins. That reaction is observed only in purified material obtained from vigorously homogenized brain tissue. Gently homogenization yields cold-stable microtubules that are responsive only to the calmodulin-dependent protein kinase. A distinguishing feature of the calmodulin-independent reaction is that it does not occur on polypeptides while they are bound to the microtubules.
Substrate-effected release of surface-located protein kinase from intact cells.
Authors: Kübler D , Pyerin W , Burow E , Kinzel V
Published: May 2006

Protein kinase activity that is independent of cAMP has been reported to exist on the surface of intact HeLa cells. Here we report that the protein kinase activity can be released by the use of casein or phosvitin within a short period of time. The discharge of the enzyme occurs from intact cells since (i) the cells do not release intracellular material and (ii) the cultures continue to grow within any morphological alteration. As shown with phosvitin, the release of protein kinase depends on substrate concentration, incubation time, and temperature. The degree of inducible release or surface protein kinase is inversely related to cell density. Four incubations with phosvitin (1 mg/ml) are sufficient to liberate most of the enzyme, thus greatly reducing the capacity of the cells to phosphorylate cellular substrates at the surface. Within approximately 24 hr after protein kinase removal, cultures have restored their surface protein kinase. Cultured cells of different origin (rat liver, mouse cerebellum, and human lung) exhibited phosvitin-induced protein kinase release from intact cells. The possible significance of these observations with respect to extracellular protein phosphorylation is discussed.
Activation of Klebsiella pneumoniae and Rhizobium meliloti nitrogenase promoters by gln (ntr) regulatory proteins.
Authors: Sundaresan V , Ow D W , Ausubel F M
Published: May 2006

We have studied the expression, in different Escherichia coli gln (ntr) mutants, of fusions (constructed in vitro) of the nifHDK (nitrogenase) promoters from Klebsiella pneumoniae and Rhizobium meliloti to E. coli lacZ. Derepression of the K. pneumoniae nifH::lacZ fusion requires the glnF (ntrA) gene product in addition to the K. pneumoniae nifA gene product, indicating that regulation of the K. pneumoniae nif genes is more closely integrated with the overall nitrogen control system than previously demonstrated. Derepression of the R. meliloti nifH::lacZ fusion in E. coli by the K. pneumoniae nifA gene product (which we had previously shown) exhibits the same requirement for glnF. Derepression of the R. meliloti nifH::lacZ fusion, but not the K. pneumoniae nifH::lacZ fusion, can be mediated by the glnG (ntrC) gene product, suggesting that the gln regulatory genes might directly regulate the symbiotic nitrogen fixation genes in Rhizobium.
In vitro synthesis of bacteriophage phi X174 by purified components.
Authors: Aoyama A , Hamatake R K , Hayashi M
Published: May 2006

An in vitro system capable of synthesizing infectious phi X174 phage particles was reconstituted from purified components. The synthesis required phi X174 supercoiled replicative form DNA, phi X174-encoded proteins A, C, J, and prohead, Escherichia coli DNA polymerase III holoenzyme, rep protein, and deoxyuridinetriphosphatase (dUTPase, dUTP nucleotidohydrolase, EC 3.6.1.23) as well as MgCl2, four deoxyribonucleoside triphosphates, and ATP. Phage production was coupled to the synthesis of viral single-stranded DNA. More than 70% of the synthesized particles sedimented at the position of mature phage in a sucrose gradient and associated with the infectivity. The simple requirement of the host proteins suggests that the mechanism of viral strand synthesis in the phage-synthesizing reaction resembles that of viral strand synthesis during the replication of replicative form DNA.
Detection of a guanine X adenine base pair in a decadeoxyribonucleotide by proton magnetic resonance spectroscopy.
Authors: Kan L S , Chandrasegaran S , Pulford S M , Miller P S
Published: May 2006

A decadeoxyribonucleotide, d(C-C-A-A-G-A-T-T-G-G) (I), forms a duplex in solution. The base pairing pattern in this duplex was studied by proton nuclear magnetic resonance spectroscopy. Five NH...N hydrogen-bonded proton resonances were observed, and they were assigned by nuclear Overhauser enhancement experiments as well as by comparison to five previously assigned NH...N hydrogen-bonded proton resonances in a self-complementary duplex of similar sequence, d(C-C-A-A-G-C-T-T-G-G) (II). The results suggest that the central -G-A- residues of I form G X A base pairs in the helical state. The fact that the H2 proton of A at the sixth position from the 5' end of I showed nuclear Overhauser enhancement when the NH...N hydrogen-bonded proton resonance of G X A was irradiated suggests that the bases of the G X A base pair are oriented in an anti-anti conformation. Comparison of the linewidths at the half height of the NH...N hydrogen-bonded proton resonances of I at 1 degree C suggest that the G X A base pairs are less stable than adjacent A X T base pairs.
Gating of ion channels made by a diphtheria toxin fragment in phospholipid bilayer membranes.
Authors: Misler S
Published: May 2006

B45, a fragment containing the major hydrophobic region of diphtheria toxin, increases the conductance of thin lipid membranes by forming ion-conducting channels that are gated by transmembrane voltage, Vm, and the bath pH. Single-channel currents show "bursting" behavior in the form of rapid transitions between a closed and an open conductance level. The average duration of a current "burst," as well as the total time a channel is actually open within a burst, decreases with increasing Vm. Analysis of these data suggests that, over a range of Vm, increases in the rate constants for transitions from the open to the closed states largely account for the decline in macroscopic conductance with increasing Vm. Increases in rate constants for transitions from a closed to an open conductance state are more likely to account for the increase in macroscopic conductance with increasing bath pH. Since several diphtheria toxin fragments and mutants are currently available, each containing various portions of the B45 region, it may be possible to study the relationship of the structure of these complex proteins to the detailed gating properties of the ion channels that they form.
Inhibition by prostaglandins of leukotriene B4 release from activated neutrophils.
Authors: Ham E A , Soderman D D , Zanetti M E , Dougherty H W , McCauley E , Kuehl F A
Published: May 2006

Chemoattractant N-formylmethionylleucylphenylalanine (fMet-Leu-Phe) in the presence of cytochalasin B stimulates the release of leukotriene B4 (LTB4), superoxide (O2-), and N-acetylglucosaminidase from elicited rat peritoneal and human peripheral neutrophils [PMN (polymorphonuclear leukocytes)]. Prostaglandins E1 and E2 (PGE1 and PGE2) inhibit LTB4 release from PMN in a dose-related manner with an IC50 of 1 X 10(-8) M. This action is associated with increased levels of cyclic AMP. The inhibitory activity of a variety of PGs on LTB4 production by rat peritoneal PMN parallels their affinity for PGE receptors in other tissues. O2- release is also suppressed by low levels of PGE1 and PGE2 in a dose-related manner and this inhibition is enhanced by theophylline. In contrast, lysosomal enzyme release is only minimally affected by physiological levels of PGs. These data are consistent with an action of PGs at the level of the PG receptor on LTB4 and O2- release from the fMet-Leu-Phe-stimulated rat peritoneal PMN. In addition, the fMet-Leu-Phe-induced adherence of PMN to endothelial cells and inhibition of this phenomenon by PGs may now be explained by PG-mediated inhibition of LTB4 formation.
Selection of functional cDNAs by complementation in yeast.
Authors: McKnight G L , McConaughy B L
Published: May 2006

Yeast cDNA was prepared in a yeast expression plasmid to generate a cDNA plasmid pool composed of approximately 40,000 members. Several yeast mutants were transformed with the cDNA plasmid pool, and the cDNAs for ADC1, HIS3, URA3, and ASP5 were isolated by functional complementation. Restriction enzyme analysis confirmed the genetic identity of the ADC1, HIS3, and URA3 cDNAs and demonstrated that the URA3 cDNA contains 5' noncoding sequences. The relative abundance of the various cDNAs in the cDNA plasmid pool paralleled the abundance of the mRNAs in total poly(A)+ RNA, which ranged from approximately 0.01% to 1%. The utility of this approach to isolate rare cDNAs from higher eukaryotes is discussed.
Efficient generation in vitro, from human peripheral blood cells, of monoclonal Epstein-Barr virus transformants producing specific antibody to a variety of antigens without prior deliberate immunization.
Authors: Winger L , Winger C , Shastry P , Russell A , Longenecker M
Published: May 2006

This paper describes a simple protocol for the efficient generation of large numbers of human monoclonal antibody-producing cells. This system is based on initial limiting-dilution culture after Epstein-Barr virus exposure of highly enriched precursors selected from peripheral blood mononuclear cells. Precursors can be enriched by using rosetting or panning approaches. Antibodies to erythrocytes, a mouse mammary carcinoma, DNA, and sperm antigens, produced without any deliberate immunization, are described. Large-scale human monoclonal antibody production may be facilitated by a combination of this protocol with a human cellular fusion system. For efficient precursor analysis and short-term (2 months or more) monoclonal antibody production, however, the system described here may be sufficient.