bluuebunny/crossref_metadata_2025 huggingface dataset:
Responses of single germinal-center B cells in T-cell-dependent microculture.
Authors: George A , Cebra J J
Published: May 2006

B cells purified from the germinal centers (GCs) of murine Peyer's patches can be stimulated in a clonal microculture containing helper T cells and dendritic cells to divide and secrete immunoglobulin. Intraclonal isotype switching occurs, and a variety of immunoglobulin isotypes, including IgA, is secreted. Memory cells, which generate clones secreting IgA exclusively, are only rarely identified in the GC B-cell subset. Such memory cells can, however, be readily identified among unfractionated Peyer's patch B cells, and in non-GC subsets of B cells. The results suggest that the GC does not contain IgA memory cells that can be restimulated in vitro to secrete only IgA. When division of GC B cells is prevented by irradiation or aphidicholin treatment, a large subset that secretes IgA as the sole immunoglobulin isotype is seen, and the output of presumably single B cells is large enough to be scored by RIA. Both helper T cells and dendritic cells are required for the phenomenon. The data indicate that commitment to IgA secretion occurs in Peyer's patch GCs and suggest that the prolific cell division known to be supported in GCs may forestall terminal differentiation of preplasmablasts to immunoglobulin secretion.
Enzymatic aminoacylation of single-stranded RNA with an RNA cofactor.
Authors: Musier-Forsyth K , Scaringe S , Usman N , Schimmel P
Published: May 2006

A chemically synthesized single-stranded ribonucleotide tridecamer derived from the 3' end of Escherichia coli alanine tRNA can be charged with alanine in the presence of short complementary RNA oligonucleotides that form duplexes with the 3' fragment. Complementary 5' oligomers of 9, 8, 6, and 4 nucleotides all confer charging of the 3' fragment. Furthermore, in the presence of limiting 5' oligomer, greater than stoichiometric amounts of the single-stranded 3' acceptor fragment can be aminoacylated. This is due to a reiterative process of transient duplex formation followed by charging, dissociation of the 5' oligomer, and then rebinding to an uncharged single-stranded ribotridecamer so as to create another transient duplex substrate. Thus, a short RNA oligomer serves as a cofactor for a charging enzyme, and it thereby makes possible the aminoacylation of single-stranded RNA. These results expand possibilities for flexible routes to the development of early charging and coding systems.
Purification of a plasma membrane-associated GTPase-activating protein specific for rap1/Krev-1 from HL60 cells.
Authors: Polakis P G , Rubinfeld B , Evans T , McCormick F
Published: May 2006

rap1/Krev-1 is a p21ras-related GTP-binding protein that has been implicated in the reversion of the ras-transformed cell phenotype. We have identified a GTPase-activating protein (GAP) specific for rap in plasma membranes isolated from differentiated HL60 cells. The rap GAP activity remained quantitatively associated with the membrane following washes with buffered 1 M LiCl containing 20 mM EDTA but was solubilized with the detergents Nonidet P-40 and deoxycholate. On the basis of size-exclusion chromatography, the membrane-associated rap GAP (rap GAPm) appeared distinct from the rap GAP detected in the cytosolic fraction from HL60 cells. The molecular sizes of the membrane and cytosolic forms were estimated to be 36 and 54 A, respectively. rap GAPm was solubilized and purified to near homogeneity by successive column chromatographies in the presence of detergent. The rap GAPm activity corresponded to a single polypeptide that migrated with a molecular mass of approximately 88 kDa on SDS/polyacrylamide gels. The purified rap GAPm was inactive toward the GTP-bound forms of p21ras, rho, G25K, and rac-1 and did not stimulate dissociation of guanine nucleotide from rap.
Primary structure of a cerulenin-binding beta-ketoacyl-[acyl carrier protein] synthase from barley chloroplasts.
Authors: Siggaard-Andersen M , Kauppinen S , von Wettstein-Knowles P
Published: May 2006

The radioactively labeled beta-ketoacyl thioester synthase inhibitor [3H] cerulenin was used to tag three dimeric barely chloroplast proteins (alpha alpha, alpha beta, and beta beta) from the stromal fraction. Oligonucleotides corresponding to amino acid sequences obtained from the purified proteins were used to generate with the polymerase chain reaction a probe for cDNAs encoding the beta subunit. cDNA sequencing revealed an open reading frame for 462 residues comprising the mature protein and a 35-amino acid transit peptide. The deduced amino acid sequence of the mature protein is homologous to the beta-ketoacyl-[acyl carrier protein] (ACP) synthase I [3-oxoacyl-ACP synthase; acyl-ACP:malonyl-ACP C-acyltransferase (decarboxylating), EC 2.3.1.41] of Escherichia coli. Under analogous experimental conditions [3H]cerulenin tagged a single dimeric protein from spinach chloroplasts.
Hormonal modulation of a gene injected into rat heart in vivo.
Authors: Kitsis R N , Buttrick P M , McNally E M , Kaplan M L , Leinwand L A
Published: May 2006

We demonstrate gene transfer into rat heart in vivo by the direct injection of plasmid DNA. Injection of gene constructs driven by retroviral and cellular promoters resulted in detectable levels of reporter gene activities. The cellular promoter and 5' flanking sequence (positions -613 to +32) were derived from the rat alpha-myosin heavy chain gene whose expression in vivo is restricted to cardiac muscle and is positively regulated by thyroid hormone. After DNA injection, activity of the firefly luciferase gene coupled to the myosin heavy chain promoter and regulatory sequence was detected in heart but not in skeletal muscle and was significantly increased in response to thyroid hormone treatment. Consequently, expression of injected genes can be targeted to specific cell types in vivo and can be modulated by the hormonal status of the animal. This approach provides a means of mapping the elements of genes that regulate their responses to complex stimuli that cannot be modeled in vitro.
The protein-folding problem: the native fold determines packing, but does packing determine the native fold?
Authors: Behe M J , Lattman E E , Rose G D
Published: May 2006

A globular protein adopts its native three-dimensional structure spontaneously under physiological conditions. This structure is specified by a stereochemical code embedded within the amino acid sequence of that protein. Elucidation of this code is a major, unsolved challenge, known as the protein-folding problem. A critical aspect of the code is thought to involve molecular packing. Globular proteins have high packing densities, a consequence of the fact that residue side chains within the molecular interior fit together with an exquisite complementarity, like pieces of a three-dimensional jigsaw puzzle [Richards, F. M. (1977) Annu. Rev. Biophys. Bioeng. 6, 151]. Such packing interactions are widely viewed as the principal determinant of the native structure. To test this view, we analyzed proteins of known structure for the presence of preferred interactions, reasoning that if side-chain complementarity is an important source of structural specificity, then sets of residues that interact favorably should be apparent. Our analysis leads to the surprising conclusion that high packing densities--so characteristic of globular proteins--are readily attainable among clusters of the naturally occurring hydrophobic amino acid residues. It is anticipated that this realization will simplify approaches to the protein-folding problem.
cDNA-derived amino acid sequence of the NADH-binding 51-kDa subunit of the bovine respiratory NADH dehydrogenase reveals striking similarities to a bacterial NAD(+)-reducing hydrogenase.
Authors: Patel S D , Aebersold R , Attardi G
Published: May 2006

A lambda gt10 bovine brain and a lambda gt11 bovine heart cDNA library were screened with oligonucleotide probes corresponding to partial protein sequences directly determined from the isolated 51-kDa subunit of the bovine respiratory-chain NADH dehydrogenase. Clones were isolated that encode a protein of 464 amino acids containing all the 11 partial tryptic peptide sequences determined from the 51-kDa subunit. The size and amino acid composition of this protein agree with those determined for the purified 51-kDa subunit. Furthermore, this protein contains a putative NADH-binding domain, a possible FMN-binding site, and a putative binding site for an iron-sulfur cluster. The above evidence indicates that the cloned protein is the 51-kDa subunit or its precursor. A search for sequence similarity with proteins in the Protein Identification Resource data base has revealed that the 51-kDa subunit has 32% amino acid sequence identity with a major portion of the alpha subunit of the soluble NAD(+)-reducing hydrogenase from Alcaligenes eutrophus. In particular, there are three segments of high sequence similarity (70-88%) between the two proteins which correspond to the three ligand-binding sites.
The short-lived MAT alpha 2 transcriptional regulator is ubiquitinated in vivo.
Authors: Hochstrasser M , Ellison M J , Chau V , Varshavsky A
Published: May 2006

The substrates of ubiquitin-dependent proteolytic pathways include both damaged or otherwise abnormal proteins and undamaged proteins that are naturally short-lived. Few specific examples of the latter class have been identified, however. Previous work has shown that the cell type-specific MAT alpha 2 repressor of the yeast Saccharomyces cerevisiae is an extremely short-lived protein. We now demonstrate that alpha 2 is conjugated to ubiquitin in vivo. More than one lysine residue of alpha 2 can be joined to ubiquitin, and some of the ubiquitin moieties form a Lys48-linked multiubiquitin chain. Overexpression of degradation-impaired ubiquitin variants was used to show that at least a significant fraction of alpha 2 degradation is dependent on its ubiquitination.
Characterization of the decay-accelerating factor gene promoter region.
Authors: Ewulonu U K , Ravi L , Medof M E
Published: May 2006

Decay-accelerating factor (DAF) expression modulates susceptibility of cells to autologous complement attack. To characterize the regulatory region controlling DAF gene transcription, genomic DNA extending from 815 base pairs (bp) upstream to approximately 4 kilobases downstream of DAF's AUG codon (designated +1) was cloned and sequenced. The 5' flanking sequence showed 59-76% G + C content (-355 to +1), at least one GC box(es) (-135 to -131), and variable length sequences (from -629 to -285) conforming to the motifs TCCTCC and TCn. Nuclease S1 digestions and primer extensions localized a major transcriptional start site to -82/-81, 38 bp downstream of a possible TATA variant, (A)TTTAA. In COS cell transfections, the sequence encompassing -815 to -67 functioned 2.5% as efficiently as the Rous sarcoma virus 3' long terminal repeat, but following deletion upstream of -355 its activity increased approximately 4-fold. Two octanucleotides exhibiting partial homology to phorbol 12-myristate 13-acetate (PMA) and cAMP responsive elements (PREs and CREs, respectively) were detected, and the respective modulators enhanced transcriptional efficiency 2- and approximately 10-fold, respectively. Thus, the DAF gene promoter (i) exhibits sequences resembling both conventional and unconventional transcriptional control elements, (ii) possesses a region with negative regulatory activity, and (iii) responds to PMA and cAMP induction presumably via PRE- and CRE-like enhancer elements.
Cardiac reperfusion damage prevented by a nitroxide free radical.
Authors: Gelvan D , Saltman P , Powell S R
Published: May 2006

Experimental evidence is presented that directly links ischemia/reperfusion injury to the formation of oxygen-derived free radicals. 2,2,6,6-Tetramethylpiperidine-N-oxyl (TEMPO)--a stable nitroxide radical that disproportionates superoxide radicals and oxidizes reduced metal ions required for OH. formation--was tested for its ability to prevent reperfusion damage in the isolated rat heart subjected to regional ischemia. Severe reperfusion arrhythmia--ventricular fibrillation and ventricular tachycardia--were prominent in control hearts, and their duration was significantly reduced by the presence of 0.4 or 1 mM TEMPO. TEMPO also repressed both postischemic release of lactate dehydrogenase and OH. formation. TEMPO slowed the heart rate, but compensatory pacing did not alter the dramatic effect of the nitroxide on reperfusion arrhythmia. TEMPO was partially protective when introduced at the end of ischemia but had no effect when added 1 min into reperfusion. It was concluded that both reperfusion arrhythmia and cell damage were directly related to oxidative damage incurred during the critical first minute of reperfusion. TEMPO strongly protected against reperfusion injury by preventing the formation of OH. and not by decreasing heart rate or by direct suppression of arrhythmia.
8-oxoguanine (8-hydroxyguanine) DNA glycosylase and its substrate specificity.
Authors: Tchou J , Kasai H , Shibutani S , Chung M H , Laval J , Grollman A P , Nishimura S
Published: May 2006

Substrate specificities of FPG protein (also known as formamidopyrimidine DNA glycosylase) and 8-hydroxyguanine endonuclease were compared by using defined duplex oligodeoxynucleotides containing single residues of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), 8-oxo-7,8-dihydro-2'-deoxyadenosine (8-oxodA), and 2,6-diamino-4-hydroxy-5-(N-methyl)formamidopyrimidine (Me-Fapy). Duplexes containing 8-oxodG positioned opposite dC, dG, or dT were cleaved, whereas single-stranded DNA and duplexes containing 8-oxodG.dA or 8-oxodA positioned opposite any of the four DNA bases were relatively resistant. Both enzymes cut duplexes containing 8-oxoG.dC 3' and 5' to the modified base but failed to cleave duplex DNA containing synthetic abasic sites, mismatches containing dG, or unmodified DNA. 8-Oxoguanine, identified by HPLC-electrochemical detection techniques, was released during the enzymatic reaction. Apparent Km values for FPG protein acting on duplex substrates containing a single Me-Fapy or 8-oxodG residue positioned opposite dC were 41 and 8 nM, respectively, and those for 8-hydroxyguanine endonuclease were 30 and 13 nM, respectively. Comparison of the properties of the two enzyme activities suggest that they are identical. In view of the widespread distribution of 8-oxodG in cellular DNA, the demonstrated miscoding and mutagenic properties of this lesion, and the existence of a bacterial gene coding for FPG protein, we propose that 8-oxodG DNA is the primary physiological substrate for a constituent glycosylase found in bacteria and mammalian cells.
Autophosphorylation of type II Ca2+/calmodulin-dependent protein kinase in cultures of postnatal rat hippocampal slices.
Authors: Molloy S S , Kennedy M B
Published: May 2006

Autophosphorylation of Thr286 on type II Ca2+/calmodulin-dependent protein kinase (CaM kinase) in vitro causes kinase activity to become partially independent of Ca2+. Here we report that Thr286 is the major CaM kinase autophosphorylation site occupied in situ in "organotypic" hippocampal cultures. Measurement of Ca(2+)-independent CaM kinase activity revealed that approximately one-third of the kinase is autophosphorylated in situ when the basal Ca2+ concentration is 15-43 nM. This proportion was substantially reduced 30 min after removal of extracellular Ca2+ or treatment of the cultures with protein kinase inhibitors and was increased by treatment with okadaic acid. Therefore, the high proportion of autophosphorylated kinase at basal Ca2+ concentrations appears to be maintained by Ca(2+)-dependent autophosphorylation. Homogenates of intact hippocampi also contain a high proportion of Ca(2+)-independent type II CaM kinase, 13-23% depending on developmental age. Thus, in hippocampal neurons, an important function of the autophosphorylation mechanism may be to produce a relatively high level of CaM kinase activity, even at basal Ca2+ concentrations, permitting both upward and downward local regulation by physiological agents.
Chloroquine inhibits heme-dependent protein synthesis in Plasmodium falciparum.
Authors: Surolia N , Padmanaban G
Published: May 2006

A cell-free protein-synthesizing system has been reconstituted using the S-30 fraction or ribosomes and the S-100 fraction from Plasmodium falciparum. Addition of heme in vitro stimulates cell-free protein synthesis strikingly. Chloroquine inhibits the heme-dependent protein synthesis in the parasite lysate. The drug has also been found to inhibit parasite protein synthesis in situ at therapeutic concentrations soon after addition to parasite cultures. Ribosomes as well as the S-100 fraction isolated from such chloroquine-treated cultures are defective in protein synthesis. Addition of hemin plus glucose 6-phosphate or high concentrations of GTP, cAMP, and an active preparation of eIF-2 to the parasite cell-free system restores protein synthesis to a significant extent in chloroquine-treated cultures. Under conditions of inhibition of protein synthesis in situ by chloroquine in the culture, the parasite eukaryotic initiation factor 2 alpha- (eIF-2 alpha) is phosphorylated in the parasite lysate to a greater extent than that observed in the control culture. Addition of hemin in vitro suppresses this phosphorylation. eIF-2 alpha kinase activity is present in the parasite lysate and is not a contaminant derived from the human erythrocytes used to culture the parasite. The heme-chloroquine interactive effects can also be demonstrated with purified eIF-2 alpha kinase from rabbit reticulocyte lysate. It is proposed that chloroquine inhibits heme-dependent protein synthesis in the parasite and this is an early event mediating the growth-inhibitory effects of the drug.
Single amino acid substitutions in the cAMP receptor protein specifically abolish regulation by the CytR repressor in Escherichia coli.
Authors: Søgaard-Andersen L , Mironov A S , Pedersen H , Sukhodelets V V , Valentin-Hansen P
Published: May 2006

Promoters in Escherichia coli that are negatively regulated by the CytR repressor are also activated by the cAMP receptor protein (CRP) complexed to cAMP; as a characteristic, these promoters encode tandem binding sites for cAMP-CRP. In one such promoter, deoP2, CytR binds to the region between the tandem CRP binding sites with a relatively low affinity; in the presence of cAMP-CRP, however, the repressor and activator bind cooperatively to the DNA. Here we have investigated this cooperativity by isolating mutants of the CRP protein that abolish CytR regulation without exhibiting a concomitant loss in their ability to activate transcription. Four different, single amino acid substitutions in CRP give rise to this phenotype. These amino acids lie in close proximity on the surface of the CRP tertiary structure in a portion of the protein that is not in contact with the DNA. In vitro analyses of one of the CRP mutants show that it interacts with the DNA in a manner indistinguishable from wild-type CRP, whereas its interaction with CytR is perturbed. These results strongly indicate that cooperative DNA binding of CytR and cAMP-CRP is achieved through protein-protein interactions.
Development of a highly efficient expression cDNA cloning system: application to oncogene isolation.
Authors: Miki T , Fleming T P , Crescenzi M , Molloy C J , Blam S B , Reynolds S H , Aaronson S A
Published: May 2006

We developed an expression cDNA cloning system capable of generating high-complexity libraries with unidirectionally inserted cDNA fragments and allowing efficient plasmid rescue. As an application of this system, a cDNA library was constructed from an NIH 3T3 transformant induced by mouse hepatocellular carcinoma DNA. Transfection of NIH 3T3 cells by the library DNA led to the detection of several transformed foci from which identical plasmids with transforming ability could be rescued. Structure and sequence analysis of the cDNA clones revealed that the oncogene was created by recombinational events involving an unknown gene and the mouse homologue of the B-raf protooncogene. Detection of the same genetic rearrangement in independent primary transformants implied that generation of the oncogene occurred within the tumor rather than during DNA transfection or cDNA library construction. The high frequency at which clones were identified and the large sizes of some of the transforming cDNA inserts isolated suggest wide applicability of this mammalian expression cloning system for isolating cDNAs of biologic interest.
CD3 eta and CD3 zeta are alternatively spliced products of a common genetic locus and are transcriptionally and/or post-transcriptionally regulated during T-cell development.
Authors: Clayton L K , D'Adamio L , Howard F D , Sieh M , Hussey R E , Koyasu S , Reinherz E L
Published: May 2006

The CD3 eta subunit of the T-cell receptor is thought to subserve an important role in signal transduction and possibly T-cell development. Herein we characterize the organization of the mouse CD3 eta gene and show that it is part of one gene locus that also encodes CD3 zeta on chromosome 1. The NH2-terminal sequence of CD3 zeta and CD3 eta, which share the same leader peptide and are identical through amino acid 122 of each mature protein, is encoded by exons 1-7. However, exons 8 and 9 are differentially spliced to give rise to CD3 zeta and CD3 eta: exons 1-8 encode CD3 zeta and exons 1-7 plus 9 encode CD3 eta. RNase protection analysis with RNA from a variety of fetal, neonatal, and adult cell types indicates that expression of both gene products is T-lineage-restricted. Importantly, expression of CD3 zeta and CD3 eta mRNA appears before or on day 16 of fetal gestation. Expression is apparently coordinate since no cell types tested express CD3 zeta or CD3 eta alone. The steady-state level of CD3 zeta mRNA is greater than or equal to 40-60 times that of CD3 eta mRNA. In immature CD4+CD8+CD3low double-positive thymocytes and CD4+CD8-CD3high or CD4-CD8+CD3high single-positive thymocytes, the respective steady-state CD3 zeta and CD3 eta mRNA levels are equivalent, whereas the amount of receptor-associated CD3 zeta and CD3 eta proteins in double-positive thymocytes is approximately 10 times less than in single-positive thymocytes. Nevertheless, the CD3 zeta/CD3 eta protein ratio remains constant in all populations (40-60:1). Furthermore, discordance between mRNA and protein levels for CD3 zeta and CD3 eta is also observed in splenic T cells. Thus, posttranscriptional and/or transcriptional regulatory mechanisms control CD3 zeta and CD3 eta expression during T-cell development.
Use of a transposon with luciferase as a reporter to identify environmentally responsive genes in a cyanobacterium.
Authors: Wolk C P , Cai Y , Panoff J M
Published: May 2006

Anabaena, a filamentous cyanobacterium, is of developmental interest because, when deprived of fixed nitrogen, it shows patterned differentiation of N2-fixing cells called heterocysts. To help elucidate its early responses to a decrease in nitrogen, we used a derivative of transposon Tn5 to generate transcriptional fusions of promoterless bacterial luciferase genes, luxAB, to the Anabaena genome. Genes that responded to removal of fixed nitrogen or to other environmental shifts by increased or decreased transcription were identified by monitoring the luminescence of colonies from transposon-generated libraries. The Tn5 derivative transposed in Anabaena at ca. 1-4 x 10(-5) per cell and permitted high-resolution mapping of its position and orientation in the genome and facile cloning of contiguous genomic DNA.
Mutation in a gene for type I procollagen (COL1A2) in a woman with postmenopausal osteoporosis: evidence for phenotypic and genotypic overlap with mild osteogenesis imperfecta.
Authors: Spotila L D , Constantinou C D , Sereda L , Ganguly A , Riggs B L , Prockop D J
Published: May 2006

Mutations in the two genes for type I collagen (COL1A1 or COL1A2) cause osteogenesis imperfecta (OI), a heritable disease characterized by moderate to extreme brittleness of bone early in life. Here we show that a 52-year-old postmenopausal woman with severe osteopenia and a compression fracture of a thoracic vertebra had a mutation in the gene for the alpha 2(I) chain of type I collagen (COL1A2) similar to mutations that cause OI. cDNA was prepared from the woman's skin fibroblast RNA and assayed for the presence of a mutation by treating DNA heteroduplexes with carbodiimide. The results indicated a sequence variation in the region encoding amino acid residues 660-667 of the alpha 2(I) chain. Further analysis demonstrated a single-base mutation that caused a serine-for-glycine substitution at position 661 of the alpha 2(I) triple-helical domain. The substitution produced posttranslational overmodification of the collagen triple helix, as is seen with most glycine substitutions that cause OI. The patient had a history of five previous fractures, slightly blue sclerae, and slight hearing loss. Therefore, the results suggest that there may be phenotypic and genotypic overlap between mild osteogenesis imperfecta and postmenopausal osteoporosis, and that a subset of women with postmenopausal osteoporosis may have mutations in the genes for type I procollagen.
A Tn3 derivative that can be used to make short in-frame insertions within genes.
Authors: Hoekstra M F , Burbee D , Singer J , Mull E , Chiao E , Heffron F
Published: May 2006

A Tn3 derivative was constructed to make small in-frame insertions within genes. The transposon contains the URA3 gene, the tetA gene, a truncated lacZ, and phage P1 loxP recombination sites at either end. Insertions that have fused lacZ to an open reading frame are lac+ because they express the truncated lacZ. In the presence of the phage P1 cyclization recombinase cre, the transposon can delete the URA3, tetA, and lacZ genes between the two loxP sites. The remaining short imperfect palindrome contains the ends of Tn3 and a loxP site and does not contain a translational termination codon in the correct reading frame. We have analyzed several insertions within the yeast HO gene. Several insertions inactivate HO and prohibit initiation of mating-type switching. In contrast, an epitope inserted in the central portion encodes a functional HO endonuclease.
Functional activation of the T-cell antigen receptor induces tyrosine phosphorylation of phospholipase C-gamma 1.
Authors: Weiss A , Koretzky G , Schatzman R C , Kadlecek T
Published: May 2006

Stimulation of the T-cell antigen receptor (TCR), which itself is not a protein-tyrosine kinase (PTK), activates a PTK and phospholipase C (PLC). Using the human T-cell leukemic line Jurkat and normal peripheral blood lymphocytes, we demonstrate that stimulation of the TCR specifically induces the recovery of PLC activity in eluates from anti-phosphotyrosine immunoprecipitates. Stimulation of the human muscarinic receptor, subtype 1, when expressed in Jurkat activates PLC through a guanine nucleotide binding protein but does not induce the recovery of PLC activity in eluates from anti-phosphotyrosine immunoprecipitates. Western blot analysis reveals that PLC-gamma 1 is tyrosine-phosphorylated in response to TCR stimulation. Nearly all of the PLC activity recovered in eluates from anti-phosphotyrosine immunoprecipitates was depleted by anti-PLC-gamma 1 antibodies. Stimulation of the TCR on mutants derived from Jurkat that are defective in TCR-induced PLC activation results in markedly reduced, if any, PLC activity recovered in phosphotyrosine immunoprecipitates and in no detectable PLC-gamma 1 tyrosine phosphorylation. Thus, the TCR functions like PTK growth factor receptors, but through an indirect interaction, to induce tyrosine phosphorylation of PLC-gamma 1. Since other studies have implicated two members of the src family of PTKs in TCR-mediated signal transduction, our findings suggest that the induction of tyrosine phosphorylation of PLC-gamma 1 by a mechanism involving a src-like kinase may be the means by which the TCR regulates PLC activity in T cells.
Hypoxia-inducible nuclear factors bind to an enhancer element located 3' to the human erythropoietin gene.
Authors: Semenza G L , Nejfelt M K , Chi S M , Antonarakis S E
Published: May 2006

Human erythropoietin gene expression in liver and kidney is inducible by anemia or hypoxia. DNase I-hypersensitive sites were identified 3' to the human erythropoietin gene in liver nuclei. A 256-base-pair region of 3' flanking sequence was shown by DNase I protection and electrophoretic mobility-shift assays to bind four or more different nuclear factors, at least two of which are induced by anemia in both liver and kidney, and the region functioned as a hypoxia-inducible enhancer in transient expression assays. These results provide insight into the molecular basis for the regulation of gene expression by a fundamental physiologic stimulus, hypoxia.
Identification of type-specific linear epitopes in the glycoproteins gp46 and gp21 of human T-cell leukemia viruses type I and type II using synthetic peptides.
Authors: Horal P , Hall W W , Svennerholm B , Lycke J , Jeansson S , Rymo L , Kaplan M H , Vahlne A
Published: May 2006

Synthetic peptides of 20-25 amino acids were employed in enzyme-linked immunosorbent assays to identify linear epitopes in the external glycoprotein gp46 and the transmembrane glycoprotein gp21 of human T-cell leukemia/lymphotropic viruses type I (HTLV-I) and II (HTLV-II). Ten linear epitopes were identified in the HTLV-I glycoproteins, seven in gp46 and three in gp21. Three major linear epitopes were identified in the gp46 of HTLV-II. Peptides representing linear epitopes of gp46 were found to be sensitive and specific for the detection of antibody and permit serological identification and differentiation of HTLV-I and HTLV-II infections.
Vascular endothelial cell growth factor (VEGF) produced by A-431 human epidermoid carcinoma cells and identification of VEGF membrane binding sites.
Authors: Myoken Y , Kayada Y , Okamoto T , Kan M , Sato G H , Sato J D
Published: May 2006

A distinct family of endothelial cell mitogens that are homologous to platelet-derived growth factor has recently been identified. Unlike other known endothelial cell mitogens, these vascular endothelial cell growth factors (VEGFs) are secreted and appear to act specifically on endothelial cells. We have purified VEGF 2083-fold to apparent homogeneity from protein-free culture medium conditioned by A-431 human epidermoid carcinoma cells. This A-431-derived VEGF was characterized as a homodimer composed of 22-kDa subunits with an N-terminal sequence that was similar to VEGFs produced by human HL-60 leukemic and U-937 histiocytic lymphoma cells. A-431 VEGF was used to identify specific and saturable binding sites for VEGF on human umbilical vein endothelial cells (HUVEC). By affinity cross-linking, VEGF-binding site complexes of 230, 170, and 125 kDa were detected on HUVEC. VEGF specifically induced the tyrosine phosphorylation of a 190-kDa polypeptide, which was similar in mass to the largest binding site detected by affinity cross-linking.
Zygotic genes that mediate torso receptor tyrosine kinase functions in the Drosophila melanogaster embryo.
Authors: Strecker T R , Yip M L , Lipshitz H D
Published: May 2006

The developmental signal that specifies the fates of cells at the anterior and posterior termini of the Drosophila embryo is transmitted by the torso receptor tyrosine kinase. This paper presents the results of a genetic interaction test for zygotic loci that act downstream of torso in the terminal genetic hierarchy. Tests of 26 zygotic mutants with defects in terminal development indicate that at least 14 reside in this hierarchy. The phenotypes associated with these genes fall into three classes, each of which represents a distinct aspect of terminal development and evolution. Four of the genes have been molecularly cloned and their products include an intercellular communication factor and three kinds of transcription factors.
Induction of apoptosis during normal and neoplastic B-cell development in the bursa of Fabricius.
Authors: Neiman P E , Thomas S J , Loring G
Published: May 2006

The lymphoid cells of embryonic bursal follicles are engaged in rapid growth and preimmune diversification of immunoglobulin genes. Disruption of follicular architecture by mechanical dispersion of these cells in short-term tissue culture was accompanied by continued cell division and extensive cell death by apoptosis. Apoptosis was suppressed in parallel cultures of intact follicles. gamma Radiation also triggered extensive apoptosis in embryonic bursal follicles within a few hours. Preneoplastic bursal stem cell populations induced by a v-myc oncogene were hypersensitive to induction of apoptosis by follicular dispersion and radiation. In contrast, tumor progression in v-myc- and v-rel-initiated bursal neoplasms was accompanied by development of resistance to induction of apoptosis. A programmed cell death pathway can be activated during normal B-cell development in the bursa, and alterations in the expression of this pathway accompany neoplastic change in this system.
Invariant chain targets HLA class II molecules to acidic endosomes containing internalized influenza virus.
Authors: Lamb C A , Yewdell J W , Bennink J R , Cresswell P
Published: May 2006

The role of the HLA class II-associated invariant chain in the intracellular trafficking of HLA-DR molecules was examined in a transient expression system using HeLa cells. In the absence of alpha and beta polypeptides, invariant chain was retained in the endoplasmic reticulum (ER). In the absence of invariant chain, intracellular alpha beta heterodimers could be detected only in the ER and the Golgi apparatus. However, when alpha and beta subunits were coexpressed with invariant chain, HLA-DR molecules were detectable in peripheral cytoplasmic vesicles, which also contained invariant chain. In addition, an antibody directed to an acid-induced conformational determinant on the influenza hemagglutinin molecule detected internalized influenza virus in the HLA-DR-containing vesicles. These findings provide direct evidence that the invariant chain targets class II molecules to an acidic endosomal compartment and that this compartment, long suspected to be the site of antigen processing, is the site where class II molecules interact with natural antigen.
Hyperimmune antisera against synthetic peptides representing the glycoprotein of human immunodeficiency virus type 2 can mediate neutralization and antibody-dependent cytotoxic activity.
Authors: Björling E , Broliden K , Bernardi D , Utter G , Thorstensson R , Chiodi F , Norrby E
Published: May 2006

Twenty-five 13- to 35-amino-acid-long peptides representing regions of human immunodeficiency virus type 2 (HIV-2), strain SBL6669, envelope proteins were evaluated for their immunogenic activity in guinea pigs. The peptides were selected to provide homologous representation of sites in the HIV-1 envelope proteins that were previously documented to have a particular immunogenic importance. A number of the HIV-2 peptides were found to be capable of inducing strain SBL6669 neutralizing and antibody-dependent cellular cytotoxicity (ADCC) antibodies. Two overlapping peptides covering amino acids 311-337 representing the central and C-terminal part of the variable third (V3) region, terminology according to Modrow et al. [Modrow, S., Hahn, B., Shaw, G. M., Gallo, R. C., Wong-Staal, F. & Wolf, H. (1987) J. Virol. 61, 570-578], showed the most pronounced capacity to induce neutralizing antibodies. One of the peptides (amino acids 318-337) also induced antibodies mediating ADCC. Two additional regions in the large glycoprotein, gp125, containing linear sites reacting with neutralizing antibodies were identified (amino acids, 119-137 and 472-509). The transmembrane protein, gp36, of HIV-2 harbored two regions of importance for induction of neutralizing antibodies (amino acids 595-614 and 714-729). ADCC activity was induced by two additional gp125-specific peptides (amino acids 291-311 and 446-461). Thus, except for the single V3-specific site there was no correlation between linear immunogenic sites stimulating neutralizing antibody and ADCC activity. These findings pave the way for development of synthetic vaccines against HIV-2 and possibly also simian immunodeficiency virus infections. The capacity of such a product to induce protective immunity can be evaluated in macaque monkeys.
Genes encoding receptors for insulin and insulin-like growth factor I are expressed in Xenopus oocytes and embryos.
Authors: Scavo L , Shuldiner A R , Serrano J , Dashner R , Roth J , de Pablo F
Published: May 2006

Insulin and insulin-like growth factor I (IGF-I) initiate their metabolic, growth, and differentiation effects through binding to the insulin receptor and the IGF-I receptor, two members of the tyrosine kinase family of receptors. To study the role of these peptides and receptors in early development, we used the polymerase chain reaction and embryo-derived RNA to generate partial cDNA sequences of the insulin receptor and IGF-I receptor from the amphibian Xenopus laevis. Three unique tyrosine kinase-related sequences were obtained. Two of the nucleotide sequences, XTK 1a and XTK 1b, corresponded to peptide that share 92% amino acid identity, and each is 89% identical to the human insulin receptor. The third sequence, XTK 2, corresponds to a peptide that has 92% amino acid identity with the human IGF-I receptor but only 80% identity with XTK 1a and XTK 1b. On the basis of these similarities, the pattern of conserved amino acids, and the tetraploid nature of the Xenopus genome, we suggest that XTK 1a and XTK 1b most likely represent the product of two different nonallelic insulin receptor genes, while XTK 2 may be one of the probable two Xenopus IGF-I receptor genes. By reverse transcription-polymerase chain reaction and gene-specific hybridization, expression of the three XTK sequences was detected in the oocyte, unfertilized egg, and embryos through gastrulation, neurulation, and tailbud stages. Competition binding assays with Xenopus membrane preparations demonstrated insulin receptors and IGF-I receptors in older tadpoles. IGF-I receptors were also present in oocytes, eggs, and gastrula embryos. By contrast, insulin binding was present but atypical in oocytes and was barely detected in eggs and gastrula embryos. The expression of receptors for insulin and IGF-I in early Xenopus embryos and their apparent distinct developmental regulation suggest that these molecules and their ligands may be important in early Xenopus development.
Normal pre-B cells express a receptor complex of mu heavy chains and surrogate light-chain proteins.
Authors: Nishimoto N , Kubagawa H , Ohno T , Gartland G L , Stankovic A K , Cooper M D
Published: May 2006

Precursors of B cells, which constitute a subpopulation of the lymphocytes in bone marrow, can be identified by their surface expression of nonimmunoglobulin markers and the absence of immunoglobulin kappa and lambda light chains. Most pre-B cells synthesize mu heavy chains but, without light-chain partners, these undergo rapid cytoplasmic degradation. In the present study, we demonstrate that late stage pre-B cells, like their neoplastic counterparts, express low levels of a surface receptor composed of mu chains paired with a surrogate light-chain complex formed by Vpre-B and lambda 5-like proteins. The data define a previously suspected but unrecognized stage in normal pre-B-cell differentiation. Expression of a clonally diverse receptor renders this population of immature B-lineage cells potentially vulnerable to clonal selection by antigens and idiotypic interactions.
Predicting chemically induced duodenal ulcer and adrenal necrosis with classification trees.
Authors: Giampaolo C , Gray A T , Olshen R A , Szabo S
Published: May 2006

Binary tree-structured statistical classification algorithms and properties of 56 model alkyl nucleophiles were brought to bear on two problems of experimental pharmacology and toxicology. Each rat of a learning sample of 745 was administered one compound and autopsied to determine the presence of duodenal ulcer or adrenal hemorrhagic necrosis. The cited statistical classification schemes were then applied to these outcomes and 67 features of the compounds to ascertain those characteristics that are associated with biologic activity. For predicting duodenal ulceration, dipole moment, melting point, and solubility in octanol are particularly important, while for predicting adrenal necrosis, important features include the number of sulfhydryl groups and double bonds. These methods may constitute inexpensive but powerful ways to screen untested compounds for possible organ-specific toxicity. Mechanisms for the etiology and pathogenesis of the duodenal and adrenal lesions are suggested, as are additional avenues for drug design.
Identification of a human gamma-glutamyl cleaving enzyme related to, but distinct from, gamma-glutamyl transpeptidase.
Authors: Heisterkamp N , Rajpert-De Meyts E , Uribe L , Forman H J , Groffen J
Published: May 2006

We have cloned a 2.4-kilobase cDNA from a human placental cDNA library by using a gamma-glutamyl transpeptidase [GGT; gamma-glutamyltransferase, (5-glutamyl)-peptide:amino acid 5-glutamyltransferase, EC 2.3.2.2] probe. The deduced amino acid sequence of this cDNA, GGT-rel, exhibited an overall similarity of 39.5% with human GGT. Sequences that could represent a heavy and a light chain, analogous to GGT, as well as a putative transmembrane region were identified in GGT-rel. Transfectants overexpressing GGT-rel were tested for their ability to catalyze cleavage of the gamma-glutamyl moiety from natural and synthetic substrates for GGT. Experiments with glutathione added to the medium suggested that GGT-rel could hydrolyze the gamma-glutamyl moiety. More definitive evidence was obtained in experiments in which this protein converted leukotriene C4 to leukotriene D4. However, GGT-rel did not convert synthetic substrates that are commonly used to assay GGT. Our results indicate that GGT can no longer be considered the only enzyme capable of cleaving the gamma-glutamyl linkage of leukotriene C4 and, most likely, of other natural compounds.
Induction of mast cell proliferation, maturation, and heparin synthesis by the rat c-kit ligand, stem cell factor.
Authors: Tsai M , Takeishi T , Thompson H , Langley K E , Zsebo K M , Metcalfe D D , Geissler E N , Galli S J
Published: May 2006

We investigated the effects of a newly recognized multifunctional growth factor, the c-kit ligand stem cell factor (SCF), on mouse mast cell proliferation and phenotype. Recombinant rat SCF164 (rrSCF164) induced the development of large numbers of dermal mast cells in normal mice in vivo. Many of these mast cells had features of "connective tissue-type mast cells" (CTMC), in that they were reactive both with the heparin-binding fluorescent dye berberine sulfate and with safranin. In vitro, rrSCF164 induced the proliferation of cloned interleukin 3 (IL-3)-dependent mouse mast cells and primary populations of IL-3-dependent, bone marrow-derived cultured mast cells (BMCMC), which represent immature mast cells, and purified peritoneal mast cells, which represent a type of mature CTMC. BMCMC maintained in rrSCF164 not only proliferated but also matured. Prior to exposure to rrSCF164, the BMCMC were alcian blue positive, safranin negative, and berberine sulfate negative; had a histamine content of 0.08 +/- 0.02 pg per cell; and incorporated [35S]sulfate into chondroitin sulfates. After 4 wk in rrSCF164, the BMCMC were predominantly safranin positive and berberine sulfate positive, had a histamine content of 2.23 +/- 0.39 pg per cell, and synthesized 35S-labeled proteoglycans that included substantial amounts (41-70%) of [35S]heparin. These findings identify SCF as a single cytokine that can induce immature, IL-3-dependent mast cells to mature and to acquire multiple characteristics of CTMC. These findings also directly demonstrate that SCF can regulate the development of a cellular lineage expressing c-kit through effects on both proliferation and maturation.
Overproduction of rat 1,25-dihydroxyvitamin D3 receptor in insect cells using the baculovirus expression system.
Authors: Ross T K , Prahl J M , DeLuca H F
Published: May 2006

The rat 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptor has been expressed at elevated levels in Spodoptera frugiperda cells using the baculovirus expression vector system. The recombinant 1,25-(OH)2D3 receptor is full-length, binds 1,25-(OH)2D3, and is recognized by a monoclonal antibody specific for 1,25-(OH)2D3 receptor. Densitometric scanning of Coomassie brilliant blue-stained SDS/polyacrylamide gels indicated a recombinant receptor protein level comprising 5% of the total soluble protein from the insect cells. The hydroxylapatite binding assay revealed average levels of 2 nmol of unoccupied 1,25-(OH)2D3 receptor per mg of protein in insect cells at 72 hr after infection with recombinant baculovirus. A measure of total 1,25-(OH)2D3 receptor using a ligand-independent, immunoradiometric assay disclosed average levels of 2.3 nmol of receptor per mg of protein produced by these same cells. A monoclonal antibody directed against the 1,25-(OH)2D3 receptor, and reported to cross-react with this receptor derived from several species, recognized the recombinant rat 1,25-(OH)2D3 receptor upon Western analysis. A monoclonal antibody directed specifically against the porcine receptor failed to recognize the recombinant rat 1,25-(OH)2D3 receptor protein. The cytosolic preparation of insect cells infected with recombinant baculovirus exhibited an equilibrium dissociation constant of 1 x 10(-11) M as determined by a 1,25-(OH)2D3 saturation analysis plotted by the method of Scatchard. This expression system provides an adequate source from which abundant quantities of 1,25-(OH)2D3 receptor can be purified for subsequent x-ray crystallographic analyses.
Maternal inheritance of centrosomes in mammals? Studies on parthenogenesis and polyspermy in mice.
Authors: Schatten G , Simerly C , Schatten H
Published: May 2006

The centrosome, the microtubule-organizing center of the cell, is introduced typically by the sperm at fertilization. In some mammals, however, this paternal pattern of inheritance appears to be violated. The hypothesis that the centrosome is maternally inherited was tested during parthenogenesis, polyspermy, and polygyny as well as after recovery from microtubule inhibition at first mitosis. During parthenogenesis the paternal contribution was absent, and in polyspermy the paternal contribution was multiplied. Haploid and diploid parthenogenotes as well as polyspermic and digynic fertilized eggs each segregated their centrosomes to organize a bipolar mitotic apparatus. Oocytes recovering from a nocodazole block formed two normal bipolar mitotic apparatus; the paternal chromosomes aligned at one spindle equator, while the maternal chromosomes were found at the other. These results show that the centrosome is maternally inherited from cytoplasmic sites in the mouse. The evolutionary switch from paternal to maternal inheritance in mammals might be related to the additional dangers that parthenogenesis represents: a threat to the life of the mother as well as to the life of the fetus.