bluuebunny/crossref_metadata_2025 huggingface dataset:
Defects in the low density lipoprotein receptor gene affect lipoprotein (a) levels: multiplicative interaction of two gene loci associated with premature atherosclerosis.
Authors: Utermann G , Hoppichler F , Dieplinger H , Seed M , Thompson G , Boerwinkle E
Published: May 2006

The lipoprotein (a) [Lp(a)] contains two nonidentical protein species, apolipoprotein (apo) B-100 and a specific high molecular weight glycoprotein, apo(a). Lp(a) represents a continuous quantitative genetic trait, the genetics of which are only poorly understood. Genetic variation at the apo(a) locus affects plasma Lp(a) levels and explains at least 40% of the variability of this trait. Lp(a) levels were found to be elevated 3-fold in the plasma from patients with the heterozygous form of familial hypercholesterolemia who have one mutant low density lipoprotein receptor gene. This elevation was not due to a higher frequency of those apo(a) types that are associated with high Lp(a) levels in familial hypercholesterolemia patients. Rather Lp(a) levels were elevated for each of the apo(a) phenotypes examined. The effects of the apo(a) and low density lipoprotein receptor genes on Lp(a) levels are not additive but multiplicative. This is a situation not commonly considered in quantitative human genetics. We conclude that Lp(a) levels in plasma may be determined by variation at more than one gene locus.
Chromatin configuration of the human CD2 gene locus during T-cell development.
Authors: Wotton D , Flanagan B F , Owen M J
Published: May 2006

To investigate the molecular basis for the tissue-specific expression of the human CD2 gene, its chromatin configuration was assessed by determining DNase I hypersensitivity and the degree of methylation during T-cell lineage commitment and development. Tissue-specific DNase I-hypersensitive sites were found within the 5' promoter region and a region 3' of the gene essential for gene expression. DNase I hypersensitivity of the 5' region correlated strictly with transcriptional activity, whereas hypersensitivity of the 3' region correlated with T-cell progenitor activity or lineage commitment but not necessarily with transcription. Hha I and Hpa II sites around the 5' and 3' regions were undermethylated in CD2-expressing T cells but were more extensively methylated in other cell types. These results define likely regulatory elements both upstream and downstream of the CD2 gene that control its tissue-specific expression. Further, they show that the 3' regulatory region adopts an open chromatin configuration prior to lineage commitment and during early stages of T-cell development before the CD2 gene is transcribed.
Interleukin 2 acts as an adjuvant to increase the potency of inactivated rabies virus vaccine.
Authors: Nunberg J H , Doyle M V , York S M , York C J
Published: May 2006

Interleukin 2 (IL-2) occupies a central position in the cascade of events involved in the immune response. We were interested in determining whether IL-2 could function as an adjuvant to vaccination, to increase the immune response to vaccine immunogens. Using the National Institutes of Health test for rabies vaccine potency, we found that daily systemic administration of IL-2 in conjunction with inactivated rabies virus can increase the potency of vaccination in outbred mice at least 25-fold, as measured by survival following challenge with virulent rabies virus. Enhanced protection is not correlated with an increase in virus-neutralizing antibody titers, and we suggest that IL-2 acts to increase the cellular immune response to vaccination.
Effect of glycyl-L-glutamine on the rate of regeneration of acetylcholinesterase in the rat gastrocnemius muscle after diisopropyl phosphorofluoridate administration.
Authors: Koelle G B , Han M S
Published: May 2006

Rats were given glycyl-L-glutamine (Gly-Gln) by intraaortic infusion with Alzet osmotic pumps during the 48-hr period following the intraaortic administration of diisopropyl phosphorofluoridate (DFP) (10 mumol/kg). The infusion of 1.2 mumol of Gly-Gln per 24 hr resulted in a significant increase in the acetylcholinesterase (AcChoEase; acetylcholine acetylhydrolase, EC 3.1.1.7) activity of the gastrocnemius muscles over that of rats that received DFP only. At a total dose of 3.6 mumol per 24 hr, a diminished result was obtained; at 0.36 mumol per 24 hr, no effect was detectable. These findings, together with earlier ones, suggest that the neurotrophic effect of Gly-Gln or a similar endogenous factor on AcChoEase synthesis is a general phenomenon at sites of cholinergic transmission.
tRNA-tRNA interactions within cellular ribosomes.
Authors: Smith D , Yarus M
Published: May 2006

We describe an assay that converts the effects of tRNA-tRNA contacts at two particular codons into a quantitative effect on beta-galactosidase level. The assay measures the separate and combined efficiency of suppression at adjacent nonsense codons in vivo using a set of specially created homologous messages. In a survey of distal anticodon arm substitutions, we find that particular mutant tRNAs occupying the P-site reduce the apparent efficiency of the suppressor tRNA reading the A-site codon by factors of 2-170. By using measured tRNA-tRNA distances and the crystallographic tRNA structure, we propose a model of the tRNA-tRNA-mRNA complex. In the model, the anticodon loops of the P-site and A-site tRNAs contact one another in a way that is consistent with our combined tRNA efficiency data. These results suggest that tRNA-tRNA interactions that modulate tRNA action are an inevitable feature of translation.
A tool for multiple sequence alignment.
Authors: Lipman D J , Altschul S F , Kececioglu J D
Published: May 2006

Multiple sequence alignment can be a useful technique for studying molecular evolution and analyzing sequence-structure relationships. Until recently, it has been impractical to apply dynamic programming, the most widely accepted method for producing pairwise alignments, to comparisons of more than three sequences. We describe the design and application of a tool for multiple alignment of amino acid sequences that implements a new algorithm that greatly reduces the computational demands of dynamic programming. This tool is able to align in reasonable time as many as eight sequences the length of an average protein.
Mismatch repair involving localized DNA synthesis in extracts of Xenopus eggs.
Authors: Brooks P , Dohet C , Almouzni G , Méchali M , Radman M
Published: May 2006

Repair of heteroduplex DNA containing G.T or A.C mismatches or containing two tandem unpaired bases occurred in vitro with Xenopus egg extracts as detected by a physical assay. The repair was accompanied by a mismatch-stimulated and mismatch-localized DNA synthesis. Repaired molecules, separated from unrepaired molecules, showed a 20- to 100-fold increase in DNA synthesis in the region of the mismatch compared to regions distant from the mismatch. The remaining unrepaired heteroduplex DNA included molecules that also displayed mismatch-stimulated DNA synthesis in the mismatch-proximal regions. These may represent intermediates in the repair process. The patterns of DNA synthesis suggest that repair begins at some distance from the mismatch and that as much as 1 kilobase or more can be involved in the mismatch-stimulated synthesis.
Sequence elements in the human osteocalcin gene confer basal activation and inducible response to hormonal vitamin D3.
Authors: Kerner S A , Scott R A , Pike J W
Published: May 2006

Osteoblast-specific expression of the bone protein osteocalcin is controlled at the transcriptional level by the steroid hormone 1 alpha,25-dihydroxyvitamin D3. As this protein may represent a marker for bone activity in human disease, we examined the regulation of its expression at the molecular level by evaluating human osteocalcin gene promoter function. We describe regions within the promoter that contribute to basal expression of the gene in osteoblast-like cells in culture. Further, we define a 21-base-pair DNA element with the sequence 5'-GTGACTCACCGGGTGAACGGG-3', which acts in cis to mediate 1 alpha,25-dihydroxyvitamin D3 inducibility of the osteocalcin gene. This response element bears sequence similarity with other short DNA segments, particularly those for estrogen and thyroid hormone, which act together with their respective trans-acting receptors to modulate gene transcription.
Exogenous ATP and other nucleoside phosphates modulate epidermal growth factor receptors of A-431 epidermoid carcinoma cells.
Authors: Hosoi K , Edidin M
Published: May 2006

The binding of epidermal growth factor (EGF) by A-431 human epidermoid carcinoma cells was reduced after exposure of the cells to low concentrations (0.01-1 mM) of ATP and other nucleoside 5'-triphosphates at 37 degrees C, but not at 0 degree C. This was due to loss of high-affinity EGF binding sites. The modulation was associated with transient increases in inositol phosphate synthesis and intracellular Ca2+ and with phosphorylation of the EGF receptor on serine and threonine. There was no evidence for entry of labeled ATP into the cells. ATP appeared to bind to specific cell surface receptors. Such binding was demonstrated directly with the nonmetabolizable ATP analogue adenosine 5'-[beta,gamma-imido]triphosphate.
Collagen fibrillogenesis in situ: fibril segments are intermediates in matrix assembly.
Authors: Birk D E , Zycband E I , Winkelmann D A , Trelstad R L
Published: May 2006

The assembly of discontinuous fibril segments and bundles was studied in 14-day chicken embryo tendons by using serial sections, transmission electron microscopy, and computer-assisted image reconstruction. Fibril segments were first found in extracytoplasmic channels, the sites of their polymerization; they also were found within fibril bundles. Single fibril segments were followed over their entire length in consecutive sections, and their lengths ranged from 7 to 15 microns. Structural differences in the ends of the fibril segments were identified, suggesting that the amino/carboxyl polarity of the fibril segment is reflected in its architecture. Our data indicate that fibril segments are precursors in collagen fibril formation, and we suggest that postdepositional fusion of fibril segments may be an important process in tendon development and growth.
Growth- and tumor-promoting effects of deregulated BCL2 in human B-lymphoblastoid cells.
Authors: Nunez G , Seto M , Seremetis S , Ferrero D , Grignani F , Korsmeyer S J , Dalla-Favera R
Published: May 2006

Human follicular B-cell lymphomas possess a t(14;18) that translocates a putative protooncogene, BCL2, into the immunoglobulin heavy chain locus. The normal BCL2 gene is quiescent in resting B cells, expressed in proliferating, but down-regulated in differentiated B cells. Inappropriately high levels of BCL2-immunoglobulin chimeric RNA are present in t(14;18) lymphomas for their mature B-cell stage. We examined the biologic effects of BCL2 deregulation in human B cells by introducing BCL2 into human B-lymphoblastoid cell lines (LCLs) with retroviral gene transfer. Although deregulated BCL2 expression as a single agent was not sufficient to confer tumorigenicity to LCLs, it consistently produced a 3- to 4-fold increment in LCL clonogenicity in soft agar. In addition, BCL2 deregulation complements the transforming effects of the MYC oncogene in LCLs. BCL2 augmented the clonogenicity of LCLs bearing exogenous MYC and increased the frequency and shortened the latency of tumor induction in immunodeficient mice. These results demonstrate a role for BCL2 as a protooncogene that affects B-cell growth and enhances B-cell neoplasia.
Mouse hematopoietic stem-cell antigen Sca-1 is a member of the Ly-6 antigen family.
Authors: van de Rijn M , Heimfeld S , Spangrude G J , Weissman I L
Published: May 2006

Recently, hematopoietic stem cells were purified to homogeneity from mouse bone marrow. The protein structure of Sca-1, the cell surface antigen used in the isolation of hematopoietic stem cells, is described here. It is shown that the Sca-1 antigen is a member of the Ly-6 antigen family. The anti-Sca-1 antibody was used in immunohistochemistry experiments to define the structures in several tissues that had previously been shown to contain Ly-6 antigens. In thymus, spleen, and kidney, specific staining of parenchymal cells can be demonstrated, whereas only vasculature reacts with anti-Sca-1 in brain, heart, and liver and possibly in lung.
Isolation and DNA sequence of a cDNA clone encoding a lymphocyte adhesion receptor for high endothelium.
Authors: Idzerda R L , Carter W G , Nottenburg C , Wayner E A , Gallatin W M , St John T
Published: May 2006

The migration of lymphocytes from the bloodstream into the secondary lymphoid organs, which is necessary for a successful immune response, occurs primarily within postcapillary venules that are characterized by high-walled endothelial cells. Lymphocyte adhesion to and extravasation at these sites is associated with the expression of specific lymphoid receptors for this specialized venule endothelium. We report here the molecular cloning from a baboon lymphoid cell line of a cDNA that encodes an adhesion receptor for HEV. The 362-amino acid protein encoded by this cDNA is not present in any of the data bases examined. The mature protein, resulting from the cleavage of a putative 20-amino acid signal peptide, has a calculated molecular mass of only 37 kDa, indicating that the 90-kDa cell surface protein is highly modified. The 342 amino acids, which lack any repeated sequences of significant length, encompass an extracellular domain (250 amino acids), a putative transmembrane domain (20 amino acids), and a cytoplasmic domain (72 amino acids).
Direct evidence that oxygen-derived free radicals contribute to postischemic myocardial dysfunction in the intact dog.
Authors: Bolli R , Jeroudi M O , Patel B S , DuBose C M , Lai E K , Roberts R , McCay P B
Published: May 2006

Electron paramagnetic resonance (EPR) spectroscopy was used to investigate whether (i) the free radicals produced in the "stunned" myocardium (myocardium with postischemic contractile dysfunction) are derived from O2, (ii) inhibition of radical reactions improves function, and (iii) i.v. spin traps are effective. Open-chest dogs undergoing a 15-min coronary occlusion received an i.v. infusion of the spin trap, alpha-phenyl N-tert-butylnitrone (PBN) (50 mg/kg). In group I (n = 6), EPR signals characteristic of radical adducts of PBN appeared in the coronary venous blood during ischemia and increased dramatically after reperfusion. In group II (n = 6), which received PBN and i.v. superoxide dismutase (SOD; 16,000 units/kg) plus catalase (12,000 units/kg), myocardial production of PBN adducts was undetectable during ischemia (delta = -100%, P less than 0.01 vs. group I) and markedly inhibited after reperfusion (delta = -86%, P less than 0.001). This effect was seen at all levels of ischemic zone flow but was relatively greater in the low-flow range. In group III (n = 8), the same dosages of SOD and catalase without PBN markedly enhanced contractile recovery (measured as systolic wall thickening) after reperfusion [P less than 0.01 at 3 hr vs. controls (group IV, n = 7)]. Systemic plasma activity of SOD and catalase averaged 127 +/- 24 and 123 +/- 82 units/ml, respectively, 2 min after reperfusion. PBN produced no apparent adverse effects and actually improved postischemic contractile recovery in group I (P less than 0.05 at 3 hr vs. controls). This study shows that (i) SOD and catalase are highly effective in blocking free radical reactions in vivo, (ii) the radicals generated in the "stunned" myocardium are derived from univalent reduction of O2, and (iii) inhibition of radical reactions improves functional recovery. The results provide direct, in vivo evidence to support the hypothesis that reactive oxygen metabolites play a causal role in the myocardial "stunning" seen after brief ischemia.
Estrogen differentially regulates neuropeptide gene expression in a sexually dimorphic olfactory pathway.
Authors: Simerly R B , Young B J , Capozza M A , Swanson L W
Published: May 2006

The posterodorsal part of the medial nucleus of the amygdala (MeAp) receives its major sensory input from the accessory olfactory bulb and projects massively to the medial preoptic nucleus and other sexually dimorphic hypothalamic nuclei thought to play key roles in mediating steroid-sensitive reproductive functions. A combined axonal transport/double-immunohistochemical method was used to show that at least one-quarter of the cholecystokinin-immunoreactive cells in the MeAp cocontain substance P and that a substantial proportion of these cells project to the medial preoptic nucleus. In situ hybridization histochemistry was then used to demonstrate that estrogen regulates the expression of preprocholecystokinin in these cells at the mRNA level in male and female rats. In contrast, levels of preprotachykinin mRNA within the MeAp do not appear to be sensitive to acute changes in circulating gonadal steroids in either sex. Although posttranscriptional regulation of mRNA stability may contribute to the observed effects, it appears likely that estrogen stimulates preprocholecystokinin expression within the MeAp by selectively inducing transcription of the corresponding gene, thereby altering the relative amounts of cholecystokinin and substance P coexpressed within individual neurons of the MeAp that project to the hypothalamus.
Risk behavior-based model of the cubic growth of acquired immunodeficiency syndrome in the United States.
Authors: Colgate S A , Stanley E A , Hyman J M , Layne S P , Qualls C
Published: May 2006

The cumulative number of cases of acquired immunodeficiency syndrome (AIDS) in the United States has grown as the cube of time rather than exponentially. We explain this by interactions involving partner choice and sexual frequency in a risk-behavior model with biased mixing. This leads to a saturation wave of infection moving from high- to low-risk groups. If this description is correct, then the decreasing growth rate of AIDS cases is not due to behavior changes; rather it is due to the intrinsic epidemiology of the disease.
Infectious potential of human immunodeficiency virus type 1 reverse transcriptase mutants with altered inhibitor sensitivity.
Authors: Larder B A , Kemp S D , Purifoy D J
Published: May 2006

There is considerable interest in the potential of human immunodeficiency virus type 1 (HIV-1) to develop drug resistance, especially as 3'-azido-3'-deoxythymidine (Retrovir) is now in widespread clinical use to treat people with AIDS and AIDS-related complex (ARC). To address this possibility, mutations in the HIV reverse transcriptase [deoxynucleoside-triphosphate:DNA deoxynucleotidyltransferase (RNA-directed), EC 2.7.7.49] gene have been introduced by site-directed mutagenesis of cloned constructs in Escherichia coli. Analysis of the recombinant mutant reverse transcriptase from a number of these constructs revealed enzymes that maintained enzyme activity but had a reduced ability to recognize inhibitors such as azidothymidine triphosphate. To assess the infectivity of these mutants, several constructs of proviral HIV clones with mutant reverse transcriptase genes have been made and used to transfect T cells. All five mutants tested have lower infectious potential, suggesting considerable levels of reverse transcriptase activity are required for efficient virus replication. Viable virus recovered from two clones showed decreased sensitivity to the antiviral compound phosphonoformate, thus demonstrating the potential for drug-resistant HIV to replicate. However, although the reverse transcriptase from these mutant viruses showed decreased sensitivity to azidothymidine triphosphate, paradoxically these viruses were hypersensitive to azidothymidine when tested in culture.
cDNA cloning reveals that the major group rhinovirus receptor on HeLa cells is intercellular adhesion molecule 1.
Authors: Tomassini J E , Graham D , DeWitt C M , Lineberger D W , Rodkey J A , Colonno R J
Published: May 2006

A 90-kDa surface glycoprotein was previously isolated and shown to be required for infection by the "major" group of human rhinovirus (HRV) serotypes. In the present work, the amino acid sequence of the receptor protein was obtained from CNBr and tryptic peptides. Using degenerate oligonucleotides predicted from the peptide sequences, we identified four cDNA clones that encode a 3-kilobase mRNA. The clones were ligated, subcloned in a simian virus 40 expression vector, and used to transfect receptor-negative Vero (monkey) cells. Results showed that transfected cells expressed receptor molecules capable of binding HRV and a monoclonal antibody which recognizes the major group HRV receptor. The cloned receptor cDNA encoded a protein with a sequence nearly identical to that of the intercellular adhesion molecule 1 (ICAM-1), indicating that the two surface proteins are one and the same. Both proteins have identical mass, carbohydrate composition, and tissue distribution. In addition, major group receptors on HeLa cells could be induced with various cytokines in a manner similar to the ICAM-1 ligand. A similar induction of the HRV "minor" group receptor was not observed.
Inhibition of human immunodeficiency virus (HIV) replication by HIV-trans-activated alpha 2-interferon.
Authors: Bednarik D P , Mosca J D , Raj N B , Pitha P M
Published: May 2006

We have prepared stable cell lines, derived from Vero cells and A3.01 cells, that express a hybrid human alpha 2-interferon gene under control of the human immunodeficiency virus (HIV) long terminal repeat. These cells constitutively produced low levels (50-150 units/ml) of alpha 2-interferon. However, high levels of interferon (10(3) units/ml) could be induced upon trans-activation by the product of the tat gene (pIIIextatIII), and de novo infection by HIV resulted in a moderate increase (400 units/ml) in alpha 2-interferon synthesis. In contrast to the fully permissive HIV replication, in transfected Vero cells or infected A3.01 cells, the transcription and replication of HIV in Vero or A3.01 cells containing the HIV long terminal repeat--alpha 2-interferon hybrid gene (VN89 and A3N89 cells, respectively) was completely inhibited. These data suggest that virus-trans-activated alpha 2-interferon synthesis can be used as a selective inhibitor of HIV replication.
Inhibition of radiation-induced neoplastic transformation by beta-lapachone.
Authors: Boothman D A , Pardee A B
Published: May 2006

Beta-lapachone is a potent inhibitor of DNA repair in mammalian cells and activates topoisomerase I. We show that beta-lapachone can prevent the oncogenic transformation of CHEF/18A cells by ionizing radiation. Potentially lethal DNA damage repair (PLDR) occurs while x-irradiated cells are held in medium containing low serum prior to replating. PLDR processes permitted survival recovery but also drastically increased the number of foci per plate (i.e., transformation) of CHEF/18A cells. By blocking PLDR with beta-lapachone, both survival recovery and enhanced transformation were prevented. At equivalent survival levels, exposure of x-irradiated cells to beta-lapachone resulted in an 8-fold decrease in the number of foci per dish as compared to the number of transformants produced after PLDR. Early PLDR-derived increases in transformation may be the result of error-prone genetic rearrangements dependent on topoisomerase I, which are thereby prevented by beta-lapachone. Beta-lapachone exposure decreased the rejoining of DNA strand breaks and produced additional double-strand breaks in x-irradiated cells during PLDR. The activation of topoisomerase I by beta-lapachone may convert repairable single-strand DNA breaks into the more repair-resistant double-strand breaks, thereby preventing PLDR and neoplastic transformation. These results suggest a new direction for the development of anticarcinogenic agents.
An apaH mutation causes AppppA to accumulate and affects motility and catabolite repression in Escherichia coli.
Authors: Farr S B , Arnosti D N , Chamberlin M J , Ames B N
Published: May 2006

apaH- mutants lack the hydrolase responsible for degradation of AppppN dinucleotides in Escherichia coli and show a greater than or equal to 16-fold increase in AppppA under nonstress conditions. These mutants lack detectable activity of sigma F, a factor required for transcription of motility and chemotaxis genes. Expression of the flbB/flaI operon, thought to encode sigma F, is decreased in apaH- mutants, and there appears to be a general decrease in expression of genes regulated by cAMP-binding protein and cAMP as well.
Structural characterization of the alpha-glycerol-3-phosphate dehydrogenase-encoding gene of Drosophila melanogaster.
Authors: von Kalm L , Weaver J , DeMarco J , MacIntyre R J , Sullivan D T
Published: May 2006

In Drosophila, multiple isoforms of alpha-glycerol-3-phosphate dehydrogenase (sn-glycerol-3-phosphate: NAD+ 2-oxidoreductase, EC 1.1.1.8) are produced in a tissue- and stage-specific manner. To understand the underlying molecular basis of these isoforms, we have sequenced a 5.8-kilobase region of the Drosophila genome that contains the entire Gpdh locus. Primer-extension and RNase protection assays show that the gene consists of eight exons and has a single transcription-start point. RNase protection mapping and comparison of the genomic sequence from three different cDNA clones reveal that three protein isoforms of glycerol-3-phosphate dehydrogenase are produced by alternative processing of 3' exons. Two of the isoforms differ from the third by the addition of either three or ten amino acids to their C-terminal ends. Transcripts corresponding to two of the isoforms are expressed during both larval and adult stages, while the third isoform is produced only in adults.
Specific chromosomal abnormalities characterize fibrosarcomas of bovine papillomavirus type 1 transgenic mice.
Authors: Lindgren V , Sippola-Thiele M , Skowronski J , Wetzel E , Howley P M , Hanahan D
Published: May 2006

In the BPV1.69 line of transgenic mice, the bovine papillomavirus type 1 genome elicits both benign dermal fibroblastic proliferation (fibromatoses) and malignant fibrosarcomas. Because these lesions arise only with time, nonviral factors appear to be involved. We have karyotyped several primary tumors as well as a series of low-passage cell lines derived both from fibromatoses and from fibrosarcomas. The fibrosarcomas, but not the preneoplastic fibromatoses, show consistent abnormalities of one or both of two chromosomes, chromosome 8 (trisomy or duplication) and chromosome 14 (monosomy or translocation). The chromosomal abnormalities are not a direct consequence of the viral integration, which we have mapped to chromosome 15 by in situ hybridization. These results suggest that transgenic mice can be used to study the role(s) of cytogenetic changes in tumorigenesis and may direct the search for genes involved in tumor progression.
Short, direct repeats at the breakpoints of deletions of the retinoblastoma gene.
Authors: Canning S , Dryja T P
Published: May 2006

We found deletions involving the retinoblastoma gene in 12 of 49 tumors from patients with retinoblastoma or osteosarcoma. After mapping the deletion breakpoints, we found that no two breakpoints coincided. Thus, our data do not support the conclusions of others regarding the existence of a "hotspot" for deletion breakpoints in this gene. In 4 of the tumors, we sequenced 200 base pairs surrounding each deletion breakpoint. Three deletions had termini within pairs of short, direct repeats ranging in size from 4 to 7 base pairs. These results indicate that the "slipped mispairing" mechanism may predominate in the generation of deletions at this locus. Our review of deletion breakpoints at other genetic loci reveals that the nature of the sequences present at deletion breakpoints (short, direct repeats versus middle repetitive elements) varies according to the genetic locus under study.
Evidence for a functional idiotypic network among natural antibodies in normal mice.
Authors: Lundkvist I , Coutinho A , Varela F , Holmberg D
Published: May 2006

We monitored in normal adult BALB/c mice the serum concentrations of four natural IgM antibodies, two of which show idiotypic complementarity in in vitro assays. In each individual, serum concentration of all four idiotypes were found to fluctuate in complex dynamical patterns with low correlation. The spectral power of some such patterns was found to be compatible with the existence of a chaotic regime. Groups of normal adult mice were injected intravenously with low (10 ng) or moderate (10 micrograms) doses of either of the two complementary idiotypes in saline. This treatment resulted in a pronounced inhibition of the fluctuation in the serum concentration of both complementary idiotypes for periods up to 3 months. Such compensations were not detected for the two unrelated natural idiotypes and were specifically induced, for they did not occur following the injection of unrelated antibodies. These results indicate the functional operation of an idiotypic network among natural antibodies.
Embryonal carcinoma cells express Qa and Tla class I genes of the major histocompatibility complex.
Authors: Ostrand-Rosenberg S , Nickerson D A , Clements V K , Garcia E P , Lamouse-Smith E , Hood L , Stroynowski I
Published: May 2006

The murine major histocompatibility complex encodes H-2K and H-2D transplantation antigens and other class I-like proteins called Qa and Tla molecules; the functions of the Qa/Tla molecules are not known. That they may participate in embryonic cell-cell interactions and/or play a role in immune responses against tumors has been speculated. We have studied two murine embryonal carcinoma tumors, 402AX and PCC4, that are rejected in vivo immunologically, although they do not express H-2K or H-2D antigens. Transplantation studies with these cells suggest that rejection is mediated by class-I-like major histocompatibility complex antigens. As a first step in evaluating Qa/Tla function(s), we have characterized expression of class I-like genes and proteins in 402AX and PCC4 cells. Northern (RNA) blot hybridizations, polymerase chain reaction studies, and cDNA cloning experiments demonstrate that EC lines transcribe genes allelic to the Tla region gene "37", Qa-2 region gene "Q7", and another, previously uncharacterized, class I-like gene. Immunoprecipitation studies show that the embryonal carcinoma tumor cells contain low levels of beta 2-microglobulin expressed in association with non-H-2K, non-H-2D class I-like proteins.
Positive selection of a T-cell subpopulation in the thymus in which it develops.
Authors: Iwabuchi K , Negishi I , Arase H , Iwabuchi C , Ogasawara K , Good R A , Onoé K
Published: May 2006

In SWR mice the expression with high-density V beta 17a (high V beta 17a) of the T-cell antigen receptors correlates with the CD4+8- subpopulation of thymocytes. By contrast, in thymocytes of SJL mice the expression of high V beta 17a is observed on the CD4+8- or CD4-8+ subpopulation. However, when the thymocytes from SWR mice have been developed in the SJL or B10 thymus but not in the H-2 compatible DBA/1 thymus, a greater proportion of thymocytes that express high V beta 17a was found to be CD4-8+. By contrast, only a small proportion of KJ23a+ thymocytes from SJL mice that had differentiated in the thymus of SWR or DBA/1 mice was CD4-8+, whereas a high proportion of CD4+8- cells expressed V beta 17a. Further, an intermediate proportion of KJ23a+ thymocytes that had derived from SJL donor mice was present on CD4-8+ thymocytes that had developed in B10.A(4R) thymus. These findings demonstrate that the appearance of a particular subpopulation of thymocytes (CD4-8+ with a beta chain of T-cell antigen receptor identified as V beta 17a) is determined by the histocompatibility complex products that are expressed in the thymic microenvironment in which the T cells develop.
Selective displacement of nuclear proteins by antitumor drugs having affinity for nucleic acids.
Authors: Bartkowiak J , Kapuscinski J , Melamed M R , Darzynkiewicz Z
Published: May 2006

The nuclear chromatin binding sites of the antitumor drugs mitoxantrone, ametantrone, doxorubicin, mithramycin, and actinomycin D and the intercalating ligand ethidium were studied by polyacrylamide gel electrophoresis of the proteins released from rat liver nuclei in the presence and absence of these drugs in buffer of low ionic strength (10 mM NaCl). At 25-50 microM free ligand concentration, each drug produced a specific and reproducible pattern of extractable proteins of different molecular weight by (i) releasing new proteins, (ii) altering the quantity of particular extracted proteins, and/or (iii) selectively entrapping other proteins in the nuclei. Ethidium, up to 100 microM, did not affect release of proteins from the nuclei. These results indicate that each ligand either has different binding site(s) in chromatin or modulates chromatin structure in a specific way by changing the affinity of different sets of proteins for their respective binding sites, resulting in their selective extraction or entrapment. The lack of effect of ethidium indicates that intercalation of the ligand to DNA, per se, does not alter the release of nuclear proteins. If patterns of nuclear proteins selectively released or retained by antitumor drugs are found to correlate with biological activity, this type of analysis may be helpful in new drug design and screening.
Deltorphins: a family of naturally occurring peptides with high affinity and selectivity for delta opioid binding sites.
Authors: Erspamer V , Melchiorri P , Falconieri-Erspamer G , Negri L , Corsi R , Severini C , Barra D , Simmaco M , Kreil G
Published: May 2006

Deltorphins are endogenous linear heptapeptides, isolated from skin extracts of frogs belonging to the genus Phyllomedusa, that have a higher affinity and selectivity for delta opioid binding sites than any other natural compound known. Two deltorphins with the sequence Tyr-Ala-Phe-Asp(or Glu)-Val-Val-Gly-NH2 have been isolated from skin extracts of Phyllomedusa bicolor. The alanine in position 2 is in the D configuration. These peptides, [D-Ala2]deltorphins I and II, show an even higher affinity for delta receptors than the previously characterized deltorphin, which contains D-methionine as the second amino acid. These peptides show some similarity to another constituent of Phyllomedusa skin, dermorphin, which is highly selective for mu-opioid receptors. These peptides all have the N-terminal sequence Tyr-D-Xaa-Phe, where D-Xaa is either D-alanine or D-methionine. While this structure seems to be capable of activating both mu and delta opioid receptors, differences in the C-terminal regions of these peptides are probably responsible for the observed high receptor selectivity of dermorphin and deltorphin.
Cholecystokinin evokes secretion of oxytocin and vasopressin from rat neural lobe independent of external calcium.
Authors: Bondy C A , Jensen R T , Brady L S , Gainer H
Published: May 2006

Cholecystokinin (CCK) and its receptors are abundantly represented in the central nervous system. However, a specific role or mechanism of action for CCK in this context has not been established. CCK coexists with oxytocin in magnocellular neurons of the hypothalamic-neurohypophysial system, sharing common neurosecretory vesicles with oxytocin in the neural lobe of the pituitary. The neural lobe, which consists primarily of oxytocin- and vasopressin-containing axons and nerve terminals and their surrounding glia, provides a relatively simple model system allowing for the study of the regulation of neurosecretion at the nerve terminal level, free from the complex array of synaptic effects present throughout the rest of the central nervous system. In this paper, we demonstrate the presence of high-affinity CCK binding sites in the rat neural lobe and show that activation of these receptors by the sulfated octapeptide, CCK-8, and related peptides causes potent secretion of oxytocin and vasopressin from the isolated nerve terminals. The secretagogue action of CCK-8, which is blocked by a CCK receptor antagonist (L-364,718), is independent of electrical stimulation and extracellular calcium and is blocked by an inhibitor of protein kinase C. Thus, the action of CCK on the neural lobe provides an example of peptide ligand-induced neurosecretion apparently mediated by second messengers rather than depolarization-induced calcium influx.
Isolation of a rat liver Golgi mannosidase II clone by mixed oligonucleotide-primed amplification of cDNA.
Authors: Moremen K W
Published: May 2006

A clone encoding Golgi mannosidase II (MII; GlcNAc-transferase I-dependent alpha 1,3(alpha 1,6) mannosidase), an enzyme involved in asparagine-linked oligosaccharide processing, was isolated from a rat liver lambda gt11 cDNA library by a method that employs a modification of the polymerase chain reaction. Specific oligonucleotide primers were designed from two regions of protein sequence and were combined in an amplification reaction with a single-stranded cDNA preparation derived from rat liver poly(A)+ RNA. Based upon mapping of the protein sequences 42 kDa apart on the MII polypeptide, the procedure was expected to generate an approximately 1150-base-pair amplification product representing a segment of the MII gene between the two primer regions. The size of the amplified product (1170 base pairs) was in close agreement with this predicted fragment size. The authenticity of the amplified fragment was confirmed by the agreement of the DNA sequence with additional protein sequence data. A 1474-base-pair clone was isolated from a cDNA library by plaque hybridization using the amplification fragment as a radiolabeled probe. The nucleotide sequence of this clone predicts a single continuous open reading frame and, based upon a polypeptide molecular mass of 117 kDa for the enzyme subunit, is consistent with the clone representing approximately 50% of the coding sequence of MII. Both the clone and the amplification product hybridized to a rat liver mRNA of approximately 8 kilobases, a message size approximately 4.7 kilobases larger than the size of the predicted open reading frame. This extensive non-coding information on the MII message is a feature common to two other Golgi processing enzymes, both of which contain most of the non-coding information on the 3' end of their messages. The function of these disproportionately large untranslated regions is not clear.
Characterization of cDNAs encoding human pyruvate dehydrogenase alpha subunit.
Authors: Ho L , Wexler I D , Liu T C , Thekkumkara T J , Patel M S
Published: May 2006

A cDNA clone (1423 base pairs) comprising the entire coding region of the precursor form of the alpha subunit of pyruvate dehydrogenase (E1 alpha) has been isolated from a human liver cDNA library in phage lambda gt11. The first 29 amino acids deduced from the open reading frame correspond to a typical mitochondrial targeting leader sequence. The remaining 361 amino acids, starting at the N terminus with phenylalanine, represent the mature mitochondrial E1 alpha peptide. The cDNA has 43 base pairs in the 5' untranslated region and 210 base pairs in the 3' untranslated region, including a polyadenylylation signal and a short poly(A) tract. The nucleotide sequence of human liver E1 alpha cDNA was confirmed by the nucleotide sequences of three overlapping fragments generated from human liver and fibroblast RNA by reverse transcription and DNA amplification by the polymerase chain reaction. This consensus nucleotide sequence of human liver E1 alpha cDNA resolves existing discrepancies among three previously reported human E1 alpha cDNAs and provides the unambiguous reference sequence needed for the characterization of genetic mutations in pyruvate dehydrogenase-deficient patients.
Mechanical properties of sensory hair bundles are reflected in their Brownian motion measured with a laser differential interferometer.
Authors: Denk W , Webb W W , Hudspeth A J
Published: May 2006

By optically probing with a focused, low-power laser beam, we measured the spontaneous deflection fluctuations of the sensory hair bundles on frog saccular hair cells with a sensitivity of about 1 pm/square root of Hz. The preparation was illuminated by two orthogonally polarized laser beams separated by only about 0.2 microns at their foci in the structure under investigation. Slight movement of the object from one beam toward the other caused a change of the phase difference between the transmitted beams and an intensity modulation at the detector where the beams interfered. Maintenance of the health of the cells and function of the transduction mechanism were occasionally confirmed by measuring the intracellular resting potential and the sensitivity of transduction. The root-mean-square (rms) displacement of approximately 3.5 nm at a hair bundle's tip suggests a stiffness of about 350 microN/m, in agreement with measurements made with a probe attached to a bundle's tip. The spectra resemble those of overdamped harmonic oscillators with roll-off frequencies between 200 and 800 Hz. Because the roll-off frequencies depended strongly on the viscosity of the bathing medium, we conclude that hair-bundle motion is mainly damped by the surrounding fluid.
Subtypes of intercalated cells in rat kidney collecting duct defined by antibodies against erythroid band 3 and renal vacuolar H+-ATPase.
Authors: Alper S L , Natale J , Gluck S , Lodish H F , Brown D
Published: May 2006

The cellular distributions of the kidney form of the erythrocyte band 3 chloride/bicarbonate exchanger and the kidney vacuolar H+-transporting ATPase were examined in rat kidney collecting duct by immunocytochemical staining of adjacent semithin sections. Polyclonal anti-peptide antibodies directed against two regions of murine erythroid band 3 gave a pattern of basolateral labeling similar to that seen with antibodies directed against the entire protein. In the medullary collecting duct almost all intercalated cells expressed basolateral membrane band 3 and displayed apical membrane H+-ATPase. In the cortical collecting duct and the connecting segment, band 3 labeling was restricted to a subpopulation of intercalcated cells. In the cortical collecting duct 46% of intercalated cells had apical H+-ATPase and basolateral band 3. Cells that had either basolateral or diffuse cytoplasmic staining for H+-ATPase were all band 3-negative and accounted for 53% of the intercalated cells. In addition, occasional intercalated cells with apical H+-ATPase appeared to lack basolateral band 3. These results demonstrate the coexpression of H+-ATPase and band 3 in opposite plasma membrane domains of a subpopulation of intercalated cells that are probably the acid-excreting (type A) cells. All other intercalated cells lacked immunoreactive band 3 and probably include the bicarbonate-excreting (type B) cells.
Activation of muscle-specific genes in pigment, nerve, fat, liver, and fibroblast cell lines by forced expression of MyoD.
Authors: Weintraub H , Tapscott S J , Davis R L , Thayer M J , Adam M A , Lassar A B , Miller A D
Published: May 2006

MyoD is a master regulatory gene for myogenesis. Under the control of a retroviral long terminal repeat, MyoD was expressed in a variety of differentiated cell types by using either a DNA transfection vector or a retrovirus. Expression of muscle-specific proteins was observed in chicken, human, and rat primary fibroblasts and in differentiated melanoma, neuroblastoma, liver, and adipocyte lines. The ability of MyoD to activate muscle genes in a variety of differentiated cell lines suggests that no additional tissue-specific factors other than MyoD are needed to activate the downstream program for terminal muscle differentiation or that, if such factors exist, they are themselves activated by MyoD expression.
Two steps in the evolution of Antennapedia-class vertebrate homeobox genes.
Authors: Kappen C , Schughart K , Ruddle F H
Published: May 2006

Antennapedia-class vertebrate homeobox genes have been classified with regard to their chromosomal locations and nucleotide sequence similarities within the 183-base-pair homeobox domain. The results of these comparisons support the view that in mammals and most likely the vertebrates, four clusters of homeobox genes exist that were created by duplications of an entire primordial gene cluster. We present evidence that this primordial cluster arose by local gene duplications of homeoboxes that were present before the divergence of arthropods and chordates. Sequence analyses indicate that the expansion of the primordial gene cluster complex was accompanied by diversification, whereas conservation predominated after the duplications of entire homeobox gene clusters.
Significant structural and functional change of an antigen-binding site by a distant amino acid substitution: proposal of a structural mechanism.
Authors: Chien N C , Roberts V A , Giusti A M , Scharff M D , Getzoff E D
Published: May 2006

To study the molecular basis for antibody diversity and the structural basis for antigen binding, we have characterized the loss of phosphocholine (P-Cho) binding both experimentally and computationally in U10, a somatic mutant of the antibody S107. Nucleotide sequencing of U10 shows a single base change in JH1, substituting Asp-101 with Ala, over 9 A distant from the P-Cho-binding pocket. Probing with antiidiotypic antibodies suggests local, not global, conformational changes. Computational results support a specific structural mechanism for the loss of P-Cho binding. The U10 mutation eliminates the charged interaction between Asp-101 and Arg-94, which allows the Arg-94 side chain to disrupt P-Cho binding sterically and electrostatically by folding into the P-Cho-binding site. These results specifically show the importance of the Arg-94 to Asp-101 side chain salt bridge in the heavy-chain CDR3 conformation and suggest that residues distant from the binding site play an important role in antibody diversity and inducible complementarity.
Detection of mRNA for the tax1/rex1 gene of human T-cell leukemia virus type I in fresh peripheral blood mononuclear cells of adult T-cell leukemia patients and viral carriers by using the polymerase chain reaction.
Authors: Kinoshita T , Shimoyama M , Tobinai K , Ito M , Ito S , Ikeda S , Tajima K , Shimotohno K , Sugimura T
Published: May 2006

Expression of human T-cell leukemia virus type I (HTLV-I) is not detectable by immunofluorescence analysis or RNA blot analysis in most fresh peripheral blood mononuclear cells of patients with adult T-cell leukemia or of asymptomatic HTLV-I carriers. However, in this work, mRNA for the HTLV-I tax1/rex1 genes was detected in fresh peripheral blood mononuclear cells of adult T-cell leukemia patients and asymptomatic HTLV-I carriers by using reverse transcription followed by the polymerase chain reaction. By using fresh peripheral blood mononuclear cells, the expression of tax1/rex1 mRNA was detected in five of the six adult T-cell leukemia patients and four of the eight HTLV-I carriers examined. The amounts of tax1/rex1 mRNA detected corresponded to approximately 10(5) to 10(6) times less than that in the HTLV-I-infected MT-2 cell line. These results indicate that, in some individuals infected with HTLV-I, the provirus in circulating blood cells is transcribed in vivo. Thus the expression of viral antigens in circulating blood cells in vivo is suggested.