bluuebunny/crossref_metadata_2025 huggingface dataset:
Determinants of the B-cell response against a transgenic autoantigen.
Authors: Skowronski J , Jolicoeur C , Alpert S , Hanahan D
Published: May 2006

The failure to induce self-tolerance of simian virus 40 large tumor antigen (T antigen) expressed in the pancreatic beta cells of transgenic mice results in an autoimmune response against this protein and the cells that synthesize it. In every transgenic mouse with delayed onset of T-antigen expression and consequent nontolerance, B cells, T cells, and macrophages are attracted to and infiltrate the pancreatic islets. In contrast, the incidence, onset, and intensity of the B-cell response to produce anti-T-antigen autoantibodies vary considerably with genetic background. Thus the initial attraction of lymphocytes to the cells synthesizing a non-self antigen can be separated from the activation of a B-cell response against it. Haplotypes of the major histocompatibility complex (MHC) differentially influence the character of the autoimmune response, with H-2d and H-2k conferring a high incidence of humoral autoimmunity. Additional non-MHC linked genes are also implicated in control of the B-cell response.
Hemophilia as a defect of the tissue factor pathway of blood coagulation: effect of factors VIII and IX on factor X activation in a continuous-flow reactor.
Authors: Repke D , Gemmell C H , Guha A , Turitto V T , Broze G J , Nemerson Y
Published: May 2006

The effect of factors VIII and IX on the ability of the tissue factor-factor VIIa complex to activate factor X was studied in a continuous-flow tubular enzyme reactor. Tissue factor immobilized in a phospholipid bilayer on the inner surface of the tube was exposed to a perfusate containing factors VIIa, VIII, IX, and X flowing at a shear rate of 57, 300, or 1130 sec-1. Factor Xa in the effluent was determined by chromogenic assay. The flux of factor Xa (moles formed per unit surface area per unit time) was strongly dependent on wall shear rate, increasing about 3-fold as wall shear rate increased from 57 to 1130 sec-1. The addition of factors VIII and IX at their respective plasma concentrations resulted in a further 2- to 3-fold increase. The direct activation of factor X by tissue factor-factor VIIa could be virtually eliminated by the lipoprotein-associated coagulation inhibitor; however, when factors VIII and IX were present at their approximate plasma concentrations, factor Xa production rates were enhanced 15- to 20-fold. These results suggest that the tissue factor pathway, mediated through factors VIII and IX, produces significant levels of factor Xa even in the presence of an inhibitor of the tissue factor-factor VIIa complex; moreover, the activation is dependent on local shear conditions. These findings are consistent both with a model of blood coagulation in which initiation of the system results from tissue factor and with the bleeding observed in hemophilia.
The distinct surface of human blood dendritic cells, as observed after an improved isolation method.
Authors: Freudenthal P S , Steinman R M
Published: May 2006

Prior studies have identified a subset of dendritic cells in human blood, as well as their stimulatory function for T-cell-mediated immune responses. However research has been limited by difficulties in isolation, since dendritic cells make up only 0.1-1% of blood mononuclear cells. We present a protocol that reliably yields preparations that are greater than 80-90% pure. The method relies on the sequential depletion of the major cell types in blood and simultaneously provides T cells, monocytes, and B plus natural killer cells for comparison with dendritic cells. The last step in the procedure is the removal of residual contaminants on the basis of expression of a CD45R epitope. The enrichment of dendritic cells is evident by three criteria, each of which is related to the surface of these antigen-presenting cells. (i) All dendritic cells are motile, constantly forming large lamellipodia or veils. (ii) When analyzed with a large panel of monoclonal antibodies and the FACS, the cells express high levels of all known polymorphic major histocompatibility complex gene products, as well as a distinct combination of receptors and adhesion molecules. Unlike monocytes, for example, dendritic cells lack Fc receptors and the colony-stimulating factor 1 receptor (c-fms) but express much higher levels of ICAM-1 and LFA-3 adhesins. (iii) In functional assays, dendritic cells are at least 100 times more potent than monocytes or lymphocytes in stimulating the primary mixed leukocyte reaction. These properties help make the trace subset of dendritic cells more amenable to further functional and clinical studies.
Promoter deletion and loss of retinoblastoma gene expression in human prostate carcinoma.
Authors: Bookstein R , Rio P , Madreperla S A , Hong F , Allred C , Grizzle W E , Lee W H
Published: May 2006

Mutational inactivation of the retinoblastoma gene (RB) is found in all retinoblastomas and in a subset of other human neoplasms, including sarcomas of bone or soft tissue and carcinomas of lung or breast. Exogenous copies of wild-type RB have been shown to suppress the tumorigenicity of several types of tumor cells with endogenous RB mutations, including a previously described human prostatic carcinoma cell line. To further support a role for RB inactivation in the genesis of prostate cancer, seven primary or metastatic prostate carcinoma specimens were examined for evidence of RB mutation. By the use of immunoblot analysis and immunostaining of histologic sections, RB-encoded protein was readily detected in tumor cells of five specimens, was equivocally detected in one specimen, and was apparently absent from tumor cells of one specimen. RB mutations in the latter case were precisely characterized as (i) a deletion of 103 nucleotides containing transcriptional start sites and (ii) loss of the second RB allele. The 103-base-pair deletion was sufficient to abolish the promoter activity of upstream DNA sequences in a heterologous expression system. These results (i) demonstrate that RB can be inactivated in vivo by mutation of its promoter, (ii) confirm the existence of RB mutations in some human prostate carcinomas, and (iii) suggest the use of immunohistochemical methods to screen for RB mutations in clinical samples of common adult neoplasms.
Inducible morphology, heterochrony, and size hierarchies in a colonial invertebrate monoculture.
Authors: Harvell C D , Padilla D K
Published: May 2006

Conditional or inducible strategies are a powerful tool for analyzing the evolution of aggressive behavior. Structures such as stolons and nematocyst-laden tentacles, induced to deter encroachment by competitors, are proportionately better represented in clonal and colonial marine invertebrates than in aclonal animals. Stolons can be produced by colonies of Membranipora membranacea (Bryozoa) within 48 hr after contact with conspecifics. Absolute size and relative size of interacting colonies determine whether stolons will be produced. Although individual stolons are eventually overgrown by conspecifics, they reduce the size of affected zooids by 27%. Since stolon production is primarily a strategy of large colonies, we suggest that stolons function to limit space occupied by small colonies and may also trigger early and localized reproduction. Thus large colonies can surround multiple small mates and, because they reproduce only locally where induced by contact with small colonies, still maintain high growth rates on free colony perimeters. Stolons appear to be juvenilized zooids and to originate through a process of heterochrony. These induced facultative polymorphisms may be one pathway by which fixed polymorphisms arise in colonial invertebrates. We attribute the unique production of inducible structures against competitors by clonal and colonial invertebrates to both unusually high levels of development plasticity and an energetically favorable architecture for perimeter defense.
Carcinogen-induced mutations in the mouse c-Ha-ras gene provide evidence of multiple pathways for tumor progression.
Authors: Brown K , Buchmann A , Balmain A
Published: May 2006

A number of mouse skin tumors initiated by the carcinogens N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methylnitrosourea (MNU), 3-methylcholanthrene (MCA), and 7,12-dimethylbenz[a]anthracene (DMBA) have been shown to contain activated Ha-ras genes. In each case, the point mutations responsible for activation have been characterized. Results presented demonstrate the carcinogen-specific nature of these ras mutations. For each initiating agent, a distinct spectrum of mutations is observed. Most importantly, the distribution of ras gene mutations is found to differ between benign papillomas and carcinomas, suggesting that molecular events occurring at the time of initiation influence the probability with which papillomas progress to malignancy. This study provides molecular evidence in support of the existence of subsets of papillomas with differing progression frequencies. Thus, the alkylating agents MNNG and MNU induced exclusively G ---- A transitions at codon 12, with this mutation being found predominantly in papillomas. MCA initiation produced both codon 13 G ---- T and codon 61 A ---- T transversions in papillomas; only the G ---- T mutation, however, was found in carcinomas. These findings provide strong evidence that the mutational activation of Ha-ras occurs as a result of the initiation process and that the nature of the initiating event can affect the probability of progression to malignancy.
Identification of essential histidine residues in the active site of Escherichia coli xylose (glucose) isomerase.
Authors: Batt C A , Jamieson A C , Vandeyar M A
Published: May 2006

Two conserved histidine residues (His-101 and His-271) appear to be essential components in the active site of the enzyme xylose (glucose) isomerase (EC 5.3.1.5). These amino acid residues were targeted for mutagenesis on the basis of sequence homology among xylose isomerases isolated from Escherichia coli, Bacillus subtilis, Ampullariella sp. strain 3876, and Streptomyces violaceus-niger. Each residue was selectively replaced by site-directed mutagenesis and shown to be essential for activity. No measurable activity was observed for any mutations replacing either His-101 or His-271. Circular dichroism measurements revealed no significant change in the overall conformation of the mutant enzymes, and all formed dimers similar to the wild-type enzyme. Mutations at His-271 could be distinguished from those at His-101, since the former resulted in a thermolabile protein whereas no significant change in heat stability was observed for the latter. Based upon these results and structural data recently reported, we speculate that His-101 is the catalytic base mediating the reaction. Replacement of His-271 may render the enzyme thermolabile, since this residue appears to be a ligand for one of the metal ions in the active site of the enzyme.
Theory for protein mutability and biogenesis.
Authors: Lau K F , Dill K A
Published: May 2006

Using an elementary physical model for protein folding, of self-avoiding short copolymer chains on two-dimensional square lattices, we address two questions regarding the evolution and origins of globular proteins. (i) How will protein native structures and stabilities be affected by single-and double-site mutations? (ii) What is the probability that a randomly chosen sequence of amino acids will be compact and globular under folding conditions? For a large number of different sequences, we search the conformational space exhaustively to find unequivocally the "native" conformation(s), of global minimum free energy, for each sequence. We find that replacing nonpolar residues in the core by polar residues is generally destabilizing, that surface sites are less sensitive than core sites, that some mutations increase the degeneracy of native states, and that overall it is most probable that a mutation will be neutral, having no effect on the native structure. These results support a "Continuity Principle," that small changes in sequence seldom have large effects on structure or stability of the native state. The simulations also show that (i) the number of "convergent" sequences (different sequences coding for the same native structure) is extremely large and (ii) most sequences become quite dense under folding conditions. This implies that the probability of formation of a globular protein from a random sequence of amino acids by prebiotic or mutational methods is significantly greater than zero.
Oligomeric structure of the human immunodeficiency virus type 1 envelope glycoprotein.
Authors: Earl P L , Doms R W , Moss B
Published: May 2006

The envelope (env) glycoprotein of human immunodeficiency virus type 1 (HIV-1) consists of two noncovalently associated subunits, gp120 and gp41, that are formed gradient sedimentation, polyacrylamide gel electrophoresis, gradient sedimentation, polyacrylamide gel electrophoresis, and chemical cross-linking, we show that gp160 is synthesized as a monomer and subsequently forms stable homodimers. The molecule remains dimeric after cleavage to gp120/gp41 but is less stable to detergent solubilization and centrifugation. Analysis of wild-type and mutated env proteins indicated that interactions between the ectodomain regions of adjoining gp41 subunits are important for dimer formation and stability. A higher-order oligomeric form was also recovered, probably a tetramer consisting of two noncovalently associated dimers. The proposed subunit composition of the HIV-1 env protein is identical to that previously observed for the paramyxovirus envelope proteins F and HN.
Pea leaf mitochondrial pyruvate dehydrogenase complex is inactivated in vivo in a light-dependent manner.
Authors: Budde R J , Randall D D
Published: May 2006

We examined the effect of light on the activity of the mitochondrial pyruvate dehydrogenase complex (mt-PDC) by using intact green pea (Pisum sativum) seedlings. Upon illumination there is an initial drop in mtPDC activity followed by oscillations that dampen during the initial period of photosynthesis to a steady-state level of one-fourth or less of the mtPDC activity measured in the dark. The initial light-dependent decrease in mtPDC activity is inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (an inhibitor of photosystem II of photosynthesis) and does not occur in etiolated seedlings. Therefore, the effect of light is indirect and most likely associated with photosynthesis and/or photorespiration. Conditions that would be unfavorable for photorespiration also inhibited the light-dependent decrease in mtPDC activity.
G-protein alpha-subunit expression, myristoylation, and membrane association in COS cells.
Authors: Mumby S M , Heukeroth R O , Gordon J I , Gilman A G
Published: May 2006

Myristoylation of seven different alpha subunits of guanine nucleotide-binding regulatory proteins (G proteins) was examined by expressing these proteins in monkey kidney COS cells. Metabolic labeling studies of cells transfected with cytomegalovirus-based expression vectors indicated that [3H]myristate was incorporated into alpha i1, alpha i2, alpha i3, alpha 0, alpha t, and alpha z but not alpha s subunits. The role of myristoylation in the association of alpha subunits with membranes was analyzed by site-directed mutagenesis and by substitution of myristate with a less hydrophobic analog, 10-(propoxy)decanoate (11-oxamyristate). Myristoylation of alpha 0 was blocked when an alanine residue was substituted for its amino-terminal glycine, as was association of the protein with membranes. Substitution of the myristoyl group with 11-oxamyristate affected the cellular distribution of a subset of acylated alpha subunits. The results are consistent with a model wherein the hydrophobic interaction of myristate with the bilayer permits continued association of the protein with the plasma membrane when G-protein alpha subunits dissociate from beta gamma.
Expression of photosynthesis-related gene fusions is restricted by cell type in transgenic plants and in transfected protoplasts.
Authors: Harkins K R , Jefferson R A , Kavanagh T A , Bevan M W , Galbraith D W
Published: May 2006

We have analyzed the expression of chimeric genes in populations of protoplasts isolated from the photosynthetic and nonphotosynthetic tissues within leaves of transgenic tobacco plants and separated by fluorescence-activated cell sorting. Expression of transcriptional gene fusions controlled by promoters from photosynthesis-associated genes showed a striking dependence on cell type. These patterns of expression were preserved when the gene fusions were transfected into normal (nontransgenic) tobacco leaf protoplasts.
Adenylyl cyclase is dispensable for vegetative cell growth in the fission yeast Schizosaccharomyces pombe.
Authors: Maeda T , Mochizuki N , Yamamoto M
Published: May 2006

Disruption of the cyr1 gene of Schizosaccharomyces pombe, which encodes adenylyl cyclase, did not confer lethality to fission yeast cells, although they grew 40% slower than wild-type strains in complete medium. These cells contained no measurable amount of cAMP and no adenylyl cyclase activity. When h+ and h- cyr1 disruptants were mixed, they underwent mating even in rich medium. Propagation of homothallic cyr1 disruptants was difficult, probably because such cells readily mate and produce asci and thus stop growing. A greater than 10-fold increase in the amount of cyr1 mRNA was observed when cloned cyr1+ was introduced into Sch. pombe cells on a multicopy plasmid. The total adenylyl cyclase activity was similarly high in these transformants. However, the level of intracellular cAMP was hardly affected. Evidence suggests that this was not due to increased phosphodiesterase activity. Thus, cAMP level in growing fission yeast cells appears to be regulated not by the amount of adenylyl cyclase protein but by a feedback mechanism at the enzyme level. The cAMP level fell by approximately 50% under nitrogen starvation, which triggers sexual development in Sch. pombe. We suggest that fission yeast controls the level of intracellular cAMP primarily to regulate sexual development rather than to drive or arrest the cell cycle.
Stimulation of a human T-cell clone with anti-CD3 or tumor necrosis factor induces NF-kappa B translocation but not human immunodeficiency virus 1 enhancer-dependent transcription.
Authors: Hazan U , Thomas D , Alcami J , Bachelerie F , Israel N , Yssel H , Virelizier J L , Arenzana-Seisdedos F
Published: May 2006

The expression of transiently transfected expression vectors under the control of the long terminal repeat (LTR) of the human immunodeficiency virus (HIV) or its enhancer sequence and the translocation of the HIV enhancer-binding protein NF-kappa B were analyzed in two human T-cell clones stimulated through their T-cell receptor complex or by tumor necrosis factor or phorbol 12-myristate 13-acetate. We found a dissociation of NF-kappa B translocation from transactivation of either the HIV LTR or the HIV enhancer. Interleukin 2 induced proliferation but not NF-kappa B translocation or LTR transactivation. Phorbol ester or specific antigen recognition induced HIV LTR transactivation, whereas stimulation with tumor necrosis factor or antibody to CD3 did not. The two latter signals were nevertheless able to induce NF-kappa B translocation with a pattern in the band-shift assay indistinguishable from that observed using phorbol ester. Our finding that induction of NF-kappa B by tumor necrosis factor or antibody to CD3 is not sufficient to induce HIV enhancer-dependent transcription in cloned T cells contrasts with results obtained in most lymphoblastoid T-cell lines and indicates that normal T lymphocytes differ from tumoral T cells in terms of requirements for HIV LTR activation. Furthermore, our results suggest that events linked to T-cell activation, in addition to NF-kappa B translocation per se, induce functional interactions of the NF-kappa B complex with the HIV enhancer.
Induction of verotoxin sensitivity in receptor-deficient cell lines using the receptor glycolipid globotriosylceramide.
Authors: Waddell T , Cohen A , Lingwood C A
Published: May 2006

Verotoxin 1 is an Escherichia coli-derived subunit toxin that specifically binds to the glycolipid globotriosylceramide and is cytotoxic for cells that contain this plasma membrane glycolipid. Glycolipid incorporation experiments have now been performed using human lymphoid cells of the B lineage that lack this receptor, to conclusively demonstrate that globotriosylceramide alone is a functional receptor for this toxin. Globotriosylceramide incorporated into the membrane of toxin-resistant cells provides intracellular access to verotoxin by receptor-mediated endocytosis. Protein synthesis is then inhibited and globotriosylceramide-containing cells are killed.
Angiotensin II stimulates both Na(+)-H+ exchange and Na+/HCO3- cotransport in the rabbit proximal tubule.
Authors: Geibel J , Giebisch G , Boron W F
Published: May 2006

Angiotensin II (AII) is a potent stimulus for HCO3- reabsorption in the rat proximal tubule in vivo. To determine the ionic mechanism of increased HCO3- reabsorption, we have examined the effect of AII on luminal Na(+)-H+ exchange and basolateral Na+/HCO3- cotransport in perfused S1 proximal tubules isolated from superficial nephrons of the rabbit kidney. Transporter activity was assessed by removing Na+ from both luminal and basolateral (i.e., bath) solutions and determining the rate at which intracellular pH (pHi) increased after Na+ was returned to only the lumen or only the bath. pHi was measured with the pH-sensitive fluorescent dye 2', 7'-bis(2-carboxyethyl)-5(and 6)-carboxyfluorescein. We found that basolateral administration of 1 nM AII not only increased the rate of luminal Na(+)-H+ exchange approximately 3.5-fold but also increased the rate of basolateral Na+/HCO3- cotransport approximately 2.5-fold. 5-(N-Ethyl-N-isopropyl)amiloride (50 microM) blocked luminal Na(+)-H+ exchange before and after stimulation by AII but had no effect on basolateral Na+/HCO3- cotransport. Conversely, 4,4'-diisothiocyanato-2,2'-stilbenedisulfonate (50 microM) blocked basolateral Na+/HCO3- cotransport before and after AII but had no effect on luminal Na(+)-H+ exchange. Our data thus indicate that, at least under the conditions of our assay, AII independently stimulates the transporters responsible for both the luminal and basolateral steps of transepithelial HCO3- reabsorption.
G protein diversity is increased by associations with a variety of gamma subunits.
Authors: Gautam N , Northup J , Tamir H , Simon M I
Published: May 2006

Guanine nucleotide-binding regulatory proteins (G proteins) are heterotrimeric proteins that transduce extracellular signals into intracellular changes. Functionally different G proteins have been identified by their different alpha subunits. The beta and gamma subunits have been assumed to constitute a common pool shared among various G protein heterotrimers. Two gamma subunits (gamma 3 and gamma 4) have been identified through molecular cloning; these are in addition to two subunits (gamma 1 and gamma 2) that were previously characterized. G proteins purified from a variety of mammalian tissues were examined with antisera specific to three of the gamma subunits. The antisera react with different gamma subunits associated with some of the purified G proteins but not all. This demonstrates that different G protein heterotrimers from different tissues carry structurally distinct members of the gamma-subunit family. Diversity in the structure of the gamma as well as the alpha and beta subunits and preferential associations between members of subunit families increase structural and possibly functional diversity of G proteins.
MyoD converts primary dermal fibroblasts, chondroblasts, smooth muscle, and retinal pigmented epithelial cells into striated mononucleated myoblasts and multinucleated myotubes.
Authors: Choi J , Costa M L , Mermelstein C S , Chagas C , Holtzer S , Holtzer H
Published: May 2006

Shortly after their birth, postmitotic mononucleated myoblasts in myotomes, limb buds, and conventional muscle cultures elongate and assemble a cohort of myofibrillar proteins into definitively striated myofibrils. MyoD induces a number of immortalized and/or transformed nonmuscle cells to express desmin and several myofibrillar proteins and to fuse into myosacs. We now report that MyoD converts normal dermal fibroblasts, chondroblasts, gizzard smooth muscle, and pigmented retinal epithelial cells into elongated postmitotic mononucleated striated myoblasts. The sarcomeric localization of antibodies to desmin, alpha-actinin, titin, troponin-I, alpha-actin, myosin heavy chain, and myomesin in these converted myoblasts are indistinguishable from in vivo and in vitro normal myoblasts. Converted myoblasts fuse into typical anisodiametric multinucleated myotubes that often contract spontaneously. Conversion and subsequent expression of the skeletal myogenic program are autonomous events, occurring in four nonmuscle microenvironments consisting of different combinations of foreign extracellular matrix molecules. Early events associated with conversion by MyoD involve (i) withdrawal from the cell cycle, (ii) down-regulation of the subverted cell's ongoing differentiation program, and (iii) initiation of desmin synthesis in presumptive myoblasts and dramatic redistribution of microtubules and desmin intermediate filaments in postmitotic myoblasts.
Long-range movement and fibril association of type X collagen within embryonic cartilage matrix.
Authors: Chen Q A , Gibney E , Fitch J M , Linsenmayer C , Schmid T M , Linsenmayer T F
Published: May 2006

A recent immunoelectron microscopic study of type X collagen in developing cartilage gave results that could be explained by movement of the molecule from one region of the cartilage matrix to another, there becoming associated with preexisting collagen fibrils. In the present study, to test the feasibility of this model we incubated pieces of nonhypertrophic, embryonic chicken sternal cartilage (which has no endogenous type X collagen) in medium with type X collagen and then used immunofluorescence and immunoelectron microscopy to evaluate movement of the molecule through the matrix. The results show that type X collagen molecules can indeed pass through embryonic sternal cartilage matrix and subsequently become fibril-associated.
When is an inhibitory synapse effective?
Authors: Qian N , Sejnowski T J
Published: May 2006

Interactions between excitatory and inhibitory synaptic inputs on dendrites determine the level of activity in neurons. Models based on the cable equation predict that silent shunting inhibition can strongly veto the effect of an excitatory input. The cable model assumes that ionic concentrations do not change during the electrical activity, which may not be a valid assumption, especially for small structures such as dendritic spines. We present here an analysis and computer simulations to show that for large Cl- conductance changes, the more general Nernst-Planck electrodiffusion model predicts that shunting inhibition on spines should be much less effective than that predicted by the cable model. This is a consequence of the large changes in the intracellular ionic concentration of Cl- that can occur in small structures, which would alter the reversal potential and reduce the driving force for Cl-. Shunting inhibition should therefore not be effective on spines, but it could be significantly more effective on the dendritic shaft at the base of the spine. In contrast to shunting inhibition, hyperpolarizing synaptic inhibition mediated by K+ currents can be very effective in reducing the excitatory synaptic potentials on the same spine if the excitatory conductance change is less than 10 nS. We predict that if the inhibitory synapses found on cortical spines are to be effective, then they should be mediated by K+ through GABAB receptors.
Molecular analysis of evolutionary changes in the expression of Drosophila esterases.
Authors: Brady J P , Richmond R C
Published: May 2006

Drosophila melanogaster transformed with the esterase 5 (Est-5) gene from Drosophila pseudoobscura were used to assess the evolutionary basis for differences in the sex- and tissue-specific expression of the esterase 5 (EST 5) enzyme in D. pseudoobscura relative to its homologue in D. melanogaster, EST 6. EST 5 is expressed in the eyes and hemolymph of transformed D. melanogaster just as it is in D. pseudoobscura, but it is not detectable in the ejaculatory duct, where the homologous enzyme, EST 6, is most abundant. EST 5 also occurs at equal levels in both sexes of the transformants and D. pseudoobscura, whereas EST 6 is more abundant in male than in female D. melanogaster. Northern analysis of transformed and untransformed flies indicates that the expression patterns of EST 5 and EST 6 are controlled at the level of transcription and suggests that regulatory differences between Est-6 and Est-5 have evolved mainly through cis-acting regulatory changes in the two loci rather than through alterations in trans-acting factors. Equal expression of EST 5 in male and female transformants also indicates that the X-chromosome-linked Est-5 gene of D. pseudoobscura, when isolated as a 4.5-kilobase restriction fragment, is not dosage compensated after integration into an autosome of D. melanogaster.
Amplification of large artificial chromosomes.
Authors: Smith D R , Smyth A P , Moir D T
Published: May 2006

Yeast artificial chromosome cloning is an attractive technology for genomic mapping studies because very large DNA segments can be readily propagated. However, detailed analyses often require the extensive application of blotting-hybridization techniques because artificial chromosomes are normally present at only one copy per haploid genome. We have developed a cloning vector and host strain that alleviate this problem by permitting copy number amplification of artificial chromosomes. The vector includes a conditional centromere that can be turned on or off by changing the carbon source. Strong selective pressure for extra copies of the artificial chromosome can be applied by selecting for the expression of a heterologous thymidine kinase gene. When this system was used, artificial chromosomes ranging from about 100 to 600 kilobases in size were readily amplified 10- to 20-fold. The selective conditions did not induce obvious rearrangements in any of the clones tested. Reactivation of the centromere in amplified artificial chromosome clones resulted in stable maintenance of an elevated copy number for 20 generations. Applications of copy number control to various aspects of artificial chromosome analysis are addressed.
Evidence for involvement of multiple forms of cytochrome P-450 in aflatoxin B1 metabolism in human liver.
Authors: Forrester L M , Neal G E , Judah D J , Glancey M J , Wolf C R
Published: May 2006

Liver cancer is a major cause of premature death in many areas of Africa and Asia and its incidence is strongly correlated with exposure to aflatoxin B1 (AFB1). Because AFB1 requires metabolic activation to achieve a biological response, there is a need for detailed knowledge of the mechanism of activation to assess individual risk. We have carried out an extensive study using a total of 19 human liver samples to determine the individual variability in the metabolism of the toxin to mutagenic or detoxification products and to identify the specific cytochrome P-450 forms involved in these processes. Metabolism to the toxic 8,9-epoxide or to products mutagenic in the Ames test was found to exhibit very large individual variation. The rates of metabolic activation were highly correlated with both the level of proteins of the P450IIIA gene family and with the total cytochrome P-450 content of the microsomes. In agreement with this, antibodies reacting with P450IIIA proteins were strong inhibitors of both the metabolism and mutagenicity in the majority of the samples. However, the inhibition varied between 50% and 100%. The expression of a protein in the P450IIC gene family also correlated with AFB1 metabolism and mutagenicity. This result therefore indicated the involvement of cytochromes other than P450IIIA in the activation of AFB1 by human liver microsomes. This hypothesis was strongly supported by the finding that antibodies to P450IA2 and P450IIA1 were also effective inhibitors of metabolism in many of the samples. These data demonstrate that, although P450IIIA probably plays an important role in AFB1 activation, several other cytochrome P-450 forms have the capacity to activate the toxin. Similar considerations apply to detoxifying metabolism to aflatoxin Q1 and aflatoxin M1. The levels of expression of many of the forms of cytochrome P-450 involved in AFB1 metabolism are known to be highly sensitive to environmental factors. This indicates that such factors will be an important determinant in individual susceptibility to the tumorigenic action of AFB1.
Identification of a gene that positively regulates expression of listeriolysin, the major virulence factor of listeria monocytogenes.
Authors: Leimeister-Wächter M , Haffner C , Domann E , Goebel W , Chakraborty T
Published: May 2006

We have isolated, by molecular cloning and genetic complementation of a listeriolysin-negative mutant, a gene required for the expression of this virulence factor in Listeria monocytogenes. The mutant strain SLCC53, which was nonhemolytic and avirulent, harbored a deletion of 450 base pairs located approximately 1500 base pairs upstream of the listeriolysin gene. No transcripts corresponding to the listeriolysin gene were detected in the mutant. DNA sequencing of this region from the hemolytic strain EGD revealed that the region deleted in the mutant would abrogate expression of a 27-kDa polypeptide. Introduction of a recombinant plasmid expressing this 27-kDa polypeptide restored hemolytic activity to the mutant and increased the hemolytic activity of the wild-type L. monocytogenes strain EGD. We have designated the gene encoding the 27-kDa polypeptide prfA, for positive regulatory factor of listeriolysin (lisA) expression. The prfA gene regulates transcription of the lisA gene positively.
Organ-specific and systemic autoimmune diseases originate from defects in hematopoietic stem cells.
Authors: Ikehara S , Kawamura M , Takao F , Inaba M , Yasumizu R , Than S , Hisha H , Sugiura K , Koide Y , Yoshida T O
Published: May 2006

Transplantation of bone marrow cells from nonobese diabetic (NOD) mice, a model for type 1 diabetes mellitus, to C3H/HeN mice, which express I-E alpha molecules and have aspartic acid at residue 57 of the I-A beta chain, induced insulitis followed by overt diabetes in the recipient C3H/HeN mice more than 40 weeks after bone marrow transplantation. When cyclosporin A, which perturbs T-cell functions, was injected intraperitoneally into [NOD----C3H/HeN] chimeric mice daily for 1 month, the chimeric mice developed insulitis and overt diabetes within 20 weeks following bone marrow transplantation. Transplantation of bone marrow cells from (NZW x BXSB)F1 mice, which develop lupus nephritis, myocardial infarction, and idiopathic thrombocytopenic purpura, into C3H/HeN or C57BL/6J mice induced in the recipient strains both lupus nephritis and idiopathic thrombocytopenic purpura more than 3 months after transplantation. Transplantation of a stem-cell-enriched population from (NZW x BXSB)F1 mice into normal mice also induced autoimmune disease in the recipients. These results indicate that both systemic autoimmune disease and organ-specific autoimmune disease originate from defects that reside within the stem cells; the thymus and environmental factors such as sex hormones appear to act only as accelerating factors.
Odor types determined by the major histocompatibility complex in germfree mice.
Authors: Yamazaki K , Beauchamp G K , Imai Y , Bard J , Phelan S P , Thomas L , Boyse E A
Published: May 2006

The major histocompatibility complex (MHC) is the prime but not exclusive determinant of genetically specific constitutive body odors, termed odor types, represented strongly in urine of the mouse. Perception of MHC-determined odor types influences reproductive behavior in the contexts of mate choice and maintenance of early pregnancy, tending to favor the propagation of one MHC type over another. How MHC genotype determines MHC odor type is unknown. One possible explanation is that differential odorants are generated by populations of commensal microorganisms whose composition is somehow geared to MHC diversity. This hypothesis was tested in the Y-maze system in which mice are trained to distinguish the urinary odors of MHC-congenic mice. First, it was shown that mice could readily be trained to distinguish the urines of germfree MHC-congenic mice. Second, it was shown that mice trained to distinguish the urines of conventionally maintained MHC-congenic mice could as readily distinguish the urines of germfree MHC-congenic mice. These results imply that MHC-determined odor types do not depend on odorants generated by microorganisms.
Identification of nuclear tau isoforms in human neuroblastoma cells.
Authors: Loomis P A , Howard T H , Castleberry R P , Binder L I
Published: May 2006

The tau proteins have been reported only in association with microtubules and with ribosomes in situ, in the normal central nervous system. In addition, tau has been shown to be an integral component of paired helical filaments, the principal constituent of the neurofibrillary tangles found in brains of patients with Alzheimer disease and of most aged individuals with Down syndrome (trisomy 21). We report here the localization of the well-characterized Tau-1 monoclonal antibody to the nucleolar organizer regions of the acrocentric chromosomes and to their interphase counterpart, the fibrillar component of the nucleolus, in human neuroblastoma cells. Similar localization to the nucleolar organizer regions was also observed in other human cell lines and in one monkey kidney cell line but was not seen in non-primate species. Immunochemically, we further demonstrate the existence of the entire tau molecule in the isolated nuclei of neuroblastoma cells. Nuclear tau proteins, like the tau proteins of the paired helical filaments, cannot be extracted in standard SDS-containing electrophoresis sample buffer but require pretreatment with formic acid prior to immunoblot analysis. This work indicates that tau may function in processes not directly associated with microtubules and that highly insoluble complexes of tau may also play a role in normal cellular physiology.
Autogenous suppression of an opal mutation in the gene encoding peptide chain release factor 2.
Authors: Kawakami K , Nakamura Y
Published: May 2006

The peptide chain release factor 2 (RF2) gene, prfB, was cloned from Salmonella typhimurium by DNA hybridization using the Escherichia coli prfB probe. The nucleotide and amino acid sequences of prfB are 87.0% and 95.6% homologous between E. coli and S. typhimurium, respectively, including an in-frame premature UGA stop codon at position 26, the site of +1 frameshift for mature RF2 synthesis. The supK584 mutation, which had been isolated as a recessive UGA suppressor in S. typhimurium, caused an opal (UGA) substitution at amino acid position 144 in the prfB gene. Complementation, reversion, and gene fusion analyses led to the conclusion that supK is a S. typhimurium RF2 mutation and this opal RF2 mutation generates a UGA suppressor activity, presumably because of inefficient translation termination due to the reduced cellular level of RF2. In fact, suppression of the supK opal mutation results from a form of autogenous control of RF2 synthesis.
Introduction of nerve growth factor (NGF) receptors into a medulloblastoma cell line results in expression of high- and low-affinity NGF receptors but not NGF-mediated differentiation.
Authors: Pleasure S J , Reddy U R , Venkatakrishnan G , Roy A K , Chen J , Ross A H , Trojanowski J Q , Pleasure D E , Lee V M
Published: May 2006

Expression of the cloned human nerve growth factor receptor (NGFR) cDNA in cell lines can generate both high- and low-affinity binding sites. Since the inability to respond appropriately to differentiation factors such as NGF may contribute to determining the malignant phenotype of neuroblastomas, we sought to determine whether the same is true of medulloblastomas. To generate a human central nervous system neuronal cell line that would respond to NGF, we infected the medulloblastoma cell line D283 MED with a defective retrovirus carrying the cDNA coding for the human NGFR. The resultant cells (MED-NGFR) expressed abundant low- and high-affinity NGFRs, and NGF treatment induced a rapid transient increase of c-fos mRNA in the NGFR-expressing cells but not in the parent line or in cells infected with virus lacking the cDNA insert. However, the MED-NGFR cells did not internalize the NGFR at high efficiency, nor did they differentiate in response to NGF. Three important conclusions emerge from this study: (i) internalization of NGFRs is not necessary for some early rapid transcriptional effects of NGF; (ii) an unknown factor(s) that cooperates with the cloned NGFR in allowing high-affinity NGF binding is found in a primitive central nervous system cell line; and (iii) NGFRs introduced into and expressed by D283 MED (i.e., MED-NGFR) cells are partially functional but are unable to induce differentiation in these primitive neuron-like tumor cells, implying that high-efficiency receptor-mediated endocytosis of NGF and its receptor may be a necessary step in the cascade of events leading to NGF-mediated differentiation.
Human monoclonal antibody that recognizes the V3 region of human immunodeficiency virus gp120 and neutralizes the human T-lymphotropic virus type IIIMN strain.
Authors: Scott C F , Silver S , Profy A T , Putney S D , Langlois A , Weinhold K , Robinson J E
Published: May 2006

We describe a human IgG1 monoclonal antibody (N701.9b) derived by Epstein-Barr virus transformation of B cells from a human immunodeficiency virus-seropositive asymptomatic donor. This antibody was shown to recognize the principal neutralizing domain contained within the V3 region of gp120 of the MN strain of human immunodeficiency virus and MN-like strains, as determined by binding to the PB-1 fragment of MN gp120 and to synthetic peptides corresponding to the V3 region of MN and related virus strains. The epitope identified by monoclonal antibody N701.9b was mapped to a segment of V3 containing at least 7 amino acids (amino acids 316-322), which is located in the "tip" and "right" side of the V3 loop of the MN strain. Furthermore, this antibody manifested potent type-specific fusion-inhibitory activity against the MN strain but not against the IIIB or RF virus strains. This antibody also neutralized four virus isolates that had MN-like V3 region sequences and failed to neutralize three other strains containing unrelated V3 region sequences. Our findings confirm that the V3 region stimulates type-specific neutralizing antibody during natural human immunodeficiency virus infection in humans. The potential clinical use of this antibody is discussed.
A DNA gyrase-binding site at the center of the bacteriophage Mu genome is required for efficient replicative transposition.
Authors: Pato M L , Howe M M , Higgins N P
Published: May 2006

We have discovered a centrally located site that is required for efficient replication of bacteriophage Mu DNA and identified it as a strong DNA gyrase-binding site. Incubation of Mu DNA with gyrase and enoxacin revealed a cleavage site 18.1 kilobases from the left end of the 37.2-kilobase genome. Two observations indicate a role for the site in Mu replication: mutants of Mu, able to grow on an Escherichia coli gyrB host that does not allow growth of wild-type Mu, were found to possess single-base changes resulting in more efficient gyrase binding and cleavage at the site. Introduction of a 147-base-pair deletion that eliminated the site from a prophage inhibited the onset of Mu replication for greater than 1 hr after induction.
Isolation of human T-cell lymphotropic virus type 2 from Guaymi Indians in Panama.
Authors: Lairmore M D , Jacobson S , Gracia F , De B K , Castillo L , Larreategui M , Roberts B D , Levine P H , Blattner W A , Kaplan J E
Published: May 2006

Human T-lymphotropic virus type I (HTLV-I) is associated with adult T-cell leukemia/lymphoma and with a chronic degenerative myelopathy. However, another major type of HTLV, HTLV-II, has been isolated only sporadically, and little is known of disease associations, transmission routes, and risk factors for HTLV-II infection. Recent studies indicate that a high percentage of certain groups of i.v. drug users and blood donors are infected with HTLV-II. Seroepidemiologic studies have found an elevated rate of seroreactivity to HTLV among Guaymi Indians from Bocas del Toro Province, Panama. To identify the cause of seroreactivity among this unique population we used HTLV-II-specific polymerase chain reaction techniques to detect HTLV genetic sequences from blood leukocytes of three seropositive Guaymi Indians. The HTLV-II primer-amplified polymerase chain reaction products from two of these subjects were partially sequenced and matched published HTLV-II nucleotide sequences in both p24 gag (94% of 107 bases) and pol (98% of 112 bases) regions. A CD4+ T-lymphocyte line established from one of these same subjects produced HTLV-II-specific proteins when tested in antigen-capture and immunoblot assays, as well as mature HTLV particles. The demonstration of HTLV-II infection in this geographically and culturally isolated Central American Indian population without typical risk factors for HTLV infection suggests that HTLV-II infection is endemic in this population and provides an important clue to potential natural reservoir for this virus.
Cultured normal human hepatocytes do not synthesize lipoprotein-associated coagulation inhibitor: evidence that endothelium is the principal site of its synthesis.
Authors: Bajaj M S , Kuppuswamy M N , Saito H , Spitzer S G , Bajaj S P
Published: May 2006

Human plasma contains a factor Xa-dependent inhibitor of tissue factor/factor VIIa complex termed lipoprotein-associated coagulation inhibitor (LACI). The present study examines the site(s) of LACI synthesis. In this study, cultured hepatocytes isolated from normal human liver were found to be essentially negative in LACI mRNA as revealed by Northern blot analysis using a full-length LACI cDNA as probe. The conditioned media from these cultures were also essentially negative for LACI activity. Similarly, poly(A)+ RNA obtained from normal human liver did not contain detectable LACI mRNA. In contrast, cultured human umbilical vein endothelial cells and human lung tissue (rich in endothelium) both contained abundant amounts of LACI mRNA. Moreover, erythrocyte lysates and culture media from normal monocytes, lymphocytes, or neutrophils did not contain measurable LACI activity; these cells were also negative for LACI mRNA. Platelets, however, contained LACI activity. The likely source of platelet LACI is the megakaryocyte cell since a megakaryocyte cell line (MEG-01) was found to contain LACI mRNA and to secrete small amounts of LACI activity. Additionally, human vascular smooth muscle cells and lung fibroblasts were also found to synthesize only small amounts of LACI. From these observations, we conclude that normal liver does not synthesize LACI and that endothelium is the principal source of plasma LACI. The undegraded LACI synthesized by endothelial cells had a molecular weight of approximately 41,000.