bluuebunny/crossref_metadata_2025 huggingface dataset:
v-abl activates embryonic globin gene expression in mouse erythroleukemia cells.
Authors: Lopez A R , Barker J , Deisseroth A B
Published: May 2006

The Philadelphia chromosome translocation, which is present in 90-95% of chronic myelogenous leukemia patients, involves translocation of the c-abl protooncogene to chromosome 22 and is accompanied by activation of embryonic globin gene expression in the K562 chronic myelogenous leukemia cell line. To test directly if the protein products of the translocated c-abl protooncogene can activate embryonic globin gene expression, we transfected the v-abl oncogene (which shares the property of autophosphorylation with the translocated c-abl protooncogene) into mouse erythroleukemia cells. v-abl-transfected mouse erythroleukemia cells, which contained multiple copies of the v-abl transgenome, exhibited activation of mouse embryonic globin gene expression. These results suggest that the translocated c-abl protooncogene of the Philadelphia chromosome translocation is central to the pathogenesis of chronic myelogenous leukemia and that it may result in the activation of embryonic globin genes in some chronic myelogenous leukemia cell lines.
Turbulent fluid shear stress induces vascular endothelial cell turnover in vitro.
Authors: Davies P F , Remuzzi A , Gordon E J , Dewey C F , Gimbrone M A
Published: May 2006

The effects of hemodynamic forces upon vascular endothelial cell turnover were studied by exposing contact-inhibited confluent cell monolayers to shear stresses of varying amplitude in either laminar or turbulent flow. Laminar shear stresses (range, 8-15 dynes/cm2; 24 hr) induced cell alignment in the direction of flow without initiating the cell cycle. In contrast, turbulent shear stresses as low as 1.5 dynes/cm2 for as short a period as 3 hr stimulated substantial endothelial DNA synthesis in the absence of cell alignment, discernible cell retraction, or cell loss. The results of these in vitro experiments suggest that in atherosclerotic lesion-prone regions of the vascular system, unsteady blood flow characteristics, rather than the magnitude of wall shear stress per se, may be the major determinant of hemodynamically induced endothelial cell turnover.
Sensitivities of rat kidney thick ascending limbs and collecting ducts to vasopressin in vivo.
Authors: Elalouf J M , Di Stefano A , de Rouffignac C
Published: May 2006

Clearance experiments were performed to characterize the sensitivity to vasopressin of the thick ascending limbs and collecting duct system of the rat kidney. The response of the thick ascending limbs was evaluated by measuring the Mg2+ excretion rate in the urine, since the [arginine-8] vasopressin-mediated effects on Mg2+ excretion are the direct result of a stimulation of Mg2+ reabsorption in this nephron segment, and the response of the collecting ducts was evaluated by changes in urine flow. To avoid the effects of parathyroid hormone, glucagon, and calcitonin, which stimulate Mg2+ reabsorption in the thick ascending limb and distal tubule, and of calcitonin, which increases the permeability of the cortical collecting ducts to water, experiments were performed on Brattleboro D. I. rats (with hereditary diabetes insipidus, due to a lack of [Arg8]vasopressin) acutely deprived of endogenous parathyroid hormone, calcitonin, and glucagon. Vasopressin infused at rates up to 5 pg/min did not reduce the Mg2+ fractional excretion rate, whereas at 5 pg/min water excretion was decreased by 50%. The half-maximal reduction of Mg2+ excretion occurred at vasopressin infusion rates 4-6 times higher than those necessary to diminish the water excretion rate to the same extent. We conclude that in vivo, two segments involved in the production of concentrated urine have different sensitivities to vasopressin and that this difference in sensitivity is very similar for the biological response in vivo and the adenylate cyclase activation in vitro. We suggest that both the magnitude and the nature of the effects of [Arg8]vasopressin on the kidney may vary according to the required antidiuretic response.
Protein kinase C as a component of a nerve growth factor-sensitive phosphorylation system in PC12 cells.
Authors: Hama T , Huang K P , Guroff G
Published: May 2006

The treatment of PC12 cells with either nerve growth factor or phorbol 12-myristate 13-acetate caused a decrease in the phosphorylation of a soluble 100-kDa protein (Nsp100). After treatment with nerve growth factor, the activity of Ca2+/phospholipid-dependent protein kinase (protein kinase C) in the cytosol was increased. When the cytosol from untreated PC12 cells was preincubated with purified protein kinase C and its cofactors, the phosphorylation of Nsp100 was decreased. The preincubation of cytosol from nerve growth factor-treated PC12 cells with protein kinase C did not decrease Nsp100 phosphorylation further. Moreover, preincubation of partially purified Nsp100 kinase with protein kinase C decreased its ability to phosphorylate Nsp100. These results suggest that the binding of nerve growth factor to its receptor on PC12 cells causes an increase in the activity of protein kinase C in the cytosol and phosphorylation of Nsp100 kinase, which in turn lowers its ability to phosphorylate Nsp100.
Frequency of 6-thioguanine-resistant T cells is inversely related to the declining T-cell activities in aging mice.
Authors: Inamizu T , Kinohara N , Chang M P , Makinodan T
Published: May 2006

The frequency of clonable 6-thioguanine-resistant (6-TGr) splenic T cells increased moderately with age in female BALB/c mice ranging in age from 3 to 32 months; however, the correlation between the frequency of clonable 6-TGr cells and age was weak. Those clonable 6-TGr T cells were deficient in hypoxanthine/guanine phosphoribosyltransferase (HGPRT) activity and sensitive to hypoxanthine/aminopterin/thymidine medium, as in the case of HGPRT-deficient L5178Y mouse lymphoma cells. When splenic T cells of individual aging mice were assessed simultaneously for the frequency of clonable 6-TGr T cells and for their ability to produce interleukin 2 or to proliferate in response to mitogenic stimulation, an inverse correlation was observed. These results indicate that the frequency of 6-TGr T cells is more closely related to physiologic age than chronologic age. This would mean that the frequency could be used as an index of physiologic age and that the T cells could serve as a cellular model relating gene alterations to physiologic age.
Primary antibody responses to a well-defined and unique hapten are not enhanced by preimmunization with carrier: analysis in a viral model.
Authors: Gupta S C , Hengartner H , Zinkernagel R M
Published: May 2006

We used a viral model to reexamine classical experiments showing that mice previously primed with a "carrier" molecule alone and then challenged with the carrier-hapten conjugate exhibited an enhanced antihapten antibody response. Mice were primed with live or UV-inactivated vesicular stomatitis virus (VSV) Indiana (Ind) serotype with or without complete Freund's adjuvant. After challenge with VSV New Jersey (NJ), these mice developed a secondary-type IgG response, measured by antibody binding in an ELISA, against both VSV-Ind and VSV-NJ. The same result was found for the reciprocal experiments where mice were primed with VSV-NJ. Similarly, when mice were primed with live VSV, UV-inactivated VSV, or purified VSV glycoprotein G of Ind or NJ serotype and later were challenged with dinitrophenyl (N2ph)-conjugated, UV-inactivated VSV or with N2ph-conjugated G protein of either serotype, they exhibited a secondary-type anti-N2ph antibody response as demonstrated by the binding of IgG to dinitrophenylated bovine serum albumin measured by ELISA. In contrast, when neutralizing antibody responses were monitored, VSV-Ind-primed mice challenged with VSV-NJ developed a strictly primary type of anti-VSV-NJ response and vice versa. We conclude that preexistent helper T cells specific for shared carrier determinants do not improve virgin B-cell responses specific for "new," unique determinants that are the target for the biologically relevant neutralizing antibodies. These findings suggest that priming of B cells rather than of helper T cells may be of importance to induce protective immunity mediated by antibodies.
Prostaglandin E2 administered via anus causes immunosuppression in male but not female rats: a possible pathogenesis of acquired immune deficiency syndrome in homosexual males.
Authors: Kuno S , Ueno R , Hayaishi O
Published: May 2006

To explain a possible pathogenesis of acquired immune deficiency syndrome (AIDS) in homosexual males, we propose the following hypothesis. Prostaglandin E2 in human semen given via anus during anal intercourse may cause an immune dysregulation in the male semen recipients; this immunosuppressive effect of prostaglandin E2 may be one of the underlying factors that stimulate AIDS-associated virus infection or that trigger the latent AIDS-associated virus. This hypothesis is supported by the following experimental results. Anal infusion of prostaglandin E2 or D2 into male rats reduced in vitro responses of T lymphocytes to phytohemagglutinin. However, the T-cell response of female rats was not reduced significantly by the anal infusion of seminal prostaglandins.
Phorbol ester-induced growth arrest of murine myelomonocytic leukemic cells with virus-disrupted myb locus is not accompanied by decreased myc and myb expression.
Authors: Shen-Ong G L , Holmes K L , Morse H C
Published: May 2006

A class of mouse neoplasms termed Abelson virus-induced plasmacytoid lymphosarcomas has previously been found to express abnormal myb transcripts due to a helper virus insertion into the 5' end of the c-myb locus. We have established clonal cell lines from these tumors and have shown that they all express markers characteristic of myelomonocytic, rather than lymphoid, cells. Treatment of the plasmacytoid lymphosarcoma lines with phorbol 12-myristate 13-acetate led to various degrees of growth arrest, presumably due to myelomonocytic differentiation. To date, characterization of myeloid cell lines with varying responses to phorbol 12-myristate 13-acetate has not been reported. The different clonal plasmacytoid lymphosarcoma lines should, therefore, provide a good model to help elucidate the role of altered myb locus and other protooncogenes in myelomonocytic leukemia. Contrary to studies on induced differentiation of myelomonocytic cells with normal myb locus, our results on plasmacytoid lymphosarcoma cells indicate that reduced myb and myc expression may not be obligatory for growth arrest to occur. The present study, however, supports the previous notion that the myb transforming ability may be restricted to cells of the myelomonocytic lineage. In addition, we found that only the more mature cells can undergo prolonged phorbol 12-myristate 13-acetate-induced growth arrest, suggesting that the maintenance of these leukemic cells in their proliferative state, presumably by the myb gene product, can be overcome with appropriate differentiation signal(s).
Spatiotemporal relationship of embryonic cholinesterases with cell proliferation in chicken brain and eye.
Authors: Layer P G , Sporns O
Published: May 2006

Close relationships between acetylcholinesterase (AcChoEase; acetylcholine acetylhydrolase, true cholinesterase, EC, 3.1.1.7) and butyrylcholinesterase (BtChoEase, acylcholine acylhydrolase, pseudocholinesterase, EC, 3.1.1.8) with cell proliferation were observed in the early chicken brain. These include the following: BtChoEase is transiently accumulating in patchy fashion on the ventricular side of the neuroepithelium shortly before AcChoEase appears in cell bodies along the opposing mantle layer. The amount of BtChoEase in retina and brain is greatest in the early phase (E3-E5, or incubation periods of 3-5 days); in retina it decreases about 2 days later than in brain. However, AcChoEase expression increases with time, in inverse order to that of BtChoEase. In both tissues decrease of cell proliferation is closely followed by decrease in BtChoEase. A double-labeling technique of cholinesterase staining together with [3H]thymidine autoradiography reveals proliferation zones that are diffusely stained by BtChoEase but not by AcChoEase. Patches intensely stained for BtChoEase accompany clusters of cells in final stages of mitosis on their way to the differentiation zone, where they begin expressing AcChoEase. By applying different thymidine pulses, we identify an 11-hr lag from the last thymidine-uptake to full AcChoEase expression. (iv) These findings are confirmed by studying lens development, where areas of proliferation and differentiation are well separated. The spatiotemporal pattern of the transition of neuroblasts from a proliferating into a differentiating state correlates with the expression of BtChoEase just before and during mitosis and that of AcChoEase about 11 hr after mitosis. Thus cholinesterases could be involved in the regulation of this transition.
Reaction of argininosuccinase with bromomesaconic acid: role of an essential lysine in the active site.
Authors: Lusty C J , Ratner S
Published: May 2006

We have undertaken studies on bovine liver argininosuccinase (L-argininosuccinate arginine-lyase, EC 4.3.2.1) with the active site-directed reagent bromo[U-14C]mesaconic acid, an analogue of fumaric acid. Reactivity, measured by enzyme inactivation, followed pseudo-first-order kinetics, and the rate increased with reagent concentration. Argininosuccinate completely protected the enzyme against inactivation, but neither arginine nor fumarate was protective. A plot of the degree of inactivation as a function of alkyl groups incorporated was extrapolated to 4 mol per mol of enzyme, or 1 mol per active site. After large-scale alkylation of the enzyme (and digestion with trypsin), two 14C-labeled tryptic peptides were isolated. These were chemically sequenced by the Edman method. The amino acid sequences proved to be identical with regions of the deduced amino acid sequences or argininosuccinases from human and yeast sources [O'Brien, W. E., McInnes, R., Kalumuck, K. & Adcock, M. (1986) Proc. Natl. Acad. Sci. USA 83, 7211-7215; Beacham, I. R., Schweitzer, B. W., Warrick, H. M. & Carbon, J. (1984) Gene 29, 271-279]. The 14C-labeled tryptic peptide in the active site region had the sequence Gly-Leu-Glu-Xaa-Ala-Gly-Leu-Leu-Thr-Lys; Xaa represents an unknown phenylthiohydantoin derivative detected in cycle 4. The corresponding amino acid was identified as lysine-51 on the basis of sequence similarity with human and yeast amino acid sequences in this region. The reaction of the enzyme with the alkylating agent and the specific protection against inactivation by argininosuccinate suggest that this lysine residue has an essential role in the binding of argininosuccinate to the enzyme and, consequently, is essential for catalysis.
Immunoglobulin A stimulates growth of the extrahepatic bile duct in BALB/c mice.
Authors: Fallon-Friedlander S , Boscamp J R , Morecki R , Lilly F , Horwitz M S , Glaser J H
Published: May 2006

BALB/c mice injected with seven 0.15-ml samples of whole rabbit serum over a 2-week period developed nonneoplastic proliferation of the extrahepatic bile duct epithelium and glandular components. Sera from other animals, including bovines, humans, pigs, goats, and chickens as well as non-serum-containing secretions such as human breast milk and bile also produced this effect. Partial purification utilizing gel filtration and affinity chromatography of the active 33-65% saturated ammonium sulfate precipitate of whole serum indicated that the distribution and characteristics of this glycoprotein showed some similarities with those of IgA. Chromatographically purified human IgA was administered to BALB/c mice and was found to induce bile duct proliferation identical to that seen with whole human serum. Purified human IgG and IgM had no activity. Since IgA-containing serum from BALB/c mice was inactive, it appears that heterologous IgA functions as a specific extrahepatic bile duct growth factor (BDGF) in BALB/c mice. Murine susceptibility to the growth-stimulating effect of serum was strain specific; genetic studies utilizing crosses of susceptible (BALB/c) and resistant (C57BL/10) strains of mice revealed that the ability to respond to the infusion of BDGF is inherited in a polygenic fashion.
Androstenedione may organize or activate sex-reversed traits in female spotted hyenas.
Authors: Glickman S E , Frank L G , Davidson J M , Smith E R , Siiteri P K
Published: May 2006

Spotted hyenas (Crocuta crocuta Erxleben) present a unique syndrome of reversal in behavioral and anatomical distinction between the sexes: females are heavier and more aggressive than males and dominant over them. The female's external genitalia include a false scrotum and a fully erectile pseudopenis through which mating and birth take place. Results of studies of circulating testosterone levels in wild spotted hyenas do not account for the "male-like" characteristics of the female. Androstenedione, however, is consistently higher in females than in males, particularly during early infancy. Experiments on rodents show that androstenedione can be a potent organizer of anatomical and behavioral differentiation. This study suggests that it may also produce the profound virilization of female spotted hyenas.
Identification of Fc and F(ab')2 IgG receptors on human platelets.
Authors: Vancura S , Steiner M
Published: May 2006

The human platelet receptors for normal, nonimmune IgG and its F(ab')2 and Fc fragments were studied by the use of a cleavable, bifunctional, photoactivable, 125I-labeled cross-linking agent. Derivatization of the ligands with N-[4-(p-azido-m-[125I]iodophenylazo) benzoyl]-3-aminopropyl-N'-oxysuccinimide ester (Denny-Jaffe reagent) reduced their binding to platelets by greater than or equal to 20%. Cleavage of the azo linkage of the Denny-Jaffe reagent, which splits the molecule so that its 125I-labeled portion becomes associated with the receptor half of the cross-linked ligand-receptor complex, was utilized to directly identify receptors for the various immunoglobulin ligands. Specificity of the binding reaction could be demonstrated by suppressing the iodination of the receptors with excess nonderivatized ligand. Two principal IgG-related receptors could be identified by high-resolution NaDodSO4/PAGE and subsequent analysis of the electrophoretically transferred peptides to nitrocellulose filters for localization of radioactivity and immunological characterization. Intact monomeric IgG and F(ab')2 fragments derived from it appeared to have the glycoprotein IIIa as the major receptor, whereas Fc fragments bound predominantly to a peptide of Mr approximately 200,000 (Mr, approximately 50,000 under rigorous reducing conditions).
Turning (Ir gene) low responders into high responders by antibody manipulation of the developing immune system.
Authors: Martinz C , Marcos M A , Pereira P , Marquez C , Toribio M , de la Hera A , Cazenave P A , Coutinho A
Published: May 2006

The ability of helper T cells directed against trinitrophenyl-modified syngeneic spleen cells to recognize low-hapten densities on target cells is under major histocompatibility complex-linked Ir gene control. Thus, BALB/c (H-2d) mice are low responders while H-2 congenic BALB.C3H (H-2k) mice are high responders. Immunization of adult BALB/c mice with the monoclonal antibody F6(51), directed to shared idiotopes by anti-trinitrophenyl antibodies and clonal receptors on anti-trinitrophenyl-self helper T cells, leads to the production of high titers of circulating idiotype, has no influence on helper T cell idiotypic profiles, but shifts to a high-responder phenotype the ability of helper T cells to recognize low-hapten densities. These effects on Ir gene phenotype are even more striking in untreated progenies from F6(51)-immunized BALB/c females, which are better responders than genetically high-responder BALB.C3H mice, although completely different in the expression of the F6(51)-defined clonotype. The general significance of these findings on Ir gene-directed T-cell repertoire selection is discussed, for they constitute formal evidence against antigen-presentation as a mechanism of Ir gene effects and strong support for the importance of maternal influences on the development of T-cell repertoires.
Monoclonal anti-idiotypic antibody mimics the CD4 receptor and binds human immunodeficiency virus.
Authors: Chanh T C , Dreesman G R , Kennedy R C
Published: May 2006

A monoclonal anti-idiotypic (anti-Id) antibody, HF1.7, was generated against anti-Leu-3a, a mouse monoclonal antibody (mAb) specific for the CD4 molecule on human helper/inducer T lymphocytes. The anti-Id nature of HF1.7 was demonstrated by the following properties. (i) It reacted in a solid-phase immunoassay with anti-Leu-3a and not with a panel of irrelevant mouse mAbs. (ii) It partially inhibited the binding of anti-Leu-3a to CD4+ T cells. (iii) It detected a common idiotype present on various anti-CD4 mAbs. Because the CD4 molecule represents the receptor site for human immunodeficiency virus (HIV), the etiologic viral agent of acquired immunodeficiency syndrome, we examined the ability of the anti-mAb HF1.7 to mimic CD4 and bind HIV. This anti-Id mAb reacted with HIV antigens in commercial HIV ELISAs and recognized HIV-infected human T cells but not uninfected cells when analyzed by flow cytofluorometry. Attesting further to the HIV specificity, the anti-Id mAb reacted with a recombinant gp160 peptide and a molecule of Mr 110,000-120,000 in immunoblot analysis of HIV-infected cell lysates. The anti-Id mAb also partially neutralized HIV infection of human T cells in vitro. These results strongly suggest that this anti-Id mAb mimics the CD4 antigenic determinants involved in binding to HIV.
Varicella-zoster virus as a live vector for the expression of foreign genes.
Authors: Lowe R S , Keller P M , Keech B J , Davison A J , Whang Y , Morgan A J , Kieff E , Ellis R W
Published: May 2006

The previous demonstration of the efficacy and tolerability of the Oka strain of varicella-zoster virus (VZV) in clinical trials involving vaccination of both normal and immunocompromised individuals has laid the foundation for its use in preventing chickenpox. In this context, VZV could be useful as a vector for vaccinating against other infectious agents as well. As an initial application, a live recombinant VZV expressing Epstein-Barr virus (EBV) membrane glycoproteins (gp350/220) was generated by inserting a gene fusion of the VZV gpI promoter and hydrophobic leader-encoding sequence with the gp350/220 coding sequence into the thymidine kinase (TK) gene of VZV (Oka). Insertion of the foreign DNA into the thymidine kinase gene was demonstrated by Southern blot analysis and the ability of the recombinant virus to replicate in the presence of bromodeoxyuridine. RNA splicing, glycosylation, and plasma membrane presentation of gp350/220 in cells infected with the recombinant virus were similar to those seen in EBV-infected cells. In addition, the expression of VZV-specific glycoproteins was unaltered by the concomitant expression of this large foreign glycoprotein. Thus, VZV can be used as a live viral vector for active immunization against EBV and other pathogens.
A protein kinase antigenically related to pp60v-src possibly involved in yeast cell cycle control: positive in vivo regulation by sterol.
Authors: Dahl C , Biemann H P , Dahl J
Published: May 2006

The effects of ergosterol, yeast's natural sterol, on cell cycling and a protein kinase antigenically related to pp60v-src were examined in a sterol auxotroph of Saccharomyces cerevisiae. Sterol-depleted cells accumulate in an unbudded, G1 state. Cell budding and proliferation are reinitiated upon addition of nonlimiting ergosterol or cholesterol with trace ergosterol, whereas cholesterol or trace ergosterol alone is less effective. Stimulation of a protein kinase associated with immune complexes of yeast protein and anti-pp60v-src shows a positive correlation with exit from the G1 phase following ergosterol addition. Ergosterol-stimulated cells also demonstrate an increase in phosphatidylinositol kinase activity. The data suggest that hormonal levels of ergosterol (effective concentration, approximately equal to 1 nM) participate in a signaling process associated with a protein kinase possibly involved in yeast cell cycle control.
Special type of morphological reorganization induced by phorbol ester: reversible partition of cell into motile and stable domains.
Authors: Dugina V B , Svitkina T M , Vasiliev J M , Gelfand I M
Published: May 2006

The phorbol ester phorbol 12-myristate 13-acetate (PMA) induced reversible alteration of the shape of fibroblastic cells of certain transformed lines--namely, partition of the cells into two types of domains: motile body actively extending large lamellas and stable narrow cytoplasmic processes. Dynamic observations have shown that stable processes are formed from partially retracted lamellas and from contracted tail parts of cell bodies. Immunofluorescence microscopy and electron microscopy of platinum replicas of cytoskeleton have shown that PMA-induced narrow processes are rich in microtubules and intermediate filaments but relatively poor in actin microfilaments; in contrast, lamellas and cell bodies contained numerous microfilaments. Colcemid-induced depolymerization of microtubules led to contraction of PMA-induced processes; cytochalasin B prevented this contraction. It is suggested that PMA-induced separation of cell into motile and stable parts is due to directional movement of actin structures along the microtubular framework. Similar movements may play an important role in various normal morphogenetic processes.
Two forms of 1B236/myelin-associated glycoprotein, a cell adhesion molecule for postnatal neural development, are produced by alternative splicing.
Authors: Lai C , Brow M A , Nave K A , Noronha A B , Quarles R H , Bloom F E , Milner R J , Sutcliffe J G
Published: May 2006

The structures of two rat brain-specific 1B236 mRNAs, alternative splice products from a single gene regulated differently during postnatal brain development, were deduced from full-length cDNA clones. The 626- and 582-amino acid-long encoded proteins are indistinguishable from two forms of myelin-associated glycoprotein, a cell adhesion molecule involved in axonal-glial and glial-glial interactions in postnatal brain development, particularly in myelination. The two proteins share a single membrane-spanning domain and a glycosylated N terminus but differ in the structures of their C termini. The N terminus consists of five domains related in sequence to each other and to immunoglobulin-like molecules, especially the neural cell adhesion molecule N-CAM, suggesting a common structure for cell adhesion molecules.
Induction of high titers of anti-IgE by immunization of inbred mice with syngeneic IgE.
Authors: Haba S , Nisonoff A
Published: May 2006

We have generated high titers (up to 1 mg/ml) of antibodies to isotypic determinants of IgE by immunization of A/J mice with syngeneic monoclonal IgE conjugated to keyhole limpet hemocyanin. As much as 3 mg of anti-idiotypic antibodies per ml was induced at the same time. In contrast to conventional rheumatoid factors, the anti-isotypic antibodies are of moderately high affinity (10(7)-10(8) M-1). Assays of the anti-IgE antisera indicated the presence of IgE, both free and in the form of immune complexes; the latter values are minimum estimates owing to masking of isotypic determinants. Regulatory effects of these high titers of anti-IgE can now be investigated. Such studies will be facilitated by the availability of monoclonal, syngeneic anti-IgE antibodies.
Synchronous oscillations in microtubule polymerization.
Authors: Carlier M F , Melki R , Pantaloni D , Hill T L , Chen Y
Published: May 2006

Under conditions where microtubule nucleation and growth are fast (i.e., high magnesium ion and tubulin concentrations and absence of glycerol), microtubule assembly in vitro exhibits an oscillatory regime preceding the establishment of steady state. The amplitude of the oscillations can represent greater than 50% of the maximum turbidity change and oscillations persist for up to 20 periods of 80 s each. Oscillations are accompanied by extensive length redistribution of microtubules. Preliminary work suggests that the oscillatory kinetics can be simulated using a model in which many microtubules undergo synchronous transitions between growing and rapidly depolymerizing phases, complicated by the kinetically limiting rate of nucleotide exchange on free tubulin.
Isolation and characterization of a nematode transposable element from Panagrellus redivivus.
Authors: Link C D , Graf-Whitsel J , Wood W B
Published: May 2006

We have isolated a transposable element, designated PAT-1, from the free-living nematode Panagrellus redivivus. P. redivivus strain C15 was found to have a high spontaneous mutation frequency compared to the standard Caenorhabditis elegans laboratory strain N2. To characterize the genetic lesions occurring in spontaneous C15 mutants, we molecularly cloned the homolog of the C. elegans unc-22 gene from wild-type P. redivivus and two strains carrying spontaneous mutations in this gene. One of these mutations resulted from the insertion of a 4.8-kilobase segment of repetitive DNA. This repetitive element (PAT-1) varies in copy number (10-50 copies) and location in different P. redivivus strains and is absent from C. elegans. The element could be useful as a transformation vector for C. elegans. Our approach is a general one that could be used to isolate additional nematode transposons from other species.
Criteria for regular spacing of reacting Mach stems
Authors: Majda Andrew J.
Published: May 2006

Nonlinear geometric optics is developed to construct translating self-similar high-frequency nonlinear wave patterns as perturbed solutions behind reacting shock waves. The geometric conditions involving these wave patterns that yield the maximum acoustic amplification lead to criteria for both the formation and regular spacing dimension for Mach stems in reacting gases.
Origin of noncoding DNA sequences: molecular fossils of genome evolution.
Authors: Naora H , Miyahara K , Curnow R N
Published: May 2006

The total amount of noncoding sequences on chromosomes of contemporary organisms varies significantly from species to species. We propose a hypothesis for the origin of these noncoding sequences that assumes that (i) an approximately equal to 0.55-kilobase (kb)-long reading frame composed the primordial gene and (ii) a 20-kb-long single-stranded polynucleotide is the longest molecule (as a genome) that was polymerized at random and without a specific template in the primordial soup/cell. The statistical distribution of stop codons allows examination of the probability of generating reading frames of approximately equal to 0.55 kb in this primordial polynucleotide. This analysis reveals that with three stop codons, a run of at least 0.55-kb equivalent length of nonstop codons would occur in 4.6% of 20-kb-long polynucleotide molecules. We attempt to estimate the total amount of noncoding sequences that would be present on the chromosomes of contemporary species assuming that present-day chromosomes retain the prototype primordial genome structure. Theoretical estimates thus obtained for most eukaryotes do not differ significantly from those reported for these specific organisms, with only a few exceptions. Furthermore, analysis of possible stop-codon distributions suggests that life on earth would not exist, at least in its present form, had two or four stop codons been selected early in evolution.
Molecular genetic characterization of the mRNA coding for an inducible suppressor factor specific for L-glutamic acid60-L-alanine30-L-tyrosine10.
Authors: Funckes-Shippy C L , Sorensen C M , Pierce C W , Levine A D
Published: May 2006

The suppressor T-cell hybridoma 1556A2.1 can be induced by the monoclonal L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT)-specific suppressor inducer 372B3.5 and soluble GAT to synthesize a disulfide-linked heterodimeric protein (GAT-TsF2), which directly suppresses a primary in vitro immune response to GAT. Induction and synthesis of the GAT-TsF2 protein is correlated with the appearance of specific mRNA, as detected by translation in vitro in a wheat germ cell-free extract of RNA isolated at various times after induction. The mRNA coding for the polypeptide chain that bears a serologically defined I-J determinant (I-J+ chain) appeared 8 hr after induction, whereas the mRNA coding for the antigen-binding chain (AB+ chain) was not detected until 16 hr after induction. The mRNAs coding for the individual chains sedimented as different species, suggesting that the two-chain factor is the product of two genes. The AB+ chain of the 1556A2.1 GAT-TsF2 was synthesized on membrane-bound polysomes, whereas the I-J+ chain was translated on free polysomes. The AB+ chain was synthesized from two independent mRNA species sedimenting at 10 S and 28 S, whereas a single 16S mRNA encoded the I-J+ chain. The in vitro translated I-J+ chain was bound by a monoclonal antibody against the I-J+ determinant of only the appropriate H-2 haplotype. These results suggest that posttranslational modification, including glycosylation, is not required for biological activity or for expression of the I-J epitope on the GAT-TsF2 molecule.
The local redshift-distance relation and spatial uniformity
Authors: Segal I. E.
Published: May 2006

Regrettably, the review of the redshift-distance relation in the review by Salpeter and Hoffman [Salpeter, E. E. & Hoffman, G. L., Jr. (1986)
Proc. Natl. Acad. Sci. USA
83, 3056-3063], appears flawed. In particular, the logically inconclusive and uncertain hypothesis of local extragalactic spatial uniformity is used in an essential way. Moreover, even in conjunction with this hypothesis, the Lundmark law fits more closely than the Hubble law,
on the basis of the data and criteria of Salpeter and Hoffman
, when a rough approximation involved in the estimation of the galaxy luminosity function is eliminated. Specifically, the assumption that all galaxies in the redshift range 500-1700 km·s
-1
are effectively at the redshift 1100 km·s
-1
is made; when this assumption is replaced by a statistically optimal procedure that uses the precise redshifts, the relative fit of the two laws is reversed.
Interferon-induced transcription of a gene encoding a 15-kDa protein depends on an upstream enhancer element.
Authors: Reich N , Evans B , Levy D , Fahey D , Knight E , Darnell J E
Published: May 2006

A human gene encoding an interferon-induced 15-kDa protein has been isolated from a genomic library. The gene appears to be single-copy and is composed of two exons, the first of which contains the ATG translation initiation codon. In vitro nuclear run-on assays showed that the transcription rate of the gene is stimulated after interferon treatment. To analyze transcriptional regulatory sequences, we constructed recombinant plasmids for use in transient transfection assays of HeLa cells. Constructs containing 115 nucleotides 5' to the transcription initiation site were found to be fully inducible by interferon. Assays of deletion mutants identified a critical element for interferon induction located between -115 and -96, just upstream of the "CCAAT box." Moreover, a DNA fragment including this region can confer interferon inducibility on a heterologous promoter (thymidine kinase) when cloned in either orientation upstream of the gene or downstream of the gene. These are properties characteristic of an enhancer element that is active only after treatment with interferon. This regulatory sequence may be shared by a group of interferon-induced genes, since a very similar sequence is present within the functional region near the RNA start site of another interferon-induced gene.
Region-specific expression of mouse homeobox genes in the embryonic mesoderm and central nervous system.
Authors: Toth L E , Slawin K L , Pintar J E , Nguyen-Huu M C
Published: May 2006

The homeobox is a 180-base-pair sequence characteristically found in homeotic and segmentation genes in Drosophila. Several copies of homeoboxes are also found in the mammalian genome, but it is not known whether these are components of morphogenetic loci in mammals as well. As a step toward understanding the function of mammalian homeoboxes, we have used in situ hybridization to define the spatial pattern of expression of two mouse homeobox genes in the midgestational mouse embryo. The two mouse homeoboxes studied here, Hox 1.2 and Hox 1.4, are located 20 kilobases apart on mouse chromosome 6. Our results demonstrate the following: (i) Hox 1.2 transcripts are localized mainly in the posterior myelencephalon, in the cervical central nervous system (CNS), and in several thoracic prevertebrae; (ii) Hox 1.4 transcripts are localized mainly in the posterior myelencephalon and in the cervical CNS; (iii) within the CNS region expressing Hox 1.4, the level of Hox 1.4 transcripts is higher in the mantle layer than in the ependymal layer and higher in the dorsal than in the ventral area. The specific localization of Hox 1.2 and Hox 1.4 transcripts in the embryonic CNS and the restricted pattern of expression along the rostrocaudal axis are strikingly reminiscent of the expression pattern of Drosophila homeoboxes in the fly embryo and larvae. Despite the different developmental strategies adopted by Drosophila and mammals, functional similarities may exist between Drosophila and mammalian homeobox genes.
Isolation and characterization of intermediates in site-specific recombination.
Authors: Hoess R , Wierzbicki A , Abremski K
Published: May 2006

Cre, the site-specific recombinase from bacteriophage P1, catalyzes a recombination reaction between specific DNA sequences designated as lox sites. The breakage and rejoining of partners during this recombination process must be highly concerted because it has not been possible to detect intermediates of the reaction with wild-type Cre. Several mutant Cre proteins have been isolated that produce significant amounts of a possible intermediate product of the recombination reaction. The product has been identified as a Holliday structure in which one set of the DNA strands of the recombining partners has been exchanged. Wild-type Cre protein is capable of acting on this structure to form recombinant products, which is consistent with this being an intermediate in the recombination reaction. Characterization of the Holliday structure indicated that one set of strands in the recombining partners was always exchanged preferentially before the other set. In addition, it has been found that certain Cre mutants that are unable to carry out recombination in vitro are able to resolve the intermediate. This suggests that these mutants are defective in a step in the reaction that precedes the formation of the Holliday intermediate.
Site-directed mutagenesis of the nitrogenase MoFe protein of
Azotobacter vinelandii
Authors: Brigle Kevin E. , Setterquist Robert A. , Dean Dennis R. , Cantwell John S. , Weiss Mary C. , Newton William E.
Published: May 2006

A strategy has been formulated for the site-directed mutagenesis of the
Azotobacter vinelandii nifDK
genes. These genes encode the α and β subunits of the MoFe protein of nitrogenase, respectively. Six mutant strains, which produce MoFe proteins altered in their α subunit by known single amino acid substitutions, have been produced. Three of these transversion mutations involve cysteine-to-serine changes (at residues 154, 183, and 275), two involve glutamine-to-glutamic acid changes (at residues 151 and 191), and one involves an aspartic acid-to-glutamic acid change (at residue 161). All three possible phenotypic responses are observed within this group— i.e., normal, slow, and no growth in the absence of a fixed-nitrogen source. Two-dimensional gel electrophoresis indicates that all mutants accumulate normal levels of the subunits of both nitrogenase component proteins. Whole-cell and crude-extract acetylene-reduction activities indicate substantial levels of Fe protein activity in all strains. In contrast, MoFe protein activities do not parallel the diazotrophic growth capability for all strains. Two strains appear to exhibit altered substrate discrimination. Such analyses should aid in the identification of metallocluster-binding sites and subunit-subunit interaction domains of the MoFe protein and also provide insight into the mechanistic roles of the various prosthetic groups in catalysis.
Calcium and calmodulin antagonists inhibit human malaria parasites (Plasmodium falciparum): implications for drug design.
Authors: Scheibel L W , Colombani P M , Hess A D , Aikawa M , Atkinson C T , Milhous W K
Published: May 2006

The malaria parasite has an obligate calcium requirement for normal intracellular growth and invasion of host erythrocytes. Calmodulin (CaM) is a vital calcium-dependent protein present in eukaryotes. We found by radioimmunoassay that free parasites contain CaM. Schizont-infected erythrocytes had CaM levels of 23.3 +/- 2.7 ng per 10(6) cells compared to normals (11.2 +/- 1.5 ng per 10(6) cells). CaM levels were proportional to parasite maturity. Immunoelectron microscopy identified CaM diffusely within the cytoplasm of mature parasites and at the apical end of merozoites within the ductule of rhoptries, which may explain the calcium requirement for invasion. Cyclosporin A (CsA) was also found by electron microscopic autoradiography to concentrate in the food vacuole, as do chloroquine and mefloquine, and to distribute within the cytoplasm of mature parasites. The binding of dansylated CsA to schizont-infected erythrocytes was higher than to normal erythrocytes as analyzed by flow cytometry. Kinetic analysis revealed that binding was saturable for normal and infected erythrocytes and possibly free parasites. Competition for binding existed between dansylated CsA and native CsA as well as the CaM inhibitor W-7 and the classic antimalarial chloroquine. The in vitro growth of Plasmodium falciparum was sensitive to CaM antagonists, and in large part inhibition of the parasite was proportional to known anti-CaM potency. Antagonism existed between combinations of these drugs in multi-drug-resistant strains of P. falciparum, suggesting possible competition for the same binding site. In addition, the malaria parasite was also susceptible to calcium antagonists.
Activation of human immunodeficiency virus by herpesvirus infection: identification of a region within the long terminal repeat that responds to a trans-acting factor encoded by herpes simplex virus 1.
Authors: Mosca J D , Bednarik D P , Raj N B , Rosen C A , Sodroski J G , Haseltine W A , Hayward G S , Pitha P M
Published: May 2006

Herpes simplex virus 1 (HSV-1) infection induces transcription of the chloramphenicol acetyltransferase (CAT) gene directed by the long terminal repeat (LTR) of human immunodeficiency virus (HIV) in both transiently and permanently transfected cells containing the HIV-LTR/CAT hybrid gene. To define the mechanism by which HSV-1 stimulates the HIV LTR, we examined the effects of isolated regulatory genes from HSV-1. The results of cotransfection assays with the immediate-early (IE) genes of HSV-1, IE110 (ICP0) and IE175 (ICP4), showed that the IE110 protein, either alone or in combination with the IE175 protein, can activate the HIV LTR. Cotransfection with the IE175 gene alone or with the Vmw65 gene (coding for a virion transcription factor) alone did not lead to HIV-LTR activation. The lack of requirement for the IE175 or Vmw65 gene products in transient-expression assays was confirmed in permanent cell lines containing the HIV-LTR/CAT hybrid gene by using temperature-sensitive mutants defective in the IE175 gene product or in uncoating functions. By deletion analysis, we localized a 73-bp-long region (positions -104 to -32) from the HIV LTR that responded to HSV-1 activation; when this region, which is distinct from the previously identified trans-activating responsive (TAR) region, was ligated to a heterologous, HSV-1-nonresponsive gene (alpha 4-interferon/CAT), it conferred inducibility by both HSV-1 infection and IE110/175 cotransfection. Both simian and human cytomegalovirus also induced the HIV-LTR/CAT hybrid gene. However, we failed to detect specific upstream sequence requirements for induction by cytomegalovirus. Our results indicate that infection with unrelated viruses can alter the expression of HIV in an infected cell.
Interaction of two nonhistone proteins with the estradiol response element of the avian vitellogenin gene modulates the binding of estradiol-receptor complex.
Authors: Feavers I M , Jiricny J , Moncharmont B , Saluz H P , Jost J P
Published: May 2006

The DNA sequence corresponding to the estradiol response element has been synthesized and tested in vitro for the binding of specific proteins. Gel retardation experiments combined with dimethyl sulfate protection experiments revealed that this region binds two nonhistone proteins (NHPs). One of them, NHP-1, has a molecular weight of 70,000 and binds specifically to the dyad symmetry sequence GGTCAGCGTGACC. The NHP-1 can be separated from the estradiol receptor chromatographically; it does not bind estradiol and does not cross-react with an antibody directed against the estradiol receptor. A series of synthetic "mutant" oligonucleotides were tested in a protein-DNA binding competition assay. Deletion of the GCG in the center of the dyad symmetry sequence suppressed the binding of NHP-1 by 90%, and the conversion of any GC pair to an AT pair decreased the affinity of the binding site for NHP-1. Methylation of the two CpGs on both strands of the dyad symmetry sequence decreased the affinity of the binding site for NHP-1 by 60%, whereas hemimethylation of the same structure did not inhibit the binding of NHP-1. NHP-1 and NHP-2, the NHP binding to the DNA next to the dyad symmetry sequence, bind exclusively to double-stranded DNA. NHP-2 has a molecular weight of 60,000. NHP-1 and NHP-2 are neither tissue nor species specific. In vitro reconstitution experiments show that NHP-1 and NHP-2 increase the binding efficiency of the estradiol-receptor complex to the estradiol response element.
Myristoylated alpha subunits of guanine nucleotide-binding regulatory proteins.
Authors: Buss J E , Mumby S M , Casey P J , Gilman A G , Sefton B M
Published: May 2006

Antisera directed against specific subunits of guanine nucleotide-binding regulatory proteins (G proteins) were used to immunoprecipitate these polypeptides from metabolically labeled cells. This technique detects, in extracts of a human astrocytoma cell line, the alpha subunits of Gs (stimulatory) (alpha 45 and alpha 52), a 41-kDa subunit of Gi (inhibitory) (alpha 41), a 40-kDa protein (alpha 40), and the 36-kDa beta subunit. No protein that comigrated with the alpha subunit of Go (unknown function) (alpha 39) was detected. In cells grown in the presence of [3H]myristic acid, alpha 41 and alpha 40 contained 3H label, while the beta subunit did not. Chemical analysis of lipids attached covalently to purified alpha 41 and alpha 39 from bovine brain also revealed myristic acid. Similar analysis of brain G protein beta and gamma subunits and of Gt (transducin) subunits (alpha, beta, and gamma) failed to reveal fatty acids. The fatty acid associated with alpha 41, alpha 40, and alpha 39 was stable to treatment with base, suggesting that the lipid is linked to the polypeptide via an amide bond. These GTP binding proteins are thus identified as members of a select group of proteins that contains myristic acid covalently attached to the peptide backbone. Myristate may play an important role in stabilizing interactions of G proteins with phospholipid or with membrane-bound proteins.