bluuebunny/crossref_metadata_2025 huggingface dataset:
Heat shock proteins and thermoresistance in lizards.
Authors: Ulmasov K A , Shammakov S , Karaev K , Evgen'ev M B
Published: May 2006

The synthesis of heat shock proteins (hsps) at normal physiological and elevated temperatures has been correlated with the natural adaptation of an organism to heat in nine lizard species studied. These species differ drastically by their adaptation to elevated temperature and represent a spectrum of forms isolated from various geographical regions of the Union of Soviet Socialist Republics. The synthesis of hsps belonging to the hsp70 family and their correspondent mRNAs have been compared at different temperature regimes. This analysis has shown that lizards inhabiting the Middle Asia deserts are characterized by a higher content of hsp70-like proteins at normal physiological temperatures (2- to 5-fold differences) when compared with the forms from central and northern regions of the European part of the Union of Soviet Socialistic Republics. Analysis of hsp70 mRNA at different temperatures substantiated these observations, showing evident correlation between adaptation of a given form to hyperthermia and the quantity of hsp70 mRNA in the cells under non-heat-shock conditions. The results obtained with a wide spectrum of ecologically different lizard species, coupled with other relevant data, enable us to propose a general rule applicable to poikilothermic organisms. This rule postulates the direct correlation between the characteristic temperature of the ecological niche of a given species and the amount of hsp70-like proteins in the cells at normal temperature.
Oligonucleotide-directed site-specific mutagenesis in Drosophila melanogaster.
Authors: Banga S S , Boyd J B
Published: May 2006

An efficient technique has been developed for performing in vivo site-directed mutagenesis in Drosophila melanogaster. This procedure involves directed repair of P-element-induced DNA lesions after injection of a modified DNA sequence into early embryos. An oligonucleotide of 50 base pairs, whose sequence spans the P-element insertion site, mediates base replacement in the endogenous gene. Restriction mapping, DNA sequencing, and polymerase chain reaction analysis demonstrate that base substitutions present in an injected oligonucleotide are incorporated into genomic sequences flanking a P insertion site in the white gene. This analysis suggests that progeny bearing directed mutations are recovered with a frequency of about 0.5 x 10(-3). Because Drosophila remains a premier organism for the analysis of eukaryotic gene regulation, this system should find strong application in that analysis as well as in the analysis of DNA recombination, conversion, repair, and mutagenesis.
Direct repeat sequences are implicated in the regulation of two Streptomyces chitinase promoters that are subject to carbon catabolite control.
Authors: Delic I , Robbins P , Westpheling J
Published: May 2006

We report the identification and partial characterization of the promoters for two chitinase genes from Streptomyces plicatus. Chitinases are a family of enzymes made by Streptomyces and other soil microbes to digest chitin, an abundant source of carbon and nitrogen in the soil. The promoter regions of two chitinases were defined by using transcriptional fusions to the xylE reporter gene. Transcription was shown to be glucose-sensitive and chitin-dependent. Each promoter contains a putative RNA polymerase binding site with a recognition sequence very similar to that observed in many eubacterial vegetatively expressed genes. In both promoters, a pair of 12-base-pair direct repeat sequences overlap the putative RNA polymerase binding sites. Further analysis of one of the promoters revealed that a single-base change within the direct repeat sequences resulted in glucose-resistant, chitin-independent expression in vivo. In addition, the promoter region that includes the direct repeat sequences was shown to interact with a sequence-specific DNA binding factor in vitro. Similar direct repeat sequences are present in other chitinase genes recently characterized, and we suggest that these repeats may be involved in repression and induction for this entire class of catabolite-controlled genes.
Insulin-like growth factor I gene expression is induced in astrocytes during experimental demyelination.
Authors: Komoly S , Hudson L D , Webster H D , Bondy C A
Published: May 2006

To investigate insulin-like growth factor I (IGF-I) and IGF-I receptor gene expression during experimental demyelination and myelin regeneration, young mice were fed cuprizone (( bis(cyclohexanone) oxaldihydrazone )). This copper-chelating agent produces demyelination in the corpus callosum and superior cerebellar peduncles, and when treatment is stopped, there is rapid remyelination. At intervals during cuprizone treatment and recovery, brain sections were hybridized with specific probes and immunostained with antibodies to determine the localization and relative amounts of IGF-I and IGF-I receptor mRNAs and peptides. In untreated littermates, IGF-I and IGF-I receptor mRNAs and peptides were not detected in white matter. In cuprizone-treated mice, high levels of both IGF-I mRNA and peptide were expressed by astrocytes in areas of myelin breakdown. Astrocyte IGF-I expression decreased rapidly during recovery and oligodendroglial expression of myelin-related genes increased. In severely demyelinated areas, immature oligodendroglia exhibited a transient increase in IGF-I receptor mRNA and peptide immunoreactivity during early recovery. This highly specific pattern of IGF-I induction in astrocytes during demyelination and the expression of the IGF-I receptor in regenerating oligodendrocytes during recovery suggest that IGF-I functions in the regulation of oligodendrocyte and myelin metabolism in vivo.
Solution conformation of the major adduct between the carcinogen (+)-anti-benzo[a]pyrene diol epoxide and DNA.
Authors: Cosman M , de los Santos C , Fiala R , Hingerty B E , Singh S B , Ibanez V , Margulis L A , Live D , Geacintov N E , Broyde S
Published: May 2006

We have synthesized, separated, and purified approximately 10 mg of a deoxyundecanucleotide duplex containing a single centrally positioned covalent adduct between (+)-anti-benzo[a]pyrene (BP) diol epoxide and the exocyclic amino group of guanosine. Excellent proton NMR spectra are observed for the (+)-trans-anti-BP diol epoxide-N2-dG adduct positioned opposite dC and flanked by G.C pairs in the d[C1-C2-A3-T4-C5-(BP)G6-C7-T8-A9-C10-C11].d[12- G13-T14-A15-G16-C17-G18-A19-T20-G 21-G22] duplex +ADdesignated (BP)G.C 11-mer+BD. We have determined the solution structure centered about the BP covalent adduct site in the (BP)G.C 11-mer duplex by incorporating intramolecular and intermolecular proton-proton distance bounds deduced from the NMR data sets as constraints in energy minimization computations. The BP ring is positioned in the minor groove and directed toward the 5' end of the modified strand. One face of the BP ring of (BP)G6 is stacked over the G18 and A19 sugar-phosphate backbone on the partner strand and the other face is exposed to solvent. A minimally perturbed B-DNA helix is observed for the d[T4-C5-(BP)G6-C7-T8].d[A15-G16-C17-G18-A19] segment centered about the adduct site with Watson-Crick alignment for both the (BP)G6.C17 pair and flanking G.C pairs. A widening of the minor groove at the adduct site is detected that accommodates the BP ring whose long axis makes an angle of approximately 45 degrees with the average direction of the DNA helix axis. Our study holds future promise for the characterization of other steroisomerically pure adducts of BP diol epoxides with DNA to elucidate the molecular basis of structure-activity relationships associated with the stereoisomer-dependent spectrum of mutational and carcinogenic activities.
Human T-cell lymphotropic virus (HTLV)-related endogenous sequence, HRES-1, encodes a 28-kDa protein: a possible autoantigen for HTLV-I gag-reactive autoantibodies.
Authors: Banki K , Maceda J , Hurley E , Ablonczy E , Mattson D H , Szegedy L , Hung C , Perl A
Published: May 2006

The presence of a human T-cell lymphotropic virus (HTLV)-related endogenous sequence, HRES-1, in the human genome has been documented. The HRES-1 genomic locus is transcriptionally active and contains open reading frames. Antibodies 232 and 233, specific for synthetic peptides pep14-24 and pep117-127, corresponding to two nonoverlapping HTLV-related regions in the longer open reading frame of HRES-1, recognize an identical 28-kDa protein in H9 human T cells. Thus, HRES-1 is a human endogenous retroviral sequence capable of protein expression. HRES-1/p28 is localized to the cytoplasm and nuclear bodies. While HTLV-I-specific antibodies react with HRES-1 peptides, antibody 233 cross-reacts with HTLV-I gag p24 protein. Three consecutive highly charged amino acid residues, Arg-Arg-Glu, present in both HRES-1 pep117-127 and HTLV-I gag p24 are likely to be the core of cross-reactive epitopes. The prevalence of antibodies to HRES-1 peptides pep14-24 and pep117-127 was determined in 65 normal blood donors and 146 patients with immunological disorders. Sera of patients with multiple sclerosis (19 out of 65, 29%), progressive systemic sclerosis (4 out of 17, 23%), systemic lupus erythematosus (4 out of 19, 21%), and Sjogren syndrome (2 out of 19, 10%) contained significantly higher HRES-1 peptide binding activity than sera of normal donors. Sera of patients with AIDS showed no specific binding to HRES-1 peptides. Nine of 30 HRES-1-seropositive patients showed immunoreactivity to HTLV-I gag p24. The data indicate that HRES-1/p28 may serve as an autoantigen eliciting autoantibodies cross-reactive with HTLV-I gag antigens.
Inactivated whole-virus vaccine derived from a proviral DNA clone of simian immunodeficiency virus induces high levels of neutralizing antibodies and confers protection against heterologous challenge.
Authors: Johnson P R , Montefiori D C , Goldstein S , Hamm T E , Zhou J , Kitov S , Haigwood N L , Misher L , London W T , Gerin J L
Published: May 2006

We tested the ability of macaques vaccinated with inactivated whole simian immunodeficiency virus (SIV) to resist challenge with either homologous or heterologous cell-free uncloned SIV administered by the intravenous route. The vaccine virus was derived from a proviral DNA clone and thus was considered genetically homogeneous. Sixteen macaques received either hepatitis B surface antigen (n = 6) or the inactivated whole-SIV vaccine (n = 10) at weeks 0, 4, and 49 of the study. All SIV vaccine recipients developed high levels of homologous and heterologous neutralizing antibodies in response to vaccination. At the time of challenge (week 53), vaccinees were further stratified to receive either homologous (n = 10) or heterologous (n = 6) uncloned live SIV. The envelope glycoproteins of the homologous and heterologous challenge viruses were 94% and 81% identical to the vaccine virus, respectively. Regardless of challenge inoculum, all vaccinees in the control group (hepatitis B surface antigen) became infected, whereas all SIV vaccinees were protected against detectable infection. These data support the concept that an efficacious vaccine for HIV might be possible, and suggest that genetic variation of HIV might not be an insurmountable obstacle for vaccine development.
Solution binding of an antigenic peptide to a major histocompatibility complex class I molecule and the role of beta 2-microglobulin.
Authors: Boyd L F , Kozlowski S , Margulies D H
Published: May 2006

The major histocompatibility complex-encoded class I molecule, a noncovalent dimer of a polymorphic 45-kDa heavy chain and a nonpolymorphic 12-kDa beta 2-microglobulin (beta 2m) light chain, binds peptide antigen prior to its interaction with T-cell antigen receptors. We report here that the binding in aqueous solution at 37 degrees C of a soluble purified murine major histocompatibility complex class I protein, H-2Lds (a soluble analogue of H-2Ld consisting of the alpha 1 and alpha 2 domains of H-2Ld, the alpha 3 domain and the C terminus of Q10b), to an antigenic peptide is controlled by the light-chain subunit beta 2m. Analysis of the equilibrium binding data favors a model in which two classes of peptide binding sites exist, the high-affinity class having an equilibrium constant for dissociation, KH, of 3.7 x 10(-7) M and accounting for 12% of the theoretically available sites. Studies of binding in the presence of excess beta 2m indicate that this increases the concentration of available high-affinity sites. These data are consistent with a ternary model in which high-affinity sites are generated by the interaction of beta 2m with the peptide-binding class I heavy chain.
Relaxin binding in the rat heart atrium.
Authors: Osheroff P L , Cronin M J , Lofgren J A
Published: May 2006

Relaxin is a member of the insulin family of polypeptides that is best known as a reproductive hormone. In an effort to elucidate the mechanism of action of relaxin we previously localized the specific binding sites of a 32P-labeled relaxin in the rat uterus and brain. These studies suggested that, in addition to its classical role in pregnancy, relaxin might have other physiological functions. In the present paper we describe the specific and high-affinity binding of relaxin to the cardiac atrium of both male and female rats. The relaxin binding could not be displaced by peptides belonging to the same family [insulin, insulin-like growth factor I (IGF-I)] or by peptides that were identified in the atrium or were known to have cardiovascular functions (atrial natriuretic peptide, angiotensin II). The dissociation constant for relaxin in the atrium was estimated to be 1.4 nM, which was similar to that found in the uterus (1.3 nM) and the brain (1.4 nM). In view of the close association of relaxin with reproduction, an experiment was also performed to compare the relaxin binding in the uterus and heart after gonadectomy and sex steroid treatment. It was found that the relaxin binding in the rat uterus was diminished by 53% overall following ovariectomy but was restored to 90% of normal levels when treated with estrogen (but not with testosterone). In contrast, the relaxin binding in the rat heart was not affected by castration or sex steroid treatment. We conclude that specific and high-affinity relaxin receptors exist in the atrium of both the male and female rat heart and that these are regulated differently than the relaxin receptors in the uterus.
Three-dimensional structure of Theiler murine encephalomyelitis virus (BeAn strain).
Authors: Luo M , He C , Toth K S , Zhang C X , Lipton H L
Published: May 2006

Depending on the strain, Theiler murine encephalomyelitis virus (TMEV) may cause acute encephalitis or chronic demyelinating disease, which is associated with viral persistence in mice. Persistent central nervous system infection and demyelination by the less-virulent TMEV has provided a useful animal model for the human demyelinating disease multiple sclerosis. The less-virulent BeAn strain of TMEV was crystallized and its atomic structure was determined by x-ray crystallography. The alpha-carbon coordinates of the closely related Mengo virus were used to calculate the initial phases to 3.5 A resolution and the interpretable electron density map was produced by 10 cycles of 30-fold noncrystallographic molecular replacement averaging. The structure revealed a high degree of overall structural similarity to Mengo virus as well as substantial differences in the surface loops. These structural changes might be correlated with TMEV host-specific recognition, pH-related stability, and neurovirulence.
Enhanced sensitivity of hippocampal pyramidal neurons from mdx mice to hypoxia-induced loss of synaptic transmission.
Authors: Mehler M F , Haas K Z , Kessler J A , Stanton P K
Published: May 2006

The gene at the Duchenne/Becker muscular dystrophy locus encodes dystrophin, a member of a protein superfamily that links the actin cytoskeleton to transmembrane plasmalemmal proteins. In mature skeletal myocytes, the absence of dystrophin is associated with decreased membrane stability, altered kinetics of several calcium channels, and increased intracellular calcium concentration. In the central nervous system, dystrophin is restricted to specific neuronal populations that show heightened susceptibility to excitotoxic damage and is localized in proximal dendrites and the neuronal somata. We report that CA1 pyramidal neurons in a hippocampal slice preparation from a dystrophin-deficient mouse genetic model of Duchenne muscular dystrophy (the mdx mouse) exhibit significant increased susceptibility to hypoxia-induced damage to synaptic transmission. This selective vulnerability was substantially ameliorated by pretreatment with diphenylhydantoin, an anticonvulsant that blocks both sodium-dependent action potentials and low-threshold transient calcium conductances. These findings suggest that dystrophin deficiency could predispose susceptible neuronal populations to cumulative hypoxic insults that may contribute to the development of cognitive deficits in Duchenne/Becker muscular dystrophy patients and that the effects of such periods of hypoxia may be pharmacologically remediable.
Rhythmogenic effects of weak electrotonic coupling in neuronal models.
Authors: Sherman A , Rinzel J
Published: May 2006

Strong gap-junctional coupling can synchronize the electrical oscillations of cells, but we show, in a theoretical model, that weak coupling can phase lock two cells 180 degrees out-of-phase. Antiphase oscillations can exist in parameter regimens where in-phase oscillations break down. Some consequences are (i) coupling two excitable cells leads to pacemaking, (ii) coupling two pacemaker cells leads to bursting, and (iii) coupling two bursters increases burst period. The latter shows that details of the fast spikes can affect macroscopic properties of the slow bursts. These effects hold in other models for bursting and may play a role in the collective behavior of cellular ensembles.
Adenovirus as an expression vector in muscle cells in vivo.
Authors: Quantin B , Perricaudet L D , Tajbakhsh S , Mandel J L
Published: May 2006

Attempting gene transfer in muscle raises difficult problems: the nuclei of mature muscle fibers do not undergo division, thus excluding strategies involving replicative integration of exogenous DNA. As adenovirus has been reported to be an efficient vector for the transfer of an enzyme encoding gene in mice, we decided to explore its potential for muscle cells. Advantages of adenovirus vectors are their independence of host cell replication, broad host range, and potential capacity for large foreign DNA inserts. We constructed a recombinant adenovirus containing the beta-galactosidase reporter gene under the control of muscle-specific regulatory sequences. This recombinant virus was able to direct expression of the beta-galactosidase in myotubes in vitro. We report its in vivo expression in mouse muscles up to 75 days after infection. The efficiency and stability of expression we obtained compare very favorably with other strategies proposed for gene or myoblast transfer in muscle in vivo.
Infectious Sindbis virus transient expression vectors for studying antigen processing and presentation.
Authors: Hahn C S , Hahn Y S , Braciale T J , Rice C M
Published: May 2006

Sindbis virus (SIN) is a small positive-strand enveloped RNA virus that infects a broad range of vertebrate and insect cells. A SIN vector (called dsSIN), designed for transient expression of heterologous RNAs and proteins, was engineered by inserting a second subgenomic mRNA promoter sequence into a nonessential region of the SIN genome. By using this vector, dsSIN recombinants have been constructed that express either bacterial chloramphenicol acetyltransferase, a truncated form of the influenza hemagglutinin (HA), or mini-genes encoding two distinct immunodominant cytotoxic T lymphocyte (CTL) HA epitopes. Infection of murine cell lines with these recombinants resulted in the expression of approximately 10(6)-10(7) chloramphenicol acetyltransferase polypeptides per cell and efficient sensitization of target cells for lysis by appropriate major histocompatibility complex-restricted HA-specific CTL clones in vitro. In addition, priming of an influenza-specific T-cell response was observed after immunizing mice with dsSIN recombinants expressing either a truncated form of HA or the immunodominant influenza CTL epitopes. This SIN expression system allows the generation of high-titered recombinant virus stocks in a matter of days and should facilitate mapping and mutational analysis of class I major histocompatibility complex-restricted T-cell epitopes expressed via the endogenous pathway of antigen processing and presentation.
Spectroscopic evidence for conformational relaxation in myoglobin.
Authors: Nienhaus G U , Mourant J R , Frauenfelder H
Published: May 2006

The time and temperature dependencies of the line area (M0) and position (M1) of band III at approximately 760 nm have been measured with Fourier-transform infrared spectroscopy in deoxymyoglobin (Mb) and continuously photolyzed carbon monoxide myoglobin (MbCO). Below 200 K, the area of band III in the photoproduct Mb* increases with time even on time scales of hours. This behavior indicates changes in the distribution of activation enthalpy barriers for ligand rebinding under extended illumination. The band position of Mb* shifts to higher wavenumbers with increasing temperature up to 100 K owing to kinetic hole burning; the same protein coordinate that controls the position of band III also determines the rebinding barrier height. The shift ceases above 100 K, implying that more than one protein coordinate affects the height of the rebinding barrier. Above 160 K, the line position in Mb* shifts again and coalesces with the value of Mb for temperatures above 200 K. The shift is accompanied by an increase of the line area, reflecting a slowing of rebinding kinetics. Both effects are explained in the framework of the model introduced by Steinbach et al. [(1991) Biochemistry 30, 3988-4001]. Above approximately 160 K, the conformational relaxation Mb*----Mb simultaneously shifts the line position of band III and increases the enthalpy barrier for ligand rebinding. Furthermore, equilibrium fluctuations lead to an averaging of the band position and the rebinding enthalpy.
Mutual relationships between chromogranins A and B and gastrin in individual gastrin cells.
Authors: Cetin Y , Bargsten G , Grube D
Published: May 2006

The chromogranins A and B (CgA and CgB, respectively), originally detected in the adrenal medulla, are present in various endocrine organs. Remarkably, their immunoreactivities vary among different endocrine cell types and also within a given endocrine cell population. With densitometric techniques at the cellular level, individual gastrin cells (n = 318) from guinea pig antral mucosa were studied to measure their content of immunoreactive CgA, CgB, and gastrin. The composition of these secretory proteins in individual gastrin cells varied considerably but with predictable components. Statistical evaluation of the data showed that immunoreactivities for gastrin and CgA correlated negatively in these cells; CgA and CgB immunoreactivities also correlated inversely. On the other hand, immunoreactivities for gastrin and CgB exhibited a high positive correlation. The mutual relationships between gastrin, CgA, and CgB suggest that under physiological conditions biosynthetic pathways of these secretory constituents are linked to each other in individual gastrin cells.
Beta-globin nonsense mutation: deficient accumulation of mRNA occurs despite normal cytoplasmic stability.
Authors: Baserga S J , Benz E J
Published: May 2006

A common mutation causing thalassemia in Mediterranean populations is an amber (UAG) nonsense mutation at the 39th codon of the human beta-globin gene, the beta-39 mutation. Studies of mRNA metabolism in erythroblasts from patients with beta-39 thalassemia and studies using heterologous transfection systems have suggested the possibility that this mutation not only affects protein synthesis but also alters mRNA metabolism. The effects of this mutation on several steps in the metabolism of mRNA have been investigated by transfection of the gene into permanent cell lines bearing a temperature-sensitive RNA polymerase II. Several RNA expression studies were performed, including analysis of transcription, mRNA stability, mRNA splicing accuracy, and mRNA polyadenylation. The results suggest that the defect in expression of the beta-39 mRNA occurs at a step prior to the accumulation of mRNA in the cytoplasm.
Hormone-induced progesterone receptor phosphorylation consists of sequential DNA-independent and DNA-dependent stages: analysis with zinc finger mutants and the progesterone antagonist ZK98299.
Authors: Takimoto G S , Tasset D M , Eppert A C , Horwitz K B
Published: May 2006

Human progesterone receptors (hPRs) are phosphorylated at multiple serine residues, first in a basal step and then in a hormone-induced step. To determine whether hormone-induced phosphorylation precedes or follows the interaction of hPRs with DNA two strategies were used. (i) DNA binding was prevented or altered with site-specific mutants of the A form of hPR; (ii) DNA binding of wild-type hPR forms A and B was prevented with the progesterone antagonist ZK98299. Two hPRA mutants were constructed: DBDCys, which lacks a critical cysteine residue in the first zinc finger, and DBDsp, which is mutated at three discriminatory amino acids to change its DNA binding specificity from a progesterone response element to an estrogen response element. Receptors were transiently expressed in PR-negative cells and were intranuclear. DBDCys did not bind DNA in vitro and DBDsp bound only the estrogen response element. Transiently expressed hPRA and DBDsp showed the upward shift in electrophoretic mobility characteristic of hormone-induced phosphorylation; it was absent with DBDCys. Hormone-induced [32P] orthophosphate incorporation into transiently expressed DBDCys was reduced 60% compared to hPRA and DBDsp but was not eliminated. ZK98299 binds hPRs but prevents their interaction with DNA. Compared to R5020, the antagonist reduced phosphorylation of hPRB and hPRA in T47D breast cancer cells by 60% and totally prevented the mobility shift. We conclude that the hormone-induced phosphorylation of hPR includes DNA-independent and DNA-dependent stages and that only DNA-dependent sites contribute to the mobility shift.
Robustness of the no-interference model for ordering genetic markers.
Authors: Speed T P , McPeek M S , Evans S N
Published: May 2006

Under the assumption of no chromatid interference, we derive constraints on the probabilities of the different recombination patterns among m + 1 genetic loci. An application of these constraints is a proof that the ordering of the loci that maximizes the likelihood under the assumption of no interference is, in fact, a consistent estimator of the true order even when there is interference.
Regulated expression of the calmodulin-related TCH genes in cultured Arabidopsis cells: induction by calcium and heat shock.
Authors: Braam J
Published: May 2006

Expression of the calmodulin-related TCH genes of Arabidopsis is strongly and rapidly up-regulated in plants after a variety of stimuli, including touch. As an approach to investigating the mechanism(s) of TCH gene regulation, a manipulable cell culture system in which TCH gene expression is regulated has been developed. In response to increased external calcium or heat shock, TCH2, -3, and -4 mRNA levels significantly increased. Significantly, these two stimuli are known to result in cytoplasmic calcium increases, therefore implicating a role for calcium itself in the regulation of calmodulin-related genes. Further, external calcium is required for maximal heat-shock induction of expression of the TCH genes but not of the 70-kDa heat shock protein; therefore, there may exist at least two distinct mechanisms of heat shock induction of gene expression. Calcium ion regulation of genes encoding calcium-binding proteins may ensure the efficacy of calcium ion as a transient second messenger and the maintenance of cellular homeostasis. This possible regulatory circuit would likely be relevant not only for plant cells but also for the great variety of animal cells that transduce extracellular stimuli, such as hormones and electrical impulses, into calcium signals.
Genes regulating the plant cell cycle: isolation of a mitotic-like cyclin from Arabidopsis thaliana.
Authors: Hemerly A , Bergounioux C , Van Montagu M , Inzé D , Ferreira P
Published: May 2006

A key element of cell cycle control in eukaryotes is the M-phase kinase, composed of p34cdc2 and cyclin. To dissect the plant cell cycle, we have previously isolated a cdc2 gene homolog from Arabidopsis thaliana. We have now cloned an Arabidopsis cDNA corresponding to cyclins. This gene (cyc1At) encodes a protein with a predicted molecular mass of 48.4 kDa and a domain homologous to the cyclin box of mitotic cyclins. However, by sequence comparison the cyc1At gene could not be assigned to the A- or B-type group. The mRNA accumulates preferentially in actively dividing cells and when these cells are blocked during the cell cycle, the amount of transcripts decreases dramatically. cyc1At mRNA is found mainly in G2-phase nuclei, suggesting that its expression is periodic in the cell cycle. Microinjection of synthetic cyc1At mRNA induced meiotic maturation in Xenopus oocytes. Cyc1At is encoded by a single gene, but the amplification by the polymerase chain reaction of other fragments homologous to cyclins indicates the presence of a family of cyclins in Arabidopsis.
Cloning and expression of the tumor-associated antigen L6.
Authors: Marken J S , Schieven G L , Hellström I , Hellström K E , Aruffo A
Published: May 2006

The L6 cell surface antigen, which is highly expressed on lung, breast, colon, and ovarian carcinomas, has attracted attention as a therapeutic target for murine monoclonal antibodies and their humanized counterparts. Its molecular nature has, however, remained elusive. Here we describe the expression cloning of a cDNA encoding the L6 antigen. COS cells transfected with this cDNA direct the expression of an approximately 24-kDa surface protein that reacts with the two anti-L6 monoclonal antibodies available. The predicted L6 peptide sequence is 202 amino acids long and contains three predicted NH2-terminal hydrophobic transmembrane regions, which are followed by a hydrophilic region containing two potential N-linked glycosylation sites and a COOH-terminal hydrophobic transmembrane region. The L6 antigen is related to a number of cell surface proteins with similar predicted membrane topology that have been implicated in cell growth. Two other members of this family of proteins, CD63 (ME491) and CO-029, are also highly expressed on tumor cells. The present findings should make it possible to further study the role of the L6-defined antigen in normal and neoplastic cells and to construct animal models for development of improved agents for active and passive cancer immunotherapy.
In vitro selection and affinity maturation of antibodies from a naive combinatorial immunoglobulin library.
Authors: Gram H , Marconi L A , Barbas C F , Collet T A , Lerner R A , Kang A S
Published: May 2006

We have used a combinatorial immunoglobulin library approach to obtain monoclonal antibodies from nonimmune adult mice, thereby establishing the principles of (i) accessing naive combinatorial antibody libraries for predetermined specificities and (ii) increasing the affinity of the selected antibody binding sites by random mutagenesis. A combinatorial Fab library expressing immunoglobulin mu and kappa light-chain fragments on the surface of filamentous phage was prepared from bone marrow of nonimmunized, adult BALB/c mice with the multivalent display vector pComb8. Phage displaying low affinity Fabs (binding constants, 10(4)-10(5) M-1) binding to a progesterone-bovine serum albumin conjugate were isolated from the library. Random mutagenesis of the heavy- and light-chain variable regions expressed in the mono-valent phage display vector pComb3 was performed by error-prone PCR, and subsequently clones with improved affinity for the hapten conjugate were selected. We demonstrate that antibodies with desirable characteristics from a nonimmune source may be selected and affinity maturation may be achieved by using the twin vectors pComb8 and pComb3, thus opening the route to obtaining specific antibodies from a generic library and bypassing immunization.
A ubiquitous 64-kDa protein is a component of a chloride channel of plasma and intracellular membranes.
Authors: Redhead C R , Edelman A E , Brown D , Landry D W , al-Awqati Q
Published: May 2006

Chloride channels are present in the plasma and intracellular membranes of most cells. Previously, using the ligand indanyloxyacetic acid (IAA), we purified four major proteins from bovine kidney cortex membrane vesicles. These proteins gave rise to chloride channel activity when reconstituted into phospholipid vesicles. Two of these proteins (97 and 27 kDa) were found to be drug-binding proteins by N-terminal sequence analysis. Antibodies raised to the 64-kDa protein stained only this protein on immunoblots, and only this protein was present after purification on an immunoaffinity column. In addition, these same antibodies were able to deplete IAA-94 inhibitable chloride channel activity from solubilized kidney membranes. Of fractions obtained from the gel filtration of solubilized kidney membranes, only those containing this 64-kDa protein exhibited measurable chloride channel activity. Immunoblots of a variety of species and cell types, both epithelial and nonepithelial, revealed that this protein is ubiquitous and highly conserved. Immunocytochemistry in CFPAC-1 cells revealed staining for this protein on the apical plasma membrane and in the membranes of intracellular organelles. These results demonstrate that the integral membrane protein p64 is a component of chloride channels present in both epithelial plasma membrane and the membranes of intracellular organelles.
Cyclosporin-mediated inhibition of bovine calcineurin by cyclophilins A and B.
Authors: Swanson S K , Born T , Zydowsky L D , Cho H , Chang H Y , Walsh C T , Rusnak F
Published: May 2006

The Ca(2+)- and calmodulin-dependent protein phosphatase calcineurin is inhibited by the immunosuppressant drug cyclosporin A in the presence of cyclophilin A or B. Of the two isoforms, cyclophilin B is more potent by a factor of 2-5 when either the phosphoprotein [32P]casein or the [32P]phosphoserine [Ser(32P)] form of the 19-residue bovine cardiac cAMP-dependent protein kinase regulatory subunit peptide RII, [Ser(32P)15]RII, is used as substrate. With [Ser(32P15]RII as substrate, the concentrations of the cyclosporin A.cyclophilin A and cyclosporin A.cyclophilin B complexes, which cause 50% inhibition of calcineurin activity, are 120 and 50 nM, respectively. Lowering the concentration of calcineurin 80% with [32P]casein as substrate lowered the apparent inhibition constant for each complex even further; 50% inhibition of calcineurin was observed at 40 nM for cyclosporin A.cyclophilin A, whereas it was less than 10 nM for cyclosporin A.cyclophilin B. In all inhibition assays with [32P]casein or [Ser(32P)15]RII, the concentration of calcineurin required for measurable phosphatase activity is such that these complexes behave as tight-binding inhibitors of calcineurin, and steady-state kinetics cannot be used to assess inhibition patterns or Ki values. Limited trypsinization of calcineurin produces a fragment that is still inhibited, indicating that the interaction of cyclosporin.cyclophilin with calcineurin does not require either calmodulin or Ca2+.
Transcriptional and posttranscriptional regulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase during liver regeneration.
Authors: Rosa J L , Tauler A , Lange A J , Pilkis S J , Bartrons R
Published: May 2006

The control of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2; EC 2.7.1.105/3.1.3.46) gene expression during liver regeneration was studied. The level of PFK-2/FBPase-2 mRNA decreased to about 5% of the control value 6 hr after partial hepatectomy. Thereafter the mRNA increased to a maximum at 48 hr and returned to normal levels by 96 hr. In sham-operated animals, only a small increase was observed during the first 4 hr. The mRNA was recognized by a 299-base-pair liver-specific cDNA probe but not by a muscle-specific probe. The time course of mRNA modulation was well correlated with PFK-2/FBPase-2 activity and with the amount of bifunctional enzyme protein determined by immunoblotting with an antibody raised against the N-terminal decapeptide of liver PFK-2/FBPase-2. No alteration in the degradation rate of PFK-2/FBPase-2 mRNA was noted after partial hepatectomy. The modulation of PFK-2/FBPase-2 gene expression during liver regeneration involved changes in the transcription rate. The rate decreased by 50% at 6 hr after liver resection. The rate increased thereafter to a maximum at 72 hr and then returned to control values by 96 hr. The transcription rate of albumin did not change, whereas that of phosphoenolpyruvate carboxykinase increased 12-fold at 6 hr. These results show that PFK-2/FBPase-2 gene transcription is specifically regulated and that this regulation is in part responsible for the alterations in hepatic metabolism seen in regenerating liver.
Antiestrogen ICI 164,384 reduces cellular estrogen receptor content by increasing its turnover.
Authors: Dauvois S , Danielian P S , White R , Parker M G
Published: May 2006

The ability of estrogens to stimulate the transcriptional activity of the estrogen receptor can be inhibited by a diverse range of estrogen antagonists. Here we show that the antiestrogen ICI 164,384, N-(n-butyl)-11-[3,17 beta-dihydroxy-estra-1,3,5(10)-trien-7 alpha-yl]N-methylundecanamide, rapidly reduces the levels of receptor protein transiently expressed in cells without affecting receptor mRNA abundance. The reduction in the levels of receptor protein is dose dependent, reversible by estradiol, and mediated by the hormone-binding domain of the receptor. Pulse-chase experiments indicate that the half-life of the receptor is reduced from approximately 5 hr in the presence of estradiol to less than 1 hr by ICI 164,384. A similar reduction in estrogen receptor levels is demonstrated in human breast cancer cells treated with ICI 164,384. We discuss the possibility that the increased turnover of the receptor might be a consequence of impaired receptor dimerization.
Manidipine regulates the transcription of cytokine genes.
Authors: Roth M , Keul R , Emmons L R , Hörl W H , Block L H
Published: May 2006

Manidipine, a Ca(2+)-channel blocker, at concentrations that lower elevated blood pressure, modulates the transcription rates of cytokine genes in the mesangial cells of humans that had been stimulated with platelet-derived growth factor BB isomer; although the transcription for mRNA of interleukin 1 beta and granulocyte/monocyte colony-stimulating factor was inhibited, the transcription of mRNA for interleukin 6 was enhanced. Additionally, the induction of c-fos, c-jun, and 3-hydroxy-3-methylglutaryl-coenzyme A reductase transcription was inhibited by manidipine. We conclude that manidipine, at nanomolar concentrations, is efficacious in modulating gene transcriptions that are involved in proinflammatory changes of mesangial cells. Thus, manidipine, at pharmacological concentrations that are one to two orders of magnitude lower than those required for inhibition of agonist- or depolarization (K+)-induced vasoconstriction, causes changes in the activity of the genes that code for inflammatory mediators.
Gene in the region of the Friedreich ataxia locus encodes a putative transmembrane protein expressed in the nervous system.
Authors: Duclos F , Boschert U , Sirugo G , Mandel J L , Hen R , Koenig M
Published: May 2006

Friedreich ataxia (FRDA) is an autosomal recessive degenerative disorder that affects the cerebellum, spinal cord, and peripheral nerves. The FRDA gene was localized in 9q13-q21 within 0.7 centimorgan of the D9S5 and D9S15 loci. One recently reported recombination event and haplotype analysis in a population with a founder effect suggested that the FRDA locus is on the D9S5 side. Using a conserved probe from the D9S5 locus, we have now identified an approximately 7-kilobase (kb) transcript and report cloning of its cDNA. The corresponding gene, X11, extends at least 80 kb in a direction opposite D9S15. The gene is expressed in the brain, including the cerebellum, but is not detectable in several nonneuronal tissues and cell lines. In situ hybridization of adult mouse brain sections showed prominant expression in the granular layer of the cerebellum. Expression was also found in the spinal cord. The cDNA contains an open reading frame encoding a 708-amino acid sequence that shows no significant similarity to other known proteins but contains a unique, 24-residue-long, putative transmembrane segment. On the basis of its genomic localization and its neuronal site of expression, particularly in the cerebellum, this "pioneer" gene represents a candidate for FRDA. Direct evidence of its involvement in FRDA will require a search for causative point mutations in patients.
Serotonin receptor cDNA cloned from Lymnaea stagnalis.
Authors: Sugamori K S , Sunahara R K , Guan H C , Bulloch A G , Tensen C P , Seeman P , Niznik H B , Van Tol H H
Published: May 2006

Serotonin (5-HT) is a major neurotransmitter that influences various behaviors, neuronal plasticity, learning, and memory in molluscs. Although the physiology of 5-HT transmission in molluscs is well studied, little is known about the pharmacology and diversity of the 5-HT receptor system. Based on the high homology of genes coding for guanine nucleotide-binding protein (G protein)-coupled receptors, we have cloned a gene for the Lymnaea stagnalis 5-HT (5HTlym) receptor. The putative receptor protein, 509 amino acids long, has highest homology with the Drosophila 5-HT receptors and mammalian 5HT1 receptors. As revealed by RNA blot-hybridization analysis, two mRNA species of 2.3 and 3.2 kb are detected in the central nervous system of Lymnaea. Transient expression of 5HTlym in COS-7 cells showed saturable [3H]lysergic acid diethylamide binding with an estimated dissociation constant of 0.9 nM. The 5HTlym receptor exhibited a mixed 5HT-like pharmacology that cannot be precisely categorized with existing mammalian classification nomenclature. However, the 5HTlym receptor does display some characteristics that have been attributed to the putative mammalian vascular 5HT1-like receptor.
Characterization of cis-acting elements regulating transcription of the human DF3 breast carcinoma-associated antigen (MUC1) gene.
Authors: Abe M , Kufe D
Published: May 2006

The present studies have examined the sequences responsible for regulating transcription of the human DF3 breast carcinoma-associated antigen (MUC1) gene. A region 1656 base pairs upstream to the DF3 transcription initiation site was fused to the chloramphenicol acetyltransferase gene. Transient expression assays using a series of deleted constructs demonstrated that the region from position -618 contains the regulatory sequences necessary for DF3 transcription in human MCF-7 breast cancer cells. Further analysis with internal deletion vectors and heterologous promoter constructs indicated the involvement of cis-acting elements in the fragment extending from positions -598 to -485. By gel retardation and DNA footprinting, we have identified a protein in MCF-7 cells that recognizes sequences between positions -505 and -485. The results of Southwestern studies demonstrate that this protein has an apparent molecular mass of 45 kDa. Taken together, these results suggest that DF3 gene transcription is regulated by a previously undescribed transacting factor.
A hepatitis B virus pre-S-retinoic acid receptor beta chimera transforms erythrocytic progenitor cells in vitro.
Authors: Garcia M , de Thé H , Tiollais P , Samarut J , Dejean A
Published: May 2006

In this report, we investigated the transforming properties of retinoic acid receptor beta (RAR beta). The v-erbA protein, which is the viral oncogenic homologue of the thyroid hormone receptor, was replaced by either the complete RAR beta (beta R) or a hepatitis B virus pre-S-RAR beta (H beta R) hybrid product in an avian erythroblastosis virus-based vector. In chicken hematopoietic cells, the H beta R protein was able to transform erythroid progenitor cells, whereas no such transformation was observed with the wild-type beta R protein. Moreover, the fully transformed phenotype was observed even in the absence of v-erbB, and H beta R-transformed erythroid cells grew independently of growth factors and transforming growth factor alpha. The analysis of erythrocytic-specific proteins revealed that the transformed cells were blocked at the colony-forming unit-erythroid stage and that the expression of the carbonic anhydrase II gene, a gene normally regulated by thyroid hormones, was repressed by the H beta R protein. Finally, hepatocarcinomas rapidly developed in some chickens infected in ovo with viruses encoding either the normal or the hybrid H beta R, suggesting that an inappropriate expression of the RAR beta gene may represent an important event in oncogenesis.
Substrate specificity of the protein tyrosine phosphatases.
Authors: Zhang Z Y , Thieme-Sefler A M , Maclean D , McNamara D J , Dobrusin E M , Sawyer T K , Dixon J E
Published: May 2006

The substrate specificity of a recombinant protein tyrosine phosphatase (PTPase) was probed using synthetic phosphotyrosine-containing peptides corresponding to several of the autophosphorylation sites in epidermal growth factor receptor (EGFR). The peptide corresponding to the autophosphorylation site, EGFR988-998, was chosen for further study due to its favorable kinetic constants. The contribution of individual amino acid side chains to the binding and catalysis was ascertained utilizing a strategy in which each amino acid within the undecapeptide EGFR988-998 (DADEpYLIPQQG) was sequentially substituted by an Ala residue (Ala-scan). The resulting effects due to singular Ala substitution were assessed by kinetic analysis with two widely divergent homogeneous PTPases. A "consensus sequence" for PTPase recognition may be suggested from the Ala-scan data as DADEpYAAPA, and the presence of acidic residues proximate to the NH2-terminal side of phosphorylation is critical for high-affinity binding and catalysis. The Km value for EGFR988-998 decreased as the pH increased, suggesting that phosphate dianion is favored for substrate binding. The results demonstrate that chemical features in the primary structure surrounding the dephosphorylation site contribute to PTPase substrate specificity.
Perturbations of the distal heme pocket in human myoglobin mutants probed by infrared spectroscopy of bound CO: correlation with ligand binding kinetics.
Authors: Balasubramanian S , Lambright D G , Boxer S G
Published: May 2006

The infrared spectra of CO bound to human myoglobin and myoglobin mutants at positions His-64, Val-68, Asp-60, and Lys-45 on the distal side have been measured between 100 and 300 K. Large differences are observed with mutations at His-64 and Val-68 as well as with temperature and pH. Although distal His-64 is found to affect CO bonding, Val-68 also plays a major role. The variations are analyzed qualitatively in terms of a simple model involving steric interaction between the bound CO and the distal residues. A strong correlation is found between the final barrier height to CO recombination and the CO stretch frequency: as compared to wild type, the barrier is smaller in those mutants that have a higher CO stretch frequency (vCO) and vice versa. Possible reasons for this correlation are discussed. It is emphasized that the temperature and pH dependence of both the kinetics and the infrared spectra must be measured to obtain a consistent picture.