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The legumin gene family: structure and evolutionary implications of Vicia faba B-type genes and pseudogenes.

We have characterized several Vicia faba genes encoding methionine residue-free group B subunits of the 11S or legumin storage proteins. The respective gene subfamily consists of 10 to 15 members, six of them having been studied by DNA sequence analysis. Four functional genes (LeB2, LeB4, LeB6, LeB7) are highly homologous in their coding region and 0.3 kb of their 3' flanking sequences. On the other hand, two pseudogenes (psi LeB1, psi LeB5) have accumulated a large number of mutations including an identical 0.7 kb internal deletion; they are both flanked by a repetitive element. Analysis of sequence changes show that transitions are nearly double as frequent as transversions. CpG is the most infrequent dinucleotide whereas TpA is significantly underrepresented in exon sequences. End points of deletions are correlated with short direct repeats and preferentially found in the two introns. Our studies indicate that the Vicia faba legumin B gene subfamily contains a group of expressed, highly homologous genes as well as more diverged pseudogenes.

Amino Acid Sequence↗

DNA regions flanking the major Arabidopsis thaliana satellite are principally enriched in Athila retroelement sequences.

An analysis of Arabidopsis thaliana heterochromatic regions revealed that genomic sequences immediately flanking the major 180 bp satellite are essentially made of middle repetitive sequences and that most of these sequences correspond to defective Athila retroelements. Using YAC and lambda clones, we evaluated the distribution of Athila elements in the Arabidopsis genome and showed that, despite the presence of numerous euchromatic copies, these elements are especially concentrated in or near heterochromatic regions. Sequencing of the various DNA transitions between satellite and Athila repeats provides strong evidence that most of the heterochromatic elements retrotransposed directly into 180 bp satellite clusters.

Arabidopsis↗

Hydration and preferential molecular adsorption on titanium in vitro.

Surface sensitive spectroscopies, Auger electron and X-ray photoelectron (XPS), were used to determine changes in titanium oxide composition, oxide stoichiometry, and adsorbed surface species as a function of exposure to human serum in a balanced electrolyte (serum/SIE) and 8.0 mM ethylenediaminetetraacetic acid in a balanced electrolyte (EDTA/SIE) at 37 degrees C. Before immersion, the oxide was near ideal TiO2, covered by two types of hydroxyl groups: acidic OH(s) with oxygens doubly coordinated to titanium, and basic Ti-OH groups singly coordinated. After extended exposure to both solutions, up to 5000 h (ca. 208 d), the surface concentration of OH groups increased and non-elemental P appeared. The P LVV Auger transition and P 2p spectra indicated the peak positions were similar to reference phosphate compounds. The adsorbed phosphate species were presumed to be either Ti-H2PO4 or Ti-HPO4-. The XPS data suggested that a lipoprotein and/or glycolipid film was adsorbed to the specimens exposed to serum/SIE. Analysis of the preferential lipoprotein/glycolipid adsorption using electrostatic bonding concepts contributed to the refinement of the hierarchical model for the Ti-tissue interface. The salient features are that Ti metal is not in direct contact with the biological milieu, rather there is a gradual transition from the bulk metal, near-stoichiometric oxide, Ca and P substituted hydrated oxide, adsorbed lipoproteins and glycolipids, proteoglycans, collagen filaments and bundles to cells.

Adsorption↗

Copper homeostasis in eukaryotes: teetering on a tightrope.

The transition metal copper is an essential trace element for both prokaryotes and eukaryotes. However, intracellular free copper has to be strictly limited due to its toxic side effects, not least the generation of reactive oxygen species (ROS) via redox cycling. Thus, all organisms have sophisticated copper homeostasis mechanisms that regulate uptake, distribution, sequestration and export of copper. From insects to mammals, metal-responsive transcription factor (MTF-1), a zinc finger transcription factor, controls expression of metallothioneins and other components involved in heavy metal homeostasis. In the fruit fly Drosophila, MTF-1 paradoxically acts as an activator under both high and low copper concentrations. Namely, under high copper conditions, MTF-1 activates metallothioneins in order to protect the cell, while under low copper conditions MTF-1 activates the copper importer Ctr1B in order to acquire scarce copper from the surroundings. This review highlights the current knowledge of copper homeostasis in eukaryotes with a focus on Drosophila and the role of MTF-1.

Animals↗

CDK activity antagonizes Whi5, an inhibitor of G1/S transcription in yeast.

Cyclin-dependent kinase (CDK) activity initiates the eukaryotic cell division cycle by turning on a suite of gene expression in late G1 phase. In metazoans, CDK-dependent phosphorylation of the retinoblastoma tumor suppressor protein (Rb) alleviates repression of E2F and thereby activates G1/S transcription. However, in yeast, an analogous G1 phase target of CDK activity has remained elusive. Here we show that the cell size regulator Whi5 inhibits G1/S transcription and that this inhibition is relieved by CDK-mediated phosphorylation. Deletion of WHI5 bypasses the requirement for upstream activators of the G1/S transcription factors SBF/MBF and thereby accelerates the G1/S transition. Whi5 is recruited to G1/S promoter elements via its interaction with SBF/MBF in vivo and in vitro. In late G1 phase, CDK-dependent phosphorylation dissociates Whi5 from SBF and drives Whi5 out of the nucleus. Elimination of CDK activity at the end of mitosis allows Whi5 to reenter the nucleus to again repress G1/S transcription. These findings harmonize G1/S control in eukaryotes.

Antibodies, Monoclonal↗

Estradiol affects androgen-binding protein expression and fertilizing ability of spermatozoa in adult male rats.

The estrogenicity of certain environmental pollutants is being increasingly correlated to decline in sperm counts and fertility of the males. Qualitative effects, if any, of estrogen(s) on terminal differentiation of spermatids have been less reported. The present study suggests that exposure to estrogen(s) can also alter the status of condensed chromatin in testicular spermatozoa and reduce their fertilizing potential. A significant reduction was evident in the serum gonadotropins, testosterone, weights of reproductive organs, sperm counts and litters sired by male rats after 10 days of estradiol exposure to a dose of 0.1mg/kg/day. Estradiol treatment led to retardation of in vitro decondensation rates of sperm chromatin, reduction in the uptake of acridine orange dye by chromatin, reduction in susceptibility of chromatin to acid denaturation in vitro, reduced uptake of thiol reactive monobromobimane dye and reduced levels of immunoreactive protamine 1 in caput epididymal sperms. Concomitantly, testicular levels of immunoreactive protamine 1, transition proteins 1/2 and cyclic adenosyl response element modulator-tau (CREMtau) were significantly reduced whilst their mRNA levels were unaffected after estradiol treatment. A significant increase was observed in the testicular mRNA levels of androgen-binding protein (ABP) in estradiol treated sires. An inverse correlation was observed between ABP mRNA levels and uptake of acridine orange by estradiol treated caput sperm chromatin. The results suggest that estradiol-induced increase in ABP mRNA underlies the mechanism(s) involved in the reduction in levels of certain proteins involved in nuclear chromatin condensation during spermiogenesis.

Androgen-Binding Protein↗

Spectroscopic approach in characterization of chromium(III), manganese(II), iron(III) and copper(II) complexes with a nitrogen donor tetradentate, 14-membered azamacrocyclic ligand.

The complexes of Cr(III), Mn(II), Fe(III) and Cu(II) were synthesized with the macrocyclic ligand i.e. 2,3,9,10-tetraketo-1,4,8,11-tetraazacyclotetradecane. The ligand was prepared by the [2 + 2] condensation reaction of diethyloxalate and 1,3-diamino propane. These complexes were found to have the general composition M(L)X3 and M'(L)X2 [where M = Mn(II) and Cu(II), M' = Cr(III) and Fe(III), L = ligand (N4) and X = Cl-, NO3-, 1/2SO4(2-) and [CH3COO-]. The ligand and its transition metal complexes were characterized by the elemental analyses, molar conductance, magnetic susceptibility, mass, IR, electronic, and EPR spectral studies. On the basis of IR, electronic and EPR spectral studies an octahedral geometry has been assigned for Cr(III), Mn(II) and Fe(III) and a tetragonal geometry for Cu(II) complexes.

Aza Compounds↗

[Several factors to distinguish anosognosia from denial after a brain injury].

This Article deals with the question of ignorance after a serious brain injury. The main purpose of this research is to present some factors that are critical for the differential diagnosis of anosognosia (organic etiology) and denial (psychological etiology) in order to improve the rehabilitation strategies of brain injuried population. In the present work we report the case study of MG (Havet-Thomassin, 2000). MG is a 32 Year old right handed man who has undergone a severe brain injury after a car accident. Neuropsychological approach of ignorance is mainly landed from questionnaire assessment of deficit. The QAM (Questionnaire of self assessment of memory performance, Van der Linden et al., 1988) and the PCRS (Patient Competency Rating Scale, Roueche et Fordyce, 1982) where used for the neuropsychological assessment of anosognosia. The neuropsychological baseline was obtained from classical executive tasks (TMT, Stroop, TOL, WCST) in order to show a possible link between executive dysfunction and anosognosia. The results point several cognitive impairments (attention, memory, executive functions) and an inadaptated behavior associated to an important anosognosia particularly at the beginning of the hospitalization (absence of self-criticism and bad compliance to rehabilitation). Furthermore, the patient was interviewed regularly in order to better dissociate denial from anosognosia. This clinical approach facilitated the understanding of the inherent psychological dynamic of MG which was particularly exacerbated by the frontal desinhibition. Characterization of identity profile and homosexuality are of great interest in this case as they were strongly reactivated by the traumatism. The brain injury leads to the reorganization of the whole identity of MG which seems no more unified but divided. Reality principle was responsible for too much anxiety which became probably acute by a narcissistic flaw. Therefore it encourages the subject to deny in order to guarantee to himself a psychic balance. Thus, at the beginning, MG denies totally his homosexuality and then, he admits it progressively declaring that it has disappeared since the accident. He progressively developed an excessive aggressiveness in regard of the homosexual community insinuated unconsciously from his discourse his feminine and passive position (slips, denegations). This denial is critical for MG's psychic integrity and that is why the priority should not be its suppression. From this work, we consider that anosognosia remains independent of the patient's will, but still linked to the dysexecutive syndrom. In opposite, the denial corresponds to defensive and strategic processes devoted to the subject adaptation to an agonizing situation. Even if those two clinical facts appear similar, it is possible to propose several factors in order to distinguish anosognosia from denial: 1) anosognosia and denial don't seem to turn on the same elements; 2) anosognosia seems to be more transitional on the contrary of the denial which appears to be more chronic; 3) behavior reaction in the case of anosognosia seems to be partially different from the denial; 4) as compared to anosognosia, denial appears less stable and more uncertain depending on the psychic cost. In conclusion, distinction between anosognosia and denial would allow the clinical psychologist to propose a more adaptated therapy for the patient. Denial must be taken in a dynamic perspective and not as a direct negative consequence of the cerebral injury. In such situation, the priority is not to suppress denial but rather to consider it is the way the subject should face laborious situations imposed by the reality.

Adult↗

Primary non-urachal adenocarcinoma of the bladder.

The clinical course of 34 patients with non-urachal adenocarcinoma of the bladder was reviewed and compared to the world experience. The 5-year survivorship was 19 per cent, with only 1 patient being free of disease. Metaplasia with formation of glandular elements reflects the unstable potential of the transitional cell and may be associated with the biologic aggressiveness of the tumor.

Adenocarcinoma↗

Catalytic assessment of the glycine-rich loop of the v-Fps oncoprotein using site-directed mutagenesis.

The three glycine residues in the glycine-rich loop of the oncoprotein, v-Fps, were mutated to determine the function of these highly conserved residues in catalysis. The kinase domains of six mutants (G928A,S, G930A,S, and G933A,S) and the wild-type enzyme were expressed and purified as fusion proteins of glutathione-S-transferase in Escherichia coli, and their catalytic properties were assessed using steady-state kinetic, inhibition, viscosity and autophosphorylation studies. Although both G928A and G930A had no detectable activity toward the substrate peptide (EAEIYEAIE), the other mutants had apparent, but varying activities. G930S lowered the rate of phosphoryl transfer by 130-fold while G928S and G933S had smaller (6-9-fold) reductions in this step. These effects on catalytic function parallel the reductions in turnover and autophosphorylation but, for G933S and G933A, net product release is still rate limiting at saturating substrate and ATP concentrations. On the basis of K(I) measurements, the effects on turnover for these mutants may be due to improved ADP affinity. While ADP affinity is reduced 2- and 3-fold for G928S and G930S, the affinity of this product is increased by 22- and 7-fold for G933S and G933A. In contrast, ATP affinity is enhanced by 5-fold for G928S and G933S and is reduced by less than 2-fold for G930S. These complex, differential effects on nucleotide binding indicate that the glycines influence the relative affinities of ADP and ATP. On the basis of the results of serine replacements, Gly-928 and Gly-930 enhance ADP affinity by 9- and 2-fold compared to ATP affinity whereas Gly-933 diminishes ADP affinity by approximately 4-fold compared to ATP affinity. These findings demonstrate that the functions of the loop lie not only in modulating the rate of the phosphoryl transfer step but also in balancing the relative affinities of ATP and ADP. These effects on nucleotide specificity may be a contributing element for the stabilization of the phosphoryl transition state and may also facilitate quick release of bound products.

Adenosine Diphosphate↗

A thiazole-containing tripodal ligand: synthesis, characterization, and interactions with metal ions and matrix metalloproteinases.

A new tripodal ligand, tris[2-(((2-thiazolyl)methylidene)amino)ethyl]amine (Tatren), has been synthesized and characterized by NMR, IR, and UV-visible absorbance spectroscopy and elemental analysis. Tatren forms stable complexes with transition metal ions (Zn(2+), 1; Mn(2+), 2; Co(2+), 3) and the alkaline earth metal ions (Ca(2+), 4; Mg(2+), 5). Single-crystal X-ray structures of 1, 2, and 5 revealed six-coordinate chelate complexes with formula [M(Tatren)](ClO(4))(2) in which the metal centers are coordinated by three thiazolyl N atoms and three acyclic imine N atoms. Crystals of 1, 2, and 5 are monoclinic, P2(1)/c space group. Crystals of 4 are triclinic, P space group. The Ca(2+) complex is eight-coordinate with all N atoms of Tatren and one water molecule coordinated to the metal ion. Spectrophotometric titrations show that formation constants for the chelates of metal ions are >>1 in methanol. Free Tatren inhibits the catalytic domain of matrix metalloproteinase-13 (MMP-13, collagenase-3) with K(i) = 3.5 +/- 0.6 microM. Molecular mechanics-based docking calculations suggest that one leg of Tatren coordinates to the catalytic Zn(2+) in MMPs-2, -9, and -13 with significant hydrogen bonding to backbone amide groups. High-level DFT calculations suggest that, in the absence of nonbonded interactions between Tatren and the enzyme, the most stable first coordination sphere of the catalytic Zn(2+) is achieved with three imidazolyl groups from His residues and two imine N atoms from one leg of Tatren. While complexes (1-3) do not inhibit MMP-13 to a significant extent, 4 does (K(i) = 30 +/- 10 microM). Hence, this study shows that tripodal chelating ligands of this class and their Ca(2+) complexes have potential as active-site inhibitors for MMPs.

Binding Sites↗

Intracellular trafficking and metabolic turnover of yeast prepro-alpha-factor-SRIF precursors in GH3 cells.

Chimeric genes coding for prepro region of yeast alpha-factor and anglerfish SRIF were expressed in rat GH3 cells to determine whether yeast signals could regulate hormone processing in mammalian cells. We report that nascent hybrid polypeptides were efficiently targeted to ER, where cleavage of signal peptides and core glycosylation occurred, and were localized mainly in Golgi. These data indicate that prepro region of yeast alpha-factor functions in sorting molecules to secretory pathway in mammalian cells. A hybrid construct with a mutated signal peptide underwent similar ER translocation, whereas such a mutation resulted in defective translocation in yeast (Cheong et al., 1997). This difference may be due to the differences in ER translocation between yeast and mammalian cells, i.e., posttranslational versus cotranslational translocation. Processing and secretion of metabolically labeled hybrid propeptides to mature SRIF peptides were assessed by HPLC. When pulse-labeled cells were chased for up to 2 h, intracellular propeptides disappeared with a half-life of approximately 25 min, showing that approximately 68% of initially synthesized propeptides were secreted constitutively. About 22% of SRIF-related products were proteolytically processed to mature SRIF, of which 38.7% were stored intracellularly with a half-life of approximately 2 h. In addition, immunocytochemical localization showed that a small proportion of SRIF molecules accumulated in secretory vesicles. All these results suggest that yeast prepropeptide could direct hybrid precursors to translocate into ER lumen and transit through secretory pathway to the distal elements of Golgi compartment, but could process and target it less efficiently to downstream in rat endocrine cells.

Animals↗

Reorganization of the Mu transpososome active sites during a cooperative transition between DNA cleavage and joining.

Transposition of mobile genetic elements proceeds through a series of DNA phosphoryl transfer reactions, with multiple reaction steps catalyzed by the same set of active site residues. Mu transposase repeatedly utilizes the same active site DDE residues to cleave and join a single DNA strand at each transposon end to a new, distant DNA location (the target DNA). To better understand how DNA is manipulated within the Mu transposase-DNA complex during recombination, the impact of the DNA immediately adjacent to the Mu DNA ends (the flanking DNA) on the progress of transposition was investigated. We show that, in the absence of the MuB activator, the 3 '-flanking strand can slow one or more steps between DNA cleavage and joining. The presence of this flanking DNA strand in just one active site slows the joining step in both active sites. Further evidence suggests that this slow step is not due to a change in the affinity of the transpososome for the target DNA. Finally, we demonstrate that MuB activates transposition by stimulating the reaction step between cleavage and joining that is otherwise slowed by this flanking DNA strand. Based on these results, we propose that the 3 '-flanking DNA strand must be removed from, or shifted within, both active sites after the cleavage step; this movement is coupled to a conformational change within the transpososome that properly positions the target DNA simultaneously within both active sites and thereby permits joining.

Bacteriophage mu↗

Effects of calcium channel blockers on the spermatogenesis and gene expression in peripubertal mouse testis.

Treatment of Ca(2+) channel blockers (CCB) to relieve hypertension causes reversible male infertility, suggesting deregulation of Ca(2+) homeostasis in testis is closely related with male infertility. To investigate the possible toxicity of therapeutic application of CCB in childhood, the effect of nifedipine and ethosuximide, an L-type and T-type CCB, respectively, on the spermatogenesis and testicular gene expression was examined. Following the intraperitoneal injection of either drug for 7 days to 18 days on old mice, the paired testes weights were significantly lower in mice treated with nifedipine (> or = 10 mg/kg/day) or ethosuximide (100 mg/kg/day) than vehicle controls. In mice given high drug dosing (100 mg/kg), seminiferous tubules showed immaturity with spermatogenic arrest at elongating spermatid stage and poorly developed lumen. Unexpectedly, the expression of activator isoform of transcription factor cAMP-responsive element modulator (CREM) mRNA increased together with transition protein 2 and protamine 2 mRNA in drug-treated mice testes, suggesting that CCB may deregulate expression of activator isoform of CREM in male germ cells and that spermatogenic defect following CCB treatment may attribute to ectopic expression of CREM-dependent gene battery in testis. Therapeutic application of CCB in childhood should be cautious because of their potential to cause spermatogenic defect and altered gene expression in testis.

Animals↗

T cell receptor-mediated negative selection of autoreactive T lymphocyte precursors occurs after commitment to the CD4 or CD8 lineages.

To identify the maturational stage(s) during which T cell receptor (TCR)-mediated positive and negative selection occurs, we followed the development of CD4+8- and CD4-8+ T cells from TCRlo CD4+8+ thymic blasts in the presence of different positive and negative selecting (major histocompatibility complex or Mls) elements. We describe novel lineage-committed transitional intermediates that are TCRmed CD4+8lo or TCRmed CD4lo8+, and that show evidence of having been positively selected. Furthermore, negative selection is not evident until after cells have attained one of the TCRmed transitional phenotypes. Accordingly, we propose that negative selection in normal mice occurs only after TCRlo CD4+8+ precursors have been positively selected into either the CD4 or CD8 lineage.

Animals↗

Dynamics of transitional endoplasmic reticulum sites in vertebrate cells.

A typical vertebrate cell contains several hundred sites of transitional ER (tER). Presumably, tER sites generate elements of the ER-Golgi intermediate compartment (ERGIC), and ERGIC elements then generate Golgi cisternae. Therefore, characterizing the mechanisms that influence tER distribution may shed light on the dynamic behavior of the Golgi. We explored the properties of tER sites using Sec13 as a marker protein. Fluorescence microscopy confirmed that tER sites are long-lived ER subdomains. tER sites proliferate during interphase but lose Sec13 during mitosis. Unlike ERGIC elements, tER sites move very little. Nevertheless, when microtubules are depolymerized with nocodazole, tER sites redistribute rapidly to form clusters next to Golgi structures. Hence, tER sites have the unusual property of being immobile, yet dynamic. These findings can be explained by a model in which new tER sites are created by retrograde membrane traffic from the Golgi. We propose that the tER-Golgi system is organized by mutual feedback between these two compartments.

Animals↗

Adenosquamous carcinoma of the lung. Clinicopathologic and immunohistochemical features.

Adenosquamous carcinomas (ASC) of the lung comprise only 1.8% of all pulmonary cancers. Eleven patients with this disease (6 men and 5 women; mean age, 62 years [range, 51 to 78 years]) were treated. On the basis of pathologic and immunohistochemical evidence, the tumors were separated into three groups: five ASCs of predominantly glandular type, which may originate from squamous metaplasia in a preexisting adenocarcinoma; three mixed type ASCs, possibly derived from the transition of undifferentiated carcinoma to two different elements; and three predominantly squamous type ASCs resembling high-grade muco-epidermoid carcinoma. The 5-year survival rate of 35% for patients with ASCs was the same as that for patients with other nonsmall cell carcinomas. For two patients with ASCs of the predominantly glandular type and for one with the mixed type, resection was incomplete because of extensive local disease. All tumors of the predominantly squamous type could be excised completely. However, differences in the malignant potential of these three types of ASCs are still controversial.

Adenocarcinoma↗

Creating maximally entangled atomic states in a Bose-Einstein condensate.

We propose a protocol to create maximally entangled pairs, triplets, quartiles, and other clusters of Bose-condensed atoms starting from a condensate in the Mott insulator state. The essential element is to drive single atom Raman transitions using laser pulses. Our scheme is simple, efficient, and can be readily applied to the recent experimental system as reported by M. Greiner 413, 44 (2002)].

Journal Article↗