Actin and spectrin-like (Mr= 260–240 000) proteins in gregarines

In Gregarina blaberae a M_r = 47 000 and a M_r = 260–240 000 doublet polypeptides reacted in immunoblotting: i) with a polyclonal monospecific rabbit antibody to frog muscular actin, a monoclonal anti-actin antibody against chicken gizzard; and ii) with polyclonal and monoclonal antibodies to human erythrocyte β-spectrin, respectively. The M_r = 47 000 actin-like protein is associated with the ghost and a contractille cytoplasmic extract. The presence of an actin-like protein in Gregarina and Lecudina and its cellular distribution in the cortex indicated that the gliding movement might involve an actin-myosin system in contrast to previous studies. Immunofluorescence showed clear differences between the anterior part of Gregarina and Lecudina which illustrated the high cell polarity of these protozoa. The M_r = 260–240 000 doublet was detected in SDS-PAGE from G. blaberae trophozoite ghosts but not in the cytoplasmic extracts or in extracts from sexual stages, indicating that the presence of these spectrin-like proteins is stage-dependent. Visualization of the M_r = 260–240 000 by immunofluorescence showed clear species differences, with rings arranged perpendicular to the longitudinal narrow folds of G. blaberae, with longitudinal lines underlying the folds of L. pellucida and with lines separating the large folds of Selenidium pendula. The cellular distribution is consistent with a stabilizer function of the spectrin-like proteins in the scaffolding of the cortex of gregarines according to the high diversity of the cell-shape and the cell motility systems in gregarines. The presence of spectrin-like proteins in protozoa and particularly in parasites from primitive arthropods indicated that ancestral spectrin genes could the M_r = 260–240 000 form.     

Alpha-aminoxy acids as building blocks for the oxime and hydroxylamine pseudopeptide links. Application to the synthesis of human elastase inhibitors.

The aminoxy acids NH2-O-C(alpha)HR-CO2H are much more easily obtained in the enantiomerically pure form than the analogous hydrazino acids NH2-NH-C(alpha)HR-CO2H, and it has been shown that the isosteric amidoxy psi[CO-NH-O] and hydrazide psi[CO-NH-NH] amide surrogates Induce two quite similar gamma-like folded structures. An aminoxy acid can also be N-coupled to a peptide aldehyde to give the aldoxime psi[CH = N-O] link or to a peptide ketone to form the ketoxime psi[CR= N-O] link. The former can be further reduced into the hydroxylamine psi[CH2-NH-O] link which gives rise to reduced amidoxy peptides. The structural properties Induced by these amide surrogates were studied, using IR and NMR spectroscopy, paying particular attention to the Z/E-isomerism of the oxime link. In order to investigate their inhibitory potency, the three amide surrogates were introduced in the Pro3-Val4 and Val4-Ala5 position of Z-Ala1-Ala2-Pro3-Val4-Ala5-Ala6-NHiPr, a substrate which is cleaved in the Val4-Ala5 position by human leukocyte elastase (HLE). The [Val4psi[CO-NH-O]Ala5] analogue was still a substrate, while the [Pro3psi[CO-NH-O]Val4] and [Val4psi[CH = N-O]Ala5] pseudopeptides acted as HLE competitive inhibitors.     

ISOZYME VARIABILITY IN TRYPANOSOMA CRUZI,THE AGENT OF CHAGAS' DISEASE: GENETICAL,TAXONOMICAL, AND EPIDEMIOLOGICAL SIGNIFICANCE

A genetic interpretation of the zymograms of 524 Trypanosoma cruzi stocks from various hosts and representing a broad geographical range (United States to Southern Brazil) reveals high genetic variability (only one monomorphic locus out of 15) and suggests that this parasite has a diploid structure. The data do not give any indication of Mendelian sexuality, although many opportunities are present for genetic exchange between extremely different genotypes. The population structure of T. cruzi appears to be multiclonal and complex. The natural clones evidenced by isozyme analysis are numerous (43 different ones are recorded among 121 stocks assayed at 15 gene loci) and exhibit a large range of genotypes, in a nonhierarchical structure; it is not possible to cluster them into a few strictly delimited groups which could represent natural taxa. The available data suggest that the genetic variability of T. cruzi reflects the long separate evolution of multiple clones. It is suggested that long clonal evolution may explain the present biological and medical variability of the causative agent of Chagas' disease.     

Further progress towards a catalogue of all Arabidopsis genes: analysis of a set of 5000 non-redundant ESTs

Nearly 7000 Arabidopsis thaliana -expressed sequence tags (ESTs) from 10 cDNA libraries have been sequenced, of which almost 5000 non-redundant tags have been submitted to the EMBL data bank. The quality of the cDNA libraries used is analysed. Similarity searches in international protein data banks have allowed the detection of significant similarities to a wide range of proteins from many organisms. Alignment with ESTs from the rice systematic sequencing project has allowed the detection of amino acid motifs which are conserved between the two organisms, thus identifying tags to genes encoding highly conserved proteins. These genes are candidates for a common framework in genome mapping projects in different plants.     

Folded Structures in Protonated Reduced Dipeptides

Reduced dipeptides with the general formula RCO-Xaa- rXbb-N⁺HR′R′′ (rXbb, reduced analogue of residue Xbb: NH-C^α HR¹ -C_r H₂) are shown to adopt a folded conformation in solution and in the solid state. The protonated reduced amide bond is an active proton donor capable of interacting with a peptide carbonyl to give a strong hydrogen bond topologically equivalent to the i+2 or i+3? i interaction. The resulting conformation is similar to the γ- or β-turn structure found in peptides and proteins.     

Tomato contains two differentially expressed genes encoding B-type phytochromes,neither of which can be considered an ortholog of Arabidopsis phytochrome B

Tomato (Solanum lycopersicon L.) contains two B-type phytochrome genes (PHYB1 and PHYB2). Fragments of these two PHYB were cloned following amplification by the polymerase chain reaction of a portion of their relatively well conserved 5 coding regions. Polypeptides encoded by these gene fragments exhibit 90% sequence identity. These two PHYB are independently expressed in organ-specific fashion. In mature plants, PHYB2 mRNA is most abundant in fruit and PHYB1 mRNA in expanded leaves. A phylogenetic analysis fails to establish which tomato PHYB is orthologous to either Arabidopsis PHYB or PHYD, the latter being a second B-type phytochrome. Instead, this analysis indicates that following the divergence of the Solanaceae and Brassicaceae from one another, a PHYB gene duplicated independently in each lineage. Consequently, Arabidopsis PHYB mutants cannot be considered strictly equivalent to the tomato tri mutants, which appear to be mutated at the PHYB1 locus. Similarly, other putative PHYB mutants might not be equivalent to those described for Arabidopsis and tomato. This situation complicates efforts to determine PHYB function because there might be no one answer to this question.Abbreviations PCR polymerase chain reaction - PHY undesignated phytochrome gene - PHYA, PHYB, etc phytochrome gene(s) of the A, B, etc. type This research was supported by USDA NRICGP grant 93-00939 and by NATO travel grant CRG 931183. It was initiated when two of us (L.H.P., M.-M.C.-P.) spent a sabbatical year at the Institut National de la Recherche Agronomique in Versailles, France. L.H.P. gratefully acknowledges support provided by a senior guest fellowship from the Ministère de l'enseignement superieur et de la recherche during his stay in Versailles. L.H.P. and M.-M.C.-P thank all of their colleagues in Versailles for their warm hospitality and their willingness to share their expertise with us. We also thank Russell Malmberg, Richard Meagher and Robert Price for helpful discussions concerning the interpretation of molecular phylogenies.     

Astroglial Cells Express Large Amounts of GABAA Receptor Proteins in Mature Brain

Abstract: GABA_A receptors were characterized in cellular fractions isolated from adult bovine brain. The fraction enriched in cortical astrocytes is very rich in high-affinity binding sites for [³H]flunitrazepam and other "central-type" benzodiazepine ligands. The amount of specific [³H]flunitrazepam binding was more than five times higher in the glial fraction than in synaptosomal and perikaryal fractions. [³H]Flunitrazepam was displaced by low concentrations of clonazepam and other specific ligands for central GABA_A receptors. Specific binding sites for GABA, flunitrazepam, barbiturates, and picrotoxin-like convulsants were characterized. Allosteric interactions between the different sites were typical of central-type GABA_A receptors. The presence of α-subunit(s), as revealed by [³H]flunitrazepam photoaffinity labeling, was demonstrated in all brain fractions at molecular mass 51–53 kDa. Photoaffinity labeling was highest in the glial fraction. However, in primary cultured astrocytes from neonate rat cortex, no photoaffinity labeling was detected. Information obtained from astrocytes in culture should thus be taken with caution when extrapolated to differentiated astroglial cells. Our results actually show that, in mature brain, most of the fully pharmacologically active GABA_A receptors are extrasynaptic and expressed in astroglia.     

Insertion mutations at the maizeOpaque2 locus induced by transposable element familiesAc, En/Spm andBg

Eight independently isolated unstable alleles of theOpaque2 (O2) locus were analysed genetically and at the DNA level. The whole series of mutations was isolated from a maize strain carrying a wild-typeO2 allele and the transposable elementActivator (Ac) at thewx-m7 allele. Previous work with another unstable allele of the same series has shown that it was indeed caused by the insertion of anAc element. Unexpectedly, the remaining eight mutations were not caused by the designatedAc element, but by other insertions that are structurally similar or identical to one of two different autonomous transposable elements. Six mutations were caused by the insertion of a transposable element of theEnhancer/Suppressor-Mutator (En/Spm) family. Two mutations were the result of the insertion of a transposable element of theBergamo (Bg) family. Genetic tests carried out with plants carrying the unstable mutations demonstrated that all were caused by the insertion of an autonomous transposable element.