Cyclostreptin derivatives specifically target cellular tubulin and further map the paclitaxel site

Cyclostreptin is the first microtubule-stabilizing agent whose mechanism of action was discovered to involve formation of a covalent bond with tubulin. Treatment of cells with cyclostreptin irreversibly stabilizes their microtubules because cyclostreptin forms a covalent bond to β-tubulin at either the T220 or the N228 residue, located at the microtubule pore or luminal taxoid binding site, respectively. Because of its unique mechanism of action, cyclostreptin overcomes P-glycoprotein-mediated multidrug resistance in tumor cells. We used a series of reactive cyclostreptin analogues, 6-chloroacetyl-cyclostreptin, 8-chloroacetyl-cyclostreptin, and [(14)C-acetyl]-8-acetyl-cyclostreptin, to characterize the cellular target of the compound and to map the binding site. The three analogues were cytotoxic and stabilized microtubules in both sensitive and multidrug resistant tumor cells. In both types of cells, we identified β-tubulin as the only or the predominantly labeled cellular protein, indicating that covalent binding to microtubules is sufficient to prevent drug efflux mediated by P-glycoprotein. 6-Chloroacetyl-cyclostreptin, 8-chloroacetyl-cyclostreptin, and 8-acetyl-cyclostreptin labeled both microtubules and unassembled tubulin at a single residue of the same tryptic peptide of β-tubulin as was labeled by cyclostreptin (219-LTTPTYGDLNHLVSATMSGVTTCLR-243), but labeling with the analogues occurred at different positions of the peptide. 8-Acetyl-cyclostreptin reacted with either T220 or N228, as did the natural product, while 8-chloroacetyl-cyclostreptin formed a cross-link to C241. Finally, 6-chloroacetyl-cyclostreptin reacted with any of the three residues, thus labeling the pathway for cyclostreptin-like compounds, leading from the pore where these compounds enter the microtubule to the luminal binding pocket.     

Description of Devosia neptuniae sp. nov. that nodulates and fixes nitrogen in symbiosis with Neptunia natans,an aquatic legume from India

Neptunia natans is a unique aquatic legume indigenous to tropical and sub-tropical regions and is nodulated symbiotically by rhizobia using an unusual infection process unlike any previously described. Previously, isolates of neptunia-nodulating rhizobia from Senegal were characterized as Allorhizobium undicola. Here we report on a different group of neptunia-nodulating rhizobia isolated from India. Sequencing of the 16S rDNA gene from two of these Indian isolates (strains J1T and J2) show that they belong in the genus Devosia rather than Allorhizobium. Currently, the only described Devosia species is D. riboflavina (family Hyphomicrobiaceae, order Rhizobiales). The complete 16S rDNA sequences of strains J1T and J2 are 95.9% homologous to the type strain, D. riboflavina LMG 2277T, suggesting that these neptunia-nodulating strains from India belong to a new Devosia species. This hypothesis was confirmed by further studies of polyphasic taxonomy (DNA-DNA hybridisation, TP-RAPD patterns, SDS-PAGE of cellular proteins, 16S rDNA RFLP patterns, carbon source utilisation, cellular fatty acid analysis and other phenotypic characterisations), all of which support the proposal that these neptunia-nodulating strains constitute a new Devosia species, which we name Devosia neptuniae sp. nov. These gram negative, strictly aerobic short rods are motile by a subpolar flagellum, positive for catalase, oxidase, urease and beta-galactosidase, can utilise several carbohydrates (but not organic acids) as carbon sources and contain C18:0 3-OH, cis-7 C18:1 11-methyl and cis-7 C18:1 as their major cellular fatty acids. Unlike D. riboflavina, the longer-chain C24:1 3-OH and C26:1 3-OH hydroxy fatty acids are not detected. The type strain of D. neptuniae is LMG 21357T (CECT 5650T). Assignment of this new taxon represents the fourth example in the literature of a non-rhizobial genus of bacteria capable of forming a bonafide dinitrogen-fixing root-nodule symbiosis with legume plants.     

Molecular Characterization of Chronic Lymphocytic Leukemia Patients with a High Number of Losses in 13q14

Background

Patients with chronic lymphocytic leukemia and 13q deletion as their only FISH abnormality could have a different outcome depending on the number of cells displaying this aberration. Thus, cases with a high number of 13q- cells (13q-H) had both shorter overall survival and time to first therapy. The goal of the study was to analyze the genetic profile of 13q-H patients.

Design and Methods:

A total of 102 samples were studied, 32 of which served as a validation cohort and five were healthy donors.

Results

Chronic lymphocytic leukemia patients with higher percentages of 13q- cells (>80%) showed a different level of gene expression as compared to patients with lower percentages (<80%, 13q-L). This deregulation affected genes involved in apoptosis and proliferation (BCR and NFkB signaling), leading to increased proliferation and decreased apoptosis in 13q-H patients. Deregulation of several microRNAs, such as miR-15a, miR-155, miR-29a and miR-223, was also observed in these patients. In addition, our study also suggests that the gene expression pattern of 13q-H cases could be similar to the patients with 11q- or 17p-.

Conclusions

This study provides new evidence regarding the heterogeneity of 13q deletion in chronic lymphocytic leukemia patients, showing that apoptosis, proliferation as well as miRNA regulation are involved in cases with higher percentages of 13q- cells.     

Genome-wide association filtering using a highly locus-specific transmission/disequilibrium test

Multimarker transmission/disequilibrium tests (TDTs) are powerful association and linkage tests used to perform genome-wide filtering in the search for disease susceptibility loci. In contrast to case/control studies, they have a low rate of false positives for population stratification and admixture. However, the length of a region found in association with a disease is usually very large because of linkage disequilibrium (LD). Here, we define a multimarker proportional TDT (mTDT _P ) designed to improve locus specificity in complex diseases that has good power compared to the most powerful multimarker TDTs. The test is a simple generalization of a multimarker TDT in which haplotype frequencies are used to weight the effect that each haplotype has on the whole measure. Two concepts underlie the features of the metric: the ‘common disease, common variant’ hypothesis and the decrease in LD with chromosomal distance. Because of this decrease, the frequency of haplotypes in strong LD with common disease variants decreases with increasing distance from the disease susceptibility locus. Thus, our haplotype proportional test has higher locus specificity than common multimarker TDTs that assume a uniform distribution of haplotype probabilities. Because of the common variant hypothesis, risk haplotypes at a given locus are relatively frequent and a metric that weights partial results for each haplotype by its frequency will be as powerful as the most powerful multimarker TDTs. Simulations and real data sets demonstrate that the test has good power compared with the best tests but has remarkably higher locus specificity, so that the association rate decreases at a higher rate with distance from a disease susceptibility or disease protective locus.     

A new cDNA sequence for the murine interleukin-2 gene.

We have amplified by PCR and sequenced the first exon of the interleukin 2 gene from the RF/J mouse strain DNA. When we compared the RF/J first exon sequence with the one reported previously, we found several differences. These differences are also reflected in the deduced amino acid sequence and they have been localized in the first 23 amino acids of the mature polypeptide. The finding of this new IL-2 sequence shows that there is more than one allele for the mouse IL-2 molecule and raises the possibility of functional differences between alleles.     

The celC gene, a new phylogenetic marker useful for taxonomic studies in Rhizobium

The celC gene codifies for a cellulase that fulfils a very significant role in the infection process of clover by Rhizobium leguminosarum. This gene is located in the celABC operon present in the chromosome of strains representing R. leguminosarum, Rhizobium etli and Rhizobium radiobacter whose genomes have been completely sequenced. Nevertheless, the existence of this gene in other species of the genus Rhizobium had not been investigated to date. In this study, the celC gene was analysed for the first time in several species of this genus isolated from legume nodules and plant tumours, in order to compare the celC phylogeny to those of other chromosomal and plasmidic genes. The results obtained showed that phylogenies of celC and chromosomal genes, such as rrs, recA and atpD, were completely congruent, whereas no relation was found with symbiotic or virulence genes. Therefore, the suitability and usefulness of the celC gene to differentiate species of the genus Rhizobium, especially those with closely related rrs genes, was highlighted. Consequently, the taxonomic status of several strains of the genus Rhizobium with completely sequenced genomes is also discussed.     

Interaction of PLP with GFP-MAL2 in the human oligodendroglial cell line HOG

The velocity of the nerve impulse conduction of vertebrates relies on the myelin sheath, an electrically insulating layer that surrounds axons in both the central and peripheral nervous systems, enabling saltatory conduction of the action potential. Oligodendrocytes are the myelin-producing glial cells in the central nervous system. A deeper understanding of the molecular basis of myelination and, specifically, of the transport of myelin proteins, will contribute to the search of the aetiology of many dysmyelinating and demyelinating diseases, including multiple sclerosis. Recent investigations suggest that proteolipid protein (PLP), the major myelin protein, could reach myelin sheath by an indirect transport pathway, that is, a transcytotic route via the plasma membrane of the cell body. If PLP transport relies on a transcytotic process, it is reasonable to consider that this myelin protein could be associated with MAL2, a raft protein essential for transcytosis. In this study, carried out with the human oligodendrocytic cell line HOG, we show that PLP colocalized with green fluorescent protein (GFP)-MAL2 after internalization from the plasma membrane. In addition, both immunoprecipitation and immunofluorescence assays, indicated the existence of an interaction between GFP-MAL2 and PLP. Finally, ultrastructural studies demonstrated colocalization of GFP-MAL2 and PLP in vesicles and tubulovesicular structures. Taken together, these results prove for the first time the interaction of PLP and MAL2 in oligodendrocytic cells, supporting the transcytotic model of PLP transport previously suggested.     

Post-translational modifications of the gamma-subunit affect intracellular trafficking and complex assembly of GlcNAc-1-phosphotransferase

GlcNAc-1-phosphotransferase plays a key role in the generation of mannose 6-phosphate, a recognition marker essential for efficient transport of lysosomal hydrolases to lysosomes. The enzyme complex is composed of six subunits (α(2)β(2)γ(2)). The α- and β-subunits are catalytically active, whereas the function of the γ-subunit is still unclear. We have investigated structural properties, localization, and intracellular transport of the human and mouse γ-subunits and the molecular requirements for the assembly of the phosphotransferase complex. The results showed that endogenous and overexpressed γ-subunits were localized in the cis-Golgi apparatus. Secreted forms of γ-subunits were detectable in media of cultured cells as well as in human serum. The γ-subunit contains two in vivo used N-glycosylation sites at positions 88 and 115, equipped with high mannose-type oligosaccharides. (35)S pulse-chase experiments and size exclusion chromatography revealed that the majority of non-glycosylated γ-subunit mutants were integrated in high molecular mass complexes, failed to exit the endoplasmic reticulum (ER), and were rapidly degraded. The substitution of cysteine 245 involved in dimerization of γ-subunits impaired neither ER exit nor trafficking through the secretory pathway. Monomeric γ-subunits failed, however, to associate with other GlcNAc-1-phosphotransferase subunits. The data provide evidence that assembly of the GlcNAc-1-phosphotransferase complex takes place in the ER and requires dimerization of the γ-subunits.     

Definition of a novel symbiovar (sv. retamae) within Bradyrhizobium retamae sp. nov., nodulating Retama sphaerocarpa and Retama monosperma

In this paper we analyze through a polyphasic approach several Bradyrhizobium strains isolated in Spain and Morocco from root nodules of Retama sphaerocarpa and Retama monosperma. All the strains have identical 16S rRNA genes and their closest relative species is Bradyrhizobium lablabi CCBAU 23086^T, with 99.41% identity with respect to the strain Ro19^T. Despite the closeness of the 16S rRNA genes, the housekeeping genes recA, atpD and glnII were divergent in Ro19^T and B. lablabi CCBAU 23086^T, with identity values of 95.71%, 93.75% and 93.11%, respectively. These differences were congruent with DNA–DNA hybridization analysis that revealed an average of 35% relatedness between the novel species and B. lablabi CCBAU 23086^T. Also, differential phenotypic characteristics of the new species were found with respect to the already described species of Bradyrhizobium. Based on the genotypic and phenotypic data obtained in this study, we propose to classify the group of strains isolated from R. sphaerocarpa and R. monosperma as a novel species named Bradyrhizobium retamae sp. nov. (type strain Ro19^T = LMG 27393^T = CECT 8261^T). The analysis of symbiotic genes revealed that some of these strains constitute a new symbiovar within genus Bradyrhizobium for which we propose the name “retamae”, that mainly contains nodulating strains isolated from Retama species in different continents.