Further evidence for the gelation ability-structure correlation in sugar-based gelators

Eight methyl glycosides of 4,6-O-benzylidene derivatives of the monosaccharides D-glucose, D-mannose, D-allose and D-altrose were synthesized to systematically study the effect of small configurational changes on the ability to gelate organic solvents. Among the beta anomers, only the D-mannose glycoside exhibits a strong gelation ability, whereas in the alpha-series the D-glucose and D-mannose derivatives act as versatile gelators. Also, as a general rule we found that the beta anomers possess a higher ability to gelate solvents than the alpha anomers. The gelation properties are discussed on the basis of SAXS, FTIR, differential scanning calorimetric (DSC) measurements and scanning electron microscopy (SEM) observations. The temperature-dependent SAXS measurements were carried out to elucidate the sol-gel transition temperature. The present study emphasizes that the saccharide family provides, not only valuable information of the structural requirements for the design of new gelators, but also for molecular assembly systems in general.     

In vivo mutagenesis by Escherichia coli DNA polymerase I. Ile(709) in motif A functions in base selection

The fidelity of DNA replication by Escherichia coli DNA polymerase I (pol I) was assessed in vivo using a reporter plasmid bearing a ColE1-type origin and an ochre codon in the beta-lactamase gene. We screened 53 single mutants within the region Val(700)-Arg(712) in the polymerase active-site motif A. Only replacement of Ile(709) yielded mutator polymerases, with substitution of Met, Asn, Phe, or Ala increasing the beta-lactamase reversion frequency 5-23-fold. Steady-state kinetic analysis of the I709F polymerase revealed reductions in apparent K(m) values for both insertion of non-complementary nucleotides and extension of mispaired primer termini. Abolishment of the 3'-5' exonuclease activity of wild-type pol I increased mutation frequency 4-fold, whereas the combination of I709F and lack of the 3'-5' exonuclease yielded a 400-fold increase. We conclude that accurate discrimination of the incoming nucleotide at the polymerase domain is more critical than exonucleolytic proofreading for the fidelity of pol I in vivo. Surprisingly, the I709F polymerase enhanced mutagenesis in chromosomal DNA, although the increase was 10-fold less than in plasmid DNA. Our findings indicate the feasibility of obtaining desired mutations by replicating a target gene at a specific locus in a plasmid under continuous selection pressure.     

A deletion of the paracellin-1 gene is responsible for renal tubular dysplasia in cattle

Various hereditary diseases analogous to particular human heritable diseases have been identified in cattle. Investigation of these cattle diseases will provide useful information regarding the pathogenesis of the corresponding human diseases. Renal tubular dysplasia is an autosomal recessive disease of Japanese black cattle characterized by renal failure and growth retardation. We have previously mapped the locus responsible for the disease within a region on bovine chromosome 1. In the present study, we further typed additional markers in this region and found that a genomic segment of bovine chromosome 1 including the microsatellite marker BMS4009 was deleted in the affected animals. Construction of a physical map covering this region with BAC clones and comparison of the nucleotide sequences of this region between normal and affected animals revealed that a region of 37 kb including exons 1 to 4 of the bovine paracellin-1 gene was deleted in the affected animals. The paracellin-1 gene, which is the causative gene for human renal hypomagnesemia with hypercaciuria and nephrocalcinosis, encodes a tight junction protein of renal epithelial cells. Therefore, we concluded that deletion of the paracellin-1 gene is responsible for renal tubular dysplasia of cattle, and the cattle disease could be a good model for the human disease.     

Pericentric Heterochromatin Generated by HP1 Protein Interaction-defective Histone Methyltransferase Suv39h1

Pericentric regions form epigenetically organized silent heterochromatin structures that accumulate histone H3 lysine 9 trimethylation (H3K9me3) and HP1. At pericentric regions, Suv39h is the major enzyme that generates H3K9me3. Suv39h also interacts directly with HP1, a methylated H3K9-binding protein. However, it is not well characterized how HP1 interaction is important for Suv39h accumulation and Suv39h-mediated H3K9me3 formation at the pericentromere. To address this, we introduced the HP1 binding-defective N-terminally truncated mouse Suv39h1 (ΔN) into Suv39h-deficient embryonic stem cells. Interestingly, pericentric accumulation of ΔN and ΔN-mediated H3K9me3 was observed to recover, but HP1 accumulation was only marginally restored. ΔN also rescued DNA methyltransferase Dnmt3a and -3b accumulation and DNA methylation of the pericentromere. In contrast, other pericentric heterochromatin features, such as ATRX protein association and H4K20me3, were not recovered. Finally, derepressed major satellite repeats were partially silenced by ΔN expression. These findings clearly showed that the Suv39h-HP1 binding is dispensable for pericentric H3K9me3 and DNA methylation, but this interaction and HP1 recruitment/accumulation seem to be crucial for complete formation of heterochromatin.     

The Hypoxia-Inducible Epigenetic Regulators Jmjd1a and G9a Provide a Mechanistic Link between Angiogenesis and Tumor Growth

Hypoxia promotes stem cell maintenance and tumor progression, but it remains unclear how it regulates long-term adaptation toward these processes. We reveal a striking downregulation of the hypoxia-inducible histone H3 lysine 9 (H3K9) demethylase JMJD1A as a hallmark of clinical human germ cell-derived tumors, such as seminomas, yolk sac tumors, and embryonal carcinomas. Jmjd1a was not essential for stem cell self-renewal but played a crucial role as a tumor suppressor in opposition to the hypoxia-regulated oncogenic H3K9 methyltransferase G9a. Importantly, loss of Jmjd1a resulted in increased tumor growth, whereas loss of G9a produced smaller tumors. Pharmacological inhibition of G9a also resulted in attenuation of tumor growth, offering a novel therapeutic strategy for germ cell-derived tumors. Finally, Jmjd1a and G9a drive mutually opposing expression of the antiangiogenic factor genes Robo4, Igfbp4, Notch4, and Tfpi accompanied by changes in H3K9 methylation status. Thus, we demonstrate a novel mechanistic link whereby hypoxia-regulated epigenetic changes are instrumental for the control of tumor growth through coordinated dysregulation of antiangiogenic gene expression.     

Cyclo(l-Pro-d-Arg): a new antibacterial and antitumour diketopiperazine from Bacillus cereus associated with a rhabditid entomopathogenic

In continuation of our search for new antimicrobial secondary metabolites from Bacillus cereus associated with rhabditid entomopathogenic nematode, a new microbial diketopiperazine, cyclo(l-Pro-d-Arg), was isolated from the ethyl acetate extract of fermented modified nutrient broth. The chemical structures of the isolated compounds were identified based on their 1D, 2D NMR and high-resolution electrospray ionisation–mass spectroscopy data. Antibacterial activity of the compound was determined by minimum inhibitory concentration and disc diffusion method against medically important bacteria, and the compound was recorded to have significant antibacterial activity against test bacteria. The highest activity was recorded against Klebsiella pneumoniae (1 μg/mL). Cyclo(l-Pro-d-Arg) was recorded to have significant antitumor activity against HeLa cells (IC₅₀ value 50 μg/mL), and this compound was recorded to have no cytotoxicity against normal monkey kidney cells (VERO) up to 100 μg/mL). To the best of our knowledge, this is the first time that cyclo(l-Pro-d-Arg) has been isolated from a microbial natural source.     

Genetic elements associated with antimicrobial resistance in enteropathogenic <Emphasis Type="Italic">Escherichia coli</Emphasis> (EPEC) from Brazil

Background  

We recently observed an association of resistance with a certain enteropathogenic Escherichia coli (EPEC) serotypes and identified a conjugative plasmid, similar to plasmid pED208, that was conserved among archival O111:H2/NM and O119:H2 strains of diverse geographical origin. In this study, we sought to determine the prevalence and distribution of this plasmid among a collection of EPEC isolates from Brazil, as well as to study the susceptibilities of these isolates to antimicrobial agents.     

Hyper-thermostability of CutA1 protein, with a denaturation temperature of nearly 150 degrees C

We found that the CutA1 protein, from Pyrococcus horikoshii (PhCutA1), has an extremely high denaturation temperature (T(d)) of nearly 150 degrees C, which exceeds the highest record determined by DSC by about 30 degrees C. To elucidate the mechanism of the ultra-high stability of PhCutA1, we analyzed the crystal structures of CutA1 proteins from three different sources, P. horikoshii, Thermus thermophilus, and Escherichia coli, with different growth temperatures (98, 75, and 37 degrees C). This analysis revealed that the remarkably increased number of ion pairs in the monomeric structure contributes to the stabilization of the trimeric structure and plays an important role in enhancing the T(d), up to 150 degrees C, for PhCutA1.