Direct force measurement of single DNA–peptide interactions using atomic force microscopy

The selective interactions between DNA and miniature (39 residues) engineered peptide were directly measured at the single‐molecule level by using atomic force microscopy. This peptide (p007) contains an α‐helical recognition site similar to leucine zipper GCN4 and specifically recognizes the ATGAC sequence in the DNA with nanomolar affinity. The average rupture force was 42.1 pN, which is similar to the unbinding forces of the digoxigenin–antidigoxigenin complex, one of the strongest interactions in biological systems. The single linear fit of the rupture forces versus the logarithm of pulling rates showed a single energy barrier with a transition state located at 0.74 nm from the bound state. The smaller k_off compared with that of other similar systems was presumably due to the increased stability of the helical structure by putative folding residues in p007. This strong sequence‐specific DNA–peptide interaction has a potential to be utilized to prepare well‐defined mechanically stable DNA–protein hybrid nanostructures. Copyright © 2013 John Wiley & Sons, Ltd.     

A 42 bp fragment of the pmas1′ promoter containing an ocs-like element confers a developmental,wound- and chemically inducible expression pattern

Synthesis of mannopine in plant tissues infected with Agrobacterium tumefaciens is controlled by a divergent promoter (pmas2 and pmas1) that in 479 bp contains all the cis-acting elements necessary to direct tissue-specific and wound-inducible expression. In this report, using transgenic tobacco plants harboring a pmas1--glucuronidase (GUS) gene fusion, we investigated the developmental expression pattern directed by pmas1 in the early stages of development and the responses of pmas1 to different chemical inducers. It was found that this promoter can respond to auxins, cytokinins, methyl jasmonate (MJ), salicylic acid (SA) and its analogue 2,6-dichloroisonicotinic acid (iNA). Treatment with chemical inducers also showed that the effects of iNA are organ-dependent, that wound-induction is a complex response mediated by at least two different chemical signals, and that MJ stimulates changes in the tissue-specific and developmental expression pattern directed by the pmas1 promoter. Using chimeric promoters we demonstrate that an ocs-like element (ocs+1) directs MJ responses in an orientation-dependent manner and that sequences around the ocs+1 are important to maintain the inducible and developmental properties of this cis-regulatory element.     

Closely related plasmids from Staphylococcus aureus and soil bacilli

Antibiotic resistance plasmids from staphylococci and soil bacilli have been isolated and compared. A tetracycline resistance (Tc^r) plasmid, indistinguishable from pT181, which is typical of Tc^r plasmids that are widely dispersed among human clinical isolates of S. aureus, has been found also in bovine mastitis isolates. This plasmid, however, shows no detectable homology to a family of related Tc^r plasmids, typified by pBC16, that is widely dispersed among aerobic spore-forming bacilli. However, and rather unexpectedly, pBC16 is highly homologous to and incompatible with pUB110, an S. aureus plasmid specifying kanamycin resistance. The two plasmids are homologous except for the region occupied by their resistance determinants, which has the appearance of a heterologous substitution. These results suggest the occurrence of natural plasmid transfer between staphylococci and soil bacilli.     

Fecundity of fishes inhabiting coastal and estuarine environments in the southwest Atlantic Ocean

The aim of this study was to estimate the fecundity of six marine fish species from the southwest Atlantic off the coast of São Paulo State, Brazil. In particular, the number of oocytes in the most advanced vitellogenic stage (NDO), batch fecundity and the number of batches that will be produced were estimated. Specimens of longfinger anchovy Anchoa filifera, atlantic anchoveta Cetengraulis edentulus, bay whiff Citharichthys spilopterus, Stellifer brasiliensis, rake stardrum S. rastrifer and southern kingcroaker Menticirrhus americanus were captured bimonthly, from June (2012) to May (2013). The mature ovaries were removed, weighed, fixed in formalin solution and histologically analysed. All histological sections were photographed and the images analysed using advanced image processing techniques. The estimated number of batches that will be recruited to the germinal vesicle migration/hydrated stock ranged from one to four batches. NDO is positively related to ovarian and female size. Small females with low ovary weight produce low NDO. The NDO in A. filifera, C. edentulus and M. americanus may represent the final number of oocytes to be spawned in the next spawning event, which could be used for batch fecundity estimation. This study contributes to a better understanding of the reproduction of species inhabiting shrimp fishery grounds which are caught as by-catch in tropical and subtropical ecosystems. Information on the maturity and fecundity of these populations is an important element in assessing stock status and the degree of fishing pressure these populations are experiencing.     

Dynamin is functionally coupled to insulin granule exocytosis

The insulin granule integral membrane protein marker phogrin-green fluorescent protein was co-localized with insulin in Min6B1 beta-cell secretory granules but did not undergo plasma membrane translocation following glucose stimulation. Surprisingly, although expression of a dominant-interfering dynamin mutant (Dyn/K44A) inhibited transferrin receptor endocytosis, it had no effect on phogringreen fluorescent protein localization in the basal or secretagogue-stimulated state. By contrast, co-expression of Dyn/K44A with human growth hormone as an insulin secretory marker resulted in a marked inhibition of human growth hormone release by glucose, KCl, and a combination of multiple secretagogues. Moreover, serial pulse depolarization stimulated an increase in cell surface capacitance that was also blocked in cells expressing Dyn/K44A. Similarly, small interference RNA-mediated knockdown of dynamin resulted in marked inhibition of glucose-stimulated insulin secretion. Together, these data suggest the presence of a selective kiss and run mechanism of insulin release. Moreover, these data indicate a coupling between endocytosis and exocytosis in the regulation of beta-cell insulin secretion.     

Analysis and Optimization of Bulk DNA Sampling with Binary Scoring for Germplasm Characterization

The strategy of bulk DNA sampling has been a valuable method for studying large numbers of individuals through genetic markers. The application of this strategy for discrimination among germplasm sources was analyzed through information theory, considering the case of polymorphic alleles scored binarily for their presence or absence in DNA pools. We defined the informativeness of a set of marker loci in bulks as the mutual information between genotype and population identity, composed by two terms: diversity and noise. The first term is the entropy of bulk genotypes, whereas the noise term is measured through the conditional entropy of bulk genotypes given germplasm sources. Thus, optimizing marker information implies increasing diversity and reducing noise. Simple formulas were devised to estimate marker information per allele from a set of estimated allele frequencies across populations. As an example, they allowed optimization of bulk size for SSR genotyping in maize, from allele frequencies estimated in a sample of 56 maize populations. It was found that a sample of 30 plants from a random mating population is adequate for maize germplasm SSR characterization. We analyzed the use of divided bulks to overcome the allele dilution problem in DNA pools, and concluded that samples of 30 plants divided into three bulks of 10 plants are efficient to characterize maize germplasm sources through SSR with a good control of the dilution problem. We estimated the informativeness of 30 SSR loci from the estimated allele frequencies in maize populations, and found a wide variation of marker informativeness, which positively correlated with the number of alleles per locus.     

Functional levels of mitochondrial anion transport proteins in non-insulin-dependent diabetes mellitus

The effect of non-insulin-dependent diabetes mellitus (i.e., NIDDM; type 2 diabetes) on the levels of functional mitochondrial anion transport proteins has been determined utilizing a chemically-induced neonatal model of NIDDM. We hypothesized that moderate insulin deficiency exacerbated by the insulin resistance, which is characteristic of NIDDM, would cause changes in mitochondrial anion transporter function that were similar to those we have previously shown to occur in insulin-dependent diabetes mellitus (i.e., IDDM; type 1 diabetes) (Arch. Biochem. Biophys. 280: 181–191, 1990). Our experimental approach consisted of the extraction of the pyruvate, dicarboxylate and citrate transport proteins from the mitochondrial inner membrane with Triton X-114 using rat liver mitoplasts (prepared from diabetic and control animals) as the starting material, followed by the functional reconstitution of each transporter in a proteoliposomal system. This strategy permitted the quantification of the functional levels of these three transporters in the absence of the complications that arise when such measurements are carried out with intact mitochondria (or mitoplasts). We found that experimental NIDDM did not cause significant changes in the extractable and reconstitutable specific (and total) transport activities of the pyruvate, dicarboxylate, and citrate transporters. These results are in marked contrast to our previous findings obtained using rats with IDDM and negated our hypothesis. The present results, in combination with our earlier findings, allow us to conclude that insulin plays an important role in the regulation of mitochondrial anion transporter function. Accordingly, in this model of NIDDM, where the level of insulin is not profoundly deficient, transporter function is unaltered, whereas in IDDM, where a profound insulinopenia exists, transporter function is altered. Furthermore, the present studies suggest that in the neonatal model of NIDDM the three mitochondrial transporters investigated are neither affected by, nor are they the sites of the well documented hepatic post-receptor insulin resistance which is characteristic of this disease.