Production and characterization of a low‐molecular‐weight bacteriocin from Bacillus licheniformis MKU3

Aims: Enhancing production and characterization of a low‐molecular‐weight bacteriocin from Bacillus licheniformis MKU3. Methods and Results: The culture supernatant of B. licheniformis MKU3 exhibited bacteriocin‐like activity against Gram‐positive and ‐negative bacteria and different fungi and yeast. SDS–PAGE analysis of the extracellular proteins of B. licheniformis MKU3 revealed a bacteriocin‐like protein with a molecular mass of 1·5 kDa. This bacteriocin activity was found to be stable under a pH range of 3·0–10·0 and at temperatures up to 100°C for 60 min, but inactivated by proteinase K, trypsin or pronase E. An experimental fractional factorial design for optimization of production medium resulted in a maximum activity of bacteriocin (11 000 AU ml^?1) by B. licheniformis MKU3. Conclusions: A low‐molecular‐weight bacteriocin‐like protein from B. licheniformis MKU3 exhibited a wide spectrum of antimicrobial activity against several Gram‐positive bacteria, several fungi and yeast. A 3·6‐fold increase in the production of bacteriocin was achieved using the culture medium optimized through a fractional factorial design. Significance and Impact of the Study: A bacteriocin with wide spectrum of activity against Gram‐positive bacterial pathogens, filamentous fungi and yeast suggested its potential clinical use. Statistical method facilitated optimization of cultural medium for the improved production of bacteriocin.     

High‐molecular‐weight poly(Gly‐Val‐Gly‐Val‐Pro) synthesis through microwave irradiation

In this study, we synthesized a polypeptide from its pentapeptide unit using microwave irradiation. Effective methods for polypeptide synthesis from unit peptides have not been reported. Here, we used a key elastin peptide, H‐GlyValGlyValPro‐OH (GVGVP), as the monomer peptide. It is difficult to obtain poly(Gly‐Val‐Gly‐Val‐Pro) (poly(GVGVP)) from the pentapeptide unit of elastin, GVGVP, via polycondensation. Poly(GVGVP) prepared from genetically recombinant Escherichia coli is a well‐known temperature‐sensitive polypeptide, and this temperature sensitivity is known as the lower critical solution temperature. When microwave irradiation was performed in the presence of various additives, the pentapeptide (GVGVP) polycondensation reaction proceeded smoothly, resulting in a product with a high molecular weight in a relatively good yield. The reaction conditions, like microwave irradiation, coupling agents, and solvents, were optimized to increase the reaction efficiency. The product exhibited a molecular weight greater than Mr 7000. Further, the product could be synthesized on a gram scale. The synthesized polypeptide exhibited a temperature sensitivity that was similar to that of poly(GVGVP) prepared from genetically recombinant E. coli. Therefore, this technique offers a facile and quick approach to prepare polypeptides in large amounts. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Elucidation of direct competition and allosteric modulation of small‐molecular‐weight protein ligands using surface plasmon resonance methods

The present work introduces a surface plasmon resonance‐based method for the discrimination of direct competition and allosteric effects that occur in ternary systems comprising a receptor protein and two small‐molecular‐weight ligands that bind to it. Fatty acid binding protein 4, fructose‐1,6‐bisphosphatase and human serum albumin were used as model receptor molecules to demonstrate the performance of the method. For each of the receptor molecules, pairs of ligand molecules were selected for which either direct competition or an allosteric effect had already been determined by other methods. The method of discrimination introduced here is based on the surface plasmon resonance responses observed at equilibrium when an immobilized receptor protein is brought into contact with binary mixtures of interacting ligands. These experimentally determined responses are compared with the responses calculated using a theoretical model that considers both direct competition and allosteric ligand interaction modes. This study demonstrates that the allosteric ternary complex model, which enables calculation of the fractional occupancy of the protein by each ligand in such ternary systems, is well suited for the theoretical calculation of these types of responses. For all of the ternary systems considered in this work, the experimental and calculated responses in the chosen concentration ratio range were identical within a five‐σ confidence interval when the calculations considered the correct interaction mode of the ligands (direct competition or different types of allosteric regulation), and in case of allosteric modulation, also the correct strength of this effect. This study also demonstrates that the allosteric ternary complex model‐based calculations are well suited to predict the ideal concentration ratio range or even single concentration ratios that can serve as hot spots for discrimination, and such hot spots can drastically reduce the numbers of measurements needed for discrimination between direct competition and distinct modulation modes (neutral, positive or negative allostery). Copyright © 2015 John Wiley & Sons, Ltd.     

The high‐molecular‐weight kininogen Domain 5 is an intrinsically unstructured protein and its interaction with ferritin is metal mediated

High‐molecular‐weight kininogen domain 5 (HK5) is an angiogenic modulator that is capable of inhibiting endothelial cell proliferation, migration, adhesion, and tube formation. Ferritin can bind to a histidine–glycine–lysine‐rich region within HK5 and block its antiangiogenic effects. However, the molecular intricacies of this interaction are not well understood. Analysis of the structure of HK5 using circular dichroism and nuclear magnetic resonance [¹H, ¹⁵N]‐heteronuclear single quantum coherence determined that HK5 is an intrinsically unstructured protein, consistent with secondary structure predictions. Equilibrium binding studies using fluorescence anisotropy were used to study the interaction between ferritin and HK5. The interaction between the two proteins is mediated by metal ions such as Co²⁺, Cd²⁺, and Fe²⁺. This metal‐mediated interaction works independently of the loaded ferrihydrite core of ferritin and is demonstrated to be a surface interaction. Ferritin H and L bind to HK5 with similar affinity in the presence of metals. The ferritin interaction with HK5 is the first biological function shown to occur on the surface of ferritin using its surface‐bound metals.     

Sinorhizobium meliloti succinylated high‐molecular‐weight succinoglycan and the Medicago truncatula LysM receptor‐like kinase MtLYK10 participate independently in symbiotic infection

The formation of nitrogen‐fixing nodules on legume hosts is a finely tuned process involving many components of both symbiotic partners. Production of the exopolysaccharide succinoglycan by the nitrogen‐fixing bacterium Sinorhizobium meliloti 1021 is needed for an effective symbiosis with Medicago spp., and the succinyl modification to this polysaccharide is critical. However, it is not known when succinoglycan intervenes in the symbiotic process, and it is not known whether the plant lysin‐motif receptor‐like kinase MtLYK10 intervenes in recognition of succinoglycan, as might be inferred from work on the Lotus japonicus MtLYK10 ortholog, LjEPR3. We studied the symbiotic infection phenotypes of S. meliloti mutants deficient in succinoglycan production or producing modified succinoglycan, in wild‐type Medicago truncatula plants and in Mtlyk10 mutant plants. On wild‐type plants, S. meliloti strains producing no succinoglycan or only unsuccinylated succinoglycan still induced nodule primordia and epidermal infections, but further progression of the symbiotic process was blocked. These S. meliloti mutants induced a more severe infection phenotype on Mtlyk10 mutant plants. Nodulation by succinoglycan‐defective strains was achieved by in trans rescue with a Nod factor‐deficient S. meliloti mutant. While the Nod factor‐deficient strain was always more abundant inside nodules, the succinoglycan‐deficient strain was more efficient than the strain producing only unsuccinylated succinoglycan. Together, these data show that succinylated succinoglycan is essential for infection thread formation in M. truncatula, and that MtLYK10 plays an important, but different role in this symbiotic process. These data also suggest that succinoglycan is more important than Nod factors for bacterial survival inside nodules.     

A SELDI‐TOF MS procedure for the detection,quantitation, and preliminary characterization of low‐molecular‐weight recombinant proteins expressed in transgenic plants

We describe a SELDI‐TOF MS procedure for the rapid detection and quantitation of low‐molecular‐weight recombinant proteins expressed in plants. Transgenic lines of potato (Solanum tuberosum L.) expressing the clinically useful protein bovine aprotinin or the cysteine protease inhibitor corn cystatin II were generated by Agrobacterium tumefaciens‐mediated transformation, and then used as test material for the analyses. Real‐time RT‐PCR amplifications and detection of the recombinant proteins by immunoblotting were first conducted for transformed potato lines accumulating the proteins in different cell compartments. Both proteins were found at varying levels in leaves, depending on their final cellular destination and transgene expression rate. These conclusions drawn from standard immunodetection assays were easily confirmed by SELDI‐TOF MS comparative profiling, after immobilizing the leaf proteins of control and transformed lines on protein biochips for weak cationic exchange. This procedure, carried out in less than 2 h, allows for the rapid comparison of recombinant protein levels in transgenic plant lines. The molecular weight of immobilized proteins can also be determined directly from the MS spectra, thus providing a simple way to assess the structural integrity and homogeneity of recombinant proteins in planta, and to identify the most suitable cellular compartments for their heterologous production.     

Expression of vimentin and high‐molecular‐weight cytokeratin (clone 34ßE12) in differentiating reactive renal tubular cells from low‐grade urothelial carcinoma cells in voided urine

H. Ohsaki, E. Hirakawa, M. Nakamura, Y. Norimatsu, H. Kiyomoto and R. Haba Expression of vimentin and high‐molecular‐weight cytokeratin (clone 34ßE12) in differentiating reactive renal tubular cells from low‐grade urothelial carcinoma cells in voided urine Objective: Reactive renal tubular cells show features of an atypical repair reaction. Differentiation between reactive renal tubular cells and low‐grade urothelial carcinoma (LG‐UC) cells can therefore be a diagnostic challenge based on morphology alone. In this study, we evaluated the diagnostic utility of vimentin and a high‐molecular‐weight cytokeratin antibody (clone 34ßE12) in differentiating reactive renal tubular cells from LG‐UC. Methods: We evaluated voided urine cytology and surgical specimens from 40 patients with renal disease, and 17 patients with LG‐UC. All slides were stained with vimentin and 34ßE12. Results: In the reactive renal tubular cells in voided urine cytology, vimentin showed strong cytoplasmic staining in 39/40 (97.5%) cases, but all were negative for 34ßE12. LG‐UC cells showed positive staining for 34ßE12 in 3/17 (17.6%) cases, whereas none were positivity for vimentin. The reactive renal tubular cells of histological specimens in the renal disease group demonstrated positive for vimentin in all 40 cases and all were negative for 34ßE12. The LG‐UC group showed abnormal staining for 34ßE12 in 4/17 (23.5%) cases, whereas none were positive for vimentin. Conclusions: Vimentin expression in urine cytology can help to distinguish reactive renal tubular cells from LG‐UC. However, 34ßE12 does not appear to be a useful adjunct to distinguish these two groups in voided urine cytology.     

The oligomeric structure of high molecular weight adiponectin

There is great interest in the structure of adiponectin as its oligomeric state may specify its biological activities. It occurs as a trimer, a hexamer and a high molecular weight complex. Epidemiological data indicate that the high molecular weight form is significant with low serum levels in type 2 diabetics but to date, has not been well-defined. To resolve this issue, characterization of this oligomer from bovine serum and 3T3-L1 adipocytes by sedimentation equilibrium centrifugation and gel electrophoresis respectively, was carried out, revealing that it is octadecameric. Further studies by dynamic light scattering and electron microscopy established that bovine and possibly mouse high molecular weight adiponectin is C1q-like in structure.     

Accessibility of low‐molecular‐mass molecules to the median eminence and arcuate hypothalamic nucleus of adult mouse

Blood‐derived molecules are able to access to the median eminence (ME) and arcuate hypothalamic nucleus (Arc) due to the lack of the blood–brain barrier. In the present study, we examined the accessibility of low‐molecular‐mass (LMM) molecules into parenchyma in the ME and Arc of adult mice by administration of Dextran 3000 (Dex3k), Dex10k, Evans blue (EB) and fluorescein isothiocyanate (FITC). In the external zone of the ME, the fluorescence of Dex3k, EB and FITC tracers generated an intensity gradient from fenestrated capillary, but that of Dex10k was detected only between the inner and outer basement membrane of pericapillary space. The fluorescence of FITC in the external zone of the ME was closely associated with axonal terminals and surrounded by cellular processes of tanycytes‐like cells and astrocytes. In the ependymal/internal zone of the ME and Arc, the fluorescence of all LMM tracers was seen at tanycytes‐like cells and neurons. The fluorescence of EB and FITC in these regions was not detected when brains were fixed during or before the administration of tracers. The inhomogeneity of accessibility for fluorescent tracers depended on routes for tracer administration. Thus, the present study indicates that the accessibility of LMM blood‐derived molecules to parenchyma depends on fenestration of the capillary in the external zone of the ME and active transport of ependymal cells in the ependymal/internal zone of the ME and Arc. Copyright © 2013 John Wiley & Sons, Ltd.     

A molecular‐genetic understanding of diapause in spider mites: current knowledge and future directions

During unfavourable conditions, many arthropods have the ability to enter into diapause and synchronize their development and reproduction to seasonal patterns. Diapause or winter hibernation in insects and mites is set off by a number of cues, with photoperiod being the most well‐defined and strongest signal. This review focuses on the current knowledge of ‘‐omics’ data and the genetics of diapause in the two‐spotted spider mite Tetranychus urticae, a member of the family Tetranychidae (Arthropoda: Chelicerata: Arachnida: Acari). This species is a serious polyphagous pest and females undergo a reproductive facultative diapause when immature stages are exposed to long nights. Winter hibernation induces different physiological processes characterized by a metabolic suppression, different energy use, increased stress tolerance and the production of cryoprotectants, all initiated by a complex signal transduction pathway. Keto‐carotenoids are known to cause the deeply orange colour typical for diapausing females. Furthermore, research with colour mutants of T. urticae has shown the need for carotenoids with respect to the induction of diapause, even though the molecular‐genetic mechanisms underlying these colour phenotypes are still unknown. In addition, marked latitudinal variation in diapause incidence among populations has been observed in nature, with modes of inheritance ranging from recessive to dominant, as well as monogenic to polygenic. We end by highlighting the emerging opportunities for functional studies that aim to unravel the complex factors underlying diapause in spider mites.     

The low‐molecular‐mass,penicillin‐binding proteins DacB and DacC combine to modify peptidoglycan cross‐linking and allow stable Type IV pilus expression in Neisseria gonorrhoeae

Neisseria gonorrhoeae is the causative agent of the sexually transmitted infection gonorrhea and is adapted to survive in humans, its only host. The N. gonorrhoeae cell wall is critical for maintaining envelope integrity, resisting immune cell killing and production of cytotoxic peptidoglycan (PG) fragments. Deletion of the N. gonorrhoeae strain FA1090 genes encoding two predicted low‐molecular‐mass, penicillin‐binding proteins (LMM PBPs), DacB and DacC, substantially altered the PG cross‐linking. Loss of the DacB peptidase resulted in global alterations to the PG composition, while loss of the DacC protein affected a much narrower subset of PG peptide components. A double ΔdacB/ΔdacC mutant resembled the ΔdacB single mutant, but had an even greater level of cross‐linked PG. While single ΔdacB or ΔdacC mutants did not show any major phenotypes, the ΔdacB/ΔdacC mutant displayed an altered cellular morphology, decreased resistance to antibiotics and increased sensitivity to detergent‐mediated death. Loss of the two proteins also drastically reduced the number of Type IV pili (Tfp), a critical virulence factor. The decreased piliation reduced transformation efficiency and correlated with increased growth rate. While these two LMM PBPs differentially alter the PG composition, their overlapping effects are essential to proper envelope function and expression of factors critical for pathogenesis.     

Morphological assessment of the Octopus vulgaris species complex evaluated in light of molecular‐based phylogenetic inferences

Cryptic species are common in the ocean, particularly among marine invertebrates such as octopuses. Delineating cryptic species is particularly problematic in octopus taxonomy where the plasticity recorded among taxonomic characters often results in low resolution at the species level. This study investigated the morphological relationships among seven phylogenetic clades (identified using cytochrome c oxidase subunit I) of the broadly distributed Octopus vulgaris species complex and close relatives. Morphological analyses in this study were successful in delimiting O. sinensis, Brazilian O. vulgaris and O. vulgaris sensu stricto, which was congruent with the molecular findings of this study. Analyses based on male morphology were successful in distinguishing 14 of 15 total pairwise comparisons and proved to be a more reliable indicator of species‐level relationships in comparison with female morphology. The majority of characters with the greatest discriminatory power were male sexual traits. Significant morphological differences were also recorded among sampling localities of conspecifics, with phenotype showing correlation with local environmental data. The findings of this study support the hypothesis that multiple O. vulgaris‐like species are currently being incorrectly treated under a single species name, O. vulgaris. Octopuses being exported globally under the name O. vulgaris are of extremely high fisheries market value and profile. Our findings have potentially significant implications for the naming and conservation of commercially harvested members of this species complex throughout their ranges.     

Brief Communication: Quantitative‐ and molecular‐genetic differentiation in humans and chimpanzees: Implications for the evolutionary processes underlying cranial diversification

Estimates of the amount of genetic differentiation in humans among major geographic regions (e.g., Eastern Asia vs. Europe) from quantitative‐genetic analyses of cranial measurements closely match those from classical‐ and molecular‐genetic markers. Typically, among‐region differences account for ～10% of the total variation. This correspondence is generally interpreted as evidence for the importance of neutral evolutionary processes (e.g., genetic drift) in generating among‐region differences in human cranial form, but it was initially surprising because human cranial diversity was frequently assumed to show a strong signature of natural selection. Is the human degree of similarity of cranial and DNA‐sequence estimates of among‐region genetic differentiation unusual? How do comparisons with other taxa illuminate the evolutionary processes underlying cranial diversification? Chimpanzees provide a useful starting point for placing the human results in a broader comparative context, because common chimpanzees (Pan troglodytes) and bonobos (Pan paniscus) are the extant species most closely related to humans. To address these questions, I used 27 cranial measurements collected on a sample of 861 humans and 263 chimpanzees to estimate the amount of genetic differentiation between pairs of groups (between regions for humans and between species or subspecies for chimpanzees). Consistent with previous results, the human cranial estimates are quite similar to published DNA‐sequence estimates. In contrast, the chimpanzee cranial estimates are much smaller than published DNA‐sequence estimates. It appears that cranial differentiation has been limited in chimpanzees relative to humans. Am J Phys Anthropol 154:615–620, 2014. © 2014 Wiley Periodicals, Inc.