First evidence of putrescine involvement in mitigating the floral malformation in mangoes: A scanning electron microscope study

Floral malformation is the most destructive disease in mangoes. To date, the etiology of this disease has not been resolved. There are indications that stress-stimulated ethylene production might be responsible for the disease. Putrescine mediates various physiological processes for normal functioning and cellular metabolism. Here, the effect of putrescine in concentration ranging from 10^?1 to 10^?3 M was evaluated on disease incidence during mango flowering seasons of 2012 and 2013. In a scanning electron microscopy (SEM) study, putrescine (10^?2 M)-treated malformed floral buds bloomed into opened flowers with separated sepals and/or petals like healthy, whereas the untreated (control) malformed buds remained deformed. Further, malformed flowers recovered upon putrescine treatment, displaying clearly bilobed anthers, enclosing a large number of normal pollen grains and functional ovary with broad stigmatic surface as compared to control. The present findings provide the first report to demonstrate the role of putrescine in reducing various adverse effects of stress ethylene via decelerating the higher pace of its biosynthesis. It stabilizes the normal morphology, development, and functions of malformed reproductive organs to facilitate successful pollination, fertilization, and, thereby, fruit set in mango flowers. However, putrescine–ethylene-mediated cell signaling network, involving various genes to trigger the response, which regulates a wide range of developmental and physiological processes leading to normal cell physiology, needs to be investigated further.     

The sensor kinase CbrA is a global regulator that modulates metabolism, virulence, and antibiotic resistance in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen that possesses a large arsenal of virulence factors enabling the pathogen to cause serious infections in immunocompromised patients, burn victims, and cystic fibrosis patients. CbrA is a sensor kinase that has previously been implied to play a role with its cognate response regulator CbrB in the metabolic regulation of carbon and nitrogen utilization in P. aeruginosa. Here it is demonstrated that CbrA and CbrB play an important role in various virulence and virulence-related processes of the bacteria, including swarming, biofilm formation, cytotoxicity, and antibiotic resistance. The cbrA deletion mutant was completely unable to swarm while exhibiting an increase in biofilm formation, supporting the inverse regulation of swarming and biofilm formation in P. aeruginosa. The cbrA mutant also exhibited increased cytotoxicity to human lung epithelial cells as early as 4 and 6 h postinfection. Furthermore, the cbrA mutant demonstrated increased resistance toward a variety of clinically important antibiotics, including polymyxin B, ciprofloxacin, and tobramycin. Microarray analysis revealed that under swarming conditions, CbrA regulated the expression of many genes, including phoPQ, pmrAB, arnBCADTEF, dnaK, and pvdQ, consistent with the antibiotic resistance and swarming impairment phenotypes of the cbrA mutant. Phenotypic and real-time quantitative PCR (RT-qPCR) analyses of a PA14 cbrB mutant suggested that CbrA may be modulating swarming, biofilm formation, and cytotoxicity via CbrB and that the CrcZ small RNA is likely downstream of this two-component regulator. However, as CbrB did not have a resistance phenotype, CbrA likely modulates antibiotic resistance in a manner independent of CbrB.     

Kinetic Investigation and Anticoagulant Activity of Amide Analogues of Isoform 2 and 3 of Antistasin

The process of blood coagulation is protecting organism from blood loss in case of surface injuries, and is ensuring blood circulation without formation of thrombus. The abnormal functioning of haemostasis is resulting in a many diseases in the recent years. Several serine proteinases and their inhibitors have a very important role in the process of the blood coagulation and are a strong potential alternative for the prevention and treatment of such illnesses. Herein, we report on anticoagulant activity, according to APTT of newly synthesized amide analogues of isoforms 2 and 3 of antistasin. Our study reveals that the replacement of carboxyl with amide function in a C-terminus of peptides is leading to significant increase of the anticoagulant activity. Additionally, some kinetic investigations on the same analogues are done. Our results show that both free acids and amides shortened analogues have a mixed type of inhibition related to serine proteinases from the blood coagulation cascade. The calculated Ki values for the model and the investigated serine proteinases show some selectivity of analogue Phe-Ile-Arg-Pro-Lys-Arg-NH₂. The obtained kinetic data correlates with the anticoagulant activity of the newly synthesized analogues.     

SCCRO3 (DCUN1D3) Antagonizes the Neddylation and Oncogenic Activity of SCCRO (DCUN1D1)

The activity of cullin-RING type ubiquitination E3 ligases is regulated by neddylation, a process analogous to ubiquitination that culminates in covalent attachment of the ubiquitin-like protein Nedd8 to cullins. As a component of the E3 for neddylation, SCCRO/DCUN1D1 plays a key regulatory role in neddylation and, consequently, cullin-RING ligase activity. The essential contribution of SCCRO to neddylation is to promote nuclear translocation of the cullin-ROC1 complex. The presence of a myristoyl sequence in SCCRO3, one of four SCCRO paralogues present in humans that localizes to the membrane, raises questions about its function in neddylation. We found that although SCCRO3 binds to CAND1, cullins, and ROC1, it does not efficiently bind to Ubc12, promote cullin neddylation, or conform to the reaction processivity paradigms, suggesting that SCCRO3 does not have E3 activity. Expression of SCCRO3 inhibits SCCRO-promoted neddylation by sequestering cullins to the membrane, thereby blocking its nuclear translocation. Moreover, SCCRO3 inhibits SCCRO transforming activity. The inhibitory effects of SCCRO3 on SCCRO-promoted neddylation and transformation require both an intact myristoyl sequence and PONY domain, confirming that membrane localization and binding to cullins are required for in vivo functions. Taken together, our findings suggest that SCCRO3 functions as a tumor suppressor by antagonizing the neddylation activity of SCCRO.     

Models of Self-Peptide Sampling by Developing T Cells Identify Candidate Mechanisms of Thymic Selection

Conventional and regulatory T cells develop in the thymus where they are exposed to samples of self-peptide MHC (pMHC) ligands. This probabilistic process selects for cells within a range of responsiveness that allows the detection of foreign antigen without excessive responses to self. Regulatory T cells are thought to lie at the higher end of the spectrum of acceptable self-reactivity and play a crucial role in the control of autoimmunity and tolerance to innocuous antigens. While many studies have elucidated key elements influencing lineage commitment, we still lack a full understanding of how thymocytes integrate signals obtained by sampling self-peptides to make fate decisions. To address this problem, we apply stochastic models of signal integration by T cells to data from a study quantifying the development of the two lineages using controllable levels of agonist peptide in the thymus. We find two models are able to explain the observations; one in which T cells continually re-assess fate decisions on the basis of multiple summed proximal signals from TCR-pMHC interactions; and another in which TCR sensitivity is modulated over time, such that contact with the same pMHC ligand may lead to divergent outcomes at different stages of development. Neither model requires that T and T are differentially susceptible to deletion or that the two lineages need qualitatively different signals for development, as have been proposed. We find additional support for the variable-sensitivity model, which is able to explain apparently paradoxical observations regarding the effect of partial and strong agonists on T and T development.     

NMR backbone resonance assignments of the prodomain variants of BDNF in the urea denatured state

Brain derived neurotrophic factor (BDNF) is a member of the neurotrophin family of proteins which plays a central role in neuronal survival, growth, plasticity and memory. A single Val66Met variant has been identified in the prodomain of human BDNF that is associated with anxiety, depression and memory disorders. The structural differences within the full-length prodomain Val66 and Met66 isoforms could shed light on the mechanism of action of the Met66 and its impact on the development of neuropsychiatric-associated disorders. In the present study, we report the backbone ¹H, ¹³C, and ¹⁵N NMR assignments of both full-length Val66 and Met66 prodomains in the presence of 2 M urea. These conditions were utilized to suppress residual structure and aid subsequent native state structural investigations aimed at mapping and identifying variant-dependent conformational differences under native-state conditions.     

Immunoregulatory activity of human bone marrow. Identification of suppressor cells possessing OKM1, SSEA-1, and HNK-1 antigens

Human bone marrow contains natural regulatory cells capable of suppressing the in vitro primary IgM response of normal tonsillar cells. The suppression is mediated by non-T cells possessing Fc receptors, OKM1, SSEA-1, and HNK-1 antigens on their surface. The suppression was abrogated by treatment of bone marrow cells (BMC) with anti-HNK-1 or anti-SSEA-1 antisera and complement. Furthermore, BMC depleted of HNK-1+ cells could respond in a primary in vitro antibody response when provided with accessory T cells and macrophages from tonsillar cells. Our findings support the idea that HNK-1+ and HNK-1- BMC populations act antagonistically in the regulation of antibody synthesis. Further, the finding of HNK-1+, SSEA-1+, and OKM1+ suppressor cells in human bone marrow may represent a precursor phenotype of mature natural killer cells with potent immunoregulatory activity.     

Codon usage in histone gene families of higher eukaryotes reflects functional rather than phylogenetic relationships

Summary The nucleic acid sequences coding for 23 H3 histone genes from a variety of species have been analyzed using a computer assisted alignment and analysis program. Although these histones are highly conserved within and between highly divergent species, they represent various classes of histones whose patterns of expression are distinctively regulated. Surprisingly, in dendrograms derived from these comparisons, H3 sequences cluster according to their modes of regulation rather than phylogenetically. These clusters are generated from highly distinctive patterns of codon usage within the functional gene classes. We suggest that one factor involved in specifying the differing codon usage patterns between functional classes is a difference in requirements for rapid translation of mRNA. In addition, the data presented here, together with structural and sequence information, suggest a heterodox evolutionary model in which genes related to the intron-bearing, basally expressed H3.3 vertebrate genes are the ancestors of the intronless H3. 1 class of genes of higher eukaryotes. The H3. 1 class must have arisen, therefore, following duplication of a primitive H3.3 gene, but prior to the plant-animal divergence. Implications of the data presented are discussed with regard to functional and evolutionary relationships.     

Pseudomonas aeruginosa outer membrane protein OprH: expression from the cloned gene and function in EDTA and gentamicin resistance.

Overexpression of major outer membrane protein OprH of Pseudomonas aeruginosa as a result of mutation (in strain H181) or adaptation to low Mg2+ concentrations (in parent strain H103) is accompanied by increased resistance to polymyxin B, gentamicin, and EDTA. A 2.8-kb EcoRI fragment containing the oprH gene was subcloned into several different expression plasmids in Escherichia coli. These experiments showed that significant levels of OprH could be produced from a promoter on the EcoRI fragment; that the cloned oprH gene was not regulated by Mg2+ deficiency; that there were no differences in the expression of OprH in any construction, regardless of whether the gene from strain H103 or its OprH-overexpressing, polymyxin B-resistant derivative, strain H181, was used; and that overexpression of OprH in E. coli to the level observed in P. aeruginosa H181 did not result in a resistance phenotype. These results favored the conclusion that the mutation in strain H181 was a regulatory rather than a promoter mutation. The oprH gene was cloned behind the benzoate-inducible pm promoter in plasmid pGB25 and transferred to P. aeruginosa H103. Overexpression of OprH from the cloned gene in H103/pGB25 resulted in EDTA resistance but not polymyxin B resistance. This result suggested that another factor, possibly lipopolysaccharide, was affected by the mutation in strain H181. Consistent with this suggestion was the demonstration that mutants of strain H181 with alterations in lipopolysaccharide had reverted to wild-type polymyxin B susceptibility but had unaltered gentamicin and EDTA resistance. These data were consistent with the hypothesis that OprH replaces outer membrane-stabilizing divalent cations.