Characterization of low-molecular-weight-glutenin subunit genes from the D-genome of Triticum aestivum,Aegilops crassa,Ae. cylindrica and Ae. tauschii

Twenty low-molecular-weight-glutenin subunit (LMW-GS) gene sequences from the D-genome from Aegilops crassa (2n = 4x = 28), Ae. cylindrica (2n = 4x = 28), Ae. tauschii (2n = 2x = 14) and Triticum aestivum (2n = 6x = 42) were obtained using five sets of specific allele primer pairs. Only the sequences of the first primer pair were complete coding sequences (cds) of LMW-GS, and had 305, 304, 306 and 305 LMW-m amino acid residues in Ae. crassa, Ae. cylindrica, Ae. tauschii and T. aestivum, respectively. The repetitive domain and repeat motif numbers of all LMW glutenin subunits showed eight conserved cysteine residues that lead to the same functional activity in different genome. Based on DNA and predicted protein sequences, phylogenetic trees for all sets of sequences were drawn. At the DNA level, the species closest to T. aestivum for the second, third, fourth and fifth set of sequences were Ae. cylindrica, Ae. tauschii and Ae. crassa, respectively. At the protein level, the species closest to T. aestivum based on the first, second and fifth set of sequences were Ae. cylindrica, Ae. crassa and Ae. crassa, respectively. For other sets of sequences, bread wheat proved to be a distinct species. The LMW-GS gene sequences have been recorded in the GenBank with accession numbers JQ726549–JQ726568JQ726549JQ726550JQ726551JQ726552JQ726553JQ726554JQ726555JQ726556JQ726557JQ726558JQ726559JQ726560JQ726561JQ726562JQ726563JQ726564JQ726565JQ726566JQ726567JQ726568.     

Dissecting Pistil Responses to Incompatible and Compatible Pollen in Self-Incompatibility <Emphasis Type="Italic">Brassica oleracea</Emphasis> Using Comparative Proteomics

Angiosperms have developed self-incompatibility (SI) systems to reject self-pollen, thereby promoting outcrossing. The Brassicaceae belongs to typical sporophytic system, having a single S-locus controlled SI response, and was chosen as a model system to study SI-related intercellular signal transduction. In this regard, the downstream factor of EXO70A1 was unknown. Here, protein two-dimensional electrophoresis (2-DE) method and coupled with matrix-assisted laser desorption ionization/time of flight of flight mass spectrometry (MALDI-TOF -MS) and peptide mass fingerprinting (PMF) was used to further explore the mechanism of SI responses in Brassica oleracea L. var. capitata L. at protein level. To further confirm the time point of protein profile change, total proteins were collected from B. oleracea pistils at 0 min, 1 h, and 2 h after self-pollination. In total 902, 1088 and 1023 protein spots were separated in 0 min, 1 h and 2 h 2-DE maps, respectively. Our analyses of self-pollination profiles indicated that proteins mainly changed at 1 h post-pollination in B. oleracea. Moreover, 1077 protein spots were separated in cross-pollinated 1 h (CP) pistil 2-DE map. MALDI-TOF-MS and PMF successfully identified 34 differentially-expressed proteins (DEPs) in SP and CP 1 h 2-DE maps. Gene ontology and KEGG analysis revealed an array of proteins grouped in the following categories: stress and defense response (35%), protein metabolism (18%), carbohydrate and energy metabolism (12%), regulation of translation (9%), pollen tube development (12%), transport (9%) and cytoskeletal (6%). Sets of DEPs identified specifically in SP or only up-regulated expressed in CP pistils were chosen for funther investigating in floral organs and during the process of self- and cross-pollination. The function of these DEPs in terms of their potential involvement in SI in B. oleracea is discussed.     

Purification and characterization of the extracellular lipase Lip2 from Yarrowia lipolytica

An extracellular lipase from Yarrowia lipolytica (YlLip2) has been purified by ion exchange chromatography on Q sepharose FF, followed by hydrophobic interaction chromatography on butyl sepharose FF. SDS-PAGE showed that the molecular weight of this lipase is about 38 kDa. N-terminal amino acid sequencing and MALDI-TOF mass spectral analysis showed that this lipase is encoded by gene LIP2 (GenBank accession no. AJ012632). Enzymatic deglycosylation showed that this lipase is a glycosylated protein which contains about 12% sugar. The corresponding deglycosylated lipase remained 88% specific activity of untreated lipase. There was a high amino acid sequence identity (91%) between YlLip2 and Candida deformans lipase CdLip1 (GenBank accession no. AJ428393). The optima temperature and pH for the purified lipase was 40 °C and 8.0, respectively. The lipase showed a preference for long chain fatty acid methyl esters (C12–C16), with the highest activity toward methyl myristate (C14). Lipase activity was stimulated by Ca²⁺ and Mg²⁺ and inhibited by Zn²⁺, Ni²⁺ and Cu²⁺, whereas EDTA had no effect on its activity. A 0.1% of Tween 80 and Span 65 increased slightly the enzyme activity and SDS inhibited it.     

Discovery of novel indole-based aroylhydrazones as anticonvulsants: Pharmacophore-based design

A number of novel melatonin derivatives, containing aroylhydrazone moieties, were synthesized and explored in vivo for anticonvulsant activity, neurotoxicity in ICR mice as well as in-vitro for cytoxicity and oxidative stress in rats. The structures and configurations were confirmed by NMR, FTIR, HRMS and crystal X-ray diffraction method. For selection of potent structures for synthesis a pharmacophore model was used. Two compounds 3e, with a 2-furyl moiety fragment and 3f with 2-thienyl fragment, showed a potency in maximal electroshock (MES) test (ED₅₀ = 50.98 mg kg⁻¹, PI > 5.88 and ED₅₀ = 108.7 mg kg⁻¹; PI > 2.76), respectively, higher than melatonin (ED₅₀ = 160.3 mg kg⁻¹, PI > 1.87). The compounds 3c, 3e, 3f and 3i suppressed psychomotor seizures in the 6 Hz test and 3c was the most potent with higher ED₅₀ = 13.98 mg kg⁻¹ and PI of > 21.46 compared to that of melatonin (ED₅₀ = 49.76 mg kg⁻¹ and PI of > 6.03) in mice. None of the compounds displayed neurotoxicity in the rota-rod test. The novel melatonin derivatives exerted weak cytotoxic effects while 3f showed the lowest hepatoxic effects comparable to that of the positive control melatonin in rats. The high affinities to the elucidated pharmacophore model of the novel melatonin compounds derived from the inclusion of aroylhydrazone moiety in the indole scaffold yielded suitable candidates with anticonvulsant activity in the MES and 6 Hz test of psychomotor seizures.     

DNA sequencing of 13 cytokine gene fragments of Aotus infulatus and Saimiri sciureus,two non-human primate models for malaria

Aotus and Saimiri are non-human primate models recommended by the World Health Organization for experimental studies in malaria, especially for vaccine pre-clinical trials. However, research using these primates is hindered by the lack of specific reagents to evaluate immune responses to infection or vaccination. As a step toward developing molecular tools for cytokine expression studies in these species, primer pairs for 18 cytokine gene fragments were designed based on human DNA sequences and used to amplify the corresponding genes in Aotus infulatus and Saimiri sciureus genomic DNA samples. IFNγ, TNFα, LTA, IL2, IL3, IL4, IL5, IL6, IL10, IL12, IL13, CSF2 and TGFβ2 gene fragments were amplified and sequenced. Primer pairs for IL8, IL17, IL18, IL27 and MIF failed to generate amplification products. When compared to the available corresponding human and non-human primate sequences, most – except IL3 and IL4 – showed identity degrees above 90%. Small variations in sequence can help to explain the failure to amplify certain genes or the amplification only at lower annealing temperatures as compared to human DNA samples for several primer pairs. The sequences made available provide the basis for designing molecular tools such as primers for real time PCR specific for A. infulatus and/or S. sciureus.The nucleotide sequences reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned accession numbers DQ985386 to DQ985389, DQ989356 to DQ989369, FJ89020 to FJ89024 and FJ89029.     

Expression profile of rrbp1 genes during embryonic development and in adult tissues of Xenopus laevis

Recent studies suggest that ribosome-binding protein 1 (RRBP1) is involved in multiple diseases such as tumorigenesis and cardiomyopathies. However, its function during embryonic development remains largely unknown. We searched Xenopus laevis database with human RRBP1 protein sequence and identified two cDNA sequences encoding Xenopus orthologs of RRBP1 including rrbp1a (NM_001089623) and rrbp1b (NM_001092468). Both genes were firstly detected at blastula stage 8 with weak signals in animal hemisphere by whole mount in situ hybridization. Evident expression of rrbp1 was mainly detected in cement gland and notochord at neurula and tailbud stages. Heart expression of rrbp1 was detected at stage 36. RT-PCR results indicated that very weak expression of rrbp1a was firstly detected in oocytes, followed by increasing expression until stage 39. Differently, very weak expression of rrbp1b was firstly observed at stage 2, and then maintained at a lower level to stage 17 followed by an intense expression from stages 19–39. Moreover, both expression profiles were also different in adult tissues. This study reports Xenopus rrbp1 expression during early embryonic development and in adult tissues. Our study will facilitate the functional analysis of Rrbp1 family during embryonic development.     

Single nucleotide polymorphism (SNP) in alpha-lactalbumin gene of Indian Jamunapari breed of Capra hircus

Alpha-lactalbumin is a major whey protein found in milk. It influences lactose synthesis by modifying the substrate specificity of galactosyl-transferase, is important to milk synthesis since lactose, an impermeable disaccharide, is the major osmole of milk. The present study was undertaken to detect polymorphism in the full coding region of alpha-lactalbumin at the genetic level and to explore allelic variability of this gene. Samples of Jamunapari breed of goat (n = 50) were included under the present study. Jamunapari is the highest milk producer among local Indian goat breeds. PCR-SSCP of all four exons of alpha-lactalbumin (ALA) revealed a total of 9 gel phenotypes of Jamunapari breed of goat. These were sequenced, analyzed and deposited in GenBank, NCBI (accession nos. EU573193–EU573195). Nucleotide and amino acid variations were searched within breeds of Indian goats and homology between caprine, ovine, bovine, bubaline and human. In the present study we describe for the first time two novel gene variants on the goat alpha-lactalbumin gene exon 4.     

Synthesis of aryl pyrazole via Suzuki coupling reaction,in vitro mushroom tyrosinase enzyme inhibition assay and in silico comparative molecular docking analysis with Kojic acid

Aryl pyrazoles are well recognized class of heterocyclic compounds found in several commercially available drugs. Owing to their significance in medicinal chemistry, in this current account we have synthesized a series of suitably substituted aryl pyrazole by employing Suzuki cross-coupling reaction. All compounds were evaluated for inhibition of mushroom tyrosinase enzyme both in vitro and in silico. Compound 3f (IC₅₀ = 1.568 ± 0.01 µM) showed relatively better potential compared to reference kojic acid (IC₅₀ = 16.051 ± 1.27 µM). A comparative docking studies showed that compound 3f have maximum binding affinity against mushroom tyrosinase (PDBID: 2Y9X) with binding energy value (−6.90 kcal/mol) as compared to Kojic acid. The 4-methoxy group in compound 3f shows 100% interaction with Cu. Compound 3f displayed hydrogen binding interaction with His61 and His94 at distance of 1.71 and 1.74 Å which might be responsible for higher activity compared to Kojic acid.     

Imidazolo and tryptophan-imidazolo hybrid derived ureas/thioureas as potent bioactive agents – SAR and molecular modelling studies

Herein, we used an imidazole derivative (IMD) which showed promising antibacterial, antifungal and antioxidant properties in our earlier investigation. Prompted by this, we converted IMD to hydrazide (IMH) by hydrazinolysis which was derivatized to various ureas (3–7) and thioureas (8–12). On the other hand, IMH was conjugated to Boc-Trp-OH as it has been shown in the past that hybridization of two molecules produced promising biologically active compounds. Boc of the conjugate was removed and further converted into several urea (14–18) and thiourea (19–23) derivatives. All the title compounds so also the starting materials and intermediates were assessed for potential biological applications. The results showed that compounds 3, 4, 8, 9, 14, 15, 19 and 20 were excellent antioxidants as revealed by DPPH, DMPD and ABTS assays. Further, certain analogues like 5–7, 10–12, 16–18 and 21–23 were found to be potent antimicrobials against pathogenic bacteria and fungi whereas good anti-inflammatory activity was obtained for molecules 5–7, 10–12, 16–18 and 21–23. All together, derivatives of the conjugates have shown superior activity over non-conjugated compounds and the former have exhibited potent activity against standard drugs in all the assays. In a quest to understand the binding interactions of the compounds with active site of tyrosine kinase (PDB ID: 2HCK), glucosamine-6-phosphate (GlcN-6-P) synthase (PDB ID: 2VF5) and cyclooxygenase-2 (PDB ID: 1CX2) enzymes, the correlation studies were conducted using molecular modelling which showed good receptor binding interactions with several amino acids of the enzymes. Overall, the current investigation may be considered for the discovery of lead compound(s) for treating multiple disorder conditions using singular molecular entity.     

The Parasites of Homo sapiens by R.W. Ashford and W. Crewe

Liverpool School of Tropical Medicine, 1998. £10.00 (viii + 128 pages)ISBN 0 9508756 9 4     

Identification of restriction fragment length polymorphisms linked to genes controlling soluble solids content in tomato fruit

Summary Gene(s) conferring high soluble solids (SS) in tomato fruit had been backcrossed previously from a wild tomato species, Lycopersicon chmielewskii LA1028 ( 10% SS), into a L. esculentum cultivar, VF36 ( 5% SS), to derive a BC₅S₅ line, LA1563, similar to VF 36 but with 7–8% SS. DNAs from these lines and a tomato breeding line, H2038, were screened for restriction fragment length polymorphisms (RFLPs) using four restriction endonucleases and sixty clones chosen at random from a tomato cDNA library. Most of the cDNA clones (56) identified the same RFLP in VF 36 and LA1563 and a different RFLP in LA1028. However, two cDNA clones identified the same RFLP in LA1563 and LA1028 and a different RFLP in VF36. To determine whether RFLPs identified by these two cDNA clones were linked to SS genes, a H2038 x LA1563 F₂ population was screened for segregation of the RFLPs and for SS content. The segregation ratios of these RFLPs were consistent with ratios expected for codominant alleles at unlinked loci. Analysis of variance of SS content for different RFLP genotypic classes indicated that RFLP alleles at one of the loci were linked to genes controlling SS content. The RFLP allele from the high SS tomato line, LA1563, was associated with significantly higher SS content and, therefore, could be useful in selecting for high SS gene(s) in a tomato breeding program.     

Why National Biomechanics Day?

National Biomechanics Day (NBD) seeks to expand the influence and impact of Biomechanics on our society by expanding the awareness of Biomechanics among young people. NBD will manifest this goal through worldwide, synchronized and coordinated celebrations and demonstrations of all things Biomechanics with high school students. NBD invites all Biomechanists to participate in NBD 2018, http://nationalbiomechanicsday.asbweb.org/.     

Human gut endogenous proteins as a potential source of angiotensin-I-converting enzyme (ACE-I)-, renin inhibitory and antioxidant peptides

It is well known that endogenous bioactive proteins and peptides play a substantial role in the body's first line of immunological defence, immune-regulation and normal body functioning. Further, the peptides derived from the luminal digestion of proteins are also important for body function. For example, within the peptide database BIOPEP (http://www.uwm.edu.pl/biochemia/index.php/en/biopep) 12 endogenous antimicrobial and 64 angiotensin-I-converting enzyme (ACE-I) inhibitory peptides derived from human milk and plasma proteins are listed. The antimicrobial peptide database (http://aps.unmc.edu/AP/main.php) lists over 111 human host-defence peptides. Several endogenous proteins are secreted in the gut and are subject to the same gastrointestinal digestion processes as food proteins derived from the diet. The human gut endogenous proteins (GEP) include mucins, serum albumin, digestive enzymes, hormones, and proteins from sloughed off epithelial cells and gut microbiota, and numerous other secreted proteins. To date, much work has been carried out regarding the health altering effects of food-derived bioactive peptides but little attention has been paid to the possibility that GEP may also be a source of bioactive peptides. In this review, we discuss the potential of GEP to constitute a gut cryptome from which bioactive peptides such as ACE-I inhibitory, renin inhibitory and antioxidant peptides may be derived.     

Design,synthesis and molecular docking of new N-4-piperazinyl ciprofloxacin-triazole hybrids with potential antimicrobial activity

New N-4-piperazinyl ciprofloxacin-triazole hybrids 6a-o were prepared and characterized. The in vitro antimycobacterial activity revealed that compound 6a experienced promising antimycobacterial activity against Mycobactrium smegmatis compared with the reference isoniazide (INH). Additionally, compound 6a exhibited broad spectrum antibacterial activity against all the tested strains either Gram-positive or Gram-negative bacteria compared with the reference ciprofloxacin. Also, compounds 6g and 6i displayed considerable antifungal activity compared with the reference ketoconazole. DNA cleavage assay of the highly active compounds 6c and 6h showed a good correlation between the Mycobactrium cleaved DNA gyrase assay and their in vitro antimycobactrial activity. Moreover, molecular modeling studies were done for the designed ciprofloxacin derivatives to predict their binding modes towards Topoisomerase II enzyme (PDB: 5bs8).     

Respiratory stimulus in Rhizobium sp. by legume lectins

High molecular weight lectins (> 100 kDa) from seeds of the legumes Canavalia brasiliensis (CnBr), Cratylia floribunda (CFL), Phaseolus vulgaris (PHA) and Vatairea macrocarpa (VML), temporarily stimulate the respiration of Rhizobium tropici-CIAT899 and R. etli-CFN42. These stimulants were significant (P < 0.05) in bacterial suspensions (> 2.85 mg dry biomass ml^–1), having at least 6200 molecules of lectins per bacteria. The VML (20 g ml^–1), induced specific O₂ demand of 2.3–2.5 M O₂ min^–1 mg dry biomass^–1, in CFN42 and CIAT899, respectively. However, CnBr, CFL and PHA induced smaller demands of O₂ (5×), in both strains. The order of affinities of the lectins was approximately VML > PHA > CFL > CnBr, with regard to respiratory stimuli in CIAT899 strain. The co-administration of 10 g VML ml^–1 and 9.8 M galactose, in CIAT899 suspensions, reduced the respiratory stimuli significantly in relation to the treatment with VML alone. These respiratory stimuli, induced by the lectins, increase the significance of the interaction lectin × Rhizobium in terms of bacterial physiology. Its understanding could be important in relation to bacterial symbiotic behaviour.     

Atul Butte discusses the divide between bioinformatics and the clinic

Atul Butte is an Assistant in Endocrinology and Informatics and Attending Physician at Children's Hospital, Boston, USA (http://www.chip.org), and is an Instructor in Paediatrics at Harvard Medical School (http://www.harvard.edu). He received his undergraduate degree in Computer Science from Brown University in 1991, and worked in several stints as a software engineer at Apple Computer and Microsoft Corporation. He graduated from the Brown University School of Medicine in 1995, during which he worked as a research fellow at National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK; http://www.niddk.nih.gov) through the Howard Hughes/NIH Research Scholars Program. He completed his residency in Paediatrics and Fellowship in Paediatric Endocrinology in 2001, both at Children's Hospital. During his research under Isaac Kohane (at Children's Hospital) he developed a novel methodology for analyzing large data sets of RNA expression, called Relevance Networks. His recent awards include the 2003 Emory University School of Medicine, Pathology Residents’ Choice Award, 2002 American Association for Clinical Chemistry Outstanding Speaker Award, 2002 Endocrine Society Travel Award based on presentation merit, 2001 American Association for Cancer Research Scholar-In-Training Award and the 2001 Lawson Wilkins Paediatric Endocrine Society Clinical Scholar Award.