Renal Cells Express Different Forms of Vimentin: The Independent Expression Alteration of these Forms is Important in Cell Resistance to Osmotic Stress and Apoptosis

Osmotic stress has been shown to regulate cytoskeletal protein expression. It is generally known that vimentin is rapidly degraded during apoptosis by multiple caspases, resulting in diverse vimentin fragments. Despite the existence of the known apoptotic vimentin fragments, we demonstrated in our study the existence of different forms of vimentin VIM I, II, III, and IV with different molecular weights in various renal cell lines. Using a proteomics approach followed by western blot analyses and immunofluorescence staining, we proved the apoptosis-independent existence and differential regulation of different vimentin forms under varying conditions of osmolarity in renal cells. Similar impacts of osmotic stress were also observed on the expression of other cytoskeleton intermediate filament proteins; e.g., cytokeratin. Interestingly, 2D western blot analysis revealed that the forms of vimentin are regulated independently of each other under glucose and NaCl osmotic stress. Renal cells, adapted to high NaCl osmotic stress, express a high level of VIM IV (the form with the highest molecular weight), besides the three other forms, and exhibit higher resistance to apoptotic induction with TNF-α or staurosporin compared to the control. In contrast, renal cells that are adapted to high glucose concentration and express only the lower-molecular-weight forms VIM I and II, were more susceptible to apoptosis. Our data proved the existence of different vimentin forms, which play an important role in cell resistance to osmotic stress and are involved in cell protection against apoptosis.     

Assessment of toxicity of volatile fatty acids to Photobacterium phosphoreum

The toxicity of four volatile fatty acids (VFAs) as anaerobic digestion (AD) intermediates was investigated at pH 7. Photobacterium phosphoreum T3 was used as an indicator organism. Binary, ternary and mixtures of AD intermediates were designated by letters A (acetic acid + propionic acid), B (acetic acid + butyric acid), C (acetic acid + ethanol), D (propionic acid + butyric acid), E (propionic acid + ethanol), F (butyric acid + ethanol), G (acetic acid + propionic acid + butyric acid), H (acetic acid + propionic acid + ethanol), I (acetic acid + butyric acid+ ethanol), J (propionic acid + butyric acid + ethanol) and K (acetic acid + propionic acid + butyric acid + ethanol) to assess the toxicity through equitoxic mixing ratio method. The IC₅₀ values of acetic acid, propionic acid, butyric acid and ethanol were 9.812, 7.76, 6.717 and 17.33 g/L respectively, displaying toxicity order of: butyric acid > propionic acid > acetic acid > ethanol being additive in nature. The toxic effects of four VFAs could be designated as synergistic and one additive in nature.     

Continuous degradation of maltose by enzyme entrapment technology using calcium alginate beads as a matrix

Maltase from Bacillus licheniformis KIBGE-IB4 was immobilized within calcium alginate beads using entrapment technique. Immobilized maltase showed maximum immobilization yield with 4% sodium alginate and 0.2 M calcium chloride within 90.0 min of curing time. Entrapment increases the enzyme–substrate reaction time and temperature from 5.0 to 10.0 min and 45 °C to 50 °C, respectively as compared to its free counterpart. However, pH optima remained same for maltose hydrolysis. Diffusional limitation of substrate (maltose) caused a declined in V_max of immobilized enzyme from 8411.0 to 4919.0 U ml^?1 min^?1 whereas, K_m apparently increased from 1.71 to 3.17 mM ml^?1. Immobilization also increased the stability of free maltase against a broad temperature range and enzyme retained 45% and 32% activity at 55 °C and 60 °C, respectively after 90.0 min. Immobilized enzyme also exhibited recycling efficiency more than six cycles and retained 17% of its initial activity even after 6th cycles. Immobilized enzyme showed relatively better storage stability at 4 °C and 30 °C after 60.0 days as compared to free enzyme.     

Peroxisomal Acyl-CoA oxidase 4 activity differs between Arabidopsis accessions

In plants, peroxisomes are the primary site of fatty acid β-oxidation. Following substrate activation, fatty acids are oxidized by Acyl-CoA Oxidase (ACX) enzymes. Arabidopsis has six ACX genes, although ACX6 is not expressed. Biochemical characterization has revealed that each ACX enzyme acts on specific chain-length targets, but in a partially overlapping manner, indicating a degree of functional redundancy. Genetic analysis of acx single and double mutants in the Columbia (Col-0) accession revealed only minor phenotypes, but an acx3acx4 double mutant from Wassileskija (Ws) is embryo lethal. In this study, we show that acx3acx4(Col) and acx1acx3acx4(Col) mutants are viable and that enzyme activity in these mutants is significantly reduced on a range of substrates compared to wild type. However, the triple mutant displays only minor defects in seed-storage mobilization, seedling development, and adult growth. Although the triple mutant is defective in the three most active and highly-expressed ACX proteins, increases in ACX2 expression may support partial β-oxidation activity. Comparison of acx mutant alleles in the Col-0 and Ws accessions reveals independent phenotypes; the Ws acx4 mutant uniquely shows increased sensitivity to propionate, whereas the Col-0 acx4 allele has sucrose-dependent growth in the light. To dissect the issues between Col-0 and Ws, we generated mixed background mutants. Although alleles with the Col-0 acx4 mutant were viable, we were unable to isolate an acx3acx4 line using the Ws acx4 allele. Reducing ACX4 expression in several Arabidopsis backgrounds showed a split response, suggesting that the ACX4 gene and/or protein functions differently in Arabidopsis accessions.     

Regulation of cotton fiber elongation by xyloglucan endotransglycosylase/hydrolase genes

Ligon lintless mutant (li1li1) with super-short fibers (5-8 mm in length) and its wild type (Li1Li1) with normal fibers (30 mm in length) were used to study the function of xyloglucan endotransglycosylase/hydrolase (XTH) genes during fiber elongation in cotton. Wild-type cotton attained the fiber elongation stage earlier (5 days post-anthesis, DPA), than the Ligon lintless mutant (12 DPA) with a higher fiber elongation velocity of about 1.76 mm/day. Xyloglucan contents in Ligon lintless mutant fibers were 5-fold higher than the wild type during 9-15 DPA. It was also observed that the activity of XTH in wild-type cotton fibers was about 2-fold higher than that of the Ligon lintless mutant with a peak at 12 DPA. DNA blot analysis indicated that the XTH gene in the Ligon lintless mutant and its wild type belonged to a multiple allelic series. However, RNA blot analysis and quantitative real-time PCR exhibited an earlier expression (10 DPA) of XTH in wild type as compared to delayed (15 DPA) expression in the Ligon lintless mutant. The study also revealed that 9-15 DPA might be a key phase for upregulation of fiber elongation via increasing XTH activity. Higher XTH activity can cleave down the xyloglucan-cellulose chains thus loosening fiber cell wall and promoting fiber cell elongation in wild type as compared to its mutant.     

Assessing the diversity of bacterial communities from marine sponges and their bioactive compounds

Symbiotic bacteria play vital roles in the survival and health of marine sponges. Sponges harbor rich, diverse and species-specific microbial communities. Symbiotic marine bacteria have increasingly been reported as promising source of bioactive compounds. A culturomics-based study was undertaken to study the diversity of bacteria from marine sponges and their antimicrobial potential. We have collected three sponge samples i.e. Acanthaster carteri, Rhytisma fulvum (soft coral) and Haliclona caerulea from north region (Obhur) of Red Sea, Jeddah Saudi Arabia. Total of 144 bacterial strains were isolated from three marine sponges using culture dependent method. Screening of isolated strains showed only 37 (26%) isolates as antagonists against oomycetes pathogens (P. ultimum and P. capsici). Among 37 antagonistic bacteria, only 19 bacterial strains exhibited antibacterial activity against human pathogens (Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 8739, Enterococcus faecalis ATCC 29212). Four major classes of bacteria i.e γ-Proteobacteria, α-Proteobacteria, Firmicutes and Actinobacteria were recorded from three marine sponges where γ-Proteobacteria was dominant class. One potential bacterial strain Halomonas sp. EA423 was selected for identification of bioactive metabolites using GC and LC-MS analyses. Bioactive compounds Sulfamerazine, Metronidazole-OH and Ibuprofen are detected from culture extract of strain Halomonas sp. EA423. Overall, this study gives insight into composition and diversity of antagonistic bacterial community of marine sponges and coral from Red Sea and presence of active metabolites from potential strain. Our results showed that these diverse and potential bacterial communities further need to be studied to exploit their biotechnological significance.     

SDF-1alpha promotes invasion of head and neck squamous cell carcinoma by activating NF-kappaB

CXCL12/stromal cell-derived factor-1alpha (SDF-1alpha), a chemokine ligand for the G protein-coupled receptor CXCR4, plays an important role in the directed movement of cells. Many studies have documented the importance of CXCR4 in tumor progression and organ-specific metastasis. Recently, several studies have implicated a role for SDF-1alpha in head and neck squamous cell carcinoma (HNSCC) metastasis, but currently there is little information about how SDF-1alpha promotes HNSCC metastasis. In this report we show that the NF-kappaB signaling pathway is activated in response to SDF-1alpha in HNSCC while primary and immortalized keratinocytes show no SDF-1alpha-mediated NF-kappaB activity. We found that SDF-1alpha-mediated NF-kappaB signaling is independent of phosphoinositide 3-kinase/Akt and ERK/MAPK pathways. We observed that SDF-1alpha induces IkappaBalpha phosphorylation and degradation and the nuclear translocation of NF-kappaB in HNSCC cell lines, suggesting that SDF-1alpha activates the classical NF-kappaB signaling pathway. Contrary to previous reports, SDF-1alpha-induced NF-kappaB activation is not mediated by tumor necrosis factor alpha. Furthermore, blocking the NF-kappaB signaling pathway with an IKKbeta inhibitor significantly reduces SDF-1alpha-mediated HNSCC invasion. Taken together, our data suggest SDF-1alpha/CXCR4 may promote HNSCC invasion and metastasis by activating NF-kappaB and that targeting NF-kappaB may provide therapeutic opportunities in preventing HNSCC metastasis mediated by SDF-1alpha.     

GC/MS Profiling,In Vitro and In Silico Pharmacological Screening and Principal Component Analysis of Essential Oils from Three Exotic and Two Endemic Plants from Mauritius

The chemical and pharmacological profiles of essential oils (EOs) hydrodistilled in yields of 0.03–0.77 % (w/w) from three exotic (Cinnamomum camphora, Petroselinum crispum, and Syzygium samarangense) and two endemic (Pittosporum senacia subsp. senacia and Syzygium coriaceum) medicinal plants were studied. GC-MS/GC-FID analysis of the EOs identified the most dominant components to be myristicin (40.3 %), myrcene (62.2 %), 1,8-cineole (54.0 %), β-pinene (21.3 %) and (E)-β-ocimene (24.4 %) in P. crispum, P. senacia and C. camphora, S. samarangense and S. coriaceum EOs, respectively. All EOs were found to possess anti-amylase (0.70–1.50 mM ACAE/g EO) and anti-tyrosinase (109.35–158.23 mg KAE/g) properties, whereas no glucosidase inhibition was displayed. Only Syzygium EOs acted as dual inhibitors of both acetyl- and butyryl-cholinesterases, while P. senacia and C. camphora EOs inhibited acetylcholinesterase selectively and P. crispum EO was inactive (AChE: 4.64–4.96 mg GALAE/g; BChE: 5.96 and 7.10 mg GALAE/g). Molecular docking revealed 1,8-cineole to present the best binding affinities with butyrylcholinesterase, amylase and tyrosinase, while both myristicin and β-pinene with acetylcholinesterase and finally β-pinene with glucosidase. In vitro antioxidant potency was also demonstrated in different assays (DPPH: 13.52–53.91 mg TE/g, ABTS: 5.49–75.62 mg TE/g; CUPRAC: 45.38–243.21 mg TE/g, FRAP: 42.49–110.64 mg TE/g; and phosphomolybdenum assay: 82.61–160.93 mM TE/g). Principal component analysis revealed the EOs to differ greatly in their bioactivities due to their chemodiversity. This study has unveiled some interesting preliminary pharmacological profiles of the EOs that could be explored for their potential applications as phytotherapeutics.     

Okra-leaf accessions of the upland cotton (Gossypium hirsutum L.): genetic variability in agronomic and fibre traits

Okra-leaf types of the upland cotton have the potential to be competitive to the normal-leaf types in yield and fibre quality, in addition to its potential resistance to insect pests and drought. Okra-leaf cotton accessions, collected at Cotton Research Institute, Faisalabad, Pakistan, were evaluated in respect of genetic variance and relative performance in half- and full-sib crosses (combining ability) for 2 years. Variation due to parents x years interaction was significant for lint percentage, seed weight and earliness index, resulting in moderately low but significant genetic variance across environments (years) for these traits. Interaction of environment with general combining ability was significant for seed yield, seed weight, and earliness index. General combining ability variation, contributed by females and males together, accounted for 71% of the total variation available for seed cotton yield, 60% of that for seed weight and height to node ratio each, and 75% of that for earliness index. Specific combining ability variation accounted for 85% and 51% of the total variation available for lint percentage and staple length, respectively. The contribution of female parents to general combining ability variation was higher than that of male parents for seed cotton yield, seed weight, height to node ratio, and earliness index. Okra-leaf accessions HR-VO-MS and HR107-NH were predicted to produce progenies having high yield, HR109-RT high lint percentage, while HR100-Okra, Gambo-Okra and HR-VO-1 were predicted to impart early crop maturity to their progenies by reason of their good general combining ability for these traits. The results also provided evidence that genes controlling high yield in HR-VO-MS and HR107-NH were different from those controlling high yield in HR109-RT. The set of genes controlling the high earliness index in HR100-Okra and that in HR-VO-MS also appeared to differ in expression.     

3D model of the Escherichia coli multidrug transporter MdfA reveals an essential membrane-embedded positive charge

MdfA is an Escherichia coli multidrug transporter of the major facilitator superfamily (MFS) of secondary transporters. Although several aspects of multidrug recognition by MdfA have been characterized, better understanding the detailed mechanism of its function requires structural information. Previous studies have modeled the 3D structures of MFS proteins, based on the X-ray structure of LacY and GlpT. However, because of poor sequence homology, between LacY, GlpT, and MdfA additional constraints were required for a reliable homology modeling. Using an algorithm that predicts the angular orientation of each transmembrane helix (TM) (kPROT), we obtained a remarkably similar pattern for the 12 TMs of MdfA and those of GlpT and LacY, suggesting that they all have similar helix packing. Consequently, a 3D model was constructed for MdfA by structural alignment with LacY and GlpT, using the kPROT results as an additional constraint. Further refinement and a preliminary evaluation of the model were achieved by correlated mutation analysis and the available experimental data. Surprisingly, in addition to the previously characterized membrane-embedded glutamate at position 26, the model suggests that Asp34 and Arg112 are located within the membrane, on the same face of the cavity as Glu26. Importantly, Arg112 is evolutionarily conserved in secondary drug transporters, and here we show that a positive charge at this position is absolutely essential for multidrug transport by MdfA.