The HOPS Proteins hVps41 and hVps39 Are Required for Homotypic and Heterotypic Late Endosome Fusion

The homotypic fusion and protein sorting (HOPS) complex is a multisubunit tethering complex that in yeast regulates membrane fusion events with the vacuole, the yeast lysosome. Mammalian homologs of all HOPS components have been found, but little is known about their function. Here, we studied the role of hVps41 and hVps39, two components of the putative human HOPS complex, in the endo‐lysosomal pathway of human cells. By expressing hemagglutinin (HA)‐tagged constructs, we show by immunoelectron microscopy (immunoEM) that both hVps41 and hVps39 associate with the limiting membrane of late endosomes as well as lysosomes. Small interference RNA (siRNA)‐mediated knockdown of hVps41 or hVps39 resulted in an accumulation of late endosomes, a depletion in the number of lysosomes and a block in the degradation of endocytosed cargo. Lysosomal pH and cathepsin B activity remained unaltered in these conditions. By immunoEM we found that hVps41 or hVps39 knockdown impairs homotypic fusion between late endosomes as well as heterotypic fusion between late endosomes and lysosomes. Thus, our data show that both hVps41 and hVps39 are required for late endosomal–lysosomal fusion events and the delivery of endocytic cargo to lysosomes in human cells.     

Expression of CspE by a Psychrotrophic Bacterium Enterobacter ludwigii PAS1, Isolated from Indian Himalayan Soil and In silico Protein Modelling, Prediction of Conserved Residues and Active Sites

Proteome analysis of Enterobacter ludwigii PAS1 provide a powerful set of tool to study the cold shock proteins along with that combination of bioinformatics is useful for interpretation of comparative results from many species. There is a considerable interest in the use of psychrotrophic bacteria for nitrogen fixation, especially at hilly regions, thus better understanding of cold adaptation mechanisms too. The psychrotrophic E. ludwigii PAS1 grown at 30 and 4 °C, isolated from Himalaya soil was undertaken for proteomic responses during optimal and cold shock conditions. Comparative proteomic analyses using two-dimensional gel electrophoresis (2-DE) and MALDI-TOF/TOF MS revealed the presence of Cold shock protein E (CspE). Three-dimensional structure of CspE of E. ludwigii PAS1 divulge the presence of five antiparallel β-sheets forming a β-barrel structure with surface exposed aromatic and basic residues that were responsible for nucleic acid binding and also reveals the presence of highly conserved nucleic acid-binding motifs RNP1 and RNP2 in Csp family.     

Length–weight relationships of nine freshwater fishes from six rivers of the Western Ghats of India

Length–weight relationships (LWRs) were estimated for nine freshwater fish species collected three times using a scoop net (mesh size 0.3–0.5 cm), cast net (mesh size 1–1.5 cm) and gillnet (mesh size 2.5–4.5 cm) from six rivers of the Western Ghats of India during August 2017–October 2018. The b values for LWRs varied from 2.862 to 3.656 (R² > 0.916 and p < 0.0001 for all species).     

Genome-wide identification and comprehensive analysis of Excretory/Secretory proteins in nematodes provide potential drug targets for parasite control

Nematodes are responsible for causing severe diseases in plants, humans and other animals. Infection is associated with the release of Excretory/Secretory (ES) proteins into host cytoplasm and interference with the host immune system which make them attractive targets for therapeutic use. The identification of ES proteins through bioinformatics approaches is cost- and time-effective and could be used for screening of potential targets for parasitic diseases for further experimental studies. Here, we identified and functionally annotated 93,949 ES proteins, in the genome of 73 nematodes using integration of various bioinformatics tools. 30.6% of ES proteins were found to be supported at RNA level. The predicted ES proteins, annotated by Gene Ontology terms, domains, metabolic pathways, proteases and enzyme class analysis were enriched in molecular functions of proteases, protease inhibitors, c-type lectin and hydrolases which are strongly associated with typical functions of ES proteins. We identified a total of 452 ES proteins from human and plant parasitic nematodes, homologues to DrugBank-approved targets and C. elegans RNA interference phenotype genes which could represent potential targets for parasite control and provide valuable resource for further experimental studies to understand host-pathogen interactions.     

pSAT vectors: a modular series of plasmids for autofluorescent protein tagging and expression of multiple genes in plants

Autofluorescent protein tags represent one of the major and, perhaps, most powerful tools in modern cell biology for visualization of various cellular processes in vivo. In addition, advances in confocal microscopy and the development of autofluorescent proteins with different excitation and emission spectra allowed their simultaneous use for detection of multiple events in the same cell. Nevertheless, while autofluorescent tags are widely used in plant research, the need for a versatile and comprehensive set of vectors specifically designed for fluorescent tagging and transient and stable expression of multiple proteins in plant cells from a single plasmid has not been met by either the industrial or the academic communities. Here, we describe a new modular satellite (SAT) vector system that supports N- and C-terminal fusions to five different autofluorescent tags, EGFP, EYFP, Citrine-YFP, ECFP, and DsRed2. These vectors carry an expanded multiple cloning site that allows easy exchange of the target genes between different autofluorescence tags, and expression of the tagged proteins is controlled by constitutive promoters, which can be easily replaced with virtually any other promoter of interest. In addition, a series of SAT vectors has been adapted for high throughput Gateway recombination cloning. Furthermore, individual expression cassettes can be assembled into Agrobacterium binary plasmids, allowing efficient transient and stable expression of multiple autofluorescently tagged proteins from a single vector following its biolistic delivery or Agrobacterium-mediated genetic transformation. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Der p 5 Crystal Structure Provides Insight into the Group 5 Dust Mite Allergens

Group 5 allergens from house dust mites elicit strong IgE antibody binding in mite-allergic patients. The structure of Der p 5 was determined by x-ray crystallography to better understand the IgE epitopes, to investigate the biologic function in mites, and to compare with the conflicting published Blo t 5 structures, designated 2JMH and 2JRK in the Protein Data Bank. Der p 5 is a three-helical bundle similar to Blo t 5, but the interactions of the helices are more similar to 2JMH than 2JRK. The crystallographic asymmetric unit contains three dimers of Der p 5 that are not exactly alike. Solution scattering techniques were used to assess the multimeric state of Der p 5 in vitro and showed that the predominant state was monomeric, similar to Blo t 5, but larger multimeric species are also present. In the crystal, the formation of the Der p 5 dimer creates a large hydrophobic cavity of ∼3000 ų that could be a ligand-binding site. Many allergens are known to bind hydrophobic ligands, which are thought to stimulate the innate immune system and have adjuvant-like effects on IgE-mediated inflammatory responses.     

Assay and inhibition of diacylglycerol lipase activity

A series of N-formyl-α-amino acid esters of β-lactone derivatives structurally related to tetrahydrolipstatin (THL) and O-3841 were synthesized that inhibit human and murine diacylglycerol lipase (DAGL) activities. New ether lipid reporter compounds were developed for an in vitro assay to efficiently screen inhibitors of 1,2-diacyl-sn-glycerol hydrolysis and related lipase activities using fluorescence resonance energy transfer (FRET). A standardized thin layer chromatography (TLC) radioassay of diacylglycerol lipase activity utilizing the labeled endogenous substrate [1″-(14)C]1-stearoyl-2-arachidonoyl-sn-glycerol with phosphorimaging detection was used to quantify inhibition by following formation of the initial product [1″-(14)C]2-arachidonoylglycerol and further hydrolysis under the assay conditions to [1-(14)C]arachidonic acid.     

Enzymatic Dehalogenation of Pentachlorophenol by <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> of the Microbial Community from Tannery Effluent

Four different bacterial isolates obtained from a stable bacterial consortium were capable of utilizing pentachlorophenol (PCP) as sole carbon and energy source. The consortium was developed by continuous enrichment in the chemostat. The degradation of PCP by bacterial strain was preceded through an oxidative route as indicated by accumulation of tetrachloro-ρ-hydroquinone and dichlorohydroquinone as determined by high performance liquid chromatography (HPLC). Among the four isolates, Pseudomonas fluorescens exhibited maximum degradation capability and enzyme production. PCP-monooxygenase enzyme was extracted from culture extract and fractionated by DEAE-cellulose ion exchange chromatography. The molecular weight of the enzyme, purified from Pseudomonas fluorescens, determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography was found to be 24,000 Da. Received: 22 July 2002 / Accepted: 23 September 2002