NHERF2 Protein Mobility Rate Is Determined by a Unique C-terminal Domain That Is Also Necessary for Its Regulation of NHE3 Protein in OK Cells

Na⁺/H⁺ exchanger regulatory factor (NHERF) proteins are a family of PSD-95/Discs-large/ZO-1 (PDZ)-scaffolding proteins, three of which (NHERFs 1-3) are localized to the brush border in kidney and intestinal epithelial cells. All NHERF proteins are involved in anchoring membrane proteins that contain PDZ recognition motifs to form multiprotein signaling complexes. In contrast to their predicted immobility, NHERF1, NHERF2, and NHERF3 were all shown by fluorescence recovery after photobleaching/confocal microscopy to be surprisingly mobile in the microvilli of the renal proximal tubule OK cell line. Their diffusion coefficients, although different among the three, were all of the same magnitude as that of the transmembrane proteins, suggesting they are all anchored in the microvilli but to different extents. NHERF3 moves faster than NHERF1, and NHERF2 moves the slowest. Several chimeras and mutants of NHERF1 and NHERF2 were made to determine which part of NHERF2 confers the slower mobility rate. Surprisingly, the slower mobility rate of NHERF2 was determined by a unique C-terminal domain, which includes a nonconserved region along with the ezrin, radixin, moesin (ERM) binding domain. Also, this C-terminal domain of NHERF2 determined its greater detergent insolubility and was necessary for the formation of larger multiprotein NHERF2 complexes. In addition, this NHERF2 domain was functionally significant in NHE3 regulation, being necessary for stimulation by lysophosphatidic acid of activity and increased mobility of NHE3, as well as necessary for inhibition of NHE3 activity by calcium ionophore 4-Br-{"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Thus, multiple functions of NHERF2 require involvement of an additional domain in this protein.     

Sex differences in kidney gene expression during the life cycle of F344 rats

Background

The kidney functions in key physiological processes to filter blood and regulate blood pressure via key molecular transporters and ion channels. Sex-specific differences have been observed in renal disease incidence and progression, as well as acute kidney injury in response to certain drugs. Although advances have been made in characterizing the molecular components involved in various kidney functions, the molecular mechanisms responsible for sex differences are not well understood. We hypothesized that the basal expression levels of genes involved in various kidney functions throughout the life cycle will influence sex-specific susceptibilities to adverse renal events.

Methods

Whole genome microarray gene expression analysis was performed on kidney samples collected from untreated male and female Fischer 344 (F344) rats at eight age groups between 2 and 104 weeks of age.

Results

A combined filtering approach using statistical (ANOVA or pairwise t test, FDR 0.05) and fold-change criteria (>1.5 relative fold change) was used to identify 7,447 unique differentially expressed genes (DEGs). Principal component analysis (PCA) of the 7,447 DEGs revealed sex-related differences in mRNA expression at early (2 weeks), middle (8, 15, and 21 weeks), and late (104 weeks) ages in the rat life cycle. Functional analysis (Ingenuity Pathway Analysis) of these sex-different genes indicated over-representation of specific pathways and networks including renal tubule injury, drug metabolism, and immune cell and inflammatory responses. The mRNAs that code for the qualified urinary protein kidney biomarkers KIM-1, Clu, Tff3, and Lcn2 were also observed to show sex differences.

Conclusions

These data represent one of the most comprehensive in-life time course studies to be published, assessing sex differences in global gene expression in the F344 rat kidney. PCA and Venn analyses reveal specific periods of sexually dimorphic gene expression which are associated with functional categories (xenobiotic metabolism and immune cell and inflammatory responses) of key relevance to acute kidney injury and chronic kidney disease, which may underlie sex-specific susceptibility. Analysis of the basal gene expression patterns of renal genes throughout the life cycle of the rat will improve the use of current and future renal biomarkers and inform our assessments of kidney injury and disease.

     

Chemodenitrification in the cryoecosystem of Lake Vida,Victoria Valley,Antarctica

Lake Vida, in the Victoria Valley of East Antarctica, is frozen, yet harbors liquid brine (~20% salt, >6 times seawater) intercalated in the ice below 16 m. The brine has been isolated from the surface for several thousand years. The brine conditions (permanently dark, ?13.4 °C, lack of O₂, and pH of 6.2) and geochemistry are highly unusual. For example, nitrous oxide (N₂O) is present at a concentration among the highest reported for an aquatic environment. Only a minor ¹⁷O anomaly was observed in N₂O, indicating that this gas was predominantly formed in the lake. In contrast, the ¹⁷O anomaly in nitrate () in Lake Vida brine indicates that approximately half or more of the present is derived from atmospheric deposition. Lake Vida brine was incubated in the presence of ¹⁵N‐enriched substrates for 40 days. We did not detect microbial nitrification, dissimilatory reduction of to ammonium (), anaerobic ammonium oxidation, or denitrification of N₂O under the conditions tested. In the presence of ¹⁵N‐enriched nitrite (), both N₂ and N₂O exhibited substantial ¹⁵N enrichments; however, isotopic enrichment declined with time, which is unexpected. Additions of ¹⁵N– alone and in the presence of HgCl₂ and ZnCl₂ to aged brine at ?13 °C resulted in linear increases in the δ¹⁵N of N₂O with time. As HgCl₂ and ZnCl₂ are effective biocides, we interpret N₂O production in the aged brine to be the result of chemodenitrification. With this understanding, we interpret our results from the field incubations as the result of chemodenitrification stimulated by the addition of ¹⁵N‐enriched and ZnCl₂ and determined rates of N₂O and N₂ production of 4.11–41.18 and 0.55–1.75 nmol L^?1 day^?1, respectively. If these rates are representative of natural production, the current concentration of N₂O in Lake Vida could have been reached between 6 and 465 years. Thus, chemodenitrification alone is sufficient to explain the high levels of N₂O present in Lake Vida.     

Design,green synthesis and pharmacological evaluation of novel 5,6-diaryl-1,2,4-triazines bearing 3-morpholinoethylamine moiety as potential antithrombotic agents*

The aim of this research work was to investigate a series of novel 5,6-diaryl-1,2,4-triazines (3a–3q) containing 3-morpholinoethylamine side chain, and to address their antiplatelet activity by in vitro, ex vivo and in vivo methods. All compounds were synthesized by environment benign route and their structures were unambiguously confirmed by spectral data. Compounds (3l) and (3m) were confirmed by their single crystal X-ray structures. Out of all the synthesized compounds, 10 were found to be more potent in vitro than aspirin; six of them were found to be prominent in ex vivo assays and one compound (3d) was found to have the most promising antithrombotic profile in vivo. Moreover, compound (3d) demonstrated less ulcerogenicity in rats as compared to aspirin. The selectivity of the most promising compound (3d) for COX-1 and COX-2 enzymes was determined with the help of molecular docking studies and the results were correlated with the biological activity.     

Genomic annotation and validation of bacterial consortium NDMC-1 for enhanced degradation of sugarcane bagasse

This study aims at designing a consortium using rumen bacterial isolates for enhancing the hydrolysis of sugarcane bagasse (SB) for efficient biofuel formation. The microbial population was screened through biochemical and molecular tools along with enzymatic activity to obtain potential isolates for diverse cellulolytic and hemicellulolytic carbohydrate active enzyme (CAZyme). Five strains (Paenibacillus, Bacillus, Enterobacter, and Microbacterium) were selected for designing the consortium NDMC-1. The hydrolytic efficiency of NDMC-1 was determined based on cellulase production with simultaneous rise in monosaccharides, oligosaccharides, and soluble chemical oxygen demand (sCOD) concentration. Cellulolytic machinery of these isolates was further explored using genome sequencing. The isolates selected for consortia NDMC-1 interacted synergistically leading to enhanced cellulase production. Maximal endoglucanase (1.67 μmol ml−1 min−1), exoglucanase (0.69 μmol ml−1 min−1), and β-glucosidase (2.03 μmol ml−1 min−1) activity were achieved with SB as a sole carbon source after 48 h of incubation. Enhancement in SB hydrolysis employing NDMC-1 was evident by the increase in sCOD from 609 to 2589 mg/l and release of 1295 mg/l reducing sugar, comprising 59.8%, 8.23%, and 6.16% of glucose, cellobiose, and cellotriose, respectively, which resulted in 5.5-fold rise in biogas production. On genome annotation, total 472 contigs from glycoside hydrolase family: 84 from Microbacterium arborescens ND21, 72 from Enterobacter cloacae ND22, 61 from Bacillus subtilis ND23, 116 from Paenibacillus polymyxa ND24, and 140 from Paenibacillus polymyxa ND25 were identified. On further analysis, total 33 cellulases, 59 hemicellulases, and 48 esterases were annotated in the reported genomes. This work proposes the application of consortia-based bioprocessing systems over the conventionally favorable single organism approach for efficient hydrolysis of cellulosic substrates to fermentable sugars.     

DYRK1A regulates the recruitment of 53BP1 to the sites of DNA damage in part through interaction with RNF169

Human Dual-specificity tyrosine (Y) Regulated Kinase 1A (DYRK1A) is encoded by a dosage dependent gene whereby either trisomy or haploinsufficiency result in developmental abnormalities. However, the function and regulation of this important protein kinase are not fully understood. Here, we report proteomic analysis of DYRK1A in human cells that revealed a novel role of DYRK1A in DNA double-strand breaks (DSBs) repair, mediated in part by its interaction with the ubiquitin-binding protein RNF169 that accumulates at the DSB sites and promotes homologous recombination repair (HRR) by displacing 53BP1, a key mediator of non-homologous end joining (NHEJ). We found that overexpression of active, but not the kinase inactive DYRK1A in U-2 OS cells inhibits accumulation of 53BP1 at the DSB sites in the RNF169-dependent manner. DYRK1A phosphorylates RNF169 at two sites that influence its ability to displace 53BP1 from the DSBs. Although DYRK1A is not required for the recruitment of RNF169 to the DSB sites and 53BP1 displacement, inhibition of DYRK1A or mutation of the DYRK1A phosphorylation sites in RNF169 decreases its ability to block accumulation of 53BP1 at the DSB sites. Interestingly, CRISPR-Cas9 knockout of DYRK1A in human and mouse cells also diminished the 53BP1 DSB recruitment in a manner that did not require RNF169, suggesting that dosage of DYRK1A can influence the DNA repair processes through both RNF169-dependent and independent mechanisms. Human U-2 OS cells devoid of DYRK1A display an increased HRR efficiency and resistance to DNA damage, therefore our findings implicate DYRK1A in the DNA repair processes.     

Evolutionary history predicts high‐impact invasions by herbivorous insects

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