Members of the eukaryotic heat shock protein 70 family (Hsp70s) are regulated by protein cofactors that contain domains homologous to bacterial DnaJ. Of the three DnaJ homologues in the yeast rough endoplasmic reticulum (RER; Scj1p, Sec63p, and Jem1p), Scj1p is most closely related to DnaJ, hence it is a probable cofactor for Kar2p, the major Hsp70 in the yeast RER. However, the physiological role of Scj1p has remained obscure due to the lack of an obvious defect in Kar2p-mediated pathways in scj1 null mutants. Here, we show that the Δscj1 mutant is hypersensitive to tunicamycin or mutations that reduce N-linked glycosylation of proteins. Although maturation of glycosylated carboxypeptidase Y occurs with wild-type kinetics in Δscj1 cells, the transport rate for an unglycosylated mutant carboxypeptidase Y (CPY) is markedly reduced. Loss of Scj1p induces the unfolded protein response pathway, and results in a cell wall defect when combined with an oligosaccharyltransferase mutation. The combined loss of both Scj1p and Jem1p exaggerates the sensitivity to hypoglycosylation stress, leads to further induction of the unfolded protein response pathway, and drastically delays maturation of an unglycosylated reporter protein in the RER. We propose that the major role for Scj1p is to cooperate with Kar2p to mediate maturation of proteins in the RER lumen.
Background: Many CpGs become hyper or hypo-methylated with age. Multiple methods have been developed by Horvath et al. to estimate DNA methylation (DNAm) age including Pan-tissue, Skin & Blood, PhenoAge, and GrimAge. Pan-tissue and Skin & Blood try to estimate chronological age in the normal population whereas PhenoAge and GrimAge use surrogate markers associated with mortality to estimate biological age and its departure from chronological age. Here, we applied Horvath's four methods to calculate and compare DNAm age in 499 subjects with type 1 diabetes (T1D) from the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) study using DNAm data measured by Illumina EPIC array in the whole blood. Association of the four DNAm ages with development of diabetic complications including cardiovascular diseases (CVD), nephropathy, retinopathy, and neuropathy, and their risk factors were investigated. Results: Pan-tissue and GrimAge were higher whereas Skin & Blood and PhenoAge were lower than chronological age (p < 0.0001). DNAm age was not associated with the risk of CVD or retinopathy over 18-20 years after DNAm measurement. However, higher PhenoAge (β = 0.023, p = 0.007) and GrimAge (β = 0.029, p = 0.002) were associated with higher albumin excretion rate (AER), an indicator of diabetic renal disease, measured over time. GrimAge was also associated with development of both diabetic peripheral neuropathy (OR = 1.07, p = 9.24E−3) and cardiovascular autonomic neuropathy (OR = 1.06, p = 0.011). Both HbA1c (β = 0.38, p = 0.026) and T1D duration (β = 0.01, p = 0.043) were associated with higher PhenoAge. Employment (β = − 1.99, p = 0.045) and leisure time (β = − 0.81, p = 0.022) physical activity were associated with lower Pan-tissue and Skin & Blood, respectively. BMI (β = 0.09, p = 0.048) and current smoking (β = 7.13, p = 9.03E−50) were positively associated with Skin & Blood and GrimAge, respectively. Blood pressure, lipid levels, pulse rate, and alcohol consumption were not associated with DNAm age regardless of the method used. Conclusions: Various methods of measuring DNAm age are sub-optimal in detecting people at higher risk of developing diabetic complications although some work better than the others.
Fluorescent molecules serve as valuable tools for the detection of a variety of biochemical phenomena. Such reagents have been employed for protein localization, quantitation of gene expression, detection of nucleic acids, cell sorting, and determination of chemical concentrations. Although fluorescence is a useful tool for detecting molecules within cells, its application in vivo has heretofore been limited. The ideal vital fluorescent tag should (1) be detectable without causing cytological damage, (2) be able to label a wide variety of cell types readily, and (3) be able to be targeted to virtually any subcellular region. The recently cloned green fluorescent protein (GFP) from the jellyfish Aequorea victoria is such a molecule. This overview describes the use of this proteinaceous fluorophore for in vivo observation of cellular phenomena, including applications and problems with the use of GFP, a discussion of mutant GFPs with altered fluorescence characteristics, and also some details on microscopy requirements.
Implementation of multicenter and/or longitudinal studies requires an effective quality assurance program to identify trends, data inconsistencies and process variability of results over time. The Diabetes Control and Complications Trial (DCCT) and the follow-up Epidemiology of Diabetes Interventions and Complications (EDIC) study represent over 30 years of data collection among a cohort of participants across 27 clinical centers. The quality assurance plan is overseen by the Data Coordinating Center and is implemented across the clinical centers and central reading units. Each central unit incorporates specific DCCT/EDIC quality monitoring activities into their routine quality assurance plan. The results are reviewed by a data quality assurance committee whose function is to identify variances in quality that may impact study results from the central units as well as within and across clinical centers, and to recommend implementation of corrective procedures when necessary. Over the 30-year period, changes to the methods, equipment, or clinical procedures have been required to keep procedures current and ensure continued collection of scientifically valid and clinically relevant results. Pilot testing to compare historic processes with contemporary alternatives is performed and comparability is validated prior to incorporation of new procedures into the study. Details of the quality assurance plan across and within the clinical and central reading units are described, and quality outcomes for core measures analyzed by the central reading units (e.g. biochemical samples, fundus photographs, ECGs) are presented.
Fluorescent molecules serve as valuable tools for the detection of numerous biochemical phenomena and have been employed for protein localization, quantitation of gene expression, detection of nucleic acids, cell sorting and determination of chemical concentrations. However, the use of such techniques generally requires significant nonphysiological perturbations to the biological system being studied; therefore, they are not always appropriate for the observation of dynamic phenomena. Green fluorescent protein (GFP), cloned from jellyfish, has been used to overcome many of these problems. It is a small, extremely stable fluorescent protein that has been successfully expressed and detected in a wide variety of organisms, both in intact form and fused to other proteins. This overview unit describes the use of this proteinaceous fluorophore for in vivo observation of cellular phenomena.
Fluorescent molecules serve as valuable tools for the detection of a variety of biochemical phenomena. Such reagents have been employed for protein localization, quantitation of gene expression, detection of nucleic acids, cell sorting, and determination of chemical concentrations. Although fluorescence is a useful tool for detecting molecules within cells, its application in vivo has been limited. The ideal vital fluorescent tag should (1) be detectable without causing cytological damage, (2) be able to label a wide variety of cell types readily, and (3) be able to be targeted to virtually any subcellular region. The recently cloned green fluorescent protein (GFP) from the jellyfish Aequorea victoria is such a molecule. This overview describes the use of this proteinaceous fluorophore for in vivo observation of cellular phenomena, including applications and problems with the use of GFP, a discussion of mutant GFPs with altered fluorescence characteristics, and also some details on microscopy requirements.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.