Isolation of a point-mutated p47 lacking binding affinity to p97ATPase

Abstractp47, a p97-binding protein, functions in Golgi membrane fusion together with p97 and VCIP135, another p97-binding protein. We have succeeded in creating p47 with a point mutation, F253S, which lacks p97-binding affinity. p47 mapping experiments revealed that p47 had two p97-binding regions and the F253S mutation occurred in the first p97-binding site. p47(F253S) could not form a complex with p97 and did not caused any cisternal regrowth in an in vitro Golgi reassembly assay. In addition, mutation corresponding to the p47 F253S mutation in p37 and ufd1 also abolished their binding ability to p97.Structured summaryMINT-7987189, MINT-7987207, MINT-7987303: p47 (uniprotkb:O35987) binds (MI:0407) to p97 (uniprotkb:Q01853) by pull down (MI:0096)MINT-7987226: p97 (uniprotkb:P46462) binds (MI:0407) to p47 (uniprotkb:O35987) by pull down (MI:0096)MINT-7987348: p97 (uniprotkb:P46462) physically interacts (MI:0915) with Ufd1 (uniprotkb:P70362) by pull down (MI:0096)MINT-7987264: p97 (uniprotkb:P46462) and p47 (uniprotkb:O35987) bind (MI:0407) by competition binding (MI:0405)MINT-7987326: p97 (uniprotkb:P46462) binds (MI:0407) to p37 (uniprotkb:Q0KL01) by pull down (MI:0096

Stepwise Assembly of Fibrinogen Is Assisted by the Endoplasmic Reticulum Lectin-Chaperone System in HepG2 Cells

The endoplasmic reticulum (ER) plays essential roles in protein folding and assembly of secretory proteins. ER-resident molecular chaperones and related enzymes assist in protein maturation by co-operated interactions and modifications. However, the folding/assembly of multimeric proteins is not well understood. Here, we show that the maturation of fibrinogen, a hexameric secretory protein (two trimers from a, b and c subunits), occurs in a stepwise manner. The ac complex, a precursor for the trimer, is retained in the ER by lectin-like chaperones, and the b subunit is incorporated into the ac complex immediately after translation. ERp57, a protein disulfide isomerase homologue, is involved in the hexamer formation from two trimers. Our results indicate that the fibrinogen hexamer is formed sequentially, rather than simultaneously, using kinetic pause by lectin chaperones. This study provides a novel insight into the assembly of most abundant multi-subunit secretory proteins

Differential expression of nuclear lamin subtypes in the neural cells of the adult rat cerebral cortex

Lamins are type V intermediate filament proteins that are located beneath the inner nuclear membrane. In mammalian somatic cells, LMNB1 and LMNB2 encode somatic lamins B1 and B2, respectively, and the LMNA gene is alternatively spliced to generate somatic lamins A and C. Mutations in lamin genes have been linked to many human hereditary diseases, including neurodegenerative disorders. Knowledge about lamins in the nervous system has been accumulated recently, but a precise analysis of lamin subtypes in glial cells has not yet been reported. In this study we investigated the composition of lamin subtypes in neurons, astrocytes, oligodendrocyte-lineage cells, and microglia in the adult rat cerebral cortex using an immunohistochemical staining method. Lamin A was not observed in neurons and glial cells. Lamin C was observed in astrocytes, mature oligodendrocytes and neurons, but not observed in oligodendrocyte progenitor cells. Microglia also did not stain positive for lamin C which differed from macrophages, with lamin C positive. Lamin B1 and B2 were observed in all glial cells and neurons. Lamin B1 was intensely positive in oligodendrocyte progenitor cells compared with other glial cells and neurons. Lamin B2 was weakly positive in all glial cells compared to neurons. Our current study might provide useful information to reveal how the onset mechanisms of human neurodegenerative diseases are associated with mutations in genes for nuclear lamin proteins

Immunization of Mice against Infection with Salmonella blegdam : Especially Protection of Mice from Typhoid Infection by Immunization with Living Rough Variants of Salmonella

It is generally known that immunization with killed bacilli has a protective effect on bacterienda. 11 But typhoid fever, paratyphoid fever and anthrax, which are also accompanied with bacierienda, have been considered to be exceptional cases. For anthrax effective immunity could not be introduced with killed vaccine, though Louis Pasteur1) invented a preventive inoculation method hi 1881, until recently low virulent living organism inoculations have been considered to be the only effectivemethod, as far as anthrax is concerned. Recently Cromartie, Watson, Bloom and others2) have proved why immunization with living bacilli is the only preventive measure for anthrax. Based on their findings, it is now possible to provide protection with vaccines containing no living organisms. Thus one of the exceptional cases has been removed, leaving mechanism of immunity of typhoid diseases as the remaining problem. In typhoid and paratyphoid fever, it is statistically known that preventive inoculation with killed bacilli has lowered morbidity and lethality. But the immunity resulting from this vaccination is not as strong and effective as in immunity after recovery Moreover its effectiveness is not completely supported, either experimentally or theoretically. This Subject has been studied at a high level in our country, particular), by Kobayashi and his collabora- tors3). According to their opinion, the protective power against typhoid and paratyphoid. infectioncan be established by vaccination with living bacilli, whether in smooth or in rough type, but not with killed vaccine. However, Hazato and his collaborators4) have reported that intravenous injectionsof killed bacilli have given satisfactory protection to mice against the infection of Salmonella enteritidis I , equal to those of living bacilli. Hosoya and others5) have recently stated that immunization with theirso-called T.T.T. or T.A.T. vaccine, which contains no living bacilli, has protective effect on infectionof Salmonella enteritidis of mice and Salmonella abortus equi of guinea-pigs. Ominaga6) has denied

Regulated motion of glycoproteins revealed by direct visualization of a single cargo in the endoplasmic reticulum

The quality of cargo proteins in the endoplasmic reticulum (ER) is affected by their motion during folding. To understand how the diffusion of secretory cargo proteins is regulated in the ER, we directly analyze the motion of a single cargo molecule using fluorescence imaging/fluctuation analyses. We find that the addition of two N-glycans onto the cargo dramatically alters their diffusion by transient binding to membrane components that are confined by hyperosmolarity. Via simultaneous observation of a single cargo and ER exit sites (ERESs), we could exclude ERESs as the binding sites. Remarkably, actin cytoskeleton was required for the transient binding. These results provide a molecular basis for hypertonicity-induced immobilization of cargo, which is dependent on glycosylation at multiple sites but not the completion of proper folding. We propose that diffusion of secretory glycoproteins in the ER lumen is controlled from the cytoplasm to reduce the chances of aggregation

Comparative Gene Analysis Focused on Silica Cell Wall Formation: Identification of Diatom-Specific SET Domain Protein Methyltransferases

Silica cell walls of diatoms have attracted attention as a source of nanostructured functional materials and have immense potential for a variety of applications. Previous studies of silica cell wall formation have identified numerous involved proteins, but most of these proteins are species-specific and are not conserved among diatoms. However, because the basic process of diatom cell wall formation is common to all diatom species, ubiquitous proteins and molecules will reveal the mechanisms of cell wall formation. In this study, we assembled de novo transcriptomes of three diatom species, Nitzschia palea, Achnanthes kuwaitensis, and Pseudoleyanella lunata, and compared protein-coding genes of five genome-sequenced diatom species. These analyses revealed a number of diatom-specific genes that encode putative endoplasmic reticulum-targeting proteins. Significant numbers of these proteins showed homology to silicanin-1, which is a conserved diatom protein that reportedly contributes to cell wall formation. These proteins also included a previously unrecognized SET domain protein methyltransferase family that may regulate functions of cell wall formation-related proteins and long-chain polyamines. Proteomic analysis of cell wall-associated proteins in N. palea identified a protein that is also encoded by one of the diatom-specific genes. Expression analysis showed that candidate genes were upregulated in response to silicon, suggesting that these genes play roles in silica cell wall formation. These candidate genes can facilitate further investigations of silica cell wall formation in diatoms

Detection of epidermal growth factor receptor mutations in exhaled breath condensate using droplet digital polymerase chain reaction

The detection of certain oncogenic driver mutations, including those of epidermal growth factor receptor (EGFR), is essential for determining treatment strategies for advanced non‑small cell lung cancer (NSCLC). The current study assessed the feasibility of testing exhaled breath condensate (EBC) for EGFR mutations by droplet digital PCR (ddPCR). Samples were collected from 12 patients with NSCLC harboring EGFR mutations that were admitted to Okayama University Hospital between June 1, 2014 and December 31, 2017. A total of 21 EBC samples were collected using the RTube™ method and EGFR mutations (L858R, exon 19 deletions or T790M) were assessed through ddPCR analysis (EBC‑ddPCR). A total of 3 healthy volunteer samples were also tested to determine a threshold value for each mutation. Various patient characteristics were determined, including sex (3 males and 9 females), age (range 54‑81 years; median, 66 years), smoking history (10 had never smoked; 2 were former smokers), histology (12 patients exhibited adenocarcinoma), clinical stage (9 patients were stage IV; 3 exhibited post‑operative recurrence) and EGFR mutation type (4 had L858R; 8 had exon 19 deletions; 8 had T790M). EBC‑ddPCR demonstrated positive droplets in 8 of the 12 patients. The sensitivity and specificity of each mutation was as follows: 27.3 and 80.0% for EGFR L858R, 30.0 and 90.9% for EGFR Ex19del, and 22.2 and 100% for EGFR T790M. EBC‑ddPCR analysis of EGFR mutations exhibited modest sensitivity and acceptable specificity. EBC‑ddPCR is a minimally invasive and replicable procedure and may be a complementary method for EGFR testing in patients where blood or tissue sampling proves difficult