scholarly journals A 57,000-mol-wt protein uniquely present in nonproliferating cells and senescent human fibroblasts.

1985 ◽  
Vol 100 (2) ◽  
pp. 545-551 ◽  
Author(s):  
E Wang

Mouse monoclonal antibody, S-30, was produced from hybridoma preparation from mice injected with the cytoskeleton extract of an in vitro aged culture of human fibroblasts derived from a 66-yr-old donor. The antibody stains positively the nuclei of the nonproliferating cells present predominantly in the senescent cultures of five selected fibroblast strains derived from donors of different age groups, whereas a negative reaction is observed in the cultures of their young counterparts. In the intermediate stage of the in vitro life span of these cell strains, a heterogeneous positive reaction for staining with S-30 antibody is observed in different subfractions of cell cultures. However, the expression of S-30 can be induced in the young fibroblasts at the early stage of their life by prolonged culturing to confluence. This induced expression of S-30 nuclear staining can be depleted upon subculturing at low cell density. Immunoelectron microscopy with colloidal gold-protein A complex demonstrates that the S-30 proteins are present in the nuclear plasma and at the region of nuclear envelope in a clustered arrangement. Immunoprecipitation of [3H]leucine labeled cell specimens shows that the antibody S-30 reacts with a protein with a molecular weight of approximately 57,000.

1998 ◽  
Vol 66 (5) ◽  
pp. 2143-2153 ◽  
Author(s):  
Mark S. Hanson ◽  
David R. Cassatt ◽  
Betty P. Guo ◽  
Nita K. Patel ◽  
Michael P. McCarthy ◽  
...  

ABSTRACT Borrelia burgdorferi, the spirochete that causes Lyme disease, binds decorin, a collagen-associated extracellular matrix proteoglycan found in the skin (the site of entry for the spirochete) and in many other tissues. Two borrelial adhesins that recognize this proteoglycan, decorin binding proteins A and B (DbpA and DbpB, respectively), have recently been identified. Infection of mice by low-dose B. burgdorferi challenge elicited antibodies against DbpA and DbpB that were sustained at high levels, suggesting that these antigens are expressed in vivo. Scanning immunoelectron microscopy showed that DbpA was surface accessible on intact borreliae. Passive administration of DbpA antiserum protected mice from infection following challenge with heterologous B. burgdorferi sensu stricto isolates, even when serum administration was delayed for up to 4 days after challenge. DbpA is the first antigen target identified that is capable of mediating immune resolution of early, localizedB. burgdorferi infections. DbpA immunization also protected mice from B. burgdorferi challenge; DbpB immunization was much less effective. DbpA antiserum inhibited in vitro growth of manyB. burgdorferi sensu lato isolates of diverse geographic, phylogenetic, and clinical origins. In combination, these findings support a role for DbpA in the immunoprophylaxis of Lyme disease and suggest that DbpA vaccines have the potential to eliminate early-stageB. burgdorferi infections.


2003 ◽  
Vol 177 (2) ◽  
pp. 249-259 ◽  
Author(s):  
JJ Gagliardino ◽  
H Del Zotto ◽  
L Massa ◽  
LE Flores ◽  
MI Borelli

The aim of this work was to study the possible relationship between pancreatic duodenal homeobox-1 (Pdx-1) and islet neogenesis-associated protein (INGAP) during induced islet neogenesis. Pregnant hamsters were fed with (S) and without (C) sucrose, and glycemia, insulin secretion in vitro, and pancreas immunomorphometric parameters were measured in their 7-day-old offspring. S offspring had significantly lower glycemic levels than C animals. Insulin release in response to increasing glucose concentrations in the incubation medium (2-16 mM glucose) did not increase in pancreata from either C or S offspring. However, pancreata from S offspring released more insulin than those from C animals. In S offspring, beta-cell mass, beta-cell replication rate and islet neogenesis increased significantly, with a simultaneous decrease in beta-cell apoptotic rate. INGAP- and Pdx-1-positive cell mass also increased in the islets and among acinar and duct cells. We found two subpopulations of Pdx-1 cells: INGAP-positive and INGAP-negative. Pdx-1/INGAP-positive cells did not stain with insulin, glucagon, somatostatin, pancreatic polypeptide, or neurogenin 3 antibodies. The increment of Pdx-1/INGAP-positive cells represented the major contribution to the Pdx-1 cell mass increase. Such increments varied among pancreas subsectors: ductal>insular>extrainsular. Our results suggested that INGAP participates in the regulation of islet neogenesis, and Pdx-1/INGAP-positive cells represent a new stem cell subpopulation at an early stage of development, highly activateable in neogenesis.


2000 ◽  
Vol 20 (15) ◽  
pp. 5690-5699 ◽  
Author(s):  
C. J. Jones ◽  
D. Kipling ◽  
M. Morris ◽  
P. Hepburn ◽  
J. Skinner ◽  
...  

ABSTRACT An initiating role for RAS oncogene mutation in several epithelial cancers is supported by its high incidence in early-stage tumors and its ability to induce proliferation in the corresponding normal cells in vitro. Using retroviral transduction of thyroid epithelial cells as a model we ask here: (i) how mutant RAS can induce long-term proliferation in an epithelial cell in contrast to the premature senescence observed in fibroblasts; and (ii) what is the “clock” which eventually triggers spontaneous growth arrest even in epithelial clones generated by mutant RAS. The early response toRAS activation in thyroid epithelial cells showed two features not seen in fibroblasts: (i) a marked decrease in expression of the cyclin-dependent kinase inhibitor (CDKI) p27kip1 and (ii) the absence of any induction of p21waf1. When proliferation eventually ceased (after up to 20 population doublings) this occurred despite undiminished expression of mutant RAS and was tightly correlated with a return to the initial high level of p27kip1 expression, together with the de novo appearance of p16ink4a. Importantly, neither the CDKI changes nor the proliferative life span of RAS-induced epithelial clones was altered by induction of telomerase activity through forced expression of the catalytic subunit, hTERT, at levels sufficient to immortalize human fibroblasts. These data provide a basis for cell-type differences in sensitivity to RAS-induced proliferation which may explain the corresponding tumor-type specificity of RAS mutation. They also show for the first time in a primary human cell model that a telomere-independent mechanism can limit not only physiological but also oncogene-driven proliferation, pointing therefore to a tumour suppressor mechanism additional, or alternative, to the telomere clock.


Marine Drugs ◽  
2021 ◽  
Vol 19 (10) ◽  
pp. 541
Author(s):  
Rute Castelo Félix ◽  
Liliana Anjos ◽  
Rita Alves Costa ◽  
Sophia Letsiou ◽  
Deborah Mary Power

Fish skin has been gaining attention due to its efficacy as a human-wound-treatment product and to identify factors promoting its enhanced action. Skin fibroblasts have a central role in maintaining skin integrity and secrete extra cellular matrix (ECM) proteins, growth factors and cytokines to rapidly repair lesions and prevent further damage or infection. The effects on scratch repair of the ubiquitous but poorly characterized ECM protein, cartilage acidic protein 1 (CRTAC1), from piscine and human sources were compared using a zebrafish SJD.1 primary fibroblast cell line. A classic in vitro cell scratch assay, immunofluorescence, biosensor and gene expression analysis were used. Our results demonstrated that the duplicate sea bass Crtac1a and Crtac1b proteins and human CRTAC-1A all promoted SJD.1 primary fibroblast migration in a classic scratch assay and in an electric cell impedance sensing assay. The immunofluorescence analysis revealed that CRTAC1 enhanced cell migration was most likely caused by actin-driven cytoskeletal changes and the cellular transcriptional response was most affected in the early stage (6 h) of scratch repair. In summary, our results suggest that CRTAC1 may be an important factor in fish skin promoting damage repair.


1992 ◽  
Vol 176 (5) ◽  
pp. 1319-1326 ◽  
Author(s):  
M Dancescu ◽  
M Rubio-Trujillo ◽  
G Biron ◽  
D Bron ◽  
G Delespesse ◽  
...  

B chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of slow-dividing and long-lived monoclonal B cells arrested at the intermediate stage of their differentiation. We previously showed that interleukin 4 (IL-4) not only inhibits but also prevents the proliferation of B-CLL cells. We report here that IL-4 protects the B-CLL cells from death by apoptosis (programmed cell death [PCD]). IL-4 inhibits spontaneous and hydrocortisone (HC)-induced PCD of highly purified B cells from 12 unselected CLL patients, as shown by sustained cell viability and lack of DNA fragmentation. IL-1, -2, -3, -5, -6, -7, tumor necrosis factor alpha, and transforming growth factor beta have no protective effect. The in vitro rescue from apoptosis by IL-4 is reflected by an increased expression of Bcl-2 protein, a proto-oncogene directly involved in the prolongation of cell survival in vivo and in vitro. Hence, IL-4-treated B-CLL cells express significantly more Bcl-2 than unstimulated, HC-treated, or fresh B-CLL cells. Furthermore, subcutaneous injection of IL-4 into one CLL patient enhances Bcl-2 protein expression in the leukemic B cells. These data may suggest that IL-4 prevents apoptosis of B-CLL cells using a Bcl-2-dependent pathway. Given our recent observations that fresh T cells from B-CLL patients express IL-4 mRNA, we propose that IL-4 has an essential role in the pathogenesis of CLL disease, by preventing both the death and the proliferation of the malignant B cells.


Molecules ◽  
2020 ◽  
Vol 25 (8) ◽  
pp. 1895
Author(s):  
Alžběta Dostálková ◽  
Kryštof Škach ◽  
Filip Kaufman ◽  
Ivana Křížová ◽  
Romana Hadravová ◽  
...  

A major structural retroviral protein, capsid protein (CA), is able to oligomerize into two different hexameric lattices, which makes this protein a key component for both the early and late stages of HIV-1 replication. During the late stage, the CA protein, as part of the Gag polyprotein precursor, facilitates protein–protein interactions that lead to the assembly of immature particles. Following protease activation and Gag polyprotein processing, CA also drives the assembly of the mature viral core. In the early stage of infection, the role of the CA protein is distinct. It controls the disassembly of the mature CA hexameric lattice i.e., uncoating, which is critical for the reverse transcription of the single-stranded RNA genome into double stranded DNA. These properties make CA a very attractive target for small molecule functioning as inhibitors of HIV-1 particle assembly and/or disassembly. Of these, inhibitors containing the PF74 scaffold have been extensively studied. In this study, we reported a series of modifications of the PF74 molecule and its characterization through a combination of biochemical and structural approaches. Our data supported the hypothesis that PF74 stabilizes the mature HIV-1 CA hexameric lattice. We identified derivatives with a higher in vitro stabilization activity in comparison to the original PF74 molecule.


2017 ◽  
Vol 2017 ◽  
pp. 1-4
Author(s):  
Ying Ying ◽  
Xi Guo ◽  
Yiping Zhong ◽  
Canquan Zhou

Background. Previously, we found women with positive anticentromere antibody showed impaired potential of oocyte maturation and embryo cleavage; the possible mechanism behind this phenomenon was still unknown. Objective. Thus, the present study aimed to preliminarily explore whether ACA could penetrate into the living embryos and impair their developmental potential via in vitro coculture with mouse embryos. Methods. Mouse embryos were collected and used for in vitro culture with polyclonal anticentromere protein A (CENP-A) antibody; then, immunofluorescence assay was performed to determine the penetration of antibody into embryos, and embryo development potential was observed. Results. All embryos cultured with anti-CENP-A antibody exhibited immunofluorescence on the nucleus, while none of the embryos from the control groups showed immunofluorescence. Additionally, embryos cultured with anti-CENP-A antibody experienced significant growth impairment compared with controls. Conclusion. Mouse embryos may be a direct target for ACA in vitro prior to implantation. However, the precise mechanism needs further clarification.


2018 ◽  
Vol 63 (1) ◽  
Author(s):  
Yiping Wang ◽  
Rupkatha Mukhopadhyay ◽  
Sujayita Roy ◽  
Arun Kapoor ◽  
Yu-Pin Su ◽  
...  

ABSTRACTArtesunate (AS), a semisynthetic artemisinin approved for malaria therapy, inhibits human cytomegalovirus (HCMV) replicationin vitro, but therapeutic success in humans has been variable. We hypothesized that the shortin vivohalf-life of AS may contribute to the different treatment outcomes. We tested novel synthetic ozonides with longer half-lives against HCMVin vitroand mouse cytomegalovirus (MCMV)in vivo. Screening of the activities of four ozonides against a pp28-luciferase-expressing HCMV Towne recombinant identified OZ418 to have the best selectivity; its effective concentration inhibiting viral growth by 50% (EC50) was 9.8 ± 0.2 µM, and cytotoxicity in noninfected human fibroblasts (the concentration inhibiting cell growth by 50% [CC50]) was 128.1 ± 8.0 µM. In plaque reduction assays, OZ418 inhibited HCMV TB40 in a concentration-dependent manner as well as a ganciclovir (GCV)-resistant HCMV isolate. The combination of OZ418 and GCV was synergistic in HCMV inhibitionin vitro. Virus inhibition by OZ418 occurred at an early stage and was dependent on the cell density at the time of infection. OZ418 treatment reversed HCMV-mediated cell cycle progression and correlated with the reduction of HCMV-induced expression of pRb, E2F1, and cyclin-dependent kinases 1, 2, 4, and 6. In an MCMV model, once-daily oral administration of OZ418 had significantly improved efficacy against MCMV compared to that of twice-daily oral AS. A parallel pharmacokinetic study with a single oral dose of OZ418 or AS showed a prolonged plasma half-life and higher unbound concentrations of OZ418 than unbound concentrations of AS. In summary, ozonides are proposed to be potential therapeutics, alone or in combination with GCV, for HCMV infection in humans.


2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 8008-8008
Author(s):  
H. M. Kluger ◽  
M. McCarthy ◽  
A. Alvero ◽  
K. Divito ◽  
R. Camp ◽  
...  

8008 Background: Chemoresistance is a major problem in treating melanoma. The X-linked inhibitor of apoptosis (XIAP) is associated with chemoresistance in other cancers. Here we characterize XIAP expression in primary and metastatic melanomas and determine whether XIAP plays a role in chemoresistance. We report differential expression of XIAP between primary and metastatic melanomas and benign nevi. We demonstrate that inhibition of XIAP expression by Phenoxodiol can reverse chemoresistance. Methods: We employed tissue microarrays containing specimens from 548 melanoma patients and 540 benign nevi. XIAP expression was evaluated by an automated method for in situ quantitative analysis of protein levels (AQUA). We use S100 to define pixels as melanoma (tumor mask) within the array spot, and measure intensity of XIAP expression using Cy5 conjugated antibodies within the mask. AQUA scores were correlated with clinical and pathological variables. Cell viability after exposure to Carboplatin and Phenoxodiol was determined in 3 melanoma cell strains using the CellTiter 96 Assay. Apoptosis was measured using the Caspase-3/7 GloTM Assay, and levels of XIAP and Caspase 2 were assessed by Western blots. Results: XIAP expression was significantly higher in melanomas than in nevi (P<0.0001), and higher in metastatic than in primary specimens (P<0.0001). All three melanoma cell strains examined were highly resistant to Carboplatin, and moderately resistant to Phenoxodiol. Pre-treatment with Phenoxodiol prior to Carboplatin induced a significant decrease in XIAP expression, which correlated with remarkable sensitization to Carboplatin. Conclusions: XIAP expression is higher in malignant melanocytes than in their benign counterparts. Expression is higher in early stage disease specimens than in metastatic specimens, suggesting an association with disease aggression. Phenoxodiol is a well tolerated drug, and has been shown to inhibit XIAP expression in ovarian cancer. Here we demonstrate that melanoma is resistant to Carboplatin, possibly due to XIAP expression. In vitro inhibition of XIAP by Phenoxodiol sensitizes melanoma cells to Carboplatin. This drug combination warrants further investigation as a therapeutic approach for metastatic melanoma. [Table: see text]


2009 ◽  
Vol 83 (7) ◽  
pp. 2862-2871 ◽  
Author(s):  
Alexandre Harari ◽  
Felicitas Bellutti Enders ◽  
Cristina Cellerai ◽  
Pierre-Alexandre Bart ◽  
Giuseppe Pantaleo

ABSTRACT Cytotoxic CD8 T cells exert their antiviral and antitumor activity primarily through the secretion of cytotoxic granules. Degranulation activity and cytotoxic granules (perforin plus granzymes) generally define CD8 T cells with cytotoxic function. In this study, we have investigated the expression of granzyme K (GrmK) in comparison to that of GrmA, GrmB, and perforin. The expression of the cytotoxic granules was assessed in virus-specific CD8 T cells specific to influenza virus, Epstein-Barr virus (EBV), cytomegalovirus (CMV), or human immunodeficiency virus type 1 (HIV-1). We observed a dichotomy between GrmK and perforin expression in virus-specific CD8 T cells. The profile in influenza virus-specific CD8 T cells was perforin− GrmB− GrmA+/− GrmK+; in CMV-specific cells, it was perforin+ GrmB+ GrmA+ GrmK−/+; and in EBV- and HIV-1-specific cells, it was perforin−/+ GrmB+ GrmA+ GrmK+. On the basis of the delineation of memory and effector CD8 T cells with CD45RA and CD127, the GrmK+ profile was associated with early-stage memory CD8 T-cell differentiation, the perforin+ GrmB+ GrmA+ profile with advanced-stage differentiation, and the GrmB+ GrmA+ Grmk+ profile with intermediate-stage differentiation. Furthermore, perforin and GrmB but not GrmA and GrmK correlated with cytotoxic activity. Finally, changes in antigen exposure in vitro and in vivo during primary HIV-1 infection and vaccination modulated cytotoxic granule profiles. These results advance our understanding of the relationship between distinct profiles of cytotoxic granules in memory CD8 T cells and function, differentiation stage, and antigen exposure.


Sign in / Sign up

Export Citation Format

Share Document