Coexpression and Accumulation of Ubiquitin +1 and ZZ Proteins in Livers of Children with α1-Antitrypsin Deficiency

2002 ◽  
Vol 5 (3) ◽  
pp. 293-298 ◽  
Author(s):  
Sandy S. Wu ◽  
Jean-Pierre de Chadarevian ◽  
Laron McPhaul ◽  
Nora E. Riley ◽  
Fred W. van Leeuwen ◽  
...  
Keyword(s):  
Neurological Disorders ◽  
Polyclonal Antibodies ◽  
Genomic Information ◽  
Double Immunofluorescence ◽  
Mutant Proteins ◽  
Explanted Liver ◽  
Immunofluorescence Studies ◽  
Antitrypsin Deficiency ◽  
Pathologic Processes ◽  
Sectioned Tissue

The ZZ variant of α1-antitrypsin deficiency (AATD) is well known to cause liver damage and cirrhosis in some affected children. Ubiquitin abnormality was recently shown to be significant in AATD in childhood cirrhosis. Molecular misreading (MM), defined as faulty transcription of genomic information from DNA into mRNA, as well as its translation into mutant proteins, has been documented in many pathologic processes where aggregation of abnormal proteins occurs. The misread protein, ubiquitin-B+1 (UBB+1), was recently identified in the hallmarks of various neurological disorders. The objective of this study was to determine whether MM of ubiquitin occurs in AATD. Twelve explanted liver specimens from AATD-affected children with cirrhosis were retrieved from archival sources, along with 10 control liver specimens obtained from autopsies of age-matched children with no clinical, gross anatomic, or histologic evidence of liver disease. Double immunofluorescence studies using rabbit polyclonal antibodies against UBB+1 and AAT were performed on consecutively sectioned tissue. UBB+1 immunoreactivity was colocalized with AAT in all cirrhotic AATD livers. The control livers were consistently negative. Ubiquitin MM is prominent in AATD-affected cirrhotic livers. This indicates that for children with AATD and cirrhosis, ubiquitin MM is a necessary cofactor to the aggregation of mutant ZZ isoform of AATD.

scholarly journals The centrin-based cytoskeleton of Chlamydomonas reinhardtii: distribution in interphase and mitotic cells.

1988 ◽  
Vol 107 (2) ◽  
pp. 635-641 ◽  
Author(s):  
J L Salisbury ◽  
A T Baron ◽  
M A Sanders
Keyword(s):  
Nuclear Envelope ◽  
Polyclonal Antibodies ◽  
Flagellar Apparatus ◽  
Immunogold Labeling ◽  
Basal Bodies ◽  
Flagellar Roots ◽  
Cell Biol ◽  
Immunofluorescence Studies ◽  
Descending Fibers

Monoclonal and polyclonal antibodies raised against algal centrin, a protein of algal striated flagellar roots, were used to characterize the occurrence and distribution of this protein in interphase and mitotic Chlamydomonas cells. Chlamydomonas centrin, as identified by Western immunoblot procedures, is a low molecular (20,000-Mr) acidic protein. Immunofluorescence and immunogold labeling demonstrates that centrin is a component of the distal fiber. In addition, centrin-based flagellar roots link the flagellar apparatus to the nucleus. Two major descending fibers extend from the basal bodies toward the nucleus; each descending fiber branches several times giving rise to 8-16 fimbria which surround and embrace the nucleus. Immunogold labeling indicates that these fimbria are juxtaposed to the outer nuclear envelope. Earlier studies have demonstrated that the centrin-based linkage between the flagellar apparatus and the nucleus is contractile, both in vitro and in living Chlamydomonas cells (Wright, R. L., J. Salisbury, and J. Jarvik. 1985. J. Cell Biol. 101:1903-1912; Salisbury, J. L., M. A. Sanders, and L. Harpst. 1987. J. Cell Biol. 105:1799-1805). Immunofluorescence studies show dramatic changes in distribution of the centrin-based system during mitosis that include a transient contraction at preprophase; division, separation, and re-extension during prophase; and a second transient contraction at the metaphase/anaphase boundary. These observations suggest a fundamental role for centrin in motile events during mitosis.

scholarly journals Tenascin-R promotes assembly of the extracellular matrix of perineuronal nets via clustering of aggrecan

2014 ◽  
Vol 369 (1654) ◽  
pp. 20140046 ◽  
Author(s):  
Markus Morawski ◽  
Alexander Dityatev ◽  
Maike Hartlage-Rübsamen ◽  
Maren Blosa ◽  
Max Holzer ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Neurological Disorders ◽  
Polyclonal Antibodies ◽  
Brain Slices ◽  
Perineuronal Nets ◽  
Wild Type ◽  
Extracellular Matrix Molecules ◽  
Histological Appearance ◽  
Organotypic Slice Cultures ◽  
Wisteria Floribunda

Perineuronal nets (PNs) in the brains of tenascin-R-deficient ( tn-r −/− ) mice develop in temporal concordance with those of wild-type ( tn-r +/+ ) mice. However, the histological appearance of PNs is abnormal in adult tn-r −/− mice. Here, we investigated whether similar defects are also seen in dissociated and organotypic cultures from hippocampus and forebrain of tn-r −/− mice and whether the structure of PNs could be normalized. In tn-r −/− cultures, accumulations of several extracellular matrix molecules were mostly associated with somata, whereas dendrites were sparsely covered, compared with tn-r +/+ mice. Experiments to normalize the structure of PNs in tn-r −/− organotypic slice cultures by depolarization of neurons, or by co-culturing tn-r +/+ and tn-r −/− brain slices failed to restore a normal PN phenotype. However, formation of dendritic PNs in cultures was improved by the application of tenascin-R protein and rescued by polyclonal antibodies to aggrecan and a bivalent, but not monovalent form of the lectin Wisteria floribunda agglutinin. These results show that tenascin-R and aggrecan are decisive contributors to formation and stabilization of PNs and that tenascin-R may implement these functions by clustering of aggrecan. Proposed approaches for restoration of normal PN structure are noteworthy in the context of PN abnormalities in neurological disorders, such as epilepsy, schizophrenia and addiction.

The pegs on the decorated tubules of the contractile vacuole complex of Paramecium are proton pumps

1995 ◽  
Vol 108 (10) ◽  
pp. 3163-3170 ◽  
Author(s):  
A.K. Fok ◽  
M.S. Aihara ◽  
M. Ishida ◽  
K.V. Nolta ◽  
T.L. Steck ◽  
...  
Keyword(s):  
Potent Inhibitor ◽  
Polyclonal Antibodies ◽  
Immunogold Labeling ◽  
Contractile Vacuole ◽  
Proton Pumps ◽  
Minimal Effect ◽  
Chromaffin Granules ◽  
B Subunit ◽  
Antigenic Sites ◽  
Immunofluorescence Studies

Our previous study has shown that the decorated tubules (collectively known as the decorated spongiome) of the contractile vacuole complex (CVC) in Paramecium are the site of fluid segregation, as the binding of microinjected monoclonal antibody (mAb) DS-1 to the tubules reduced the CVC's fluid output. In this study, we showed by immunogold labeling on cryosections that the antigenic sites for mAb DS-1 were located on the 15 nm ‘pegs’ protruding from the cytosolic surface of the decorated tubules. In immunofluorescence studies, both polyclonal antibodies against the subunits of the V-ATPase of Dictyostelium discoideum and against the 57 kDa B-subunit of the V-ATPase of chromaffin granules gave identical labeling patterns to that produced by mAb DS-1. On cryosections, all three antigens were located most consistently near or on the pegs of the decorated tubules. These data support the notion that the pegs on the membrane of the decorated tubules represent the V1 complex of a proton pump. Concanamycin B, a potent inhibitor of V-ATPase activity and of acidification of lysosomes and endosomes, strongly and reversibly inhibited fluid output from the CVC but had minimal effect on the integrity of the decorated spongiome as observed by immunofluorescence. Such inhibition suggests that a V-ATPase is intimately involved in fluid segregation. Exposing Paramecium to 12 degrees C or 1 degrees C for 30 minutes resulted in the dissociation of the decorated tubules from the smooth spongiome that borders the collecting canals; thus the DS-1-reactive A4 antigen, the 75 kDa and 66 kDa antigens were all found dispersed in the cytosol.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Precision Autism: Genomic Stratification of Disorders Making Up the Broad Spectrum May Demystify its "Epidemic Rates"

2021 ◽  
Author(s):  
Elizabeth B Torres
Keyword(s):  
Neurological Disorders ◽  
Genetic Information ◽  
Self Regulation ◽  
Compact Set ◽  
Human Tissues ◽  
Brain Disorders ◽  
Overlapping Genes ◽  
Genomic Information ◽  
Genes Expression ◽  
Exponential Rates

In the last decade, Autism has broadened and often shifted its diagnostics criteria, allowing several neuropsychiatric and neurological disorders of known etiology. This has resulted in a highly heterogeneous spectrum with apparent exponential rates in prevalence. We ask if it is possible to leverage existing genetic information about those disorders making up Autism today and use it to stratify this spectrum. To that end, we combine genes linked to Autism in the SFARI database and genomic information from the DisGeNet portal on 25 diseases, inclusive of non-neurological ones. We use the GTEx data on genes' expression on 54 human tissues and ask if there are overlapping genes across those associated to these diseases and those from Autism-SFARI. We find a compact set of genes across all brain-disorders which express highly in tissues fundamental for somatic-sensory-motor function, self-regulation, memory, and cognition. Then, we offer a new stratification that provides a distance-based orderly clustering into possible Autism subtypes, amenable to design personalized targeted therapies within the framework of Precision Medicine. We conclude that viewing Autism through this physiological (Precision) lens, rather than from a psychological behavioral construct, may make it a more manageable condition and dispel the Autism epidemic myth.

Double immunofluorescence studies of glomerular sialic acids in patients with diabetic nephropathy

1987 ◽  
Vol 1 (2) ◽  
pp. 61-64 ◽  
Author(s):  
Yasuhiko Tomino ◽  
Mitsunori Yagame ◽  
Wataru Inoue ◽  
Seiichi Watanabe ◽  
Hideaki Kaneshige ◽  
...  
Keyword(s):  
Diabetic Nephropathy ◽  
Sialic Acids ◽  
Double Immunofluorescence ◽  
Immunofluorescence Studies

scholarly journals beta1 Integrin and alpha-dystroglycan binding sites are localized to different laminin-G-domain-like (LG) modules within the laminin alpha5 chain G domain

2003 ◽  
Vol 371 (2) ◽  
pp. 289-299 ◽  
Author(s):  
Hao YU ◽  
Jan F. TALTS
Keyword(s):  
Binding Sites ◽  
Mammalian Cells ◽  
Solid Phase ◽  
Polyclonal Antibodies ◽  
Matrix Proteins ◽  
High Affinity ◽  
Immunofluorescence Studies ◽  
Beta1 Integrin ◽  
Alpha Dystroglycan ◽  
Tandem Arrays

Laminins are a group of extracellular-matrix proteins important in development and disease. They are heterotrimers, and specific domains in the different chains have specialized functions. The G domain of the α5 chain has now been produced in transfected mammalian cells as single modules and two tandem arrays, α5LG1–3 and α5LG4–5 (LG is laminin G domain-like). Using these fragments we produced specific polyclonal antibodies functional in immunoblotting and immunofluorescence studies and in solid-phase assays. Both α5LG tandem arrays had physiologically relevant affinities for sulphated ligands such as heparin and sulphatides. Cells adhered to these fragments and acquired a spread morphology when plated on α5LG1–3. Binding of integrins α3β1 and α6β1 was localized to the α5LG1–3 modules, and α-dystroglycan binding was localized to the α5LG4–5 modules, thus locating these activities to different LG modules within the laminin α5 G domain. However, both these activities were of relatively low affinity, indicating that integrin-mediated cell adhesion to the laminin 10/11 α5G domain depends on contributions from the other chains of the heterotrimer and that high-affinity α-dystroglycan binding could be dependent on specific Ca2+-ion-co-ordinating amino acids absent from α5LG4–5.

Isolation and full-genome sequencing of porcine sapelovirus strain in piglets from Hainan province, China

2020 ◽  
Author(s):  
Hechao Zhu ◽  
Xinxin Li ◽  
Jingyi Xiong ◽  
Xiangmin Li ◽  
Huanchun Chen ◽  
...  
Keyword(s):  
Neurological Disorders ◽  
Genomic Sequence ◽  
Polyclonal Antibodies ◽  
Hainan Province ◽  
Rt Pcr ◽  
Full Genome Sequencing ◽  
The Family ◽  
Swine Population ◽  
Porcine Sapelovirus ◽  
Reference Strains

Abstract Background: Porcine sapelovirus (PSV) is a species of the genus Sapelovirus within the family Picornaviridae, which associated with facute diarrhoea, respiratory distress, reproductive failure, and severe neurological disorders in swine. The first isolate strian of PSV was reported in Hainan province, China in 2019. Results: We report the isolation, genomic sequence of PSV isolated from pig diarrhea samples. The PSV strain was correctly identified by RT-PCR, IFA, WB assays, which appeared spherical with a diameter of approximately 25 nm by TEM. We named the strain PSV HaN01-CH2019, and its full genomes were 7,551 bp nucleotides in length. Phylogenetic analysis revealed that PSV HaN01-CH2019 and Chinese HuN01 strain are related in comparing with other reference strains. Conclusions: We successfully isolated the first PSV strain in Hainan province and prepared polyclonal antibodies. It is evident that PSV infection has occurred in Hainan province, and therefore, active molecular and serological investigation is important to swine population. Moreover, veterinarians must pay attention to this diarrhoea and reinforce biosecurity measures to prevent PSV spread.

scholarly journals Precision Autism: Genomic Stratification of Disorders Making Up the Broad Spectrum May Demystify Its “Epidemic Rates”

2021 ◽  
Vol 11 (11) ◽  
pp. 1119
Author(s):  
Elizabeth B. Torres
Keyword(s):  
Neurological Disorders ◽  
Genetic Information ◽  
Self Regulation ◽  
Compact Set ◽  
Human Tissues ◽  
Brain Disorders ◽  
Overlapping Genes ◽  
Genomic Information ◽  
Genes Expression ◽  
Exponential Rates

In the last decade, Autism has broadened and often shifted its diagnostics criteria, allowing several neuropsychiatric and neurological disorders of known etiology. This has resulted in a highly heterogeneous spectrum with apparent exponential rates in prevalence. I ask if it is possible to leverage existing genetic information about those disorders making up Autism today and use it to stratify this spectrum. To that end, I combine genes linked to Autism in the SFARI database and genomic information from the DisGeNET portal on 25 diseases, inclusive of non-neurological ones. I use the GTEx data on genes’ expression on 54 human tissues and ask if there are overlapping genes across those associated to these diseases and those from SFARI-Autism. I find a compact set of genes across all brain-disorders which express highly in tissues fundamental for somatic-sensory-motor function, self-regulation, memory, and cognition. Then, I offer a new stratification that provides a distance-based orderly clustering into possible Autism subtypes, amenable to design personalized targeted therapies within the framework of Precision Medicine. I conclude that viewing Autism through this physiological (Precision) lens, rather than viewing it exclusively from a psychological behavioral construct, may make it a more manageable condition and dispel the Autism epidemic myth.

scholarly journals Distribution of the Na(+)-Ca2+ exchange protein in mammalian cardiac myocytes: an immunofluorescence and immunocolloidal gold-labeling study

1992 ◽  
Vol 117 (2) ◽  
pp. 337-345 ◽  
Author(s):  
JS Frank ◽  
G Mottino ◽  
D Reid ◽  
RS Molday ◽  
KD Philipson
Keyword(s):  
Monoclonal Antibodies ◽  
Guinea Pig ◽  
Immunoelectron Microscopy ◽  
Polyclonal Antibodies ◽  
Heart Cells ◽  
Spleen Cells ◽  
Cardiac Sarcolemma ◽  
Immunofluorescence Studies ◽  
Tubular System ◽  
Mouse Myeloma

The present study reports on the location of the Na(+)-Ca2+ exchanger in cardiac sarcolemma with immunofluorescence and immunoelectron microscopy. Both polyclonal and monoclonal antibodies to the Na(+)-Ca2+ exchanger were used. The mAb was produced from a hybridoma cell line generated by the fusion of mouse myeloma NS-1 cells with spleen cells from a mouse repeatedly immunized with isolated reconstituted canine cardiac Na(+)-Ca2+ exchanger (Philipson, K. D. S. Longoni, and R. Ward. 1988. Biochim. Biophys. Acta. 945:298-306). The polyclonal antibody has been described previously and reacts with three proteins (70, 120, 160 kD) in cardiac sarcolemma associated with the Na(+)-Ca2+ exchanger (Nicoll, D. A., S. Longoni, and K. D. Philipson. 1990. Science (Wash. DC). 250:562-565). Both the monoclonal and the polyclonal antibodies appear to react with extracellular facing epitopes in the cardiac sarcolemma. Immunofluorescence studies showed labeling of the transverse tubular membrane and patchy labeling of the peripheral sarcolemma. The immunofluorescent labeling clearly delineates the highly interconnected T-tubular system of guinea pig myocytes. This localization of the exchanger to the sarcolemma, with an apparent high density in the transverse tubules, was also seen with immunoelectron microscopy. It is of great interest that the Na(+)-Ca2+ exchanger, as the main efflux route for Ca2+ in heart cells, would be abundantly located in sarcolemma closest to the release of Ca2+.

Mutagenesis of theN-glycosylation site of hNaSi-1 reduces transport activity

2003 ◽  
Vol 285 (5) ◽  
pp. C1188-C1196 ◽  
Author(s):  
Hongyan Li ◽  
Ana M. Pajor
Keyword(s):  
Xenopus Oocytes ◽  
Polyclonal Antibodies ◽  
Membrane Vesicles ◽  
Glycosylation Site ◽  
Transport Activity ◽  
Wild Type ◽  
Glutathione S Transferase ◽  
Mutant Proteins ◽  
Amino Acid Peptide

The human Na+-sulfate cotransporter (hNaSi-1) belongs to the SLC13 gene family, which also includes the high-affinity Na+-sulfate cotransporter (hSUT-1) and the Na+-dicarboxylate cotransporters (NaDC). In this study, the location and functional role of the N-glycosylation site of hNaSi-1 were studied using antifusion protein antibodies. Polyclonal antibodies against a glutathione S-transferase fusion protein containing a 65-amino acid peptide of hNaSi-1 (GST-Si65) were raised in rabbits, purified, and then used in Western blotting and immunofluorescence experiments. The antibodies recognized native NaSi-1 proteins in pig and rat brush-border membrane vesicles as well as the recombinant proteins expressed in Xenopus oocytes. Wild-type hNaSi-1 and two N-glycosylation site mutant proteins, N591Y and N591A, were functionally expressed and studied in Xenopus oocytes. The apparent mass of N591Y was not affected by treatment with peptide- N-glycosylase F, in contrast to the mass of wild-type hNaSi-1, which was reduced by up to 15 kDa, indicating that Asn591is the N-glycosylation site. Although the cell surface abundance of the two glycosylation site mutants, N591Y and N591A, was greater than that of wild-type hNaSi-1, both mutants had greatly reduced Vmax, with no change in Km. These results suggest that Asn591and/or N-glycosylation is critical for transport activity in NaSi-1.

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