Search PubMed⌕ Search

Biomedical subjects

K Hsiao

Publications and source records attributed to K Hsiao.

At least 37 records · Page 2Linked to original sources
PubMed1995

X-ray structure of calcineurin inhibited by the immunophilin-immunosuppressant FKBP12-FK506 complex.

The X-ray structure of the ternary complex of a calcineurin A fragment, calcineurin B, FKBP12, and the immunosuppressant drug FK506 (also known as tacrolimus) has been determined at 2.5 A resolution, providing a description of how FK506 functions at the atomic level. In the structure, the FKBP12-FK506 binary complex does not contact the phosphatase active site on calcineurin A that is more than 10 A removed. Instead, FKBP12-FK506 is so positioned that it can inhibit the dephosphorylation of its macromolecular substrates by physically hindering their approach to the active site. The ternary complex described here represents the three-dimensional structure of a Ser/Thr protein phosphatase and provides a structural basis for understanding calcineurin inhibition by FKBP12-FK506.

Amino Acid Sequence↗
PubMed1995

A novel and simple method to assay the activity of individual protein kinases in a crude tissue extract.

Protein kinases and phosphatases play an important role in a variety of cellular functions. Thus, it is of interest to develop an assay system that can be used to quantify the activity of individual enzymes specifically in a crude cellular extract, is simple to perform, and is amenable to automation. Here we report on the development of a protein kinase assay that addresses these points and circumvents the pitfalls of existing methodologies. The assay is based on the high affinity and strong binding of streptavidin toward biotin-linked peptide substrates. The biotinylated peptide substrate is phosphorylated by the cognate protein kinase using [gamma-32P]ATP under optimal enzyme condition, and the phosphorylated peptide product is then captured by a streptavidin-linked disk. After removal of free [gamma-32P]ATP, the 32P incorporated into the peptide substrate can be used as an expression of enzyme activity. In contrast to the commonly used phosphocellulose method, only the phospho-, biotinylated peptide (and not other phosphorylated proteins present in the extract) will bind to the disks, thus giving a true estimate of enzymatic activity. In addition to specificity, this assay does not require the peptide substrate to contain basic amino acids or to be modified by the addition of basic amino acid residues as required for the phosphocellulose method which may result in altered specificity of the substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Three distinct messenger RNAs can encode the human immunosuppressant-binding protein FKBP12.

FKBP12 is an 11.8-kDa protein that binds the potent immunosuppressants FK506 and rapamycin. When bound to FK506, FKBP12 forms an inhibitory complex with calcineurin and interferes with signal transduction in activated T lymphocytes. In studying human FKBP12 cDNAs and the human FKBP12 gene, we found that three distinct transcripts can encode human FKBP12. The transcripts, which we designate FKBP 12A, 12B and 12C, contain identical open reading frames, but vary in abundance and are distinguished by unique 3' untranslated regions. The mature transcripts derive from either four or five exons and are generated by the differential use of one splice junction and three cleavage-polyadenylation sites within FKBP12. FKBP12A and 12B populations increase in abundance and/or stability when T-cell populations are mitogenically activated in vitro, implying that one result of T-cell stimulation is increased demand for the FKBP12 message. These transcripts are also present in a variety of human tissues, suggesting that FKBP12 and/or the mRNAs encoding it might affect physiological function(s) in a diverse array of cells.

Alternative Splicing↗
PubMed1994

A proposed strategic research program for Alzheimer's disease.

This article has recommended four priorities for future AD research in the United States over the next 10 years: 1. Complete the development of transgenic animal models of AD. 2. Integrate studies in aging with molecular biology studies on the genetics of AD in transgenic paradigms. 3. Reexamine the nature and role of amyloid plaques and neurofibrillary tangles in AD. 4. Investigate the age-dependent role of APP in AD using transgenic and aged nontransgenic animals. These recommendations are derived from my own studies and from a close review of the current literature on AD. There are undoubtedly many intriguing and even promising lines of research possible that are not mentioned here and that might deserve citation. However, with finite resources and limited time, the National Institute of Aging (NIA) should concentrate its funding on those research strategies that are most closely tied to the role of age and aging in AD. In conclusion, it may be useful to think of the future NIA agenda for AD research as a series of concentric rings, somewhat like a target. At the center and of highest priority should be purely aging-related studies, such as those just recommended. At the intermediate rings and of lower priority should be those studies that are less directly age related. Nonaging-related approaches to the study of AD would be placed at the outer-most rings and would receive funding and support from NIA only if they did not detract from critical support for the work adjudged to have higher priority, i.e., the age-related studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1993

Overexpression of p59-HBI (FKBP59), full length and domains, and characterization of PPlase activity.

It has been previously proposed that the rabbit p59-HBI (Heat shock protein Binding Immunophilin) or rFKBP59 (FK506 Binding Protein), found associated with the 90 kDa heat shock protein in nontransformed steroid receptor complexes, has three domains structurally related to hFKBP12 (Callebaut, I., Renoir, J.M., Lebeau, M.C., Massol, N., Burny, A., Baulieu, E.E. and Mornon, J.P. (1992) Proc. Natl. Acad. Sci., USA 89, 6270-6274). Here we report the overexpression, as fusion proteins in E. coli, of the full length p59-HBI and a series of p59-HBI mutants delimiting these domains and their respective peptidyl prolyl cis trans isomerase (PPlase) activity. The PPlase activity of p59-HBI is comparable to that of hFKBP12 and is due to domain p59-HBI I which displays the highest homology with this immunophilin. The residual enzymatic activity found in domain p59-HBI II is discussed.

Amino Acid Isomerases↗
PubMed1992

Linkage of the Indiana kindred of Gerstmann-Sträussler-Scheinker disease to the prion protein gene.

The Indiana kindred variant of Gerstmann-Sträussler-Scheinker disease has amyloid plaques that contain prion protein (PrP), but is atypical because neurofibrillary tangles like those of Alzheimer disease are present. To map the position of the disease causing gene, we used three markers for linkage analyses. A missense mutation at codon 198 of the PrP gene (PRNP) is found in all definitely affected individuals and yields a maximum lod score of 6.37 (theta = 0). The disease also is concordant with the two other PRNP-region markers. These results demonstrate tight linkage of the disease-causing gene to PRNP and support the hypothesis that the codon 198 mutation is the cause of IK-GSS. Our studies also suggest that methionine/valine heterozygotes at PRNP codon 129 have a later age of onset of the disease than codon 129 valine/valine homozygotes.

Adult↗
PubMed1992

Mutant prion proteins in Gerstmann-Sträussler-Scheinker disease with neurofibrillary tangles.

Two families with Gerstmann-Sträussler-Scheinker disease (GSS) are atypical in possessing neocortical neurofibrillary tangles (NFTs), which are few or absent in other kindreds with GSS, in addition to amyloid plaques that react with prion protein (PrP) antibodies and protease-resistant PrP accumulation in the brain. A leucine substitution at PrP codon 102 has been genetically linked to GSS in some families. We examined the PrP gene in these families. A serine for phenylalanine substitution was found at codon 198 in the Indiana patients; arginine for glutamine substitution at codon 217 in the Swedish patients. These mutations in PrP are the first to be associated with the appearance of both PrP amyloid plaques and neocortical NFTs in GSS patients.

Adult↗
PubMed1991

Mutation of the prion protein in Libyan Jews with Creutzfeldt-Jakob disease.

BACKGROUND: Creutzfeldt-Jakob disease is a transmissible neurodegenerative disorder that occurs more than 100 times more frequently among Libyan Jews than in the worldwide population. We examined 11 patients with the disease--10 Libyan Jews from Israel and 1 Libyan Jew from Italy--to determine whether abnormalities of the prion protein could be detected in them. Abnormal forms of this host-encoded protein are the predominant if not sole components of the transmissible agent that causes the disease. METHODS: The prion-protein open-reading frame in peripheral-leukocyte DNA from the Italian patient was amplified with the polymerase chain reaction and sequenced. Allele-specific oligonucleotide hybridization was used to assess a prion-protein codon 200 lysine mutation in the 10 Israeli patients and 37 control subjects. RESULTS: The prion-protein sequence in DNA from the Italian patient revealed a single nucleotide change (G----A) at the first position of codon 200 that resulted in a substitution of lysine for glutamate. This substitution was detected in all 10 Israeli patients, 8 of whom had a positive family history of Creutzfeldt-Jakob disease. One patient was homozygous for the lysine mutation, and her clinical course did not differ from that of the patients heterozygous for the mutation. The lysine mutation was not found in one Moroccan Jew from Israel with Creutzfeldt-Jakob disease. CONCLUSIONS: The codon 200 lysine mutation of the prion-protein gene is consistently present among Libyan Jews with Creutzfeldt-Jakob disease, strongly supporting a genetic pathogenesis of their illness. The similarity of the clinical courses of the patient homozygous for this mutation and the patients heterozygous for it argues that familial Creutzfeldt-Jakob disease is a true dominant disorder.

Adult↗
PubMed1991

Genetics of prion infections.

Although the infectious prions causing scrapie and several human transmissible neurodegenerative diseases resemble viruses in many respects, molecular and genetic analyses indicate that prions are fundamentally different from viruses in their structure and the mechanisms by which they cause disease. The only macromolecule that has been identified in infectious prion preparations is a disease-specific isoform of the prion protein, which is encoded by a host gene. A growing body of data supports the contention that prion infections represent a novel host-pathogen interaction.

Animals↗
PubMed1991

Molecular genetics and transgenic model of Gertsmann-Sträussler-Scheinker disease.

Gerstmann-Sträussler-Scheinker disease (GSS) is a rare, dominantly inherited neurodegenerative disease that can sometimes be transmitted to experimental animals through intracerebral inoculation of brain homogenates from patients. Substitution of leucine for proline at codon 102 of the prion protein gene has been found in several families with the disease; this mutation is genetically linked to GSS. Mice containing murine prion protein transgenes with this mutation spontaneously develop neurologic symptoms of ataxia, lethargy, and rigidity accompanied by spongiform degeneration throughout the brain. Thus, many of the clinical and pathological features of the GSS have been reproduced in this transgenic mouse paradigm; to our knowledge, this study illustrates, for the first time, that a neurodegenerative process similar to a human disease can be genetically modeled in animals. Whether or not this transgenic mouse model of GSS may facilitate the understanding of common neurodegenerative disorders such as Alzheimer disease remains to be established.

Alzheimer Disease↗
PubMed1991

Spontaneous neurodegeneration in transgenic mice with prion protein codon 101 proline----leucine substitution.

Gerstmann-Sträussler-Scheinker syndrome (GSS) is an autosomal, dominantly inherited, human neurodegenerative disease that can sometimes be transmitted to non-human primates and rodents through intracerebral inoculation of brain homogenates from patients. Recent studies of GSS demonstrated significant genetic linkage between GSS and a leucine substitution at codon 102 of the human prion protein (PrP) gene. Transgenic mice were created to test the biologic activity of this mutation. Spontaneous neurologic disease with spongiform degeneration developed in one of three lines of transgenic mice containing murine PrP genes with a leucine substitution at codon 101 (homologous to codon 102 in humans). Transmission studies of brain homogenates from affected mice are in progress. These results indicate that some of the clinical and pathologic features of GSS can be reproduced in a transgenic mouse paradigm; this represents the first time a dominantly inherited, neurodegenerative process similar to a human disease has been genetically modeled in an experimental animal (Hsiao and Prusiner 1990).

Amino Acid Sequence↗
PubMed1990

Spin filter perfusion system for high density cell culture: production of recombinant urinary type plasminogen activator in CHO cells.

We have used a 20 liter stirred tank fermentor, equipped with a 127 mesh ethylene-tetrafluoroethylene rotating screen for cell recycle, for the continuous production of recombinant single chain urokinase-type plasminogen activator (rscu-PA) from Chinese hamster ovary (CHO) cells. Viable cell densities between 60 and 74 million per ml were maintained at medium perfusion rates of 3.0 to 4.0 fermentor volumes per day. Cells were retained by the 120 micron nominal opening filter through the formation of "clumped" cell aggregates of 200 to 600 microns in size, which did not foul the filter. In 31 days of culture, a total of 51 grams of rscu-PA were produced in 1,000 liters of medium. The rscu-PA produced over the course of this continuous culture was purified and characterized both in vitro and in vivo and shown to be comparable to natural scu-PA produced from the transformed human kidney cell line, TCL-598.

Animals↗
PubMed1989

Linkage of a prion protein missense variant to Gerstmann-Sträussler syndrome.

Gerstmann-Sträussler syndrome is a rare familial neurodegenerative condition that is vertically transmitted, in an apparently autosomal dominant way. It can also be horizontally transmitted to non-human primates and rodents through intracerebral inoculation of brain homogenates from patients with the disease. The exact incidence of the syndrome is unknown but is estimated to be between one and ten per hundred million. Patients initially suffer from ataxia or dementia and deteriorate until they die, in one to ten years. Protease-resistant prion protein (PrP) and PrP-immunoreactive amyloid plaques with characteristic morphology accumulate in the brains of these patients. Current diagnostic criteria for Gerstmann-Sträussler syndrome incorporate clinical and neuropathological features, as animal transmission studies can be unreliable. PrP is implicated in the pathogenesis and transmission of the condition and in scrapie, an equivalent animal disease. It was discovered by enriching scrapie-infected hamster brain fractions for infectivity. Because there is compelling evidence that the scrapie isoform of PrP is a necessary component of the infectious particle, it seemed possible that the PrP gene on the short arm of human chromosome 20 in Gerstmann-Sträussler syndrome might be abnormal. We show here that PrP codon 102 is linked to the putative gene for the syndrome in two pedigrees, providing the best evidence to date that this familial condition is inherited despite also being infectious, and that substitution of leucine for proline at PrP codon 102 may lead to the development of Gerstmann-Sträussler syndrome.

Chromosome Mapping↗