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Biomedical subjects

S Li

Publications and source records attributed to S Li.

At least 451 records · Page 25Linked to original sources

Immune response in Stat2 knockout mice.

Type I IFNs induce gene expression through Stat1 and Stat2, which can in turn associate either to form Stat1 homodimers or the transcription factor ISGF-3. Stat1 homodimers also transduce signals for IFN-gamma. To explore the unique properties of Stat2 and ISGF-3 in type I IFN signaling, its gene was targeted for deletion. Stat2 null mice exhibit a number of defects in immune response. This includes an increased susceptibility to viral infection and the loss of a type I IFN autocrine/ paracrine loop, which in turn regulates several aspects of immune response. Intriguingly, Stat2-deficient fibroblasts exhibit a more significant defect in their response to type I IFNs than macrophages, highlighting tissue-specific differences in the response to this family of ligands.

Animals↗

Sequence-specific transcriptional corepressor function for BRCA1 through a novel zinc finger protein, ZBRK1.

BRCA1 has been implicated in the transcriptional regulation of DNA damage-inducible genes that function in cell cycle arrest. To explore the mechanistic basis for this regulation, a novel human gene, ZBRK1, which encodes a 60 kDa protein with an N-terminal KRAB domain and eight central zinc fingers, was identified by virtue of its interaction with BRCA1 in vitro and in vivo. ZBRK1 binds to a specific sequence, GGGxxx CAGxxxTTT, within GADD45 intron 3 that supports the assembly of a nuclear complex minimally containing both ZBRK1 and BRCA1. ZBRK1 represses transcription through this recognition sequence in a BRCA1-dependent manner. These results thus reveal a novel corepressor function for BRCA1 and provide a mechanistic basis for the biological activity of BRCA1 through sequence-specific transcriptional regulation.

Amino Acid Sequence↗

Fabrication inside microchannels using fluid flow.

This Account summarizes techniques for carrying out microfabrication of structures with dimensions down to 10 microm in microchannels that are 0.02-2 mm wide. These methods are largely based on the exploitation of laminar flow at low Reynolds number (Re) to control the spatial delivery of reagents. These methods are illustrated by fabrication of fibers, microelectrode arrays, arrays of crystals, and patterns of proteins and cells.

Crystallization↗

Selective enzymatic degradations of poly(L-lactide) and poly(epsilon-caprolactone) blend films.

Solution cast films were prepared from poly(L-lactide) (PLLA) and poly(epsilon-caprolactone) (PCL) as well as from three blends, namely B75, B50, and B25 with PLLA/PCL proportions of 75/25, 50/50, and 25/75, respectively. The enzymatic degradation of square samples (10 x 10 x 0.2 mm) cut from the films was investigated at 37 degrees C in a pH = 8.6 Tris buffer containing proteinase K or in a pH = 7.0 phosphate buffer containing Pseudomonas lipase. It was confirmed that proteinase K can degrade amorphous domains of PLLA, but cannot degrade crystalline PLLA or PCL. In contrast, Pseudomonas lipase can degrade both amorphous and crystalline PCL but cannot degrade PLLA. The two faces of solution cast films showed different morphologies due to the solvent evaporation process. The lower face appeared more crystalline than the upper face because of the plasticizing effect of solvent entrapped inside which allowed crystallization to proceed. Therefore, the lower face was more resistant to enzymatic attack by proteinase K in the cases of PLLA and the blends. The two polymers in the blends exhibited well separated crystalline domains. PCL seemed to constitute the continuous phase of the blends with formation of large size spherulites when the PCL content was over 50%. The selective degradation of PCL or PLLA components revealed the inner morphology of the blends where microspherelike or islandlike patterns were observed.

Calorimetry, Differential Scanning↗

Study of conformational rearrangement and refinement of structural homology models by the use of heteronuclear dipolar couplings.

For an increasing fraction of proteins whose structures are being studied, sequence homology to known structures permits building of low resolution structural models. It is demonstrated that dipolar couplings, measured in a liquid crystalline medium, not only can validate such structural models, but also refine them. Here, experimental 1H-15N, 1Halpha-13Calpha, and 13C'-13Calpha dipolar couplings are shown to decrease the backbone rmsd between various homology models of calmodulin (CaM) and its crystal structure. Starting from a model of the Ca2+-saturated C-terminal domain of CaM, built from the structure of Ca2+-free recoverin on the basis of remote sequence homology, dipolar couplings are used to decrease the rmsd between the model and the crystal structure from 5.0 to 1.25 A. A better starting model, built from the crystal structure of Ca2+-saturated parvalbumin, decreases in rmsd from 1.25 to 0.93 A. Similarly, starting from the structure of the Ca2+-ligated CaM N-terminal domain, experimental dipolar couplings measured for the Ca2+-free form decrease the backbone rmsd relative to the refined solution structure of apo-CaM from 4.2 to 1.0 A.

Amino Acid Sequence↗

Nonviral gene therapy: promises and challenges.

The last 10 years have seen substantial progress in the development and application of nonviral vectors in gene therapy. However, many problems remain to be resolved before nonviral gene therapy can become a standard clinical practice. This review highlights the major breakthroughs in this field. The problems and future research directions are also discussed. Gene Therapy (2000) 7, 31-34.

Chimera↗

MHC class I pathway is not required for the development of crescentic glomerulonephritis in mice.

MHC II and CD4+ T cells are required for anti-glomerular basement membrane (GBM) globulin-initiated crescentic glomerulonephritis (GN) in mice, but the role of MHC I and CD8+ T cells is unclear. The cytolytic function of CD8+ T cells requires recognition of peptide antigens presented on MHC I. CD8+ T cells can also perform helper functions via cytokine production. The contribution of MHC I to crescentic GN was investigated using TAP-1 gene knock out (TAP-1-/-) mice, which have deficient MHC I antigen presentation. Heterozygous TAP-1 mice have normal MHC I expression and developed GN with crescents in 42 +/- 4% of glomeruli (normal 0%), proteinuria (9.1 +/- 1.6 mg/20 h, normal 1.5 +/- 0.3 mg/20 h) and impaired renal function (creatinine clearance 110 +/- 8 microl/min, normal 193 +/- 10 microl/min) following administration of sheep anti-mouse GBM globulin. TAP-1-/- mice, which have extremely low MHC I expression and reduced CD8+ T cells, developed similar GN with 39 +/- 3% crescents, proteinuria (12.7 +/- 4.3 mg/20 h) and impaired renal function (creatinine clearance 123 +/- 20 microl/min). In vivo antibody-induced CD8 depletion did not attenuate crescent formation or protect renal function in C57Bl/6 mice developing GN, although significant reduction in proteinuria (5.3 +/- 1.2 mg/20 h, P = 0. 012) and glomerular recruitment of CD4+ T cells and macrophages were observed compared with control treated mice with GN. These data demonstrate that MHC I is not required for development of crescentic GN in mice. The MHC I-independent contribution of CD8+ T cells to proteinuria and inflammatory cell recruitment suggests that they may serve a 'helper' rather than cytolytic role in this disease.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

A diminished activation capacity of the interferon-inducible protein kinase PKR in human T lymphocytes.

The double-stranded (ds) RNA activated protein kinase PKR is an interferon (IFN)-inducible serine/threonine protein that regulates protein synthesis through the phosphorylation of the alpha subunit of translation initiation factor 2 (eIF-2alpha). PKR activation in cells is induced by virus infection or treatment with dsRNA and is modulated by a number of viral and cellular factors. To better understand the mechanisms of PKR action we have analyzed and compared the mode of PKR activation in a number of cell lines of different histological origin. Here we show that PKR activation and phosphorylation of eIF-2alpha are both diminished in various virus-transformed and nontransformed human T cells. Priming of T cells with IFN does not restore PKR activation. In vitro kinase assays show that the diminished PKR activation in T cells correlates with the presence of a 60-kDa (p60) phosphoprotein coimmunoprecipitated with PKR. P60 is absent from PKR immunoprecipitates from non T cells. Incubation of active PKR with T cell extracts results in inhibition of PKR autophosphorylation, which is proportional to the amount of phosphorylated p60 in the kinase reactions. Treatment of T cells with proteasome inhibitors or incubation of PKR immunoprecipitates with phosphatase inhibitors does not restore PKR activation. However, phosphorylation of p60 is enhanced upon treatment with the phosphatase inhibitor microcystin. These data show that the impaired activation capacity of PKR in human T cells is exerted at the post-translational levels in a manner that is independent of cell transformation or virus infection.

Adult↗

Hematocrit levels and associated Medicare expenditures.

Clinical studies and the National Kidney Foundation-Dialysis Outcomes Quality Initiative guidelines suggest that a target hematocrit of 33% to less than 36% is appropriate for patient benefit. Previous studies have shown an association of lower risks for death and hospitalization when hematocrits were 33% to less than 36%. In this study, we assessed the relationship between hematocrit value and associated Medicare expenditures, analyzing incident Medicare hemodialysis patients from January 1, 1991, through June 30, 1995. All patients survived at least 90 days to normalize eligibility and an additional 6-month entry period to assess comorbidity and hematocrit values. All patients were followed up from July 1, 1991, through December 31, 1996. We assessed the association between hematocrit values in the 6-month entry period and the Medicare-allowable Part A and Part B per-member-per-month (PMPM) expenditures in the follow-up period, controlling for other variables, including patient demographic characteristics, comorbid conditions, and severity of disease. We found that hematocrits of 33% to less than 36% and 36% and higher were associated with lower Medicare-allowable payments in the follow-up period. Compared with reference patients with hematocrits of 30% to less than 33%, the Medicare-allowable PMPM expenditures were significantly greater for patients with hematocrits less than 27% and 27% to less than 30% (12. 7% and 5.3%, respectively), and the Medicare-allowable PMPMs were significantly less for patients with hematocrits of 33% to less than 36% and 36% and higher (6.0% and 8.2%, respectively). Although these findings suggest that the treatment of anemia may be associated with significant savings in total patient Medicare expenditures, caution should be considered because these findings are associations and should not be deemed as showing causality.

Adult↗

Action of locally administered NMDA and AMPA/kainate receptor antagonists in spinal cord injury.

NMDA or AMPA/kainate receptor antagonists have been shown to provide neuroprotection following in vitro spinal cord injury, but the mechanisms by which these agents improve behavioral recovery and protect axonal function remains unclear. We hypothesized that treatment of spinal cord injury with these drugs would attenuate glutamate excitatory transmission by blocking the effects of glutamate receptors at the injury site or would improve spinal cord blood flow. To test these hypotheses, we observed the effects of locally administered MK-801 (30 nmol) or NBQX (5 or 15 nmol) into the injured spinal cord on axonal conduction and post-traumatic ischemia of the cord. The outcome measures were multimodality evoked potentials and blood flow in an acute compression injury model in rats. We found that locally administered MK-801 or NBQX 15 min after spinal cord injury attenuated the amplitude, delayed the latency of sensory evoked potentials and increased the sensory conduction time across the injury site, but did not improve blood flow during the 4-h period of observation. These results demonstrate that the NMDA and non-NMDA receptor antagonists produced a blockade of glutamate excitatory transmission in the afferent pathways at the injury site. It is suggested that the neuroprotection provided by these agents following spinal cord injury is mediated through blockade of glutamate ionotropic receptors in the injured spinal cord, but is not related to improvement of SCBF.

Animals↗

Pyrogen sensing and signaling: old views and new concepts.

Fever is thought to be caused by endogenous pyrogenic cytokines, which are elaborated and released into the circulation by systemic mononuclear phagocytes that are activated by exogenous inflammatory agents and transported to the preoptic-anterior hypothalamic area (POA) of the brain, where they act. Prostaglandin (PG) E2 is thought to be an essential, proximal mediator in the POA, and induced by these cytokines. It seems unlikely, however, that these factors could directly account for early production of PGE2 following the intravenous administration of bacterial endotoxic lipopolysaccharides (LPS), because PGE2 is generated before the cytokines that induce it are detectable in the blood and the before cyclooxygenase-2, the synthase that they stimulate, is expressed. Hence other, more quickly evoked mediators are presumed to be involved in initiating the febrile response; moreover, their message may be conveyed to the brain by a neural rather than a humoral pathway. This article reviews current conceptions of pyrogen signalling from the periphery to the brain and presents new, developing hypotheses about the mechanism by which LPS initiates fever.

Brain↗

Improved detection and characterization of paroxysmal nocturnal hemoglobinuria using fluorescent aerolysin.

Paroxysmal nocturnal hemoglobinuria (PNH) is caused by a somatic mutation in the gene PIGA, which encodes an enzyme essential for the synthesis of glycosylphosphatidylinositol (GPI) anchors. The PIGA mutation results in absence or marked deficiency of more than a dozen proteins on PNH blood cells. Current flow cytometric assays for PNH rely on the use of labeled antibodies to detect deficiencies of specific GPI anchor proteins, such as CD59. However, because no single GPI anchor protein is always expressed in all cell lineages, no one monoclonal antibody can be used with confidence to diagnose PNH. We describe a new diagnostic test for PNH, based on the ability of a fluorescently labeled inactive variant of the protein aerolysin (FLAER) to bind selectively to GPI anchors. We compared GPI anchor protein expression in 8 patients with PNH using FLAER and anti-CD59. In all cases, FLAER detected similar or higher proportions of PNH monocytes and granulocytes compared with anti-CD59. Because of the increased sensitivity of detection, FLAER could detect small abnormal granulocyte populations in patients to a level of about 0.5%; samples from healthy control subjects contained substantially fewer FLAER-negative cells. FLAER gives a more accurate assessment of the GPI anchor deficit in PNH.

Anemia, Aplastic↗

Cerebral palsy and multiple births in China.

BACKGROUND: A population-based study on prevalence of cerebral palsy in multiple births has not been carried out in China. The purpose of this paper was to determine the prevalence of cerebral palsy in multiple births and to explore the influence of multiple pregnancy on cerebral palsy after controlling for birthweight. METHODS: A cross-sectional study of cerebral palsy was carried out among 388,192 children aged <7 years in seven cities of Jiangsu province in China. Information about birthweight and plurality was obtained from routine health care records. Pediatricians at city level diagnosed all cases. All the doctors involved had taken part in a training programme held by Beijing Medical University. Stratified analysis by birthweight and its standard normal deviate was employed to compare the prevalence of cerebral palsy in multiples and singletons. RESULTS: The prevalence of cerebral palsy for children aged <7 years in multiples was 9.7 per 1000 children (95% confidence interval [CI]: 6.5-14.0), which was 6.5 times that in singletons (95% CI: 4.4-9.3). The overall neonatal mortality rate was 60.9 per 1000 liveborn multiples, being highest (944.4 per 1000) in the 500-999 g birthweight groups. Most liveborn multiples weighing <1500 g at birth probably died from diseases related to very low birthweight prior to this study. The prevalence of cerebral palsy in multiple births was likely to be higher than that reported in developed countries for children weighing 1500-2499 g even though our data were from a cross-sectional study. When stratified by birthweight, the prevalence of cerebral palsy in multiples weighing <2500 g had tended to be lower than that of singletons in the same birthweight group. In contrast, in normal birthweight categories multiple births had a higher prevalence of cerebral palsy than singletons. When stratified by birthweight normal deviate, the prevalence of cerebral palsy in multiple births was uniformly higher than that in singletons in all birthweight strata and the prevalence of cerebral palsy among multiples appeared to be augmented as birthweight increased. CONCLUSION: The prevalence of cerebral palsy in multiples, 9.7 per 1000 children, is 6.5 times that in singletons. Survival of low birthweight infants is lower in China than in developed countries and survival quality of Chinese children weighing 1500-2499 g needs to be further improved. In terms of birthweight multiples and singletons may be heterogeneous. It might be difficult to directly use actual birthweight specific prevalence to compare the prevalence of cerebral palsy in multiples and singletons. Birthweight normal deviate specific prevalence of cerebral palsy suggests that multiple pregnancy is an independent risk factor for cerebral palsy in all birthweight groups. Multiples are in adverse circumstances very early in gestation and as the foetus matures the risk of cerebral palsy increases.

Birth Weight↗

Aberrant T cell responses to myelin antigens during clinical exacerbation in patients with multiple sclerosis.

Multiple sclerosis (MS) is a demyelinating disease of presumed T cell autoimmunity against self myelin. We hypothesized that if myelin-reactive T cells are associated with the disease processes, they may undergo activation and expansion during acute exacerbation. In this study, we examined the precursor frequency, epitope recognition and cytokine profile of myelin-reactive T cells in 14 relapsing/remitting MS patients during exacerbation and remission. The study revealed that T cells recognizing the immunodominant peptides of candidate myelin antigens, including myelin basic protein (MBP), proteolipid protein and myelin oligodendrocyte glycoprotein, occurred at increased precursor frequency during acute exacerbation. The T cell responses to MBP focused on the immunodominant regions (residues 83-99 and 151-170) during exacerbation and shifted toward other epitopes of MBP at the time of remission. Furthermore, there was a marked increase in the production of T(h)1 cytokines among T cell lines obtained during exacerbation compared to those obtained during remission. The study demonstrated that myelin-reactive T cells underwent selective activation and expansion during acute MS exacerbation. In contrast, myelin-reactive T cells found during remission in the same patients generally resembled those identified in healthy controls with some discrepancies. The findings suggest potential association of aberrant myelin-reactive T cell responses with acute exacerbation in MS, which may reflect transient activation of myelin-reactive T cell populations of pathogenic potential.

Adult↗

Purification, characterization, and primary structure of a novel cell wall hydrolytic amidase, CwhA, from Achromobacter lyticus.

A novel bacteriolytic enzyme CwhA (cell wall hydrolytic amidase) was purified by ion exchange and gel-filtration chromatographies from a commercial bacteriolytic preparation from Achromobacter lyticus. CwhA exhibited optimal pH at 8.5 and lysed CHCl(3)-treated Escherichia coli more efficiently than Micrococcus luteus, Staphylococcus aureus, Enterococcus faecalis, and Pediococcus acidilactici. The enzyme was inhibited by 1,10-phenanthroline strongly and by EDTA to a lesser extent, suggesting that it is probably a metalloenzyme. Amino acid composition and mass spectrometric analyses for the CwhA-derived M. luteus muropeptides revealed that CwhA is N-acetylmuramoyl-L-alanine amidase [EC 3.5.1. 28]. The complete amino acid sequence of CwhA was established by a combination of Edman degradation and mass spectrometry for peptides obtained by Achromobacter protease I (API) digestion and cyanogen bromide (CNBr) cleavage. The enzyme consists of a single polypeptide chain of 177 amino acid residues with one disulfide bond, Cys114-Cys121. CwhA was found to be homologous to N-acetylmuramoyl-L-alanine amidase from bacteriophage T7 (BPT7). Its sequence identity with BPT7 is 35%, but the amino acid residues functioning as zinc ligands in BPT7 are absent in CwhA. These results suggest that CwhA is a new type of N-acetylmuramoyl-L-alanine amidase.

Alcaligenes↗

Molecular cloning and characterization of functional domains of a human testis-specific isoform of calpastatin.

Human serum containing sperm-agglutinating antibodies was used to screen a testis cDNA expression library to identify the cognate antigens that may be responsible for this biological effect. The longest positive phage clone (1.9 kb) was sequenced and found to be a testis-specific isoform of calpastatin (tCAST). The testis-specific segment of tCAST is encoded by a single exon within intron 14 of the calpastatin gene. A unique protein isoform is produced that differs in domain structure from the somatic calpastatins (sCAST). Human sCAST most commonly has an N-terminal domain L plus the four functional calpain inhibitory domains. Human tCAST consists of a 40-amino-acid N-terminal T domain plus a part of domain II and all of domains III and IV from the somatic isoform. Our data show that the T domain can target cytosolic localization and membrane association of tCAST, whereas domain I of sCAST exhibits a nuclear localization function. Calpastatin is the endogenous inhibitor of calpain. The calpain/calpastatin system is involved in membrane fusion events for several cell types, and calpain has been localized to the sperm acrosome. We detected tCAST in human sperm and testes extracts by Western blotting with specific antisera. These observations suggest that tCAST may modulate calpain in the calcium-mediated acrosome reaction that is required for fertilization.

Amino Acid Sequence↗

A novel N-terminal domain directs membrane localization of mouse testis-specific calpastatin.

Multiple isoforms of calpastatin have been identified with unique N-terminal regions followed by identical calpain inhibitory domains (II-IV). In many instances the isoforms are cell-type specific, although the precise functional differences among these N-terminal regions are largely unknown. Here we report a germ cell-specific isoform of calpastatin (tCAST) that consists of a novel N-terminal peptide of 40 amino acids (domain T) followed by domains II to IV of somatic calpastatin (sCAST). Domain T is responsible for membrane association of tCAST through a protein modification by myristylation. Mutation of the myristylation site eliminates membrane targeting. Unlike most of the isoforms of calpastatin that are generated through alternative RNA splicing or post-translational proteolysis, the testis-specific isoform is transcribed from an intronic promoter in haploid germ cells of the testis. The intronic promoter directs specific expression of a reporter transgene in developing germ cells of the mouse testis.

Animals↗

The VMAT2 gene in mice and humans: amphetamine responses, locomotion, cardiac arrhythmias, aging, and vulnerability to dopaminergic toxins.

Monoamine compartmentalization in monoaminergic neurons uses serial action of the plasma membrane and vesicular monoamine (VAMT2) transporters. We can now define the sequences of the genes encoding these transporters in mice and humans, examine influences of deletions of this gene and alteration in its expression levels in transgenic mice, and identify sequence polymorphisms in the human VMAT2 gene. Examination of VMAT2 variants can provide potential insights into roles for allelic variants at these loci in variant drug responses and in diseases linked to monoaminergic systems, including substance abuse and Parkinson's disease.

Aging↗