Biomedical subjects

H Li

Publications and source records attributed to H Li.

At least 577 records · Page 32Linked to original sources

PubMed2000

Multiplex genotype analysis of invasive carcinoma and accompanying proliferative lesions microdissected from breast tissue.

To understand the genetic basis of breast cancer in a comprehensive way, purported precursor lesions need to be analyzed at a large number of genetic marker loci and compared with each other and with the invasive components. However, the microscopic size of most of these lesions and the very small amount of material that can be obtained through microdissection limit the number of loci that can be included in the analysis. To address this issue, a multiplex genotyping approach has been developed. With this approach, polymorphic sequences at 28 marker loci were amplified simultaneously from the micro-dissected components in 5-microm paraffin-embedded breast tissue sections. The genotypes of the lesions were determined after resolving the amplified allelic products by denaturing gradient gel electrophoresis. Because the material isolated from each lesion in a single 5-microm section was sufficient for several 28-locus assays and several successive tissue sections with the same set of lesions may be prepared, it is possible to determine the genotype of each lesion at hundreds of genetic marker loci that may well cover the human genome. Analyzing a sufficient number of cases may yield information that could be used to understand the genetic basis of breast cancer development in a comprehensive way.

Alleles↗

PubMed2000

Endogenous parathyroid hormone-related peptide enhances proliferation and inhibits differentiation in the osteoblast-like cell line ROS 17/2.8.

To investigate potential effects of endogenous parathyroid hormone-related peptide (PTHrP) on osteoblast function, ROS 17/2.8 cells were transfected with full-length PTHrP cDNA in a sense or antisense orientation to alter PTHrP production. Compared with vector-transfected control cells, PTHrP-overproducing (sense-transfected) cells showed increased DNA synthesis ([(3)H]-thymidine incorporation) and increased growth (cell number). The extent of apoptosis was compared for the different clones using the terminal deoxynucleotide-mediated dUTP nick-end-labeling assay (TUNEL) and Hoechst staining. No differences in percentages of apoptotic cells were found under basal culture conditions or after 3 days of serum deprivation, which, itself, markedly increased numbers of apoptotic cells. The effect of PTHrP on osteoblast differentiation was assessed by examining two protein markers of differentiation, alkaline phosphatase, and bone morphogenetic protein (BMP)-2. Alkaline phosphatase activity was decreased in sense-transfected cells and increased in antisense-transfected cells, compared with cells transfected with empty vector. PTHrP-overproducing cells also showed decreased numbers of BMP-2-positive cells, whereas antisense-transfected cells showed no difference compared with vector control. The results indicate that: (a) endogenously produced PTHrP can increase growth of these osteoblastic cells by stimulating proliferation while not affecting apoptosis; and (b) the increased cell proliferation produced by PTHrP was accompanied by a reduction in activity or amount of two proteins normally expressed by differentiated osteoblasts.

Alkaline Phosphatase↗

PubMed2000

Effects of age at onset on the power of the affected sib pair and transmission/disequilibrium tests.

Information on the age of a patient at disease onset, an important feature of complex diseases, is often collected in studies designed to map the disease genes. Penetrance-model-free methods, requiring no specification of penetrance functions, have been used extensively for detecting linkage and association between marker and disease loci. In this paper, we conduct an analytical study to examine the effects of incorporation of age at onset information on the power of two commonly used penetrance-model-free methods, the affected sib-pair (ASP) and transmission/disequilibrium tests (TDT). Assuming a Cox model with a major gene effect for the age at onset, we quantify analytically how age at onset affects the identity by descent (IBD) probabilities, the mean IBD values, and the expected numbers of alleles transmitted from heterozygous parents to affected children under various genetic models. We show that the power of the mean IBD test and the TDT can be greatly affected by the ages at onset of affected siblings or children used in the study. Generally, the most powerful test for ASPs is that based on affected sib pairs both having early disease onset and for TDT analyses is that based on trios with early-onset children. Naively combining affected sib pairs with different ages at onset or parent-children trios with different ages at onset of affected children can result in reduced power for detecting linkage or association. These results may be used to guide collection and analysis of sib pairs or families for diseases with variable age at onset.

Adult↗

PubMed2000

Predictors of poor outcome in patients with a spontaneous cerebellar hematoma.

BACKGROUND AND PURPOSE: The authors studied the clinical and neuroimaging features of cerebellar hematomas to predict poor outcome using comprehensive statistical models. METHODS: We retrospectively reviewed clinical and neuroimaging features in 94 patients with spontaneous cerebellar hematomas to identify predictive features for a poor neurologic outcome, defined as death or dismissal to long-term care facility. Data were analyzed using chi square and Fisher's exact test with calculation of odd's ratios together with 95% confidence intervals. RESULTS: Clinical and neuroradiologic predictors for a poor outcome at p < 0.05 were admission systolic blood pressure > 200 mm Hg, hematoma size > 3 cm, visible brain stem distortion, and acute hydrocephalus. Presenting findings predicting subsequent death at p < 0.05 were abnormal corneal and oculocephalic responses, Glasgow coma sum score less than 8, motor response less than localization to pain, acute hydrocephalus and intraventricular hemorrhage. CONCLUSION: A tree-based analysis model using binary recursive partitioning showed that cornea reflex, hydrocephalus, doll's eyes, age, and size were the most important discriminating factors. Absent corneal reflexes on admission highly predicts poor outcome (86 percent, confidence limits 67-96 percent). When a cornea reflex is present, acute hydrocephalus predicts poor outcome but only when doll's eyes are additionally absent.

Adult↗

PubMed2000

A new twist in trypanosome RNA metabolism: cis-splicing of pre-mRNA.

It has been known for almost a decade and a half that in trypanosomes all mRNAs are trans-spliced by addition to the 5' end of the spliced leader (SL) sequence. During the same time period the conviction developed that classical cis-splicing introns are not present in the trypanosome genome and that the trypanosome gene arrangement is highly compact with small intergenic regions separating one gene from the next. We have now discovered that these tenets are no longer true. Poly(A) polymerase (PAP) genes in Trypanosoma brucei and Trypanosoma cruzi are split by intervening sequences of 653 and 302 nt, respectively. The intervening sequences occur at identical positions in both organisms and obey the GT/AG rule of cis-splicing introns. PAP mRNAs are trans-spliced at the very 5' end as well as internally at the 3' splice site of the intervening sequence. Interestingly, 11 nucleotide positions past the actual 5' splice site are conserved between the T. bruceiand T. cruzi introns. Point mutations in these conserved positions, as well as in the AG dinucleotide of the 3' splice site, abolish intron removal in vivo. Our results, together with the recent discovery of cis-splicing introns in Euglena gracilis, suggest that both trans- and cis-splicing are ancient acquisitions of the eukaryotic cell.

Amino Acid Sequence↗

PubMed2000

Oxidative metabolites of 5-S-cysteinylnorepinephrine are irreversible inhibitors of mitochondrial complex I and the alpha-ketoglutarate dehydrogenase and pyruvate dehydrogenase complexes: possible implications for neurodegenerative brain disorders.

The major initial product of the oxidation of norepinephrine (NE) in the presence of L-cysteine is 5-S-cysteinylnorepinephrine which is then further easily oxidized to the dihydrobenzothiazine (DHBT) 7-(1-hydroxy-2-aminoethyl)-3,4-dihydro-5-hydroxy-2H-1, 4-benzothiazine-3-carboxylic acid (DHBT-NE-1). When incubated with intact rat brain mitochondria, DHBT-NE-1 evokes rapid inhibition of complex I respiration without affecting complex II respiration. DHBT-NE-1 also evokes time- and concentration-dependent irreversible inhibition of NADH-coenzyme Q(1) (CoQ(1)) reductase, the pyruvate dehydrogenase complex (PDHC), and alpha-ketoglutarate dehydrogenase (alpha-KGDH) when incubated with frozen and thawed rat brain mitochondria (mitochondrial membranes). The time dependence of the inhibition of NADH-CoQ(1) reductase, PDHC, and alpha-KGDH by DHBT-NE-1 appears to be related to its oxidation, catalyzed by an unknown component of the inner mitochondrial membrane, to electrophilic intermediates which bind covalently to active site cysteinyl residues of these enzyme complexes. The latter conclusion is based on the ability of glutathione to block inhibition of NADH-CoQ(1) reductase, PDHC, and alpha-KGDH by scavenging electrophilic intermediates, generated by the mitochondrial membrane-catalyzed oxidation of DHBT-NE-1, forming glutathionyl conjugates, several of which have been isolated and spectroscopically identified. The possible implications of these results to the degeneration of neuromelanin-pigmented noradrenergic neurons in the locus ceruleus in Parkinson's disease are discussed.

Animals↗

PubMed2000

Characterization of a novel silencer element in the human aromatase gene PII promoter.

Approximately two thirds of breast cancer patients have estrogen-dependent carcinomas. The biosynthesis of estrogens is catalyzed by the microsomal enzyme aromatase. Mechanisms controlling human aromatase gene expression are complicated by the existence of multiple tissue specific promoters. The most proximally located Pll promoter is mainly active in ovarian granulosa cells. PlI can be switched on in human breast cancer cells. Since there are strong silencer elements located within the 3' portion of the PlI promoter, we propose that the function of these silencer elements could be reversed by breast cancer cell specific signals/factors, resulting in aberrant expression of aromatase. We have identified and characterized a novel silencer element, S2, which is upstream of S1, a silencer element recently identified by another group. S2, a 54-bp fragment 100% conserved between humans and rodents, functions in both orientation- and promoter-independent manners. The core region of S2 contains two consensus binding sites for members of the GATA transcription factors. GATA-4 was found to be expressed in three out of four human breast cancer cell lines examined by RT-PCR, and transfection with GATA-4 partially reversed the repressive function of S2. However, we were unable to demonstrate that DNA-protein complexes formed between nuclear extracts of human breast and ovarian cancer cells and S2 contain GATA-4 using a supershifting approach. We suggest that the expression of GATA-4, and more importantly, other yet to be identified GATA or GATA-related factor(s), are implicated in provoking aberrant expression of aromatase, and therefore, the biosynthesis of estrogens, in human breast cancer cells.

Aromatase↗

PubMed2000

Cytotoxic T-lymphocyte responses elicited to Wilms' tumor gene WT1 product by DNA vaccination.

We recently have reported that Wilms' tumor gene WT1 is highly expressed not only in leukemias but also in various types of solid tumors and that WT1 protein is a novel tumor antigen against which cytotoxic T lymphocytes (CTLs) can be elicited by immunization with 9-mer WT1 peptides capable of binding to major histocompatibility complex (MHC) class I molecules. In the present study, plasmid DNA encoding murine full-length WT1 protein was injected intramuscularly into C57BL/6 mice. The mice vaccinated with the WT1 plasmid DNA elicited CTLs against the WT1 protein, and the CTLs specifically killed WT1-expressing tumor cells in a MHC class I-restricted manner. Furthermore, the vaccinated mice rejected the challenges of WT1-expressing tumor cells and survived with no signs of autoimmunity caused by the CTLs. These results demonstrated that vaccination with the WT1 plasmid DNA can elicit CTL responses specific for the WT1 protein, resulting in the acquisition of rejection activity against challenges of WT1-expressing tumor cells. This WT1 DNA vaccination may find clinical application for various types of solid tumors as well as leukemias.

Animals↗

PubMed2000

Transcatheter cryoablation of ventricular myocardium in dogs.

INTRODUCTION: Surgical cryoablation, a highly effective technique used during antiarrhythmic surgery, produces voluminous, histologically uniform and discreet myocardial lesions. In contrast, radiofrequency (RF) catheter ablation, which as a result of its less invasive nature has largely supplanted antiarrhythmic surgery, produces smaller, histologically heterogeneous myocardial lesions. Since small lesion size and heterogeneity may reduce antiarrhythmic efficacy, we sought to reproduce the large, histologically homogeneous lesions created by surgical cryoablation, using a catheter cryoablation system (Cryogen, Inc., San Diego, CA) in the canine ventricle. METHODS AND RESULTS: In seven dogs, nineteen ventricular lesions (two right and seventeen left) were created with a 10F cryoablation catheter with either a 2 or 6 mm tip. In one dog AV node ablation was also performed. For each 'freeze', catheter tip nadir temperature, lesion width, depth, and transmurality were recorded, and lesion volume calculated. Average tip nadir temperature was -79.6+/-4.9 degrees C. Cooler nadir tip temperature was associated with deeper (p=.007) and more voluminous lesions (p=.042), and a greater likelihood of lesion transmurality (p=.034). Average lesion volume was 500+/-356 mm(3). No other variables predicted lesion volume or transmurality. Histologically, the catheter cryoablation lesions were sharply demarcated and homogeneous. The single freeze performed at the AV junction produced complete AV block. One complication, catheter rupture following its repetitive use, resulted in a coronary air embolus and death. CONCLUSION: Catheter cryoablation of canine ventricular myocardium produced voluminous, discrete, transmural lesions, which might be effective for ablation of ventricular tachycardia. Lesion volume and transmurality were dependent on catheter tip nadir temperature.

Analysis of Variance↗

PubMed2000

Pressure alters electronic orbital overlap in hydrogen bonds.

Pressure-induced changes in 3hJ(NC') scalar couplings through hydrogen bonds were investigated in the immunoglobulin binding domain of streptococcal protein G. 1H, 15N and 13C triple-resonance NMR spectroscopy coupled with the on-line high pressure cell technique was used to monitor 3hJ(NC') scalar couplings at 30 and 2000 bar in uniformly labeled 15N and 13C protein isotopes. Both increased and decreased 3hJ(NC') scalar couplings were observed at high pressure. No correlation with secondary structure was apparent. The difference in coupling constants as well as pressure-induced chemical shift data suggests a compaction of the helix ends and an increase of the helix pitch at its center in response to pressure. Our data provides the first direct evidence that the electronic orbital overlap in protein backbone hydrogen bonds is altered by pressure.

Bacterial Proteins↗

PubMed2000

An immunohistochemical study of matrix molecules associated with barrier membrane-mediated periodontal wound healing.

Guided tissue regeneration (GTR) is a clinical procedure developed to facilitate periodontal regeneration by using barrier membranes to selectively promote the repopulation of a periodontal defect by periodontal ligament and bone cells at the expense of epithelial and gingival connective tissue cells. The aim of this study was to gain insight into the biological events occurring during membrane mediated periodontal wound healing by examining the immunohistochemical expression of a number of extracellular matrix components in tissues treated via the GTR technique. Experimental periodontal defects were created around the second premolar tooth in 4 dogs and wound closure was achieved by application of expanded polytetrafluoroethylene membranes around each tooth and flap positioning coronal to the cementoenamel junction. The dogs were sacrificed after a 4-wk healing period, block dissections of the part of the mandible containing the experimental tooth were obtained and paraffin sections were prepared. Using standard immunohistochemical techniques, the sections were stained with a monoclonal antibody against bone morphogenetic proteins 2 and 4 (BMP-2 and -4) and polyclonal antibodies against collagen I, collagen II, decorin, biglycan, bone sialoprotein, osteopontin and osteocalcin. Collagen I was predominantly localized within the regenerating bone, whereas collagen III staining was more abundant in the soft connective tissues of the defect. Decorin and biglycan staining was faint within the extracellular matrix of the regenerating defect, although both proteoglycans exhibited intense intracellular localization within some of the cells inhabiting the defect. The staining for BMP-2 and -4 was weak within the bone but strong within the extracellular matrix of the regenerating soft tissue. Osteopontin and bone sialoprotein were strongly localized in the regenerating bone and cementum found within the defect. Osteocalcin staining was present in both the regenerating and mature cementum and associated cementoblasts, and it was relatively weaker in the regenerating bone compared to the mature bone. The observed pattern of immunolocalization of the extracellular matrix macromolecules suggests that the heterogeneous cell population filling the GTR wound had created an environment that was conducive to periodontal regeneration.

Alveolar Process↗

PubMed2000

Nitric oxide synthase type-II is synthesized by human gingival tissue and cultured human gingival fibroblasts.

Nitric oxide is known to be an important inflammatory mediator, and is implicated in the pathophysiology of a range of inflammatory disorders. The aim of this study was to determine the localization and distribution of endothelial NOS (NOS-II) in human gingival tissue, and to ascertain if human gingival fibroblasts express NOS-II when stimulated with interferon gamma (IFN-gamma) and bacterial lipopolysaccharide (LPS). The distribution of NOS-II in inflamed and non-inflamed specimens of human gingivae was studied using a monoclonal antibody against nitric oxide synthase II. Cultures of fibroblasts derived from healthy human gingivae were used for the cell culture experiments. The results from immunohistochemical staining of the tissues indicated an upregulation of NOS-II expression in inflamed compared to non-inflamed gingival tissue. Fibroblasts and inflammatory cells within the inflamed connective tissue were positively stained for NOS-II. In addition, basal keratinocytes also stained strongly for NOS-II, in both healthy and inflamed tissue sections. When cultured human gingival fibroblasts were stimulated by INF-gamma and Porphyromonas gingivalis LPS, NOS-II was more strongly expressed than when the cells were exposed to LPS or IFN-gamma alone. These data suggest that, as for other inflammatory diseases, NO plays a role in the pathophysiology of periodontitis.

Cells, Cultured↗

PubMed2000

Expression and distribution of cell-surface proteoglycans in the normal Lewis rat molar periodontium.

Cell-surface proteoglycans participate in several biological functions such as cell cell and cell-matrix interactions, cell adhesion, the binding to various growth factors as co-receptors and repair. To understand better the expression and distribution of cell-surface proteoglycans in the periodontal tissues, an immunohistochemical evaluation of the normal Lewis rat molar periodontium using panels of antibodies for syndecan-1, -2, -4, glypican and betaglycan was carried out. Our results demonstrated the expression and distribution of all proteoglycans in the suprabasal gingival epithelium, soft and hard connective tissues. Both cellular and matrix localization was evident within the various periodontal compartments. The presence of these cell-surface proteoglycans indicates the potential for roles in the process of tissue homeostasis, repair or regeneration in periodontium of which each function requires further study.

Animals↗

PubMed2000

Distribution and synthesis of elastin in porcine gingiva and alveolar mucosa.

Histologically, there are variations in the distribution of the types of elastic fibers within the tissues of the masticatory mucosa. For example, the alveolar mucosa has heavy deposits of elastin which decrease in quantity and size as it merges with the attached gingiva. These differences in elastin content may result from the tissues' different physiological workload modulating the expression of tropoelastin mRNA. Alternatively, these differences could be due to the presence of phenotypically different fibroblasts that have different basal levels of tropoelastin mRNA expression. The aim of this study was to determine whether the differences in the distribution of elastin between alveolar mucosa and attached gingiva could be due to phenotypic differences between fibroblasts derived from these tissues. Histochemical (Miller's elastin stain) and immunohistochemical staining were used to localize elastin within these tissues. Elastin production by cultured fibroblasts derived from alveolar mucosa and attached gingiva was assessed by Western blot analyses, and Northern blot analyses were used to detect levels of elastin mRNA by these cells. The results from this study have confirmed that elastin is richly expressed in oral mucosa and poorly expressed in attached gingiva. In vitro, the cells demonstrated a capacity to synthesize and secrete tropoelastin, however this was not found to differ between cells from the two different sources. These findings indicate that while some heterogeneity in fibroblast populations may exist within various tissues of the periodontium, other factors, such as functional requirements of the tissues in which they reside, may also play an important role in the phenotypic expression of these cells.

Analysis of Variance↗

PubMed2000

PDGF-C is a new protease-activated ligand for the PDGF alpha-receptor.

Platelet-derived growth factors (PDGFs) are important in many types of mesenchymal cell. Here we identify a new PDGF, PDGF-C, which binds to and activates the PDGF alpha-receptor. PDGF-C is activated by proteolysis and induces proliferation of fibroblasts when overexpressed in transgenic mice. In situ hybridization analysis in the murine embryonic kidney shows preferential expression of PDGF-C messenger RNA in the metanephric mesenchyme during epithelial conversion. Analysis of kidneys lacking the PDGF alpha-receptor shows selective loss of mesenchymal cells adjacent to sites of expression of PDGF-C mRNA; this is not found in kidneys from animals lacking PDGF-A or both PDGF-A and PDGF-B, indicating that PDGF-C may have a unique function.

Animals↗

PubMed2000

A mouse model for spinal muscular atrophy.

The survival motor neuron gene is present in humans in a telomeric copy, SMN1, and several centromeric copies, SMN2. Homozygous mutation of SMN1 is associated with proximal spinal muscular atrophy (SMA), a severe motor neuron disease characterized by early childhood onset of progressive muscle weakness. To understand the functional role of SMN1 in SMA, we produced mouse lines deficient for mouse Smn and transgenic mouse lines that expressed human SMN2. Smn-/- mice died during the peri-implantation stage. In contrast, transgenic mice harbouring SMN2 in the Smn-/- background showed pathological changes in the spinal cord and skeletal muscles similar to those of SMA patients. The severity of the pathological changes in these mice correlated with the amount of SMN protein that contained the region encoded by exon 7. Our results demonstrate that SMN2 can partially compensate for lack of SMN1. The variable phenotypes of Smn-/-SMN2 mice reflect those seen in SMA patients, providing a mouse model for this disease.

Animals↗

PubMed2000

Directed vascular expression of the thromboxane A2 receptor results in intrauterine growth retardation.

Thromboxane (Tx) A2 is a platelet agonist, smooth muscle cell constrictor, and mitogen. Urinary Tx metabolite (Tx-M) excretion is increased in syndromes of platelet activation and early in both normal pregnancies and in pregnancy-induced hypertension. A further increment occurs in patients presenting with severe preeclampsia, in whom Tx-M correlates with other indices of disease severity. TxA2 exerts its effects through a membrane receptor (TP), of which two isoforms (alpha and beta; refs. 5,6) have been cloned. Overexpression of TP in the vasculature under the control of the pre-proendothelin-1 promoter results in a murine model of intrauterine growth retardation (IUGR), which is rescued by timed suppression of Tx synthesis with indomethacin. IUGR is commonly associated with maternal diabetes or cigarette smoking, both conditions associated with increased TxA2 biosynthesis.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed2000

Amino-terminal fragments of mutant huntingtin show selective accumulation in striatal neurons and synaptic toxicity.

Huntington disease (HD) is caused by expansion of a glutamine repeat in the amino-terminal region of huntingtin. Despite its widespread expression, mutant huntingtin induces selective neuronal loss in striatal neurons. Here we report that, in mutant mice expressing HD repeats, the production and aggregation of N-terminal huntingtin fragments preferentially occur in HD-affected neurons and their processes and axonal terminals. N-terminal fragments of mutant huntingtin form aggregates and induce neuritic degeneration in cultured striatal neurons. N-terminal mutant huntingtin also binds to synaptic vesicles and inhibits their glutamate uptake in vitro. The specific processing and accumulation of toxic fragments of N-terminal huntingtin in HD-affected striatal neurons, especially in their neuronal processes and axonal terminals, may contribute to the selective neuropathology of HD.

Animals↗