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Biomedical subjects

H Yang

Publications and source records attributed to H Yang.

At least 613 records · Page 34Linked to original sources
PubMed1997

Regulatory volume decrease by SV40-transformed rabbit corneal epithelial cells requires ryanodine-sensitive Ca2+-induced Ca2+ release.

The relationship between relative cell volume and time-dependent changes in intracellular Ca2+ concentration ([Ca2+]i) during exposure to hypotonicity was characterized in SV-40 transformed rabbit corneal epithelial cells (tRCE) (i). Light scattering measurements revealed rapid initial swelling with subsequent 97% recovery of relative cell volume (characteristic time (tauvr) was 5.9 min); (ii). Fura2-fluorescence single-cell imaging showed that [Ca2+]i initially rose by 216% in 30 sec with subsequent return to near baseline level after another 100 sec. Both relative cell volume recovery and [Ca2+]i transients were inhibited by either: (a) Ca2+-free medium; (b) 5 mM Ni2+ (inhibitor of plasmalemma Ca2+ influx); (c) 10 microM cyclopiazonic acid, CPA (which causes depletion of intracellular Ca2+ content); or (d) 100 microM ryanodine (inhibitor of Ca2+ release from intracellular stores). To determine the temporal relationship between an increased plasmalemma Ca2+ influx and the emptying of intracellular Ca2+ stores during the [Ca2+]i transients, Mn2+ quenching of fura2-fluorescence was quantified. In the presence of CPA, hypotonic challenge increased plasmalemma Mn2+ permeability 6-fold. However, Mn2+ permeability remained unchanged during exposure to either: 1.100 microM ryanodine; 2.10 microM CPA and 100 microM ryanodine. This report for the first time documents the time dependence of the components of the [Ca2+]i transient required for a regulatory volume decrease (RVD). The results show that ryanodine sensitive Ca2+ release from an intracellular store leads to a subsequent increase in plasmalemma Ca2+ influx, and that both are required for cells to undergo RVD.

Animals↗
PubMed1997

A conducting salt-based amperometric biosensor for measurement of extracellular lactate accumulation in ischemic myocardium.

In this paper, fabrication, characterization, and physiological application of a miniaturized amperometric lactate biosensor are described. The sensor is based on cross-linked lactate oxidase and tetrathiafulvalene-tetracyano-quinodimethane (TTF-TCNQ) charge transfer complex. The sensor was developed for continuous quantitative measurement of the lactate accumulation in ischemic myocardium under severe depletion of oxygen. The sensor was evaluated in vitro at an applied potential of 0.15 V vs Ag/AgCl; it proved to combine all the performance characteristics desired for the present application, such as proper response in absence of oxygen, good operational stability, good accuracy and precision (103.5 +/- 1.2%), adequate response time (t95% = 80 s), and wide linear dynamic range up to 27 mM (r = 0.9998) in N2-saturated solutions and at 37 degrees C. The prepared sensors (n = 12) showed sensitivity of 380 +/- 90 nA/mM, and a background current of 240 +/- 50 nA. The lower limit of detection is 0.4 +/- 0.15 mM with a S/N ratio equal to 3. Results obtained for direct lactate monitoring in ischemic rabbit papillary muscle under no-flow conditions and PO2 < 6 mm Hg are presented.

Animals↗
PubMed1997

Long-term function of fish islet xenografts in mice by alginate encapsulation.

BACKGROUND: Large, anatomically discrete pancreatic islets, Brockmann bodies (BBs), exist in certain teleost fish. When transplanted under the renal capsules of streptozotocin-diabetic athymic nude mice, BB grafts produce uniform normoglycemia for 50 days and mammalian-like glucose tolerance profiles; however, these very discordant islets reject in 7-8 days when transplanted into euthymic BALB/c mice. METHODS: In the present study, BBs were mass harvested, minced into <1-mm tissue fragments, and encapsulated in alginate-based macrospheres (5 mm diameter) or noodles (0.5x3 cm). Nonencapsulated and encapsulated BB fragments were transplanted intraperitoneally into streptozotocin-diabetic (nonfasting blood glucose >400 mg/dl) nu/nu and BALB/c mice. Glucose levels were monitored at least 3 times a week. RESULTS: Encapsulated BB grafts uniformly survived >50 days (10/10) or >100 days (3/3) in nu/nu recipients. The mean graft survival time was 27+/-13 days in BALB/c recipients (n=7). Daily intraperitoneal administration of 2.5 mg/kg 15-deoxyspergualin, in combination with encapsulation, resulted in uniform long-term BB graft function in BALB/c recipients (n=5). Similarly, long-term function was achieved in four of six BALB/c recipients with daily intraperitoneal administration of 10 mg/kg cyclosporine (two grafts failed after 39 and 45 days). Nonencapsulated BB grafts transplanted intraperitoneally into BALB/c or nu/nu recipients functioned for <7 days; immunosuppression alone did not permit graft survival in BALB/c recipients. In all cases of graft survival of >50 days, grafts were surgically removed from the peritoneal cavity, and blood sugar levels returned to a diabetic state within a few days. Historical sections of grafts, stained with hematoxylin and eosin and immunoperoxidase for insulin, showed viable, well-granulated BB tissue. CONCLUSIONS: This study demonstrates that tilapia BBs are suitable for encapsulation and that encapsulated BBs can be made to function long term in diabetic mice.

Alginates↗
PubMed1997

A "knockdown" mutation created by cis-element gene targeting reveals the dependence of erythroid cell maturation on the level of transcription factor GATA-1.

The hematopoietic-restricted transcription factor GATA-1 is required for both mammalian erythroid cell and megakaryocyte differentiation. To define the mechanisms governing its transcriptional regulation, we replaced upstream sequences including a DNase I hypersensitive (HS) region with a neomycin-resistance cassette by homologous recombination in mouse embryonic stem cells and generated mice either harboring this mutation (neoDeltaHS) or lacking the selection cassette (DeltaneoDeltaHS). Studies of the consequences of these targeted mutations provide novel insights into GATA-1 function in erythroid cells. First, the neoDeltaHS mutation leads to a marked impairment in the rate or efficiency of erythroid cell maturation due to a modest (4- to 5-fold) decrease in GATA-1 expression. Hence, erythroid differentiation is dose-dependent with respect to GATA-1. Second, since expression of GATA-1 from the DeltaneoDeltaHS allele in erythroid cells is largely restored, transcription interference imposed by the introduced cassette must account for the "knockdown" effect of the mutation. Finally, despite the potency of the upstream sequences in conferring high-level, developmentally appropriate expression of transgenes in mice, other cis-regulatory elements within the GATA-1 compensate for its absence in erythroid cells. Our work illustrates the usefulness of targeted mutations to create knockdown mutations that may uncover important quantitative contributions of gene function not revealed by conventional knockouts.

Alleles↗
PubMed1997

Multifocal defects in immune responses in RelB-deficient mice.

Mice with a targeted disruption of the Rel/nuclear factor-kappaB family member RelB develop a complex inflammatory phenotype, myeloid hyperplasia, and splenomegaly due to extramedullary hemopoiesis. In this work, we report that RelB-deficient mice, in addition to the pathologic changes, were highly susceptible to infection by the facultative intracellular bacterium Listeria monocytogenes. RelB binds transcriptionally active kappaB motifs in the TNF-alpha promoter in normal cells, and in vitro studies with macrophages isolated from RelB-deficient animals revealed impaired production of TNF-alpha in response to LPS and IFN-gamma. RelB-deficient mice also were unable to mount a protective immune response against lymphocytic choriomeningitis virus. These results indicate a defective T cell-macrophage interaction and cytotoxic T cell response, respectively, in mice lacking RelB. Analysis of resting and specific Ab production demonstrated that while RelB is not required for the secretion of Ig isotypes that result from heavy chain class switching, it is necessary for normal production of Ag-specific IgG in response to T cell-dependent and -independent stimuli. Thus, RelB is not only essential for a normal hemopoietic system in the unchallenged animal, but also involved in various specific and nonspecific immune responses.

Animals↗
PubMed1997

Anterior iliac crest bone graft. Anatomic considerations.

STUDY DESIGN: A morphologic study of the anterior part of the iliac crest was performed. OBJECTIVE: To define the anatomic characteristics of the anterior part of the ilium and to determine an optimal area to harvest the iliac bone graft from the anterior iliac crest. SUMMARY OF BACKGROUND DATA: Stress fracture or avulsion fracture of the anterior cut for anterior iliac crest graft have been noted previously. However, there is insufficient published information on the morphology of the anterior part of the ilium relative to the optimal location of harvesting the bone graft. METHODS: Direct measurements using digital calipers were taken from 30 dried human pelves and 10 cadaveric pelves. The thickness of the anterior part of the ilium was measured, with different starting points on the iliac crest. The length of the bicortical iliac bone graft also was determined. RESULTS: The thickest portion of the ilium was 18.9 +/- 2.3 mm at the iliac tubercle, which was 45% thicker than at a point 3 cm posterior to the anterior superior iliac spine. The thick region of the anterior iliac crest extended 54.0 +/- 10.2 mm posteriorly from a point 3 cm posterior to the anterior superior iliac spine. The mean length of a 10 mm thick bicortical iliac tubercle bone graft was 36.8 +/- 8.7 mm. CONCLUSIONS: The region around the iliac tubercle is suitable for harvesting bicortical or tricortical bone graft.

Bone Transplantation↗
PubMed1997

Involvement of MAP kinase in angiotensin II-induced phosphorylation and intracellular targeting of neuronal AT1 receptors.

MAP kinase stimulation is a key signaling event in the AT1 receptor (AT1R)-mediated chronic stimulation of tyrosine hydroxylase and norepinephrine transporter in brain neurons by angiotensin II (Ang II). In this study, we investigated the involvement of MAP kinase in AT1R phosphorylation to further our understanding of these persistent neuromodulatory actions of Ang II. Ang II caused a time-dependent phosphorylation of neuronal AT1R. This phosphorylation was associated with internalization and translocation of AT1R into the nucleus. MAP kinase also stimulated phosphorylation of neuronal AT1R. The conclusion that MAP kinase participates in neuronal AT1R phosphorylation and its targeting into the nucleus is supported further by the following. (1) MAP kinase-mediated phosphorylation of AT1R was blocked by the AT1R antagonist losartan; (2) AT1R co-immunoprecipitated with MAP kinase; (3) MAP kinase-kinase inhibitor PD98059 attenuated Ang II-induced phosphorylation of AT1R; and (4) PD98059 blocked Ang II-induced nuclear translocation of AT1Rs. In summary, these observations demonstrate that Ang II-induced phosphorylation of AT1R is mediated by its activation of MAP kinase. A possible role of AT1R translocation into the nucleus on persistent neuromodulatory actions of Ang II has been discussed.

1-Sarcosine-8-Isoleucine Angiotensin II↗
PubMed1997

Functional expression of a cDNA to human acyl-coenzyme A:cholesterol acyltransferase in yeast. Species-dependent substrate specificity and inhibitor sensitivity.

We have identified two yeast genes with similarity to a human cDNA encoding acyl-coenzyme A:cholesterol acyltransferase (ACAT). Deletion of both yeast genes results in a viable cell with undetectable esterified sterol (Yang, H., Bard, M., Bruner, D. A., Gleeson, A., Deckelbaum, R. J., Aljinovic, G., Pohl, T., Rothstein, R., and Sturley, S. L. (1996) Science 272, 1353-1356). Here, we expressed the human cDNA in the yeast double mutant, resulting in high level production of ACAT protein, but low in vivo esterification of ergosterol, the predominant yeast sterol. The activity of the human enzyme was increased by incubation of these cells with 25-hydroxy, cholesterol, an established positive regulator of mammalian sterol esterification. In contrast, the yeast enzymes were unaffected by this reagent. In vitro microsomal assays indicated no sterol esterification in extracts from the double mutant. However, significant activity was detected from strains expressing human ACAT when cholesterol was equilibrated with the microsomal membranes. The human enzyme in yeast utilized cholesterol as the preferred sterol and was sensitive to competitive (S58035) and non-competitive (DuP 128) ACAT inhibitors. The yeast esterifying enzymes exhibited a diminished sterol substrate preference and were sensitive only to S58035. Human ACAT had a broad acyl-CoA substrate specificity, the other substrate for this reaction. By contrast, the yeast enzymes had a marked preference for specific acyl-CoAs, particularly unsaturated C18 forms. These results confirm the yeast genes as functional homologs of the human gene and demonstrate that the enzymes confer substrate specificity to the esterification reaction in both organisms.

Cholesterol↗
PubMed1997

Arabidopsis thaliana ECP63 encoding a LEA protein is located in chromosome 4.

DCECP63 is a carrot embryogenic cell protein. To perform its genetic analysis, we isolated an Arabidopsis thaliana (At) ECP63 cDNA. The cDNA encoded a polypeptide of 449 amino acids (aa). Its deduced aa sequence showed extensive similarity with DCECP63, DCDC8 and BPBP8 which are embryonic proteins isolated from carrot and birch, respectively. The aa sequence contained eight well-conserved tyrosine phosphorylation sites and 16 repeats of 11 aa. Southern analysis showed that the AtECP63 gene might belong to a small multigene family. The AtECP63 transcripts accumulated specifically in mature seeds, and exogenous abscisic acid (ABA) induced its expression in immature siliques, but not in vegetative tissues. These results suggested that the AtECP63 gene encoding a putative phosphotyrosine protein belonging to late embryogenesis abundant (LEA) protein in group 3 might be involved in maturation and desiccation tolerance of seeds. Restriction fragment length polymorphism (RFLP) and cleaved amplified polymorphic sequence (CAPS) mapping showed that AtECP63 gene was present in the South part of chromosome 4.

Amino Acid Sequence↗
PubMed1997

Novel signal transduction and peptide specificity of glucagon-like peptide receptor in 3T3-L1 adipocytes.

Glucagon-like peptide-1 (7-36) amide (GLP-1), in addition to its well known effect of enhancing glucose-mediated insulin release, has been shown to have insulinomimetic effects and to enhance insulin-mediated glucose uptake and lipid synthesis in 3T3-L1 adipocytes. To elucidate the mechanisms of GLP-1 action in these cells, we studied the signal transduction and peptide specificity of the GLP-1 response. In 3T3-L1 adipocytes, GLP-1 caused a decrease in intracellular cAMP levels which is the opposite to the response observed in pancreatic beta cells in response to the same peptide. In 3T3-L1 adipocytes, free intracellular calcium was not modified by GLP-1. Peptide specificity was examined to help determine if a different GLP receptor isoform was expressed in 3T3-L1 adipocytes vs. beta cells. Peptides with partial homology to GLP-1 such as GLP-2, GLP-1 (1-36), and glucagon all lowered cAMP levels in 3T3-L1 adipocytes. In addition, an antagonist of pancreatic GLP-1 receptor, exendin-4 (9-39), acted as an agonist to decrease cAMP levels in 3T3-L1 adipocytes as did exendin-4 (1-39), a known agonist for the pancreatic GLP-1 receptor. Binding studies using 125I-GLP-1 also suggest that pancreatic GLP-1 receptor isoform is not responsible for the effect of GLP-1 and related peptides in 3T3-L1 adipocytes. Based on these results, we propose that the major form of the GLP receptor in 3T3-L1 adipocytes is functionally different from the pancreatic GLP-1 receptor.

3T3 Cells↗
PubMed1997

Factors influencing the identification of major genes in a complex disease genome scan.

A two-stage linkage strategy was employed to identify major genes for a simulated complex disease via a genome scan. The importance of several approaches for improving the ability to locate major genes has been illustrated. These approaches are: adjusting for covariates, ascertaining through multiple affected family members, increasing the sample size, and using multipoint linkage analysis.

Chromosome Mapping↗
PubMed1997

Functional comparison of mouse, rat, and fish islet grafts transplanted into diabetic nude mice.

Equal volumes of teleost fish (tilapia), Lewis rat, or CD-1 mouse islets were transplanted under the kidney capsules of streptozotocin-diabetic athymic nude mice. Nonfasting blood glucose levels were monitored in recipient mice over a period of 30 days. Mean nonfasting blood glucose levels in recipients of tilapia (n = 7), rat (n = 8), and murine (n = 8) islets were 78.8, 77.0, and 115 mg/dl, respectively. Mean blood glucose levels were significantly higher in recipients of murine islets than in recipients of fish and rat islets. After Day 30, intraperitoneal glucose tolerance tests were performed on recipient mice. Mean fasted blood glucose levels in mouse, rat, and fish islet recipients were 113.3, 89.8, and 72.7 mg/dl, respectively. All three groups of recipient mice had similar glucose tolerance profiles with mean glucose disappearance rates (K values) between 4.3 and 5.7. Tilapia islet grafts resulted in a significantly lower baseline for blood glucose values than either rat or mouse islet grafts.

Animals↗
PubMed1997

A blind testing design for authenticating ancient DNA sequences.

Reproducibility is a serious concern among researchers of ancient DNA. We designed a blind testing procedure to evaluate laboratory accuracy and authenticity of ancient DNA obtained from closely related extant and extinct species. Soft tissue and bones of fossil and contemporary museum proboscideans were collected and identified based on morphology by one researcher, and other researchers carried out DNA testing on the samples, which were assigned anonymous numbers. DNA extracted using three principal isolation methods served as template in PCR amplifications of a segment of the cytochrome b gene (mitochondrial genome), and the PCR product was directly sequenced and analyzed. The results show that such a blind testing design performed in one laboratory, when coupled with phylogenetic analysis, can nonarbitrarily test the consistency and reliability of ancient DNA results. Such reproducible results obtained from the blind testing can increase confidence in the authenticity of ancient sequences obtained from postmortem specimens and avoid bias in phylogenetic analysis. A blind testing design may be applicable as an alternative to confirm ancient DNA results in one laboratory when independent testing by two laboratories is not available.

Animals↗
PubMed1997

Rat alveolar macrophage cytokine production and regulation of neutrophil recruitment following acute ozone exposure.

The alveolar macrophage generation of interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) cytokines has been implicated in the recruitment of neutrophils into acutely injured lungs. To examine the role of these cytokines in neutrophil chemotaxis, cytokine mRNA transcripts and content were examined in macrophages lavaged from rats immediately following 6 hr exposure to air or 1 ppm ozone. Ozone exposure enhanced the number of lavaged macrophages demonstrating mRNA transcripts and immunocytochemical staining for IL-1 beta and TNF-alpha. These changes occurred prior to ozone-induced increases in permeability and lavageable neutrophils. The supernatant from in vitro macrophage cultures demonstrated ozone-associated enhancements in neutrophil chemotactic activity and in IL-1 beta and TNF-alpha levels. However, treatment of the macrophage-conditioned media with anti-IL-1 beta and anti-TNF-alpha antibodies separately and in combination demonstrated that these cytokines were not directly responsible for the observed neutrophil chemoattraction. However, coculturing the macrophages with anti-IL-1 beta and anti-TNF-alpha together, but not separately, resulted in a 44% inhibition of media chemotactic activity, suggesting that maximal macrophage generation of chemoattractants was dependent on either IL-1 beta or TNF-alpha. The mRNA transcripts for the neutrophil chemoattractants macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (CINC) were found to be enhanced in cultured macrophages from ozone-exposed rats, but reduced on incubation with anti-IL-1 beta and anti-TNF-alpha together. These results demonstrated that ozone-induced enhancements in IL-1 beta and TNF-alpha productions appear not to be associated directly with neutrophil chemoattraction, but are more likely involved in stimulating the generation of the neutrophil chemoattractants MIP-2 and CINC.

Animals↗
PubMed1997

Anatomic considerations of the principal nutrient foramen and artery on internal surface of the ilium.

Direct measurements of the nutrient foramen of thirty dried ilia using digital calipers and observations of the nutrient a. from ten cadaveric specimens were made in the present study. The nutrient foramen was situated 12.5 +/- 2.7 mm lateral to the anterosuperior sacroiliac joint line but perpendicular to this line and 23.5 +/- 5.8 mm above the pelvic brim parallel to the sacroiliac joint line. The nutrient a. originated from the iliolumbar a. as it coursed across the anterosuperior aspect of the sacroiliac joint. The present anatomic study indicates that the nutrient a. on the internal surface of the ilium is prone to injury as a result of traumatic disruption of the sacroiliac joint, sacral alar fractures and during the anterior approach to the sacroiliac joint.

Aged↗
PubMed1997

Anatomic considerations of the second sacral vertebra and dorsal screw placement.

Direct measurements and measurements from images of axial cross-sections on 20 cadaveric sacra that had been scanned on computer were used in this study. The measurements, including parameters from the vertebral body, lateral mass and spinal canal of the second sacral vertebra (S2) were performed. The length of the screw path and the optimal angulation of the screw placement for dorsal sacral internal fixation were also included. The mean values of height, anteroposterior diameter, width and breadth of the S2 were 25.0 mm, 13.5 mm, 29.4 mm and 83.0 mm, respectively. The mean values of the mid-sagittal diameter, maximum transverse diameter and area of the S2 spinal canal were 10.3 mm, 23.1 mm and 162.4 mm2, respectively. The mean transpedicular screw length of the S2 and optimal medial angle were 25.2 mm and 30.0 degrees, respectively. The mean lateral mass screw length of the S2 and optimal lateral angle were 32.8 mm and 22.0 degrees, respectively. The present study provides quantitative anatomic data of the second sacral vertebra. All parameters indicate that, compared with our previous study, S2 is smaller than S1. When S2 lateral mass screw fixation is intended, anchoring the anterior cortex may violate the iliac vessels or lumbosacral trunk; therefore, understanding the unique anatomy of the S2 is imperative.

Aged↗
PubMed1997

Regulation by tetracycline of gene expression in Saccharomyces cerevisiae.

A convenient system for the control of gene expression in Saccharomyces cerevisiae was developed. Tetracycline-responsive promoters were constructed by fusing the tetracycline operator (tetO) to the S. cerevisiae HOP1 promoter. When fused to the tetracycline repressor (tetR), trans-activation domains of both GAL4 and HAP4 were capable of promoting transcription from the tetO-HOP1 chimeric promoter, but the tetR-HAP4 fusion activator was the more efficient transcriptional activator. Addition of tetracycline nearly completely repressed activator-dependent transcription from the tetO-HOP1 promoter. Moreover, tetracycline-dependent repression of YEF3, CDC28 and RAM2 expression impaired cell growth. Thus, this system is useful for the elucidation of gene function in S. cerevisiae.

Gene Expression Regulation, Fungal↗