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Biomedical subjects

H Lin

Publications and source records attributed to H Lin.

At least 433 records · Page 24Linked to original sources
PubMed1991

Muscle-specific expression of the troponin I gene requires interactions between helix-loop-helix muscle regulatory factors and ubiquitous transcription factors.

The quail fast skeletal troponin I (TnI) gene is a member of the contractile protein gene set and is expressed exclusively in differentiated skeletal muscle cells. TnI gene transcription is controlled by an internal regulatory element (IRE), located within the first intron, that functions as a muscle-specific enhancer. Recent studies have shown that the TnI IRE may interact directly with the muscle regulatory factors MyoD, myogenin, and Myf-5 to produce a muscle-specific expression pattern, since these factors trans-activate cotransfected TnI gene constructs in C3H10T1/2 fibroblasts. In this study, we have examined the protein-IRE interactions that are responsible for transcriptionally activating the TnI gene during skeletal muscle development. We demonstrate that the helix-loop-helix muscle regulatory factors MyoD, myogenin, Myf-5, and MRF4, when complexed with the immunoglobulin enhancer-binding protein E12, interact with identical nucleotides within a muscle regulatory factor-binding site (MRF site) located in the TnI IRE. The nuclear proteins that bind to the MRF site are restricted to skeletal muscle cells, since protein extracts from HeLa, L, and C3H10T1/2 fibroblasts do not contain similar binding activities. Importantly, the TnI MRF site alone is not sufficient to elicit the full enhancer activity associated with the IRE. Instead, two additional regions (site I and site II) are required. The proteins that interact with site I and site II are expressed in both muscle and nonmuscle cell types and by themselves are ineffective in activating TnI gene expression. However, when the MRF site is positioned upstream or downstream of site I and site II, full enhancer activity is restored. We conclude that helix-loop-helix muscle regulatory factors must interact with ubiquitously expressed proteins to generate the active TnI transcription complex that is present in differentiated muscle fibers.

Animals↗
PubMed1991

pHi regulation in frog retinal pigment epithelium: two apical membrane mechanisms.

This study demonstrates that the apical membrane of frog retinal pigment epithelium (RPE) contains two intracellular pH (pHi) regulatory mechanisms, an electrogenic Na-HCO3 cotransporter blocked by DIDS and an amiloride-inhibitable Na-H antiporter. pHi was studied using the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). In these cells resting pHi equals 7.26 +/- 0.09 (n = 58). After an acid load (NH4Cl prepulse), pHi recovery required apical extracellular Na concentration ([Na]o) in HCO3 or HCO3-free Ringer. In HCO3 Ringer recovery was completely blocked by 1 mM apical DIDS (n = 5) but was not affected by absence of Cl. In HCO3-free Ringer, recovery was completely blocked by 1 mM apical amiloride (n = 3). At resting pHi, the intrinsic pH-buffering capacity of the cell is approximately 7.1 mM/pH and rises monotonically as pHi decreases. In HCO3 Ringer, the initial rate of acidification caused by apical Na removal, 0.39 +/- 0.03 pH/min (n = 26), was 80-90% inhibited by apical DIDS (n = 5) and 16% inhibited by 1 mM apical amiloride (n = 7), but not affected by absence of Cl. In HCO3 Ringer, initial rates of acidification induced by apical DIDS or amiloride were 0.11 +/- 0.06 (n = 5) and 0.03 +/- 0.02 pH/min (n = 7), respectively. These results indicate that the Na-HCO3 cotransporter accounts for 80-90% of the acid extrusion from frog RPE cells. Increasing apical [K]o from 2 to 5 mM approximates the in vivo apical [K]o changes during a light-dark transition and alkalinizes the cells. [K]o-induced alkalinization had an initial rate of 0.11 +/- 0.02 pH/min (n = 16), which was approximately 75% inhibited by apical DIDS (to 0.04 +/- 0.01 pH/min, n = 7) and completely blocked by HCO3/CO2 removal from both bathing solutions. [K]o-induced pHi changes alter RPE transport mechanisms and may affect RPE-photoreceptor interactions.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1991

Nasopharyngeal carcinoma and lymphoinfiltration.

Originally referred to as 'lymphoepithelioma', undifferentiated and poorly differentiated nasopharyngeal carcinoma (NPC) tissues showed intense lymphoinfiltration. In a study of cryosections from 15 NPC tissues, we found that infiltrating lymphoid elements were comprised predominantly of lymphocytes, but plasma cells, follicular dendritic cells, and eosinophils were also commonly seen. Subpopulations of lymphocytes having the same phenotypes tend to aggregate, forming clusters or secondary follicles in stromatous tissues. The tumor areas were mainly infiltrated by T cells. Tumor cells and/or apparently normal epithelium in the paratumorous areas frequently expressed CD21, CD23, CD40 and a B lymphocytes carcinoma cross-reacting antigen (BLCa), all of which are involved in B cell activation and proliferation. CD21 and BLCa were strongly expressed near the surface of both squamous and columnar epithelium by those epithelial cells which are at advanced stage of differentiation, while CD40 was expressed by epithelial cells at earlier stages of differentiation located at or near the basement membrane. CD23 was mainly expressed by columnar cells and basal cells underlying squamous epithelium, but not, or weakly so, by flattened squamous cells or reserve cells underlying columnar epithelium. The large majority of tumor cells expressed CD40 and BLCa. A substantial proportion of them also expressed CD23, but the tumor cells were not reactive for CD21. Despite eosinophilic infiltration, IL-6 was not detected in tumor tissues. IL-1 was, however, detected in abundance in the cytoplasm of follicular dendritic-like cells and in the intercellular spaces in tumor areas and surrounding stromatous tissues. The immunobiology of NPC is discussed in the light of these observations.

Antibodies, Monoclonal↗
PubMed1991

The in situ expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA at the maternal-fetal interface.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is produced by cells in the placenta, is known to be a growth factor for trophoblast cells in vitro and when injected into pregnant mice at risk for mid-gestation fetal resorption, dramatically lowers the fetal death rate while stimulating placental and fetal growth. We describe here the localization of GM-CSF mRNA expression in murine placenta by in situ hybridization. It is found in small round cells (lymphoid-like) and endothelial cells in the maternal decidua. In addition, GM-CSF transcripts are located in cells of the spongiotrophoblast zone (trophoblast-like cells), but not in the labyrinthine zone. These results indicate that GM-CSF may be influencing the growth and function of the fetal placenta in a paracrine-autocrine manner. These results support earlier observations that link GM-CSF production during pregnancy to decidual T-lymphocytes and further suggest a placental source within the invasive trophoblast.

Animals↗
PubMed1990

Structure and function of the enhancer 3' to the human A gamma globin gene.

An enhancer is located immediately 3' to the A gamma globin gene. We have used DNase I footprinting to map the sites of interaction of nuclear proteins with the DNA sequences of this enhancer. Eight footprints were discovered, distributed over 600 base pairs of DNA. Three of these contain a consensus binding site for the erythroid specific factor GATA-I. Each of these GATA-1 sites had an enhancer activity when inserted into a reporter plasmid and tested in human erythroleukemia cells. Other footprints within the enhancer contained consensus binding sequences for the ubiquitous, positive regulatory proteins AP2 and CBP-1. An Sp1-like recognition sequence was also identified. Synthetic oligonucleotides encompassing two of the footprints generated a slowly migrating complex in gel mobility shift assays. The same complex forms on a fragment of the human gamma globin gene promoter extending from -260 to -200. The DNaseI footprint of this protein complex with the enhancer overlapped a sequence, AGGAGGA, found within the binding site for a protein that interacts with the chicken beta globin promoter and enhancer, termed the stage selector element. We propose that this complex of proteins may be involved in the human gamma globin promoter-enhancer interaction.

Animals↗
PubMed1990

Membrane localization of the pertussis toxin-sensitive G-protein subunits alpha i-2 and alpha i-3 and expression of a metallothionein-alpha i-2 fusion gene in LLC-PK1 cells.

The renal epithelial cell line LLC-PK1 has topographically distinct regulatory roles for the alpha subunits of pertussis toxin-sensitive guanine nucleotide regulatory proteins (alpha i subunit); these include the inhibition of adenylyl cyclase at the basolateral membrane and the stimulation of Na+ channel activity at the apical membrane. We now report that LLC-PK1 cells contain two members of the alpha i protein family, alpha i-2 and alpha i-3, which have distinct cellular locations consistent with their diverse functional roles. By using specific alpha i antibodies and immunofluorescence, the alpha i-2 subunit was found to be localized to the basolateral membrane, whereas the alpha i-3 subunit was concentrated in the Golgi and was also detectable at low levels on apical membranes in some cells. Induction of a chimeric mouse metallothionein 1-rat or canine alpha i-2 gene stably transfected into the LLC-PK1 cells produced an increase in the content of the alpha i-2 subunit, which was targeted only to the basolateral membrane. These findings suggest that alpha i subunit specificity for effectors may be achieved in polarized renal epithelial cells by their geographic segregation to different cellular membranes. The LLC-PK1 cell stably transfected with the metallothionein-alpha i-2 fusion gene will provide a model for the study of guanine nucleotide regulatory protein function in epithelia.

Animals↗
PubMed1990

Expression of recombinant genes in myocardium in vivo after direct injection of DNA.

The ability to program recombinant gene expression in cardiac myocytes in vivo holds promise for the treatment of many inherited and acquired cardiovascular diseases. In this report, we demonstrate that a recombinant beta-galactosidase gene under the control of the Rous sarcoma virus promoter can be introduced into and expressed in adult rat cardiac myocytes in vivo by the injection of purified plasmid DNA directly into the left ventricular wall. Cardiac myocytes expressing recombinant beta-galactosidase were detected histochemically in rat hearts for at least 4 weeks after injection of the beta-galactosidase gene. These results demonstrate the potential of this method of somatic gene therapy for the treatment of cardiovascular disease.

Animals↗
PubMed1990

[Synthesis, cloning and expression of human alpha-atrial natriodiuretic peptide gene in yeast system].

Human alpha-ANP was synthesised using a yeast system. In order to obtain correct expression of the human alpha-ANP gene, a few bases on one strand were substituted by the corresponding yeast sequence, but those bases were not replaced in the complementary so a few unpaired bases existed. The gene was cloned into a shuttle vector (with alpha-factor). The recombinants containing different human alpha-ANP DNA sequences were separated by BglII restriction analysis. We compared the expression level of human alpha-ANP from two kinds of genes and the results showed that the ANP expression level by the gene containing yeast sequences was lower (0.5-0.7 mg/L) than that expressed by the original gene (0.8-1.0 mg/L).

Atrial Natriuretic Factor↗
PubMed1990

Influence of dopamine on the liver assessed by changes in arterial ketone body ratio in brain-dead dogs.

The influence of dopamine on liver metabolism in the state of brain death was assessed by measuring arterial ketone body ratio (AKBR) in dogs. Mean arterial blood pressure (MABP) was significantly decreased, from 137.4 +/- 3.7 to 64.7 +/- 2.8 mm Hg, 1 hour after completion of brain death (p less than 0.01). In the control group AKBR was maintained at the near control value of 1.07 thereafter, concomitant with a significant decrease in serum lactate levels, despite marked hypotension (p less than 0.05). Dopamine infusion at rates of 5 and 10 micrograms/kg/min sustained both AKBR and MABP at near control values. In contrast, dopamine given at doses greater than 15 micrograms/kg/min caused a significant reduction of AKBR, to less than 0.66 +/- 0.12 (p less than 0.01), although MABP was restored to near-normal levels. In addition, serum levels of alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase were significantly elevated, reflecting liver cell injury. It is suggested that the liver is primarily tolerant to hypotension in the state of brain death and that dopamine administered at a rate of 15 micrograms/kg/min or more impairs liver metabolism by reducing the redox state (free nicotinamide-adenine dinucleotide/reduced nicotinamide-adenine dinucleotide) of liver mitochondria.

Animals↗
PubMed1990

Prognostic implications of postoperative suppression of arterial ketone body ratio: time factor involved in the suppression of hepatic mitochondrial oxidation-reduction state.

To determine the tolerance limit of the liver in the critically suppressed mitochondrial oxidation-reduction state, the arterial ketone body ratio (acetoacetate/3-hydroxybutyrate), which reflects hepatic mitochondrial oxidation-reduction potential, was measured 1319 times in 161 patients during the postoperative critical period. Because patients who showed arterial ketone body ratios between 0.40 and 0.25 had a higher incidence of postoperative complications than had those who showed ratios above 0.40, this was designated as the critical zone of the arterial ketone body ratio. When duration in the critical zone was less than 2 days, 90% of the patients were able to tolerate the condition and survive. By contrast, when an arterial ketone body ratio below 0.40 was prolonged for more than 5 days, there was a high incidence of multiple organ failure and a 100% mortality rate, with the average survival period after a 5-day suppression being estimated as 5.7 +/- 2.4 days. It is suggested that the arterial ketone body ratio in the critical zone must be returned to normal values within 2 days to obtain a good prognosis.

Adolescent↗
PubMed1989

A 165-base pair sequence between the dihydrofolate reductase gene and the divergently transcribed upstream gene is sufficient for bidirectional transcriptional activity.

The dihydrofolate reductase gene encodes a key enzyme of one-carbon metabolism and is constitutively expressed in all cells. Recently, transcripts initiated at 89 base pairs upstream from the transcriptional initiation site of the dihydrofolate reductase gene and transcribed from the opposite strand have been identified and shown to encode for a protein with homology to a bacterial DNA mismatch repair enzyme (Fujii, H., and Shimada, T. (1989) J. Biol. Chem. 264, 10057-10064). Therefore, the two genes are organized in a head-to-head configuration separated by an 89-base pair segment. The promoter activities of this short spacer sequence were studied in a transient assay using the chloramphenicol acetyltransferase and the guanine phosphoribosyltransferase genes as reporters. A 165-base pair fragment from -111 to +54 relative to the dihydrofolate reductase initiation site was shown to be sufficient for transcriptional activity in either direction, suggesting that expression of the two divergent genes is regulated by a bidirectional promoter that may use common regulatory elements.

Animals↗
PubMed1989

Cloning and analysis of fs(1) Ya, a maternal effect gene required for the initiation of Drosophila embryogenesis.

The maternal effect locus fs(1) Ya is required for the fusion of the apposed sperm and egg pronuclei (syngamy) following fertilization in Drosophila. It is tightly linked to another complementation group, fs(1) Yb, needed for both oogenesis and embryogenesis. We have isolated a set of overlapping cloned sequences in the 3B4-6 region of the X chromosome encompassing the fs(1) Ya-fs(1) Yb region. A single 2.4 kb maternal transcript is encoded within this region, and an 8.5 kb DNA fragment that contains this transcript complements both fs(1) Ya and fs(1) Yb mutations. Northern and in situ hybridization analyses show that the maternal transcript is only present in nurse cells and oocytes beginning in previtellogenic stages, and is evenly distributed in the cytoplasm of 0-2 h syncytial embryos. The transcript is not detected in later stages of embryonic development. This expression pattern correlates closely with the genetic and developmental characteristics expected of the fs(1) Ya gene product.

Animals↗
PubMed1989

Responses of the working rat heart to carbon monoxide.

The effects of carbon monoxide (CO) were studied in the isolated working rat heart. Hearts removed from male laboratory rats were perfused via the left atrium with Krebs-Henseleit solution (KH) oxygenated with 95% O2-5% CO2 (O2). Heart rate and arterial pressures were measured by a transducer inserted in the aortic outflow line and connected to a data logger. Aortic flow was determined by collecting the effluent from the aortic bubble trap in a graduated cylinder. Coronary flow through the pulmonary cannula was collected and measured in a graduated cylinder. After 30 min, the hearts were challenged with solutions containing either CO (5% CO-90% O2-5% CO2) or N2 (5% N2-90% O2-5% CO2) for 10 min (Challenge I). After recovery in O2, the hearts were challenged with the alternate test solution (Challenge II). CO increased coronary flow (CF) and coronary flow as a percent of cardiac output (CF%) 13 and 16% respectively. N2 had no significant effect on CF or CF%. CO and N2 had no significant effect on heart rate, cardiac output, oxygen consumption or on aortic flow or pressure. These results indicate that vasodilation is the major response of the working heart to CO, and this response is not mediated by simple hypoxia.

Animals↗