Biomedical subjects

Y Song

Publications and source records attributed to Y Song.

At least 37 records · Page 2Linked to original sources

PubMed2001

In vivo glycosylation suppresses the aggregation of amyloidogenic hen egg white lysozymes expressed in yeast.

The mutant hen egg white lysozymes Ile55Thr and Asp66His, corresponding to human amyloidogenic mutant lysozymes Ile56Thr and Asp67His, respectively, were secreted in Saccharomyces cerevisiae. The amyloidogenic mutants (I55T and D66H) of hen egg white lysozymes were remarkably less soluble than that of the wild-type protein. To enhance the secretion of these mutants, we constructed the glycosylated amyloidogenic lysozymes (I55T/G49N and D66H/G49N) having the N-glycosylation signal sequence (Asn-X-Ser) by the substitution of glycine with asparagine at position 49. The secretion of these glycosylated mutant proteins is greatly increased in S. cerevisiae, compared with that of non-glycosylated type. Both the glycosylated mutants retained about 40% enzymatic activity when incubated at pH 7.4 for 1 h at the physiological temperature of 37 degrees C whereas the non-glycosylated proteins eventually lost all activity under these conditions. These results suggest that the glycosylated chains could mask the beta-strand of amyloidogenic lysozymes from the intermolecular cross-beta-sheet association, thus improving the solubility of amyloidogenic lysozymes.

Amyloid↗

PubMed2001

Importance of internal regions and the overall length of tropomyosin for actin binding and regulatory function.

Tropomyosin (Tm) binds along actin filaments, one molecule spanning four to seven actin monomers, depending on the isoform. Periodic repeats in the sequence have been proposed to correspond to actin binding sites. To learn the functional importance of length and the internal periods we made a series of progressively shorter Tms, deleting from two up to six of the internal periods from rat striated alpha-TM (dAc2--3, dAc2--4, dAc3--5, dAc2--5, dAc2--6, dAc1.5--6.5). Recombinant Tms (unacetylated) were expressed in Escherichia coli. Tropomyosins that are four or more periods long (dAc2--3, dAc2--4, and dAc3--5) bound well to F-actin with troponin (Tn). dAc2--5 bound weakly (with EGTA) and binding of shorter mutants was undetectable in any condition. Myosin S1-induced binding of Tm to actin in the tight Tm-binding "open" state did not correlate with actin binding. dAc3--5 and dAc2--5 did not bind to actin even when the filament was saturated with S1. In contrast, dAc2--3 and dAc2--4 did, like wild-type-Tm, requiring about 3 mol of S1/mol of Tm for half-maximal binding. The results show the critical importance of period 5 (residues 166--207) for myosin S1-induced binding. The Tms that bound to actin (dAc2--3, dAc2--4, and dAc3--5) all fully inhibited the actomyosin ATPase (+Tn) in EGTA. In the presence of Ca(2+), relief of inhibition by these Tms was incomplete. We conclude (1) four or more actin periods are required for Tm to bind to actin with reasonable affinity and (2) that the structural requirements of Tm for the transition of the regulated filament from the blocked-to-closed/open (relief of inhibition by Ca(2+)) and the closed-to-open states (strong Tm binding to actin-S1) are different.

Actins↗

PubMed2001

Non-oxidative mechanisms are responsible for the induction of mutagenesis by reduction of Cr(VI) with cysteine: role of ternary DNA adducts in Cr(III)-dependent mutagenesis.

Intracellular reduction of carcinogenic Cr(VI) generates Cr-DNA adducts formed through the coordination of Cr(III) to DNA phosphates (phosphotriester-type adduct). Here, we examined the role of Cr(III)-DNA adducts in mutagenesis induced by metabolism of Cr(VI) with cysteine. Reduction of Cr(VI) caused a strong oxidation of 2', 7'-dichlorofluoroscin (DCFH) and extensive Cr-DNA binding but no DNA breakage. Cr-DNA adducts induced unwinding of supercoiled plasmids and structural distortions in the DNA helix as detected by decreased ethidium bromide binding. Propagation of Cr-treated pSP189 plasmids in human fibroblasts led to a dose-dependent formation of the supF mutants and inhibition of replication. Blocking of Cr(III)-DNA binding by occupation of DNA phosphates with Mg(2+) or by sequestration of Cr(III) by inorganic phosphate or EDTA eliminated mutagenic responses and restored a normal yield of replicated plasmids. Dissociation of Cr(III) from DNA by a phosphate-based reversal procedure returned mutation frequency to background levels. The mutagenic responses at the different phases of the reduction reaction were unrelated to the amount of reduced Cr(VI) but reflected the number and the spectrum of Cr(III)-DNA adducts that were formed. Ternary cysteine-Cr(III)-DNA adducts were approximately 4-5 times more mutagenic than binary Cr(III)-DNA adducts. Although intermediate reaction products (CrV/IV, thiyl radicals) were capable of oxidizing DCFH, they were insufficiently reactive to damage DNA. Single-base substitutions at G/C pairs were the predominant type of Cr-induced mutations. The majority of mutations occurred at the sites where G had adjacent purine in the 3' or 5' position. Overall, our results present the first evidence that Cr(III)-DNA adducts play the dominant role in the mutagenicity caused by the metabolism of Cr(VI) by a biological reducing agent.

Base Sequence↗

PubMed2001

Chemical structure and chain conformation of the water-insoluble glucan isolated from Pleurotus tuber-regium.

A water-insoluble polysaccharide (TM8) was isolated from sclerotium of Pleurotus tuber-regium by extraction with 0.5M NaOH aqueous solutions at 120 degrees C. Its chemical structure was confirmed by infrared, high performance liquid chromatography, gas chromatography, and (13)C NMR in dimethylsulfoxide (DMSO) to be composed of beta-(1 --> 3)-D-glucan backbone chain linked with a branched glucose, one out of every three glycosyl units being substituted at C6 position. The glucan TM8 in DMSO was fractionated by nonsolvent addition method into ten fractions, and the solution properties were studied by size exclusion chromatography combined with multiangle laser light scattering (SEC-MALLS) and viscometry in DMSO at 30 degrees C. The dependencies of intrinsic viscosity [eta] and radius of gyration [(s(2)(1/2)(z-2)] on weight-average molecular mass M(w) for this glucan were found to be [eta] = (9.24 +/- 0.2) x 10(-2)M(w)(0.51 +/- 0.02) (cm(3)g(-1)) and [(s(2)(1/2)(z-2)] = (3.67 +/- 0.3) x 10(-2)M(w)(0.56 +/- 0.02) (nm) in the range of M(w) from 1.07 x 10(4) to 77.4 x 10(4). Based on current theories for a wormlike chain, the conformational parameters of the glucan TM8 were found to be 408 (nm(-1)) for M(L), 3.1 (nm) for q, and 16.8 for C(infinity), suggesting that the polysaccharide exists as a dense random-coil chain in DMSO, due to branched structure.

Biopolymers↗

PubMed2001

Effect of osteogenic protein 1/collagen composite combined with impacted allograft around hydroxyapatite-coated titanium alloy implants is moderate.

This study evaluated the effects of osteogenic protein 1/collagen composite (OP-1/col) mixed with impacted allograft around hydroxyapatite (HA)-coated titanium alloy implants in a canine model. The aim of the study was to test different doses of OP-1 growth factor in a collagen composite for stimulatory effect on allograft incorporation around an implant. Unloaded implants were inserted in each proximal humerus of 16 skeletally mature dogs. The cylindrical implants (4 x 9 mm) coated with HA were initially surrounded by a 3-mm gap into which allograft mixed with OP-1/col was impacted. Two different doses of OP-1 were investigated. In eight animals 325 mg OP-1 protein and 130 mg bovine collagen type I as carrier were mixed with the allograft chips. This composite is identical to the clinically used OP-1 device called Novus. In another eight animals a lower dose of 65 mg OP-1 protein and 130 mg bovine collagen type I was used. Control implants placed in the contralateral humerus were surrounded by allograft mixed with collagen carrier only. The dogs were euthanized at 6 weeks. Implant fixation was determined by push-out testing. Bone ingrowth and bone formation were evaluated by quantitative histomorphometry on serial sections of the bone-implant interface. Impacted allograft together with low-dose OP-1 enhanced bone volume in a zone adjacent to HA-coated titanium alloy implants. The high dose had no effect on bone formation. Mechanical fixation, bone ingrowth, and bone volume in the gap near the original trabecular bone were unaffected by both low and high OP-1/col composite. In this model and observation period, the low dose of OP-1/col composite mixed with impacted allograft has a moderate effect on bone healing around HA-coated implants and no effect on implant fixation.

Animals↗

PubMed2001

Promotion of S-phase entry and cell growth under serum starvation by SAG/ROC2/Rbx2/Hrt2, an E3 ubiquitin ligase component: association with inhibition of p27 accumulation.

The sensitive-to-apoptosis gene (SAG) was initially identified as a redox-inducible, apoptosis-protective protein and subsequently found to be the second family member of regulator of cullins (ROC)/RING box protein (Rbx)/Hrt, which acts as a component of E3 ubiquitin ligase. We report here that SAG promoted cell growth under serum starvation. Microinjection of SAG mRNA into quiescent NIH/3T3 cells induced S-phase entry as determined by [(3)H]-thymidine incorporation. Likewise, overexpression of SAG by either adenovirus infection of immortalized human epidermal keratinocytes (Rhek-1) or DNA transfection of SY5Y human neuroblastoma cells induced cell proliferation under serum starvation. Because cyclin-dependent kinase inhibitors (CKIs), including p21, p27, and p57, are degraded through the ubiquitin pathway, we tested whether SAG-induced cell growth is associated with CKI degradation. Although there was no significant difference in the levels of p21 and p57 between the vector controls and SAG-overexpressing cells, serum starvation induced 10- to 18-fold accumulation of p27 in control Rhek-1 cells. Accumulation of p27 was remarkably inhibited (only 2 to 5-fold) in SAG-infected cells. Inhibition of p27 accumulation was also observed in stably SAG-overexpressing SY5Y cells. Significantly, SAG-associated inhibition of p27 accumulation was largely abolished by the treatment with a proteasome inhibitor. In vivo binding of SAG and Skp2, an F-box protein that promotes p27 ubiquitination, was detected, and the binding was enhanced in SAG-overexpressing cells grown under serum starvation. Thus, SAG-induced growth with serum withdrawal appears to be associated with SAG-mediated p27 degradation. Mol. Carcinog. 30:37-46, 2001.

Animals↗

PubMed2001

Effect of 'pH' on the rate of asparagine deamidation in polymeric formulations: 'pH'-rate profile.

The rate of Asn deamidation of a model hexapeptide (L-Val-L-Tyr-L-Pro-L-Asn-Gly-L-Ala) was measured as a function of effective pH ('pH') in glassy and rubbery polymeric solids containing poly(vinyl pyrrolidone) (PVP) and in solution controls at 70 degrees C. The reaction exhibited pseudo-first-order kinetics in all samples over a wide 'pH' range (0.5 < 'pH' < 12); the formation of similar products suggests that the reaction mechanism is unaffected by matrix type. Rates of deamidation were comparable for the polymeric and solution samples in the acidic range ('pH' < 4). Solution-state rates were faster than those in polymeric solids at neutral 'pH' (6 < 'pH' < 8), increasing to a > 10,000-fold difference in the basic range ('pH' > 8). Specific base catalysis was observed in solution and in the polymeric solids under neutral conditions (6 < 'pH' < 8). In solution, the reaction exhibited general base catalysis for 'pH' > 8, whereas the reaction was 'pH'-independent in the polymeric solids in this range. The 'pH'-rate profile and supporting buffer catalysis data are consistent with a change in the rate-determining step in the basic range from 'pH'-dependent attack of the deprotonated backbone amide nitrogen on the Asn side chain in solution to 'pH'-independent ammonia expulsion in the polymeric solids. The results suggest that polymer matrix incorporation not only affects the magnitude of the deamidation rate constant but also the 'pH' dependency of the reaction and the rate-determining step in the basic 'pH' range.

Amides↗

PubMed2001

Mutagen sensitivity as an indicator of soft tissue sarcoma risk.

The etiology of soft tissue sarcoma is poorly understood. Exposure to environmental chemicals may play a role, but the data are not clear. We compared a group of soft tissue sarcoma patients with healthy controls to determine whether the mutagen sensitivity assay, a simple chromosome aberration assay using the radiomimetic bleomycin, might be useful to identify patients at risk for soft tissue sarcoma. Patients with a diagnosis of soft tissue sarcoma at Memorial Sloan-Kettering's outpatient clinic signed informed consent and donated 30 ml of blood. Controls were selected from the general population of Connecticut by random digit dialing. Unrepaired DNA damage was assessed for 100 metaphase spreads for each individual, with the number of breaks in chromatids being counted as breaks per cell (b/c). The 20 cases with soft tissue sarcoma had 1.03 mean b/c and the controls had 0.88 b/c (P = 0.16). Patients with soft tissue sarcoma were 5.7 times more likely to be mutagen sensitive than controls (P = 0.01), as determined after dividing subjects into sensitive or not sensitive groups based on the median b/c among controls. As mutagen sensitivity has been shown to be associated with a number of cancers and appears to reflect genetic susceptibility, this assay may be an appropriate biomarker for radiation sensitivity or it may be a marker of susceptibility to soft tissue sarcoma. Larger studies should be undertaken to assess these possibilities.

Adult↗

PubMed2001

Formaldehyde production by Tris buffer in peptide formulations at elevated temperature.

This technical note provides evidence for the degradation of Tris buffer in a peptide formulation stored at elevated temperature (70 degrees C). The buffer degrades to liberate formaldehyde, which is shown to react with the peptide tyrosine residue. Those involved in peptide/protein formulation should be aware of the possible instability in this common biological buffer.

Formaldehyde↗

PubMed2001

TNF receptor I mediates chemokine production and neutrophil accumulation in the lung following systemic lipopolysaccharide.

BACKGROUND: Tumor necrosis factor (TNF)-alpha is a critical effector of lipopolysaccharide (LPS)-induced acute lung injury, and its effects are mediated by two structurally related receptors, RI and RII. Cellular adhesion molecules and C-X-C chemokines (Keratinocyte chemoattractant (KC) and macrophage inflammatory protein [MIP]-2) regulate tissue neutrophil polymorphonuclear neutrophil (PMN) accumulation in a multitude of inflammatory states. We hypothesized that TNFRI signaling dictates PMN accumulation in the lung via regulation of chemokine molecule production. Therefore, the purposes of this study were to (1) delineate LPS-induced lung TNF-alpha production and (2) characterize the contribution of both TNF receptors to lung chemokine production and neutrophil influx following systemic LPS. METHODS: Wild-type or TNFRI and TNFRII knockout (KO) mice were injected with vehicle (saline) or LPS (Escherichia coli 0.5 mg/kg intraperitoneally). After 2, 4, 6, or 24 h, lungs were analyzed for TNF-alpha and chemokine (KC and MIP-2) protein expression (enzyme-linked immunosorbent assay) and PMN accumulation (myeloperoxidase assay). RESULTS: There was an increase in total lung TNF-alpha (vehicle, 5.0 +/- 1.2 pg/mg total protein vs LPS, 950 +/- 318; P < 0.05) after LPS. Lung chemokine production and PMN accumulation were also increased compared to vehicle-injected mice. Lung chemokine production and PMN accumulation were significantly lower in TNFRI KO, but not TNFRII KO, mice, despite no difference in TNF-alpha production (TNFRI KO, 925 +/- 301 vs TNFRII KO, 837 +/- 267, P = 0.82). CONCLUSIONS: Acute lung injury following systemic LPS administration is characterized by increased lung (1) TNF-alpha production, (2) C-X-C chemokine production, and (3) neutrophil accumulation. The maximal effect of LPS-induced lung neutrophil accumulation appears to be dependent upon the TNFRI receptor but not the TNFRII receptor. .

Animals↗

PubMed2001

Anti-viral activity of hairpin ribozyme directed against HBV core region in vitro.

To study the preparation and cleavage of hairpin ribozyme (HpRz) directed against the transcript of HBV core region in vitro, HRz gene designed by computer targeting the transcript of HBV core gene was cloned into the vector p1.5 between 5'-cis-Rz and 3'-cis-Rz. 32P-labeled HpRz transcript proved whether the vector fit for the preparation of hairpin ribozyme. 32P-labeled pKC transcript containing HBV core region as targets-RNA was transcribed by using T7 RNA polymerase and purified by PAGE. Cold HpRz transcript was incubated with 32P-labeled target-RNAs under different conditions and radioautographed after denaturing polyacrylamide gel electrophoresis. The results showed that HpRz had the ability of cleavage at 37 degrees C and 12 mmol/L MgCl2 and the design of ribozyme was correct. It is concluded that HpRz prepared in vitro possesses specific catalytic activity, indicating that it is possible for HpRz to intracellularly inhibit the replication of HBV. It may be developed into a nucleic acid drug in the treatment of hepatitis B in the future.

Antiviral Agents↗

PubMed2001

Validation of denaturing high performance liquid chromatography as a rapid detection method for the identification of human INK4A gene mutations.

The incidence of melanoma is increasing rapidly in western countries. Genetic predisposition in familial and in some sporadic melanomas has been associated with the presence of INK4A gene mutations. To better define the risk for developing sporadic melanoma based on genetic and environmental interactions, large groups of cases need to be studied. Mutational analysis of genes lacking hot spots for sequence variations is time consuming and expensive. In this study we present the application of denaturing high performance liquid chromatography (DHPLC) for screening of mutations. Exons 1alpha, 2, and 3 were amplified from 129 samples and 13 known mutants, yielding 347 products that were examined at different temperatures. Forty-two of these amplicons showed a distinct non-wild-type profile on the chromatogram. Independent sequencing analysis confirmed 16 different nucleotide variations in Leu32Pro; Ile49Thr; 88 del G; Gln50Arg; Arg24Pro; Met53Ile; Met53Thr; Arg58stop; Pro81Leu; Asp84Ala; Arg80stop; Gly101Trp; Val106Val; Ala148Thr; and in positions (-2) in intron 1 (C --> T); and in the 3' UTR, nucleotide 500 (C --> G). No false negatives or false positives were obtained by DHPLC in samples with mutations or polymorphisms. We conclude that the DHPLC is a fast, sensitive, cost-efficient, and reliable method for the scanning of INK4A somatic or germline mutations and polymorphisms of large number of samples.

Buffers↗

PubMed2001

A fast neutron deletion mutagenesis-based reverse genetics system for plants.

A new reverse genetics method has been developed to identify and isolate deletion mutants for targeted plant genes. Deletion mutant libraries are generated using fast neutron bombardment. DNA samples extracted from the deletion libraries are used to screen for deletion mutants by polymerase chain reaction (PCR) using specific primers flanking the targeted genes. By adjusting PCR conditions to preferentially amplify the deletion alleles, deletion mutants were identified in pools of DNA samples, each pool containing DNA from 2592 mutant lines. Deletion mutants were obtained for 84% of targeted loci from an Arabidopsis population of 51 840 lines. Using a similar approach, a deletion mutant for a rice gene was identified. Thus we demonstrate that it is possible to apply this method to plant species other than Arabidopsis. As fast neutron mutagenesis is highly efficient, it is practical to develop deletion mutant populations with more complete coverage of the genome than obtained with methods based on insertional mutagenesis. Because fast neutron mutagenesis is applicable to all plant genetic systems, this method has the potential to enable reverse genetics for a wide range of plant species.

Arabidopsis↗

PubMed2001

Vascular cell adhesion molecule--1 expression is obligatory for endotoxin-induced myocardial neutrophil accumulation and contractile dysfunction.

BACKGROUND: Sepsis-induced cardiac dysfunction occurs commonly in critically ill patients and is associated with high mortality rates. Neutrophils play a central role in sepsis-induced lung and liver injury; however, the mechanism of sepsis-induced cardiac dysfunction remains unclear. Vascular cell adhesion molecule-1 (VCAM-1) has been implicated in neutrophil-mediated liver injury during endotoxemia and is also expressed in myocardium. The purposes of this study were to examine the temporal relationship of myocardial VCAM-1 expression with neutrophil accumulation during endotoxemia and to determine whether VCAM-1 mediates neutrophil accumulation and cardiac dysfunction during endotoxemia. METHODS: Mice were subjected to lipopolysaccharide (LPS; 0.5 mg/kg, intraperitoneally). Myocardial VCAM-1 expression and neutrophil accumulation were determined by immunofluorescence staining. Cardiac performance with or without VCAM-1 blocking antibody (5 mg/kg, intravenously) was determined by the Langendorff technique. RESULTS: LPS caused a time-dependent increase in both myocardial VCAM-1 expression and neutrophil accumulation. At 6 hours after LPS, the immunofluorescent intensity for VCAM-1 increased from 2.5 +/- 0.6 x 10(6) in saline solution controls to 19.9 +/- 3.5 x 10(6) (P <.05, analysis of variance), and neutrophil count increased from 2.4 +/- 1.7/mm(2) in saline solution controls to 13.0 +/- 2.5/mm(2) (P <.05). Left ventricular developed pressure was decreased maximally at 6 hours after LPS compared with saline solution controls (29.1 +/- 1.1 mm Hg vs 53.1 +/- 3.9 mm Hg; P <.05). Treatment with VCAM-1 monoclonal antibody abrogated both myocardial neutrophil accumulation and cardiac dysfunction during endotoxemia. CONCLUSIONS: LPS-induced myocardial dysfunction is associated with increased expression of VCAM-1 and with neutrophil accumulation. Blockade of VCAM-1 abrogates myocardial neutrophil accumulation and preserves cardiac function during endotoxemia, which supports a role for VCAM-1 as a therapeutic target for myocardial protection during sepsis.

Animals↗

PubMed2001

Airway surface liquid osmolality measured using fluorophore-encapsulated liposomes.

The airway surface liquid (ASL) is the thin layer of fluid coating the luminal surface of airway epithelial cells at an air interface. Its composition and osmolality are thought to be important in normal airway physiology and in airway diseases such as asthma and cystic fibrosis. The determinants of ASL osmolality include epithelial cell solute and water transport properties, evaporative water loss, and the composition of secreted fluids. We developed a noninvasive approach to measure ASL osmolality using osmotically sensitive 400-nm-diam liposomes composed of phosphatidylcholine/cholesterol/polyethylene glycol-phosphatidylcholine (1:0.3:0.08 molar ratio). Calcein was encapsulated in the liposomes at self-quenching concentrations (30 mM) as a volume-sensitive marker, together with sulforhodamine 101 (2 mM) as a volume-insensitive reference. Liposome calcein/sulforhodamine 101 fluorescence ratios responded rapidly (< 0.2 s) and stably to changes in solution osmolality. ASL osmolality was determined from calcein/sulforhodamine 101 fluorescence ratios after addition of microliter quantities of liposome suspensions to the ASL. In bovine airway epithelial cells cultured on porous supports at an air-liquid interface, ASL thickness (by confocal microscopy) was 22 microm and osmolality was 325 +/- 12 mOsm. In anesthetized mice in which a transparent window was created in the trachea, ASL thickness was 55 microm and osmolality was 330 +/- 36 mOsm. ASL osmolality was not affected by pharmacological inhibition of CFTR in airway cell cultures or by genetic deletion of CFTR in knockout mice. ASL osmolality could be increased substantially to > 400 mOsm by exposure of the epithelium to dry air; the data were modeled mathematically using measured rates of osmosis and evaporative water loss. These results establish a ratio imaging method to map osmolality in biological compartments. ASL fluid is approximately isosmolar under normal physiological conditions, but can become hyperosmolar when exposed to dry air, which may induce cough and airway reactivity in some patients.

Animals↗

PubMed2001

Role of aquaporin water channels in airway fluid transport, humidification, and surface liquid hydration.

Several aquaporin-type water channels are expressed in mammalian airways and lung: AQP1 in microvascular endothelia, AQP3 in upper airway epithelia, AQP4 in upper and lower airway epithelia, and AQP5 in alveolar epithelia. Novel quantitative methods were developed to compare airway fluid transport-related functions in wild-type mice and knockout mice deficient in these aquaporins. Lower airway humidification, measured from the moisture content of expired air during mechanical ventilation with dry air through a tracheotomy, was 54-56% efficient in wild-type mice, and reduced by only 3-4% in AQP1/AQP5 or AQP3/AQP4 double knockout mice. Upper airway humidification, measured from the moisture gained by dry air passed through the upper airways in mice breathing through a tracheotomy, decreased from 91 to 50% with increasing ventilation from 20 to 220 ml/min, and reduced by 3-5% in AQP3/AQP4 knockout mice. The depth and salt concentration of the airway surface liquid in trachea was measured in vivo using fluorescent probes and confocal and ratio imaging microscopy. Airway surface liquid depth was 45 +/- 5 microm and [Na(+)] was 115 +/- 4 mM in wild-type mice, and not significantly different in AQP3/AQP4 knockout mice. Osmotic water permeability in upper airways, measured by an in vivo instillation/sample method, was reduced by approximately 40% by AQP3/AQP4 deletion. In doing these measurements, we discovered a novel amiloride-sensitive isosmolar fluid absorption process in upper airways (13% in 5 min) that was not affected by aquaporin deletion. These results establish the fluid transporting properties of mouse airways, and indicate that aquaporins play at most a minor role in airway humidification, ASL hydration, and isosmolar fluid absorption.

Adsorption↗

PubMed2001

DNA hypermethylation is a mechanism for loss of expression of the HLA class I genes in human esophageal squamous cell carcinomas.

The three human leukocyte antigen (HLA) class I antigens, HLA-A, HLA-B and HLA-C, play important roles in the elimination of transformed cells by cytotoxic T cells. Frequent loss of expression of these antigens at the cell surface has been observed in many human cancers. Various mechanisms for post-transcriptional regulation have been proposed and tested but the molecular mechanisms for transcriptional regulation are not clear. We show by immunohistochemistry that the HLA class I antigens are absent in 26 of 29 (89%) samples of human esophageal squamous cell carcinomas (ESCC). Eleven of the 26 ESCC samples lost mRNA expression for at least one of the HLA genes, as shown by RT-PCR. DNA from the 29 pairs of ESCC and neighboring normal epithelium were examined for CpG island hypermethylation, homozygous deletion, microsatellite instability (MSI) and loss of heterozygosity (LOH). DNA from normal epithelial tissues had no detectable methylation of the CpG islands of any of these gene loci. Thirteen of 29 ESCC samples (45%) exhibited methylation of one or more of the three HLA loci and six samples (21%) exhibited methylation of all three loci. The HLA-B gene locus was most frequently methylated (38%). HLA-B mRNA expression in an ESCC cell line, where HLA-B was hypermethylated and did not express mRNA, was activated after treatment with 5-aza-2'-deoxycytidine. Homozygous deletion of these three gene loci was not observed. Relatively low rates of LOH and MSI were observed for the microsatellite markers D6S306, D6S258, D6S273 and D6S1666, close to the HLA-A, -B and -C loci, although a high ratio of LOH was observed at a nearby locus (represented by the markers D6S1051 and D6S1560), where the tumor suppressor gene p21(Waf1) resides. A strong correlation between genetic alterations and mRNA inactivation was observed in the ESCC samples. Our results indicate that HLA class I gene expression was frequently down-regulated in ESCC at both the protein and mRNA levels and that hypermethylation of the promoter regions of the HLA-A, -B and -C genes is a major mechanism of transcriptional inactivation.

Azacitidine↗

PubMed2001

Effects of calnexin deletion in Saccharomyces cerevisiae on the secretion of glycosylated lysozymes.

Disruption of the calnexin gene in Saccharomyces cerevisiae did not lead to gross effects on the levels of cell growth and secretion of wild-type hen egg white lysozymes (HEWL). To investigate the function of calnexin in relation to the secretion of glycoproteins, we expressed both stable and unstable mutant glycosylated lysozymes in calnexin-disrupted S. cerevisiae. The secreted amounts of stable mutant glycosylated lysozymes (G49N and S91T/G49N) were almost the same in both wild-type and calnexin-disrupted S. cerevisiae. In contrast, the secretion of unstable mutant glycosylated lysozymes (K13D/G49N, C76A/G49N, and D66H/G49N) greatly increased in calnexin-disrupted S. cerevisiae, although their secretion was very low in the wild-type strain. This indicates that calnexin may act in the quality control of glycoproteins. We further investigated the expression level of the mRNA of the molecular chaperones BiP and PDI, which play a major role in the protein folding process in the ER, when glycosylated lysozymes were expressed in wild-type and calnexin-disrupted S. cerevisiae. The mRNA concentrations of BiP and PDI were evidently increased when the glycosylated lysozymes were expressed in calnexin-disrupted S. cerevisiae. This observation indicates that BiP and PDI may be induced by the accumulation of unfolded glycosylated lysozymes due to the deletion of calnexin.

Calcium-Binding Proteins↗