Biomedical subjects

W Guo

Publications and source records attributed to W Guo.

At least 73 records · Page 4Linked to original sources

PubMed2000

Requirement for either a host- or pectin-induced pectate lyase for infection of Pisum sativum by Nectria hematococca.

Fungal pathogens usually have multiple genes that encode extracellular hydrolytic enzymes that may degrade the physical barriers in their hosts during the invasion process. Nectria hematococca, a plant pathogen, has two inducible pectate lyase (PL) genes (pel) encoding PL that can help degrade the carbohydrate barrier in the host. pelA is induced by pectin, whereas pelD is induced only in planta. We show that the disruption of either the pelA or pelD genes alone causes no detectable decrease in virulence. Disruption of both pelA and pelD drastically reduces virulence. Complementation of the double disruptant with pelD gene, or supplementation of the infection droplets of the double disruptant with either purified enzyme, PLA, or PLD, caused a recovery in virulence. These results show that PL is a virulence factor. Thus, we demonstrate that disruption of all functionally redundant genes is required to demonstrate the role of host barrier-degrading enzymes in pathogenesis and that dismissal of the role of such enzymes based on the effects of single-gene disruption may be premature.

Ascomycota↗

PubMed2000

Esterification of free fatty acids in adipocytes: a comparison between octanoate and oleate.

Medium-chain triacylglycerols (MCT) are present in milk, coconut oil and other foods, and are used therapeutically in special diets for certain disorders of lipid and glucose utilization. Recently, it has become apparent that MCT are not only oxidized in the liver, but are also present in lymph and fat tissue, particularly after chronic treatment. To evaluate the influence of MCT on metabolism in fat cells, we compared incorporation of octanoate and oleate into cellular triacylglycerols of 3T3-L1 adipocytes as well as their effects on preadipocyte differentiation. We found that less octanoate than oleate was stored and that more octanoate than oleate was oxidized. Octanoate was esterified to a greater extent at the sn-1,3 position of glyceryl carbons than at the sn-2 position, whereas the opposite was true for oleate. Glycerol release from fat cells pre-treated with octanoate was also greater than from cells pre-treated with oleate, presumably related to the preferential release of octanoate from the sn-1,3 position. Octanoate was not incorporated into lipids in undifferentiated cells and did not induce differentiation in these cells, whereas oleate was readily stored and actually induced differentiation. Incorporation of octanoate into lipids increased as cells differentiated, but reached a maximum of about 10% of the total stored fatty acids. If these effects in vitro also occur in vivo, substitution of octanoate for oleate or other long-chain fatty acids could have the beneficial effect of diminishing fat-cell number and lipid content.

3T3 Cells↗

PubMed2000

Functional consequences of elimination of i(to,f) and i(to,s): early afterdepolarizations, atrioventricular block, and ventricular arrhythmias in mice lacking Kv1.4 and expressing a dominant-negative Kv4 alpha subunit.

It was recently reported that the slow transient outward K(+) current, I(to, s), that is evident in mouse left ventricular septal cells is eliminated in mice with a targeted deletion of the Kv1.4 gene (Kv1.4(-/-)). The rapidly inactivating transient outward K(+) current, I(to, f), in contrast, is selectively eliminated in ventricular myocytes isolated from transgenic mice expressing a dominant-negative Kv4 alpha subunit, Kv4.2W362F. Expression of Kv4. 2W362F results in marked prolongation of action potentials and QT intervals. In addition, a slow transient outward K(+) current, that is similar to I(to,s) in wild-type mouse left ventricular septal cells, is evident in all Kv4.2W362F-expressing (left and right) ventricular cells. To test directly the hypothesis that upregulation of Kv1.4 alpha subunit underlies the appearance of this slow transient outward K(+) current in Kv4.2W362F-expressing ventricular cells and to explore the functional consequences of elimination of I(to,f) and I(to,s), mice expressing Kv4.2W362F in the Kv1.4(-/-) background (Kv4.2W362FxKv1.4(-/-)) were generated. Histological and echocardiographic studies revealed no evidence of structural abnormalities or contractile dysfunction in Kv4.2W362FxKv1.4(-/-) mouse hearts. Electrophysiological recordings from the majority (approximately 80%) of cells isolated from the right ventricle and left ventricular apex of Kv4.2W362FxKv1.4(-/-) animals demonstrated that both I(to, f) and I(to,s) are eliminated; action potentials are prolonged significantly; and, in some cells, early afterdepolarizations were observed. In addition, in vivo telemetric ECG recordings from Kv4.2W362FxKv1.4(-/-) animals revealed marked QT prolongation, atrioventricular block, and ventricular tachycardia. These observations demonstrate that upregulation of Kv1.4 contributes to the electrical remodeling evident in the ventricles of Kv4.2W362F-expressing mice and that elimination of both I(to,f) and I(to,s) has dramatic functional consequences.

Action Potentials↗

PubMed2000

Differential expression of a novel C-terminally truncated splice form of SMAD5 in hematopoietic stem cells and leukemia.

SMADs are evolutionarily conserved transducers of the differentiation and growth arrest signals from the transforming growth factor/BMP (TGF/BMP) family of ligands. Upon receptor activation, the ligand-restricted SMADs(1-35) are phosphorylated in the C-terminal MH2 domain and recruit the common subunit SMAD4/DPC-4 gene to the nucleus to mediate target gene expression. Frequent inactivating mutations of SMAD4, or less common somatic mutations of SMAD2 seen in solid tumors, suggest that these genes have a suppressor function. However, there have been no identified mutations of SMAD5, although the gene localizes to the critical region of loss in chromosome 5q31.1 (chromosome 5, long arm, region 3, band 1, subband 1) in myelodysplasia (MDS) and acute myelogenous leukemia (AML). A ubiquitously expressed novel isoform, SMAD5beta, encodes a 351 amino acid protein with a truncated MH2 domain and a unique C-terminal tail of 18 amino acids, which may be the functional equivalent of inactivating mutations. The levels of SMAD5beta transcripts are higher in the undifferentiated CD34(+) hematopoietic stem cells than in the terminally differentiated peripheral blood leukocytes, thereby implicating the beta form in stem cell homeostasis. Yeast 2-hybrid interaction assays reveal the lack of physical interactions between SMAD5beta and SMAD5 or SMAD4. The expression of SMAD5beta may represent a novel mechanism to protect pluripotent stem cells and malignant cells from the growth inhibitory and differentiation signals of BMPs. (Blood. 2000;95:3945-3950)

Alternative Splicing↗

PubMed2000

Chromosomal mapping and genomic organization of an evolutionarily conserved zinc finger gene ZNF277.

A novel C2H2 zinc finger gene, ZNF277, has been localized to human chromosome 7q31.1. The gene is encoded by 12 exons in a genomic fragment of >100 kb between the microsatellite markers D7S523 and D7S471, deleted in a number of malignancies. The predicted open reading frame (ORF) of 438 amino acids shows an overall homology of 50% to the putative ORF F46B6.7 of Caenorhabditis elegans. The presence of a 30-amino-acid coiled-coil domain in both the C. elegans ORF F46B6.7 and ZNF277 is suggestive of functional similarities. ESTs for the murine orthologue ZFP277 are found in early embryonic stem cells, 16-cell stage embryo, and blastocysts. The evolutionary conservation and the expression profile suggest ZNF277 to be a critical regulator of development and differentiation.

Amino Acid Sequence↗

PubMed2000

Structure of the complex of Cdc42Hs with a peptide derived from P-21 activated kinase.

Cdc42Hs is a member of the Ras superfamily of GTPases and initiates a cascade that begins with the activation of several kinases, including p21-activated kinase (PAK). We have previously used a 46 amino acid fragment of PAK (PBD46) to define the binding surface on Cdc42Hs [Guo et al. (1998) Biochemistry 37, 14030-14037]. Here we describe the three-dimensional solution structure of the Cdc42Hs. GMPPCP-PBD46 complex. Heteronuclear NMR methods were used to assign resonances in the complex, and approximately 2400 distance and dihedral restraints were used to calculate a set of 20 structures using a combination of distance geometry, simulated annealing, and chemical shift and Ramachandran refinement. The overall structure of Cdc42Hs in the complex differs from the uncomplexed structure in two major aspects: (1) the first alpha helix is reoriented to accommodate the binding of the peptide and (2) the regions corresponding to switch I and switch II are less disordered. As suggested by our previous work (Guo et al., 1998) and similar to the complex between Cdc42Hs and fACK [Mott et al. (1999) Nature 399, 384-388], PBD46 forms an intermolecular beta-sheet with beta2 of Cdc42Hs and contacts both switch I and switch II. The extensive binding surface between PBD46 and Cdc42Hs can account for both the high affinity of the complex and the inhibition by PBD46 of GTP hydrolysis.

Amino Acid Sequence↗

PubMed2000

T2Rs function as bitter taste receptors.

Bitter taste perception provides animals with critical protection against ingestion of poisonous compounds. In the accompanying paper, we report the characterization of a large family of putative mammalian taste receptors (T2Rs). Here we use a heterologous expression system to show that specific T2Rs function as bitter taste receptors. A mouse T2R (mT2R-5) responds to the bitter tastant cycloheximide, and a human and a mouse receptor (hT2R-4 and mT2R-8) responded to denatonium and 6-n-propyl-2-thiouracil. Mice strains deficient in their ability to detect cycloheximide have amino acid substitutions in the mT2R-5 gene; these changes render the receptor significantly less responsive to cycloheximide. We also expressed mT2R-5 in insect cells and demonstrate specific tastant-dependent activation of gustducin, a G protein implicated in bitter signaling. Since a single taste receptor cell expresses a large repertoire of T2Rs, these findings provide a plausible explanation for the uniform bitter taste that is evoked by many structurally unrelated toxic compounds.

Afferent Pathways↗

PubMed2000

Antiangiogenic effects of S-nitrosocaptopril crystals as a nitric oxide donor.

Angiogenesis is the formation of new capillaries from preexisting vessels by migration and proliferation of endothelial cells, which produce a cellular signaling messenger, nitric oxide (NO). The purpose of the present study was to examine the effects of exogenous NO donors on angiogenesis by using a novel crystalline NO donor, S-nitrosocaptopril. The characteristic X-ray diffraction pattern of S-nitrosocaptopril was demonstrated for the first time. On primary capillary endothelial cells pretreated with vascular endothelium growth factor (VEGF), S-nitrosocaptopril (1-500 microM), but not captopril, produced a dose-dependent inhibition of endothelial proliferation. On chick embryos of entire living eggs, gelatin sponges adsorbed with VEGF were implanted on the embryo chorioallantoic membrane to promote vascular growth activity within the sponges. Addition of S-nitrosocaptopril crystals (0.1 mg) to the gelatin sponges markedly reduced vascular density around the sponges, whereas captopril did not inhibit neovascularization. The vascular hemoglobin content surrounding each of the gelatin sponges was determined as a confirmatory test. S-nitrosocaptopril, but not captopril, significantly decreased the hemoglobin content of the embryo tissues immediately surrounding the gelatin sponges. In conclusion, S-nitrosocaptopril exerts an inhibitory effect on angiogenesis. This newly discovered function of S-nitrosocaptopril appears to be governed by distinct structural NO moiety.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed2000

Physical properties of the transmembrane signal molecule, sn-1-stearoyl 2-arachidonoylglycerol. Acyl chain segregation and its biochemical implications.

sn-1,2-diacylglycerol (DAG), a key intermediate in lipid metabolism, activates protein kinase C and is a fusogen. Phosphoinositides, the main sources of DAG in cell signaling, contain mostly stearoyl and arachidonoyl in the sn-1 and -2 positions, respectively. The polymorphic behavior of sn-1-stearoyl-2-arachidonoylglycerol (SAG) was studied by differential scanning calorimetry, x-ray powder diffraction, and solid state magic angle spinning (MAS) (13)C NMR. Three alpha phases were found in the dry state. X-ray diffraction indicated that the acyl chains packed in a hexagonal array in the alpha phase, and the two sub-alpha phases packed with pseudo-hexagonal symmetry. In the narrow angle range strong diffractions of approximately 31 and approximately 62 A were present. High power proton-decoupled MAS (13)C NMR of isotropic SAG gave 16 distinct resonances of the 20 arachidonoyl carbons and 5 distinct resonances of the 18 stearoyl carbons. Upon cooling, all resonances of stearoyl weakened and vanished in the sub-alpha(2) phase, whereas arachidonoyl carbons from 8/9 to 20 gave distinct resonances in the frozen phases. Remarkably, the omega-carbon of the two acyl chains had different chemical shifts in alpha, sub-alpha(1), and sub-alpha(2) phases. Large differences in spin lattice relaxation of the stearoyl and arachidonoyl methene and methyl groups were demonstrated by contact time (cross-polarization) MAS (13)C NMR experiments in the solid phases alpha, sub-alpha(1), and sub-alpha(2). This shows that stearoyl and arachidonoyl in SAG have different environments in the solid states (alpha, sub-alpha(1), and sub-alpha(2) phases) and may segregate during cooling. The NMR and long spacing x-ray diffraction results suggest that SAG does not pack in a conventional double layer with the two acyls in a hairpin fashion. Our findings thus provide a physicochemical basis for DAG hexagonal phase domain separation within membrane bilayers.

Calorimetry, Differential Scanning↗

PubMed2000

Determination of nicotinamide (NA) using its polarographic catalytic wave in the presence of KIO3.

Nicotinamide (NA) yields a polarographic catalytic wave with a peak potential -1.38 V (vs. SCE) in 0.1 mol/L HAc-NaAc (pH 4.7)/4 x 10(-3) mol/L KIO3 buffer solution. The sensitivity of the catalytic wave increased in one order of magnitude as compared to that of the responding reduction wave without KIO3. Based on this observation, a new method for the determination of NA was recommended. The second order derivative peak current was proportional to the NA concentration in the range of 5 x 10(-8)- 6 x 10(-7) mol/L. 0.11-fold vitamin B1, 0.13-fold B2, 0.14-fold B6 and 8-fold nicotinic acid amounts do not interfere the determination of 1 x 10(-6) mol/L NA. The proposed method was used to determine the NA content in multivitamin tablets, with good agreement to the declared amount.

Hydrogen-Ion Concentration↗

PubMed2000

Effects of inescapable stress and treatment with pyridostigmine bromide on plasma butyrylcholinesterase and the acoustic startle response in rats.

Pyridostigmine bromide (PB) is a reversible, peripherally active inhibitor of acetylcholinesterase (AChE) activity, and is recommended by the military as a pretreatment against potential nerve gas exposure. Recent evidence suggests that exposure to inescapable stressors allows PB to cross the blood-brain barrier, and thereby affect central AChE activity in mice. Here, we evaluated the functional impact of a stress/PB treatment interaction on acoustic startle responding and plasma butyrylcholinesterase (BuChE) activity in male Sprague-Dawley rats. To model the treatment protocol used by the military, PB was delivered in the drinking water of rats for 7 consecutive days. The morning after the start of PB treatment, and for the next 6 days, half the rats were exposed to 1 h of supine restraint stress. We therefore employed a 2 x 2 (stress x PB treatment) between-groups design. Exposure to supine stress alone induced a persistent decrease in plasma BuChE activity. Further decreases in BuChE activity were not observed in rats exposed to supine restraint and PB treatment. Exposure to stress also induced an exaggerated startle response, evident on the last day of stress and 24 h after stressor cessation. Treatment with PB alone produced an exaggerated startle response over the same time period, albeit to a lesser degree. Although treatment with PB concurrent with stress did not produce further changes in either BuChE activity or acoustic startle responding, stress-induced alterations in drinking behavior (and thereby the dose of PB ingested) may have affected these results. Persistent stress-induced reductions in BuChE activity may increase the risk of adverse reactions to cholinomimetics.

Acoustic Stimulation↗

PubMed2000

Protein complexes in transport vesicle targeting.

The transport of material between membrane-bounded organelles in eukaryotic cells requires the accurate delivery of different classes of carrier vesicles to specific target compartments. Recent studies indicate that different targeting reactions involve distinct protein complexes that act to mark the target organelle for incoming vesicles. This review focuses on the proteins and protein complexes that have been implicated in various targeting reactions.

Biological Transport↗

PubMed2000

Efficient gene delivery using anionic liposome-complexed polyplexes (LPDII).

Synthetic gene transfer vectors based on polyplexes complexed to anionic liposomes (LPDII vectors) were characterized for their transfection efficiency in cultured mammalian cells. The effects of polycation to DNA ratio, lipid to DNA ratio, choice of polycation and lipid composition were systematically evaluated in human oral carcinoma KB cells, using a luciferase reporter gene. For LPDII formulations containing poly-L-lysine and dioeoylphosphatidylethanolamine/cholesteryl hemisuccinate (DOPE/CHEMS) anionic liposomes, at a constant lipid to DNA ratio, an increase in the polycation/DNA (N/P) ratio resulted in an increase in transfection activity. Meanwhile, the optimal lipid to DNA ratio for efficient gene delivery was influenced by the N/P ratio used, and was increased at higher N/P ratios. For the DNA condensing agent, poly-L-lysine could be replaced by polyethylenimine (PEI) as the DNA condensing agent in the formulations. For the lipidic components, CHEMS could be replaced by other anionic lipids including oleic acid, dicetylphosphate and phosphatidylserine, but DOPE, a fusogenic helper lipid, could not be replaced by dioleolyphosphatidylcholine. LPDII formulation showed significantly less cytotoxicity compared to the commonly used cationic lipsomes or PEI mediated transfection and several cell lines were transfected with high efficiency. LPDII vectors avoid the use of toxic cationic lipids and may have potential application in gene therapy.

Anions↗

PubMed2000

Exocyst is involved in cystogenesis and tubulogenesis and acts by modulating synthesis and delivery of basolateral plasma membrane and secretory proteins.

Epithelial cyst and tubule formation are critical processes that involve transient, highly choreographed changes in cell polarity. Factors controlling these changes in polarity are largely unknown. One candidate factor is the highly conserved eight-member protein complex called the exocyst. We show that during tubulogenesis in an in vitro model system the exocyst relocalized along growing tubules consistent with changes in cell polarity. In yeast, the exocyst subunit Sec10p is a crucial component linking polarized exocytic vesicles with the rest of the exocyst complex and, ultimately, the plasma membrane. When the exocyst subunit human Sec10 was exogenously expressed in epithelial Madin-Darby canine kidney cells, there was a selective increase in the synthesis and delivery of apical and basolateral secretory proteins and a basolateral plasma membrane protein, but not an apical plasma membrane protein. Overexpression of human Sec10 resulted in more efficient and rapid cyst formation and increased tubule formation upon stimulation with hepatocyte growth factor. We conclude that the exocyst plays a central role in the development of epithelial cysts and tubules.

Animals↗

PubMed2000

Structural time series models with feedback mechanisms.

Structural time series models have applications in many different fields such as biology, economics, and meteorology. A structural times series model can be represented as a state-space model where the states of the system represent the unobserved components and the structural parameters have clear interpretations. This paper introduces a class of structural time series models that incorporate feedback from the latent components of the history. An iterative procedure is proposed for estimation. These models allow flexible and robust feedback mechanisms, have clear interpretations, and have a computationally efficient estimation procedure. They are applied to hormone data to characterize hormone secretion and to explore a potential feedback mechanism.

Animals↗

PubMed2000

Selective inhibition of splicing at the avian sarcoma virus src 3' splice site by direct-repeat posttranscriptional cis elements.

The direct-repeat elements (dr1) of avian sarcoma virus (ASV) and leukosis virus have the properties of constitutive transport elements (CTEs), which facilitate cytoplasmic accumulation of unspliced RNA. It is thought that these elements represent binding sites for cellular factors. Previous studies have indicated that in the context of the avian sarcoma virus genome, precise deletion of both ASV dr1 elements results in a very low level of virus replication. This is characterized by a decreased cytoplasmic accumulation of unspliced RNA and a selective increase in spliced src mRNA. Deletion of either the upstream or downstream dr1 results in a delayed-replication phenotype. To determine if the same regions of the dr1 mediate inhibition of src splicing and unspliced RNA transport, point mutations in the upstream and downstream elements were studied. In the context of viral genomes with single dr1 elements, the effects of the mutations on virus replication and increases in src splicing closely paralleled the effects of the mutations on CTE activity. For mutants strongly affecting CTE activity and splicing, unspliced RNA but not spliced RNA turned over in the nucleus more rapidly than wild-type RNA. In the context of wild-type virus containing two dr1 elements, mutations of either element that strongly affect CTE activity caused a marked delay of virus replication and a selective increase in src splicing. However, the turnover of the mutant unspliced RNA as well as the spliced mRNA species did not differ significantly from that of the wild type. These results suggest the dr1 elements in ASV act to selectively inhibit src splicing and that both elements contribute to the fitness of the wild-type virus. However, a single dr1 element is sufficient to stabilize unspliced RNA.

Animals↗

PubMed2000

Quantification of cholesteryl esters in human and rabbit atherosclerotic plaques by magic-angle spinning (13)C-NMR.

Accumulation of cholesteryl esters (CEs) is a key event in the formation of atherosclerotic plaques. More recent work suggests a role for CEs in plaque rupture leading to thrombosis, which can result in an acute event such as myocardial infarction or stroke. In this study, we present nuclear magnetic resonance (NMR) protocols for quantification of CEs in plaques in situ. Total CEs quantified by (13)C magic-angle spinning (MAS) NMR in excised plaques from human carotid arteries and rabbit aortic arteries were in good agreement with the amounts determined by subsequent standard chemical assays. The latter analysis is disadvantageous because it requires that plaque lipids be extracted from the tissue, resulting in the loss of all phase information of CEs as well as other major plaque components. With our MAS-NMR protocol, the plaque components are preserved in their native phases. Combining MAS and off-MAS NMR, we were able to quantitatively distinguish isotropic (liquid) CEs from anisotropic (liquid-crystalline) CEs in plaque tissues. In a recent study, we applied a different (13)C MAS-NMR protocol to quantify crystalline cholesterol monohydrate in plaques. Together, these 2 studies describe a new, noninvasive MAS-NMR strategy for the identification and quantification of the major lipid components in plaques in situ. This approach will be useful for investigation of the relationship between plaque rupture and specific lipids in their biologically relevant phases.

Animals↗

PubMed2000

Quantification in situ of crystalline cholesterol and calcium phosphate hydroxyapatite in human atherosclerotic plaques by solid-state magic angle spinning NMR.

Because of renewed interest in the progression, stabilization, and regression of atherosclerotic plaques, it has become important to develop methods for characterizing structural features of plaques in situ and noninvasively. We present a nondestructive method for ex vivo quantification of 2 solid-phase components of plaques: crystalline cholesterol and calcium phosphate salts. Magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra of human carotid endarterectomy plaques revealed (13)C resonances of crystalline cholesterol monohydrate and a (31)P resonance of calcium phosphate hydroxyapatite (CPH). The spectra were obtained under conditions in which there was little or no interference from other chemical components and were suitable for quantification in situ of the crystalline cholesterol and CPH. Carotid atherosclerotic plaques showed a wide variation in their crystalline cholesterol content. The calculated molar ratio of liquid-crystalline cholesterol to phospholipid ranged from 1.1 to 1.7, demonstrating different capabilities of the phospholipids to reduce crystallization of cholesterol. The spectral properties of the phosphate groups in CPH in carotid plaques were identical to those of CPH in bone. (31)P MAS NMR is a simple, rapid method for quantification of calcium phosphate salts in tissue without extraction and time-consuming chemical analysis. Crystalline phases in intact atherosclerotic plaques (ex vivo) can be quantified accurately by solid-state (13)C and (31)P MAS NMR spectroscopy.

Animals↗