Functional expression of horseradish peroxidase in E. coli By directed evolution
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Journal Article↗
Biomedical subjects
Z Lin
Publications and source records attributed to Z Lin.
Postprandial response of gastric slow waves: correlation of serosal recordings with the electrogastrogram.
Controversial interpretations have been given to the postprandial increase in the dominant power (amplitude) of the electrogastrogram (EGG). The aim of this study was to find an appropriate interpretation of the postprandial EGG power changes. Simultaneous serosal and cutaneous recordings of gastric myoelectrical activity were made in 11 patients with gastroparesis in the fasting state and after the ingestion of 8 oz of water. The dominant frequency and corresponding power of the recording before and after water were computed using the power spectral analysis method. It was found that the dominant frequency of the EGG was the same as that of the serosal recording in 10 patients. One patient showed a substantial amount of dysrhythmia and no obvious dominant frequency was noted. A decrease in the dominant frequency was found in these 10 patients after the ingestion of water. Tachygastria of higher than 4 cycles/min was observed in one of 11 patients both in the prewater and postwater states. Consistent changes in amplitude after a drink of water were noted in both serosal recording and EGG. Statistical analysis demonstrated that the dominant power change after water computed from the EGG was correlated with that observed in the serosal recording (r = 0.757, P = 0.007). In conclusion, exogenous stimulation, such as ingestion of water, may change the amplitude of the gastric slow wave and this change is reflected in the EGG, suggesting that the change of the slow-wave amplitude is an important contributing factor to the postprandial change in the EGG dominant power.
Adult↗
Aberrant SP-B mRNA in lung tissue of patients with congenital alveolar proteinosis (CAP).
Mutations in the surfactant protein (SP)-B gene are responsible for SP-B deficiency in congenital alveolar proteinosis (CAP) (Nogee et al. J Clin Invest 1994: 93: 1860-1883; Lin et al. Mol Genet Metab 1998: 64: 25-35; Klein et al. Pediatrics 1998: 132: 244-248; Ballard et al. Pediatrics 1995: 96: 1046-1052). The multigenerational consanguineous pedigree under study does not carry any of the known mutations, although this pedigree had 14 infant deaths following respiratory distress at birth. Immunostaining of the lungs from three such infants revealed decreased or absent SP-B. By sequencing of SP-B exons, exon-intron junctions, and the 5' and 3' flanking regions, nine polymorphisms were found in this pedigree, but none of them could explain the observed SP-B deficiency. Further analysis of SP-B mRNA by reverse transcription-polymerase chain reaction from paraffin-embedded lung tissue of CAP patients showed that SP-B mRNA is not intact. Although the sequence of mRNA from exon 1-exon 7 and from exon 8-exon 10 could be amplified, the region between exons 7 and 8 could not. From fluorescence in situ hybridization of the short arm of chromosome 2p, only 2 signals were identified, eliminating the possibility of translocation as the cause of the SP-B mRNA aberrance. Although the nature of the genetic basis of SP-B deficiency in this family is currently unknown, the existence of aberrant SP-B mRNA may, at least in part, be responsible for the SP-B deficiency in this pedigree.
Chromosomes, Human, Pair 2↗
Polymorphisms of human SP-A, SP-B, and SP-D genes: association of SP-B Thr131Ile with ARDS.
An allele association study of 19 polymorphisms in surfactant proteins SP-A1, SP-A2, SP-B, and SP-D genes in acute respiratory distress syndrome (ARDS) was carried out. Trend-test analysis revealed differences (p < 0.05) in the frequency of alleles for some of the microsatellite markers flanking SP-B, and for one polymorphism (C/T) at nucleotide 1580 [C/T (1580)], within codon 131 (Thr131Ile) of the SP-B gene. The latter determines the presence or absence of a potential N-linked glycosylation site. Multivariate analysis revealed significant differences only for the C/T (1580) polymorphism. When the ARDS population was divided into subgroups, idiopathic (i.e., pneumonia, etc.) or exogenic (i.e., trauma, etc.), significant differences were observed for the C/T (1580), for the idiopathic ARDS group, and the frequency of the C/C genotype was increased in this group. Based on the odds ratio, the C allele may be viewed as a susceptibility factor for ARDS. Although the expression of both C and T alleles occurs in heterozygous individuals, it is currently not known whether these alleles correspond to similar levels of SP-B protein. These data suggest that SP-B or a linked gene contributes to susceptibility to ARDS.
Alleles↗
Atomic force microscopy analysis of intermediates in cobalt hexammine-induced DNA condensation.
The packaging pathway of cobalt hexammine-induced DNA condensation on the surface of mica was examined by varying the concentration of Co(NH3)6(3+) in a dilute DNA solution and visualizing the condensates by atomic force microscopy (AFM). Images reveal that cobalt hexammine-induced DNA condensation on mica involves well-defined structures. At 30 microM Co(NH3)6(3+), prolate ellipsoid condensates composed of relatively shorter rods with linkages between them are formed. At 80 microM Co(NH3)6(3+), the condensed features include toroids with average diameter of approximately 240 nm as well as U-shaped and rod-like condensates with nodular appearances. The results imply that the condensates, whether toroids, U-shaped or rod-like structures have similar intermediate state which includes relatively shorter rod-like segments. The average size of the condensed toroids after incubated at room temperature for 5 h (approximately 240 nm) is much larger than that incubated for 0.5 h (approximately 100 nm). The results indicate that the condensation of DNA by Co(NH3)6(3+) is a kinetic-controlled process.
Adsorption↗
Functional expression of horseradish peroxidase in Saccharomyces cerevisiae and Pichia pastoris.
The ability to engineer proteins by directed evolution requires functional expression of the target polypeptide in a recombinant host suitable for construction and screening libraries of enzyme variants. Bacteria and yeast are preferred, but eukaryotic proteins often fail to express in active form in these cells. We have attempted to resolve this problem by identifying mutations in the target gene that facilitate its functional expression in a given recombinant host. Here we examined expression of HRP in Saccharomyces cerevisiae. Through three rounds of directed evolution by random point mutagenesis and screening, we obtained a 40-fold increase in total HRP activity in the S.cerevisiae culture supernatant compared with wild-type, as measured on ABTS ¿2, 2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (260 units/l/OD(600)). Genes from wild-type and two high-activity clones were expressed in Pichia pastoris, where the total ABTS activity reached 600 units/l/OD(600) in shake flasks. The mutants show up to 5.4-fold higher specific activity towards ABTS and 2.3-fold higher specific activity towards guaiacol.
Base Sequence↗
Dopamine transporter proline mutations influence dopamine uptake, cocaine analog recognition, and expression.
Analyses of mutation effects can aid in understanding how large proteins act. The dopamine transporter (DAT) mediates complex actions in recognizing cocaine and in recognizing and translocating dopamine, sodium, and chloride. DAT proline residues, especially those in transmembrane (TM) domains, are good candidates for involvement in these DAT actions. We now report production of mutants substituting alanine and/or glycine residues for 16 prolines located in or near putative DAT TM domains. We examine effects of these modifications on DAT expression, dopamine uptake, and cocaine analog binding. Mutants in prolines located in five DAT TM domains and four connecting loops alter apparent DAT membrane targeting. Five mutations decrease dopamine affinities more than threefold without significantly decreasing cocaine analog affinities. One decreases cocaine analog affinity without decreasing dopamine affinity. Two mutations decrease affinities for both dopamine and cocaine analog. P101 is especially implicated in dopamine uptake. Alanine substitution for this proline yields dopamine V(max) values of less than 3% of wild-type values despite dopamine affinities more than fourfold higher than wild-type and normal Na(+) and Cl(-) dependence. These DAT proline mutants identify DAT regions likely for dopamine translocation and for recognition of dopamine and cocaine.
Amino Acid Sequence↗
The VMAT2 gene in mice and humans: amphetamine responses, locomotion, cardiac arrhythmias, aging, and vulnerability to dopaminergic toxins.
Monoamine compartmentalization in monoaminergic neurons uses serial action of the plasma membrane and vesicular monoamine (VAMT2) transporters. We can now define the sequences of the genes encoding these transporters in mice and humans, examine influences of deletions of this gene and alteration in its expression levels in transgenic mice, and identify sequence polymorphisms in the human VMAT2 gene. Examination of VMAT2 variants can provide potential insights into roles for allelic variants at these loci in variant drug responses and in diseases linked to monoaminergic systems, including substance abuse and Parkinson's disease.
Aging↗
Simulation of field-induced structural formation and transition in electromagnetorheological suspensions.
A computer simulation method has been used to study the three-dimensional structural formation and transition of electromagnetorheological (EMR) suspensions under compatible electric and magnetic fields. When the fields are applied simultaneously and perpendicularly to each other, the particles rapidly arrange into single layer structures parallel to both fields. In each layer, there is a two-dimensional hexagonal lattice. The single layers then combine together to form thicker sheetlike structures. With the help of the thermal fluctuations, the thicker structures relax into three-dimensional close-packed structures, which may be face-centered cubic (fcc), hexagonal close-packed (hcp) lattices, or, more probably, the mixture of them, depending on the initial configurations and the thermal fluctuations. On the other hand, if the electric field is applied first to induce the body-centered tetragonal (bct) columns in the system, and then the magnetic field is applied in the perpendicular direction, the bct to fcc structure transition is observed in a very short time. Following that, the structure keeps on evolving due to the demagnetization effect and finally forms close-packed structures with fcc and hcp lattice character. The simulation results are in agreement with the theoretical and experimental results.
Journal Article↗
Antisense inhibition of the Nr gene restores normal ripening to the tomato Never-ripe mutant, consistent with the ethylene receptor-inhibition model.
The hormone ethylene regulates many aspects of plant growth and development, including fruit ripening. In transgenic tomato (Lycopersicon esculentum) plants, antisense inhibition of ethylene biosynthetic genes results in inhibited or delayed ripening. The dominant tomato mutant, Never-ripe (Nr), is insensitive to ethylene and fruit fail to ripen. The Nr phenotype results from mutation of the ethylene receptor encoded by the NR gene, such that it can no longer bind the hormone. NR has homology to the Arabidopsis ethylene receptors. Studies on ethylene perception in Arabidopsis have demonstrated that receptors operate by a "receptor inhibition" mode of action, in which they actively repress ethylene responses in the absence of the hormone, and are inactive when bound to ethylene. In ripening tomato fruit, expression of NR is highly regulated, increasing in expression at the onset of ripening, coincident with increased ethylene production. This expression suggests a requirement for the NR gene product during the ripening process, and implies that ethylene signaling via the tomato NR receptor might not operate by receptor inhibition. We used antisense inhibition to investigate the role of NR in ripening tomato fruit and determine its mode of action. We demonstrate restoration of normal ripening in Nr fruit by inhibition of the mutant Nr gene, indicating that this receptor is not required for normal ripening, and confirming receptor inhibition as the mode of action of the NR protein.
Blotting, Northern↗
Noninvasive feature-based detection of delayed gastric emptying in humans using neural networks.
Radioscintigraphy is currently the gold standard for gastric emptying test which involves radiation exposure and is considerably expensive. We present a feature-based detection approach using neural networks for the noninvasive diagnosis of delayed gastric emptying from the cutaneous electrogastrogram (EGG). Simultaneous recordings of the EGG and scintigraphic gastric emptying test were made in 152 patients with symptoms suggestive of delayed gastric emptying. Spectral analyses were performed to derive EGG parameters which were used as the input of the neural network. The result of scintigraphic gastric emptying was used as the gold standard for the training and testing of the neural network. A correct classification of 85% (a specificity of 89% and a sensitivity of 82%) was achieved using the proposed method.
Adolescent↗
Dopamine transporter tryptophan mutants highlight candidate dopamine- and cocaine-selective domains.
Cocaine blocks the normal role of the dopamine transporter (DAT) in terminating dopamine signaling and in restricting its spatial spread through molecular interactions that remain largely obscure. Cocaine analog structure-activity studies suggest roles for cationic and hydrophobic interactions between DAT, dopamine, cocaine, and the sodium and chloride ions whose gradients power uptake processes. Tryptophan residues lying in putative DAT transmembrane domains could contribute to both aromatic and cationic interactions between DAT and dopamine or cocaine. We thus produced mutant DATs with alanine substitutions for tryptophans lying in or near putative DAT transmembrane domains. We have focused analyses on mutations that exert selective influences on affinities for dopamine or the cocaine analog CFT [(-)-2-beta-carbomethoxy-3-beta-(4-fluorophenyl)tropane]. Substitutions W162A, W255A, and W310A reduced dopamine uptake affinities. 5W266A, 12W555A, and 12W561A each reduced dopamine superficial recognition affinities by more than 3-fold and all retained affinity for CFT. W406A, W496A and W523A each reduced CFT affinity, and W84A increased CFT affinity. None of these four mutations decreased dopamine uptake affinity. These data, current provisional DAT structural models, and results from parallel studies of other mutants identify candidate dopamine-selective DAT domains for transmembrane dopamine permeation and regions in which mutations selectively lower CFT affinities. Tryptophan residues may contribute more extensively to these selective domains than other previously studied DAT amino acids. These sites provide tempting targets for selective blockers of cocaine recognition by DAT.
Alanine↗
GLUT-1 reduces hypoxia-induced apoptosis and JNK pathway activation.
Many studies have suggested that enhanced glucose uptake protects cells from hypoxic injury. More recently, it has become clear that hypoxia induces apoptosis as well as necrotic cell death. We have previously shown that hypoxia-induced apoptosis can be prevented by glucose uptake and glycolytic metabolism in cardiac myocytes. To test whether increasing the number of glucose transporters on the plasma membrane of cells could elicit a similar protective response, independent of the levels of extracellular glucose, we overexpressed the facilitative glucose transporter GLUT-1 in a vascular smooth muscle cell line. After 4 h of hypoxia, the percentage of cells that showed morphological changes of apoptosis was 30.5 +/- 2.6% in control cells and only 6.0 +/- 1.1 and 3.9 +/- 0.3% in GLUT-1-overexpressing cells. Similar protection against cell death and apoptosis was seen in GLUT-1-overexpressing cells treated for 6 h with the electron transport inhibitor rotenone. In addition, hypoxia and rotenone stimulated c-Jun-NH(2)-terminal kinase (JNK) activity >10-fold in control cell lines, and this activation was markedly reduced in GLUT-1-overexpressing cell lines. A catalytically inactive mutant of MEKK1, an upstream kinase in the JNK pathway, reduced hypoxia-induced apoptosis by 39%. These findings show that GLUT-1 overexpression prevents hypoxia-induced apoptosis possibly via inhibition of stress-activated protein kinase pathway activation.
Animals↗
Polymorphisms of surfactant protein gene A, B, D, and of SP-B-linked microsatellite markers in COPD of a Mexican population.
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Genetic Predisposition to Disease↗
The impact of workplace smoking ordinances in California on smoking cessation.
OBJECTIVES: The effect of local workplace smoking laws in California was assessed to determine whether such laws increase smoking cessation. METHODS: Workplace smoking ordinance data from 1990 were appended to 1990 California Tobacco Survey data from 4680 adult indoor workers who were current cigarette smokers or reported smoking in the 6 months before the survey. Ordinance effects on cigarette smoking and worksite policy were estimated by using multiple logistic regression controlling for sociodemographic variables. RESULTS: Smokers who worked in localities with a strong workplace ordinance (compared with no workplace ordinance) were more likely to report the existence of a worksite smoking policy (odds ratio [OR] = 1.6; 95% confidence interval [CI] = 1.2, 2.2) and to report quitting smoking in the prior 6 months (OR = 1.5; 95% CI = 1.1, 1.7). In communities with strong ordinances, an estimated 26.4% of smokers quit smoking within 6 months of the survey and were abstinent at the time of the survey, compared with an estimated 19.1% in communities with no ordinance. CONCLUSIONS: Workplace smoking ordinances increased smoking cessation among employed smokers, indicating that these laws may benefit smokers as well as nonsmokers.
Adolescent↗
Protocol for genomic DNA preparation from fresh or frozen serum for PCR amplification.
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DNA↗
Progress in the development of inhibitors of SH2 domains.
SH2 domains are discrete structural motifs common to a variety of critical intracellular signaling proteins. Inhibitors of specific SH2 domains have become important therapeutic targets in the treatment and/or prevention of restenosis, cancers (including small cell lung), cardiovascular disease, osteoporosis, apoptosis among others. Considering the social and economic impact of these diseases significant attention has been focused on the development of potent and selective inhibitors of specific SH2 domains. In particular, considerable research has been performed on Src, PI 3-kinase, Grb2 and more recently, Lck. In this review, we will focus on progress in the development of inhibitors for these specific SH2 domains and evaluate potential future targets.
Amino Acid Sequence↗
Impact of Guglielmi detachable coils on outcomes of patients with intracranial aneurysms treated by a multidisciplinary team at a single institution.
OBJECT: The goal of this study was to investigate the impact of the introduction of the Guglielmi detachable coil (GDC) therapeutic option on the overall management outcome of intracranial aneurysms. The authors accomplished this by assessing patient morbidity and mortality, inflation-adjusted hospital charges, lengths of stay in the hospital and the intensive care unit (ICU), and treatment efficacy. METHODS: The authors conducted a retrospective analysis of consecutive cases of intracranial intradural aneurysms managed by a single multidisciplinary neurovascular team at a tertiary care, academic referral center during the 24 months preceding the introduction of the GDC procedure (Group I or pre-GDC era, 77 patients) and during the first 24 months after its introduction (Group II or GDC era, 99 patients). Treatment with GDCs was considered for cases of higher clinical grade or poor surgical risk, or in response to patient preference (27 [27%] of 99 patients in Group II). Host and lesion parameters in our cohort were validated against outcome parameters by using univariate and multivariate analyses. The pre-GDC and GDC subgroups of patients were comparable for major disease severity parameters (patient age, lesion location, clinical grade, and hemorrhage severity). There was no significant difference in clinical outcome at 6 months, infarcts on computerized tomography scanning, or aneurysm obliteration rates before and after introduction of GDC treatment. Decreasing trends in duration of hospital and ICU stay and in inflation-adjusted hospital charges occurred well before and thus were unrelated to the introduction of the GDC therapeutic option. CONCLUSIONS: The results of this study do not demonstrate any significant impact of integration of the GDC modality on clinical outcome, mortality, morbidity, or effectiveness of treatment. Ongoing improvements in hospital charges and length of hospital stay appeared unrelated to the introduction of the GDC option.
Adult↗