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Biomedical subjects

D W Wong

Publications and source records attributed to D W Wong.

At least 19 recordsLinked to original sources
PubMed2001

Cloning and characterization of two cellulase genes from Lentinula edodes.

Lentinula edodes has traditionally been grown on fallen logs. It produces a wide array of enzymes to digest the lignocellulolytic substrate for nutrients. Thus, this organism represents a rich source of potentially potent lignocellulolytic enzymes that can be harnessed for conversion of biomass to simple sugars. These sugars can then be used as feedstock for ethanol production or other chemical syntheses. We have cloned two cellulase genes from L. edodes grown on a wood substrate without the use of genomic or cDNA libraries by using a PCR-based strategy employing degenerate primers directed at the cellulose-binding domain. cel7A encoded a 516-amino acid protein that belonged to glycosyl hydrolase family 7 and had sequence similarities to cbhI genes from other fungi. cel6B encoded a 444-amino acid protein that belonged to glycosyl hydrolase family 6 and had sequence similarities to cbhII genes from other fungi. We demonstrated that cel7A and cel6B transcript levels were positively correlated to L. edodes growth in the presence of crystalline cellulose.

Amino Acid Sequence↗
PubMed2001

An E. coli expression system for the extracellular secretion of barley alpha-amylase.

Libraries of modified genes are often screened during the process of genetically engineering enzymes with specifically tailored activities. It is important, therefore, to create expression systems which allow for the rapid screening of many clones. We developed an Escherichia coli expression system which will secrete enzymes into the growth medium. We describe the first reported expression of barley alpha-amylase in E. coli. The enzyme is secreted onto solid media containing starch to produce easily visualized halos. In addition, the enzyme is secreted into liquid media in an intact, active form.

Culture Media↗
PubMed2001

Characterization of active barley alpha-amylase 1 expressed and secreted by Saccharomyces cerevisiae.

Recombinant barley alpha-amylase 1 isozyme was constitutively secreted by Saccharomyces cerevisiae. The enzyme was purified to homogeneity by ultrafiltration and affinity chromatography. The protein had a correct N-terminal sequence of His-Gln-Val-Leu-Phe-Gln-Gly-Phe-Asn-Trp, indicating that the signal peptide was efficiently processed. The purified alpha-amylase had an enzyme activity of 1.9 mmol maltose/mg protein/min, equivalent to that observed for the native seed enzyme. The kcat/Km was 2.7 x 10(2) mM(-1) x s(-1), consistent with those of alpha-amylases from plants and other sources.

Hordeum↗
PubMed2000

Polypoidal choroidal vasculopathy masquerading as central serous chorioretinopathy.

OBJECTIVE: To differentiate polypoidal choroidal vasculopathy (PCV) from central serous chorioretinopathy (CSC). DESIGN: A retrospective, observational case series. PARTICIPANTS: Thirteen patients originally diagnosed with CSC proved to have PCV after more extensive evaluation and follow-up. METHODS: A clinical and angiographic review of patients with manifestations of CSC, including macular detachment. MAIN OUTCOME MEASURES: Demographic data, funduscopic examination, and fluorescein and indocyanine green (ICG) angiographic findings. RESULTS: Thirteen patients initially suspected of having CSC were ultimately diagnosed as having PCV. These eyes had exudative macular detachments secondary to a small caliber, polypoidal choroidal vascular abnormality or so-called polypoidal choroidal neovascularization. The clinical manifestations in the fundus varied. They included multiple, variably sized serous pigment epithelial detachments, neurosensory retinal detachment, lipid deposition, patchy atrophy of the pigment epithelium and indistinct staining from decompensation of the posterior blood-retinal barrier on fluorescein angiography. In reality, the suspected PEDs proved to be polypoidal lesions of PCV when imaged with ICG angiography. CONCLUSIONS: The clinical diagnosis of CSC or PCV generally poses little challenge to the experienced retinal specialist. However, in CSC with persistent and/or recurrent exudation, a myriad of retinal pigment epithelial changes may evolve that make it difficult to differentiate these two entities. In such patients, ICG angiography is useful in differentiating CSC from PCV. An accurate clinical diagnosis is important since each of these entities, CSC and PCV, may differ in terms of their risk factors, natural course, and visual prognosis.

Adult↗
PubMed2000

Microassay for rapid screening of alpha-amylase activity.

A microassay was developed for measuring the activity of alpha-amylases in the nanogram enzyme concentration range, based on the use of dye-labeled cross-linked starch as the substrate, and the release of soluble colored fragments formed in enzyme hydrolysis. Reaction conditions were optimized to generate a linear correlation between the increase in absorbance and a reaction time of 0-10 min, as well as enzyme concentrations in the range of 0-50 ng. A standard curve for the conversion of absorbance to enzyme activity units was constructed. The protocol developed was applied to monitoring the production of ultralow concentrations of recombinant barley alpha-amylase in yeast cells.

Coloring Agents↗
PubMed2000

A functional raw starch-binding domain of barley alpha-amylase expressed in Escherichia coli.

The mature form of barley seed low-pI alpha-amylase (BAA1) possesses a raw starch-binding site in addition to the catalytic site. A truncated cDNA encoding the C-terminal region (aa 281-414) and containing the proposed raw starch-binding domain (SBD) but lacking Trp278/Trp279, a previously proposed starch granule-binding site, was synthesized via PCR and expressed in Escherichia coli as an N-terminal His-Tag fusion protein. SBD was produced in the form of insoluble inclusion bodies that were extracted with urea and successfully refolded into a soluble form via dialysis. To determine binding, SBD was purified by affinity chromatography with cycloheptaamylose as ligand cross-linked to Sepharose. This work demonstrates that a SBD is located in the C-terminal region and retains sufficient function in the absence of the N-terminal, catalytic, and Trp278/279 regions.

Amino Acid Sequence↗
PubMed2000

Isolation of a raw starch-binding fragment from barley alpha-amylase.

Barley alpha-amylase was purified by ammonium sulfate fraction, ion-exchange, ultrafiltration, and gel filtration to homogeneity. The purified enzyme was partially digested with trypsin, and the reaction mixture was applied to a cyclohepta-amylose epoxy Sepharose 6B column. Bound fragments were eluted by free cyclohepta-amylose, lyophilized, and separated on Tricine gels. Four fragments were shown to interact with beta-cyclodextrin. The fragment that could be identified on the gel with the lowest molecular weight (11 kDa) was electroblotted onto PVDF membrane for sequencing. The N-terminal sequence of this fragment was determined with the N-terminal amino acid corresponding to Ala283 in the whole protein. The trypsin cleavage was at Lys282/Ala283 and the C-terminal cleavage occurred at Lys354/Ile355 to give a fragment size of 11 kDa as estimated by SDS-PAGE. The fragment would be located at the C-terminal region, forming a majority of the antiparallel beta-sheets in domain C and the alpha7- and alpha8-helices of the (alpha/beta)8 domain.

Amino Acid Sequence↗
PubMed1999

Polypoidal choroidal vasculopathy and neovascularized age-related macular degeneration.

OBJECTIVE: To determine the nature and frequency of polypoidal choroidal vasculopathy (PCV) in a series of patients suspected of having neovascularized age-related macular degeneration (AMD). METHODS: A prospective analysis of 167 consecutive, newly diagnosed patients aged 55 years or older with presumed neovascularized AMD was performed. All patients were examined with fundus biomicroscopy as well as fluorescein and indocyanine green angiography. RESULTS: Choroidal neovascularization secondary to AMD was diagnosed in 154 (92.2%) of 167 patients; 13 (7.8%) patients had PCV. The patients affected by PCV were younger than those with AMD (P = .01). Peripapillary choroidal neovascularization was seen in 3 (1.9%) of 154 patients with AMD and 3 (23.1%) of 13 patients with PCV (P = .006). Significant drusen were present in 63 (70%) of 90 fellow eyes with unilateral AMD compared with only 1 (16.7%) of 6 eyes with PCV (P = .02). Only 5 patients with AMD (3.2%) were nonwhite compared with 3 patients with PCV (23.1%) (P = .02). CONCLUSIONS: A measurable number of elderly patients with findings suggestive of neovascularized AMD and serosanguineous macular manifestations will instead have PCV. Polypoidal choroidal vasculopathy can occur in any sex or race, but is more commonly seen in the peripapillary area, without associated drusen, and in nonwhite patients. It is important to differentiate AMD from PCV because there are significant differences in the demographic risk profile, natural course, visual prognosis, and management of these patients.

Aged↗
PubMed1999

Combinatorial chemistry and its applications in agriculture and food.

Combinatorial chemistry has become a major focus of research activity in the pharmaceutical industry for development new therapeutic compounds. The same techniques could be potentially applied to benefit agricultural and food research. This article reviews the various procedures used in combinatorial chemistry, outlines some of the strengths and limitations of the various methods, and proposes potential areas in agriculture and food that could be benefited by this technology. These areas include developing new antimicrobial agents, antioxidants, and other additives, creating antigen-binding molecules for the detection or removal of food pathogens or toxicants, engineering food proteins and enzymes for specific functions, and modifying biosynthetic pathways for the production of novel natural products.

Chemistry, Agricultural↗
PubMed1999

Phage-displayed peptide ligands for pancreatic alpha-amylase cross-react with barley alpha-amylase.

Peptide ligands that bind to pancreatic alpha-amylase were isolated from bacteriophage libraries displaying random 15-mer peptides by iterative affinity selection and amplification. The DNA sequences of selected clones from the final round of biopanning were determined. The two phage-display ligands with high-binding activities contained a high content of Arg, Tyr, and Trp residues with the short consensus sequence Arg-X-Tyr-Trp. These clones were shown to exhibit comparable binding interactions toward barley alpha-amylase based on transducing units titering and measurement of the dissociation constants.

Amino Acid Sequence↗
PubMed1998

Cutting seton versus two-stage seton fistulotomy in the surgical management of high anal fistula.

BACKGROUND: The aim of this study was to compare the clinical results obtained with the cutting seton and the two-stage seton fistulotomy (TSSF) in the surgical management of high anal fistula. METHODS: The case records of 59 patients with high anal fistula of cryptoglandular origin treated with cutting seton (n = 12) or TSSF (n = 47) over a 5-year period were retrospectively reviewed. There was no difference between the groups in age, sex distribution, or estimated percentage of anal sphincter involved by the fistula. Follow-up was by a mailed questionnaire inquiring about fistula recurrence, incontinence, and degree of satisfaction. Mean follow-up was similar in both groups (27 months for cutting seton versus 33 months for TSSF). Comparisons were made by Student t and chi 2 tests, as required. RESULTS: There were no differences in the rate of fistula recurrence between the groups treated with cutting seton or TSSF (one of 12 versus four of 47), difficulty holding gas (six of 12 versus 25 of 47), underwear staining (six of 12 versus 18 of 47), stool incontinence (three of 12 versus 12 of 27), overall incontinence (eight of 12 versus 31 of 47) and mean incontinence score (4.9 versus 4.2). The fistula healing time and degree of satisfaction with the operation were not significantly different between the groups. One-half of the patients treated by TSSF had the seton removed under general or epidural anaesthesia. CONCLUSION: Both techniques are equally effective in eradicating the fistula, and both are associated with a similar rate of incontinence.

Colectomy↗
PubMed1997

Harvesting a lamellar graft from a corneoscleral button: a new technique.

PURPOSE: To describe a new technique for harvesting a lamellar graft from a corneoscleral button in the absence of an artificial anterior chamber. METHODS: Two layers of sterile fine-weave fabric were wrapped tightly around a glass orbital implant. The corneoscleral button was sutured firmly at its scleral rim onto the fabric. The lamellar graft then was dissected in the regular fashion. RESULTS: The lamellar graft was successfully obtained. During the dissection, the donor cornea and its supporting fabric-covered glass ball were easily handled, and there was minimal risk of perforation of the posterior lamella of the donor cornea. CONCLUSION: This simple technique allows a lamellar graft to be easily obtained from a corneoscleral button without specialized and expensive equipment.

Cornea↗
PubMed1997

Rapid detection of acute appendicitis with Tc-99m-labeled intact polyvalent human immune globulin.

BACKGROUND: Acute appendicitis remains problematic for emergency clinicians. A rapid and definitive test is needed for detecting acute appendicitis before surgical intervention. The purpose of this clinical trial was to determine the efficacy of Tc-99m-labeled intact polyvalent human immune globulin (Tc-99m IgG) in the evaluation of acute appendicitis. STUDY DESIGN: Thirty-five patients with clinically suspected acute appendicitis were evaluated with Tc-99m IgG. After the intravenous injection of 25 mCi (92.5 MBq) of Tc-99m IgG, anterior flow, single photon emission computerized tomography (SPECT) and planar delayed images of the abdomen were obtained. Any abnormal focal uptake of Tc-99m IgG in the right lower quadrant was considered to be a positive scan. RESULTS: Twenty-one patients with a positive Tc-99m IgG scan underwent laparotomy and were found to have acute appendicitis. Of the 14 patients who had negative scans, 7 underwent surgery. In this series, Tc-99m IgG study yielded 21 true-positive, 12 true-negative, and 2 false-negative results with a sensitivity, specificity, and accuracy of 91%, 100%, and 94%, respectively. The positive and negative predictive values were 100% and 86%, respectively. There were no false-positive results. CONCLUSIONS: Tc-99m IgG scintigraphy can provide the clinicians a simple, rapid, and definitive test for the diagnosis of acute appendicitis.

Acute Disease↗
PubMed1997

Analysis of cross-reactive allergens from American and German cockroaches by human IgE.

Serum from atopics hypersensitive to the American cockroach were examined for their specific IgE to American and German cockroaches by the fluoroallergosorbent test (FAST). Of 44 sera tested, 86.4% (38/44) contained IgE to American and German cockroaches, and 13.6% (6/44) were found to be positive for American cockroach alone by FAST. Nine individual sera containing IgE antibodies to both cockroaches were used to analyze the cross-reacting allergens in the crude American and German cockroach extracts by FAST inhibition and immunoblotting FAST-inhibition studies showed various degrees but similar inhibition of binding of human IgE to solid-phase American cockroach extract. Proteins from both cockroach extracts were separated on SDS-PAGE followed by immunoblotting, and the results showed considerable heterogeneity in the IgE-binding patterns with each of the cockroach extracts for the same nine individual sera. Components with apparent molecular weights of 60, 52, 49, 38, and 12 kDa from both the American and German cockroaches were able to bind IgE antibody. These results suggest the presence of cross-reactive allergens in the American and the German cockroaches.

Adolescent↗
PubMed1996

Technetium-99m IgG immunodetection of acute appendicitis in rats.

The efficacy of Tc-99m-labeled intact polyvalent human immune globulin (IgG) for the early localization and detection of acute appendicitis was evaluated by an animal model. Acute appendicitis was surgically produced in adult Sprague-Dawley white rats by ligating a 3-cm segment of the caecum and inoculating with E. coli. After 4-5 days, nuclear scintigraphy was performed following intravenous injection of 5 mCi of Tc-99m IgG via the tail vein. Appendiceal and colonic infections were clearly visualized by Tc-99m IgG scans within 30 min postinjection and confirmed by autopsy and tissue distribution data. With the exception of the liver, the kidneys, and the urinary bladder, abdominal tissue background activity was minimal. These findings suggest that Tc-99m-labeled intact IgG may have potential clinical application in the early detection and diagnosis of acute appendicitis in emergency medicine.

Acute Disease↗
PubMed1996

Structures and functionalities of milk proteins.

During the last decade, marked progress has been made in the study of the fine details of the structures of milk proteins such as caseins, beta-lactoglobulin, alpha-lactalbumin, and lactotransferrin. Many of the functional properties of the individual milk proteins, as well as the milk protein products, may be described at the molecular level. This article is an attempt to thoroughly review the three-dimensional structures of major milk proteins, and to correlate them with the functional aspects of these proteins as food ingredients.

Amino Acid Sequence↗
PubMed1996

American cockroach Cr-PI allergen induces lymphocyte proliferation and cytokine production in atopic patients.

BACKGROUND: Previous studies have shown cockroach-induced antigen-specific IgE-mediated asthma. In cockroach-infested areas, more then 50% of asthmatic subjects may have positive skin reactions to this allergen. Partial purified Cr-PI allergen from American cockroaches contains allergens with molecular weights of 72 and 78 kDa; however, little is known about its effect on the lymphocyte proliferation and cytokine production. OBJECTIVE: IgE synthesis is known to be regulated by interleukin-4 (IL-4) and interferon gamma (IFN gamma). Therefore, we studied Cr-PI allergen-induced cytokine production in atopic patients and healthy normal controls to understand each factors' role in the disease. METHODS: Peripheral blood mononuclear cells (PBMC) from cockroach skin-sensitive patients and controls were stimulated with mitogen and Cr-PI for proliferative response and cytokine production. Cr-PI antigen-specific T-cell cultures of atopic patients and healthy normal controls were used to test Cr-PI-induced proliferation and cytokine mRNA expression. RESULTS: PMBC of atopic subjects showed a significantly (P < 0.01) higher stimulation index for Cr-PI induced proliferation (SI = 11.8 +/- 3.7) when compared with that of non-atopic subjects (SI = 4.1 +/- 0.8) and cord bloods (SI = 2.1 +/- 0.4). Cr-PI-induced IL-4 was observed only in the PBMC of atopic patients, whereas Cr-PI-induced IFN gamma was detected in both atopic patients and normal controls. Likewise, Cr-PI-induced IL-4 mRNA expression in T-cell cultures was detected in all atopics but only one of nine controls. CONCLUSION: IL-4 mRNA expression and IL-4 production in PBMC and T-cell cultures of atopic patients showed good correlation with clinical symptoms, skin-reactivity, specific IgE and proliferative response to Cr-PI. These results suggests that cockroach allergen may be a hidden cause of asthma and other atopic diseases.

Adult↗