Biomedical subjects

Y W Hwang

Publications and source records attributed to Y W Hwang.

At least 19 recordsLinked to original sources

PubMed2003

Nitrification and denitrification using a single biofilter packed with granular sulfur.

This study was performed to develop a granular sulfur packed nitrification/denitrification process employing a uniquely designed single biofilter, which treated a relatively low carbon loaded domestic wastewater taken from a primary clarifier at a municipal wastewater treatment facility. The system was tested on varying experimental conditions, e.g. inflow flow, organic load and nitrogen load. Regardless of flow rate being increased, SS and COD was unvaryingly removed up to 90 and 80%, respectively. Moreover, TKN was also decomposed up to 90%. Increase in COD load gradually led to escalating level of non-biodegradable compounds observed in effluent. Nitrification was accomplished as high as 92%, whereas denitrification was achieved up to approximately 87%. For a while, nitrification and denitrification were observed at 0.65 and 0.55 kg/m3 x day, respectively. Eventually, T-N was decomposed as high as 46%. It was concluded that granular sulfur can be used for not only electron donor, but also for a media to properly treat low carbon loaded wastewater and to filter SS efficiently.

Biodegradation, Environmental↗

PubMed2000

Two distinct mechanisms for regulation of nonmuscle myosin assembly via the heavy chain: phosphorylation for MIIB and mts 1 binding for MIIA.

In search of the regulation mechanisms for isoform specific myosin assembly, we have used the COOH-terminal fragments of nonmuscle myosin isoforms MIIA and MIIB (MIIA(F46) and MIIB(alpha)(F47)) as a model system. Phosphorylation by protein kinase C (PK C) or casein kinase II (CK II) within or near the nonhelical tail-end domain inhibits assembly of MIIB(alpha)(F47) [Murakami, N., et al. (1998) Biochemistry 37, 1989]. In the study presented here, we mutated the kinase sites to analyze the inhibition mechanisms of MIIB assembly by phosphorylation. Replacement of the CK II or PK C sites with Asp (MIIB(alpha)(F47)-CK-5D or -PK-4D) strongly inhibited the filament assembly, with or without Mg(2+), by significantly increasing the critical concentrations for assembly. Without Mg(2+), MIIB(alpha)(F47)-CK-5D or -PK-4D inhibited the assembly of wild-type (wt) MIIB(alpha)(F47) by either mixing as homofragments or forming heterofragments. With 2.5 mM Mg(2+), MIIB(alpha)(F47)-wt promoted assembly of MIIB(alpha)(F47)-CK-5D and -PK-4D in homofragment mixtures, but not by forming heterofragments. MIIA(F46) coassembled with MIIB(alpha)(F47)-wt and -CK-5D and altered their assembly patterns. In contrast, assembly of MIIB(alpha)(F47)-PK-4D was unchanged by MIIA(F46). A metastasis-associated protein, mts 1, bound in a Ca(2+)-dependent manner to MIIA(F46), but not appreciably to MIIB(alpha)(F47). At 0.15 M NaCl, mts 1-Ca(2+) not only inhibited MIIA(F46) assembly but also disassembled the MIIA(F46) filaments. Mts 1, however, did not affect the assembly of MIIB(alpha)(F47) in MIIA(F46) and MIIB(alpha)(F47) mixtures, indicating that mts 1 is an inhibitor specific to MIIA assembly. Our results suggest strongly that assembly of MIIA and MIIB is regulated by distinct mechanisms via tail-end domains: phosphorylation of MIIB and mts 1 binding to MIIA. These mechanisms may also function to form MIIA or MIIB homofilaments by selectively inhibiting MIIB or MIIA assembly.

Actin Cytoskeleton↗

PubMed2000

Comparison of serum specific IgE antibodies to staphylococcal enterotoxins between atopic children with and without atopic dermatitis.

BACKGROUND: The skin of patients with atopic dermatitis (AD) exhibits a striking susceptibility to colonization and infection by Staphylococcus aureus. The exotoxins secreted by S. aureus can act as superantigens and classic allergens, inducing the production of functionally relevant specific IgE antibodies. The aim of this study was to compare the levels and positive rates of serum staphylococcal enterotoxin A (SEA)- and staphylococcal enterotoxin B (SEB)-specific IgE between atopic children with and without AD. METHODS: Sixty children with AD, 55 children with respiratory allergy without AD, and 24 nonatopic healthy children were studied. The levels and positive rates of serum SEA- and SEB-specific IgE were compared among three groups. The correlation between the levels or positive rates of serum SEA/SEB-specific IgE and the severity of AD or the presence of previous skin infections was studied. RESULTS: The children with AD had significantly higher levels and positive rates of serum SEA- and SEB-specific IgE than the atopic children without AD (P < 0.001) and the nonatopic children (P < 0.001). There was no significant difference in the levels and positive rates of serum SEA- and SEB-specific IgE between the atopic children without AD and the nonatopic children. With or without adjustment for the potential confounding effect of total serum IgE levels, the levels and positive rates of serum SEA- and SEB-specific IgE were significantly correlated with severity of AD (P <0.005), but they were not significantly different between AD children with and without previous skin infections. CONCLUSIONS: SEA and SEB may contribute to chronic inflammation and exacerbation of AD through the IgE-mediated immune response.

Adolescent↗

PubMed1997

The role of cysteinyl residues in the activity of bacterial elongation factor Ts, a guanosine nucleotide dissociation protein.

The modification of E.coli elongation factor Ts (EF-Ts) by NEM and other sulfhydryl reagents inactivates the protein's ability to bind EF-Tu.GDP and to catalyze GDP exchange. The reactive residue was found to be Cys-22. Replacement of Cys-22 by Ser or Gly only partially impairs the binding or catalytic properties of EF-Ts while it completely protects EF-Ts from the inactivation by NEM. Cys-22 of EF-Ts is not located at the EF-Ts.EF-Tu interface, yet it can be modified only when EF-Ts is not bound to EF-Tu. These results support the proposal that the conformation change around Cys-22 in the amino terminus of EF-Ts rather than Cys-22 itself is essential for binding EF-Tu. Apparently, modification of Cys-22 by NEM disrupts the conformation change and inactivates EF-Ts. The return of EF-Ts to its native conformation may provide the driving force for the rate-determining step in the catalytic cycle, the dissociation of EF-Ts from EF-Tu.GNP.

Amino Acid Substitution↗

PubMed1997

TNF receptor-2-triggered apoptosis is associated with the down-regulation of Bcl-xL on activated T cells and can be prevented by CD28 costimulation.

Stimulation of recently activated T cells results in apoptosis of the responding cells, a process referred to as activation-induced cell death. This process is believed to play an important role in the regulation of immune homeostasis and is suggested to be mediated mainly by interactions between Fas and Fas ligand. Recent evidence indicates that TNF-alpha and TNFR2 interaction plays an important role in down-regulating activated T cells. The role of TNFR2 signaling in activation-induced cell death, however, has not been directly examined. We demonstrate here that 48-h activated T cells are most sensitive to TNFR2-induced apoptosis. Cross-linking of TNFR2 on activated T cells results in down-regulated protein and mRNA expression of Bcl-xL. Furthermore, CD28 costimulation can prevent anti-TNFR2-induced apoptosis and restore Bcl-xL expression. These results have potential implications for understanding the role of TNFR2 signaling in the regulation of T cell responses.

Animals↗

PubMed1996

The dominant negative effects of H-Ras harboring a Gly to Ala mutation at position 60.

v-H-Ras harboring the Gly-60 to Ala mutation (G60A) lacks the ability to induce germinal vesicle breakdown in Xenopus oocytes. Moreover, this mutant is capable of inhibiting the activity of v-H-Ras to induce oocyte germinal vesicle breakdown when co-injected. The duration and the extent of inhibition depends on the molar ratio of v-H-Ras(G60A) to v-H-Ras. The inhibition is not due to a general toxicity of v-H-Ras(G60A) to oocytes because oocytes injected with v-H-Ras(G60A) can be readily induced to mature by other mitogenic agents, such as insulin, insulin-like growth factor 1, insulin-like growth factor 2, and phosphatidylcholine-specific phospholipase C. The dominant negative effect of v-H-Ras(G60A) requires proper membrane attachment of v-H-Ras(G60A). By using a competition assay, it was concluded that the dominant negative phenotype of v-H-Ras(G60A) resulted from sequestering H-Ras downstream effector(s). Raf-1 was identified as one of the sequestered targets.

Animals↗

PubMed1996

The differential effects of the Gly-60 to Ala mutation on the interaction of H-Ras p21 with different downstream targets.

We examined the effects of the Gly-60 to Ala mutation on the interaction of H-Ras with Ras GTPase activating protein (GAP), neurofibromin 1 (NF1), Raf-1, and ral guanine nucleotide dissociation stimulator (ralGDS), factors that interact with GTP-bound form of H-Ras. Previous study has shown that the G60A mutation perturbs GTP-induced conformational changes of H-Ras. We found that the G60A mutation decreases GTPase activity of H-Ras without significantly affecting GTP/GDP binding. The reduction in GTPase activity is most dramatic in the presence of GAP or NF1. Interestingly, the G60A mutation does not appear to alter the affinity of H-Ras for GAP or NF1. The G60A mutation moderately reduces the binding of H-Ras to Raf-1 Ras binding domain; however, the binding of H-Ras to ralGDS Ras binding domain was more significantly affected by the same mutation. These results indicate that although GAP, NF1, Raf-1, and ralGDS all interact with H-Ras in a GTP-dependent manner and they are able to compete against each other for binding to H-Ras, these factors share overlapping but not identical binding domains on H-Ras. The significance of our findings is discussed in the light of the GTP-induced conformational change model.

Alanine↗

PubMed1996

Ras interaction with two distinct binding domains in Raf-1 may be required for Ras transformation.

Although Raf-1 is a critical Ras effector target, how Ras mediates Raf-1 activation remains unresolved. Raf-1 residues 55-131 define a Ras-binding domain essential for Raf-1 activation. Therefore, our identification of a second Ras-binding site in the Raf-1 cysteine-rich domain (residues 139-184) was unexpected and suggested a more complex role for Ras in Raf-1 activation. Both Ras recognition domains preferentially associate with Ras-GTP. Therefore, mutations that impair Ras activity by perturbing regions that distinguish Ras-GDP from Ras-GTP (switch I and II) may disrupt interactions with either Raf-1-binding domain. We observed that mutations of Ras that impaired Ras transformation by perturbing its switch I (T35A and E37G) or switch II (G60A and Y64W) domain preferentially diminished binding to Raf-1-(55-131) or the Raf-1 cysteine-rich domain, respectively. Thus, these Ras-binding domains recognize distinct Ras-GTP determinants, and both may be essential for Ras transforming activity. Finally, since Ha-Ras T35A and E37G mutations prevent Ras interaction with full-length Raf-1, we suggest that Raf-Cys is a cryptic binding site that is unmasked upon Ras interaction with Raf-1-(55-131).

3T3 Cells↗

PubMed1995

Switching nucleotide specificity of Ha-Ras p21 by a single amino acid substitution at aspartate 119.

We examined c-Ha-Ras harboring an aspartate to asparagine substitution at position 119 (mutation D119N). The Asp-119 is part of the conserved NKXD motif shared by members of the regulatory GTPase family. This asparagine residue has been proposed to participate in direct bonding to the guanine ring and to determine the guanine-nucleotide binding specificity. The D119N mutation was found to alter nucleotide specificity of Ha-Ras from guanine to xanthine, an observation that directly supports the essential role of hydrogen bonding between the side chain of the aspartic acid residue and the guanine ring in nucleotide binding specificity. Besides nucleotide binding specificity, the D119N mutation has little or no effect on the interaction of Ha-Ras with SDC25C, SOS1, GAP, or Raf. Neither does it affect the hydrolysis of nucleotide triphosphate. Like xanthine-nucleotide-specific EF-Tu, xanthine-nucleotide-specific Ras and related proteins will be useful tools for elucidating cellular systems containing multiple regulatory GTPases.

Aspartic Acid↗

PubMed1995

Mutagenesis of the H-ras p21 at glycine-60 residue disrupts GTP-induced conformational change.

The function of Gly-60, the conserved glycine in the DXXG domain of v-H-ras, was examined by site-directed mutagenesis. It was found that while the G60A (Gly-60 to Ala substitution) mutation has little effect on the interaction of H-ras with guanine nucleotides, it completely abolishes the biological activity of v-H-ras. The G60A mutation also exerts little effect on the interaction of H-ras with SDC25C (a guanine nucleotide exchange factor) and GAP. However, the G60A mutation does lower the ability of H-ras to bind Raf. GTP induces an enhancement of fluorescence emission in complexes consisting of H-ras and the fluorescent dye 8-anilino-1-naphthalenesulfonic acid. This enhancement is blocked by the G60A mutation. On the basis of these observations, we propose that the GTP-induced conformational change of H-ras, a process required for H-ras activities, is impaired by the G60A mutation.

3T3 Cells↗

PubMed1993

Biological activity of a K-ras mutant that contains the 12R/59T/116Y mutations.

The 12R/59T/116Y mutations have been shown to confer a dominant negative activity on H-ras oncogene (H-ras 116Y). To determine whether this event is unique for H-ras, we introduced the same mutations into K-ras oncogene. This mutant, K-ras 116Y, suppressed transformed phenotypes induced by overexpression of H-ras proto-oncogene. NIH3T3 cells expressing K-ras 116Y were resistant to transformation by v-fes oncogene. Analysis of chimaeras between H- and K-ras 116Y showed that the C-terminal variable region determines the level of suppressor activity. These results suggest that these mutations are applicable to other GDP/GTP binding proteins.

3T3 Cells↗

PubMed1993

Inhibition of SDC25 C-domain-induced guanine-nucleotide exchange by guanine ring binding domain mutants of v-H-ras.

Guanine-nucleotide exchange is the reaction that controls the activation of H-ras p21. This reaction is stimulated by the guanine-nucleotide exchange factor. In this study we show that H-ras p21 harboring guanine ring binding domain (the conserved NKXD motif) mutations, such as N116I or K117E, are potent inhibitors of H-ras p21 guanine-nucleotide exchange reaction promoted by SDC25C (Saccharomyces cerevisiae SDC25 C-domain gene product), a guanine-nucleotide exchange factor. The inhibition is due to the formation of a stable but catalytically inactive complex consisting of the H-ras mutant and SDC25C. By examining the interaction of v-H-ras(N116I) or v-H-ras(K117E) with SDC25C at different concentrations of guanine-nucleotide, we demonstrate that the mechanism of SDC25C-promoted guanine-nucleotide exchange proceeds through the following pathway. First, SDC25C binds to H-ras and forms an intermediate H-ras.SDC25C complex; subsequently, an incoming guanine-nucleotide dissociates the complex, releasing SDC25C from H-ras and causes guanine-nucleotide exchange. This mechanism is similar to the one proposed for Escherichia coli elongation factor Ts-catalyzed guanine-nucleotide exchange.

Fungal Proteins↗

PubMed1992

The identification of a domain in Escherichia coli elongation factor Tu that interacts with elongation factor Ts.

A method has been developed to search for the elongation factor Tu (EF-Tu) domain(s) that interact with elongation factor Ts (EF-Ts). This method is based on the suppression of Escherichia coli EF-Tu-dominant negative mutation K136E, a mutation that exerts its effect by sequestering EF-Ts. We have identified nine single-amino acid- substituted suppression mutations in the region 146-199 of EF-Tu. These mutations are R154C, P168L, A174V, K176E, D181G, E190K, D196G, S197F, and I199V. All suppression mutations but one (R154C) significantly affect EF-Tu's ability to interact with EF-Ts under equilibrium conditions. Moreover, with the exception of mutation A174V, the GDP affinity of EF-Tu appears to be relatively unaffected by these mutations. These results suggest that the domain of residues 154 to 199 on EF-Tu is involved in interacting with EF-Ts. These suppression mutations are also capable of suppressing dominant negative mutants N135D and N135I to various degrees. This suggests that dominant negative mutants N135D and N135I are likely to have the same molecular basis as the K136E mutation. The method we have developed in this study is versatile and can be readily adapted to map other regions of EF-Tu. A model of EF-Ts-catalyzed guanine-nucleotide exchange is discussed.

Amino Acid Sequence↗

PubMed1992

Detection of point mutations in codon 331 of mitochondrial NADH dehydrogenase subunit 2 in Alzheimer's brains.

Point mutations in codon 331 of mitochondrial NADH dehydrogenase subunit 2 (ND2) were detected in 10 of 19 Alzheimer's brains but not in 11 normal brains. The same mutations were also detected in 2 of 6 patients with amyotrophic lateral sclerosis (ALS). However, neurofibrillary tangles and neuritic plaques characteristic of Alzheimer's disease were found histologically in the brain of one ALS patient who was positive of the mutation. The finding suggests that a point mutation in ND2 is a potential risk factor for Alzheimer's disease.

Adult↗

PubMed1990

trans-Dominant suppressor mutations of the H-ras oncogene.

Site-directed mutagenesis of the conserved sequence motifs of p21 generated a group of mutant p21s defective in GTP binding. Some of these mutants were highly transforming, whereas others were transformation defective. Among the latter group, we found two mutants, derived from the v-H-ras oncogene by substituting the asparagine-116 with tyrosine and isoleucine, that exhibited a trans-dominant activity of suppressing the transformed phenotype of NIH3T3 cells induced by a long terminal repeat-linked c-H-ras and a wild-type v-H-ras. They caused reduction of the colony-forming efficiency in soft agar (78% in c-ras-transformed cells; 55% in v-ras cells) and morphological reversion of ras transformants. Subclones of revertants expressed a great excess of mutant p21 relative to the c-ras p21 present in these cells. These mutants were not lethal to NIH3T3 cells. Apparently, defective proteins encoded by suppressor mutants sequestered vital targets for ras function. Suppressor mutants also induced morphological reversion of NIH3T3 cells transformed by src, fes/flp, sis, and fms oncogenes, suggesting that these oncogenes function upstream to ras in the signaling pathways. Cells transformed by mos and a chemical carcinogen were unaffected.

Animals↗

PubMed1989

Site-directed mutagenesis of the GDP binding domain of bacterial elongation factor Tu.

The tertiary structure model of EF-Tu predicts that the amino acid sequence Val-Asp-His-Gly-Lys-Thr-Thr-Leu (residues 20-27) forms a pocket that binds the pyrophosphate group. To test this model we used site-directed mutagenesis to produce forms of EF-Tu altered in this region. The following mutations were constructed: Gly-20, Val-23, Glu-24, Ile-25, and Pro-27. Each protein was labeled with [35S]Met and was tested for its ability to interact with guanosine nucleotides and EF-Ts. The in vivo activity of each altered protein was tested by determining its ability to confer aurodox sensitivity to a resistant host. Mutations at residues 23, 24, 25, and 27 eliminated the ability of EF-Tu to interact with either guanosine nucleotides or EF-Ts in vitro, and these forms were also inactive in vivo. In contrast, the Gly-20 form was nearly as active as wild-type EF-Tu in vitro and in vivo. This mutation is theoretically equivalent to reversion of the Gly to Val transforming mutation of the cellular form of the ras gene product p21, a protein proposed to be structurally similar to EF-Tu in the GDP binding domain. In contrast to its effect in the ras gene, the Val to Gly conversion did not affect the endogenous GTPase of EF-Tu. We conclude that the tertiary structure model is correct in its assignment of the pyrophosphate binding site to residues 23-27; however, there are likely to be some significant differences between the configurations of the GTPases of EF-Tu and p21.

Amino Acid Sequence↗

PubMed1989

Mutagenesis of bacterial elongation factor Tu at lysine 136. A conserved amino acid in GTP regulatory proteins.

We have studied the effects of specific amino acid replacements in EF-Tu upon the protein's interactions with guanine nucleotides and elongation factor Ts (EFTs). We found that alterations at the lysine residue of the Asn-Lys-Cys-Asp sequence, the guanine ring-binding sequence, differentially affect the protein's ability to bind guanine nucleotides. Wild type EF-Tu (Lys-136) binds GDP and GTP much more tightly than do many of the altered proteins. Replacing lysine by arginine lowers the protein's affinity for GDP by about 20-fold relative to the change in its affinity for EF-Ts. Substitutions at residue 136 by glutamine (K136Q) and glutamic acid (K136E) further lower the protein relative affinity for GDP by factors of about 4 and 10, respectively. In contrast, replacement of the residue by isoleucine (K136I) eliminates guanine nucleotide binding as well as EF-Ts binding. Apparently, the distortion of this loop by substitution at residue 136 of a bulky hydrophobic residue can hamper the binding for both substrates or disrupt the folding of the protein. All altered proteins except EF-Tu(K136I) are able to bind tRNA(Phe); however, they require much higher concentrations of GTP than wild type EF-Tu. In minimal media, Escherichia coli cells harboring plasmids encoding EF-Tu(K136E) or EF-Tu(K136Q) suffer growth retardation relative to cells bearing the same plasmid encoding wild type EF-Tu. Co-transformation of these cells with a compatible plasmid bearing the EF-Ts gene reverses this growth problem. The growth retardation effect of some of the altered proteins can be explained by their sequestering EF-Ts. These results indicate that EF-Ts is essential to the growth of E. coli and suggest a technique for studying EF-Ts mutants as well as for identifying other guanine nucleotide exchange enzymes.

Amino Acid Sequence↗

PubMed1987

A mutation that alters the nucleotide specificity of elongation factor Tu, a GTP regulatory protein.

A single amino acid substitution (Asp to Asn) at position 138 of Escherichia coli elongation factor Tu (EF-Tu) was introduced in the tufA gene clone by oligonucleotide site-directed mutagenesis. The mutated tufA gene was then expressed in maxicells. The properties of [35S]methionine-labeled mutant and wild type EF-Tu were compared by in vitro assays. The Asn-138 mutation greatly reduced the protein's affinity for GDP; however, this mutation dramatically increased the protein's affinity for xanthosine 5'-diphosphate. The mutant protein forms a stable complex with Phe-tRNA and xanthosine 5'-triphosphate, which binds to ribosomes, whereas it does not form a complex with Phe-tRNA and GTP (10 microM). These results suggest that in EF-Tu.nucleoside diphosphate complexes, amino acid residue 138 must interact with the substituent on C-2 of the purine ring. Thus, in wild type EF-Tu, Asp-138 would hydrogen bond to the 2-amino group of GDP, and in the mutant EF-Tu, Asn-138 would form an equivalent hydrogen bond with the 2-carbonyl group of xanthosine 5'-diphosphate. Aspartic acid 138 is conserved in the homologous sequences of all GTP regulatory proteins. This mutation would allow one to specifically alter the nucleotide specificity of other GTP regulatory proteins.

Asparagine↗