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W Huang

Publications and source records attributed to W Huang.

At least 559 records · Page 31Linked to original sources
PubMed1994

Expansion of CREB's DNA recognition specificity by Tax results from interaction with Ala-Ala-Arg at positions 282-284 near the conserved DNA-binding domain of CREB.

The transactivator protein of human T-lymphotropic virus type I (HTLV-I), Tax, forms multiprotein complexes with the ubiquitous transcription factor CREB and the CREB/ATF-1 heterodimer. The interaction between Tax and CREB is highly specific and results in increased binding of the Tax/CREB complexes to the HTLV-I 21-bp repeats. Despite the extensive sequence similarities between CREB and ATF-1, Tax interacts with ATF-1 only marginally. Compared with CREB, Tax/CREB exhibits greatly increased DNA recognition specificity and preferentially assembles on a consensus binding site, GGGGG(T/A)TGACG(T/C)(A/C)TA(T/C)C-CCCC, homologous to the HTLV-I 21-bp repeats. Here we report that Tax affects CREB binding to the Tax-inducible DNA elements by interacting with the basic-leucine zipper (bZip) domain of CREB. We show by domain switching that the basic region in CREB bZip can confer on c-Jun and ATF-1 leucine zippers the ability to interact with Tax in vitro. Mutational analyses further demonstrate that the amino acid residues of CREB critical for Tax/CREB interaction are Ala-Ala-Arg at positions 282-284 (AAR284), immediately upstream of the highly conserved DNA-binding domain (R/K)XX(R/K) N(R/K)XAAXX(S/C)RX(R/K)(K/R) characteristic of all bZip proteins. Specific amino acid substitutions in AAR284 of CREB weakened or abolished Tax/CREB interaction, whereas reciprocal changes in ATF-1 allowed it to interact with Tax. These results support a model in which the specific interaction between Tax and the AAR284 residues near the DNA-binding domain of CREB results in a multiprotein complex with altered DNA recognition property. This protein complex assembles selectively on the viral Tax-responsive 21-bp repeats to augment transcription.

Activating Transcription Factor 1↗
PubMed1994

Crystal structure of an ATP-dependent carboxylase, dethiobiotin synthetase, at 1.65 A resolution.

BACKGROUND: In Escherichia coli, the enzymes of the biotin biosynthesis pathway are encoded by the bio operon. One of these enzymes, ATP-dependent dethiobiotin synthetase, catalyzes the carboxylation of 7,8-diaminopelargonic acid leading to the formation of the ureido ring of biotin. The enzyme belongs to the class of ATP-dependent carboxylases and we present here the first crystal structure determined for this class of enzyme. RESULTS: We have determined the crystal structure of homodimeric dethiobiotin synthetase to 1.65 A resolution. The subunit consists of a seven-stranded parallel beta-sheet, surrounded by alpha-helices. The sheet contains the classical mononucleotide-binding motif with a fingerprint peptide Gly-X-X-X-X-X-Gly-Lys-Thr. The mononucleotide binding part of the structure is very similar to the GTP-binding protein H-ras-p21 and thus all GTP-binding proteins. A comparison reveals that some of the residues, which in H-ras-p21 interact with the nucleotide and the metal ion, are conserved in the synthetase. CONCLUSIONS: The three-dimensional structure of dethiobiotin synthetase has revealed that ATP-dependent carboxylases contain the classical mononucleotide-binding fold. Considerable similarities to the structure of the GTP-binding protein H-ras-p21 were found, indicating that both proteins might have evolved from a common ancestral mononucleotide-binding fold.

Adenosine Triphosphate↗
PubMed1994

Identification of biologically active mutants by combinatorial cassette mutagenesis: exclusion of wild-type codon from degenerate codons.

A degenerate codon (N)(N)(G+C) is often used in cassette mutagenesis to encode all 20 natural amino acids at the target mutation site. However, the presence of the wild-type codon in the degenerate codon presents some inconvenience in screening and identification of catalytically active mutants. The wild-type enzyme will always be identified as catalytically active in a screening process and in most cases can only be distinguished from active mutants by DNA sequencing. Sequencing of background wild-type enzyme represents wasted effort in the identification of active mutants. This paper describes a simple approach for exclusion of the wild-type codon in degenerate codons through the synthesis of two or three oligonucleotide mixtures. The minimum number of individual colonies required to achieve a high degree of certainty of including all possible codons for screening of catalytic activity can be estimated using a statistical procedure. The use of degenerate codons that exclude the wild-type amino acid facilitates the screening process and saves time and expense in DNA sequencing.

Amino Acids↗
PubMed1994

Crystallization and preliminary X-ray analysis of phenol hydroxylase from Trichosporon cutaneum.

Recombinant phenol hydroxylase from the soil yeast Trichosporon cutaneum has been crystallized with PEG 4000 as precipitant. The crystals are monoclinic, space group P2(1) with cell dimensions a = 101.8 A, b = 153.0 A, c = 116.0 A and beta = 114.8 degrees. The crystal asymmetric unit most likely contains two dimers of phenol hydroxylase corresponding to a packing density in the crystals of 2.54 A 3/Da. The self-rotation function is consistent with the packing of two dimers in the asymmetric unit. The observed diffraction pattern extends beyond 2.8 A resolution and the crystals are well suited for structural analysis by X-ray diffraction methods.

Crystallization↗
PubMed1994

Isolation and characterization of a novel acetyl-CoA carboxylase kinase from rat liver.

Acetyl-CoA carboxylase is regulated allosterically by citrate and covalently by a phosphorylation/dephosphorylation mechanism. We have isolated and purified from rat livers a novel kinase that phosphorylates and inactivates the carboxylase. This kinase is bound to the carboxylase and can be eluted in salt-rich solution. The native kinase exists as high molecular weight aggregates of a subunit that has a molecular weight of 40,000. The phosphorylation sites of the carboxylase were determined after tryptic and cyanogen bromide digestions of 32P-labeled carboxylase and separation of the peptides by various chromatographic procedures. Amino acid analyses of the phosphopeptides showed that the Ser77 and Ser1200 residues were the sites of phosphorylation. Treating the phosphorylated carboxylase with the Mn(2+)-dependent acetyl-CoA carboxylase phosphatase 2 removed the phosphate and reactivated the carboxylase. These results suggest that both this kinase and the acetyl-CoA carboxylase phosphatase 2 act at the same site(s) in the acetyl-CoA carboxylase molecule. Citrate dramatically inhibits the kinase-mediated phosphorylation of the carboxylase, suggesting that the allosteric modification and activation by citrate render the phosphorylation sites inaccessible to the kinase and therefore maintain high carboxylase activity. This observation indicates that there is a close interplay between the citrate effect on and phosphorylation of the carboxylase in regulating its activity.

Acetyl-CoA Carboxylase↗
PubMed1994

In vivo transcription from the adenovirus E2 early promoter by RNA polymerase III.

We have previously reported that the subgroup C adenovirus E2 early (E2E) RNA polymerase II promoter can specify efficient in vitro transcription by RNA polymerase III. We now show that promoter proximal sequences of the E2E transcription unit are also transcribed by RNA polymerase III in nuclei isolated from adenovirus-infected cells. Small E2E RNA species that possessed the same properties as in vitro synthesized RNA polymerase III E2E transcripts were detected in cytoplasmic RNA populations from infected cells by using blotting, primer extension, and RNase protection assays. The 3' termini of these RNAs were mapped to thymidine-rich sequences typical of RNA polymerase III termination sites. These results demonstrate that a single gene can be transcribed by both RNA polymerase II and RNA polymerase III in vivo.

Adenovirus E2 Proteins↗
PubMed1994

Analysis of FAS3/ACC regulatory region of Saccharomyces cerevisiae: identification of a functional UASINO and sequences responsible for fatty acid mediated repression.

We have determined the sequence of the FAS3/ACC regulatory region and mapped the transcription initiation site. In this sequence, there are two putative UASINO sequences. Deletion and mutation analyses revealed that the UASINO sequence at nucleotides -719 to -710 is functional. The expression of FAS3-lacZ reporter genes and the measurement of mRNA levels in regulatory mutants of phospholipid biosynthesis clearly indicated that FAS3 is regulated by inositol and choline. Previous studies have shown that the genes coding for fatty acid synthase, FAS1 and FAS2, are regulated by inositol (Chirala, S.S. [1992] Proc. Natl. Acad. Sci. USA 89, 10232-10236). Thus all three genes involved in saturated fatty acid biosynthesis are coordinately regulated with phospholipid biosynthesis. Comparison of the UASINO sequences present in FAS1, FAS2, and FAS3 suggested that the functional sequence of this UAS element is YTTCACATG. However, even when the functional UASINO was mutated, substantial expression of the FAS3-lacZ reporter gene was observed. Deletion analysis, electrophoretic mobility shift assays, and expression using a heterologous reporter gene showed that the region between nucleotides -840 and -736 has two UAS elements. The same sequence seems to be responsible for fatty acid-mediated repression of FAS3. The presence of these additional UAS sequences explains why yeast does not require fatty acids even when repressing amounts of inositol and choline are present in the medium.

Amino Acid Sequence↗
PubMed1994

Fluorescence resonance energy transfer within the regulatory light chain of myosin.

Rabbit skeletal muscle myosin regulatory light chain-2 (LC2) contains two reactive cysteine residues, Cys125 and Cys154, and one tryptophan at position 137. Using wild-type rabbit LC2 or its genetically engineered mutant with Cys125-->Arg (C125R), these residues can be selectively modified with fluorescent or chromophoric probes for spectroscopic studies. We have bound suitable donor/acceptor probe pairs to the two cysteine residues and Trp137 in LC2 or C125R, and measured the distance in solution between the probes by fluorescence resonance energy transfer spectroscopy. C125R was made to facilitate specific labelling of the less reactive Cys154, thus allowing the distance between Cys154 and Trp137 to be measured. Our measurements show that these residues are in close proximity to each other, the distance between them ranging from 1.7 nm (between Cys125 and Trp137) to 2.7 nm (Cys125 and Cys154). These results suggest that Cys125, Trp137 and Cys154, spanning up to 29 residues in the sequence of LC2, are spatially close, consistent with these residues residing within a C-terminal globular domain. The distances we obtained are in agreement with previous crosslinking studies [Huber, P. A., Brunner, U.T. & Schaub, M. C. (1989) Biochemistry 28, 9116-9123; Saraswat, L. & Lowey, S. (1991) J. Biol. Chem. 266, 19777-19785] and structure predictions of LC2. LC2 is located at the head-rod junction of the myosin crossbridge, and provides the primary regulatory mechanism in molluscan and smooth muscle. In skeletal muscle, its functional role is unclear, although it has been implicated in modulating actomyosin interaction [Metzger, J. M. & Moss, R. L. (1992) Biophys. J. 63, 460-468]. The incorporation of spectroscopic probes onto the light chains of myosin in solution or in fibres has become a valuable tool for evaluating the dynamic properties of the crossbridge during force generation.

Amino Acid Sequence↗
PubMed1994

High risk HLA-DR/DQ genotypes for IDD confer susceptibility to autoantibodies but DQB1*0602 does not prevent them.

Although HLA class II genes are important in insulin-dependent diabetes (IDD), their influence on the expression of IDD-associated autoantibodies (aAb) is unclear. We compared HLA-DRB1 and DQB1 gene frequencies in several Caucasian groups: 191 normal controls, 378 IDD patients, and 357 non-diabetic relatives of which 250 had no aAb, 107 had at least one aAb (79 ICA+, 31 GAD65+ and 49 IAA+), and 23 had both ICA+ and IAA+. We found that the frequencies of DR3/4 or DQB1*0201/0302 heterozygotes were significantly higher in aAb+ relatives compared to aAb- relatives. The frequencies of DR4/4 or DR4/X (X = non 3 or 4) and DQB1*0302/X (X = 0201 or 0302) in aAb+ relatives were not different from the aAb- relatives (which were enriched for these haplotypes), but were significantly higher than normal controls. The frequencies of DR3/X or DQB1*0201/X were decreased in both aAb+ relatives and IDD patients. Interestingly, the dominant IDD-protective DQB1*0602 allele allowed the development of individual aAbs (10% of ICA+ and 8% IAA+ relatives had the allele), but was not observed in any high risk double aAb+, or GAD65Ab+ relatives. The latter finding was similar to that in our patients with IDD, in that only two of them (0.5%) had a DQB1*0602 allele. In conclusion, HLA-encoded susceptibilities to disease-relevant autoantibody production and IDD are concordant with the susceptibility alleles, but discordant for the protective DQB1*0602. Thus HLA genotyping for DQB1*0602 would impact on the selection of aAb+ relatives for disease prevention trials.

Adolescent↗
PubMed1994

D2 receptors mediate dopamine suppression of irANF release and pro-ANF mRNA expression of rat hypothalamic neurons in culture.

Although N-terminal truncated forms of atrial natriuretic factor (ANF) are produced and released from rat hypothalamic neurons, the intrahypothalamic regulation of these processes remains unclear. Employing a well-characterized hypothalamic cell culture system, we report here that dopamine, mediating through D2 receptors, inhibits the synthesis and release of ANF. In long-term cultures of hypothalamic neurons, daily treatment for 4 days with quinpirole, a D2 agonist, significantly suppressed the basal irANF release in a time-related and a dose-dependent manner. The ED50 and Emax of the drugs were 9.1 x 10(-8) M and 10(-5) M, respectively. This effect of quinpirole was mimicked by 10(-7) M of dopamine, a physiological ligand for D2 receptor. Furthermore, the suppressing effects of both quinpirole and dopamine were abolished by sulpiride, a D2 antagonist. Whereas 10(-6) M of forskolin treatment consistently enhanced the release of irANF through activating the adenylyl cyclase-cAMP system, this stimulatory effect was suppressed by quinpirole in a dose-related manner. In addition, the application of pertussis toxin, a bacterial toxin which inactivated G1 protein activity, reversed the suppressing effect of quinpirole or dopamine on irANF release. These immunoassay findings were accompanied by corresponding changes in the abundance of pro-ANF mRNA in the cultures as determined by colorimetric Northern blot analysis. By combining the techniques of in situ hybridization and immunocytochemistry, the mRNA of D2 receptor was colocalized with irANF at a single cell level by double fluorescent staining.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Enhancement of adenylate cyclase activity in S49 lymphoma cell membranes by the toxin thionin from Pyrularia pubera.

We investigated the mode of action of Pyrularia thionin on adenylate cyclase in S49 lymphoma cell membranes. Thionin increased the forskolin-induced cAMP production in both wild-type and cyc- variant cell membranes. Thionin increased the apparent efficacy, but not potency, of the stimulation of adenylate cyclase by forskolin. Our results also indicated that the stimulatory G protein (Gs) was not involved in the action of thionin on adenylate cyclase; however, thionin appeared to reduce the effect of Gi on adenylate cyclase. We examined the effect of thionin on the temperature dependence of adenylate cyclase activity. The effect of thionin was not quantitatively the same at all temperatures. The effect was greatest in the range of about 30 degrees C to 50 degrees C. In addition, cardiotoxin, a structurally distinct peptide that alters the properties of biological membranes similarly to thionin, also enhanced the activity of adenylate cyclase. It seems reasonable to suspect that thionin influences the activity of adenylate cyclase indirectly by acting on the membrane.

Adenylyl Cyclases↗
PubMed1994

Lipophilic multiple antigen peptide system for peptide immunogen and synthetic vaccine.

We describe the development and structural requirements of a new lipophilic multiple antigen peptide (lipoMAP) system for immunogens that contains a built-in lipophilic adjuvant and has the ability to elicit cytotoxic T-lymphocytes (CTLs). In addition to the peptide antigens of choice at the amino terminus, the basic lipoMAP design consists of three components: a tetravalent symmetrical core matrix containing two levels of branching beta-alanyl-lysine as a building unit, a hydrophilic Ser-Ser dipeptide linker, and at the carboxyl terminus, palmitoyl lysines (PL) with alternating chirality. An 18-residue peptide from the third variable region in the gp120 of HIV-1 was used as antigen in eight models for a structure-function study. Alternating palmitoyl lysine (PL) was introduced as the lipid anchor and built-in adjuvant because D and L Lys (Pal) was found via molecular modeling to best mimic phosphatidylcholine and thus provide the most stable peptide antigens on the ordered lipid membranes. The requirements of the palmitoyl lysines and the L-Ser-L-Ser linker were crucial, since replacement with palmitoyl serines or L-Ser-D-Ser linkers led to a marked decrease in immune response. The stoichimetric ratio of PL vs MAP was also important. Multiple antigen peptide (MAP) constructs without the lipophilic PLs, those that were underlipidated and contained one PL, or those that were overlipidated containing four PLs, were ineffective. LipoMAPs containing three palmitic acids elicited significant humoral responses in oil-based emulsion and liposomes, but not in water or alum formulations. LipoMAP containing only two PLs was found best to be incorporated in liposomes and elicited a significant immune response and cytotoxic T-lymphocytes (CTLs). These models were compared favorably with a preparation using tripalmitoyl-S-glyceryl cysteine (P3C) as the lipid anchor. We also developed a modular synthesis of MAP-P3C that incorporated P3C as a premade unit containing a thiopyridine, which simplified the overall scheme and minimized oxidation during stepwise peptide synthesis. This lipoMAP model is a new addition to the design of our macromolecular assemblage approach mimicking peptide antigens on the surface of micro-organisms. It may be a potentially useful approach to the design of a synthetic vaccine for humans.

Amino Acid Sequence↗
PubMed1994

Survival of hemodialysis patients in the United States is improved with a greater quantity of dialysis.

The mortality rate for hemodialysis patients in the United States is higher than in other industrialized countries. Some attribute this to insufficient quantities of prescribed and delivered dialysis. A multicenter study in Dallas dialysis centers (Dallas Nephrology Associates) was begun in 1989 to assess the impact of increasing the delivered quantity of dialysis on mortality in subsequent years. Dialysis dose was measured by urea kinetic modeling. Kt/V, reflecting the fractional volume of body water clearance of urea during a dialysis treatment, was purposefully increased from 1.18 starting in 1989 to 1.46 in 1992. Additionally, the dialysis dose measured by the urea reduction ratio, the fractional reduction of blood urea nitrogen concentration caused by a dialysis treatment, increased from 63.0% to 69.6% between 1990 and 1992. Outcome analytical methods included both crude and standardized mortality rates and mortality ratios standardized to large end-stage renal disease databases at the United States Renal Data System and at National Medical Care, Inc. Crude mortality rates at Dallas Nephrology Associates decreased from 22.5% in 1989 to 18.1% in 1992. In comparison, between 1990 and 1992 the urea reduction ration in National Medical Care facilities increased from 57.1% to 62.5%. During that time crude mortality rates decreased from 21.8% to 19.5%. Crude mortality in the United States remained essentially unchanged in the same time period. By 1992, Dallas Nephrology Associates and National Medical Care had standardized mortality ratios of 0.77 and 0.74, respectively, compared with the US dialysis population, indicating almost 30% fewer observed deaths than expected. Monitoring dialysis dose by urea kinetic modeling or urea reduction ratio are equally effective in predicting improvement in patient survival. Improved survival is possible in the US end-stage renal disease program with greater amounts of dialysis. This strategy can save an estimated 8,000 to 16,000 lives per year.

Age Factors↗
PubMed1994

Effects of neurohypophyseal antagonists in postnephrectomy natriuresis in male rats.

Acute unilateral nephrectomy (AUN) in anesthetized male Lewis x DA rats induced rapid and consistent increases in electrolyte and fluid excretion by the remaining kidney during the first hours. Continuous infusion of a vasopressin (AVP) V1-receptor antagonist d(CH2)5Tyr(Me)AVP (V1-ant) reduced renal electrolyte and fluid excretion before and after AUN to a similar extent, whereas an oxytocin (OT)-receptor antagonist [Mpa1,D-Tyr(Et)2,Thr4,Orn8]-OT (CAP) at the same dose selectively attenuated the increase in sodium excretion after AUN. The plasma concentration of OT rose significantly after AUN (9.16 +/- 1.4 to 21.45 +/- 5.07 pg.ml-1). A similar OT level obtained by infusion of OT mimicked the renal responses to AUN without elevating blood pressure; however, only CAP but not V1-ant efficiently reversed OT-induced natriuresis. Also, the infusion of CAP at the same dose produced no effects on the rise of blood pressure caused by AVP while the infusion of the V1-ant prevented such a rise. Thus, CAP reduced the natriuresis after AUN by interfering with OT- and not V1-receptors. In conclusion, evidence is presented, for the first time, concerning the major role of OT receptors in the acute readjustment of the renal sodium excretion after AUN, and a synergistic role for AVP in terms of the general magnitude of renal excretion.

Animals↗