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J C Hu

Publications and source records attributed to J C Hu.

At least 37 records · Page 2Linked to original sources
PubMed2000

Localization of EMSP1 expression during tooth formation and cloning of mouse cDNA.

Enamel matrix serine proteinase 1 (EMSP1) is a proteolytic enzyme that has been isolated from the developing enamel of pig teeth. Its apparent function is to degrade the organic matrix in preparation for enamel maturation. The expression of EMSP1 has never been investigated in another organism besides the pig, and EMSP1 expression in the enamel organ has never been specifically demonstrated in ameloblasts. Here we report the expression of recombinant pig EMSP 1 (rpEMSP 1), the generation of rabbit polyclonal antibodies against rpEMSP1, the characterization of the antibodies and EMSP1 expression by Western blot and immunohistochemical analyses, the cloning and characterization of a full-length cDNA encoding mouse EMSP1, and the localization of EMSP1 expression in ameloblasts in mouse day 14 first and second molars by in situ hybridization. The full-length mouse EMSP1 cDNA clone has 1,237 nucleotides, excluding the poly(A+) tail, and encodes a preproprotein of 255 amino acids. Mouse EMSP1 shares 75% amino acid identity with pig EMSP1 and has three potential N-linked glycosylation sites, two of which are conserved in the pig homologue. Western blot analysis shows that the polyclonal antibodies are specific for EMSP1 and do not cross-react with trypsin. Immunohistochemistry of pig incisors shows discrete staining in the surface enamel at the earliest part of the maturation stage. In mouse molars, in situ hybridization gives a distinct and specific signal in maturation-stage ameloblasts, and in the junctional epithelium following tooth eruption. We conclude that EMSP1 is expressed by pig and mouse ameloblasts during the early maturation stage of amelogenesis.

Amino Acid Sequence↗
PubMed2000

Two-hybrid system for characterization of protein-protein interactions in E. coli.

The yeast two-hybrid system has been used to characterize many protein-protein interactions. A two-hybrid system for E. coli was constructed in which one hybrid protein bound to a specific DNA site recruits another to an adjacent DNA binding site. The first hybrid comprises a test protein, the bait, fused to a chimeric protein containing the 434 repressor DNA binding domain. In the second hybrid, a second test protein, the prey, is fused downstream of a chimeric protein with the DNA binding specificity of the lambda repressor. Reporters were designed to express cat and lacZ under the control of a low-affinity lambda operator. At low expression levels, lambda repressor hybrids weakly repress the reporter genes. A high-affinity operator recognized by 434 repressor was placed nearby, in a position that does not yield repression by 434 repressor alone. If the test proteins interact, the 434 hybrid bound to the 434 operator stabilizes the binding of the lambda repressor hybrid to the lambda operator, causing increased repression of the reporter genes. Reconstruction experiments with the fos and jun leucine zippers detected protein-protein interactions between either homodimeric or heterodimeric leucine zippers.

Amino Acid Sequence↗
PubMed1999

Genetic selection of short peptides that support protein oligomerization in vivo.

An important goal in protein engineering is to control associations between designed proteins. This is most often done by fusing known, naturally occurring oligomerization modules, such as leucine zippers [1] [2] [3], to the proteins of interest [4] [5] [6]. It is of considerable interest to design or discover new oligomerization domains that have novel binding specificities [7] [8] [9] [10] [11] in order to expand the 'toolbox' of the protein engineer and also to eliminate associations of the designed proteins with endogenous factors. We report here a simple genetic selection scheme through which to search libraries for peptides that are able to mediate homodimerization or higher-order self-oligomerization of a protein in vivo. We found several peptides that support oligomerization of the lambda repressor DNA-binding domain in Escherichia coli cells, some of them as efficiently as the endogenous dimerization domain or the GCN4 leucine zipper. Many are very small, comprising as few as six residues. This study strongly supports the notion that peptide sequence space is rich in small peptides, which might be useful in protein engineering and other applications.

Amino Acid Sequence↗
PubMed1998

Effect of the gasoline additives on PAH emission.

PAH emission from the powered engines fueled by a 95 leadfree gasoline (95-LFG), a 92 leadfree gasoline (92-LFG) and a Premium leaded gasoline (PLG) with two gasoline additives (SA and SB) were collected using a PAH sampling system with a particulate interception device. Twenty one PAHs were analyzed primarily by an GC/MS, while eight metal elements were determined mainly by an ICP-AES. This investigation showed that the gasoline additives contain more amounts of carcinogenic PAHs than gasolines do. Blending these additives do raise the PAH content in the gasolines, simultaneously, will emit more amount of PAHs from the tailpipe of engine exhaust. It is suggested that before a gasoline additive is commercialized, an assessment on its PAH emission should be evaluated to make sure that the additive will not emit more PAHs and cause adverse effect on public health.

Carcinogens↗
PubMed1998

The association of selected pathological features with prostate cancer in a single-needle biopsy accession.

Isolated high-grade prostatic intraepithelial neoplasia (PIN) has been shown to be a positive predictor of prostate cancer (PCa) on follow-up biopsy. However, the incidence of isolated high-grade PIN in needle biopsy specimens has been reported with a highly variable frequency of 1% to 15%. The current study examined the relationship of various pathological features with PCa on a single biopsy accession. A study population of 388 community-based consecutive needle biopsy accessions was prospectively recorded by a single pathologist (T.M.W.). All of the individual biopsy specimens were coded for the presence of PCa, high-grade PIN, low-grade PIN, chronic inflammation (CI), intraluminal prostatic crystalloids (IPC) in benign glands, and mucinous metaplasia (MM). One hundred twenty-nine (33%) of the patients were diagnosed with PCa. The 8% incidence of isolated high-grade PIN was consistent with previous studies. The incidence of other pathological features were as follows: high-grade PIN, 14%; low-grade PIN, 13%; CI, 30%; IPC, 4%; and MM, 8%. Of the patients with high-grade PIN, 47% had PCa on a separate core biopsy, whereas 31% of patients without high-grade PIN were observed to have PCa (P=.021). Of the patients with CI, 21% were found to have PCa on a separate core, whereas 38% of patients without CI were found to have PCa (P=.0009). None of the other pathological features surveyed showed any significant association with PCa. High-grade PIN was a relatively common finding (14%) in this study and was positively associated with PCa on a separate core from the same accession biopsy. The negative association of CP with PCa within the same accession has not been reported previously and may be an artifact related to the clinical indications for a prostatic biopsy.

Adenocarcinoma↗
PubMed1997

Gene expression from plasmids containing the araBAD promoter at subsaturating inducer concentrations represents mixed populations.

Gene expression from plasmids containing the araBAD promoter can be regulated by the concentration of arabinose in the growth medium. Guzman et al. [Guzman, L.-M., Belin, D., Carson, M. J. & Beckwith, J. (1995) J. Bacteriol. 177, 4121-4130] showed that expression of a cloned gene could be modulated over several orders of magnitude in cultures grown in the presence of subsaturating concentrations of arabinose. We constructed plasmids expressing a fast-folding mutant Aequorea victoria green fluorescent protein from the araBAD promoter to examine the distribution of expressed gene products in individual cells at intermediate induction levels. Microscopic examination of cells grown at low arabinose concentrations shows mixtures of brightly fluorescent and dark cells, suggesting that intermediate expression levels in cultures reflect a population average of induced and uninduced cells. The kinetics of green fluorescent protein induction suggest that this reflects an "autocatalytic" induction mechanism due to accumulation of the inducer by active transport. This mechanism, which is analogous to the induction of the lac operon at subsaturating inducer concentrations in lacY+ cells, was described 40 years ago by Novick and Weiner [Novick, A. & Weiner, M. (1957) Proc. Natl. Acad. Sci. USA 43, 553-566].

Gene Expression↗
PubMed1997

Buried asparagines determine the dimerization specificities of leucine zipper mutants.

Regulation of gene expression by many transcription factors is controlled by specific combinations of homo- and heterodimers through a short alpha-helical coiled-coil known as a leucine zipper. The dimer interface of a leucine zipper involves side chains of the residues at the a, d, e, and g positions of the (abcdefg)n heptad repeat. To understand the basis for the specificity of dimer formation, we characterized GCN4 leucine zipper mutants with all 16 possible permutations and combinations of isoleucines and asparagines at four a positions in the dimer interface, using a genetic test for the specificity of dimer formation by lambda repressor-leucine zipper fusions. Heterodimers were detected by loss of repressor activity in the presence of a fusion to a dominant-negative mutant form of the DNA-binding domain of repressor. Reconstruction experiments using leucine zippers from GCN4, Jun, Fos, and C/EBP showed that this assay distinguishes pairs that form heterodimers from those that do not. We found that the mutants have novel dimerization specificities determined by the positioning of buried asparagine residues at the a positions. The pattern of buried polar residues could also explain the dimerization specificities of some naturally occurring leucine zippers. The altered specificity mutants described here should be useful for the construction of artificial regulatory circuitry.

Amino Acid Sequence↗
PubMed1997

Detection of tetramerization domains in vivo by cooperative DNA binding to tandem lambda operator sites.

Chimeric proteins comprising the N-terminal DNA binding domain of lambda repressor fused to a fragment of a foreign protein have been used to detect oligomerization of the latter. Fusions containing dimeric and tetrameric leucine zipper domains can be distinguished based on their in vivo repressor activities on a pair of cat-lacZ reporter strains. Repressor fusions are unable to efficiently repress transcription from a synthetic promoter that overlaps a weak operator site; repression by tetrameric, but not dimeric, fusion proteins is increased by the presence of a strong, upstream operator site. To construct reporters we developed a shuttle system that allows rapid construction of single-copy operon fusions in E. coli, with both cat and lacZ as reporters.

Bacteriophage lambda↗
PubMed1997

Oligomerization properties of GCN4 leucine zipper e and g position mutants.

Putative intersubunit electrostatic interactions between charged amino acids on the surfaces of the dimer interfaces of leucine zippers (g-e' ion pairs) have been implicated as determinants of dimerization specificity. To evaluate the importance of these ionic interactions in determining the specificity of dimer formation, we constructed a pool of > 65,000 GCN4 leucine zipper mutants in which all the e and g positions are occupied by different combinations of alanine, glutamic acid, lysine, or threonine. The oligomerization properties of these mutants were evaluated based on the phenotypes of cells expressing lambda repressor-leucine zipper fusion proteins. About 90% of the mutants do not form stable homooligomers. Surprisingly, approximately 8% of the mutant sequences have phenotypes consistent with the formation of higher-order (> dimer) oligomers, which can be classified into three types based on sequence features. The oligomerization states of mutants from two of these types were determined by characterizing purified fusion proteins. The Type I mutant behaved as a tetramer under all tested conditions, whereas the Type III mutant formed a variety of higher-order oligomers, depending on the solution conditions. Stable homodimers comprise less than 3% of the pool; several g-e' positions in these mutants could form attractive ion pairs. Putative repulsive ion pairs are not found among the homodimeric mutants. However, patterns of charged residues at the e and g positions do not seem to be sufficient to predict either homodimer or heterodimer formation among the mutants.

Amino Acid Sequence↗
PubMed1997

Oriented DNA binding by one-armed lambda repressor heterodimers and contacts between repressor and RNA polymerase at P(RM).

Bacteriophage lambda repressor activates transcription from P(RM) by contacting the sigma subunit of E. coli RNA polymerase. Although mutations in repressors that are defective in activation affect exposed residues in the repressor-operator co-crystal, the subunit in repressor dimers that is responsible for activation has not been determined experimentally. Here, we describe an oriented heterodimer approach using one-armed repressor-leucine zipper fusion proteins to resolve this question. Protection against methylation of operator DNA in vitro by one-armed and two-armed repressor-leucine zipper heterodimers shows that the subunit containing the arm in a one-armed repressor heterodimer binds preferentially in the consensus half-site of lambda operators. The patterns of activation of the P(RM) promoter in vivo by one-armed heterodimers containing the pc2 mutation in only one subunit show that RNA polymerase at P(RM) contacts the N-terminal domain of the promoter-proximal subunit of the repressor dimer.

Bacteriophage lambda↗
PubMed1995

Operator binding by lambda repressor heterodimers with one or two N-terminal arms.

The first 6 amino acids (NH2-Ser1-Thr2-Lys3-Lys4-Lys5-Pro6) of bacteriophage lambda cI repressor form a flexible arm that wraps around the operator DNA. Homodimeric lambda repressor has two arms. To determine whether both arms are necessary or only one arm is sufficient for operator binding, we constructed heterodimeric repressors with two, one, or no arms by fusing the DNA binding domain of lambda repressor to leucine zippers from Fos and Jun. Although only one arm is visible in the cocrystal structure of the N-domain-operator complex, our results indicate that both arms are required for optimal operator binding and normal site discrimination.

Amino Acid Sequence↗
PubMed1995

The role of platelet-derived growth factor in the development of mouse molars.

Platelet-derived growth factor (PDGF) is a potent mitogen that functions in cytodifferentiation and wound healing. PDGF receptor-alpha (PDGFR-alpha) is required for the normal development of the dental ectomesenchyme (Stephenson et al., Proc. Natl. Acad. Sci. USA 88: 6-10, 1991). To investigate the regulatory potential of PDGF on tooth development, the expression of PDGF-AA was determined by reverse transcription-polymerase chain reaction (RT-PCR), the PDGF ligands and receptors were localized by immunohistochemistry. The growth-promoting effects of exogenous PDGF-AA on molar explants were determined by assaying for tritiated thymidine incorporation, the presence of type I collagen and amelogenin messenger RNA transcripts, and total DNA, RNA and protein accumulation. Based upon the temporal and spatial localization, PDGFs and their receptors are present in the enamel epithelia and pulpal mesenchyme of developing mouse molars. Exogenous PDGF-AA administration increased the total protein accumulation of mouse molar explants but produces no discernible effect on amelogenin and type I collagen expression.

Animals↗
PubMed1993

Probing the roles of residues at the e and g positions of the GCN4 leucine zipper by combinatorial mutagenesis.

Combinatorial mutagenesis with an alphabet limited to alanine, glutamic acid, lysine, and threonine was used to probe the role of interactions involving surface residues in stabilizing a short alpha-helical coiled coil. The residues at eight e and g positions in the leucine zipper of the Saccharomyces cerevisiae transcription factor GCN4 were randomized to these four residues in a lambda repressor-leucine zipper fusion protein, resulting in 65,536 possible residue combinations. Roughly three-fourths of these combinations allowed stable coiled-coil formation as assayed by DNA binding by the fusion protein. To understand the basis for the activity differences, functional and non-functional mutants were sequenced and statistical tests were applied to identify structure/function correlations. Helix-forming propensity and favorable intrasubunit and intersubunit charge-charge interactions were positively correlated with activity. These studies suggest that the identities of surface side chains at the e and g positions of coiled coils contribute modestly to stability; by comparison with previous work, however, the e and g positions are far less critical than residues at the a and d positions, which form the hydrophobic core of the dimer interface.

Amino Acid Sequence↗
PubMed1991

Disinfecting activities of non-peroxide soft contact lens cold disinfection solutions.

The antimicrobial activities of three non-peroxide soft contact lens chemical disinfection systems--ReNu Multi-Purpose Solution (0.00005% polyaminopropyl biguanide), Opti-Soft Disinfecting Solution (0.001% polyquaternium-1), and Opti-Free Rinsing, Disinfecting & Storage Solution (0.001% polyquaternium-1)--were compared to Soft Mate Disinfecting Solution (0.005% chlorhexidine digluconate). Each product was separately inoculated with each of five microorganisms at approximately 10(6) microorganisms per mL. All of the solutions demonstrated excellent disinfecting activity against Pseudomonas aeruginosa and Staphylococcus epidermidis, with complete disinfection occurring within 4 hours. Only Soft Mate disinfected Serratia marcescens within 4 hours. ReNu reduced the microorganisms to 10-100 cells/mL and Opti-Soft and Opti-Free reduced the number to 10(2)-10(3) cells/mL. For the fungal species, Soft Mate showed excellent activity against Candida albicans (disinfection in 4 hours) and reduced Aspergillus fumigatus to 10(3) spores/mL in 4 hours. After 4 hours ReNu, Opti-Soft, and Opti-Free had reduced C. albicans only slightly, to 10(5) cells/mL and displayed virtually no disinfecting activity against A. fumigatus. For these newer chemical disinfection systems, diligent cleaning and rinsing of the soft contact lenses are the most important steps in the patient care regimen.

Bacteria↗
PubMed1990

Sequence requirements for coiled-coils: analysis with lambda repressor-GCN4 leucine zipper fusions.

A genetic system was developed in Escherichia coli to study leucine zippers with the amino-terminal domain of bacteriophage lambda repressor as a reporter for dimerization. This system was used to analyze the importance of the amino acid side chains at eight positions that form the hydrophobic interface of the leucine zipper dimer from the yeast transcriptional activator, GCN4. When single amino acid substitutions were analyzed, most functional variants contained hydrophobic residues at the dimer interface, while most nonfunctional sequence variants contained strongly polar or helix-breaking residues. In multiple randomization experiments, however, many combinations of hydrophobic residues were found to be nonfunctional, and leucines in the heptad repeat were shown to have a special function in leucine zipper dimerization.

Amino Acid Sequence↗