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Biomedical subjects

J C Hu

Publications and source records attributed to J C Hu.

At least 19 recordsLinked to original sources
PubMed2006

Disruption of granules by hydrodynamic force in internal circulation anaerobic reactor.

A process of granule disruption by hydrodynamic force is discussed in this paper. Shear force and attrition among granules originated from hydrodynamic force are the main causes of the disruption. Since it is positively correlated to the attrition force, the shear force is utilized to describe the effect of hydrodynamic force on granule disruption. In the experiment, when increase rate of average shear rate (IRgamma) of the 1st stage is about 0.2 s(-1) x d(-1), the granules are disrupted; while re-granulation could develop when IRgamma is about 0.07 s(-1) x d(-1); even when the shear rate is as high as about 30s(-1), the granulation rate keeps stably at a relatively high level, which shows that granules could bear the high hydrodynamic force only if it increases by low increase rate. The experimental results would be valuable for the operation and controlling of the upflow reactors.

Aerobiosis↗
PubMed2001

A molecular approach to dominance in hypophosphatasia.

Hypophosphatasia is an inherited disorder characterized by defective bone mineralization and a deficiency of tissue-nonspecific alkaline phosphatase (TNSALP) activity. The disease is highly variable in its clinical expression, because of various mutations in the TNSALP gene. In approximately 14% of the patients tested in our laboratory, only one TNSALP gene mutation was found, despite exhaustive sequencing of the gene, suggesting that missing mutations are harbored in intron or regulatory sequences or that the disease is dominantly transmitted. The distinction between these two situations is of importance, especially in terms of genetic counseling, but dominance is sometimes difficult to conclusively determine by using familial analysis since expression of the disease may be highly variable, with parents of even severely affected children showing no or extremely mild symptoms of the disease. We report here the study of eight point mutations (G46 V, A99T, S164L, R167 W, R206 W, G232 V, N461I, I473F) found in patients with no other detectable mutation. Three of these mutations, G46 V, S164L, and I473F, have not previously been described. Pedigree and/or serum alkaline phosphatase data suggested possible dominant transmission in families with A99T, R167 W, and G232 V. By means of site-directed mutagenesis, transfections in COS-1 cells, and three-dimensional (3D) modeling, we evaluated the possible dominant effect of these eight mutations. The results showed that four of these mutations (G46 V, A99T, R167 W, and N461I) exhibited a negative dominant effect by inhibiting the enzymatic activity of the heterodimer, whereas the four others did not show such inhibition. Strong inhibition resulted in severe hypophosphatasia, whereas partial inhibition resulted in milder forms of the disease. Analysis of the 3D model of the enzyme showed that mutations exhibiting a dominant effect were clustered in two regions, viz., the active site and an area probably interacting with a region having a particular biological function such as dimerization, tetramerization, or membrane anchoring.

Adolescent↗
PubMed2001

Model systems: Studying molecular recognition using bacterial n-hybrid systems.

Since the first description of the yeast two-hybrid system, related genetic assays for protein-protein interactions have become popular and powerful tools for structure-function analysis on the scale of individual proteins or whole proteomes. After a somewhat surprising lag, similar systems have recently been described for use in bacterial hosts. n-hybrid modifications of the original yeast system have been used to examine interactions with DNA, RNA and small molecules, and other modifications have improved throughput for genomic applications. Bacterial n-hybrid systems are being designed for a similar array of uses. Will the bacterial systems be as popular as the yeast n-hybrid systems? Only time will tell.

Bacteria↗
PubMed2001

A comparison of enamelin and amelogenin expression in developing mouse molars.

Amelogenin and enamelin are structural proteins in the enamel matrix of developing teeth. The temporal and spatial patterns of enamelin expression in developing mouse molars have not been characterized, while controversy remains with respect to amelogenin expression by odontoblasts and cementoblasts. Here we report the results of in situ hybridization analyses of amelogenin and enamelin expression in mouse molars from postnatal days 1, 2, 3, 7, 9, 14, and 21. Amelogenin and enamelin mRNA in maxillary first molars was first observed in pre-ameloblasts on the cusp slopes at day 2. The onsets of amelogenin and enamelin expression were approximately synchronous with the initial accumulation of predentin matrix. Both proteins were expressed by ameloblasts throughout the secretory, transition, and early maturation stages. Enamelin expression terminated in maturation stage ameloblasts on day 9, while amelogenin expression is still detected in maturation stage ameloblasts on day 14. No amelogenin expression was observed in day 21 mouse molars. Amelogenin and enamelin RNA messages were restricted to ameloblasts. No expression was observed in pulp, bone, or along the developing root. We conclude that amelogenin and enamelin are enamel-specific and do not directly participate in the formation of dentin or cementum in developing mouse molars.

Ameloblasts↗
PubMed2001

An engineered leucine zipper a position mutant with an unusual three-state unfolding pathway.

The leucine zipper is a dimeric coiled-coil structural motif consisting of four to six heptad repeats, designated (abcdefg)(n). In the GCN4 leucine zipper, a position 16 in the third heptad is occupied by an Asn residue whereas the other a positions are Val residues. Recently, we have constructed variants of the GCN4 leucine zipper in which the a position Val residues were replaced by Ile. The folding and unfolding of the wild-type GCN4 leucine zipper and the Val to Ile variant both adhere to a simple two-state mechanism. In this study, another variant of the GCN4 leucine zipper was constructed by moving the single Asn residue from a position 16 to a position 9. This switch causes the thermal unfolding of the GCN4 leucine zipper to become three state. The unfolding pathway of this variant was determined by thermal denaturation, limited proteinase K digestion, and sedimentation equilibrium analysis. Our data are consistent with a model in which the variant first unfolds from its N terminus and changes the oligomerization specificity from a native dimer to a partially unfolded intermediate containing a mixture of dimers and trimers and then completely unfolds to unstructured monomers.

Amino Acid Sequence↗
PubMed2001

Cloning and characterization of the mouse and human enamelin genes.

Enamelin is likely to be essential for proper dental enamel formation. It is secreted by ameloblasts throughout the secretory stage and can readily be isolated from the enamel matrix of developing teeth. The gene encoding human enamelin is located on the long arm of chromosome 4, in a region previously linked to an autosomal-dominant form of amelogenesis imperfecta (AI). To gain information on the structure of the enamelin gene and to facilitate the future assessment of the role of enamelin in normal and diseased enamel formation, we have cloned and characterized the mouse and human enamelin genes. Both genes are about 25 kilobases long. The enamelin gene has 10 exons interrupted by 9 introns. Translation initiates in exon 3 and terminates in exon 10. All of the intron/exon junctions within the mouse and human enamelin coding regions are between codons, so there are no partial codons in any exon, and deletion of one or more coding exons by alternative RNA splicing would not shift the downstream reading frame.

Ameloblasts↗
PubMed2001

Pubovaginal sling using cadaveric fascia and bone anchors: disappointing early results.

PURPOSE: Pubovaginal sling procedures offer highly effective treatment for patients with female stress urinary incontinence. A recent modification of this technique is the use of cadaveric fascia lata as a sling material supported with titanium anchors placed bilaterally in the pubic bone. We reviewed our experience with this procedure and assessed our outcome. MATERIALS AND METHODS: A total of 154 consecutive patients underwent a bone anchored, cadaveric fascia pubovaginal sling procedure by a single surgeon from July 1998 to June 1999. All patients were evaluated preoperatively with a detailed history, pelvic examination and radiographic or multichannel video urodynamic studies to diagnose stress urinary incontinence. Our technique begins with the nonincision placement of titanium bone anchors transvaginally into the pubic bone bilaterally. A 2 cm. wide tunnel is created bluntly beneath the vaginal epithelium between the 2 puncture sites with a right angle clamp. A 2 x 7 cm. strip of cadaveric fascia is then passed through the tunnel, into the retropubic space and secured to 2-0 polypropylene sutures attached to the anchors. After securing the sling, the transvaginal puncture sites are closed with 2-0 polyglactin sutures. Patients were seen postoperatively at 6 weeks, and 3 and 6-month followup. Patient age averaged 60 years (range 38 to 85), with an overall average length of followup from surgery of 10.6 months (range 6 to 16). All patients were mailed a self-administered questionnaire and participated in a telephone interview with an office nurse to retrospectively assess outcome and evaluate for recurrent stress urinary incontinence. Recurrent stress urinary incontinence was graded as 0-none, 1-rare, 2-moderate and 3-severe. Repeat pubovaginal sling procedure that was performed in patients with grades 2 to 3 stress urinary incontinence was considered a failure for the purpose of our study. RESULTS: Of all 154 patients 58 (37.6%) had recurrent moderate to severe (grades 2 to 3) stress urinary incontinence at followup. A total of 26 patients underwent a second pubovaginal sling procedure for a reoperation rate of 16.9%. Intraoperative findings at reoperation revealed the titanium anchors to be in position, the polypropylene sutures to be intact, and retropubic fibrosis and scarring of the urethropelvic ligament suggesting appropriate retropubic placement of the sling in all cases. Uniformly all allogenic cadaveric fascia used for sling material appeared to be fragmented, attenuated or simply absent. Average time to reoperation was 9 months (range 3 to 15). CONCLUSIONS: Early results using a bone anchored cadaveric fascia pubovaginal sling procedure were discouraging. Based on findings at reoperation, we attribute this result to the failure of our sling material and have abandoned the use of cadaveric fascia allografts in all pubovaginal slings at our institution.

Adult↗
PubMed2001

Dental enamel formation and its impact on clinical dentistry.

The nature of tooth enamel is of inherent interest to dental professionals. The current-day clinical practice of dentistry involves the prevention of enamel demineralization, the promotion of enamel remineralization, the restoration of cavitated enamel where demineralization has become irreversible, the vital bleaching of dental enamel that has become discolored, and the diagnosis and treatment of developmental enamel malformations, which can be caused by environmental or genetic factors. On a daily basis, dental health providers make diagnostic and treatment decisions that are influenced by their understanding of tooth formation. A systemic condition during tooth development, such as high fever, can produce a pattern of enamel defects in the dentition. Knowing the timing of tooth development permits estimates about the timing of the disturbance. The process of enamel maturation continues following tooth eruption, so that erupted teeth can become less susceptible to decay over time. Mutations in the genes encoding enamel proteins lead to amelogenesis imperfecta, a collection of inherited diseases having enamel malformations as the predominant phenotype. Defects in the amelogenin gene cause X-linked amelogenesis imperfecta, and genes encoding other enamel proteins are candidates for autosomal forms. Here we review our current understanding of dental enamel formation, and relate this information to clinical circumstances where this understanding may be particularly relevant.

Amelogenesis↗
PubMed2000

The contribution of buried polar groups to the conformational stability of the GCN4 coiled coil.

The dimeric interface of the leucine zipper coiled coil from GCN4 has been used to probe the contributions of hydrophobic and hydrogen bonding interactions to protein stability. We have determined the energetics of placing Ile or Asn residues at four buried positions in a two-stranded coiled coil. As expected, Ile is favored over Asn at these buried positions, but not as much as predicted by considering only the hydrophobic effect. It appears that interstrand hydrogen bonds form between the side-chains of the buried Asn residues and these contribute to the conformational stability of the coiled-coil peptides. However, these contributions are highly dependent on the locations of the Asn pairs. The effect of an Ile to Asn mutation is greatest at the N terminus of the peptide and decreases almost twofold as we move the substitution from the N to C-terminal heptads.

Amino Acid Sequence↗
PubMed2000

Characterization of the mouse and human PRSS17 genes, their relationship to other serine proteases, and the expression of PRSS17 in developing mouse incisors.

The human PRSS17 (serine protease 17) gene, which is located on chromosome 19q in a cluster of genes encoding serine proteases, has been variously designated enamel matrix serine proteinase 1 (EMSP1), prostase, KLK4, and KLK-L1. We have cloned and characterized the mouse and human PRSS17 genes. Both have six exons and five introns. The mouse PRSS17 gene sequence is 10134bp; the human sequence is 7115bp. Computer analysis of the mouse PRSS17 gene sequence upstream of the translation initiation codon identified two potential transcription initiation sites, at nucleotides 2878 and 2336. The first nucleotide of the reported mouse PRSS17 cDNA sequence corresponds to position 2352 on the gene, only 16 bases downstream from one of the putative transcription initiation sites. Repetitive DNA sequences from the MSR1 family are found in both the mouse and human PRSS17 genes. Additionally, the human PRSS17 gene contains Tigger2, MER8, and Alu repetitive sequences. Phylogenetic analyses of human and rodent proteases suggest that the PRSS17 protein is not a member of the kallikrein family of serine proteases but that the PRSS17 gene may have originated prior to the divergence of the kallikrein and trypsin families of proteases. To better characterize the timing of PRSS17 expression in developing teeth, we performed in-situ hybridization on postnatal day 3 developing mouse mandibular incisors. PRSS17 mRNA was not detected in secretory stage ameloblasts but could be detected in odontoblasts, while transition-stage and maturation-stage ameloblasts were strongly positive. This pattern supports a role for the PRSS17 protein in the degradation of enamel proteins.

Amino Acid Sequence↗
PubMed2000

Escherichia coli one- and two-hybrid systems for the analysis and identification of protein-protein interactions.

Genetic methods based on fusion proteins allow the power of a genetic approach to be applied to the self-assembly of proteins or protein fragments, regardless of whether or not the normal function of the fused assembly domains is either known or amenable to selection or screening. The widespread adoption of variations of the yeast two-hybrid system originally described by S. Fields and O. Song (1989, Nature 340, 245-246) demonstrates the usefulness of these kinds of assays. This review describes some of the many systems used to select or screen for protein-protein interactions based on the regulation of reporter constructs by hybrid proteins expressed in bacteria, including recent implementations of generalizable two-hybrid systems for Escherichia coli.

Amino Acid Sequence↗
PubMed2000

Chemically induced dimerization of dihydrofolate reductase by a homobifunctional dimer of methotrexate.

BACKGROUND: Chemically induced dimerization (CID) can be used to manipulate cellular regulatory pathways from signal transduction to transcription, and to create model systems for study of the specific interactions between proteins and small-molecule chemical ligands. However, few CID systems are currently available. The properties of, and interactions between, Escherichia coli dihydrofolate reductase (DHFR) and the ligand methotrexate (MTX) meet many of the desired criteria for the development of a new CID system. RESULTS: BisMTX, a homobifunctional version of MTX, was synthesized and tested for its ability to induce dimerization of DHFR. Gel-filtration analysis of purified DHFR confirmed that, in vitro, the protein was a monomer in the absence of dimerizer drug; in the presence of bisMTX, a complex of twice the monomeric molecular weight was observed. Furthermore, the off-rate was found to be 0.0002 s(-1), approximately 100 times slower than that reported for DHFR-MTX. Interestingly, the addition of excess bisMTX did not result in formation of the binary complex (1 protein:1 dimerizer) over the ternary complex (2 proteins:1 dimerizer), which suggests cooperative binding interactions (affinity modulation) between the two DHFR molecules in the bisMTX:DHFR(2) ternary complex. CONCLUSIONS: The combination of DHFR and bisMTX provides a new CID system with properties that could be useful for applications in vivo. Formation of the bisMTX:DHFR(2) ternary complex in vitro is promoted over a wide range of dimerizer concentrations, consistent with the idea that formation of the ternary complex recruits energetically favorable interactions between the DHFR monomers in the complex.

Binding Sites↗
PubMed2000

Characterization of a family with dominant hypophosphatasia.

A kindred with dominant hypophosphatasia resulting from an alanine to threonine substitution at position 99 of the alkaline phosphatase protein is described. The clinical findings of individual members of the kindred were assessed by oral and physical examinations, or from the descriptions of multiple family members. The proband displayed enamel hypoplasia and premature loss of fully rooted primary anterior teeth, which were shown by histological examination to lack cementum. Serum alkaline phosphatase (ALP) and a vitamin B6 panel, and urine phosphoethanolamine (PEA) were measured on 21 family members. Based upon the clinical and laboratory tests, affected and unaffected status was assigned. Parametric linkage analysis of the kindred using different dominant models and frequency distributions for the disease allele and the mutation gave lodscores > 4.2 and confirmed the strong linkage between the disease and the mutation. Assuming the defined mutation causes the disease, the reliability of clinical and laboratory tests is assessed.

Alanine↗
PubMed2000

Resistance to methotrexate due to AcrAB-dependent export from Escherichia coli.

Many laboratory strains of Escherichia coli are resistant to methotrexate (MTX), a folate analogue that binds dihydrofolate reductase (DHFR). Mutations that inactivate either tolC or acrA confer MTX sensitivity. Further, overexpression of a fusion protein with DHFR activity reverses this sensitivity by titrating out intracellular MTX. These results suggest that MTX accumulates in cells where mutations in acrA or tolC have inactivated the TolC-dependent AcrAB multidrug resistance efflux pump.

ATP Binding Cassette Transporter, Subfamily B, Mem↗