Biomedical subjects

Ping Hu

Publications and source records attributed to Ping Hu.

82 records · Page 5Linked to original sources

PubMed2002

Role of membrane proteins in permeability barrier function: uroplakin ablation elevates urothelial permeability.

Although water, small nonelectrolytes, and gases are freely permeable through most biological membranes, apical membranes of certain barrier epithelia exhibit extremely low permeabilities to these substances. The role of integral membrane proteins in this barrier function has been unclear. To study this problem, we have ablated the mouse gene encoding uroplakin III (UPIII), one of the major protein subunits in urothelial apical membranes, and measured the permeabilities of these membranes. Ablation of the UPIII gene greatly diminishes the amounts of uroplakins on the apical urothelial membrane (Hu P, Deng FM, Liang FX, Hu CM, Auerbach AB, Shapiro E, Wu XR, Kachar B, and Sun TT. J Cell Biol 151: 961-972, 2000). Our results indicate that normal mouse urothelium exhibits high transepithelial resistance and low urea and water permeabilities. The UPIII-deficient urothelium exhibits a normal transepithelial resistance (normal 2,024 +/- 122, knockout 2,322 +/- 114 Omega. cm(2); P > 0.5). However, the UPIII-deficient apical membrane has a significantly elevated water permeability (normal 0.91 +/- 0.06, knockout 1.83 +/- 0.14 cm/s x 10(-5); P < 0.05). The urea permeability of the UPIII-deficient membrane also increased, although to a lesser extent (normal 2.22 +/- 0.24, knockout 2.93 +/- 0.31 cm/s x 10(-6); P = 0.12). These results indicate that reduced targeting of uroplakins to the apical membrane does not significantly alter the tight junctional barrier but does double the water permeability. We provide the first demonstration that integral membrane proteins contribute to the apical membrane permeability barrier function of urothelium.

Animals↗

PubMed2002

Identification of six novel genes by experimental validation of GeneMachine predicted genes.

In silico gene identification from finished and unfinished human genome sequence has become critically important in many projects seeking to gain insights into the gene content of genomic regions implicated in diseases. To establish limitations and criteria for in silico gene identification, and to identify novel genes of potential relevance to human prostate cancer and melanoma, 3 Mb of chromosome 1 sequence have been analyzed using GeneMachine. This program is a software suite comprising of sequence similarity programs and four gene identification programs. A total of 49 potential transcripts were selected and 37 of them were selected for experimental validation. We verified 16 of the predicted genes by experimental analysis. The comparison of the predicted transcripts with their cloned forms helped to refine predicted gene models as well as to identify splice variants for several of them. Although sequences matching with ten of our verified genes have been recently deposited in the GenBank, six of them remain novel. Our studies support the feasibility of identifying novel genes from regions of interest using draft human genome sequence.

Chromosomes, Human, Pair 1↗

PubMed2002

Capillary array electrophoresis-MALDI mass spectrometry using a vacuum deposition interface.

We previously introduced a vacuum deposition interface for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF MS) on a moving surface (e.g., quartz wheel, Mylar tape, metal target). In our present work, the approach has been extended to demonstrate parallel analysis for multiple on-line infusion MALDI MS and capillary array electrophoresis (CAE)-MALDI MS. In the infusion mode, individual peptide samples were simultaneously deposited on a Mylar tape cartridge using an array of eight capillaries, yielding eight parallel traces. For CAE-MALDI/TOF MS, the same number of separation capillaries were coupled with an array of eight infusion capillaries using a common liquid junction, containing matrix solution. A fast-scanning mirror was employed to traverse the beam of the desorption laser across the Mylar tape to probe one trace at a time. The positions of the eight sample traces formed on the tape were automatically determined, and all samples were analyzed in rapid sequence using a kilohertz repetition rate laser and a high-throughput data acquisition system. The instrumentation was operated with CAE MS for high-throughput analysis without compromising data quality. The principles of parallel separation-vacuum deposition should be generally applicable to MALDI/TOF MS analysis for proteomics and other areas where separation and high throughput are required.

Angiotensins↗

PubMed2002

Physical and transcript map of the hereditary prostate cancer region at xq27.

We have recently mapped a locus for hereditary prostate cancer (termed HPCX) to the long arm of the X chromosome (Xq25-q27) through a genome-wide linkage study. Here we report the construction of an approximately 9-Mb sequence-ready bacterial clone contig map of Xq26.3-q27.3. The contig was constructed by screening BAC/PAC libraries with markers spaced at approximately 85-kb intervals. We identified overlapping clones by end-sequencing framework clones to generate 407 new sequence-tagged sites, followed by PCR verification of overlaps. Contig assembly was based on clone restriction fingerprinting and the landmark information. We identified a minimal overlap contig for genomic sequencing, which has yielded 7.7 Mb of finished sequence and 1.5 Mb of draft sequence. The transcriptional mapping effort localized 57 known and predicted genes by database searching, STS content mapping, and sequencing, followed by sequence annotation. These transcriptional units represent candidate genes for HPCX and multiple other hereditary diseases at Xq26.3-q27.3.

Chromosome Mapping↗

PubMed2002

Study on the three-dimensional proliferation of rabbit articular cartilage-derived chondrocytes on polyhydroxyalkanoate scaffolds.

Polymer scaffold systems consisting of poly(hydroxybutyrate-co-hydroxyhexanoate) (PHBHHx)/polyhydroxybutyrate (PHB) (PHBHHx/PHB) were investigated for possible application as a matrix for the three-dimensional growth of chondrocyte culture. Blend polymers of PHBHHx/PHB were fabricated into three-dimensional porous scaffolds by the salt-leaching method. Chondrocytes isolated from rabbit articular cartilage (RAC) were seeded on the scaffolds and incubated over 28 days, with change of the culture medium every 4 days. PHB scaffold was taken as a control. Methylthiazol tetrazolium (MTT) (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltertra-zolium bromide) assay was used to quantitatively examine the proliferation of chondrocytes. Results showed that chondrocytes proliferated better on the PHBHHx/PHB scaffolds than on PHB one. The maximal cell densities were all observed after 7 days of incubation. As for the blend polymers, cells grew better on scaffolds consisting of PHBHHx/PHB in ratios of 2:1 and 1:2 than they did on PHBHHx/PHB of 1:1. Scanning electron microscopy (SEM) also showed that large quantities of chondrocytes grew initially on the surface of the scaffold. After 7 days, they further grew into the open pores of the blend polymer scaffolds. Morphologically, cells found on the surface of the scaffold exhibited a flat appearance and slowly form confluent cell multilayers starting from 14 to 28 days of the growth. In contrast, cells showed rounded morphology, formed aggregates and islets inside the scaffolds. In addition, chondrocytes proliferated on the scaffold and preserved their phenotype for up to 28 days.

Animals↗

PubMed2002

Experimental design issues for the early detection of disease: novel designs.

This paper investigates two experimental designs which have been used to evaluate the benefit of the early detection of breast cancer. They have some advantages over a classical design (the screening program versus usual medical care) in that subjects in a control group may benefit by participating in the study. We refer to the two experimental designs as the up-front (UFD) and close-out (COD) designs. The UFD consists of offering an initial exam to all participants. Then they can be randomized to a usual care group or a screening group receiving one or more special examinations. If the outcome of the initial examination is included in the analysis, then the study can answer the question of the benefit of an additional screening program after an initial examination. If the analysis excludes all the cases diagnosed at the initial examination, then the analysis evaluates the benefit of a screening program after elimination of the prevalent cases. These prevalent cases are most likely to be affected by length bias sampling and consequently will tend to have less aggressive disease and live longer. As a result, the UFD can answer two scientific questions. The COD consists of randomizing subjects to a usual care group and a screened group. However, the usual care group receives an examination which coincides at the time of the last exam in the study group. In this paper the power of these two designs have been evaluated. In both cases the power is severely reduced compared to the usual control group receiving no special exams. The power is a function of the sensitivity of the exam, the number and spacings of the exams given to the screened group as well as the sample size, disease incidence of the population and the survival distribution. The theoretical results on power are applied to the Canadian National Breast Cancer Study (ages 40-49) which used an UFD and the Stockholm Mammography Breast Cancer Screening Trial which utilized a COD.

Journal Article↗

PubMed2002

Genetic variability of Yersinia pestis isolates as predicted by PCR-based IS100 genotyping and analysis of structural genes encoding glycerol-3-phosphate dehydrogenase (glpD).

A PCR-based genotyping system that detects divergence of IS100 locations within the Yersinia pestis genome was used to characterize a large collection of isolates of different biovars and geographical origins. Using sequences derived from the glycerol-negative biovar orientalis strain CO92, a set of 27 locus-specific primers was designed to amplify fragments between the end of IS100 and its neighboring gene. Geographically diverse members of the orientalis biovar formed a homogeneous group with identical genotype with the exception of strains isolated in Indochina. In contrast, strains belonging to the glycerol-positive biovar antiqua showed a variety of fingerprinting profiles. Moreover, strains of the biovar medievalis (also glycerol positive) clustered together with the antiqua isolates originated from Southeast Asia, suggesting their close phylogenetic relationships. Interestingly, a Manchurian biovar antiqua strain Nicholisk 51 displayed a genotyping pattern typical of biovar orientalis isolates. Analysis of the glycerol pathway in Y. pestis suggested that a 93-bp deletion within the glpD gene encoding aerobic glycerol-3-phosphate dehydrogenase might account for the glycerol-negative phenotype of the orientalis biovar. The glpD gene of strain Nicholisk 51 did not possess this deletion, although it contained two nucleotide substitutions characteristic of the glpD version found exclusively in biovar orientalis strains. To account for this close relationship between biovar orientalis strains and the antiqua Nicholisk 51 isolate, we postulate that the latter represents a variant of this biovar with restored ability to ferment glycerol. The fact that such a genetic lesion might be repaired as part of the natural evolutionary process suggests the existence of genetic exchange between different Yersinia strains in nature. The relevance of this observation on the emergence of epidemic Y. pestis strains is discussed.

Bacterial Proteins↗

PubMed2002

Characterization of human RNA polymerase III identifies orthologues for Saccharomyces cerevisiae RNA polymerase III subunits.

Unlike Saccharomyces cerevisiae RNA polymerase III, human RNA polymerase III has not been entirely characterized. Orthologues of the yeast RNA polymerase III subunits C128 and C37 remain unidentified, and for many of the other subunits, the available information is limited to database sequences with various degrees of similarity to the yeast subunits. We have purified an RNA polymerase III complex and identified its components. We found that two RNA polymerase III subunits, referred to as RPC8 and RPC9, displayed sequence similarity to the RNA polymerase II RPB7 and RPB4 subunits, respectively. RPC8 and RPC9 associated with each other, paralleling the association of the RNA polymerase II subunits, and were thus paralogues of RPB7 and RPB4. Furthermore, the complex contained a prominent 80-kDa polypeptide, which we called RPC5 and which corresponded to the human orthologue of the yeast C37 subunit despite limited sequence similarity. RPC5 associated with RPC53, the human orthologue of S. cerevisiae C53, paralleling the association of the S. cerevisiae C37 and C53 subunits, and was required for transcription from the type 2 VAI and type 3 human U6 promoters. Our results provide a characterization of human RNA polymerase III and show that the RPC5 subunit is essential for transcription.

Amino Acid Sequence↗

PubMed2002

[Bilirubin induced apoptosis of human neuroblastoma cell line SH-SY5Y and affected the mitochondrial membrane potential].

OBJECTIVE: To study whether bilirubin induces apoptosis of human neuroblastoma cell line SH-SY5Y and affects the mitochondrial membrane potential. METHODS: SH-SY5Y cells were exposed to bilirubin, then morphological changes of nucleus were assayed by Hoechst 33258 staining. The percentage of apoptotic cells was measured by flow cytometry. The mitochondrial membrance potential(MMP) was detected by flow cytometry through Rhodamine123 staining. RESULTS: MMP of SH-SY5Y cells significantly decreased from (16.6 +/- 1.556) U to (7.11 +/- 0.701) U (P < 0.01) after exposed to 0.02 g/L bilirubin for 1 h. The percentage of apoptotic cells was 19.4% after exposed for 3 h, and significantly increased to 76.4% after 4 h, but typical apoptotic nuclear changes occured after 6 h. CONCLUSION: Bilirubin could early cause disruption of MMP, then induce the apoptosis of SH-SY5Y cells.

Apoptosis↗

PubMedDate not supplied

Off-line coupling of high-resolution capillary electrophoresis to MALDI-TOF and TOF/TOF MS.

High-resolution capillary electrophoresis has been coupled to MALDI-TOF and TOF/TOF MS through off-line vacuum deposition onto standard stainless steel MALDI targets. This off-line approach allowed the decoupling of the separation from the MS analysis, thus allowing each to be independently optimized in terms of time. Using BSA tryptic digest as a model sample, the deposited streaks, roughly 100-microm wide, were first analyzed in the MS mode, consuming only a fraction of the sample. After data analysis, segments of the deposited trace, containing unidentified peptides, as well as several species chosen for sequence confirmation, were reanalyzed in the MS/MS mode using MALDI-TOF/TOF MS. Additionally, it is shown that the shot-to-shot reproducibility of the vacuum-deposited trace (5% RSD) is 1 order of magnitude lower than that found for the standard dried droplet method. Moreover, a linear dependence of signal intensities (relative to an internal standard) over 3 orders of magnitude was found for a peptide sample with concentrations ranging from 1 to 1000 nM. This paper demonstrates the potential of off-line coupling of high-resolution separations to MALDI-MS and MALDI-MS/MS using vacuum deposition for the analysis of complex peptide mixtures from protein digests.

Electrophoresis, Capillary↗