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Biomedical subjects

P Hu

Publications and source records attributed to P Hu.

At least 19 recordsLinked to original sources
PubMed2000

Distinct sprouting responses of sympathetic and peptidergic sensory axons proximal to a sciatic nerve transection in guinea pigs and rats.

Sprouting of sympathetic and peptidergic sensory neurones proximal to nerve lesions may reflect upregulation of growth factors around damaged dorsal root ganglion (DRG) cells. Axons containing noradrenaline or calcitonin gene-related peptide were visualized in DRGs and spinal roots of guinea pigs and rats. After sciatic transection in rats, varicose terminals of both types appeared around large DRG somata. These neurones were surrounded by proliferated satellite cells expressing glial fibrillary acidic protein (GFAP) and p75. This did not occur in guinea pigs. Instead, sympathetic axons grew through the DRG and centrally along the dorsal roots (which contained p75-positive glia), avoiding the DRG somata. Thus the glial reaction to peripheral injury differs between species such that, in guinea pigs, the environment in the spinal roots rather than in the DRGs favours sympathetic sprouting.

Animals↗
PubMed2000

Capillary electrophoresis--matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using a vacuum deposition interface.

An improved vacuum deposition interface for coupling capillary electrophoresis with MALDI-TOF MS has been developed. Liquid samples consisting of analyte and matrix were deposited on a moving tape in the evacuated source chamber of a TOF mass spectrometer, enabling 24 h of uninterrupted analysis. The vacuum deposition procedure was compared with the dried-droplet method, and it was found that vacuum deposition generated significantly more reproducible signal intensity, eliminating the need for "sweet spot" searching. A concentration detection limit in the low-nanomolar range has been achieved with a low-attomole amount of sample consumed per spectrum. In addition, ion suppression caused by hydrophobicity differences in the analytes was reduced. To minimize ion suppression further, separation prior to MALDI MS analysis was employed. The performance of capillary electrophoresis (CE)-MALDI-TOF MS using the vacuum deposition interface was evaluated with a peptide mixture injected at low-femtomole levels. All peptides were baseline resolved with separation efficiencies in the range of 250000-400000 plates/m (2-3-s band half-width), demonstrating the high separation efficiency of the CE-MALDI MS coupling. A fast (approximately 40 s) CE separation of a mixture of angiotensins was found to reduce significantly ion suppression and enable trace level detection. It was also shown, for the analysis of an enolase digest, that sequence coverage of 65% was obtained using CE separation compared to 52% using step-elution solid-phase extraction and 44% in the control experiment using an unseparated mixture.

Electrophoresis, Capillary↗
PubMed2000

Molecular cloning and mapping of the brain-abundant B1gamma subunit of protein phosphatase 2A, PPP2R2C, to human chromosome 4p16.

Protein phosphatase 2A (PP2A) is one kind of serine/ threonine protein phosphatase regulating mainly cell growth and division. It comprises three subunits, A, B, and C. The B-subunit is involved in enzyme activity and substrate specificity. The B-subunit family is of great diversity and is divided into three classes, the B1, B2, and B3 subfamilies. Until now, two members of the B1 subfamily, B1alpha and B1beta, have been identified in human. In this report, the third member of the sub-family, B1gamma, was identified, and its cDNA was isolated from a human brain cDNA library. This novel cDNA is 4,120 bp in length and contains an open reading frame (nt 55-1,398) encoding 447 amino acid residues. The putative protein shares 81 and 85% identity with B1alpha (PPP2R2A) and B1beta (PPP2R2B), respectively, and was named PPP2R2C for its high level of homology to the other two isoforms. One remarkable characteristic of this novel gene is that it is highly expressed in brain with a 4.7-kb transcript while it is nearly undetectable in other tissues. In addition, the PPP2R2C gene was localized to human chromosome 4p16 between markers D4S2925 and D4S3007 with 5.45 cR (LOD > 14) and 2.63 cR (LOD > 15) RH distance, respectively, by radiation hybrid panel mapping.

Amino Acid Sequence↗
PubMed2000

Regions of genomic instability on 22q11 and 11q23 as the etiology for the recurrent constitutional t(11;22).

The constitutional t(11;22)(q23;q11) is the only known recurrent, non-Robertsonian translocation. To analyze the genomic structure of the breakpoint, we have cloned the junction fragments from the der(11) and der(22) of a t(11;22) balanced carrier. On chromosome 11 the translocation occurs within a short, palindromic AT-rich region (ATRR). Likewise, the breakpoint on chromosome 22 has been localized within an ATRR that is part of a larger palindrome. Interestingly, the 22q11 breakpoint falls within one of the 'unclonable' gaps in the genomic sequence. Further, a sequenced chromosome 11 BAC clone, spanning the t(11;22) breakpoint in 11q23, is deleted within the palindromic ATRR, suggesting instability of this region in bacterial clones. Several unrelated t(11;22) families demonstrate similar breakpoints on both chromosomes, indicating that their translocations are within the same palindrome. It is likely that the palindromic ATRRs produce unstable DNA structures in 22q11 and 11q23 that are responsible for the recurrent t(11;22) translocation.

Base Sequence↗
PubMed2000

Molecular cloning and functional expression of contortrostatin, a homodimeric disintegrin from southern copperhead snake venom.

Contortrostatin is a unique dimeric disintegrin isolated from southern copperhead snake venom. Through antagonism of integrins alphaIIbbeta3, alpha5beta1, alphavbeta3, and alphavbeta5, contortrostatin inhibits platelet aggregation and disrupts cancer cell adhesion and invasion. We cloned cDNA from a library made from the venom gland cells of Agkistrodon contortrix contortrix using polymerase chain reaction. We found that the contortrostatin gene is part of a precursor composed of proprotein, metalloproteinase, and disintegrin domains. The precursor cDNA is 2027 bp with a 1449-bp open reading frame. The disintegrin domain is 195 bp encoding 65 amino acids. Like other members of the disintegrin family, each subunit of contortrostatin has an RGD site, and the cysteine alignment is conserved. The disintegrin domain of the cDNA has been expressed in a eukaryotic expression system as a homodimeric fusion protein with an immunoglobulin. The recombinant protein is recognized by an antiserum against native contortrostatin in Western blot. Both the native and recombinant proteins bind to integrins alphavbeta3 and alphavbeta5. Like native contortrostatin, the recombinant fusion protein inhibits platelet aggregation, blocks cancer cell adhesion to fibronectin and vitronectin, and prevents invasion of cancer cells through a Matrigel barrier. The success of functional expression not only validates the cDNA cloning of this disintegrin, but also provides adequate material for functional studies of contortrostatin.

Agkistrodon↗
PubMed2000

Chromosome 22-specific low copy repeats and the 22q11.2 deletion syndrome: genomic organization and deletion endpoint analysis.

The 22q11.2 deletion syndrome, which includes DiGeorge and velocardiofacial syndromes (DGS/VCFS), is the most common microdeletion syndrome. The majority of deleted patients share a common 3 Mb hemizygous deletion of 22q11.2. The remaining patients include those who have smaller deletions that are nested within the 3 Mb typically deleted region (TDR) and a few with rare deletions that have no overlap with the TDR. The identification of chromosome 22-specific duplicated sequences or low copy repeats (LCRs) near the end-points of the 3 Mb TDR has led to the hypothesis that they mediate deletions of 22q11.2. The entire 3 Mb TDR has been sequenced, permitting detailed investigation of the LCRs and their involvement in the 22q11.2 deletions. Sequence analysis has identified four LCRs within the 3 Mb TDR. Although the LCRs differ in content and organization of shared modules, those modules that are common between them share 97-98% sequence identity with one another. By fluorescence in situ hybridization (FISH) analysis, the end-points of four variant 22q11.2 deletions appear to localize to the LCRs. Pulsed-field gel electrophoresis and Southern hybridization have been used to identify rearranged junction fragments from three variant deletions. Analysis of junction fragments by PCR and sequencing of the PCR products implicate the LCRs directly in the formation of 22q11.2 deletions. The evolutionary origin of the duplications on chromosome 22 has been assessed by FISH analysis of non-human primates. Multiple signals in Old World monkeys suggest that the duplication events may have occurred at least 20-25 million years ago.

Animals↗
PubMed2000

Fine mapping of the constitutional translocation t(11;22)(q23;q11).

Translocation t(11;22)(q23;q11) is the most common constitutional reciprocal translocation in man. Balanced carriers are phenotypically normal, except for decreased fertility, an increased spontaneous abortion rate and a possible predisposition to breast cancer in some families. Here, we report the high resolution mapping of the t(11;22)(q23;q11) breakpoint. We have localised the breakpoint, by using fluorescence in situ hybidisation (FISH) walking, to a region between D11S1340 and WI-8564 on chromosome 11, and D22S134 and D22S264 on chromosome 22. We report the isolation of a bacterial artificial chromosome (BAC) clone spanning the breakpoint in 11q23. We have narrowed down the breakpoint to an 80-kb sequenced region on chromosome 11 and FISH analysis has revealed a variation of the breakpoint position between patients. In 22q11, we have sequenced two BACs (BAC2280L11 and BAC41C4) apparently mapping to the region; these contain low copy repeats (LCRs). Southern blot analysis with probes from BAC2280L11 has revealed different patterns between normal controls and translocation carriers, indicating that sequences similar/identical to these probes flank the translocation breakpoint. The occurrence of LCRs has previously been associated with genomic instability and "unclonable" regions. Hence, the presence of such repeats renders standard translocation breakpoint cloning techniques ineffective. Thus, we have used high resolution fiber-FISH to study this region in normal and translocation cases by using probes from 22q11, LCRs and 11q23. We demonstrate that the LCR containing the gap in 22q11 is probably substantially larger than the previous estimates of 100 kb. Using fiber-FISH, we have localised the breakpoint in 22q11 to approximately 20-40 kb from the centromeric border of the LCR (i.e. the telomeric end of AC006547) and have confirmed the breakpoint position on 11q23.

Base Sequence↗
PubMed2000

Breakdown of the blood-retinal barrier induced by activated T cells of nonneural specificity.

The cellular and microvascular responses of JC Lewis rats to an intravenous injection of activated T cells specific for ovalbumin were examined with the retinal whole mount technique. The retina was examined at various times post-injection (pi) with the use of antibodies to the alphabeta T cell receptor (TCR) or to major histocompatibility complex class II (MHC II), the monoclonal antibody ED1, and intravascular tracers. By 12 hours pi, small numbers of TCR(+), ED1(+), and MHC II(+) cells were present within the lumen of retinal vessels, and minor breakdown of the blood-retinal barrier (BRB) and microglial activation were evident. The intensity of these responses had increased by 1 day pi, when small numbers of TCR(+) cells had also undergone extravasation. By 2 to 3 days pi, the numbers of TCR(+), ED1(+), and MHC II(+) cells in the retinal parenchyma had increased, but the BRB breakdown and microglial activation had subsided. Thus, in the absence of target antigen, activated T cells induced limited and transient breakdown of the BRB, microglial activation, and the extravasation of ED1(+), MHC II(+) monocytes. In contrast, the retina of rats that received an intraocular injection of ovalbumin in addition to the intravascular injection of T cells showed massive cellular recruitment and breakdown of the BRB. These results indicate that an increase in the number of activated T cells in the circulation, such as that which occurs during viral or bacterial infection, has the potential to result in transient breakdown of the BRB and a mild local microglial response.

Animals↗
PubMed2000

Depression among needle exchange program and methadone maintenance clients.

The objective of this study was to compare the prevalence of major depression in two cohorts of injection drug users, those enrolled in a Rhode Island Methadone Maintenance Treatment Program (MMTP) and those enrolled in a Rhode Island Needle Exchange Program (NEP) using cross-sectional interviews. Symptomatic and duration criteria for major depression in the last 6 months were identified using the Structured Clinical Interview for DSM-III-R (SCID). Among 528 persons interviewed, 54% of those in NEP and 42% of those in MMTP met criteria for major depression. Using multivariate logistic regression, women (odds ratio [OR] 2.5; 95% confidence interval [CI] 1.7-3.7), persons with alcohol use disorders (OR 1.7; 95% CI 1.1-2. 7), and persons without a current partner (OR 1.8; 95% CI 1.2-2.6) were more likely to be depressed controlling for age, race, education and HIV status. Persons enrolled in MMTP were less likely to be depressed (OR 0.6; 95% CI 0.4-0.8) than NEP. Higher rates of depression were found among NEP attendees than among those enrolled in MMTP. Mental health referrals should be part of the growing number of needle exchanges in the United States.

Adult↗
PubMed2000

Regional oral absorption, hepatic first-pass effect, and non-linear disposition of salmon calcitonin in beagle dogs.

The dose-dependent disposition, first pass hepatic elimination, and absorption pharmacokinetics (PK) of salmon calcitonin (sCT) were investigated in a canine Intestinal Vascular Access Port (IVAP) model. The PK of sCT were determined after intravenous (IV), subcutaneous (SC), portal venous (PV), and oral (PO) administration of sCT. Regional oral absorption of unformulated sCT was also evaluated by direct administration into the duodenum (ID), ileum (IL), and colon (IC) by means of surgically implanted, chronic catheters. Plasma samples were collected and analyzed by radioimmunoassay (RIA). Salmon calcitonin PK were evaluated using 2-compartmental and model independent methods. Intravenous sCT PK were non-linear over the dose range studied. High dose groups (100-1000 microg) demonstrated higher total plasma clearance (CL) and V(dss) than the low dose groups (1-25 microg). However, the MRT did not change for doses ranging from 10 to 1000 microg. After SC administration, the absorption of sCT was rapid with bioavailability (BA) varying from 21.4 to 52.9%. However, the BA of sCT was low after ID, IL, and IC administration (0.039, 0.064, and 0.021%, respectively). The role of hepatic first-pass elimination was negligible. The results of these studies demonstrate that the elimination of sCT is rapid but does not occur in the liver. Enhanced sCT clearance at higher doses was indicated by increasing V(dss) values, and it is hypothesized that increased renal blood flow and/or saturated plasma protein binding may contribute to the non-linear behavior. The IVAP canine model was found to have utility for probing the absorption and disposition PK of sCT. The combination of high oral bioavailability variability and non-linear disposition of sCT may produce highly variable therapeutic effects. The practical impact of the non-linear disposition of sCT remains to be determined. Based on the current results it appears that the rate-limiting step to the successful oral administration of sCT is its delivery into the portal vein since hepatic metabolism was negligible.

Administration, Oral↗
PubMed2000

Collisionally activated dissociations of aminocyclitol-aminoglycoside antibiotics and their application in the identification of a new compound in tobramycin samples.

Several aminocyclitol-aminoglycoside antibiotics have been studied by tandem mass spectrometry. Glycosidic bond cleavages were the major reactions in the low energy collisionally activated decomposition (CAD) of the protonated antibiotics. Only the glycoside residing on the C6-O of the 2-deoxystreptamine was observed to undergo significant decomposition at the C2-C3 and O-C1 bonds. The comprehension of the CAD of known aminoglycosides aided in the identification of an unknown impurity in tobramycin. The unknown compound was initially detected by reverse phase high-performance liquid chromatography following dinitrofluorobenzene derivatization of the amino groups. The molecular weight of the dinitrobenzene derivative measured by LC mass spectrometry (MS) led to the detection of two isomeric impurities in tobramycin by LC-MS using an amino column. Their CAD spectra were subsequently acquired by LC-MS/MS. One of the two compounds was determined to be a known compound, 6"-O-carbamyltobramycin with the carbamyl group substituted on the glycoside residing on the C6-O of 2-deoxystreptamine. The fragmentation pattern of the other compound was interpreted as that the unknown was also a carbamyltobramycin. The carbamyl group was, however, substituted on 2-deoxystreptamine. It was speculated that the carbamyl group was substituted at the C1 amino group. This compound, to our knowledge, has not been reported before.

Anti-Bacterial Agents↗
PubMed2000

Adherence to antiretroviral therapy among HIV-infected methadone patients: effect of ongoing illicit drug use.

Methadone maintenance patients infected with human immunodeficiency virus (HIV) currently receiving antiretroviral therapy had HIV RNA testing and were surveyed regarding their adherence to their treatment regimens. Adherence was measured using self-report on four questions relating to medication use in the last day and last month and whether the patient took "drug holidays." Of the patients (N = 42), 52% were receiving two-drug antiretroviral therapy and 48% were receiving triple therapy that included a protease inhibitor. Persons on triple therapy reported higher rates of adherence on all measures and were more likely to have undetectable HIV RNA levels than persons on dual therapy (60% vs. 50%). Ongoing illicit drug injection was the only factor significantly associated (p < .05) with multiple measure nonadherence; however, it was not associated with undetectable HIV RNA level. Levels of nonadherence were comparable to estimates from other chronic diseases, but this finding has important implications for patients receiving highly active antiretroviral therapy.

Antiretroviral Therapy, Highly Active↗
PubMed2000

A plasmid partition system of the P1-P7par family from the pMT1 virulence plasmid of Yersinia pestis.

The complete sequence of the virulence plasmid pMT1 of Yersinia pestis KIM5 revealed a region homologous to the plasmid partition (par) region of the P7 plasmid prophage of Escherichia coli. The essential genes parA and parB and the downstream partition site gene, parS, are highly conserved in sequence and organization. The pMT1parS site and the parA-parB operon were separately inserted into vectors that could be maintained in E. coli. A mini-P1 vector containing pMT1parS was stably maintained when the pMT1 ParA and ParB proteins were supplied in trans, showing that the pMT1par system is fully functional for plasmid partition in E. coli. The pMT1par system exerted a plasmid silencing activity similar to, but weaker than those of P7par and P1par. In spite of the high degree of similarity, especially to P7par, it showed unique specificities with respect to the interactions of key components. Neither the P7 nor P1 Par proteins could support partition via the pMT1parS site, and the pMT1 Par proteins failed to support partition with P1parS or P7parS. Typical of other partition sites, supernumerary copies of pMT1parS exerted incompatibility toward plasmids supported by pMT1par. However, no interspecies incompatibility effect was observed between pMT1par, P7par, and P1par.

Bacterial Proteins↗
PubMed2000

Comparison of chemotherapy with chemohormonal therapy as first-line therapy for metastatic, hormone-sensitive breast cancer: An Eastern Cooperative Oncology Group study.

PURPOSE: Although hormonal therapy represents standard therapy for metastatic hormone-sensitive disease, many patients receive initial chemotherapy because of the location, bulk, or aggressiveness of their disease. It is uncertain whether simultaneous hormonal therapy provides additional benefit compared with chemotherapy alone. Eastern Cooperative Oncology Group trial E3186 was initiated to explore this question. PATIENTS AND METHODS: Between January 1988 and December 1992, 231 patients with estrogen receptor (ER)-positive or ER-unknown metastatic breast cancer were randomized to receive either chemotherapy (cyclophosphamide, doxorubicin, and fluorouracil ¿CAF) or chemohormonal therapy (CAF plus tamoxifen and Halotestin ¿fluoxymesterone; Pharmacia-Upjohn, Kalamazoo, MI ¿CAFTH) as front-line therapy for metastatic breast cancer. Patients who experienced a complete response to induction therapy either received or did not receive maintenance cyclophosphamide, methotrexate, fluorouracil, prednisone, and TH as a secondary randomization. RESULTS: The response rates (complete response and partial response) of patients who received CAF and CAFTH were similar (69.2% v 68.9%, respectively; P =.99). Time to treatment failure (TTF) was slightly longer for patients who received chemohormonal therapy compared with chemotherapy alone patients (13.4 months v 10.3 months, respectively; P =.087), and TTF was significantly longer in ER-positive compared with ER-negative patients (17.4 months v 10.3 months, respectively; P =.048). However, ER status had no effect on overall survival (30.0 months for CAF v 29.3 months for CAFTH). CONCLUSION: In patients with potentially hormone-sensitive metastatic breast cancer, chemohormonal therapy prolongs TTF for ER-positive patients without improving overall survival.

Adult↗
PubMed2000

[Refined chromosome assignment of human novel H-RalGDS gene on chromosome 9q34.1 by using radiation hybrid genebridge 4 panel].

OBJECTIVE: Refined chromosome assignment of a novel Ras-related H-RalGDS gene (the human homology of rat RalGDS, Ral guanine nucleotide dissociation stimulator recently cloned and characterized by our laboratory) by using human/rodent radiation somatic cell hybrid panel GB4. METHODS: According to the sequence of the 3'-untranslated region of H-RalGDS cDNA, forward and reverse primers were designed and used to amplify the human/rodent somatic radiation hybrid Genebridge 4 panel. The PCR result of each cell line of the panel was scored in a certain way and put into the related internet site of WI/MIT Radiation Hybrid Mapper for RH mapping and comprehensive analysis. RESULTS: The H-RalGDS gene was successfully localized to the framework of chromosome 9. After referring to literature and further integrated mapping analysis of the physical, genetic linkage and cytological mappings, the gene was assigned precisely between the markers SURF5 and RPL7A which had already been assigned to human chromosome 9q34.1. CONCLUSION: The localization of H-RalGDS not only contributed much to the construction of refined gene map and cytogenetic map within the region of chromosome 9q34, but also made clear that RH mapping of human novel genes is a simple, convenient and reliable method which can provide even more genetic information of genes within chromosomal regions.

Animals↗
PubMed2000

Single amino acid substitution in the Fc region of chimeric TNT-3 antibody accelerates clearance and improves immunoscintigraphy of solid tumors.

UNLABELLED: Recent studies in antibody catabolism have identified residues at the CH2-CH3 interface of the IgG heavy chain critical for serum persistence of immunoglobulins. Amino acid substitutions in the Fc region of murine IgG1 were shown to drastically accelerate antibody clearance in mice. Our laboratory has previously described a human-mouse chimeric TNT-3 (chTNT-3) monoclonal antibody directed against a universal nuclear antigen that has potential for the radioimmunotherapy of many solid tumors. In the current study, we engineered a chTNT-3 mutant containing a single amino acid substitution, to determine whether a more rapid clearance profile would make the antibody suitable for diagnostic imaging. METHODS: A single amino acid substitution in the CH2 domain of the human gamma1 constant region was made by polymerase chain reaction mutagenesis. High-level expression was achieved using the Glutamine Synthetase Gene Amplification System, and the chTNT-3 mutant was purified by protein A affinity and ion-exchange chromatography. A radioimmunoassay was performed to examine antigen binding, and in vivo studies were undertaken to evaluate clearance and tumor targeting in human tumor xenograft models. RESULTS: The chTNT-3 mutant retained the high affinity of chTNT-3, with a binding constant of 1.5 x 10(-9) mol/L. The mutant was eliminated rapidly from BALB/c mice, with a beta-phase half-life of 33.8 h, compared to 134.2 h for chTNT-3. Moreover, biodistribution studies in human colon tumor-bearing nude mice reflected this accelerated clearance. Tumor levels of the mutant were, respectively, 65%, 39%, and 36% of the tumor levels achieved with the parental chTNT-3 6, 12, and 24 h postinjection. The rapid clearance of the chTNT-3 mutant from the blood resulted in higher tumor-to-normal organ ratios for many normal tissues. Imaging of tumor-bearing mice with 99mTc-labeled chTNT-3 mutant demonstrated early visualization of tumors in 3 different solid tumor xenograft models. CONCLUSION: The accelerated clearance produced by a single amino acid substitution in the Fc region of chTNT-3 leads to improved imaging in tumor-bearing mice. These studies suggest that a rapidly clearing antibody generated by this approach may be useful for the immunoscintigraphy of human tumors.

Adenocarcinoma↗
PubMed1999

A role for PML and the nuclear body in genomic stability.

The PML gene of acute promyelocytic leukemia (APL) encodes a cell-growth and tumor suppressor. PML localizes to discrete nuclear bodies (NBs) that are disrupted in APL cells. The Bloom syndrome gene BLM encodes a RecQ DNA helicase, whose absence from the cell results in genomic instability epitomized by high levels of sister-chromatid exchange (SCE) and cancer predisposition. We show here that BLM co-localizes with PML to the NB. In cells from persons with Bloom syndrome the localization of PML is unperturbed, whereas in APL cells carrying the PML-RARalpha oncoprotein, both PML and BLM are delocalized from the NB into microspeckled nuclear regions. Treatment with retinoic acid (RA) induces the relocalization of both proteins to the NB. In primary PML-/- cells, BLM fails to accumulate in the NB. Strikingly, in PML-/- cells the frequency of SCEs is increased relative to PML+/+ cells. These data demonstrate that BLM is a constituent of the NB and that PML is required for its accumulation in these nuclear domains and for the normal function of BLM. Thus, our findings suggest a role for BLM in APL pathogenesis and implicate the PML NB in the maintenance of genomic stability.

Adenosine Triphosphatases↗