Biomedical subjects

F Chang

Publications and source records attributed to F Chang.

At least 55 records · Page 3Linked to original sources

PubMed1999

[Clinical observation on treatment of bile regurgitational gastritis with danwei capsule].

OBJECTIVE: To evaluate the therpeutic effect of Danwei capsule in treating bile regurgitational gastritis. METHODS: One hundred and thirty-four patients with bile regurgitational gastritis were divided into two groups. Danwei capsule was used in therapeutic group and Motilium was used in control group. RESULTS: The total effective rate in therapeutic group was 92.59%, and 71.70% in control group (P < 0.01). The improvement in symptoms, physical findings and bile regurgitation in therapeutic group was better than that of control. CONCLUSION: Curative effect of Danwei capsule was better than that of Motilium, which should be used widely.

Adult↗

PubMed1998

Profiling expression patterns and isolating differentially expressed genes by cDNA microarray system with colorimetry detection.

A high-density cDNA microarray with colorimetry detection system to simultaneously monitor the expression of many genes on nylon membrane is described and characterized. To quantify the expression of genes and to isolate differentially expressed genes, the southern hybridization process on filter membranes was employed. The levels of gene expression were represented by color intensities generated by colorimetric reactions in place of hazardous radioisotopes or costly laser-induced fluorescence detection. The gene expression patterns on nylon membranes were digitized by devices such as an economical flatbed scanner or a digital camera. The quantitative information of gene expression was retrieved by image analysis software. Quantitative comparison of the northern dot-blotting method with the microarray system is described. Applications employing single-color detection as well as dual-color detection to isolate differentially expressed genes among thousands of genes are demonstrated.

Blotting, Northern↗

PubMed1998

Thoracoscopic anterior spinal procedures in children.

METHODS: From February 1996 through July 1997, 20 patients were determined to be appropriate for a thoracoscopic approach for the anterior portion of their spinal surgery. Ages ranged from 8 to 17 years of age and weight from 20 to 70 kg. The diagnosis included severe idiopathic and neurogenic scoliosis or kyphosis (n = 20) and congenital hemivertebra (n = 4). The procedures performed included diskectomy and anterior release from two to nine vertebral levels, hemivertebrectomy, and epiphysiodesis. Four patients also had an open lumbar exposure and all had a posterior fusion. RESULTS: All procedures were completed successfully endoscopically. Surgical times for the thoracoscopic portion of the procedure averaged 106 minutes and total procedure times were comparable with the standard open technique. Three patients were extubated at the end of the procedure with the majority extubated on postoperative day 1. Average intensive care unit stay was 1.8 days, and chest tubes were removed between postoperative day 1 and 5 (average, 2.2 days). In follow-up, surgical correction was deemed to be acceptable and equivalent to open techniques in all cases. CONCLUSION: This technique has proven to be safe and effective in children and appears to be associated with less pain and morbidity as evidenced by earlier extubation and chest tube removal and shorter ICU stay.

Adolescent↗

PubMed1998

Labeling peptides with technetium-99m using a bifunctional chelator of a N-hydroxysuccinimide ester of mercaptoacetyltriglycine.

UNLABELLED: A modified mercaptoacetyltriglycine (MAG3) chelator, which has acetyl S-protection and which is derivitized with N-hydroxysuccinimide (NHS) ester for conjugation, has been used to radiolabel four small (approximately 6- to 7-kDa) peptides, bovine pancreatic trypsin inhibitor, epidermal growth factor, human neutrophil elastase inhibitor and plasmin inhibitor, with 99mTc. METHODS: Each peptide was specifically labeled at the MAG3 chelation sites at ambient temperature and neutral pH. Specific activities of 100-150 mCi/mg were achieved at labeling efficiencies of about 50%, but specific activities of 3500 mCi/micromol could be attained. RESULTS: By a variety of assays, protein activity was unimpaired by the conjugation and labeling for two of the four peptides. The activities for plasmin of the plasmin inhibitor and bovine pancreatic trypsin inhibitor were reduced by conjugation, presumably because of a sensitive lysine residue in the structure of each of these two peptides. Multiple peaks were present in the high-performance liquid chromatography radiochromatograms, especially of human neutrophil elastase inhibitor; however, most peaks could be shown to be labeled active peptide. Stability during cysteine challenge at modest cysteine-to-peptide molar ratios and during incubation in serum was observed in each case. Large differences among the labeled peptides were apparent in the 3-hr biodistributions of 99mTc in normal mice. CONCLUSION: The use of NHS-S-acetyl-MAG3 may be a convenient method of radiolabeling peptides with 99mTc.

Animals↗

PubMed1997

Early results in the irrational design of new bifunctional chelators.

BACKGROUND: The development of a simple route for the synthesis of the N-hydroxysuccinimide (NHS) ester of S-acetyl-protected mercaptoacetyltriglycine (MAG3) has opened the possibility of preparing novel bifunctional N3S chelators for technetium-99m (99mTc) and other radionuclides. In particular, the synthesis may be applied to a vast number of tripeptides in place of triglycine, to provide a "library" of bifunctional N3S chelators, each with unique properties related to the particular amino acid residues within each tripeptide. METHODS: The authors have synthesized by this simple route the NHS esters of four N3S chelators by reacting NHS-S-acetylthioglycolic acid with ala-gly-gly, phe-gly-gly, pro-gly-gly, and ser-ser-ser, in addition to gly-gly-gly. Each bifunctional chelator was conjugated to biocytin as a model primary amine and radiolabeled with 99mTc. The properties of the four chelators were compared with MAG3 with respect to the stability of the label in saline and serum, the extent of serum protein binding, and the instability to cysteine challenge. RESULTS: A range of values was observed. Labeled mercaptoacetyltriserine showed stability towards transchelation to cysteine similar to that of MAG3 as well as lower serum protein binding; labeled mercaptoacetylalanyldiglycine showed slightly higher serum protein binding than labeled MAG3 but greater stability to cysteine challenge. CONCLUSIONS: The authors concluded that this simple synthesis and evaluation scheme may be used to prepare and screen a large library of bifunctional chelators for those with useful properties.

Blood Proteins↗

PubMed1997

Pretargeting using peptide nucleic acid.

BACKGROUND: Pretargeting studies in animals and humans have usually involved (strept)avidin and biotin. Depending on the particular strategy, endogenous biotin can adversely influence localization when these molecules are used. METHODS: As an alternative to (strept)avidin and biotin, we have explored the use of a single-stranded peptide nucleic acid (PNA) bound to a protein administered first and followed by the complementary single-stranded PNA radiolabeled with 99mTc. Target localization of the PNA-bound protein in a mouse infection and a mouse tumor model occurred by passive diffusion while the radiolabeled complementary PNA localized by in vivo hybridization. The PNA-streptavidin was prepared by adding biotin-conjugated PNA to streptavidin; the complementary PNA, derivatized with a primary amine, was conjugated with acetyl S-protected NHS-MAG3 bifunctional chelator and radiolabeled with 99mTc. RESULTS: In both the infection and tumor mouse models, increased localization of radiolabel was achieved in animals receiving both injectates compared with control animals receiving only the radiolabeled PNA. In the infection model, the infected to normal thigh radioactivity ratio was 3.5 for the study animals compared with 1.7 for control animals (P = 0.0001). In the tumor model, these values were 1.7 versus 1.2 (P = 0.003). CONCLUSIONS: We conclude that PNA may be considered an alternative to (strept)avidin and biotin for pretargeting studies.

Animals↗

PubMed1997

Detection of medulloblastoma and astrocytoma-associated ganglioside GD3 in cerebrospinal fluid.

Shedding of gangliosides by tumor cells may enhance tumor development. We recently showed that cells of the human brain tumor, medulloblastoma, shed gangliosides in vitro and have therefore examined ganglioside shedding by pediatric brain tumors into the cerebrospinal fluid (CSF). GD3, a major ganglioside in medulloblastoma and astrocytoma, was the target for detection in the CSF by immunostaining using the monoclonal antibody R24 and enhanced chemiluminescence detection. Mean CSF GD3 levels in patients with medulloblastomas (n = 9) and astrocytomas (n = 10) were significantly higher than those of controls (mean +/- SD 44.7 +/- 8.4 versus 18.2 +/- 1.9 pmol/ml, n = 20, P < 0.0002). Mass spectrometric analysis showed that tumor-derived ganglioside GD3 contained heterogeneous ceramide structures and, interestingly, the ceramide subspecies with shorter fatty acyl chains were selectively shed. The elevated CSF GD3 concentrations in patients with medulloblastoma and astrocytoma support the concept that ganglioside shedding, which may have significant biological consequences, is characteristic of human brain tumors.

Astrocytoma↗

PubMed1997

Shedding of gangliosides by human medulloblastoma cells.

Shedding of immunosuppressive gangliosides is an important characteristic of both experimental and human tumors. Using a medulloblastoma cell line, Daoy, with a very high ganglioside expression (141 +/- 13 nmol/10(8) cells) and a well-characterized ganglioside complement, we have now studied ganglioside shedding by human brain tumor cells. Shedding of gangliosides, quantified by metabolic radiolabeling, was significant (169 pmol/10(8) cells/h) and was generalized with respect to the major ganglioside carbohydrate structures (G(M2), G(M3), and G(D1a)). For each ganglioside, however, shedding was selective for ceramide structures containing shorter fatty acyl chains. Rapid and ceramide-selective shedding was confirmed in two additional human medulloblastoma cell lines, D341 Med and D283 Med (112 and 59 pmol/10(8) cells/h). Significant ganglioside shedding is therefore a common characteristic of human medulloblastoma cells and may influence the biological behavior of this tumor, in view of immunosuppressive and other biological properties of shed gangliosides.

Cell Division↗

PubMed1997

cdc12p, a protein required for cytokinesis in fission yeast, is a component of the cell division ring and interacts with profilin.

As in many other eukaryotic cells, cell division in fission yeast depends on the assembly of an actin ring that circumscribes the middle of the cell. Schizosaccharomyces pombe cdc12 is an essential gene necessary for actin ring assembly and septum formation. Here we show that cdc12p is a member of a family of proteins including Drosophila diaphanous, Saccharomyces cerevisiae BNI1, and S. pombe fus1, which are involved in cytokinesis or other actin-mediated processes. Using indirect immunofluorescence, we show that cdc12p is located in the cell division ring and not in other actin structures. When overexpressed, cdc12p is located at a medial spot in interphase that anticipates the future ring site. cdc12p localization is altered in actin ring mutants. cdc8 (tropomyosin homologue), cdc3 (profilin homologue), and cdc15 mutants exhibit no specific cdc12p staining during mitosis. cdc4 mutant cells exhibit a medial cortical cdc12p spot in place of a ring. mid1 mutant cells generally exhibit a cdc12p spot with a single cdc12p strand extending in a random direction. Based on these patterns, we present a model in which ring assembly originates from a single point on the cortex and in which a molecular pathway for the functions of cytokinesis proteins is suggested. Finally, we found that cdc12 and cdc3 mutants show a synthetic-lethal genetic interaction, and a proline-rich domain of cdc12p binds directly to profilin cdc3p in vitro, suggesting that one function of cdc12p in ring assembly is to bind profilin.

Actins↗

PubMed1997

Tissue distribution and abundance of human FKBP51, and FK506-binding protein that can mediate calcineurin inhibition.

We previously described the isolation of an FK506-binding protein, FKBP51, that is predominantly expressed in murine T cells and is capable of mediating drug-dependent calcineurin inhibition in vitro. In addition, the gene for FKBP51 is induced by glucocorticoids. Screening of a human thymus cDNA library resulted in the identification of the human homologue of FKBP51. Expression of the 3.7 kb mRNA corresponding to FKBP51 is induced by glucocorticoids in the human T cell line, C7TK.4. The 51.2 kDa protein encoded by this gene shares 87% identity to murine FKBP51 and demonstrates a similar IC50 value for the FK506-mediated inhibition of calcineurin phosphatase in vitro. The distribution and abundance of FKBP51 and FKBP12 in seventeen human tissues were compared by Western analysis. Unlike its murine counterpart, the human FKBP51 is abundantly expressed in numerous tissues and in many cases, is in molar excess over FKBP12.

Amino Acid Sequence↗

PubMed1997

Developmental change in the voltage-dependence of the pacemaker current, if, in rat ventricle cells.

Myocytes were isolated from newborn and adult rat ventricle. Using the whole-cell patch clamp, the two cell populations were compared for the presence of the hyperpolarization-activated pacemaker current if. As in other mammalian species, the threshold voltage in acutely dissociated adult rat myocytes was extremely negative (-113 +/- 5 mV; n=12). In contrast, threshold in newborn cells was relatively positive, regardless of whether measured in acutely dissociated (-72 +/- 2 mV; n=6) or cultured cells (-70 +/- 2 mV; n=9). Current density was not reduced in the adult. These results suggest that with development the ventricle assumes its non-pacemaker function, at least in part, by a shift of the voltage dependence of if outside the physiological range.

Animals↗

PubMed1997

The influence of temperature and alkaline pH on the labeling of free and conjugated MAG3 with technetium-99m.

Benzoyl-protected mercaptoacetyltriglycine (S-benzoyl MAG3) is radiolabeled by tartrate transchelation at elevated temperatures or basic pH. The object of this investigation was to establish whether the same 99mTc labeled species are formed when S-acetyl (S-acetyl MAG3)-conjugated compounds are radiolabeled by tartrate transchelation at ambient temperature and neutral pH in contrast to labeling at 95 degrees C and pH 11. S-acetyl MAG3 was conjugated to biocytin and to the amine-derivitized oligomers DNA and PNA. Along with free S-acetyl MAG3, these were radiolabeled under the different conditions. Although labeling efficiencies were always lower when labeled at ambient temperature and neutral pH relative to labeling at 95 degrees C or pH 11 (free S-acetyl MAG3 could not be labeled at all), size exclusion and reverse phase HPLC showed no difference with labeling conditions in the radiochemical profiles for labeled DNA and biocytin. In the case of DNA, a cysteine challenge also failed to demonstrate a difference. However, in the case of PNA, some important differences were observed in the size exclusion HPLC radiochromatograms. In addition, PNA labeled at ambient temperature and neutral pH was less stable to transchelation to cysteine. In conclusion, S-acetyl MAG3 conjugated compounds may be radiolabeled at ambient temperature and neutral pH. In most cases, the radiochemical species produced appear to be identical to those formed when labeling is accomplished at 95 degrees C or pH 11.

Chelating Agents↗

PubMed1997

Preparation and use of NHS-MAG3 for technetium-99m labeling of DNA.

The chelator mercaptoacetylglycylglycylglycine (MAG3) is on of several amidothiols that have been used successfully to radiolabeled proteins and other molecules with 99mTc. Prior to radiolabeling, the sulfur in these amidothiols is usually protected by a benzoyl group (i.e. S-benzoyl MAG3) which requires extreme alkaline pH or boiling water temperatures for rapid deprotection. As a result, the benzoyl-protected chelator is radiolabeled prior to conjugation (i.e. preconjugation labeling) in the case of carriers such as proteins or polypeptides which cannot withstand harsh conditions. We have employed a simple, two-step, synthesis of the N-hydroxysuccinimide ester of MAG3 in which the sulfur is protected with an acetyl group (i.e. S-acetyl NHS-MAG3). A single-stranded amine-derivitized DNA was coupled with NHS-S-acetyl MAG3. Radiolabeling was accomplished at room temperature and neutral pH by transchelation from 99mTc-tartrate. In comparison to labeled SHNH-DNA, the labeled MAG3-DNA was unstable to cysteine transchelation, however, in contrast to SHNH-DNA, no evidence for serum protein binding of the labeled MAG3-DNA was observed. We conclude that the S-acetyl NHS MAG3 bifunctional chelator may prove to be an attractive alternative method of radiolabeling DNA and other biologically important molecules with 99mTc.

Chelating Agents↗

PubMed1997

Human medulloblastoma gangliosides.

To establish a model system for the study of ganglioside metabolism of the human brain tumor, medulloblastoma, we have chemically characterized the gangliosides of the Daoy cell line. These cells contain a high concentration of gangliosides (143 +/- 13 nmol LBSA/10(8) cells). The major species have been structurally confirmed to be GM2 (65.9%), GM3 (13.0%), and GD1a (10.3%). Isolation of individual gangliosides homogeneous in both carbohydrate and ceramide moieties by reversed-phase HPLC and analysis by negative-ion fast atom bombardment collisionally activated dissociation tandem mass spectrometry have allowed us to unequivocally characterize ceramide structures. In the case of GM2, 10 major ceramide subspecies were identified: d18:1-hC16:0, d18:1-C16:0, d18:0-C16:0, d18:1-C18:0, d18:1-C20:0, d18:1-C22:0, d18:2-C24:1, d18:1-C23:1, d18:1-C24:1, and d18:1-C24:0. Taken together with previous studies, these findings in buman medulloblastoma cells support the view that high expression and marked heterogeneity of ceramide structure are general characteristics of tumor gangliosides, molecules which are shed by the tumor cells and which are biologically active in vivo.

Ceramides↗

PubMed1997

Technetium-99m labeled epidermal growth factor-tumor imaging in mice.

We have shown previously that the epidermal growth factor peptide (EGF) may be radiolabeled with 99mTc at room temperature and neutral pH by using the N-hydroxysuccinimide ester of S-acetyl mercaptoacetyltriglycine (MAG3) as a bifunctional chelator. By a competition binding assay, we found that MAG3-conjugated EGF retained biological activity. Furthermore, the labeled peptide exhibited saturation binding to EGF receptor-positive tumor cell lines which could be inhibited by presaturation of the cells with unlabeled, native EGF. Biodistribution in normal mice at 3 h postadministration showed rapid clearance with minimal retention of the label in sampled organs. We have now investigated the tumor localization properties in mice of this labeled peptide. Nude mice implanted with the EGF receptor-positive tumors A431 and LS-174T were administered labeled EGF and a labeled control peptide (BPTI, aprotinin). Tumor uptake at 12 h postadministration was 0.44% injected dose/g for EGF/g vs. 0.09 for the control. Pretreatment of tumored mice with unlabeled EGF blocked about half the tumor uptake. Animals were also administered an anti-EGF receptor antibody labeled with 99mTc via MAG3. Relative to the antibody, tumor-to-muscle ratios were improved from 6 to 15 and tumor-to-blood ratios from 0.4 to 7 with EGF. These favorable results along with documented evidence of overexpression of the EGF receptor in many human tumors suggest that 99mTc-EGF should be considered further for tumor detection.

Animals↗

PubMed1997

Integration specificities of two lambdoid phages (21 and e14) that insert at the same attB site.

It was shown previously that phage 21 and the defective element e14 integrate at the same site within the icd gene of Escherichia coli K-12 but that 21 integrase and excisionase excise e14 in vivo very infrequently compared to excision of 21. We show here that the reverse is also true: e14 excises itself much better than it excises an adjacent 21 prophage. In vitro integrase assays with various attP substrates delimit the minimal attP site as somewhere between 366 and 418 bp, where the outer limits would include the outermost repeated dodecamers suggested as arm recognition sites by S. J. Schneider (Ph.D. dissertation, Stanford University, Stanford, Calif., 1992). We speculate that the reason 21 attP is larger than lambda attP (240 bp) is because it must include a 209-bp sequence homologous to the 3' end of the icd transcript in order to allow icd expression in lysogens. Alteration of portions of 21 attP to their e14 counterparts shows that 21 requires both the arm site and core site sequences of 21 but that replacements by e14 sequences function in some positions. Consistent with Schneider's in vivo results, and like all other known integrases from lambdoid phages, 21 requires integration host factor for activity.

Bacterial Proteins↗

PubMed1997

In vivo hybridization of technetium-99m-labeled peptide nucleic acid (PNA).

UNLABELLED: Hybridization of a radiolabeled single-stranded DNA oligonucleotide with its single-stranded complement in vivo has not yet been convincingly demonstrated. A contributing factor may be unfavorable in vivo properties of the phosphodiester and phosphorothioate DNAs. Peptide nucleic acid (PNA) oligomers have been reported to possess in vivo properties more suitable for radiopharmaceutical applications. METHODS: We have radiolabeled an amine-derivatized 15-base PNA oligomer with 99mTc through a modified MAG3 chelator. RESULTS: The ability of the PNA to hybridize in vitro with its complement appeared to be unimpaired after conjugation and radiolabeling. Size-exclusion, high-performance liquid chromatography (HPLC) analysis of 37 degrees C serum after 24 hr of incubation showed the radiolabel to be present predominately as labeled PNA with indications of labeled serum proteins and a low molecular weight catabolite. Whole-body clearance in mice was rapid, with 50% of the label eliminated in about 2 hr. After 2.5 hr, the highest uptake (kidneys) was only 1.5% of the injected dose/g; less than 0.07%/g was present in all sampled tissues at 24 hr. To evaluate in vivo hybridization, beads were implanted subcutaneously in both thighs of normal mice. In the left thigh only, the beads were conjugated with complementary single-stranded PNA. At 23 hr following intraperitoneal administration of the labeled PNA, the left/right thigh radioactivity ratio was 6:1. Whole-body images at this time showed only bladder, kidneys and the left thigh. CONCLUSION: Unlike the radiolabeled DNAs investigated in this laboratory, 99mTc-PNA displays stability and pharmacokinetic properties suitable for eventual use as radiopharmaceuticals.

Animals↗

PubMed1996

Cell division: why daughters cannot be like their mothers.

A cell-fate determinant that segregates asymmetrically at cell division has been identified in budding yeast. Possible mechanisms for this asymmetric segregation are suggested by the identification of mutants in genes encoding cortically localized proteins.

Animals↗