Microtubule-dependent nuclear positioning and nuclear-dependent septum positioning in the fission yeast Schizosaccharomyces [correction of Saccharomyces] pombe.
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Benzimidazoles↗
Biomedical subjects
F Chang
Publications and source records attributed to F Chang.
Tandem duplication polymorphism upstream of the dopamine D4 receptor gene (DRD4).
The dopamine D4 receptor (DRD4) is a member of the D2-like dopamine receptor family. Polymorphisms at the DRD4 gene have been examined for association with a wide range of neuropsychiatric disorders and normal behavioral variation. The DRD4 gene is unusual in its high amount of expressed polymorphism in humans. Here we study the identification of a polymorphic tandem duplication of 120 bp located 1.2 kb upstream of the initiation codon. The duplicated region contains consensus sequences of binding sites for several known transcription factors, suggesting that different alleles may differ in their transcriptional activity. Because chimpanzees, gorillas, and orangutans lack the duplication, the duplicated allele is inferred to be derived. The frequency of this derived duplication allele ranges from 0.40-0.81 in the 11 populations from around the world typed for this polymorphism. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 88:705-709, 1999.
Alleles↗
An interlaboratory study to determine the presence of human papillomavirus DNA in esophageal carcinoma from China.
Esophageal-carcinoma samples originating from the high-incidence area of China were tested in 2 different laboratories, each using a different degenerate PCR approach. Results confirmed the notion that none of the PCR approaches available for HPV-DNA detection today, is optimal for detecting all known HPV types at equal sensitivity and specificity. In combining results obtained in both laboratories, HPV DNA was demonstrated in 20/117 (17.1%) esophageal-carcinoma samples analyzed. HPV DNA was detected in 3/70 (4.3%) diagnostic biopsies, 7/23 (30.4%) surgical specimen and 10/24 (41.6%) cytological scrapings originating from the entire surface of the esophagus. Mucosotropic HPV types were present in 7/117 (6%) samples, only 3 being of the high-risk types (HPV 16, 18, 33). Other mucosal types found were HPV 6, 11, 13, 53 and 54. Cutaneous HPV types were present in 14/117 (12.0%) samples. HPVs 20 and 38 were present in 3 (2.6%) of the total samples and, in each case, together with another HPV type within one lesion. Two putative new HPV types, DL347 and DL 369, were identified.
Adult↗
NHS-MAS3: a bifunctional chelator alternative to NHS-MAG3.
This laboratory uses an N-hydroxysuccinimide derivative of S-acetylmercaptoacetyltriglycine (NHS-MAG3) to conjugate amines for subsequent labeling with 99mTc. However, the synthesis from triglycerine is general and not restricted to this tripeptide. We had earlier selected a small number of alternative tripeptides and synthesized the corresponding NHS derivatives. Each was then evaluated in a search for bifunctional chelators with properties superior to NHS-MAG3, such as lower serum protein binding or improved stability to cysteine challenge. Based on these preliminary results, NHS-S-acetylmercaptoacetyltriserine (NHS-MAS3) was selected for further investigation. We have now conjugated this bifunctional chelator to an biocytin and to an amine-derivatized peptide nucleic acid (PNA). Both carriers were also conjugated with NHS-MAG3 under identical conditions and all were labeled with 99mTc at neutral pH and at boiling temperature while the conjugated PNAs were radiolabelled at neutral pH and at room temperature. Regardless of the chelator, reverse phase HPLC radiochromatograms of the labeled biotins and PNAs after purification showed a single peak. However, by size exclusion HPLC, the radiochromatograms always showed several peaks even after purification, but the MAS3 radiochromatograms were less complicated. For biotin and PNA both, radiolabeling via MAS3 showed improved 99mTc stability in 37 degrees C serum and in cysteine solution. The four preparations were administered to mice implanted in one thigh with avidin beads (biotins) or complementary PNA beads (PNAs). At 5 h post-administration, no significant differences were observed in the targeting of PNA beads between the two chelators, however the target thigh/normal thigh ratio was significantly higher for MAS3-biotin compared to MAG3-biotin. We conclude that labeling biocytin and amine-derivatized PNA with NHS-MAS3 compared to NHS-MAG3 provides simpler radiochromatographic profiles, improved stability of the label in serum and cysteine solution and can improve in vivo targeting.
Animals↗
Technetium-99m labeled peptides--an investigation of multiple HPLC peaks.
This laboratory, and others, have reported multiple radioactive peaks in the size exclusion high performance liquid chromatographic (HPLC) analysis of 99mTc-labeled peptides. In the case of one 99mTc-MAG3-labeled peptide studied in this laboratory, human neutrophil elastase inhibitor, all five radioactive peaks were shown to be due to active peptide rather than radiocontaminants. By a variety of experiments, the nature of these peaks have now been examined. A high molecular weight UV peak could be generated by heating the MAG3 coupled, but not the native, peptide. Furthermore, this UV peak did not appear upon heating the peptide if the sulfur within the MAG3 chelator was replaced with oxygen. This peak may therefore be due to polymers resulting from intermolecular disulfide bond formation between sulfurs in the MAG3 chelate and the peptide. Several peaks with apparent lower molecular weights were absent on analysis with a different size exclusion column with superior resolution in their molecular weight range. More importantly, they were also absent on analysis by SDS polyacrylamide gel electrophoresis. These "low" molecular weight radioactive peaks may therefore be due to interactions between the 99mTc-MAG3 chelate and the peptide which produce multiple molecular configurations of identical molecular weight but differing in shape, charge, isomerism or lipophilicity such that they are resolved under the conditions of certain analyses. In support of this possibility, lengthening the linker between MAG3 and the peptide reduced the number of radioactive peaks, while encouraging the interaction by replacing MAG3 with the shorter MAG2 seemed to increase the number of radioactive peaks. Finally, that the three "low" molecular weight radioactive peaks reappeared when a single peak fraction was reanalyzed suggests that the species responsible are in rapid equilibrium. One conclusion from this investigation is that the appearance of a single peak by any HPLC analysis offers no assurance that multiple peaks would not appear on alternative HPLC analyses. Evidence that each species is due to radiolabeled active peptide and not to radiocontaminants is therefore potentially more important than evidence of a single peak.
Chelating Agents↗
Signal transduction, cell cycle regulatory, and anti-apoptotic pathways regulated by IL-3 in hematopoietic cells: possible sites for intervention with anti-neoplastic drugs.
Over the past decade, there has been an exponential increase in our knowledge of how cytokines regulate signal transduction, cell cycle progression, differentiation and apoptosis. Research has focused on different biochemical and genetic aspects of these processes. Initially, cytokines were identified by clonogenic assays and purified by biochemical techniques. This soon led to the molecular cloning of the genes encoding the cytokines and their cognate receptors. Determining the structure and regulation of these genes in normal and malignant hematopoietic cells has furthered our understanding of neoplastic transformation. Furthermore, this has allowed the design of modified cytokines which are able to stimulate multiple receptors and be more effective in stimulating the repopulation of hematopoietic cells after myelosuppressive chemotherapy. The mechanisms by which cytokines transduce their regulatory signals have been evaluated by identifying the involvement of specific protein kinase cascades and their downstream transcription factor targets. The effects of cytokines on cell cycle regulatory molecules, which either promote or arrest cell cycle progression, have been more recently examined. In addition, the mechanisms by which cytokines regulate apoptotic proteins, which mediate survival vs death, are being elucidated. Identification and characterization of these complex, interconnected pathways has expanded our knowledge of leukemogenesis substantially. This information has the potential to guide the development of therapeutic drugs designed to target key intermediates in these pathways and effectively treat patients with leukemias and lymphomas. This review focuses on the current understanding of how hematopoietic cytokines such as IL-3, as well as its cognate receptor, are expressed and the mechanisms by which they transmit their growth regulatory signals. The effects of aberrant regulation of these molecules on signal transduction, cell cycle regulatory and apoptotic pathways in transformed hematopoietic cells are discussed. Finally, anti-neoplastic drugs that target crucial constituents in these pathways are evaluated.
Animals↗
Multicenter evaluation of implantable cardioverter defibrillator testing after implant: the Post Implant Testing Study (PITS).
To reassess the function of the implantable cardioverter defibrillator (ICD) many electrophysiology centers perform a second test after the initial test at implant. A prospective multicenter study evaluated the necessity and yield of routine postimplant defibrillator testing. The results of 843 postimplant defibrillator tests were collected from 31 centers. The 764 routine tests in which ventricular fibrillation was successfully induced were analyzed. Variables examined included patient age, presenting arrhythmia, underlying heart disease, left ventricular ejection fraction, defibrillator age, make and model of ICD, electrode system, defibrillation threshold, polarity, and waveform. The overall failure rate was 3.1% (24/764). Units tested later than 365 days after implant tended to have a higher failure rate than those tested within the first month or the next eleven months (6.5%, 3.0%, 2.3%, respectively, P = 0.374). The failure rate was higher in patients with left ventricular ejection fraction < 40% than those with higher ejection fractions (3.8% vs 2.0%, P = 0.167). These trends did not reach statistical significance. No other baseline characteristic was associated with higher failure rates. Routine testing of ICDs reveals an overall failure rate of 3.1%. While the rate was low, defibrillator failure places the patient at high risk for sudden cardiac death. As any failure in this population is associated with a high risk of sudden cardiac death, routine defibrillator testing may be justified.
Adolescent↗
Dynamic confocal imaging of interphase and mitotic microtubules in the fission yeast, S. pombe.
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Interphase↗
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↗
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↗
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↗
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↗
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↗
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↗
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↗
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↗
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↗
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↗