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Biomedical subjects

P Chang

Publications and source records attributed to P Chang.

At least 19 recordsLinked to original sources
PubMed2000

Phase I study in advanced cancer patients of a diversified prime-and-boost vaccination protocol using recombinant vaccinia virus and recombinant nonreplicating avipox virus to elicit anti-carcinoembryonic antigen immune responses.

PURPOSE: This trial sought to determine, for the first time, the validity in human vaccinations of using two different recombinant vaccines in diversified prime-and-boost regimens to enhance T-cell responses to a tumor antigen. PATIENTS AND METHODS: Eighteen patients with advanced tumors expressing carcinoembryonic antigen (CEA) were randomized to receive either recombinant vaccinia (rV)-CEA followed by three avipox-CEA vaccinations, or avipox-CEA (three times) followed by one rV-CEA vaccination. Subsequent vaccinations in both cohorts were with avipox-CEA. Immunologic monitoring was performed using a CEA peptide and the enzyme-linked immunospot assay for interferon gamma production. RESULTS: rV-CEA followed by avipox-CEA was superior to the reverse order in the generation of CEA-specific T-cell responses. Further increases in CEA-specific T-cell precursors were seen when local granulocyte-macrophage colony-stimulating factor (GM-CSF) and low-dose interleukin (IL)-2 were given with subsequent vaccinations. The treatment was extremely well tolerated. Limited clinical activity was seen using vaccines alone in this patient population. Antibody production against CEA was also observed in some of the treated patients. CONCLUSION: rV-CEA was more effective in its role as a primer of the immune system; avipox-CEA could be given up to eight times with continued increases in CEA T-cell precursors. Future trials should use rV-CEA first followed by avipox-CEA. Vaccines specific to CEA are able to generate CEA-specific T-cell responses in patients without significant toxicity. T-cell responses using vaccines alone may be inadequate to generate significant anticancer objective responses in patients with advanced disease. Cytokines such as GM-CSF and IL-2 may play a key role in generating such responses.

Adult↗
PubMed2000

Cloning of a sugar utilization gene cluster in Aspergillus parasiticus.

At one end of the 70 kb aflatoxin biosynthetic pathway gene cluster in Aspergillus parasiticus and Aspergillus flavus reported earlier, we have cloned a group of four genes that constitute a well-defined gene cluster related to sugar utilization in A. parasiticus: (1) sugR, (2) hxtA, (3) glcA and (4) nadA. No similar well-defined sugar gene cluster has been reported so far in any other related Aspergillus species such as A. flavus, A. nidulans, A. sojae, A. niger, A. oryzae and A. fumigatus. The expression of the hxtA gene, encoding a hexose transporter protein, was found to be concurrent with the aflatoxin pathway cluster genes, in aflatoxin-conducive medium. This is significant since a close linkage between the two gene clusters could potentially explain the induction of aflatoxin biosynthesis by simple sugars such as glucose or sucrose.

Aflatoxins↗
PubMed2000

Nuclei and microtubule asters stimulate maturation/M phase promoting factor (MPF) activation in Xenopus eggs and egg cytoplasmic extracts.

Although maturation/M phase promoting factor (MPF) can activate autonomously in Xenopus egg cytoplasm, indirect evidence suggests that nuclei and centrosomes may focus activation within the cell. We have dissected the contribution of these structures to MPF activation in fertilized eggs and in egg fragments containing different combinations of nuclei, centrosomes, and microtubules by following the behavior of Cdc2 (the kinase component of MPF), the regulatory subunit cyclin B, and the activating phosphatase Cdc25. The absence of the entire nucleus-centrosome complex resulted in a marked delay in MPF activation, whereas the absence of the centrosome alone caused a lesser delay. Nocodazole treatment to depolymerize microtubules through first interphase had an effect equivalent to removing the centrosome. Furthermore, microinjection of isolated centrosomes into anucleate eggs promoted MPF activation and advanced the onset of surface contraction waves, which are close indicators of MPF activation and could be triggered by ectopic MPF injection. Finally, we were able to demonstrate stimulation of MPF activation by the nucleus-centriole complex in vitro, as low concentrations of isolated sperm nuclei advanced MPF activation in cycling cytoplasmic extracts. Together these results indicate that nuclei and microtubule asters can independently stimulate MPF activation and that they cooperate to enhance activation locally.

Animals↗
PubMed2000

Identification of a region of Escherichia coli DnaB required for functional interaction with DnaG at the replication fork.

The fundamental activities of the replicative primosomes of Escherichia coli are provided by DnaB, the replication fork DNA helicase, and DnaG, the Okazaki fragment primase. As we have demonstrated previously, DnaG is recruited to the replication fork via a transient protein-protein interaction with DnaB. Here, using site-directed amino acid mutagenesis, we have defined the region on DnaB required for this protein-protein interaction. Mutations in this region of DnaB affect the DnaB-DnaG interaction during both general priming-directed and phiX174 complementary strand DNA synthesis, as well as at replication forks reconstituted in rolling circle DNA replication reactions. The behavior of the purified mutant DnaB proteins in the various replication systems suggests that access to the DnaG binding pocket on DnaB may be restricted at the replication fork.

Amino Acid Substitution↗
PubMed2000

The basic helix-loop-helix transcription factor capsulin controls spleen organogenesis.

Formation of numerous internal organs involves reciprocal epithelial-mesenchymal signaling and subsequent patterning and growth of the organ primordium. Capsulin is a basic helix-loop-helix transcription factor expressed in mesenchymal cells that encapsulate the epithelial primordia of internal organs, including the kidney and lung, as well as the epicardium, which gives rise to the coronary arteries. Capsulin is also expressed in the mesothelium that gives rise to the spleen. We demonstrate that mice homozygous for a capsulin null mutation fail to form a spleen. The homeobox genes Hox11 and Bapx1, shown previously to be essential regulators of spleen organogenesis, and a lacZ reporter introduced into the capsulin locus, were expressed in the early splenic primordium, derived from the splanchnic mesoderm, of homozygous mutant embryos. However, this primordium failed to develop beyond an initial group of precursor cells and underwent rapid apoptosis. The phenotype of capsulin mutant mice demonstrates that capsulin acts within a subpopulation of splanchnic mesodermal cells to control an essential early step in spleen organogenesis that is likely to represent a point of regulatory convergence of the capsulin, Hox11, and Bapx1 genes.

Animals↗
PubMed2000

Epidermal growth factor receptor activation of calpain is required for fibroblast motility and occurs via an ERK/MAP kinase signaling pathway.

To become migratory, cells must reorganize their connections to the substratum, and during locomotion they must break rear attachments. The molecular and biochemical mechanisms underlying these biophysical processes are unknown. Recent studies have implicated both extracellular signal-regulated kinase/mitogen-activated protein (ERK/MAP) kinase and calpain (EC 3.4.22.17) in these processes, but it is uncertain whether these are two distinct pathways acting on different modes of motility. We report that cell deadhesion involved in epidermal growth factor (EGF) receptor-mediated fibroblast motility requires activation of M-calpain downstream of ERK/MAP kinase signaling. NR6 fibroblasts expressing full-length wild type epidermal growth factor receptor required both calpain and ERK activation, as demonstrated by pharmacological inhibitors (calpeptin and calpain inhibitor I and PD98059, respectively) for EGF-induced deadhesion and motility. EGF induced rapid activation of calpain that was preventable by molecular inhibition of the Ras-Raf-MEK but not phospholipase Cgamma signaling pathway, and calpain was stimulated by transfection of constitutively active MEK. Enhanced calpain activity was not mirrored by increased calpain protein levels or decreased levels of its endogenous inhibitor calpastatin. The link between ERK/MAP kinase signaling and cell motility required the M-isoform of calpain (calpain II), as determined by specific antisense-mediated down-regulation. These data promote a previously undescribed signaling pathway of ERK/MAP kinases activating calpain to destabilize cell-substratum adhesions in response to EGF stimulation.

Animals↗
PubMed2000

Molecular heterogeneity of protein kinase C expression in human ventricle.

OBJECTIVE: Although activation of protein kinase C (PKC) modulates the function of normal cardiac myocytes and likely plays a role in the pathogenesis of cardiomyopathic disease states, the molecular basis of PKC expression in human ventricle has not been examined in detail. METHODS: We have performed Western analysis and immunohistochemistry on explanted human cardiac tissue from nondiseased and diseased specimens using isoform-specific antibodies directed against all known PKC isozymes. RESULTS: In homogenates from left and right ventricle, all isoforms except PKC-gamma and theta were detected by immunoblotting, with confirmation using a second antibody directed against a different epitope when possible. For PKC-betaII, delta, and epsilon, data indicated that these isoforms were variably phosphorylated in vivo, resulting in multiple bands during immunoblotting. Because of potential antibody cross-reactivity, reverse transcriptase polymerase chain reaction (RT-PCR) was performed which confirmed expression of PKC-alpha, betaI, and zeta. Immunohistochemistry demonstrated that all isoforms detected in ventricular homogenate by Western analysis could be localized to cardiac myocytes. From a methodologic standpoint, significant degradation of PKC isoforms could be demonstrated when samples were either frozen or allowed to remain at room temperature, compared to immediate subcellular fractionation. CONCLUSIONS: These findings indicate that the PKC expression in human ventricular myocytes is remarkably diverse, with multiple conventional, novel, and atypical isoforms present, and highlight the importance of sample preparation in comparative studies of PKC isoform expression.

Adolescent↗
PubMed2000

Characterization of bovine collagens using capillary electrophoresis.

A capillary electrophoresis method was developed and characterized for analyzing the spectrum of collagen subspecies in collagen preparations. The Bio-Rad CE-SDS protein kit was used for the dynamic sieving separation of collagen subspecies in this CE method (DSCE). The optimized method utilized a 36 cm (or 24 cm) x 50 microm uncoated capillary, electrophoretic injection at 10 kV for 10 s, a run voltage of 15 kV, a capillary temperature of 20 degrees C, and UV detection at 220 nm. A preliminary validation of the method was performed. The assay had good repeatability (RSDs for peaks were 15%), and responses were linear for assay solutions with collagen concentrations from 0.125 to 1.25 mg/ml. The DSCE electropherogram of bovine skin collagen provided a profile of subspecies similar in number and relative abundance to that generated by scanning of Coomassie-stained SDS-PAGE gels.

Animals↗
PubMed2000

Protocol for quantitative and qualitative analyses of the in vitro aggregation of synthetic beta-amyloid. A method applicable to the identification of substances that may have therapeutic efficacy for Alzheimer's disease.

Biochemical studies of beta-amyloid (Abeta) aggregation have been hampered by the lack of a simple method to examine simultaneously the extent of aggregation and the structural nature of the aggregates. Consequently, often only the extent of aggregation is analyzed and reported. This means that there is often no knowledge of whether the aggregates consist of fibrils, a structure crucial for their neurotoxicity. Here we describe the development of a protocol for quantitatively and qualitatively evaluating the in vitro aggregation of synthetic Abeta. Specifically, a fluorescein derivatized Abeta(1-42) peptide (FITC-Abeta(1-42)) was used as the aggregation material. We found that the fluorescent Abeta peptide aggregated as efficiently as the native ones and the extent of their aggregation could be determined accurately by fluorescence spectrophotometry. Significantly, our approach is also qualitative because it allows a direct structural examination of Abeta fibrils by fluorescence microscopy. In addition, this protocol can also be applied to the identification of substance(s) capable of inhibiting Abeta aggregation and further the assessment of the nature of such inhibition.

Alzheimer Disease↗
PubMed2000

Delta-tubulin and epsilon-tubulin: two new human centrosomal tubulins reveal new aspects of centrosome structure and function.

The centrosome organizes microtubules, which are made up of alpha-tubulin and beta-tubulin, and contains centrosome-bound gamma-tubulin, which is involved in microtubule nucleation. Here we identify two new human tubulins and show that they are associated with the centrosome. One is a homologue of the Chlamydomonas delta-tubulin Uni3, and the other is a new tubulin, which we have named epsilon-tubulin. Localization of delta-tubulin and epsilon-tubulin to the centrosome is independent of microtubules, and the patterns of localization are distinct from each other and from that of gamma-tubulin. Delta-tubulin is found in association with the centrioles, whereas epsilon-tubulin localizes to the pericentriolar material. epsilon-Tubulin exhibits a cell-cycle-specific pattern of localization, first associating with only the older of the centrosomes in a newly duplicated pair and later associating with both centrosomes. epsilon-Tubulin thus distinguishes the old centrosome from the new at the level of the pericentriolar material, indicating that there may be a centrosomal maturation event that is marked by the recruitment of epsilon-tubulin.

Antineoplastic Agents↗
PubMed2000

Magnetic resonance imaging results in patients with central electronystagmography findings.

The objective of this study was to determine the findings on magnetic resonance imaging (MRI) in patients identified as having central vestibular abnormalities on electronystagmography (ENG) testing, to discuss the issue of 'gold standard' in the investigation of central oculo-vestibular system diseases and to present a model for understanding this area. A retrospective review of the case notes of patients (n = 23) found to have central ENG findings at vestibular assessment and for whom MRI scanning data was available was undertaken. Each patient underwent a full ENG evaluation, including gaze, ocular-motor and caloric testing, and MRI. Only seven of the patients with central ENG findings had abnormal MRI scans. Thus, the incidence of the identification of structural abnormality on MRI in patients with central ENG findings is low. These investigations are complementary in the investigation of balance disorder patients.

Brain↗
PubMed2000

Unresectable primary and recurrent head and neck tumors: effect of hyperthermia and carboplatin--preliminary experience.

PURPOSE: To perform a single-arm study to determine the effectiveness of and potential toxic reactions to local hyperthermia and systemic carboplatin (cis-diammine-1,1-cyclobutane dicarboxylate platinum II) for the treatment of advanced or recurrent squamous cell carcinomas of the head and neck. MATERIALS AND METHODS: Eight patients with squamous cell carcinoma of the head and neck and stage IV disease (N2 or N3 neck adenopathy) or recurrent local-regional disease and who were previously and definitively treated were included in the study. Thermochemotherapy was administered every 4 weeks. Recorded end points were tumor response, duration of response, incidence of distant metastases, survival, cause of death, and toxic reactions. RESULTS: One patient had a complete response to therapy, and two had a partial response. Five patients had no response or developed progressive disease during therapy. Six patients died after 4-13 months of progressive disease. Two long-term survivors received radiation therapy; one also underwent surgical resection for residual neck disease. Each thermochemotherapeutic session was well tolerated, with minimal discomfort. Toxic reactions included hypotension, vomiting, hyponatremia, anemia, thrombocytopenia, and infection at the site of administration. There were no life-threatening toxic reactions. CONCLUSION: The combined use of hyperthermia and carboplatin shows potential in the management of unresectable head and neck tumors and is safe and well tolerated. Further studies on thermochemotherapy are warranted to assess its potential.

Aged↗
PubMed2000

Complex enterocutaneous fistula: closure with rectus abdominis muscle flap.

Most enterocutaneous fistulas are caused by complications of abdominal surgery that may result from anastomotic failure, poor blood supply, or iatrogenic bowel injuries. Mortality rates are high when associated sepsis and malnutrition are uncontrolled. Fistulas that occur late and those that recur spontaneously present more difficulty and may close spontaneously in less than 30% of cases. Mortality rates in patients with complex enterocutaneous fistulas may reach 60% to 80%. When traditional conservative surgeries of fistulous tract excision, bowel mobilization, and resection with primary end-to-end anastomosis fail, a more aggressive approach is required. The rectus abdominis muscle flap has been extensively studied and used in a wide variety of abdominal, vaginal, and perineal repairs. We report successful closure of complex enterocutaneous fistulas with a rectus abdominis muscle flap in a complicated case.

Cutaneous Fistula↗
PubMed2000

Renal malformations in children with Turner's syndrome.

Between 1988 and 1999, renal sonography and intravenous urography were performed to detect renal malformations in 54 patients with Turner's syndrome (TS). The mean age of these patients at diagnosis of TS was 9.2 +/- 4.6 years. Renal malformations were detected in 21 patients by intravenous urography and there was no significant difference in the frequency of renal malformations among different karyotype groups. Horseshoe kidney was the most common renal malformation, followed by duplex kidney. Fifteen of 21 renal malformations were not detected by renal sonography. We conclude that these TS patients had a high frequency of renal malformations, and that the detection rate of horseshoe kidney and duplex kidney by renal sonography was not satisfactory. Although renal sonography alone can be used to detect more severe renal malformations that may need further management, it may underestimate the frequency of renal malformation in children with TS.

Adolescent↗
PubMed1999

Organisation of Xenopus oocyte and egg cortices.

The division of the Xenopus oocyte cortex into structurally and functionally distinct "animal" and "vegetal" regions during oogenesis provides the basis of the organisation of the early embryo. The vegetal region of the cortex accumulates specific maternal mRNAs that specify the development of the endoderm and mesoderm, as well as functionally-defined "determinants" of dorso-anterior development, and recognisable "germ plasm" determinants that segregate into primary germ cells. These localised elements on the vegetal cortex underlie both the primary animal-vegetal polarity of the egg and the organisation of the developing embryo. The animal cortex meanwhile becomes specialised for the events associated with fertilisation: sperm entry, calcium release into the cytoplasm, cortical granule exocytosis, and polarised cortical contraction. Cortical and subcortical reorganisations associated with meiotic maturation, fertilisation, cortical rotation, and the first mitotic cleavage divisions redistribute the vegetal cortical determinants, contributing to the specification of dorso-anterior axis and segregation of the germ line. In this article we consider what is known about the changing organisation of the oocyte and egg cortex in relation to the mechanisms of determinant localisation, anchorage, and redistribution, and show novel ultrastructural views of cortices isolated at different stages and processed by the rapid-freeze deep-etch method. Cortical organisation involves interactions between the different cytoskeletal filament systems and internal membranes. Associated proteins and cytoplasmic signals probably modulate these interactions in stage-specific ways, leaving much to be understood.

Animals↗
PubMed1999

Mutant huntingtin expression in clonal striatal cells: dissociation of inclusion formation and neuronal survival by caspase inhibition.

Neuronal intranuclear inclusions are found in the brains of patients with Huntington's disease and form from the polyglutamine-expanded N-terminal region of mutant huntingtin. To explore the properties of inclusions and their involvement in cell death, mouse clonal striatal cells were transiently transfected with truncated and full-length human wild-type and mutant huntingtin cDNAs. Both normal and mutant proteins localized in the cytoplasm, and infrequently, in dispersed and perinuclear vacuoles. Only mutant huntingtin formed nuclear and cytoplasmic inclusions, which increased with polyglutamine expansion and with time after transfection. Nuclear inclusions contained primarily cleaved N-terminal products, whereas cytoplasmic inclusions contained cleaved and larger intact proteins. Cells with wild-type or mutant protein had distinct apoptotic features (membrane blebbing, shrinkage, cellular fragmentation), but those with mutant huntingtin generated the most cell fragments (apoptotic bodies). The caspase inhibitor Z-VAD-FMK significantly increased cell survival but did not diminish nuclear and cytoplasmic inclusions. In contrast, Z-DEVD-FMK significantly reduced nuclear and cytoplasmic inclusions but did not increase survival. A series of N-terminal products was formed from truncated normal and mutant proteins and from full-length mutant huntingtin but not from full-length wild-type huntingtin. One prominent N-terminal product was blocked by Z-VAD-FMK. In summary, the formation of inclusions in clonal striatal cells corresponds to that seen in the HD brain and is separable from events that regulate cell death. N-terminal cleavage may be linked to mutant huntingtin's role in cell death.

Amino Acid Chloromethyl Ketones↗
PubMed1999

Review: emotional and behavioral functioning in phenylketonuria.

OBJECTIVE: To examine 17 studies of the psychological sequelae of early-treated phenylketonuria (PKU) with emphasis on the impact of dietary control on functioning. Two questions are addressed: (1) What is the typical psychological profile associated with PKU? (2) Is emotional and behavioral disturbance more prevalent in PKU-affected individuals compared to appropriate controls? METHOD: Computerized searches of PsycINFO identified studies using behavioral, personality, and diagnostic measures. RESULTS: Findings converge upon a profile including attentional difficulties, depression, anxiety, and low self-esteem. Methodological constraints limit conclusions regarding the nature and severity of observed difficulties. A single study has used comparison groups appropriate for the simultaneous examination of the questions posed (Waisbren and Levy, 1991). CONCLUSIONS: We discuss results using a biopsychosocial framework, addressing the factors and processes that may influence emotional and behavioral functioning in this neurodevelopmental disorder. We outline potential lines of new investigation that address critical methodological factors.

Child↗