Biomedical subjects

C Wu

Publications and source records attributed to C Wu.

At least 19 recordsLinked to original sources

PubMed1992

Rapid changes in ascorbate and dopamine release in rat nucleus accumbens after intracerebroventricular administration of NMDA.

In vivo voltammetry at electrochemically pretreated carbon fibre electrodes was used to investigate the effect of intracerebroventricular (i.c.v.) administration of N-methyl-D-aspartic acid (NMDA) on neuronal activity in rat nucleus accumbens. Infusion of a low dose of NMDA (1 nmol) was followed a few minutes later by rapid changes in both Peak 1 and Peak 2 heights indicating large but short-lived increases in the extracellular concentrations of ascorbate and catecholamines, respectively. These responses did not seem to be dependent on the dose infused since infusion of NMDA for a longer time period neither changed the amplitude nor the time-course of these effects. The increase in Peak 2 height was resistant to pargyline pretreatment indicating that this response mainly reflected the release of dopamine. The administration of NMDA was followed by behavioural activation in the animals but not convulsions. Co-administration of the competitive NMDA receptor antagonist, CPP (1 nmol), completely blocked these effects while the acetylcholine receptor antagonist, atropine (1.5 nmol), and the GABA receptor antagonist, picrotoxin (1 nmol), failed in this respect. The phenomenon spreading depression is discussed as a possible explanation of these results.

Animals↗

PubMed1992

IL-4 induces conformational change of CD20 antigen via a protein kinase C-independent pathway. Antagonistic effect of anti-CD40 monoclonal antibody.

The CD20 molecule is a unique phosphoprotein exclusively expressed on B cells during most stages of B cell ontogeny. We here report that rIL-4 down-regulates the expression of CD20 with anti-Leu-16 mAb (clone L27) on both unstimulated and anti-mu preactivated normal and leukemic B cells. None of the other recombinant lymphokines tested (IL-1, IL-2, IL-3, IL-6, IFN-alpha, and IFN-gamma, granulocyte/macrophage-CSF, transforming growth factor-beta, TNF-alpha, and lymphotoxin) decreased CD20 expression. Incubation of unstimulated or anti-mu preactivated B cells with IL-4 did not affect the steady state CD20 mRNA, suggesting that IL-4 exerted its effect mainly at a nontranscriptional level. Hence, IL-4 selectively down-regulates the CD20 epitope recognized by clone L27 without affecting seven other different epitopes, indicating that IL-4 acts by modifying the conformation of the CD20 molecule rather than by inhibiting its production or inducing its internalization. IL-4 most likely utilizes a protein kinase C-independent signal transduction pathway to modify CD20 molecule inasmuch as staurosporine, an inhibitor of protein kinase C, antagonizes phorbol esters (PMA) but not IL-4-induced CD20 down-regulation. In contrast, anti-CD40 mAb reverses the IL-4 but not the PMA inhibitory effect on CD20 expression. Given that CD20 may be part of a Ca2+ ion channel and plays a role in B cell activation and proliferation, it is proposed that the ability of anti-CD40 mAb to maintain the CD20 molecule in a given epitopic configuration on IL-4-stimulated B cells may be related to the long term proliferation of normal B cells that are strictly dependent on the presence of IL-4 and cross-linked anti-CD40 mAb for their continuous growth.

Antibodies, Monoclonal↗

PubMed1992

Protein classification artificial neural system.

A neural network classification method is developed as an alternative approach to the large database search/organization problem. The system, termed Protein Classification Artificial Neural System (ProCANS), has been implemented on a Cray supercomputer for rapid superfamily classification of unknown proteins based on the information content of the neural interconnections. The system employs an n-gram hashing function that is similar to the k-tuple method for sequence encoding. A collection of modular back-propagation networks is used to store the large amount of sequence patterns. The system has been trained and tested with the first 2,148 of the 8,309 entries of the annotated Protein Identification Resource protein sequence database (release 29). The entries included the electron transfer proteins and the six enzyme groups (oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases), with a total of 620 superfamilies. After a total training time of seven Cray central processing unit (CPU) hours, the system has reached a predictive accuracy of 90%. The classification is fast (i.e., 0.1 Cray CPU second per sequence), as it only involves a forward-feeding through the networks. The classification time on a full-scale system embedded with all known superfamilies is estimated to be within 1 CPU second. Although the training time will grow linearly with the number of entries, the classification time is expected to remain low even if there is a 10-100-fold increase of sequence entries. The neural database, which consists of a set of weight matrices of the networks, together with the ProCANS software, can be ported to other computers and made available to the genome community. The rapid and accurate superfamily classification would be valuable to the organization of protein sequence databases and to the gene recognition in large sequencing projects.

Computers, Mainframe↗

PubMed1992

Investigations of N-linked macrocycles for 111In and 90Y labeling of proteins.

To simplify the synthesis of macrocyclic chelators, commercially available macrocyclic amines were condensed with halogenated acetic acid to prepare the five chelators 12N4 (DOTA), 14N4 (TETA), 15N4, 9N3 and 12N3. Only 12N4 and 9N3 showed efficient labeling of the free chelator with 111In and 90Y. Serum stability studies at 37 degrees C with In-labeled DTPA, 12N4 and 9N3 showed no loss of label over 2 days whereas, with 90Y, only 12N4 showed stabilities comparable to DTPA. The 12N4 chelator was derivatized by attaching biotin on one N-acetate group to stimulate the attachment to protein. The serum stability for both 111In and 90Y was identical to that of biotin derivatized DTPA and lower than that of the free chelators. Biodistribution studies in normal mice of a model protein (avidin) labeled with 90Y via biotinylated 12N4 and biotinylated DTPA showed identical distribution at 1 day except in bone where the %ID/g for the macrocyclic-conjugated protein (3.4 +/- 0.5, N = 8) was significantly (P less than 0.001) lower than that of the DTPA-conjugated protein (9.4 +/- 0.9, N = 7). In conclusion, macrocycles may be readily synthesized from the macrocyclic amines and several show useful stabilities with In and Y. When N-linked to a protein, the Y biodistribution was found to be superior to that of the corresponding DTPA-coupled protein.

Amines↗

PubMed1992

Assembly of extracellular matrix.

A great challenge in understanding how different extracellular matrices assemble is to sort through the vast number of possible interactions between and among matrix molecules. The most profound insights are likely to come from patients with defined defects of matrix molecules and the use of transgenic mice or other experimental technologies that mimic the complexity of the human system.

Amino Acid Sequence↗

PubMed1992

The stability of 99Tcm directly labelled to an Fab' antibody via stannous ion and mercaptoethanol reduction.

The anti-CEA FO23C5 F(ab')2 antibody was directly radiolabelled with 99mTcm by two methods (stannous ion and mercaptoethanol reduction) and compared in vitro and in vivo for label stability. By both methods, reduction of the F(ab')2 fragment produced primarily Fab' fragments. By both methods, the label was stable to 99Tcm-pertechnetate formation in vitro. Analysis by high performance liquid chromatography (HPLC) of serum, urine, kidney and liver homogenates from mice injected with 99Tcm-antibodies by both methods consistently showed a prominent radiolabelled peak with an estimated molecular weight of about 300 daltons. An identical peak was observed in the analysis of patient samples in a related investigation from this laboratory. Cysteine was radiolabelled with reduced 99Tcm and analysed by HPLC and thin layer chromatography (TLC); one of the 99Tcm-cysteine species so produced showed the same chromatographic behaviour as that of the 300 dalton species. In conclusion, the FO23C5 and other antibodies are stably labelled with 99Tcm via either stannous ion or mercaptoethanol reduction. In mice and in patients, the labelled proteins are either catabolized or, more likely, the 99Tcm label is transchelated such that the label is present on several low molecular weight species, the most prominent of which is postulated to be 99Tcm-cysteine.

Animals↗

PubMed1992

Cell-free system for assembly of transcriptionally repressed chromatin from Drosophila embryos.

We describe a cell-free system, derived from preblastoderm Drosophila embryos, for the efficient assembly of cloned DNA into chromatin. The chromatin assembly system utilizes endogenous core histones and assembly factors and yields long arrays of regularly spaced nucleosomes with a repeat length of 180 bp. The assembly system is also capable of complementary-strand DNA synthesis accompanied by rapid nucleosome formation when the starting template is single-stranded circular DNA. Chromatin assembled with the preblastoderm embryo extract is naturally deficient in histone H1, but exogenous H1 can be incorporated during nucleosome assembly in vitro. Regular spacing of nucleosomes with or without histone H1 is sufficient to maximally repress transcription from hsp70 and fushi tarazu gene promoters. The Drosophila assembly system should be particularly useful for in vitro studies of chromatin assembly during DNA synthesis and for elucidating the action of transcription factors in the context of native chromatin.

Animals↗

PubMed1992

Regulated tissue- and cell-specific expression of the human renin gene in transgenic mice.

Transgenic mice containing the human renin gene were constructed with the aim of examining the tissue- and cell-specific expression of human renin. The human renin transgene used consisted of a genomic sequence extending approximately 900 bp upstream and 400 bp downstream of the coding region and included all exon and intron sequences. Two assays were developed to differentiate human renin transcripts from endogenous mouse renin transcripts at the whole-tissue level. High level human renin expression was evident in the kidney, adrenal gland, ovary, testis, lung, and adipose tissue of all four transgenic lines examined. Human renin mRNA could also be detected at lower levels in the submandibular gland and heart of two different individual lines. No expression was evident in the liver or brain of any line tested. In situ hybridization revealed the human renin mRNA to be localized and exquisitely restricted to renal juxtaglomerular cells. Treatment of transgenic mice with captopril resulted in an increase in the accumulation of renal renin mRNAs derived from both the mouse and human renin genes. Plasma renin activity assays using synthetic human renin substrate clearly demonstrated the elaboration of active human renin into the systemic circulation of transgenic mice. These data strongly suggest that the human renin transgene exhibits both tissue- and cell-specific expression in transgenic mice. Its expression is entrained to the same regulatory signals as the endogenous renin gene in kidney, and active human renin is released into the plasma of the transgenic mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

[Solid phase clean-up and determination of vesnarinone in plasma by high performance liquid chromatography].

Vesnarinone, a new positive inotropic agent, was synthesized by Tominaga et al of the Otsuka Pharmaceutical Co. Ltd. Institute in 1982. Determination of Vesnarinone plasma level in dogs by reversed-phase HPLC was developed and presented in this paper. A plasma sample preparation was carried out by solid phase cleaned-up through neutral aluminum column (10 x 1 cm I.D.) eluting with methanol. The chromatography consisted of a slim-pack CLC ODS column (150 x 6.0 mm I.D.) with methanol: 1 mmol/L HAc (55:45) as the mobile phase at a flow rate of 0.8 ml/min. Spectrophotometric detection was at 271 nm, and column temperature was at 25 degrees C. Using 3,4-dihydro-6-(4-(4-methoxybenzoyl)-1-piperazinyl)-2 (1H) quinolinone as an internal standard. The cleaned-up procedure is simple, rapid and satisfactory. The detection limit of vesnarinone was 0.5 ng/ml (R/N, 3:1). The calibration curve was liner (r = 0.99998) in the concentration range of 5-500 ng/10 microliters. Within-day and day-to-day precisions (CV%) were 1.56 and 1.98. The recoveries obtained from cleaned-up and spiked plasma samples were up to 86.52 +/- 2.77% and 96.26 +/- 5.46%, respectively.

Animals↗

PubMed1992

[Determination of bendazac lysine by RP-HPLC].

Bendazac (BD) was separated on a CLC-ODS column, 150 x 6 mm id (Shimadzu), using methanol acetic acid 0.1 mol/L (67:33) as the mobile phase. The flow rate was 1.0 ml/min and the detection wavelength, 254 nm. The detection limit of BD was 0.1 ng (R/N, 3:1), which was determined by the peak area measurement using an external standard method. The calibration curve was linear in the range of 5-10 micrograms (r = 0. 9998). The average recovery of BDL was 99.46 +/- 0.59% (n = 9). This method has been applied satisfactorily to the determination of BDL samples.

Chromatography, High Pressure Liquid↗

PubMed1992

[The genetic analysis of long-term index selection--a simulation experiment].

The genetic effects of long-term index selection were investigated by means of Monte Carlo Method. The response and the effect on genetic structure of population to selection using four methods, that is theoretical index, emperical index, restricted index and single-trait selection, were compared. Results obtained from 50 generations demonstrated that much greater response was achieved in large populations, and selection in small ones led to a decrease in selection limit. Genetic correlation played an important role on response in small populations. Emperical index selection was more effective than theoretical index, restricted index had less response in the early period of selection but greater response in later period and also had less effect on genetic structure of population even if the genetic correlation was intense. Meanwhile, index selection could cause decrease in additive genetic variance and beritabilities while genetic correlation changed depending on the coefficients of index formulae. The suggestion is that emperical index or restricted index should be used for long-term selection for multi-traits.

Computer Simulation↗

PubMed1991

Stress-induced oligomerization and chromosomal relocalization of heat-shock factor.

The induction of heat-shock transcription factor (HSF) binding to DNA is accomplished by a heat-induced oligomerization. The transition to the induced state is accompanied by a chromosomal redistribution of HSF to the heat-shock puff sites. Over 150 additional chromosomal sites also accumulate HSF, including developmental loci that are repressed during heat shock. These findings suggest an unforeseen role for HSF as a repressor of normal gene activity during heat stress.

Animals↗

PubMed1991

Potential role of entactin in hemostasis. Specific interaction of entactin with fibrinogen A alpha and B beta chains.

The interactions between the endothelial basement membrane, platelets, and coagulation factors play essential roles in hemostasis. Entactin is an integral and ubiquitous component of the basement membrane. Experiments were designed to study the interactions between entactin and blood coagulation factors. We have demonstrated, for the first time, that entactin interacts with fibrinogen in a specific manner. Entactin binding sites have been localized to the A alpha and B beta chains of fibrinogen. The binding of entactin to either the A alpha chain or the B beta chain was divalent cation-independent. The binding of 35S-labeled entactin to the immobilized fibrinogen A alpha or B beta chain was concentration-dependent and saturable and could be inhibited by unlabeled entactin, soluble fibrinogen, anti-entactin antiserum, or anti-fibrinogen antiserum. In addition, we have provided evidence that entactin can be cross-linked to itself, and probably also to fibrin(ogen), by transglutaminase. These novel properties, together with its cell binding, chemotactic, and phagocytic promoting activities and ubiquitous distribution in basement membranes, suggest that entactin may play important roles in hemostasis and wound healing.

Chromatography, Affinity↗

PubMed1991

Entactin forms a complex with fibronectin and co-localizes in the extracellular matrix of the embryonal carcinoma-derived 4CQ cell line.

A novel extracellular matrix that consists of a complex of fibronectin and entactin was synthesized by the embryonal carcinoma-derived cell line 4CQ. The matrix was devoid of laminin. High steady state levels of the messenger RNAs for fibronectin, entactin, and the B2 chain of laminin were detected in these cells. Laminin B1 message was several fold lower while laminin A chain message was undetectable. In contrast, in the sister embryonal carcinoma-derived cell M1536-B3 there were high levels of message for all three chains of laminin and for entactin but very little for fibronectin. The data suggest that the synthesis and deposition of laminin and fibronectin are inversely related. The direct binding of entactin and fibronectin was also demonstrated by affinity column chromatography and solid phase assay.

Animals↗

PubMed1991

Molecular cloning and expression of a human heat shock factor, HSF1.

Human cells respond to heat stress by inducing the binding of a preexisting transcriptional activator (heat shock factor, HSF) to DNA. We have isolated recombinant DNA clones for a human HSF (HSF1) by screening cDNA libraries with a human cDNA fragment. The human HSF1 probe was produced by the PCR with primers deduced from conserved amino acids in the Drosophila and yeast HSF sequences. The human HSF1 mRNA is constitutively expressed in HeLa cells under nonshock conditions and encodes a protein with four conserved leucine zipper motifs. Like its counterpart in Drosophila, human HSF1 produced in Escherichia coli in the absence of heat shock is active as a DNA binding transcription factor, suggesting that the intrinsic activity of HSF is under negative control in human cells. Surprisingly, an independently isolated human HSF clone, HSF2, is related to but significantly different from HSF1 [Schuetz, T. J., Gallo, G. J., Sheldon, L., Tempst, P. & Kingston, R. E. (1991) Proc. Natl. Acad. Sci. USA 88, 6911-6915].

Amino Acid Sequence↗

PubMed1991

Heat shock-regulated transcription in vitro from a reconstituted chromatin template.

To investigate the mechanisms of transcriptional regulation of Drosophila heat shock genes we studied the activity of a heat shock promoter in vitro after reconstitution into chromatin. Increasing the duration of nucleosome assembly progressively inactivated a plasmid template when it was transcribed with extracts of either unshocked or heat-shocked Drosophila embryos, despite induction of the transcriptional activator heat shock factor. Addition of the general transcription factor IID (TFIID) before nucleosome assembly did not significantly relieve nucleosomal inhibition, but TFIID potentiated the promoter to be responsive to activation by heat shock factor in the heat shock transcription extract. The potentiation by TFIID could be related to the nucleosome-free, hypersensitive state of heat shock promoters previously observed in vivo before heat shock induction and may be necessitated by the need to expedite activation of heat shock genes in response to environmental stress.

Animals↗

PubMed1991

FTZ-F1, a steroid hormone receptor-like protein implicated in the activation of fushi tarazu.

The Drosophila homeobox segmentation gene fushi tarazu (ftz) is expressed in a seven-stripe pattern during early embryogenesis. This characteristic pattern is largely specified by the zebra element located immediately upstream of the ftz transcriptional start site. The FTZ-F1 protein, one of multiple DNA binding factors that interacts with the zebra element, is implicated in the activation of ftz transcription, especially in stripes 1, 2, 3, and 6. An FTZ-F1 complementary DNA has been cloned by recognition site screening of a Drosophila expression library. The identity of the FTZ-F1 complementary DNA clone was confirmed by immunological cross-reaction with antibodies to FTZ-F1 and by sequence analysis of peptides from purified FTZ-F1 protein. The predicted amino acid sequence of FTZ-F1 revealed that the protein is a member of the nuclear hormone receptor superfamily. This finding raises the possibility that a hormonal ligand affects the expression of a homeobox segmentation gene early in embryonic development.

Amino Acid Sequence↗

PubMed1991

Repression of the Drosophila fushi tarazu (ftz) segmentation gene.

The striped expression of the Drosophila segmentation gene fushi tarazu in alternate parasegments of the early embryo is controlled by the 740 bp zebra element. Among multiple protein factors that bind to the zebra element, FTZ-F2 behaves as a transcriptional repressor of ftz. Point mutations in the zebra element which disrupt FTZ-F2 binding to DNA cause ectopic expression of zebra-lacZ activity in transformed embryos. The mutant constructs are expressed from the zygotic genome in preblastoderm embryos as early as the third nuclear division cycle. This unprecedented early transcription suggests that ftz requires active repression during initial nuclear division cycles, a novel type of embryonic gene regulation. A putative FTZ-F2 cDNA clone isolated by recognition site screening of an expression library was found to be identical in sequence with the zinc finger protein tramtrack (Harrison and Travers, 1990).

Animals↗