Biomedical subjects
E Lee
Publications and source records attributed to E Lee.
Pax-7 immunoreactivity in the post-natal chicken central nervous system.
In this immunocytochemical study on the constitutive expression of Pax-7 protein in the postnatal chicken brain, Pax-7 showed region and cell type specific expression. In the optic tectum, only cells in grey matter showed positive immunoreactivities (IRs), whereas those in the white matters did not show any IRs. In thalamic nuclei and several pontine nuclei, we also localized Pax-7 positive IRs. On the contrary, in the cerebellum, Pax-7 was mainly localized within the Bergmann glia, whereas Purkinje cells did not show any IRs. In double immunolabelling studies, most of the Pax-7 IRs did not originate from neuroglial cells such as oligodendrocytes, microglia or astrocytes, but from neurons, with the exception of Bergmann glia in the cerebellum. The presence of Pax-7 IRs in the adult chicken brain could suggest that Pax-7 might play a role in maintaining normal physiological function in some postnatal chicken brain cells.
High dose rate (192)Ir afterloading brachytherapy for cancer of the vagina.
We report results of brachytherapy for carcinoma of the vagina, utilizing a Nucletron high dose rate system for Delclos Vaginal Applicators (cylinder) and Syed Template Applicators (interstitial). The linear quadratic (LQ) model was used to determine the optimum time-dose-fractionation schedules. Interstitial doses were determined at the isodose line that included gross tumour. Cylinder doses were determined either at the vaginal surface (5 cases), at 0.5 cm depth (5 cases), or at 1.0 cm depth (1 case). For the first treatment (n=19), interstitial templates were utilized in 8 patients and vaginal cylinders in 11. 11 patients received second treatments: 6 templates and 5 cylinders. The median dose of external beam radiation (n=15) was 40.0 Gy followed, after a median 23 day interval, by high dose rate brachytherapy (HDRB) of 4 fractions in 30-42 h; then a median interval gap of 25 days, followed by repeat HDRB. The median total fractionated HDRB dose per patient was 23.0 Gy (range: 6.9 Gy to 40.4 Gy; calculated low dose rate equivalent of 29.8 Gy). Tumour histologies included 14 squamous cell carcinomas, 2 adenocarcinomas, 2 melanomas, and 1 small cell tumour. Three patients experienced early brachytherapy-related complications (diarrhoea, dysuria and labial dermatitis). Three patients (15.8%) developed serious/late complications including ureteral stenosis, painful vaginal necrosis and small bowel obstruction. The first of these patients received 2 templates, the second a cylinder followed by a template and a cylinder, and the third a single cylinder. The 2 year progression-free survival was 39.3% (median 15.7 months), while the 2 year overall survival was 66.1% (median 29.9 months). (192)Ir afterloading HDRB is a feasible approach to women with vaginal cancer with acceptable toxicity and tumour response. Potential advantages include patient preference, outpatient cost-effectiveness in the case of cylinder technique, and no radiation exposure to hospital personnel. Long-term follow-up is needed to further assess late complications, and larger studies are needed to confirm our results.
Characterization and baculovirus-directed expression of a myosuppressin encoding cDNA from the true armyworm, Pseudaletia unipuncta.
Insect myosuppressins are a highly conserved sub-family of peptides which are primarily characterized by the ability to suppress contraction of visceral muscles in a variety of insect species. We have isolated a cDNA from the true armyworm, Pseudaletia unipuncta, that encodes a prohormone containing a peptide identical to ManducaFLRFamide. We have shown that this myosuppressin gene appears to be expressed in late larval and adult insects. In Manduca sexta, a number of extended-FLRFamide peptides have previously been purified including ManducaFLRFamide, F7D (DPSFLRFamide), F7G (GNSFLRFamide) and two larger peptides F24 and F39 that contain the shorter ManducaFLRFamide sequence at their C-terminus. Comparison with the true armyworm prepropeptide characterized here identifies F24 and F39 as partially processed products from the same precursor. Expression in the true armyworm was shown by in situ hybridization to occur in over 150 cells throughout the adult brain and nerve cord, and also to occur in both open and closed endocrine type cells of the gut. Overexpression of the P. unipuncta FLRFamide cDNA from a baculovirus vector in cabbage looper caterpillars was used to assess the potential for myosuppressin expression as a means of enhancing virus efficacy. Viral expression of the armyworm prohormone cDNA resulted in raised levels of RFamide-like products in the hemolymph of infected insects, but the products were found to be chemically distinguishable from authentic mature peptide and probably represent partially processed hormone.
Antisense Bcl2 oligonucleotide in cisplatin-resistant bladder cancer cell lines.
OBJECTIVE: To determine the change of expression of Bcl2 in cisplatin-resistant bladder cancer cell lines and the reversibility of chemoresistance to cisplatin with antisense oligonucleotide against Bcl2, as higher expression of Bcl2 is associated with drug resistance in many different cancer cell lines. MATERIALS AND METHODS: In the cisplatin-resistant bladder tumour cell lines T24R1 and T24R2, the expression of Bcl2 was determined by reverse transcription-polymerase chain reaction and Western blot assay, and antisense oligonucleotide targeting of the Bcl2 coding sequence was administered with lipofectin. RESULTS: The expression of Bcl2 mRNA and protein was greater in T24R1 and T24R2 cells than in the parent T24 cells. Short-term exposure to cisplatin up-regulated Bcl2 mRNA and protein expression in parent T24 cells. Treatment with antisense oligonucleotide down-regulated Bcl2 protein expression and significantly enhanced the cytotoxicity of cisplatin. CONCLUSIONS: Up-regulation of Bcl2 protein expression might be one of the mechanisms of cisplatin resistance in bladder cancer cells, and antisense Bcl2 oligonucleotide may be helpful in chemotherapy for bladder cancer by reversing cisplatin resistance.
Reduction in nocturnal functional bladder capacity is a common factor in the pathogenesis of refractory nocturnal enuresis.
OBJECTIVE: To evaluate the diurnal and nocturnal bladder reservoir function in patients with refractory primary nocturnal enuresis (PNE). PATIENTS AND METHODS: Ninety-five children (68 boys, 27 girls, mean age 9.3 years) with significant PNE (>/=3 wet nights/week) that was refractory to treatment with desmopressin +/- an enuretic alarm were assessed using detailed recording of voiding frequency and urinary volume both day and night, natural filling cystometry during the day and continuous cystometry with simultaneous electroencephalogram monitoring during sleep at night. RESULTS: Patients could be broadly categorized into two groups. Group A comprised those with normal daytime urodynamics and functional bladder capacity (FBC) on detailed frequency-volume recording, but who developed marked detrusor instability associated with a significant reduction in nocturnal FBC and small-volume voiding only after sleep at night (33 patients, 35%); and group B, those with abnormal daytime urodynamics and with reduced FBC and small-volume voiding both day and night, but who somehow managed to mask their bladder symptoms during the day (62 patients, 65%). There was no evidence of nocturnal polyuria in either group and the ratios of day : night urinary output volumes for type A and type B patients were 1.48 and 1.99, respectively. CONCLUSION: A reduction in nocturnal FBC, either occurring only after sleep at night in association with the appearance of detrusor instability in patients with normal daytime urodynamics and FBC, or as a manifestation of occult voiding dysfunction or bladder outlet obstruction that affects the bladder reservoir function both day and night, appears to be a common factor and probably the main cause for a mismatch between nocturnal urine output and bladder storage capacity in patients with severe bed-wetting that was refractory to treatment.
Improving the use of mixing factors for dilution ventilation design.
In specifying dilution ventilation flow rate, a safety factor, K, is often used to provide a margin of safety and to compensate for uncertainties and health impact severity. In current practice, the selection of K is very subjective. Here the component of K accounting for imperfect mixing, Km, was studied to develop more effective and efficient design procedures. Air flow and contaminant distribution in a 10 m x 3 m x 7 m room with a single contaminant source on a 1-m high table were simulated for steady, isothermal conditions using computational fluid dynamics. A series of 10 simulations explored factorial combinations of air exchange rates (1, 2,4, 8, 16 ACH) and inlet types (a high wall jet and a ceiling diffuser). Nine additional simulations explored exhaust opening location effects and 13 other simulations investigated source location effects. Km was calculated at each of 25,600 grid locations within the room by linear regression of emission rate/flow rate (G/Q) on concentration (C). The linear relationship between C and G/Q at each of the points was nearly perfect (R2 > 0.97). For the simulations with varying dilution flow rate, Km ranged from 0.19 to 2.86 for the wall jet and from 0.94 to 4.34 for the ceiling diffuser. Holding G/Q at 100 ppm and varying source and exhaust location produced room average concentrations from 55.7 to 173 ppm. Unlike orthodox design approaches, this work suggests that air monitoring data often can be used to calculate dilution flow rate requirements. Also, dilution flow rate requirements may be reduced by enhancing room mixing with fans or altering air inlet configuration. However, mixing should not be increased if the altered room air currents could transport contaminant to an occupant's breathing zone or interfere with other control methods that depend on segregation of incoming air and contaminant.
Single ventricle, total transposition, and hypoplastic aorta in a calf.
A purebred Ayrshire calf was born with multiple congenital cardiac defects. The major defects included absence of an interventricular septum (i.e., single ventricle), transposition of the aorta and pulmonary trunk, interatrial septal defect, patent ductus arteriosus, and aortic arch anomalies. Despite the severity of the anomalies, the calf survived to 5 days of age, when it was found dead. This particular combination of cardiac defects has not been previously described in domestic animals.
Human kinesin superfamily member 4 is dominantly localized in the nuclear matrix and is associated with chromosomes during mitosis.
In a previous study, we identified the human counterpart of murine kinesin superfamily member 4 (KIF4), a microtubule-based motor protein [Oh, Hahn, Torrey, Shin, Choi, Lee, Morse and Kim (2000) Biochim. Biophys. Acta 1493, 219-224]. As an initial step to understand the function(s) of human KIF4, its subcellular localization in HeLa cells was examined by using immunocytochemical and subcellular fractionation methods, and it was found that most KIF4 is localized in the nucleus. Since murine KIF4 is known to transport cytoplasmic vesicles, dominant nuclear localization of the human counterpart was somewhat surprising. Subsequent subnuclear fractionation revealed predominant association of KIF4 with the nuclear matrix. These results clearly indicate that human KIF4 is, at least, a nuclear protein. In further confirmation of this conclusion, the hexapeptide PKLRRR (amino acids 773-778) in the molecule was found to function as a nuclear localization signal. During the mitotic phase of the cell cycle, human KIF4 was associated with the chromosomes, suggesting that human KIF4 might be a microtubule-based mitotic motor, with DNA as its cargo.
Chromosomal integration and expression of the Escherichia coli K88 gene cluster in Salmonella enterica ser. Choleraesuis strain 54 (SC54).
Attempts to develop live vaccines to protect against enterotoxigenic Escherichia coli (ETEC) infection by induction of both cell-mediated and mucosal immunity, and serum antibody responses have included use of recombinant Salmonella strains that produce K88 fimbrial antigens (Hone et al., 1988; Attridge et al., 1988; Morona et al., 1994). However, none of the recombinant Salmonella vectors has been licensed by the United States Department of Agriculture (USDA) for use as a live vaccine in pigs in the United States. A variant of Salmonella enterica ser. Choleraesuis strain 54 (SC54) is currently used as a safe and effective intranasal attenuated live vaccine in pigs. In order to expand the efficacy of this live vaccine strain, we sought to modify strain SC54 to express the K88 antigens of ETEC. To accomplish this, a plasmid-based system was used to integrate the K88 gene cluster into the chromosome of strain SC54 by site-specific recombination. The K88 antigens were expressed by strain SC54, and the gene cluster was stably maintained in the host.
Synthesis and In vitro binding affinities of 1-azabicyclic compounds as muscarinic ligands.
Two series of compounds, 2 and 3, were synthesized and their binding affinities were evaluated for the human recombinant muscarinic M(1) receptor subtype expressed in CHO cells. Comparing their binding affinities for the NMS binding sites and the Oxo-M binding sites, they were assumed as agonists. In particular, compound 2e was a good ligand for the agonist binding sites with an IC(50) of 23 nM, which represents over 1585 times stronger binding than for the antagonist binding sites.
Anti-apoptotic effect of cGMP in cultured astrocytes: inhibition by cGMP-dependent protein kinase of mitochondrial permeable transition pore.
Reperfusion of cultured astrocytes with normal medium after exposure to H(2)O(2)-containing medium causes apoptosis. We have recently shown that ibudilast, which has been used for bronchial asthma and cerebrovascular disorders, attenuated the H(2)O(2)-induced apoptosis of astrocytes via the cGMP signaling pathway. This study examines the mechanism underlying the protective effect of cGMP. The membrane-permeable cGMP analog dibutyryl-cGMP attenuated the H(2)O(2)-induced decrease in cell viability, DNA ladder formation, nuclear condensation, reduction of the mitochondrial membrane potential, cytochrome c release from mitochondria, and caspase-3 activation in cultured astrocytes. These effects of dibutyryl-cGMP were almost completely inhibited by the cGMP-dependent protein kinase (PKG) inhibitor KT5823. In isolated rat brain mitochondria, cGMP in the presence of cytosolic extract from astrocytes inhibited the mitochondrial permeability transition pore (PTP) as determined by monitoring Ca(2+)-induced mitochondrial swelling. This ability of the cytosolic extract was inactivated by heat treatment and was mimicked by exogenous PKG. The effect of cGMP on the mitochondrial swelling was blocked by KT5823. The PTP inhibitors cyclosporin A and bongkrekic acid prevented the H(2)O(2)-induced decrease in cell viability and caspase-3 activation. These findings demonstrate that cGMP inhibits the mitochondrial PTP via the activation of PKG, and the prevention of mitochondrial dysfunction contributes to its anti-apoptotic effect.
Total synthesis of pamamycin-607.
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IL-10 improves skin disease and modulates endothelial activation and leukocyte effector function in patients with psoriatic arthritis.
Psoriatic arthritis (PsA) provides an ideal disease model in which to investigate the bioactivities of potentially therapeutic cytokines at multiple sites of tissue inflammation. We investigated the effects of IL-10, an antiinflammatory cytokine, given s.c. for 28 days in a double-blind, placebo-controlled study in PsA patients. Synovial/skin biopsies, peripheral blood leukocytes, articular magnetic resonance images, and clinical disease activity scores were obtained sequentially. Modest, but significant clinical improvement in skin, but not articular disease activity scores with only minor adverse effects was observed. Type 1, but not type 2 T cell cytokine production in vitro was suppressed in human rIL-10 compared with placebo recipients. Similarly, monokine production in vitro was reduced, whereas serum soluble TNFRII levels were elevated, indicating suppression of monocyte function. Decreased T cell and macrophage infiltration in synovial tissues was accompanied by reduced P-selectin expression. Moreover, suppressed synovial enhancement on magnetic resonance imaging and reduced alpha(v)beta(3) integrin expression on von Willebrand factor(+) vessels were observed. Together these data demonstrate that a short course of IL-10 modulates immune responses in vivo via diverse effects on endothelial activation, and leukocyte recruitment and effector function. Such biological changes may result in clinically meaningful improvement in disease activity.
Monitoring transfected cells without selection agents by using the dual-cassette expression EGFP vectors.
Conventional methods of selecting gene transfected cells by toxic agents may yield ambiguous results. It is difficult to determine whether cell death is due to selection agents or gene transfection, owing to the substantial overlap of the time-courses for both effects. Therefore, to determine transfection-induced cell toxicity, the mammalian expression vector pEGFP-N1 (CLONTECH Lab., Palo Alto, CA, USA) has been modified to the dual-cassette expression vectors named pEGFP-Ks by the relocation of its EGFP expression cassette. We have precisely monitored the cells transfected with this vector on our custom culture dishes, thereby bypassing the need for selection agent or fluorescent cell sorting. This is a useful method to screen genes encoding potential toxic or useful proteins without performing undesirable selection agent and also can be used to monitor the transfected cells for various purposes, either the inhibition or proliferation of mammalian cells for applications in biotechnology.
Substrate recognition by the Cdc20 and Cdh1 components of the anaphase-promoting complex.
The specificity of ubiquitin-mediated protein degradation with regards to the selection of substrates to be polyubiquitinated has only been determined rather recently. Substrate targeting by the N-end rule and HECT (homology to E6AP carboxyl terminus) domain ubiquitin ligases occurs through substrate-specific binding domains. In contrast, the SCF complex recruits substrates through a substrate adaptor protein, the F-box subunit. Despite evidence showing that Cdc20 and Cdh1 bind and activate the anaphase-promoting complex (APC) in a substrate-specific manner, there is no evidence that the activating protein and substrate interact directly; hence, no clear model exists for the mechanism of APC activation or recruitment of substrates. We show here that the activators Cdc20 and Cdh1 can associate with substrates via their N termini. In the absence of APC, Cdc20 and Cdh1 bind substrates reflecting Cdc20-APC and Cdh1-APC specificity. The N termini of Cdc20 and Cdh1 provide specificity functionally, as demonstrated by the generation of active chimeras that display the specificity corresponding to their N termini. Thus, Cdc20 and Cdh1 act as both substrate recognition and activating modules for APC.
Physiological regulation of [beta]-catenin stability by Tcf3 and CK1epsilon.
The wnt pathway regulates the steady state level of beta-catenin, a transcriptional coactivator for the Tcf3/Lef1 family of DNA binding proteins. We demonstrate that Tcf3 can inhibit beta-catenin turnover via its competition with axin and adenomatous polyposis for beta-catenin binding. A mutant of beta-catenin that cannot bind Tcf3 is degraded faster than the wild-type protein in Xenopus embryos and extracts. A fragment of beta-catenin and a peptide encoding the NH2 terminus of Tcf4 that block the interaction between beta-catenin and Tcf3 stimulate beta-catenin degradation, indicating this interaction normally plays an important role in regulating beta-catenin turnover. Tcf3 is a substrate for both glycogen synthase kinase (GSK) 3 and casein kinase (CK) 1epsilon, and phosphorylation of Tcf3 by CKIepsilon stimulates its binding to beta-catenin, an effect reversed by GSK3. Tcf3 synergizes with CK1epsilon to inhibit beta-catenin degradation, whereas CKI-7, an inhibitor of CK1epsilon, reduces the inhibitory effect of Tcf3. Finally, we provide evidence that CK1epsilon stimulates the binding of dishevelled (dsh) to GSk3 binding protein (GBP) in extracts. Along with evidence that a significant amount of Tcf protein is nonnuclear, these findings suggest that CK1epsilon can modulate wnt signaling in vivo by regulating both the beta-catenin-Tcf3 and the GBP-dsh interfaces.
Inhibition of Cdh1-APC by the MAD2-related protein MAD2L2: a novel mechanism for regulating Cdh1.
Exit from mitosis requires the degradation of regulatory proteins including the mitotic cyclins and securin through ubiquitination by the anaphase promoting complex (APC) bound to Cdc20 or Cdh1. Cdc20-APC is regulated through inhibition by the spindle assembly checkpoint protein MAD2. Knowledge of Cdh1-APC regulation is limited to the phosphorylation-dependent dissociation of Cdh1 from APC. We report a novel means of regulating Cdh1 by the MAD2-related gene, MAD2L2. MAD2L2 specifically binds and inhibits Cdh1-APC, paralleling the effect of MAD2 on Cdc20. We suggest that MAD2L2 and MAD2 inhibit the release of substrates from APC and propose a mechanism of inhibition.