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J K Wu

Publications and source records attributed to J K Wu.

At least 19 recordsLinked to original sources

Gap junctions: the "kiss of death" and the "kiss of life".

Cells expressing herpes simplex-thymidine kinase (HSV-tk) can be killed "in vitro" within 5 days of treatment with 20 microM ganciclovir (GCV) and transmit this toxicity to adjacent cells lacking HSV-tk; this phenomenon was termed "bystander effect" or "kiss of death". On testing a large number of cell lines in vitro, a wide range of sensitivity to GCV-mediated bystander killing has been reported. Although intercellular transfer of GCV metabolites through gap junction channels seems to be a likely mechanism for the "kiss of death", some studies suggest that other pathways may contribute to induced apoptosis of neighboring cells. To further investigate the mechanism underlying cell death mediated by HSV-tk and to evaluate the efficacy of gap junction channels formed by different connexins in this process, we have stably transfected a virtually uncoupled mouse neuroblastoma cell line (N2A cells) with different connexin-types expressed by neural cells (Cx32, Cx37, Cx40, Cx43) and co-cultured these cells with N2A cells stably transfected with Cx37 and HSV-tk. Here, we confirm our previous studies and those of others that the extent of cell death and sensitivity to GCV depend on the degree of connexin expression in transfectants. Further, we show that the bystander effect also depends on which connexin is expressed; reported disparities regarding the extent of GCV-mediated cellular apoptosis are likely due both to the degree of functional coupling and the type of connexin expressed. These results support the notion that gap junction hemichannels formed of certain connexins are more likely than others to pair functionally with Cx37, and suggest co-transfection strategies that might prove effective in sensitizing tumor cell populations to GCV. In addition, potential applications are discussed for use of the "good Samaritan effect", a mechanism by which bystander cells have been suggested to prevent cytotoxicity.

Animals↗

Inhibition of IMP-1 metallo-beta-lactamase and sensitization of IMP-1-producing bacteria by thioester derivatives.

IMP-1 metallo-beta-lactamase is a transferable carbapenem-hydrolyzing enzyme found in some clinical isolates of Pseudomonas aeruginosa, Serratia marcescens and Klebsiella pneumoniae. Bacteria that express IMP-1 show significantly reduced sensitivity to carbapenems and other beta-lactam antibiotics. A series of thioester derivatives has been shown to competitively inhibit purified IMP-1. As substrates for IMP-1, the thioesters yielded thiol hydrolysis products which themselves were reversible competitive inhibitors. The thioesters also increased sensitivity to the carbapenem L-742,728 in an IMP-1-producing laboratory stain of Escherichia coli, but will need further modification to improve their activity in less permeable organisms such as Pseudomonas and Serratia. Nonetheless, the thioester IMP-1 inhibitors offer an encouraging start to overcoming metallo-beta-lactamase-mediated resistance in bacteria.

Bacteria↗

A non-invasive immobilization system and related quality assurance for dynamic intensity modulated radiation therapy of intracranial and head and neck disease.

PURPOSE: To develop and implement a non-invasive immobilization system guided by a dedicated quality assurance (QA) program for dynamic intensity-modulated radiotherapy (IMRT) of intracranial and head and neck disease, with IMRT delivered using the NOMOS Corporation's Peacock System and MIMiC collimator. METHODS AND MATERIALS: Thermoplastic face masks are combined with cradle-shaped polyurethane foaming agents and a dedicated quality assurance program to create a customized headholder system (CHS). Plastic shrinkage was studied to understand its effect on immobilization. Fiducial points for computerized tomography (CT) are obtained by placing multiple dabs of barium paste on mask surfaces at intersections of laser projections used for patient positioning. Fiducial lines are drawn on the cradle along laser projections aligned with nasal surfaces. Lateral CT topograms are annotated with a crosshair indicating the origin of the treatment planning and delivery coordinate system, and with lines delineating the projections of superior-inferior field borders of the linear accelerator's secondary collimators, or with those of the fully open MIMiC. Port films exposed with and without the MIMIC are compared to annotated topograms to measure positional variance (PV) in superior-inferior (SI), right-left (RL), and anterior posterior (AP) directions. MIMiC vane patterns superposed on port films are applied to verify planned patterns. A 12-patient study of PV was performed by analyzing positions of 10 anatomic points on repeat CT topograms, plotting histograms of PV, and determining average PV. RESULTS AND DISCUSSION: A 1.5+/-0.3 mm SD shrinkage per 70 cm of thermoplastic was observed over 24 h. Average PV of 1.0+/-0.8, 1.2+/-1.1, and 1.3+/-0.8 mm were measured in SI, AP, and RL directions, respectively. Lateral port films exposed with and without the MIMiC showed PV of 0.2+/-1.3 and 0.8+/-2.2 mm in AP and SI directions. Vane patterns superimposed on port films consistently verified the planned patterns. CONCLUSION: The CHS provided adequately reproducible immobilization for dynamic IMRT, and may be applicable to decrease PV for other cranial and head and neck external beam radiation therapy.

Brain Neoplasms↗

Noninvasive evaluation of blunt abdominal trauma: prospective study using diagnostic algorithms to minimize nontherapeutic laparotomy.

A prospective study was performed to investigate the feasibility and benefit of evaluating blunt abdominal trauma (BAT) without diagnostic peritoneal lavage (DPL) or other invasive methods. Diagnostic algorithms were designed by using ultrasonography (US) as a screening method. For unstable patients, a free fluid >/= 2 mm thickness on US images over any one of the intraperitoneal spaces (bilateral subphrenic, Morrison, and Douglas pouch) was used as an indicator for laparotomy. For stable patients, any intraabdominal free fluid detected by US was used as an indicator for further investigations. Computed tomography served as a principal complementary method. To further clarify the clinical results, the rate of nontherapeutic laparotomy (NTL) was compared with that from a previous 5-year review done before this study. During studying period of 1 year, 170 patients were consecutively enrolled. There was no delayed diagnosis, and 66 patients were found to have BAT; 17 patients were initial unstable, among whom 15 had free fluid shown by US and 13 patients had confirmed BAT. Eight of the unstable patients with free fluid on US were saved from NTL, of whom six had retroperitoneal hematoma. There was no NTL in unstable patients. Twenty-two patients underwent laparotomy. Two laparotomies done for a suspicion of bowel injury turned out to be NTL. The rate of NTL in the present study was significant lower than that in a previous review (9.1% vs. 32.2%, p = 0.025). Hence following well designed algorithms, noninvasive evaluation of BAT can proceed with safety, and NTL is minimized.

Abdominal Injuries↗

Connexins are expressed in primary brain tumors and enhance the bystander effect in gene therapy.

OBJECTIVE: Experimental brain tumor gene therapy with the herpes simplex virus thymidine kinase (HSV-tk) gene has demonstrated that not only HSV-tk transduced but surrounding non-HSV-tk transduced cells are killed when given ganciclovir. This so-called bystander effect has recently been shown to be dependent on connexin-mediated intercellular communication. To assess potential susceptibility to the bystander effect, we examined levels of connexin-26 and connexin-43 expression in a series of primary brain tumors. Connexin-26 expression has not previously been studied in primary brain tumors and connexin-43 expression has not been studied in nonastrocytic primary brain tumors. We also attempted to enhance the bystander effect in vitro by overexpressing connexin in tumor cells with high basal levels of connexin expression. METHODS: Western blot analysis and immunohistochemistry were used to determine levels of connexin-26 and connexin-43 expression in a series of primary brain tumors. Wild-type 9L gliosarcoma cells were transfected in vitro with the connexin-43 gene and the HSV-tk gene or the HSV-tk gene alone. The bystander effect of each transfectant was then assessed and compared. RESULTS: Most of the primary brain tumors tested, including low-grade astrocytomas, anaplastic astrocytomas, glioblastomas, oligodendrogliomas, gangliogliomas, meningiomas, and medulloblastomas, showed connexin-26 and connexin-43 expression. Bystander experiments revealed a significant enhancement of the bystander effect in the gliosarcoma cells transfected with connexin-43 and HSV-tk, as compared with gliosarcoma cells transfected with HSV-tk alone. CONCLUSION: Most primary brain tumors express connexin-26 and connexin-43. This suggests that most primary brain tumors may be susceptible to the bystander effect of HSV-tk gene therapy. The bystander effect can be enhanced in vitro by overexpression of connexin-43 in a cell line with a high basal level of connexin-43 expression.

Animals↗

Clonal analysis of a case of multiple meningiomas using multiple molecular genetic approaches: pathology case report.

OBJECTIVE: Multiple meningiomas are uncommon brain tumors occurring concurrently in several intracranial locations in the same patient. In the present study, we determined the clonality, methylation status of deoxyribonucleic acid, and relationship of genetic alterations in eight meningiomas from one female patient. METHODS: Six molecular genetic techniques, including two methylation-based clonality assays and one transcription-based clonality assay, methylation analysis of CpG islands by methylation-specific polymerase chain reaction, loss of heterozygosity, microsatellite instability, and mutational analysis of the NF2 gene on chromosome 22, were used in comparative investigations on clonality and genetic alterations. RESULTS: The presence of clonal tumor cells was demonstrated by 1) loss of the same copy of chromosome 22 in all eight tumors; 2) transcription of the human AR gene from the same allele in six of eight tumors; 3) a common unmethylated allele at the AR locus in all eight tumors; and 4) the identical single-basepair insertion mutation in exon 9 of the NF2 gene in six of eight tumors. In addition, loss of a copy of the X chromosome in one tumor nodule and microsatellite instability in another nodule were observed. CONCLUSION: Taken together, this case of multiple meningiomas was most likely monoclonal in origin. Loss of chromosome 22 was an early event during the development of multiple meningiomas and was followed by mutations at the NF2 locus. Later events, including loss of the X chromosome, variation of AR gene expression, or microsatellite instability, may also have played a role in the development of multiple meningiomas in this patient.

Brain↗

Unanticipated inhibition of the metallo-beta-lactamase from Bacteroides fragilis by 4-morpholineethanesulfonic acid (MES): a crystallographic study at 1.85-A resolution.

As part of a structure-aided effort to design clinically useful inhibitors of metallo-beta-lactamases, the X-ray crystal structure of a complex between the metallo-beta-lactamase from Bacteroides fragilis and 4-morpholinoethanesulfonic acid (MES) has been determined and a model for the structure has been refined to a crystallographic R-factor of 0.151 for data between 10.0- and 1.85-A resolution. Although the binding of MES was an adventitious result of the use of MES as a buffer in the crystallization mixture, MES was subsequently shown to be a competitive inhibitor of the enzyme, with a Ki of 23 +/- 5 mM. MES binds in the same fashion to both of the molecules in the crystallographic asymmetric unit; both direct and solvent-mediated hydrogen bonds to the protein and to the binuclear zinc cluster are observed, involving the oxygens of the sulfonic acid group and the nitrogen of the morpholino ring. In addition, there are hydrophobic interactions between the morpholino ring and residues in the flexible beta-strand of the enzyme between residues 26 and 36. Comparison of this structure with the previously reported unliganded structures of the same enzyme [Concha, N. O., Rasmussen, B. A., Bush, K., and Herzberg, O. (1996) Structure 4, 823-836; Carfi, A., Duée, E., Paul-Soto, R., Galleni, M., Frère, J. -M., and Dideberg, O. (1998) Acta Crystallogr. D54, 47-57] reveals that although the overall conservation of structure in the three different crystal lattices is very high, binding of MES is correlated with a significant change in the conformation of this beta-strand. The flexibility of this beta-strand will be an important consideration in the design of inhibitors of the metallo-beta-lactamases.

Alkanesulfonic Acids↗

Dosimetric verification of the dynamic intensity-modulated radiation therapy of 92 patients.

PURPOSE: To verify that optimized dose distributions provided by an intensity-modulated radiation therapy (IMRT) system are delivered accurately to human patients. METHODS AND MATERIALS: Anthropomorphic phantoms are used to measure IMRT doses. Four types of verification are developed for: I) system commissioning with beams optimized to irradiate simulated targets in phantoms, II) plans with patient-optimized beams directed to phantoms simulating the patient, III) patient-phantom hybrid plans with patient-optimized beams calculated in phantom without further optimization, and IV) in vivo measurements. Phantoms containing dosimeters are irradiated with patient-optimized beams. Films are scanned and data were analyzed with software. Percent difference between verified and planned maximum target doses is defined as "dose discrepancy" (deltavp). The frequency distribution of type II deltavp from 204 verification films of 92 IMRT patients is fit to a Gaussian. Measurements made in vivo yield discrepancies specified as deltaivp, also fit to a Gaussian. RESULTS AND DISCUSSION: Verification methods revealed three systematic errors in plans that were corrected prior to treatment. Values of [deltavp] for verification type I are <2%. Type II verification discrepancies are characterized by a Gaussian fit with a peak 0.2% from the centroid, and 158 [deltavp] <5%. The 46 values of [deltavp] >5% arise from differences between phantom and patient geometry, and from simulation, calculation, and other errors. Values of [deltavp] for verification III are less than half of the values of [deltavp] for verification II. A Gaussian fit of deltaivp from verification IV shows more discrepancy than the fit of deltavp, attributed to dose gradients in detectors, and exacerbated by immobilization uncertainty. CONCLUSIONS: Dosimetric verification is a critical step in the quality assurance (QA) of IMRT. Hybrid Verification III is suggested as a preliminary quality standard for IMRT.

Brain Neoplasms↗

Soluble penicillin-binding protein 2a: beta-lactam binding and inhibition by non-beta-lactams using a 96-well format.

High level methicillin resistance in Staphylococcus aureus is dependent upon the acquisition of the mecA gene encoding penicillin-binding protein 2a (PBP2a). PBP2a is a member of a family of peptidoglycan biosynthetic enzymes involved in assembly of the cell wall in bacteria and is poorly inactivated by beta-lactam antibiotics. We describe a 96-well-filter binding assay using recombinant, soluble PBP2a which allows for kinetic measurement of penicillin binding. The deacylation rate constant for the PBP2a-penicillin G covalent complex was found to be 5.7 +/- 1.0 x 10(-5) s-1 at 30 degrees C (half-life of approximately 200 min). For the PBP2a acylation reaction, the value of K(m) (penicillin G) = 0.5 +/- 0.1 mM and kcat = 1 x 10(-3) s-1, which yields a second-order rate constant (kcat/K(m)) for inactivation of 2.0 M-1 s-1. Using this assay, several non-beta-lactam inhibitors including Cibacron blue have been found which exhibit IC50 values between 10 and 30 microM. The binding affinities of several carbapenems and beta-lactams correlated well between the filter binding assay described in this report and an electrophoretic assay for PBP2a using membranes prepared form methicillin-resistant S. aureus.

Anti-Bacterial Agents↗

Screening for loss of heterozygosity and microsatellite instability in oligodendrogliomas.

To assess the frequency of loss of heterozygosity (LOH) and microsatellite instability (MI) in oligodendrogliomas, we performed an extensive screening of 16 oligodendrogliomas and nine anaplastic oligodendrogliomas by using 132 microsatellite markers on chromosomes 1 through 12 and 15 through 21. In total, 3,135 loci were examined in 25 tumor samples. Only 33/1,965 (1.7%) of oligodendroglioma (low-grade) and 11/1,070 (1.0%) of anaplastic oligodendroglioma (high-grade) loci exhibited MI. High-frequency LOH regions were identified on chromosome arms 1p (31-73% for oligodendrogliomas and 60-100% for anaplastic oligodendrogliomas) and 19q (23-69% for oligodendrogliomas and 100% for anaplastic oligodendrogliomas). In addition, regions on chromosomes 4, 6, and 11 were found to be lost in 30-80% of both oligodendrogliomas and anaplastic oligodendrogliomas. Increased LOH frequency of chromosome 17 (38-40%) was found only in high-grade oligodendrogliomas. The differences in LOH frequencies between low-grade and high-grade oligodendrogliomas in all loci combined and at three loci (DIS447, DIS226, and DIS252) on chromosome arm 1p were determined to be statistically significant [chi 2(1) = 20.2, P < 0.0001, and Fisher's exact test respective P values: 0.01, 0.03, and 0.02]. Our results provide evidence that microsatellite instability does not play an important role in the development of oligodendrogliomas. Furthermore, high LOH frequency on chromosomes 6 and 11 in addition to that identified previously on chromosomes 1, 19, and 4 suggests that multiple candidate tumor suppressor genes on these chromosomes may underlie the processes of initiation and/or progression in oligodendroglioma tumorigenesis.

Adult↗

Antibiotic sensitization using biphenyl tetrazoles as potent inhibitors of Bacteroides fragilis metallo-beta-lactamase.

BACKGROUND: High level resistance to carbapenem antibiotics in gram negative bacteria such as Bacteroides fragilis is caused, in part, by expression of a wide-spectrum metallo-beta-lactamase that hydrolyzes the drug to an inactive form. Co-administration of metallo-beta-lactamase inhibitors to resistant bacteria is expected to restore the antibacterial activity of carbapenems. RESULTS: Biphenyl tetrazoles (BPTs) are a structural class of potent competitive inhibitors of metallo-beta-lactamase identified through screening and predicted using molecular modeling of the enzyme structure. The X-ray crystal structure of the enzyme bound to the BPT L-159,061 shows that the tetrazole moiety of the inhibitor interacts directly with one of the two zinc atoms in the active site, replacing a metal-bound water molecule. Inhibition of metallo-beta-lactamase by BPTs in vitro correlates well with antibiotic sensitization of resistant B. fragilis. CONCLUSIONS: BPT inhibitors can sensitize a resistant B. fragilis clinical isolate expressing metallo-beta-lactamase to the antibiotics imipenem or penicillin G but not to rifampicin.

Bacteroides fragilis↗

A technique for accurate planning of stereotactic brain implants prior to head ring fixation.

PURPOSE: A two-step procedure is described for accurate planning of stereotactic brain implants prior to head-ring fixation. METHODS AND MATERIALS: Approximately 2 weeks prior to implant a CT scan without the head ring is performed for treatment-planning purposes. An entry point and a reference point, both marked with barium and later tattooed, facilitate planning and permit correlation of the images with a later CT scan. A plan is generated using a conventional treatment-planning system to determine the number and activity of I-125 seeds required and the position of each catheter. I-125 seed anisotropy is taken into account by means of a modification to the treatment planning program. On the day of the implant a second CT scan is performed with the head ring affixed to the skull and with the same points marked as in the previous scan. The planned catheter coordinates are then mapped into the coordinate system of the second CT scan by means of a manual translational correction and a computer-calculated rotational correction derived from the reference point coordinates in the two scans. RESULTS: The rotational correction algorithm was verified experimentally in a Rando phantom before it was used clinically. For analysis of the results with individual patients a third CT scan is performed 1 day following the implant and is used for calculating the final dosimetry. CONCLUSION: The technique that is described has two important advantages: 1) the number and activity of seeds required can be accurately determined in advance; and 2) sufficient time is allowed to derive the best possible plan.

Brachytherapy↗

Response of intracranial melanoma metastases to stereotactic radiosurgery.

We analyzed our recent stereotactic radiosurgery (SRS) experience to determine the radiographic response of intracranial metastatic melanomas to SRS. Twelve patients with 21 intracranial melanoma metastases treated with SRS were evaluated. Fifteen (72%) metastases were hemispheric, 3 (14%) were cerebellar, and 3 (14%) were in the basal ganglion or thalamus. All lesions were 2.5 cm or less in maximum diameter. Eleven patients also had whole brain external beam radiotherapy. Mean SRS dosage was 1,800 cGy to the 85% isodose surface and median dose was 1,800 cGy to the 80% isodose surface (range 1,100-3, 100 cGy at the 80-95% isodose surface). Overall, 12 (57%) lesions showed decrease or stabilization of tumor volume (i.e., local control), while 9 (43%) showed enlargement. Division of metastases into small (< or = 1.0 cm diameter) and large (> 1.0 cm diameter) tumors showed that the small tumors were more likely to regress than the large tumors (chi-square test; P < 0.03). Only 1 of 9 (11%) large lesions regressed as opposed to 7 of 12 (58%) small lesions regressed with SRS. We conclude that SRS is suited for small melanoma brain metastases, but lesions between 1.0 and 2.5 cm in diameter, while still generally considered appropriate for SRS, may not be as responsive to SRS at currently employed dosages.

Adult↗

High-yield expression, purification, and characterization of active, soluble Bacteroides fragilis metallo-beta-lactamase, CcrA.

The gene from Bacteroides fragilis encoding a metallo-beta-lactamase, ccrA, was expressed in Escherichia coli BL21(DE3) containing the wild-type disulfide bond-catalyzing system dsb as an active, soluble enzyme in quantities exceeding 100 mg/liter using both rich and minimal media. Both the nonfusion and a glutathione S-transferase fusion enzyme lacking the periplasmic signal sequence were purified to homogeneity. Characteristics of the purified nonfusion enzyme are shown to be similar to those of the renatured enzyme previously reported. Thermal denaturation studies using circular dichroism and fluorescence spectroscopy show that CcrA undergoes a transition at approximately 50 degrees C which corresponds to the transition temperature of catalytic activity. The secondary structure of the protein and the catalytic apparatus are thus intimately linked.

Bacterial Proteins↗

The bystander effect exerted by tumor cells expressing the herpes simplex virus thymidine kinase (HSVtk) gene is dependent on connexin expression and cell communication via gap junctions.

To elucidate the role gap junctions play in the bystander effect, we examined the cytotoxic effect of herpes simplex virus thymidine kinase (HSVtk) modified tumor cells on gap junction communication-deficient tumor cells and their connexin transfectants. Communication competent Walker 256 tumor cells engineered to express the HSVtk gene (Walker-tk+) when cocultured with N2A mouse neuroblastoma and PC12 rat pheochromocytoma cells with absent endogenous junctional conductance showed no bystander cytotoxicity. Transfection of N2A cells with the rat connexin37 gene (5Q) and PC12 cells with the human connexin43 gene rendered them susceptible to bystander cell death. Additionally, communication-deficient N2A cells transfected with the HSVtk gene failed to exert a bystander effect, whereas N2A transfectants coexpressing the connexin37 and HSVtk genes (5Qtk+ cells) exerted bystander cytotoxicity on gap junction communication-competent 5Q but not on communication-deficient N2A cells in vitro. In vivo experiments also showed tumor growth inhibition of communication-competent 5Q but not communication-incompetent N2A cells by 5Qtk+ cells. In conclusion, these results indicate that in several cellular environments the bystander effect is dependent on connexin expression and gap junctional communication between HSVtk-positive and HSVtk-negative cells.

Animals↗

Evidence for clonal origin of neoplastic neuronal and glial cells in gangliogliomas.

Gangliogliomas are rare tumors of the central nervous system that account for approximately 1% of all brain tumors. Histologically, gangliogliomas are composed of intimately admixed glial and neuronal components, the pathological origins of which remain to be characterized. Clonal analysis through examination of the pattern of the X chromosome inactivation allows one to distinguish monoclonal differentiation of a genetically abnormal progenitor cell from parallel, but independent, clonal expansion of two different cell types during tumorigenesis in biphasic neoplasms, such as gangliogliomas. In the present study, we investigated the clonality of eight gangliogliomas from female patients using both methylation- and transcription-based clonality assays at the androgen receptor locus (HUMARA) on the X chromosome. Among tumors from seven patients who were heterozygous at the HUMARA locus, five were identified as monoclonal with the methylation-based clonality assay, and the results were confirmed by the transcription-based method, whereas two were shown to be polyclonal by the methylation-based clonality assay but monoclonal by transcription-based clonality analysis. We conclude that the predominant cell types in most gangliogliomas are monoclonal in origin and derive from a common precursor cell that subsequently differentiates to form neoplastic glial and neuronal elements.

Adult↗

[Clinical study of effect of yinji capsule on left ventricular systolic function of angina pectoris patients with blood stasis syndrome].

OBJECTIVE: To explore the Yinji Capsule (YJC) in improving the left ventricular systolic function of angina pectoris patients with Blood Stasis Syndrome. METHODS: The systolic function of left ventricle (LV) in cardiac cycle of 28 angina pectoris patients with Blood Stasis Syndrome was examined with three-dimensional echocardiograph (3-DE) before and after treatment with YJC. RESULTS: The total symptomatic effective rate was 85.7%. The changes of LV systolic function were those: left ventricle ejection fraction (LVEF) increased from 45.0 +/- 4.9% to 48.2 +/- 3.5% (P < 0.05); EF on early stage and late stage increased from 22.6 +/- 2.1%, 8.3 +/- 1.2% to 28.1 +/- 3.0% and 10.3 +/- 0.9% respectively (P < 0.01, P < 0.05), myocardial region with segment systole (SS) < 5% decreased significantly (P < 0.01). CONCLUSION: YJC could improve LV systolic function on early stage and late stage in cardiac cycles, and mainly improve the systolic function of the region with low SS of LV.

Aged↗