Role of the TRAF binding site and NF-kappaB activation in Epstein-Barr virus latent membrane protein 1-induced cell gene expression

In this study, we investigated the induction of cellular gene expression by the Epstein-Barr Virus (EBV) latent membrane protein 1 (LMP1). Previously, LMP1 was shown to induce the expression of ICAM-1, LFA-3, CD40, and EBI3 in EBV-negative Burkitt lymphoma (BL) cells and of the epidermal growth factor receptor (EGF-R) in epithelial cells. We now show that LMP1 expression also increased Fas and tumor necrosis factor receptor-associated factor 1 (TRAF1) in BL cells. LMP1 mediates NF-kappaB activation via two independent domains located in its C-terminal cytoplasmic tail, a TRAF-interacting site that associates with TRAF1, -2, -3, and -5 through a PXQXT/S core motif and a TRADD-interacting site. In EBV-transformed B cells or transiently transfected BL cells, significant amounts of TRAF1, -2, -3, and -5 are associated with LMP1. In epithelial cells, very little TRAF1 is expressed, and only TRAF2, -3, and -5, are significantly complexed with LMP1. The importance of TRAF binding to the PXQXT/S motif in LMP1-mediated gene induction was studied by using an LMP1 mutant that contains alanine point mutations in this motif and fails to associate with TRAFs. This mutant, LMP1(P204A/Q206A), induced 60% of wild-type LMP1 NF-kappaB activation and had approximately 60% of wild-type LMP1 effect on Fas, ICAM-1, CD40, and LFA-3 induction. In contrast, LMP1(P204A/Q206A) was substantially more impaired in TRAF1, EBI3, and EGF-R induction. Thus, TRAF binding to the PXQXT/S motif has a nonessential role in up-regulating Fas, ICAM-1, CD40, and LFA-3 expression and a critical role in up-regulating TRAF1, EBI3, and EGF-R expression. Further, D1 LMP1, an LMP1 mutant that does not aggregate failed to induce TRAF1, EBI3, Fas, ICAM-1, CD40, and LFA-3 expression confirming the essential role for aggregation in LMP1 signaling. Overexpression of a dominant form of IkappaBalpha blocked LMP1-mediated TRAF1, EBI3, Fas, ICAM-1, CD40, and LFA-3 up-regulation, indicating that NF-kappaB is an important component of LMP1-mediated gene induction from both the TRAF- and TRADD-interacting sites.     

Salbutamol enhances isotonic contractile properties of rat diaphragm muscle

The effects of the beta2-adrenoceptor agonist salbutamol (Slb) on isometric and isotonic contractile properties of the rat diaphragm muscle (Diamus) were examined. A loading dose of 25 microg/kg Slb was administered intracardially before Diamus excision to ensure adequate diffusion. Studies were then performed with 0.05 microM Slb in the in vitro tissue chamber. cAMP levels were determined by radioimmunoassay. Compared with controls (Ctl), cAMP levels were elevated after Slb treatment. In Slb-treated rats, isometric twitch and maximum tetanic force were increased by approximately 40 and approximately 20%, respectively. Maximum shortening velocity increased by approximately 15% after Slb treatment, and maximum power output increased by approximately 25%. During repeated isotonic activation, the rate of fatigue was faster in the Slb-treated Diamus, but both Slb-treated and Ctl Diamus fatigued to the same maximum power output. Still, endurance time during repetitive isotonic contractions was approximately 10% shorter in the Slb-treated Diamus. These results are consistent with the hypothesis that beta-adrenoceptor stimulation by Slb enhances Diamus contractility and that these effects of Slb are likely mediated, at least in part, by elevated cAMP.     

Transcrystalline morphology of nylon 6 on the surface of aramid fibers

In this paper, the isothermal crystallization of nylon 6 in the presence of Kevlar 129 fiber was investigated by polarized optical microscopy (POM). The formation of a transcrystalline domain was found to be mainly controlled by crystallization conditions, such as the temperature of the isothermal crystallization, residual time at melting temperature and the cooling rate of the melt. The nucleation rate of nylon 6 on the fibers was mainly affected by the crystallization temperature. The interfacial transcrystallinity of nylon 6 occurred on the surface of Kevlar 129 fiber in the temperature range 130–190 °C. The reason for the formation of interfacial transcrystalline morphology is discussed from the molecular level, based on the understanding of the packing mode of nylon 6 chains around fibers and the interaction between matrix and fibers. It was found that the lattice matching and hydrogen‐bonding between nylon 6 and poly(p‐phenylene terephthalamide) (PPTA) crystals play an important role in the epitaxial crystallization. Copyright © 2004 Society of Chemical Industry     

Specific, high affinity binding of tissue inhibitor of metalloproteinases-4 (TIMP-4) to the COOH-terminal hemopexin-like domain of human gelatinase A. TIMP-4 binds progelatinase A and the COOH-terminal domain in a similar manner to TIMP-2

The binding properties of the newly described tissue inhibitor of metalloproteinases-4 (TIMP-4) to progelatinase A and to the COOH-terminal hemopexin-like domain (C domain) of the enzyme were examined. We present evidence for the first time of a specific, high affinity interaction between TIMP-4 and the C domain of human gelatinase A and show that TIMP-4 binds both progelatinase A and the C domain in a similar manner to that of TIMP-2. Saturable binding of recombinant C domain to TIMP-4 and to TIMP-2 but not to TIMP-1 was demonstrated using a microwell protein binding assay. The recombinant collagen binding domain of gelatinase A, comprised of the three fibronectin type II-like repeats, did not bind to TIMP-4, indicating that binding is mediated selectively by the C domain. Binding to TIMP-4 was of high affinity with an apparent Kd of 1.7 x 10(-7) M but slightly weaker than that to TIMP-2 (apparent Kd of 0.66 x 10(-7) M). Affinity chromatography confirmed the TIMP-4-C domain interaction and also showed that the complex could not be disrupted by 1 M NaCl or 10% dimethyl sulfoxide, thereby further demonstrating the tight binding. To verify the biological significance of this interaction, binding of full-length progelatinase A to TIMP-4 was investigated. TIMP-4 and TIMP-2 but not TIMP-1 bound specifically to purified TIMP-2-free human recombinant full-length progelatinase A and to full-length rat proenzyme from the conditioned culture medium of ROS 17/2.8 cells. Preincubation of the C domain with TIMP-2 was found to reduce subsequent binding to TIMP-4 in a concentration-dependent manner. Competition between TIMP-2 and TIMP-4 for a common or overlapping binding sites on the gelatinase A C domain may occur; alternatively TIMP-2 may prevent the binding of TIMP-4 by steric hindrance or induction of a conformational change in the C domain. We propose that the binding of progelatinase A to TIMP-4 represents a third TIMP-progelatinase interaction in addition to that of progelatinase A with TIMP-2 and progelatinase B with TIMP-1 described previously. This new phenomenon may be of important physiological significance in modulating the cell surface activation of progelatinase A.     

One-stage hypospadias repair with stent or suprapubic diversion: which is better?

PURPOSE: The authors report on 105 consecutive patients who underwent one-stage hypospadias repair based on use of suprapubic diversion or transurethral drainage with stenting. METHODS: The surgical procedures included 52 metal-based flap urethroplasty (Mathieu) for coronal, subcoronal, and distal shaft hypospadias; 32 transverse island pedicle graft (Duckett) for mid and proximal shaft hypospadias; 21 transverse island pedicle (Duckett) plus rolled midline tube (Thierchs) for penoscrotal and scrotal hypospadias. To accomplish urinary drainage, suprapubic diversion (cystofix) was used in 28 of 52 Mathieu operations, in 17 of 32 Duckett operations, and in 11 of 21 transverse island pedicle graft plus rolled midline tube operations. In the rest of the cases, transurethral drainage with stenting was used. RESULTS: All children had excellent cosmetic and functional outcomes. But the rates of complications such as fistula and meatal stenosis were significantly different between the groups in which suprapubic tube or urethral stent was used. In 56 of the 105 patients in whom suprapubic diversion was used, four (7.14%) had fistulas and three (5.35%) had meatal stenosis, in contrast to a fistula rate of 14.28% and meatal stenosis rate of 12.24% in patients that urethral stent is used for urinary drainage. CONCLUSION: The authors believe that the use of suprapubic diversion is advantageous for the outcome of one-stage hypospadias repair in relation to fistula occurrence and meatal stenosis.     

Immunological detection of membrane-associated human luteinizing hormone correlates with gene expression in cultured human cancer and fetal cells

We have demonstrated the expression of membrane-associated hCG and its subunits and fragments by cells from 78 human cancer cell lines of different types and origins, indicating that such expression is a common phenotypic characteristic of cultured human malignant cells. Because human (h) LH beta has 80% homology with hCG beta and is coded by one of the seven genes in the gene cluster located in chromosome 19, it was important to determine whether hLH and its beta-subunit are also expressed as membrane-associated proteins by cells from human cancer cell lines. Thus, 11 cancer cell lines of different types and origins were adapted to grow in serumless medium, with Nutridoma-HU or SP as serum substitute, and analyzed by flow cytometry using two monoclonal antibodies directed to different conformational epitopes of intact hLH and a monoclonal antibody reacting with an epitope of hLH beta-free. The cells were also analyzed simultaneously for the expression of hCG and its subunits and fragments. Determination of translatable levels of hLH beta and hCG beta messenger RNAs (mRNAs) was performed in cells from some of the cancer cell lines, including the JEG-3 choriocarcinoma cell line, and in cells from a human fetal lung cell line. The analytical flow cytometry studies showed that in addition to the expression of membrane-associated hCG in all of its forms, expression of membrane-associated intact (holo) hLH and its free beta-subunit occurred in every case. These findings were corroborated by the presence of translatable levels of hLH beta and hCG beta mRNAs in all of the cancer cell lines analyzed, indicating that the expression of these membrane-associated glycoproteins is a phenotypic characteristic of human cancer cells and that the activation of the hCG beta-hLH beta gene cluster is nonselective. The presence of translatable levels of hCG beta-hLH beta mRNAs in the cultured human fetal lung cells punctuates once more the in vivo and in vitro biochemical similarities between fetal and cancer cells.     

Hair analysis does not support hypothesized arsenic and chromium exposure from drinking water in Woburn, Massachusetts

We hypothesized that residents of Woburn, Massachusetts, had been exposed to as much as 70 microg/l of arsenic (As) and 240 microg/l of chromium (Cr) in drinking water from municipal supply wells G and H. To test this hypothesis, we measured the concentrations of As and Cr in 82 hair samples donated by 56 Woburn residents. Thirty-six samples were cut between 1964 and 1979, the period during which wells G and H were in operation. The remainder were cut either before 1964 (1938-1963; n = 26) or after 1979 (1982-1994; n = 20). Washed hair samples were analyzed by instrumental neutron activation. Exposure to the well water--measured as access--was estimated using well pumping records and a model of the Woburn water distribution system. Our results show that access to wells G and H water was not significantly correlated (95% confidence interval) with As and Cr concentrations measured in the hair of Woburn residents, but As concentrations have declined significantly over the last half century. Linear regression of As concentrations (micrograms per gram) upon year of hair cut and access to wells G and H water yielded a standard coefficient for year of -0. 0074 +/- 0.0017 (standard error; p = 2.5 -multiple- 10(-5)) and -0.12 +/- 0.10 (p = 0.22) for access. The r2 value for the model was 0.19. The geometric mean concentrations (geometric standard deviation) of As and Cr in the hair of residents who had access (i.e., relative access estimate >0) to wells G and H water (n = 27) were 0.14 (2.6) and 2.29 (1.8) microg/g, respectively; the geometric mean concentrations of As and Cr in all of the hair samples from residents who did not have access (1938-1994; n = 55) were 0.13 (3.0) and 2.19 (2.0) microg/g, respectively.