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Chemoresistance is a major cause of treatment failure and poor outcome in neuroblastoma. In this study, we investigated the expression and function of dual-specificity phosphatase 26 (DUSP26), also known as mitogen-activated protein kinase phophatase-8, in human neuroblastoma. We found that DUSP26 was expressed in a majority of neuroblastoma cell lines and tissue specimens. Importantly, we found that DUSP26 promotes the resistance of human neuroblastoma to doxorubicin-induced apoptosis by acting as a p53 phosphatase to downregulate p53 tumor suppressor function in neuroblastoma cells. Inhibiting DUSP26 expression in the IMR-32 neuroblastoma cell line enhanced doxorubicin-induced p53 phosphorylation at Ser20 and Ser37, p21, Puma, Bax expression as well as apoptosis. In contrast, DUSP26 overexpression in the SK-N-SH cell line inhibited doxorubicin-induced p53 phosphorylation at Ser20 and Ser37, p21, Puma, Bax expression and apoptosis. Using in vitro and in vivo assays, we found that DUSP26 binds to p53 and dephosphorylates p53 at Ser20 and Ser37. In this report, we show that DUSP26 functions as a p53 phosphatase, which suppresses downstream p53 activity in response to genotoxic stress. This suggests that inhibition of this phosphatase may increase neuroblastoma chemosensitivity and DUSP26 is a novel therapeutic target for this aggressive pediatric malignancy.

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AIM: To determine the five-year success rates, site or sites of failure, prognostic indicators and lower lip morbidity associated with molar apicectomy using amalgam root-end filling. DESIGN: Multicentre, prospective study. SETTING: The departments of oral and maxillo-facial surgery in two district general hospitals. METHOD: One thousand and seven molar apicectomy procedures, combined with amalgam root-end filling were expedited during the period 1974-1995. A five-year review of each operated tooth was carried out or attempted between 1979-2000. RESULTS: Of the 790 (78%) operated molars successfully reviewed at 5 years or later 451 (57%) exhibited 'complete healing' and 39 (5%) 'uncertain healing'. Three hundred (38%) were classified as 'unsatisfactory healing' (failures), and these included 12 which were assumed to be of periodontal origin. Whilst longitudinal root fracture, perforation and/or infection in the furcation, periodontal disease or a non-restorable crown accounted for treatment failure and often the need to remove teeth subsequently, the study probably pointed to the apical ends of the roots rather than the furcation as being the major sites at which 'unsatisfactory healing' occurred. Mandibular first molars attracted the highest 'complete healing' rate (60%) and mandibular second molars the lowest (46%). 'Good' root canal treatment (RCT) at the outset improved the prognosis of a root-end filling (REF) whilst the absence of RCT compromised it. Cystic change pointed to a better prognosis than apical granulomatous change as did a deep compared with a shallow 'bone cuff'. Disease at the furcation suggested a worse prognosis. Teeth which showed 'complete healing' at 1 year had a 75% probability of maintaining this outcome at 5 years. Sensory disturbance of variable duration occurred in the lower lip following 20-21% of mandibular molar procedures. In the majority of cases (79-80%) this had remitted within 3 months. A permanent deficit occurred in 8 patients (1%) where the apicectomy could definitely be incriminated as causative. Four were associated with first molar apicectomy and four with second molar apicectomy. CONCLUSIONS: Molar apicectomy with amalgam root-end filling attracts an overall 'complete healing' rate at 5 years of 57%, the results being best with mandibular first molars and worst with mandibular second molars. The prognosis is also better where there is 'good' initial orthograde root filling, an associated radicular cyst as compared with granulomatous change and where the buccal sulcus is deep rather than shallow. It is worse when orthograde root filling is absent and when there is disease in the furcation. 'Complete healing' at 1 year can be expected to be maintained at 5 years in 75% of cases. The commonest site of subsequent periradicular rarefaction seems to be 'apical' whilst failure at the furcation is probably comparatively rare. There is a threefold increase in the occurrence of permanent lower lip sensory impairment following second molar surgery in comparison with first molar surgery, the overall incidence being 1%.

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The present study identified and characterized a unique operon (spl) encoding six serine protease-like proteins. In addition, native Spl proteins were isolated and characterized. Typical of most exoproteins, the spl gene products contain putative 35- or 36-amino-acid signal peptides. The Spl proteins share 44 to 95% amino acid sequence identity with each other and 33 to 36% sequence identity with V8 protease. They also contain amino acids found in catalytic triads of enzymes in the trypsin-like serine protease family, and SplB and SplC were shown to degrade casein. The spl operon is transcribed on a 5.5-kb transcript, but several nonrandom degradation products of this transcript were also identified. Similar to other S. aureus exoprotein genes, the spl operon is maximally expressed during the transition into stationary phase and is positively controlled by the Agr virulence factor regulator. The Sar regulatory system did not affect spl operon expression. PCR analysis revealed the presence of the spl operon in 64% of the S. aureus isolates tested, although one spl operon-negative isolate was shown to contain at least two of the spl genes. Finally, intraperitoneal injection of an spl operon deletion mutant revealed no major differences in virulence compared to the parental strain.

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In this study, we demonstrate that the mechanism by which Staphylococcus aureus induces apoptosis in bovine mammary epithelial (MAC-T) cells involves caspases 8 and 3, two key components of a proteolytic cascade leading to apoptosis. In addition, internalized S. aureus induces expression of the inflammatory cytokines tumor necrosis factor alpha and interleukin-1beta by MAC-T cells. These data suggest that the internalization of S. aureus could induce specific cellular responses in vivo that may ultimately impact the course of infection.

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Staphylococcus aureus was recently shown to be internalized by and to induce apoptosis in a bovine mammary epithelial cell line, suggesting that these processes could be involved in staphylococcal pathogenesis or persistence. To examine the role of virulence factor regulators during internalization, mutant agr and sar strains of S. aureus were analyzed for their abilities to enter and induce apoptosis in epithelial cells. Like a previously characterized bovine mastitis isolate, the standard laboratory strain, RN6390 (wild type), entered the epithelial cells and subsequently induced apoptosis. In contrast, the mutant strains RN6911 (agr), ALC136 (sar), and ALC135 (agr sar) were internalized by the cultured cells at levels reproducibly greater than that for RN6390 but failed to induce apoptosis. The internalization of S. aureus was affected by growth phase, suggesting a role for agr-regulated surface proteins in this process. Furthermore, the ability to induce apoptosis required metabolically active intracellular bacteria. These data indicate that the ability of S. aureus to enter mammalian cells and induce apoptosis is dependent on factors regulated by Agr and Sar. Since transcriptional control by these global regulators is mediated by quorum-sensing and environmental factors, staphylococci may have the potential to induce several alternative effects on cells from an intracellular environment. A model for the function of the agr locus in the context of internalization, intracellular persistence, and dissemination is proposed.

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Calsequestrin, the major Ca2+ storage protein of muscle, coordinately binds and releases 40-50 Ca2+ ions per molecule for each contraction-relaxation cycle by an uncertain mechanism. We have determined the structure of rabbit skeletal muscle calsequestrin. Three very negative thioredoxin-like domains surround a hydrophilic center. Each monomer makes two extensive dimerization contacts, both of which involve the approach of many negative groups. This structure suggests a mechanism by which calsequestrin may achieve high capacity Ca2+ binding. The suggested mechanism involves Ca2+-induced collapse of the three domains and polymerization of calsequestrin monomers arising from three factors: N-terminal arm exchange, helix-helix contacts and Ca2+ cross bridges. This proposed structure-based mechanism accounts for the observed coupling of high capacity Ca2+ binding with protein precipitation.

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We examined the invasion of an established bovine mammary epithelial cell line (MAC-T) by a Staphylococcus aureus mastitis isolate to study the potential role of intracellular survival in the persistence of staphylococcal infections. S. aureus cells displayed dose-dependent invasion of MAC-T cells and intracellular survival. An electron microscopic examination of infected cells indicated that the bacteria induced internalization via a mechanism involving membrane pseudopod formation and then escaped into the cytoplasm following lysis of the endosomal membrane. Two hours after the internalization of S. aureus, MAC-T cells exhibited detachment from the matrix, rounding, a mottled cell membrane, and vacuolization of the cytoplasm, all of which are indicative of cells undergoing programmed cell death (apoptosis). By 18 h, the majority of the MAC-T cell population exhibited an apoptotic morphology. Other evidence for apoptosis was the generation of MAC-T cell DNA fragments differing in size by increments of approximately 180 bp and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling of the fragmented nuclear DNA of the infected host cells. These results demonstrate that after internalization S. aureus escapes the endosome and induces apoptosis in nonprofessional phagocytes.

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A 56kDa protein with high similarity in its N-terminal amino acid sequence to animal calreticulin and 100% homology with the N-terminal amino acids of spinach calreticulin has been identified in seeds of the pea plant (Pisum sativum). A new purification procedure is described by which the calreticulin-like protein was selectively solubilized by incubation with deoxycholate and HgCl2 from microsomes enriched for endoplasmic reticulum. Following Mono Q ion exchange chromatography of the deoxycholate extract by fast protein liquid chromatography, the calreticulin-like protein was obtained in nearly pure form. This purified protein is similar to animal calreticulin in apparent mass, characteristic blue staining with Stains-all dye and calcium-binding ability. In addition, this protein is recognized only by affinity purified antibodies against rabbit calreticulin and is not recognized by anti-calsequestrin antibodies. Our data suggested that calreticulin rather than calsequestrin functions as the Ca(2+)-storage protein in the endoplasmic reticulum of pea plants.

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Ovine pregnancy-specific protein B (oPSPB) was isolated from sheep placentas. Antiserum to oPSPB was developed in rabbits. A quantitative RIA was developed and used to assay the serum concentrations of oPSPB during and after pregnancy in ewes bearing single or twin fetuses. Suffolk and Panama ewes, kept with rams equipped with a marking harness, were checked daily for breeding marks as an indication of estrus and bled daily between 10 and 30 d after marking. Ovine PSPB became detectable at 19.7 +/- .14 (mean +/- SE) d after breeding and increased steadily to d 30. Panama oPSPB concentration increased at a greater rate than that of Suffolks (breed x day interaction, P < .01). Ten ewes were bled twice weekly 3 wk before their expected date of lambing and weekly for 7 wk postpartum. Serum concentrations differed considerably between prepartum ewes, but concentrations remained stable within the period of 20 d prepartum. Following parturition, oPSPB concentrations dropped rapidly. In nine ewes, oPSPB was last detectable at 12.78 +/- 2.26 (mean +/- SE) d postpartum. In the 10th ewe, oPSPB was .65 ng/mL at the last sample on d 46 postpartum. To determine the effect of fetal number on oPSPB concentrations, ewes in which estrus was synchronized were bled at d 18, 25, 38, 60, 90, and 120 after breeding. Ewes were killed at d 60, 90, 120, and 148 and fetal number determined. There was a significant (P < .01) difference in the log of oPSPB concentrations according to number of fetuses, day postbreeding, and the day x fetal number interaction.(ABSTRACT TRUNCATED AT 250 WORDS)

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A recombinant gene fusion was created and cloned using a previously constructed gene encoding a monodomain IgG Fc binding protein and the gene coding for bacterial alkaline phosphatase. The construct was able to express and secrete a fusion protein that exhibited both IgG binding and alkaline phosphatase enzymatic activities. Greater than 60% of the protein demonstrating both biological activities was detected from periplasmic space preparations. Nanogram concentrations of the Fc binding--alkaline phosphatase fusion protein allowed primary IgG antibody detection without the use of conjugated secondary antibodies. Removal of the domain coding for alkaline phosphatase resulted in decreased resistance of the protein to proteolytic degradation and the loss of IgG Fc binding ability. Using affinity-purified fusion protein, the specificity of binding to IgG, IgM and IgA was examined; binding was strong to IgG and barely detectable against IgM or IgA. Affinity for binding of the fusion protein to IgG (Kd = 6.7 x 10(-8) M) was determined to be equal to or greater than previously reported for protein A.

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Calsequestrin is an intermediate affinity, high capacity Ca(2+)-binding protein found in the lumen of the sarcoplasmic reticulum of both skeletal and cardiac muscle cells. Previous sequence analysis suggested that calsequestrin may contain a hydrophobic binding site for the drug trifluoperazine, a site shared by the calmodulin family and shown to play a role in calmodulin/calmodulin receptor interaction. Previous studies showed that, upon Ca2+ binding, calsequestrin undergoes a conformational change, burying the trifluoperazine-binding site, folding into a more compact structure that is trypsin-resistant, and increasing the negative ellipticity of the circular dichroism spectrum. In this study, the structural and functional roles of the trifluoperazine-binding site in the Ca(2+)-induced conformational change of calsequestrin are further studied using the calmodulin antagonists trifluoperazine and melittin. If trifluoperazine or melittin is added to calsequestrin prior to Ca2+ addition, then Ca(2+)-induced folding is inhibited as determined by the changes in circular dichroism spectra and protein sensitivity to trypsin digestion. If, however, Ca2+ is added prior to trifluoperazine or melittin, calsequestrin remains resistant to trypsin digestion, just as if the calmodulin antagonists are not present, suggesting that the conformational change is not affected. Aggregates of calsequestrin that exhibit high Ca2+ binding capacity have previously been shown to occur at high Ca2+ and calsequestrin concentrations. By preventing a prerequisite folding step, trifluoperazine or melittin also prevents the Ca(2+)-induced aggregation of calsequestrin, thus decreasing the maximal Ca2+ binding by calsequestrin. These data suggest that the trifluoperazine-binding site is critically involved in the Ca(2+)-induced intramolecular folding step required for the intermolecular interactions leading to high capacity Ca(2+)-binding by calsequestrin.

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A semi-isolated cockroach heart preparation was used to rapidly determine the activity of cobra cardiotoxin, monitored as a direct response on heart rate. This preparation produced a dose-response curve in the presence of active cardiotoxin and demonstrated that cardiotoxin retained its biological activity after boiling, although cardiotoxin activity was destroyed by heating in the presence of dithiothreitol. Experiments that cross-linked radiolabeled cardiotoxin to solubilized cockroach heart membranes suggested that cardiotoxin bound specifically to a 59,000 mol. wt membrane protein in this tissue.

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This investigation focused on the effects of two independent variables: (a) teacher-developed goals and monitoring systems versus a curriculum-based measurement (CBM) goal and monitoring system; and (b) individual expert versus group follow-up consultation. The dependent data were academic achievement measures. Subjects were 55 special education, elementary school students with mild and moderate disabilities randomly assigned to one of four treatment conditions: A, teacher-developed goal and progress monitoring with individual expert follow-up consultation; B, CBM goal and progress monitoring with individual expert follow-up consultation; C, teacher-developed goal and progress monitoring with group follow-up consultation; and D, CBM goal and progress monitoring with group follow-up consultation. Results showed that groups employing CBM and group consultation generally out-performed the other groups. Implications included expanded use of CBM goals and progress monitoring and continued study of collaboration as a method of CBM program implementation.

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The bronchodilator and side effects of fenoterol and isoproterenol were evaluated in 19 asthmatic adults in a double-blind study. The study demonstrated that fenoterol has an onset of action which is just as rapid as isoproterenol and a duration of action that is markedly superior. Side effects were minimal.

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