Nitrophenol Reduction with Silver Oxide Nanostructures as a Sustainable Approach to Environmental Remediation.

We propose silver oxide as a cost-effective and sustainable alternative to noble metals for the catalytic reduction of nitroaromatics. In the present investigation, we adopt a facile and green synthetic route for the synthesis of silver oxide nanostructures. The prepared nanostructures were found to crystallize in the cuprite phase and exhibit absorbance across the entire visible range of the electromagnetic spectrum. The catalytic potential of the silver oxide was evaluated by following the kinetics of nitrophenol reduction under ambient conditions and is observed to follow pseudo-first order kinetics with the apparent rate constant k a p p = 4 . 24 × 10 - 3 ${{k}_{app}=4.24\ \times {10}^{-3}}$ s-1 at minimum concentration of the catalyst. We attribute the observed catalytic activity to the freshly generated catalytic surface featuring a partially reduced form of silver oxide during reaction. The findings highlight the efficacy of silver oxide in mitigating the environmental pollution originating from the recalcitrant nitroarenes.

A pathogenic CtBP1 missense mutation causes altered cofactor binding and transcriptional activity.

We previously reported a pathogenic de novo p.R342W mutation in the transcriptional corepressor CTBP1 in four independent patients with neurodevelopmental disabilities [1]. Here, we report the clinical phenotypes of seven additional individuals with the same recurrent de novo CTBP1 mutation. Within this cohort, we identified consistent CtBP1-related phenotypes of intellectual disability, ataxia, hypotonia, and tooth enamel defects present in most patients. The R342W mutation in CtBP1 is located within a region implicated in a high affinity-binding cleft for CtBP-interacting proteins. Unbiased proteomic analysis demonstrated reduced interaction of several chromatin-modifying factors with the CtBP1 W342 mutant. Genome-wide transcriptome analysis in human glioblastoma cell lines expressing -CtBP1 R342 (wt) or W342 mutation revealed changes in the expression profiles of genes controlling multiple cellular processes. Patient-derived dermal fibroblasts were found to be more sensitive to apoptosis during acute glucose deprivation compared to controls. Glucose deprivation strongly activated the BH3-only pro-apoptotic gene NOXA, suggesting a link between enhanced cell death and NOXA expression in patient fibroblasts. Our results suggest that context-dependent relief of transcriptional repression of the CtBP1 mutant W342 allele may contribute to deregulation of apoptosis in target tissues of patients leading to neurodevelopmental phenotypes.

The Cellular Protein Complex Associated with a Transforming Region of E1A Contains c-MYC.

UNLABELLED: The cell-transforming activity of human adenovirus 5 (hAd5) E1A is mediated by the N-terminal half of E1A, which interacts with three different major cellular protein complexes, p300/CBP, TRRAP/p400, and pRb family members. Among these protein interactions, the interaction of pRb family proteins with conserved region 2 (CR2) of E1A is known to promote cell proliferation by deregulating the activities of E2F family transcription factors. The functional consequences of interaction with the other two protein complexes in regulating the transforming activity of E1A are not well defined. Here, we report that the E1A N-terminal region also interacted with the cellular proto-oncoprotein c-MYC and the homolog of enhancer of yellow 2 (ENY2). Our results suggested that these proteins interacted with an essential E1A transforming domain spanning amino acid residues 26 to 35 which also interacted with TRRAP and p400. Small interfering RNA (siRNA)-mediated depletion of TRRAP reduced c-MYC interaction with E1A, while p400 depletion did not. In contrast, depletion of TRRAP enhanced ENY2 interaction with E1A, suggesting that ENY2 and TRRAP may interact with E1A in a competitive manner. The same E1A region additionally interacted with the constituents of a deubiquitinase complex consisting of USP22, ATXN7, and ATXN7L3 via TRRAP. Acute short hairpin RNA (shRNA)-mediated depletion of c-MYC reduced the E1A transforming activity, while depletion of ENY2 and MAX did not. These results suggested that the association of c-MYC with E1A may, at least partially, play a role in the E1A transformation activity, independently of MAX. IMPORTANCE: The transforming region of adenovirus E1A consists of three short modules which complex with different cellular protein complexes. The mechanism by which one of the transforming modules, CR2, promotes cell proliferation, through inactivating the activities of the pRb family proteins, is better understood than the activities of the other domains. Our analysis of the E1A proteome revealed the presence of the proto-oncoprotein c-MYC and of ENY2. We mapped these interactions to a critical transforming module of E1A that was previously known to interact with the scaffolding molecule TRRAP and the E1A-binding protein p400. We showed that c-MYC interacted with E1A through TRRAP, while ENY2 interacted with it independently. The data reported here indicated that depletion of c-MYC in normal human cells reduced the transforming activity of E1A. Our result raises a novel paradigm in oncogenic transformation by a DNA viral oncogene, the E1A gene, that may exploit the activity of a cellular oncogene, the c-MYC gene, in addition to inactivation of the tumor suppressors, such as pRb.

Incidence of proximal junctional kyphosis with magnetic expansion control rods in early onset scoliosis.

BACKGROUND: The introduction of magnetic expansion control growth rods for the surgical management of EOS has gained popularity. However, there are no published studies on the incidence of proximal junctional kyphosis (PJK) using this technique. PURPOSE: The aim of this study is to report the incidence of PJK following treatment with magnetic growth rods in EOS. METHODS: Retrospective review of data from 21 cases (12 males, 9 females) over 3 years. PJK was obtained from whole spine X-rays pre-op, immediate post-op and last follow-up. Cobb angle was measured between the superior end plate of vertebra two levels above the upper instrumented vertebra (UIV) and the inferior end plate of the UIV. A difference of >10° between the pre-operative x-rays and the last follow-up X-rays was recorded as PJK. RESULTS: 6/21 (28.6 %) had proximal junctional kyphosis of more than 10° at last follow-up. Average age was 5.3. Average follow-up was 32.5 months. All the patients with PJK were syndromic. Four out of these six patients were males (66 %). Average PJK angle was 25.55°. Average pre-operative kyphosis was 52.5°. Average number of distractions was 7.4. All six patients had syndromic association. 3/6 patients (50 %) were conversion cases treated with traditional growth rods previously (TGR). None of the patients required unplanned surgery for PJK. CONCLUSION: The incidence of PJK in EOS patients treated with magnetic rods is favourably comparable to that reported with traditional growth rods. Also, children who are male, syndromic, hyperkyphotic, and younger must be monitored closely.

Functional similarity between E6 proteins of cutaneous human papillomaviruses and the adenovirus E1A tumor-restraining module.

The adenovirus E1A C-terminal region restrains oncogenic transformation through interaction with three distinct cellular protein complexes that include the DYRK1A/1B/HAN11 complex. The E6 proteins of beta-human papillomaviruses (beta-HPVs) also interact with the DYRK1/HAN11 complex. A variant of HPV5 E6 frequently found in epidermodysplasia verruciformis skin lesions interacted less efficiently with DYRK1A/HAN11. The E6 variant and E7 of HPV5 efficiently coimmortalized primary epithelial cells, suggesting that naturally arising variants may contribute potential oncogenic activities of beta-HPV E6 proteins.

Adenovirus type 5 E1A and E6 proteins of low-risk cutaneous beta-human papillomaviruses suppress cell transformation through interaction with FOXK1/K2 transcription factors.

The adenovirus (Adv) oncoprotein E1A stimulates cell proliferation and inhibits differentiation. These activities are primarily linked to the N-terminal region (exon 1) of E1A, which interacts with multiple cellular protein complexes. The C terminus (exon 2) of E1A antagonizes these processes, mediated in part through interaction with C-terminal binding proteins 1 and 2 (CtBP1/2). To identify additional cellular E1A targets that are involved in the modulation of E1A C-terminus-mediated activities, we undertook tandem affinity purification of E1A-associated proteins. Through mass spectrometric analysis, we identified several known E1A-interacting proteins as well as novel E1A targets, such as the forkhead transcription factors, FOXK1/K2. We identified a Ser/Thr-containing sequence motif in E1A that mediated interaction with FOXK1/K2. We demonstrated that the E6 proteins of two beta-human papillomaviruses (HPV14 and HPV21) associated with epidermodysplasia verruciformis also interacted with FOXK1/K2 through a motif similar to that of E1A. The E1A mutants deficient in interaction with FOXK1/K2 induced enhanced cell proliferation and oncogenic transformation. The hypertransforming activity of the mutant E1A was suppressed by HPV21 E6. An E1A-E6 chimeric protein containing the Ser/Thr domain of the E6 protein in E1A interacted efficiently with FOXK1/K2 and inhibited cell transformation. Our results suggest that targeting FOXK1/K2 may be a common mechanism for certain beta-HPVs and Adv5. E1A exon 2 mutants deficient in interaction with the dual-specificity kinases DYRK1A/1B and their cofactor HAN11 also induced increased cell proliferation and transformation. Our results suggest that the E1A C-terminal region may suppress cell proliferation and oncogenic transformation through interaction with three different cellular protein complexes: FOXK1/K2, DYRK(1A/1B)/HAN11, and CtBP1/2.

Human iPSC-Derived Neuronal Cells From CTBP1-Mutated Patients Reveal Altered Expression of Neurodevelopmental Gene Networks.

A recurrent de novo mutation in the transcriptional corepressor CTBP1 is associated with neurodevelopmental disabilities in children (Beck et al., 2016, 2019; Sommerville et al., 2017). All reported patients harbor a single recurrent de novo heterozygous missense mutation (p.R342W) within the cofactor recruitment domain of CtBP1. To investigate the transcriptional activity of the pathogenic CTBP1 mutant allele in physiologically relevant human cell models, we generated induced pluripotent stem cells (iPSC) from the dermal fibroblasts derived from patients and normal donors. The transcriptional profiles of the iPSC-derived "early" neurons were determined by RNA-sequencing. Comparison of the RNA-seq data of the neurons from patients and normal donors revealed down regulation of gene networks involved in neurodevelopment, synaptic adhesion and anti-viral (interferon) response. Consistent with the altered gene expression patterns, the patient-derived neurons exhibited morphological and electrophysiological abnormalities, and susceptibility to viral infection. Taken together, our studies using iPSC-derived neuron models provide novel insights into the pathological activities of the CTBP1 p.R342W allele.

Ingestion of subthreshold doses of environmental toxins induces ascending Parkinsonism in the rat.

Increasing evidence suggests that environmental neurotoxicants or misfolded α-synuclein generated by such neurotoxicants are transported from the gastrointestinal tract to the central nervous system via the vagus nerve, triggering degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and causing Parkinson's disease (PD). We tested the hypothesis that gastric co-administration of subthreshold doses of lectins and paraquat can recreate the pathology and behavioral manifestations of PD in rats. A solution containing paraquat + lectin was administered daily for 7 days via gastric gavage, followed by testing for Parkinsonian behavior and gastric dysmotility. At the end of the experiment, brainstem and midbrain tissues were analyzed for the presence of misfolded α-synuclein and neuronal loss in the SNpc and in the dorsal motor nucleus of the vagus (DMV). Misfolded α-synuclein was found in DMV and SNpc neurons. A significant decrease in tyrosine hydroxylase positive dopaminergic neurons was noted in the SNpc, conversely there was no apparent loss of cholinergic neurons of the DMV. Nigrovagally-evoked gastric motility was impaired in treated rats prior to the onset of parkinsonism, the motor deficits of which were improved by l-dopa treatment. Vagotomy prevented the development of parkinsonian symptoms and constrained the appearance of misfolded α-synuclein to myenteric neurons. These data demonstrate that co-administration of subthreshold doses of paraquat and lectin induces progressive, l-dopa-responsive parkinsonism that is preceded by gastric dysmotility. This novel preclinical model of environmentally triggered PD provides functional support for Braak's staging hypothesis of idiopathic PD.

A six-year observational study of 31 children with early-onset scoliosis treated using magnetically controlled growing rods with a minimum follow-up of two years.

Aims: Magnetically controlled growing rod (MCGR) systems use non-invasive spinal lengthening for the surgical treatment of early-onset scoliosis (EOS). The primary aim of this study was to evaluate the performance of these devices in the prevention of progression of the deformity. A secondary aim was to record the rate of complications. Patients and Methods: An observational study of 31 consecutive children with EOS, of whom 15 were male, who were treated between December 2011 and October 2017 was undertaken. Their mean age was 7.7 years (2 to 14). The mean follow-up was 47 months (24 to 69). Distractions were completed using the tailgating technique. The primary outcome measure was correction of the radiographic deformity. Secondary outcomes were growth, functional outcomes and complication rates. Results: The mean Cobb angle was 54° (14° to 91°) preoperatively and 37° (11° to 69°) at the latest follow-up (p < 0.001). The mean thoracic kyphosis (TK) was 45° (10° to 89°) preoperatively and 42° (9° to 84°) at the latest follow-up. The mean T1-S1 height increased from 287 mm (209 to 378) to 338 mm (240 to 427) (p < 0.001) and the mean sagittal balance reduced from 68 mm (-76 to 1470) preoperatively to 18 mm (-32 to 166) at the latest follow-up. The mean coronal balance was 3 mm (-336 to 64) preoperatively and 8 mm (-144 to 64) at the latest follow-up. The mean increase in weight and sitting and standing height at the latest follow-up was 45%, 10% and 15%, respectively. The mean Activity Scale for Kids (ASKp) scores increased in all domains, with only personal care and standing skills being significant at the latest follow-up (p = 0.02, p = 0.03). The improvements in Cobb angle, TK and T1-S1 heights were not related to gender, the aetiology of the EOS, or whether the procedure was primary or conversion from a conventional growing rod system. A total of 21 children developed 23 complications at a rate of 0.23 per patient per year. Seven developed MCGR-specific complications. Complications developed at a mean of 38 months (3 to 67) after the initial surgery and required 22 further procedures. Children who developed a complication were more likely to be younger, have syndromic EOS, and have a single-rod construct (6.9 versus 9.3 years, p = 0.034). Conclusion: The progression of EOS can be controlled using MCGRs allowing growth and improved function. Younger and syndromic children are more likely to develop complications following surgery. Cite this article: Bone Joint J 2018;100-B:1187-1200.

An NIHR-approved two-year observational study on magnetically controlled growth rods in the treatment of early onset scoliosis.

Aims: The primary aim of this study was to evaluate the performance and safety of magnetically controlled growth rods in the treatment of early onset scoliosis. Secondary aims were to evaluate the clinical outcome, the rate of further surgery, the rate of complications, and the durability of correction. Patients and Methods: We undertook an observational prospective cohort study of children with early onset scoliosis, who were recruited over a one-year period and followed up for a minimum of two years. Magnetically controlled rods were introduced in a standardized manner with distractions performed three-monthly thereafter. Adverse events which were both related and unrelated to the device were recorded. Ten children, for whom relevant key data points (such as demographic information, growth parameters, Cobb angles, and functional outcomes) were available, were recruited and followed up over the period of the study. There were five boys and five girls. Their mean age was 6.2 years (2.5 to 10). Results: The mean coronal Cobb angle improved from 57.6° (40° to 81°) preoperatively, 32.8° (28° to 46°) postoperatively, and 41° (19° to 57°) at two years. Five children had an adverse event, with four requiring return to theatre, but none were related to the device. There were no neurological complications or infections. No devices failed. One child developed a proximal junctional kyphosis. The mean gain in spinal column height from T1 to S1 was 45.4 mm (24 to 81) over the period of the study. Conclusion: Magnetically controlled growth rods provide an alternative solution to traditional growing rods in the surgical management of children with early onset scoliosis, supporting growth of the spine while controlling curve progression. Their use has clear psychosocial and economic benefits, with the reduction of the need for repeat surgery as required with traditional growing rods. Cite this article: Bone Joint J 2018;100-B:507-15.

An adenovirus 2-coded tumor antigen located on the endoplasmic reticulum and nuclear envelope is required for growth of transformed cells in Ca2+-deficient media.

Rat embryo cell lines containing the adenovirus 2 E1a region together with normal or mutant forms of the N-terminal half of the E1b region (HindIII G fragment) were generated by using a dominant selection marker, neo. Biochemically transformed cells containing a nonmutated HindIII G fragment proliferated more rapidly in Ca2+-deficient media, whereas cells containing a specific deletion within the E1b-encoded, 175-amino-acid (175R) (19-kilodalton) T-antigen gene and nontransformed cells grew at a slower rate. Furthermore, transformed cells that did not express the 175R T antigen and untransformed cells could not replicate their DNA efficiently in low-Ca2+ medium. Our results suggest that Ca2+ ions may provide an important stimulus for cell proliferation in adenovirus-transformed cells through a mechanism that involves the functions of the 175R T antigen.

Quantifying the 'law of diminishing returns' in magnetically controlled growing rods.

AIMS: Magnetically controlled growing rods (MCGRs) allow non-invasive correction of the spinal deformity in the treatment of early-onset scoliosis. Conventional growing rod systems (CGRS) need repeated surgical distractions: these are associated with the effect of the 'law of diminishing returns'. The primary aim of this study was to quantify this effect in MCGRs over sequential distractions. PATIENTS AND METHODS: A total of 35 patients with a maximum follow-up of 57 months were included in the study. There were 17 boys and 18 girls with a mean age of 7.4 years (2 to 14). True Distraction (TD) was determined by measuring the expansion gap on fluoroscopy. This was compared with Intended Distraction (ID) and expressed as the 'T/I' ratio. The T/I ratio and the Cobb angle were calculated at several time points during follow-up. RESULTS: The mean follow-up was 30 months (6 to 57). There was a significant decrease in the mean T/I ratio over time (convex rod at 3 months 0.81, sd 0.58 vs 51 months 0.17, sd 0.16, p = 0.0001; concave rod at 3 months 0.93, sd 0.67 vs 51 months 0.18, sd 0.15, p = 0.0001). A linear decline of the mean T/I ratios was noted for both convex rods (r2 = 0.90, p = 0.004) and concave rods (r2 = 0.81, p = 0.015) over 51 months. At the 24-month follow-up stage, there was a significant negative correlation between the mean T/I ratio of the concave rod with weight (r = -0.59, p = 0.01), age (r = -0.59, p = 0.01), and BMI of the child (r = -0.54, p = 0.01). CONCLUSIONS: The 'law of diminishing returns' is also seen after serial distraction using MCGR. Compared to previously published data for CGRS, there is a gradual linear decline rather than a rapid initial decline in lengthening. In older, heavier children a reduced distraction ratio in the concave rod of the MCGR device is noted over time. Cite this article: Bone Joint J 2017;99-B:1658-64.

Characterization of Chlorhexidine-Loaded Calcium-Hydroxide Microparticles as a Potential Dental Pulp-Capping Material.

This study explores the delivery of novel calcium hydroxide [Ca(OH)2] microparticles loaded with chlorhexidine (CHX) for potential dental therapeutic and preventive applications. Herein, we introduce a new approach for drug-delivery to deep dentin-surfaces in the form of drug-loaded microparticles. Unloaded Ca(OH)2 [Ca(OH)2/Blank] and CHX-loaded/Ca(OH)2 microparticles were fabricated by aqueous chemical-precipitation technique. The synthesized-microparticles were characterized in vitro for determination of surface-morphology, crystalline-features and thermal-properties examined by energy-dispersive X-ray scanning and transmission electron-microscopy (EDX-SEM/TEM), Fourier-transform infrared-spectroscopy (FTIR), X-ray diffraction (XRD), thermogravimetric analysis (TGA) and differential scanning-calorimetry (DSC). Time-related pH changes, initial antibacterial/biofilm-abilities and cytotoxicity of CHX-loaded/Ca(OH)2 microparticles were evaluated. Microparticles were delivered to dentin-surfaces with subsequent SEM examination of treated dentin-substrates. The in vitro and ex vivo CHX-release profiles were characterized. Ca(OH)2/Blank were hexagonal-shaped with highest z-average diameter whereas CHX-inclusion evidenced micro-metric spheres with distinguishable surface "rounded deposits" and a negative-shift in diameter. CHX:Ca(OH)2/50 mg exhibited maximum encapsulation-efficiency with good antibacterial and cytocompatible properties. SEM examination revealed an intact layer of microparticles on exposed dentin-surfaces with retention of spherical shape and smooth texture. Microparticles loaded on dentin-surfaces showed prolonged release of CHX indicating substantial retention on dentin-substrates. This study validated the inherent-applicability of this novel drug-delivery approach to dentin-surfaces using micro-metric CHX-loaded/Ca(OH)2 microparticles.

Mitochondrial localization of p53 during adenovirus infection and regulation of its activity by E1B-19K.

Recent results have revealed that the p53 tumor suppressor protein possesses a direct transcription-independent apoptotic activity. During apoptosis induced by genotoxic stress, a small fraction of p53 is targeted to mitochondria where it initiates apoptosis by causing mitochondrial dysfunction. In adenovirus-infected cells, the expression of E1A protein enhances the accumulation of p53 during early phases of infection and during late times after infection, it is targeted for degradation by the combined action of E1B-55K and E4-orf6 proteins. The functional significance of E1A-mediated accumulation of p53 during early phases of viral replication is not known. Our studies with isogenic epithelial cell lines that differ only on the status of p53 indicate that Ad infection induces apoptosis by p53-dependent and -independent pathways and both pathways are suppressed by E1B-19K. We show that during early phase of Ad infection, a fraction of p53 is targeted to the mitochondria. In virus infected cells, a large fraction of the viral antiapoptosis protein E1B-19K is also localized in mitochondria during early and late phases of infection. Coimmunoprecipitation analysis has revealed that p53 and E1B-19K form a complex in mitochondria. The interaction of 19K involves two noncontiguous regions located around amino-acid residues 14-15 and 123-124. On p53, the mutations within the DNA-binding domain reduce interaction with E1B-19K. Our studies also suggest that 19K may additionally complex with the multidomain mitochondrial proapoptotic protein BAK, thereby reducing the level of p53 interaction with BAK. We suggest that p53-induced apoptosis may be important for efficient cell lysis and viral spread and that E1B-19K may neutralize the apoptotic activity of p53 at multiple levels.

bfl-1, a bcl-2 homologue, suppresses p53-induced apoptosis and exhibits potent cooperative transforming activity.

The bcl-2 family of genes code for proteins that contain anti-apoptotic or pro-apoptotic activity. The human bfl-1 gene contains an open reading frame for a 175-amino acid Bcl-2 family protein. Among the various Bcl-2 family members, the Bfl-1 protein shares the highest homology with the mouse A1 protein. These two proteins share three conserved domains, Bcl homology (BH)1, BH2, and BH3, with other Bcl-2 family proteins. Unlike other Bcl-2 family members, Bfl-1 contains a GIn-rich NH2-terminal region and lacks an NH (19K homology) domain 1. We demonstrate that the Bfl-1 protein suppresses apoptosis induced by the p53 tumor suppressor protein in a manner similar to other Bcl-2 family members such as Bcl-2, Bcl-xL and EBV-BHRF1. In addition, the bfl-I gene cooperates efficiently with the Ela oncogene in transformation of primary rodent epithelial cells. Our results suggest that the human bfl-1 gene may play an important role in carcinogenesis.

Deletion of a nonconserved region of Bcl-2 confers a novel gain of function: suppression of apoptosis with concomitant cell proliferation.

The Bcl-2 protein coded by the proto-oncogene bcl-2 is expressed in a variety of embryonic and postnatal tissues and is overproduced in several types of tumours. Bcl-2 expression suppresses apoptosis induced by a multitude of stimuli in diverse cell types without exerting significant effects on cell proliferation, and is believed to contribute to oncogenesis by extending cell survival. In certain B-cell lymphomas, chromosomal translocations result in a gain of function of Bcl-2 by overexpression. Here, we report that a deletion of a nonconserved region of human Bcl-2 (residues 51-85) confers a novel gain of function that not only suppresses apoptosis induced by the tumor suppressor protein p53 and the Myc oncoprotein but also permits continued cell proliferation. Our result raises the possibility that mutations within the bcl-2 gene may contribute to oncogenesis by both suppressing apoptosis and facilitating cell proliferation.

Separation of immortalization and T24-ras oncogene cooperative functions of adenovirus E1a.

An adenovirus 2 E1a gene coding for a protein of 243 (243R) amino acids can efficiently immortalize primary rat kidney (BRK) cells and cooperate with the activated cellular ras oncogene (T24 ras). A mutant (47-0) of the 243R gene that maps between amino acid residues 47-50 within a region that is highly conserved among the various adenovirus serotypes was found to be severely defective in immortalization. Despite the defect in immortalization, mutant 47-0 had the ability to cooperate with T24 ras in oncogenic transformation. These results suggest that the immortalization and the oncogene cooperation functions of the 243R are separable. Our results further suggest that the requirement for a separate immortalization function can be circumvented by oncogenic transformation and that the immortalization of cells transformed by E1a and T24 ras may be a secondary consequence of transformation by these two oncogenes.

Functional substitution identifies a cell survival promoting domain common to adenovirus E1B 19 kDa and Bcl-2 proteins.

The adenovirus E1B 19 kDa protein provides a cell survival function during adenovirus infection and facilitates efficient virus replication by preventing premature cell death. These functions resemble those performed by the protein coded by the proto-oncogene Bcl-1. The Bcl-1 protein, which provides a survival function in cells exposed to a number of cell death-inducing stimuli, can substitute for the 19 kDa protein during adenovirus infection. Although these two survival- promoting proteins are not overtly related by primary amino acid sequence, they appear to share short homologous sequences. In order to determine if these sequence motifs constitute common functional domains, we carried out domain exchanges between the 19 kDa and Bcl-2 proteins. Our results indicate that a seven amino acid region of the Bcl-2 protein (108-YRRDFAE-114) can efficiently substitute for the corresponding 19 kDa domain (47-YKWEFEE-53). Mutagenizing this domain in Bcl-2 abolishes the survival promoting activity of Bcl-2. Substitution of the kDa sequences into Bcl-2 restores the survival promoting activity, albeit at reduced levels. Our results suggest that this domain (designated NH1) may constitute a common functional domain for the 19 kDa protein and survival promoting members of the Bcl-2 family of proteins.

Requirement of the C-terminal region of adenovirus E1a for cell transformation in cooperation with E1b.

We have previously reported that adenovirus E1a mutants lacking the C-terminal 61 or 67 amino acids were severely defective in immortalization, but cooperated more efficiently (than wt E1a) with activated T24 ras oncogene in transformation of primary rat kidney (BRK) cells (Subramanian et al., 1989; Oncogene, 4:415-420). Here, we show that in contrast to these previous results, transformation of BRK cells in cooperation with the Ad2 E1b region is dependent on the C-terminal region of E1a. Mutational analysis of the C-terminal region has revealed that a region located between residues 266 and 276 may be important for E1a/E1b cooperative transformation. Like E1a/T24 ras cooperative transformation, E1a/E1b cooperative transformation also requires two essential domains involved in the binding of two cellular proteins (300K and 105K) within the N-terminal half of E1a. E1a/E1b cooperative transformation therefore requires an additional E1a activity encoded within the C-terminal region of E1a.