ECF-Type ATP-Binding Cassette Transporters.

Energy-coupling factor (ECF)-type ATP-binding cassette (ABC) transporters catalyze membrane transport of micronutrients in prokaryotes. Crystal structures and biochemical characterization have revealed that ECF transporters are mechanistically distinct from other ABC transport systems. Notably, ECF transporters make use of small integral membrane subunits (S-components) that are predicted to topple over in the membrane when carrying the bound substrate from the extracellular side of the bilayer to the cytosol. Here, we review the phylogenetic diversity of ECF transporters as well as recent structural and biochemical advancements that have led to the postulation of conceptually different mechanistic models. These models can be described as power stroke and thermal ratchet. Structural data indicate that the lipid composition and bilayer structure are likely to have great impact on the transport function. We argue that study of ECF transporters could lead to generic insight into membrane protein structure, dynamics, and interaction.

A mycobacterial ABC transporter mediates the uptake of hydrophilic compounds.

Mycobacterium tuberculosis (Mtb) is an obligate human pathogen and the causative agent of tuberculosis1-3. Although Mtb can synthesize vitamin B12 (cobalamin) de novo, uptake of cobalamin has been linked to pathogenesis of tuberculosis2. Mtb does not encode any characterized cobalamin transporter4-6; however, the gene rv1819c was found to be essential for uptake of cobalamin1. This result is difficult to reconcile with the original annotation of Rv1819c as a protein implicated in the transport of antimicrobial peptides such as bleomycin7. In addition, uptake of cobalamin seems inconsistent with the amino acid sequence, which suggests that Rv1819c has a bacterial ATP-binding cassette (ABC)-exporter fold1. Here, we present structures of Rv1819c, which reveal that the protein indeed contains the ABC-exporter fold, as well as a large water-filled cavity of about 7,700 Å3, which enables the protein to transport the unrelated hydrophilic compounds bleomycin and cobalamin. On the basis of these structures, we propose that Rv1819c is a multi-solute transporter for hydrophilic molecules, analogous to the multidrug exporters of the ABC transporter family, which pump out structurally diverse hydrophobic compounds from cells8-11.

Attention-deficit/hyperactivity disorder and other neurodevelopmental disorders in offspring of parents with depression and bipolar disorder.

BACKGROUND: Offspring of parents with major mood disorders (MDDs) are at increased risk for early psychopathology. We aim to compare the rates of neurodevelopmental disorders in offspring of parents with bipolar disorder, major depressive disorder, and controls. METHOD: We established a lifetime diagnosis of neurodevelopmental disorders [attention-deficit/hyperactivity disorder (ADHD), autism spectrum disorder, communication disorders, intellectual disabilities, specific learning disorders, and motor disorders] using the Kiddie Schedule for Affective Disorders and Schizophrenia, Present and Lifetime Version in 400 participants (mean age 11.3 + s.d. 3.9 years), including 93 offspring of parents with bipolar disorder, 182 offspring of parents with major depressive disorder, and 125 control offspring of parents with no mood disorder. RESULTS: Neurodevelopmental disorders were elevated in offspring of parents with bipolar disorder [odds ratio (OR) 2.34, 95% confidence interval (CI) 1.23-4.47, p = 0.010] and major depressive disorder (OR 1.87, 95% CI 1.03-3.39, p = 0.035) compared to controls. This difference was driven by the rates of ADHD, which were highest among offspring of parents with bipolar disorder (30.1%), intermediate in offspring of parents with major depressive disorder (24.2%), and lowest in controls (14.4%). There were no significant differences in frequencies of other neurodevelopmental disorders between the three groups. Chronic course of mood disorder in parents was associated with higher rates of any neurodevelopmental disorder and higher rates of ADHD in offspring. CONCLUSIONS: Our findings suggest monitoring for ADHD and other neurodevelopmental disorders in offspring of parents with MDDs may be indicated to improve early diagnosis and treatment.

Observed psychopathology in offspring of parents with major depressive disorder, bipolar disorder and schizophrenia.

BACKGROUND: Children of parents with mood and psychotic disorders are at elevated risk for a range of behavioral and emotional problems. However, as the usual reporter of psychopathology in children is the parent, reports of early problems in children of parents with mood and psychotic disorders may be biased by the parents' own experience of mental illness and their mental state. METHODS: Independent observers rated psychopathology using the Test Observation Form in 378 children and youth between the ages of 4 and 24 (mean = 11.01, s.d. = 4.40) who had a parent with major depressive disorder, bipolar disorder, schizophrenia, or no history of mood and psychotic disorders. RESULTS: Observed attentional problems were elevated in offspring of parents with major depressive disorder, bipolar disorder and schizophrenia (effect sizes ranging between 0.31 and 0.56). Oppositional behavior and language/thought problems showed variable degrees of elevation (effect sizes 0.17 to 0.57) across the three high-risk groups, with the greatest difficulties observed in offspring of parents with bipolar disorder. Observed anxiety was increased in offspring of parents with major depressive disorder and bipolar disorder (effect sizes 0.19 and 0.25 respectively) but not in offspring of parents with schizophrenia. CONCLUSIONS: Our results suggest that externalizing problems and cognitive and language difficulties may represent a general manifestation of familial risk for mood and psychotic disorders, while anxiety may be a specific marker of liability for mood disorders. Observer assessment may improve early identification of risk and selection of youth who may benefit from targeted prevention.

Low-temperature decomposition and segregation on a surface in carbide-containing solid solutions of the zirconium-niobium-carbon system and in related ternary systems.

The crystal structure and microstructure of pseudobinary (ZrC0.96)1-x(NbC0.97)x carbide solid solutions has been studied. It was found that monocrystalline grains of zirconium carbide are spontaneously isolated on the surface of diluted solid solutions of the pseudobinary system ZrCy-NbCy' containing less than 2.0 mol% of zirconium carbide. It is shown that the appearance of zirconium carbide is a consequence of the solid-phase decomposition of these solid solutions and anisotropy of elastic properties of monocrystalline ZrC carbide grains. The model of subregular solutions was used in the temperature interval from 300 to 3900 K to calculate and plot an equilibrium phase diagram of the ternary Zr-Nb-C system. It is shown that at temperatures above 1210 K in the Zr-Nb-C ternary system, nonstoichiometric carbides ZrCy and NbCy' have unlimited mutual solubility and form a continuous series of (ZrCy)1-x(NbCy')x solid solutions with 0.6 ≤ y ≤ 0.98, 0.7 ≤ y' ≤ 1.0, and 0 ≤ x ≤ 1.0. At temperatures below 1200 K, under equilibrium conditions, a discontinuity of the miscibility of the solid solutions ZrCy-NbCy' is observed and there appears a region of solid phase decomposition. The anisotropy of elastic properties of monocrystalline grains of ZrC was considered. It is predicted that solid-phase decomposition and surface segregation can be observed in such related carbide systems as HfCy-NbCy', HfCy-TaCy', ZrCy-TaCy', VCy-TaCy' and VCy-NbCy'.

Deep machine learning interatomic potential for liquid silica.

The use of machine learning to develop neural network potentials (NNP) representing the interatomic potential energy surface allows us to achieve an optimal balance between accuracy and efficiency in computer simulation of materials. A key point in developing such potentials is the preparation of a training dataset of ab initio trajectories. Here we apply a deep potential molecular dynamics (DeePMD) approach to develop NNP for silica, which is the representative glassformer widely used as a model system for simulating network-forming liquids and glasses. We show that the use of a relatively small training dataset of high-temperature ab initio configurations is enough to fabricate NNP, which describes well both structural and dynamical properties of liquid silica. In particular, we calculate the pair correlation functions, angular distribution function, velocity autocorrelation functions, vibrational density of states, and mean-square displacement and reveal a close agreement with ab initio data. We show that NNP allows us to expand significantly the time-space scales achievable in simulations and thus calculating dynamical and transport properties with more accuracy than that for ab initio methods. We find that developed NNP allows us to describe the structure of the glassy silica with satisfactory accuracy even though no low-temperature configurations were included in the training procedure. The results obtained open up prospects for simulating structural and dynamical properties of liquids and glasses via NNP.

Effectiveness of hand hygiene practices in preventing influenza virus infection in the community setting: A systematic review.

BACKGROUND: Hand hygiene is known to be an effective infection prevention and control measure in health care settings. However, the effectiveness of hand hygiene practices in preventing influenza infection and transmission in the community setting is not clear. OBJECTIVE: To identify, review and synthesize available evidence on the effectiveness of hand hygiene in preventing laboratory-confirmed or possible influenza infection and transmission in the community setting. METHODS: A systematic review protocol was established prior to conducting the review. Three electronic databases (MEDLINE, Embase and the Cochrane Library) were searched to identify relevant studies. Two reviewers independently screened the titles, abstracts and full-texts of studies retrieved from the database searches for potential eligibility. Data extraction and quality assessment of included studies were performed by a single reviewer and validated by a second reviewer. Included studies were synthesized and analyzed narratively. RESULTS: A total of 16 studies were included for review. Studies were of low methodological quality and there was high variability in study design, setting, context and outcome measures. Nine studies evaluated the effectiveness of hand hygiene interventions or practices in preventing laboratory-confirmed or possible influenza infection in the community setting; six studies showed a significant difference, three studies did not. Seven studies assessed the effectiveness of hand hygiene practices in preventing laboratory-confirmed or possible influenza transmission in the community setting; two studies found a significant difference and five studies did not. CONCLUSION: The effectiveness of hand hygiene against influenza virus infection and transmission in the community setting is difficult to determine based on the available evidence. In light of its proven effectiveness in other settings, there is no compelling evidence to stop using good hand hygiene practice to reduce the risk of influenza infection and transmission in the community setting.

PIKE-A is a proto-oncogene promoting cell growth, transformation and invasion.

PIKE-A (phosphoinositide 3-kinases (PI 3)-kinase enhancer) is a ubiquitously expressed GTPase, which binds to and enhances protein kinase B (Akt) kinase activity in a guanine nucleotide-dependent manner. PIKE-A is one of the components of the CDK4 amplicon that is amplified in numerous human cancers. However, whether PIKE-A itself can mediate cell transformation, proliferation and migration remains unknown. Here, we show that PIKE-A is overexpressed in various human cancer samples, escalates U87MG glioblastoma invasion and provokes NIH3T3 cell transformation. Overexpression of wild-type (WT) PIKE-A enhances NIH3T3 and U87MG cell growth, which is further increased by cancer cell-derived PIKE-A active mutants. In contrast, both the dominant-negative mutant and the phosphoinositide lipids interaction-defective mutant antagonize cell proliferation. Moreover, PIKE-A and its active and inactive mutants similarly enhance or antagonize U87MG cell survival and invasion, and their ability to do so is coupled with the catalytic effect they have on Akt activation. Furthermore, PIKE-A WT and its active mutants significantly elicit NIH3T3 cell transformation. Thus, our findings support the concept that PIKE-A acts as a proto-oncogene, promoting cell transformation through Akt activation.

Cysteine-mediated decyanation of vitamin B12 by the predicted membrane transporter BtuM.

Uptake of vitamin B12 is essential for many prokaryotes, but in most cases the membrane proteins involved are yet to be identified. We present the biochemical characterization and high-resolution crystal structure of BtuM, a predicted bacterial vitamin B12 uptake system. BtuM binds vitamin B12 in its base-off conformation, with a cysteine residue as axial ligand of the corrin cobalt ion. Spectroscopic analysis indicates that the unusual thiolate coordination allows for decyanation of vitamin B12. Chemical modification of the substrate is a property other characterized vitamin B12-transport proteins do not exhibit.

Loss of heterozygosity for loci on chromosome 10 is associated with morphologically malignant meningioma progression.

Meningioma is a common tumor of the central nervous system which displays morphological heterogeneity. In order to determine whether this phenotypic variability is associated with distinct or overlapping genetic lesions, we compared genotypes at several loci defined by allele length polymorphism in tumor and normal tissues from patients with meningioma. In particular, we concentrated on loci on chromosomes 22 and 10 because these genomic regions have previously been shown to be altered in the former in sporadic and familial meningiomas and in the latter as a late stage event in progression of another common brain tumor, astrocytoma. We examined 38 tumors which were classified as benign, atypical, or malignant by morphological criteria, invasive characteristics, or both. We found that loss of heterozygosity (LOH) for loci on chromosome 22 occurred in 5 of 15 benign, 2 of 2 atypical, and 5 of 10 malignant meningiomas. Similar alterations of chromosome 10 were found in 0 of 20 benign, 1 of 2 atypical, and 4 of 13 malignant meningiomas. Among the malignant tumors, LOH for loci on chromosome 10 occurred in 2 of 4 morphologically malignant tumors and in 2 of 4 morphologically and invasively malignant tumors. In contrast, LOH was not observed for any of the 5 informative tumors classified as malignant by invasive characteristics only. LOH for loci on chromosome 22 accompanied (but was not restricted to) allelic loss of loci on chromosome 10. These data suggest that the progression of meningiomas from arachnoidal cells to the morphologically malignant phenotype may, in part, entail the loss of a tumor suppressor gene(s) on chromosome 22 early in the process and that this may be compounded by alterations of chromosome 10, the LOH of which is associated with morphological signs of malignancy.

Turcot's syndrome of glioma and polyposis occurs in the absence of germ line mutations of exons 5 to 9 of the p53 gene.

The term "Turcot's syndrome" has been used to describe approximatively 55 patients with an association of colonic polyposis and primary neuroepithelial tumors of the central nervous system. The p53 tumor suppressor gene is a possible candidate underlying the syndrome because (a) mutations in the p53 gene are ubiquitous in human cancer, including colon carcinoma and gliomas, and (b) somatic or germ line mutations of the p53 tumor suppressor gene cause the Li-Fraumeni syndrome, which is characterized by the association of breast and soft tissue tumors. We determined the DNA sequence of the conserved regions of the p53 gene (exons 5 to 9) in the tumor tissues and lymphocytes of two patients with glioma-polyposis and found that mutations did occur as independent tumor-specific alterations but did not involve the germ line of these patients, suggesting that p53 may play a role in progression but not initiation of the disease.

Cathepsin B expression and localization in glioma progression and invasion.

The poor prognosis of human malignant gliomas is due to their invasion and recurrence, the molecular mechanisms of which remain poorly characterized. We have accumulated substantial evidence implicating the cysteine protease cathepsin B in human glioma malignancy. Increases in cathepsin B expression were observed throughout progression. In primary brain tumor tissue, transcript abundance (Northern blot analysis) increased in low-grade astrocytoma to high-grade glioblastoma from 3- to 6-fold, respectively, above normal brain levels. This increase correlated with increases in protein abundance (from + to ) as measured by immunohistochemistry. Furthermore, in glioblastoma cell lines increases in transcript abundance (ranging from 3- to 12-fold) were accompanied by increases in enzyme activity (44-133 nmol/min x mg protein). Altered subcellular localization was observed both immunohistochemically and by indirect immunofluorescence confocal microscopy and was found to correlate with increased grade. In addition, this increase in cathepsin B expression and altered subcellular localization correlated with histomorphological invasion and clinical evidence of invasion as detected by magnetic resonance imaging. These data support the hypothesis that cathepsin B plays a role in human glioma progression and invasion.

Outbreak of acute hepatitis B virus infection associated with exposure to acupuncture.

BACKGROUND: The most common risk factors for acute hepatitis B virus (HBV) infection are sexual contact, injection drug use and perinatal, or nosocomial exposure. Acupuncture, used in China for over 2,500 years, has been gaining popularity as an alternative medical therapy in the western world, but when associated with poor infection control practices, is also a risk for blood-borne infections. OBJECTIVE: To describe the outbreak investigation following detection of two cases of acute HBV infection associated with acupuncture services from the same provider within four months of symptom onset. METHODS: The outbreak investigation included genotyping of HBV from the identified cases, on-site assessment of the acupuncturist's infection prevention and control practices and chart review of known clients. RESULTS: Both cases had HBV genotype D1 with an identical fingerprint and both clients had visited the clinic on the same day denying other recent risk exposures. Inspection of the acupuncturist's practice revealed high-risk re-use and inappropriate storage of disposable needles. The Regional Health Authority ordered cessation of clinic practice until infection control measures were remediated. A public service announcement and mailed notifications to clients identified from practitioner records recommended that all clients be tested for HBV, human immunodeficiency virus (HIV) and hepatitis C. CONCLUSIONS: A clear epidemiological linkage of these two acute HBV infections to the same acupuncture clinic, evidence of substandard infection control practice in the clinic and identical HBV molecular and genotypic profiles of the two cases are highly suggestive that contaminated acupuncture needles likely resulted in at least two cases of acute HBV infection. This is the first known reported transmission of HBV from acupuncturists re-use of disposable needles and the first HBV outbreak associated with exposure to acupuncture reported this century in an industrialized country. Increased provider oversight and patient education may prevent future outbreaks.

Novel regions of allelic deletion on chromosome 18p in tumors of the lung, brain and breast.

Lung cancer is now the number one cause of cancer death for both men and women. An age-adjusted analysis over the past 25 years shows that in women specifically, lung cancer incidence is on the rise. It is estimated that 10-20 genetic events including the alteration of oncogenes and tumor suppressor genes will have occurred by the time a lung tumor becomes clinically evident. In an effort to identify regions containing novel cancer genes, chromosome 18p11, a band not previously implicated in disease, was examined for loss of heterozygosity (LOH). In this study, 50 matched normal and NSCLC tumor samples were examined using six 18p11 and one 18q12.3 PCR-based polymorphic markers. In addition, LOH was examined in 29 glioblastoma pairs and 14 paired breast carcinomas. This analysis has revealed potentially two regions of LOH in 18p11 in up to 38% of the tumor samples examined. The regions of LOH identified included a 2 cm area between markers D18S59 and D18S476, and a more proximal, 25 cm region of intermediate frequency between D18S452 and D18S453. These results provide evidence for the presence of one or more potential tumor suppressor genes on the short arm of chromosome 18 which may be involved in NSCLC, brain tumors and possibly breast carcinomas as well.

SPARC: a potential diagnostic marker of invasive meningiomas.

SPARC, a secreted, extracellular matrix-associated protein implicated in the modulation of cell adhesion and migration, was evaluated as a marker for invasive meningiomas. Although the majority of meningiomas are clinically and morphologically benign, approximately 10% progress into atypical and malignant tumors, according to the standard criteria. However, a subset of meningiomas presents as histomorphologically benign tumors (WHO grade I), but they are clinically invasive. It has been suggested that these tumors should be classified as malignant, and that the patients may require adjuvant therapy and closer follow up. Unfortunately, a significant number of these tumors may not be recognized because the surgical specimen used to assess the grade of a tumor lacks the infiltrative interface with the brain, which is currently necessary to determine its invasive character. Therefore, a marker of heightened invasiveness would greatly facilitate the identification of this subset of patients. In this study, the immunohistochemical expression of SPARC in benign, noninvasive primary meningiomas was compared with its expression in invasive, aggressive, primary and recurrent meningiomas. SPARC was not expressed in the 9 benign, noninvasive tumors, but was highly expressed in the 20 invasive tumors, regardless of the grade. The findings suggest that SPARC is a potential diagnostic marker of invasive meningiomas and is capable of distinguishing the histomorphologically benign noninvasive from the histomorphologically benign but invasive meningiomas, in the absence of the infiltrative interface.

Carboxyamido-triazole induces apoptosis in bovine aortic endothelial and human glioma cells.

Carboxyamido-triazole (CAI), an inhibitor of non-voltage-gated calcium channels, has been studied in Phase I/II clinical trials following the identification of its inhibitory effects on tumor cell invasion and motility. It has also been reported to inhibit human endothelial cell proliferation, migration, and adhesion to the basement membrane. In glioma, biological assays have shown CAI to be active in inhibiting the phenotypes of invasion and angiogenesis. The exact mechanism of action is not clearly understood, although it appears to work via inhibition of calcium influx in several signal transduction pathways that inhibit cell cycle progression. Recent evidence implicates apoptosis as a contributing mechanism of chemotherapy-induced tumor cytotoxicity. Therefore, we studied the effects of CAI on apoptosis in bovine aortic endothelial cells and a human glioma cell line (U251N) using a variety of methods, including: (a) cell morphology; (b) terminal deoxynucleotidyl transferase-mediated nick end labeling analysis of in situ DNA strand breaks; (c) agarose gel electrophoresis to visualize DNA fragmentation; and (d) flow cytometry. Here we report that the kinetics of CAI-induced apoptosis in bovine aortic endothelial cells and glioma cells was determined to be both dose and time dependent in micromolar concentrations achievable in brain tissue in vivo.

Identification and localization of the cytokine SDF1 and its receptor, CXC chemokine receptor 4, to regions of necrosis and angiogenesis in human glioblastoma.

Glioblastoma multiforme (GBM) tumors display extensive histomorphological heterogeneity, with great variability in the extent of invasiveness, angiogenesis, and necrosis. The identification of genes associated with these phenotypes should further the molecular characterization, permitting better definition of glioma subsets that may ultimately lead to better treatment strategies. Therefore, we performed a differential mRNA display analysis comparing six GBM-derived primary cell cultures from patients having tumors with varied histomorphological features. We identified stromal cell-derived factor 1 (SDF1) as a gene with varied expression. SDF1 (cytokine) and CXC chemokine receptor 4 (CXCR4) interactions are implicated in modulating cell migration. They are also implicated in modulating the immune response in AIDS patients by macrophage-mediated T-cell apoptosis. GBM patients also fail to mount an immune response, although their tumors are seemingly exposed to immune cells in regions of angiogenesis, where the blood-brain barrier is absent, or in areas of necrosis. To determine whether the expression and localization of SDF1 and CXCR4 are consistent with such a role in these brain tumors, immunohistochemical analyses of these proteins were performed on normal brain and astrocytomas (grades II-IV). In normal brain tissue, low levels of SDF1 (0.5+) were observed in astrocytic processes, in neurons, and in the occasional phagocytic cells around vessels. CXCR4 expression was negative in brain tissue but was observed in phagocytic cells within the vessel lumen. In tumors, SDF1 and CXCR4 expression was colocalized when both were expressed, and SDF1 and CXCR4 expression increased with increasing tumor grade (from 0.5+ to 6+). Additionally, CXCR4 was expressed in neovessel endothelial cells. The proteins were expressed in regions of angiogenesis and degenerative, necrotic, and microcystic changes. Those tumors displaying greater amounts of these features had greater staining intensity of the proteins. The expression of SDF1 and CXCR4 did not colocalize with the proliferation marker MIB-1. Thus, our data suggest that SDF1 and CXCR4 expressions: (a) increase with increasing grade; (b) colocalize to regions within these tumors where their interaction may contribute to angiogenesis and/or modulation of the immune response; and (c) may serve to characterize subsets of GBMs.

Surveillance summary of hospitalized pediatric enterovirus D68 cases in Canada, September 2014.

BACKGROUND: Enterovirus D68 (EV-D68) has been detected infrequently and has not been associated with severe disease in Canada. In the early fall of 2014, following an unusual case increase in the United States, clusters of EV-D68 among children and some adults manifesting severe symptoms were reported in Canada. OBJECTIVE: To provide an initial epidemiological summary of pediatric cases hospitalized with EV-D68 in Canada. METHODS: A time-limited surveillance pilot was conducted collecting information on pediatric cases (less than 18 years of age) hospitalized with EV-D68 between September 1 and 30, 2014. RESULTS: In total, 268 cases were reported from Ontario (n=210), Alberta (n=45), and British Columbia (n=13). Of the 268 reported cases, 64.9% (n=174) were male; the sex difference was statistically significant (p<0.01). Age was reported for 255 cases, with a mean age for males of 5.4 years and for females of 5.3 years. For cases with data available, 6.8% (18/266) were admitted to an intensive care unit. Of those where clinical illness was recorded, respiratory illness alone was present in 98.3% (227/231), neurologic illness alone was present in 0.4% (n=1), and both illnesses were present in 0.9% of cases (n=2); cases with neither respiratory nor neurologic illness were rare (n=1). Of the 90 cases with additional clinical information available, 43.3% were reported as having asthma. No deaths were reported among the 268 cases. CONCLUSION: The EV-D68 outbreak in Canada in September 2014 represents the beginning of a novel outbreak associated with severe illness in children. These findings provide the first epidemiological summary of severe cases of EV-D68 as an emergent respiratory pathogen in Canada. The continued investigation of this pathogen is necessary to build on these results and capture the full spectrum of associated illness.

Dynein- and microtubule-mediated translocation of adenovirus serotype 5 occurs after endosomal lysis.

Modified viruses are used as gene transfer vectors because of their ability to transfer genetic material efficiently to the nucleus of a target cell. To better understand intracellular translocation of adenovirus serotype 5 (Ad), fluorophores were covalently conjugated to Ad capsids, and movement of fluorescent Ad within the cytoplasm was observed during the first hour of infection of a human lung epithelial carcinoma cell line (A549). Ad translocation was characterized with respect to its ability to achieve nuclear envelope localization as well as directed movement in the cytoplasm. Whereas Ad achieved efficient nuclear localization 60 min after infection of A549 cells under control conditions, depolymerization of the microtubule cytoskeleton by addition of 25 microM nocodazole reversibly inhibited development of nuclear localization. In contrast, depolymerization of microfilaments by addition of 1 microM cytochalasin D had no effect on nuclear localization. Direct video observation of Ad motility showed that nocodazole, but not cytochalasin D, caused a reversible decrease in rapid linear translocations of Ad in the cytoplasm of A549 cells. Microinjection of function-blocking antibodies against the microtubule-dependent motor protein, cytoplasmic dynein, but not kinesin, blocked nuclear localization of Ad, consistent with net minus end-directed motility indicated by accumulation of Ad at mitotic spindles. Fluorescence ratio imaging revealed a neutral pH in the environment of translocating Ad, leading to a model in which the interaction of Ad with an intact microtubule cytoskeleton and functional cytoplasmic dynein occurs after escape from endosomes and is a necessary prerequisite to nuclear localization of adenovirus serotype 5.

SPARC: a signal of astrocytic neoplastic transformation and reactive response in human primary and xenograft gliomas.

In an attempt to identify genetic alterations occurring early in astrocytoma progression, we performed subtractive hybridization between astrocytoma and glioblastoma cDNA libraries. We identified secreted protein acidic and rich in cysteine (SPARC), a protein implicated in cell-matrix interactions, as a gene overexpressed early in progression. Northern blot and immunohistochemical analyses indicated that transcript and protein were both elevated in all tumor specimens (grades II-IV) examined when compared with levels in normal brain. The level of SPARC expression was found to be tumor-dependent rather than grade-related. Immunohistochemically, SPARC protein was found to be overexpressed in 1) cells in the less cellularly dense regions within the tumor mass, 2) histomorphologically neoplastic-looking cells in adjacent normal brain at the tumor/brain interface, 3) neovessel endothelial cells in both the tumor and adjacent normal brain, and 4) reactive astrocytes in normal brain adjacent to tumor. Using a combination of DNA in situ hybridization and protein immunohistochemical analyses of the human/rat xenograft, SPARC expression was observed in the human glioma cells within the tumor mass, and in cells that invaded along vascular basement membranes and individually into the rat brain parenchyma, suggesting it may be an invasion-related gene. While it remains to be determined whether SPARC functionally contributes to tumor cell invasion, these data suggest that the early onset of increased SPARC expression, though complex, may serve as a signal indicative of neoplastic astrocytic transformation and reactive response to tumor-induced stress.