Deletion of creB in Aspergillus oryzae increases secreted hydrolytic enzyme activity.

Aspergillus oryzae has been used in the food and beverage industry for centuries, and industrial strains have been produced by multiple rounds of selection. Targeted gene deletion technology is particularly useful for strain improvement in such strains, particularly when they do not have a well-characterized meiotic cycle. Phenotypes of an Aspergillus nidulans strain null for the CreB deubiquitinating enzyme include effects on growth and repression, including increased activity levels of various enzymes. We show that Aspergillus oryzae contains a functional homologue of the CreB deubiquitinating enzyme and that a null strain shows increased activity levels of industrially important secreted enzymes, including cellulases, xylanases, amylases, and proteases, as well as alleviated inhibition of spore germination on glucose medium. Reverse transcription-quantitative PCR (RT-qPCR) analysis showed that the increased levels of enzyme activity in both Aspergillus nidulans and Aspergillus oryzae are mirrored at the transcript level, indicating transcriptional regulation. We report that Aspergillus oryzae DAR3699, originally isolated from soy fermentation, has a similar phenotype to that of a creB deletion mutant of the RIB40 strain, and it contains a mutation in the creB gene. Collectively, the results for Aspergillus oryzae, Aspergillus nidulans, Trichoderma reesei, and Penicillium decumbens show that deletion of creB may be broadly useful in diverse fungi for increasing production of a variety of enzymes.

Persistent increased DNA-binding and expression of serum response factor occur with epilepsy-associated long-term plasticity changes.

We have previously shown that NMDA receptor activation during status epilepticus (SE) is required to produce epilepsy in in vitro and in vivo models. As in human symptomatic epilepsy, the epilepsy in these models is permanent, suggesting that the pathological activation of NMDA receptors causes permanent plasticity changes in the brain. Ca(2+) influx through NMDA receptors is known to transiently activate a key transcription factor, serum response factor (SRF). Thus, we investigated whether this factor, in terms of its expression and ability to bind to the consensus serum response element, was altered long term in the pilocarpine model of epilepsy. In hippocampal nuclear extracts, SRF binding to DNA was significantly increased over saline-injected control rats at 24 hr and at 8 weeks after the onset of SE. This increase was shown to be the result of significantly elevated levels of SRF. DNA binding was also persistently increased in the cortical, but not in the cerebellar, extracts. Hippocampal expression of SRF was localized to neurons using immunohistochemistry. NMDA receptor activation during SE was required for these changes to take place, and the spontaneous seizures seen in epileptic rats did not appear to be responsible for the increase in SRF. The results demonstrate that SRF is persistently elevated after SE in the pilocarpine model of epilepsy and support the theory that long-term gene changes in this model occur and are associated with the long-lasting plasticity changes that are initiated during epileptogenesis.

Diffusing capacity is not measurably affected by routine lung perfusion scanning.

Lung scanning with radiolabeled macroaggregated albumin (MAA) has caused cardiovascular collapse and death in patients with extensive pulmonary vascular disease. These adverse reactions have been suggested to be secondary to MAA embolic occlusion of a significant portion of the remaining pulmonary circulation. The single breath diffusing capacity for carbon monoxide (SBDLCO) is heavily dependent on the status of the pulmonary microcirculation and is reduced in clinical pulmonary embolism. The effect of MAA particles on the lung microcirculation was measured by SBDLCO in 11 patients undergoing clinically indicated lung perfusion scanning. SBDLCO was measured before and immediately after injection of 256,000 to 448,000 20-40 micron particles of [99mTc]MAA. Mean SBDLCO prior to injection was 18.9 +/- 1.7 (s.e.m.) and immediately after injection was unchanged at 19.0 +/- 1.6 ml/min/mmHg. The lowest pre-injection SBDLCO values were 11.5 and 6.2 ml/min/mmHg (54% and 28% of predicted, respectively); in neither of these patients was there a detectable change in SBDLCO measured after injection of MAA. Thus occlusion of as many as 448,000 20-40 micron pulmonary vessels by MAA is without detectable impact on the transfer of carbon monoxide even in patients with sufficient pulmonary disease to lower the SBDLCO to 28% of predicted.

Localization of upstream silencer elements involved in the expression of cone transducin alpha-subunit (GNAT2).

PURPOSE: To localize cis-acting elements involved in the expression of the cone-specific G-protein, cone transducin alpha-subunit (GNAT2). METHODS: In this study, the authors used a genomic clone, HGLG3, to sequence 3139 base pairs of the upstream region of the GNAT2 gene and to localize cis-acting elements involved in the expression of GNAT2. Upstream elements were localized functionally by transfection of chloramphenicol acetyltransferase gene constructs containing nested deletions of this upstream region into WERI-Rb1 cells. Cell specificity of the localized elements was determined by transfection of the HeLa cells. Trans-acting factor-binding sites to functional cis-acting elements were determined by DNasel footprinting. Cell specificity of protein interaction with footprinted regions was tested by electrophoretic mobility shifts with nuclear extracts from WERI-Rb1 and HeLa cells. RESULTS: Transfection of WERI-Rb1 and HeLa cells revealed the presence of a strong, noncell-specific silencer region between -1130 and -23, a weak, cell-specific promoter between -151 and -10, and a stronger, noncell-specific element between +143 and +167. DNaseI footprinting showed three major footprints (S1, S2, and S3) between -807 and -176, indicating the binding sites for putative negative trans-acting factors. Individual footprinted sequences had similar electrophoretic mobility shifts when they were incubated with nuclear extracts from either WERI-Rb1 or HeLa cells, suggesting that these cells express the same negative factors. CONCLUSIONS: The expression of the GNAT2 gene is controlled by a strong silencer region, a weak upstream cell-specific promoter, and a strong downstream element. The silencer region interacts with similar proteins from retina- and nonretina-derived cell lines.

Degree of polymer organization decreases the binding of a monoclonal antibody raised against the beta-chain amino terminus of fibrin.

UNLABELLED: Accurate non-invasive diagnosis of deep venous thrombosis and pulmonary embolism remains an elusive goal. Radiolabeled antibodies specific for the epitope exposed on the beta-chain of fibrin after fibrino-peptide B release (anti-beta) enabled in situ imaging of thrombi in experimental subjects with nuclear medicine techniques. When used in patients anticoagulated for thrombo-embolic disease, however, the antibody was unable to reliably image the thrombi. We postulated that the neoepitope on the beta-chain of fibrin is covered up as fibrin organizes into a polymer network and is therefore exposed to the antibody only during active incorporation of fibrin subunits. We determined the equilibrium binding kinetics of an anti-beta monoclonal antibody to fibrin in various stages of organization. The concentration of exposed epitopes on immobilized fibrin monomers was equal to the molar concentration of fibrin beta-chains. The percentage of beta-chains exposed to the antibodies markedly decreased as the fibrin network was allowed to organize, a process catalyzed by calcium. CONCLUSIONS: The beta-chain amino terminus of fibrin is exposed transiently as subunits are added to the enlarging fibrin network. Anti-beta antibodies bind preferentially to actively enlarging fibrin polymers.

Chronic DeltaFosB expression and increased AP-1 transcription factor binding are associated with the long term plasticity changes in epilepsy.

NMDA receptor activation during status epilepticus (SE) has previously been shown to be required for epileptogenesis as well as the persistent upregulation of serum response factor (SRF) in the in vivo pilocarpine model of epilepsy. SRF is established as a regulator of the FosB gene which expresses FosB and DeltaFosB components of the AP-1 transcription factor complex. Therefore we investigated whether DeltaFosB expression and AP-1 DNA binding were also persistently elevated in pilocarpine-treated rats which chronically displayed spontaneous seizures. Using hippocampal nuclear extracts, DeltaFosB expression and AP-1 DNA binding were significantly elevated for up to one year in the epileptic animals. The expression of other fos and jun proteins was not persistently altered in epilepsy. Neuronal upregulation of DeltaFosB was correlated with regions of the brain that were involved in seizure generation and propagation. The increase in AP-1 DNA binding was shown to be dependent on NMDA receptor activation during SE. Hippocampal DeltaFosB immunostaining was seen predominately in the neuronal nuclei as opposed to other cell types. The data indicate that recurrent seizures which persistently occur in this model were not responsible for the increased DeltaFosB expression. Chronic DeltaFosB expression in epilepsy may be playing a role in the altered expression of other genes in this model and may be involved in some of the neuronal plasticity changes associated with epileptogenesis.

Parenchymal scarring is associated with restrictive spirometric defects in patients with chronic thromboembolic pulmonary hypertension.

UNLABELLED: The finding of a restrictive pulmonary defect may divert clinicians from considering the diagnosis of chronic thromboembolic pulmonary hypertension because lung volumes are usually normal in this disorder. We have, however, encountered a significant number of these patients with reduced lung volumes. Furthermore, we have observed many patients who have developed parenchymal scars and/or pleural thickening. To determine whether such findings are associated with lung volume restriction, we analyzed patients evaluated at our institution for chronic thromboembolic pulmonary hypertension over a 20-month period in whom thoracic high-resolution CT scans and pulmonary function testing had been performed. Patients with obstructive or restrictive lung disease from another cause were excluded. We compared the presence of lung restriction (total lung capacity below 80% of predicted) with the extent of parenchymal scarring, pleural thickening, and pulmonary artery diameter on CT scans. Of 191 patients evaluated, 51 met criteria for entry. Eleven patients (22%) had lung restriction. Parenchymal scarring was highly associated with lung restriction (p = 0.01). Neither pleural thickening (p = 0.08) nor pulmonary artery diameter (p = 0.80) was associated with lung restriction. CONCLUSIONS: A significant number of patients with chronic thromboembolic pulmonary hypertension may have restrictive lung defects. The restriction may be due to parenchymal scarring.

The structure of medical informatics journal literature.

OBJECTIVE: Medical informatics is an emergent interdisciplinary field described as drawing upon and contributing to both the health sciences and information sciences. The authors elucidate the disciplinary nature and internal structure of the field. DESIGN: To better understand the field's disciplinary nature, the authors examine the intercitation relationships of its journal literature. To determine its internal structure, they examined its journal cocitation patterns. MEASUREMENTS: The authors used data from the Science Citation Index (SCI) and Social Science Citation Index (SSCI) to perform intercitation studies among productive journal titles, and software routines from SPSS to perform multivariate data analyses on cocitation data for proposed core journals. RESULTS: Intercitation network analysis suggests that a core literature exists, one mark of a separate discipline. Multivariate analyses of cocitation data suggest that major focus areas within the field include biomedical engineering, biomedical computing, decision support, and education. The interpretable dimensions of multidimensional scaling maps differed for the SCI and SSCI data sets. Strong links to information science literature were not found. CONCLUSION: The authors saw indications of a core literature and of several major research fronts. The field appears to be viewed differently by authors writing in journals indexed by SCI from those writing in journals indexed by SSCI, with more emphasis placed on computers and engineering versus decision making by the former and more emphasis on theory versus application (clinical practice) by the latter.

Approaching equity in consumer health information delivery: NetWellness.

The growing public interest in health and wellness information stems from many sources, including social changes related to consumers' rights and women's health movements, and economic changes brought about by the managed health care revolution. Public, hospital, and medical center libraries have been ill-equipped to meet the increasing need for consumer-oriented materials, even though a few notable programs have been established. The "Information Superhighway" could be an effective tool for sharing health information if access to telecomputing equipment and training were available to those with an information need. The University of Cincinnati Medical Center, with its libraries in the leading role, is delivering NetWellness, an electronic consumer health library service, to residents of 29 counties in three midwestern states. Users connect directly through the Internet, through regional Free-Nets, and by visiting one of 43 public access sites where networked workstations have been installed. The continued success of the project depends on developing partnerships, providing quality content and maintaining fair access.

Anti-thrombotic efficacies of enoxaparin, dalteparin, and unfractionated heparin in venous thrombo-embolism.

BACKGROUND: Few data exist by which the anti-thrombotic efficacy of different anticoagulants may be compared. We used a radiolabeled antibody specific for polymerizing fibrin to compare the in vivo anti-thrombotic potencies of different systemic anticoagulants (enoxaparin, dalteparin, and unfractionated heparin). METHODS AND RESULTS: Deep venous thrombi (DVTs) were induced in dogs' femoral veins. The dogs were then treated with one of the following subcutaneous regimens: enoxaparin 100 units/kg (1.0 mg/kg) every 12 hours (n=4), dalteparin 200 units/kg every 24 hours (n=4), or unfractionated heparin 240 units/kg every 8 hours with dose adjustment via aPTT (n=3). 111Indium-labeled anti-fibrin antibodies, specific for propagating thrombi, were given intravenously and nuclear scans of the legs were taken over the following 24 hours. Thrombus propagation was estimated by the ratio of gamma emissions from the legs containing DVTs divided by the emissions from the contralateral "control" legs. DVTs accumulated labeled anti-fibrin antibodies at the same rates in both the enoxaparin group and the dalteparin group (gamma emissions 171+/-6% and 168+/-36% of control by 24 hours, respectively). DVTs in the adjusted dose unfractionated heparin group tended to accumulate antibodies at a slower rate (129+/-19% of control by 24 hours). CONCLUSIONS: Enoxaparin and dalteparin inhibited propagation of pre-formed thrombi to the same degree. Subcutaneous unfractionated heparin, adjusted every 8 hours by aPTT, tended to suppress ongoing thrombosis more than either LMWH.

Management of lone star ticks (Acari: Ixodidae) in recreational areas with acaricide applications, vegetative management, and exclusion of white-tailed deer.

A project on management of lone star ticks, Amblyomma americanum (L.), at Land Between the Lakes, a Tennessee Valley Authority recreational area in Kentucky-Tennessee, during 1984-1988, demonstrated the effectiveness and economics of three control technologies. Acaricide applications (chlorpyrifos at 0.28 kg [AI]/ha), vegetative management (mowing and removal of 40% overstory and 90-100% of midstory, understory, and leaf litter), and host management (white-tailed deer exclusion from a 71-ha campground with a single-line fence) provided 75, 70, and 64% mean controls of all life stages of the lone star tick, respectively. Combinations of acaricide applications + vegetative management, acaricide applications + host management, and acaricide applications + vegetative management + host management produced 94, 89, and 96% mean control of all life stages, respectively. The costs of acaricide applications (two per year), vegetative management (two mowings per year), and white-tailed deer exclusion (single-line fence) were $45, $150, and $30/ha/yr, respectively. Results of this project are used to design management strategies that could be considered for use against lone star ticks in recreational areas.

Intravascular bronchioloalveolar tumor of the lung presenting as pulmonary thromboembolic disease and pulmonary hypertension.

Intravascular bronchioloalveolar tumor, the pulmonary counterpart of epithelioid hemangioendothelioma, typically presents as bilateral pulmonary nodules in young women. We report a case of intravascular bronchioloalveolar tumor that clinically mimicked acute pulmonary thromboembolic disease initially and was subsequently proven to have pulmonary hypertension with right ventricular dysfunction by angiography. The diagnosis of intravascular bronchioloalveolar tumor was confirmed by immunohistochemical and ultrastructural studies after it was suspected on routine histologic examination. In addition, the tumor cells expressed glycoprotein cell adhesion molecule CD44, which has been implicated in increased tumor invasiveness and metastasis in various carcinomas and several aggressive non-Hodgkin's lymphomas.

Phosphine generation by mixed- and monoseptic-cultures of anaerobic bacteria.

A microbial basis for bioreductive generation of phosphine is proposed, which could account at least in part for the presence of this toxic gas in natural anaerobic environments and in sewage and landfill gases. Phosphine generation under anaerobic growth conditions was dependent upon both the culture inoculum source (animal faeces) and enrichment culture conditions. Phosphine was detected in headspace gases from mixed cultures under conditions promoting fermentative growth of mixed acid and butyric acid bacteria, either in the presence or absence of methane generation. Monoseptic cultures of certain mixed acid fermentors (Escherichia coli, Salmonella gallinarum, and Salmonella arizonae) and solvent fermentors (Clostridium sporogenes, Clostridium acetobutyricum and Clostridium cochliarium) also generated phosphine. Such fermentative bacteria participate in the multi-stage process of methanogenesis in nature. Generation of phosphine by these bacteria, rather than by methanoarchaea themselves, could explain the apparent correlation between methanogenesis and the formation of phosphine in nature.

Tinidazole or pivampicillin in third molar surgery.

There is an increasing body of evidence implicating oral anaerobic bacteria in the aetiology of post-surgical dentoalveolar infections. This information has lead to several studies demonstrating the usefulness of specific anaerobicidal drugs in the prevention and treatment of dento-alveolar infection. One such study utilised a single 2 g preoperative oral dose of tinidazole which was found to be significantly better than placebo in preventing infective sequelae after removal of impacted mandibular third molars. The present study was designed to compare a high-dose short-term broad spectrum penicillin, (pivampicillin), with the previously described regimen, using tinidazole in order to discern the existence or otherwise of any practical difference between an anaerobicidal and a broad spectrum antibiotic when local infection was considered.

Evaluation of cot mattress inner foam as a potential site for microbial generation of toxic gases.

Recent reports of biovolatilisation of phosphorus and antimony by anaerobic bacteria and of leaching of phosphorus and antimony fire-retardant additives from PVC cot mattress covers, indicate that the polyurethane inner-foam of cot mattresses could be a site for generation of toxic gases of group 15 elements. A toxic gas hypothesis for sudden infant death syndrome (SIDS) involving polyurethane foam of cot mattresses was proposed and tested experimentally. Levels of antimony, phosphorus, arsenic and bismuth were determined at four sites for 44 SIDS and 50 control (no death) cot mattress foams. There was no evidence to suggest that the levels of these elements in cot mattress foam have a causal relation to SIDS. Leaching of antimony trioxide from PVC mattress covers could account for detectable levels of this element in 52% of the cot mattress samples analysed. Volatile forms of antimony, phosphorus, arsenic and bismuth was not detected in the headspace of mixed or monoseptic cultures of anaerobic bacteria containing polyurethane foam. Past microbial activity had given rise to involatile methylated species of antimony in some of the cot mattress foams tested (61%, n = 24). Abiotic oxidation of biogenic trimethylantimony together with physical adsorption of methylantimony forms to the polyurethane foam matrix could account for the apparent absence of "escaped" volatile antimony species in culture headspaces of incubation vial. There was no evidence to suggest that levels of trimethylantimony or total methylantimony forms in cot mattress foams have a causal relation to SIDS.

Fanconi anemia deficiency stimulates HPV-associated hyperplastic growth in organotypic epithelial raft culture.

Fanconi anemia (FA) is a recessive genome instability syndrome characterized by heightened cellular sensitivity to DNA damage, aplastic anemia and cancer susceptibility. Leukemias and squamous cell carcinomas (SCCs) are the most predominant FA-associated cancers, with the latter exhibiting markedly early disease onset and aggressiveness. Although studies of hematopoietic cells derived from FA patients have provided much insight into bone marrow deficiencies and leukemogenesis, molecular transforming events in FA-deficient keratinocytes, which are the cell type of origin for SCC, are poorly understood. We describe here the growth and molecular properties of FANCA-deficient versus FANCA-corrected HPV E6/E7 immortalized keratinocytes in monolayer and organotypic epithelial raft culture. In response to DNA damage, FANCA-deficient patient-derived keratinocyte cultures displayed a G2/M phase arrest, senescence and apoptosis. Organotypic raft cultures exhibited DNA repair-associated defects with more 53BP1 foci and TdT-mediated dNTP nick end labeling-positive cells over their corrected counterparts. Interestingly, together with reduced rates of DNA damage, FA correction resulted in a marked decrease in epithelial thickness and the presence of fewer cell layers. The observed FANCA-mediated suppression of hyperplasia correlated with the detection of fewer cells transiting through the cell cycle in the absence of gross differentiation abnormalities or apoptotic differences. Importantly, the knockdown of either FANCA or FANCD2 in HPV-positive keratinocytes was sufficient for increasing epithelial hyperplasia. Our findings support a new role for FA pathways in the maintenance of differentiation-dependent cell cycle exit, with the implication that FA deficiencies may contribute to the high risk of FA patients for developing HPV-associated SCC.

Structural relationships within medical informatics.

This study seeks to increase our understanding of the structure of Medical Informatics. In particular, it focuses on the relationships between information science and information technology on the one hand, and biomedical research, clinical practice, and medical education on the other, that have defined "medical informatics." Using indexing terms and MeSH tree structures assigned to medical informatics literature covered by MEDLINE, co-occurrence analysis provides a "map" of the field. Major research and application focuses arrayed within the map elucidate a finer structure than reported previously. Dimensions "Techniques vs. Systems" and "Signs & Symptoms vs. Processes" form the two axes of the map and relate to the relationships underlying the indexing assignments given to the literature studied. Related studies underway using the INSPEC database will provide a complementary perspective on the structure of medical informatics as a field.