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In the process of sepsis, excessive occurrence of pyroptosis, a form of programmed cell death acting as a defense mechanism against pathogens, can disrupt immune responses, thus leading to tissue damage and organ dysfunction. Chitosan oligosaccharide (COS), derived from chitosan degradation, has demonstrated diverse beneficial effects. However, its impact on sepsis-induced pyroptosis remains unexplored. In the present study, ATP/LPS was utilized to induce canonical-pyroptosis in THP-1 cells, while bacterial outer membrane vesicles (OMV) were employed to trigger non-canonical pyroptosis in RAW264.7 cells. Our results revealed a dose-dependent effect of COS on both types of pyroptosis. This was evidenced by a reduction in the expression of pro-inflammatory cytokines, as well as crucial regulatory proteins involved in pyroptosis. In addition, COS inhibited the cleavage of caspase-1 and GSDMD, and reduced ASC oligomerization. The underlying mechanism revealed that COS acts an antioxidant, reducing the release of pyroptosis-induced ROS and malondialdehyde (MDA) by upregulation the expression and promoting the nuclear translocation of nuclear factor erythroid-2-related factor 2 (Nrf2), which led to an elevation of glutathione peroxidase 4 (GPX4) and superoxide dismutase (SOD). Notably, the actions of COS were completely reversed by the Nrf2 inhibitor. Consequently, COS intervention increased the survival rate of sepsis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objective: To investigate the early predictors of respiratory depression in patients with glufosinate poisoning, and provide reference for clinicians to make decisions. Methods: In March 2022, the clinical data of patients with glufosinate poisoning admitted to the intensive care unit of the Affiliated Xiangshan Hospital of Wenzhou Medical University from March 2018 to January 2022 were retrospectively analyzed. The patients were divided into respiratory depression group and non-respiratory depression group according to the occurrence of respiratory depression during hospitalization. The clinical data such as age, gender, past history, intake, initial treatment and laboratory examination were compared between the two groups. Multivariate logistic regression was used to analyze the predictors of respiratory depression in patients with glufosinate poisoning, and its predictive value was analyzed by receiver operating characteristic (ROC) curve. Results: A total of 34 patients with glufosinate poisoning were enrolled, including 13 patients in non-respiratory depression group and 21 patients in respiratory depression group. There were significant differences in intake, blood amylase and bicarbonate radical in arterial blood gas between the two groups ( P <0.05). Respiratory depression occurred at 6.5-48.0 h after ingestion, with a median of 15.0 (9.5, 24.0) h. Multivariate logistic regression analysis showed that the intake of glufosinate ( OR =1.440, 95% CI : 1.033-2.009, P =0.032) and bicarbonate radical in arterial blood gas ( OR =0.199, 95% CI : 0.040-0.994, P =0.049) were predictors of respiratory depression in patients with glufosinate poisoning, and the area under the curve (AUC) of ROC curves were 0.936 and 0.842. The optimal cut-off values were 15.0 g (sensitivity=95.2%, specificity=76.9%) and 17.6 mmol/L (sensitivity=71.4%, specificity=84.6%), respectively. Conclusion: The intake of glufosinate and bicarbonate radical in arterial blood gas have good prediction effects on the occurrence of respiratory depression in patients with glufosinate poisoning.

For longitudinal overdispersed Poisson data sets, estimators of the intra-, inter-, and total concordance correlation coefficient through variance components have been proposed. However, biased estimators of quadratic forms are used in concordance correlation coefficient estimation. In addition, the generalized estimating equations approach has been used in estimating agreement for longitudinal normal data and not for longitudinal overdispersed Poisson data. Therefore, this paper proposes a modified variance component approach to develop the unbiased estimators of the concordance correlation coefficient for longitudinal overdispersed Poisson data. Further, the indices of intra-, inter-, and total agreement through generalized estimating equations are also developed considering the correlation structure of longitudinal count repeated measurements. Simulation studies are conducted to compare the performance of the modified variance component and generalized estimating equation approaches for longitudinal Poisson and overdispersed Poisson data sets. An application of corticospinal diffusion tensor tractography study is used for illustration. In conclusion, the modified variance component approach performs outstandingly well with small mean square errors and nominal 95% coverage rates. The generalized estimating equation approach provides in model assumption flexibility of correlation structures for repeated measurements to produce satisfactory concordance correlation coefficient estimation results.

The Ti-6Al-4V alloy is the most common biomaterial used for bone replacements and reconstructions. Despite its advantages, the Ti-6Al-4V has a high stiffness that can cause stress-shielding. In this work, we demonstrated that the selective laser melting (SLM) technology could be used to fabricate porosity in Ti-6Al-4V extra low interstitial (ELI) to reduce its stiffness while improving cell adhesion and proliferation. With a porosity of 14.04%, the elastic modulus of the porous Ti-6Al-4V ELI was reduced to 80 GPa. The compressive stress and the 3-point-bending flexural tests revealed that the porous Ti-6Al-4V ELI possessed a brittle characteristic. The additional pores within the beams of the lattice structures of porous Ti-6Al-4V ELI increased its surface arithmetic average roughness, R a  = 3.94 μm. The in vitro cytocompatibility test showed that the SLM printing process and the post-processes did not cause any toxicity in the MC3T3-E1 cells. The in vitro cell proliferation test also showed that the porous Ti-6Al-4V ELI increased the proliferation rate of osteogenic induced MC3T3-E1 cells on Day 7. The findings from this study would provide engineers and researchers with both the mechanical information and biological understanding of SLM printed porous Ti-6Al-4V ELI, and SLM printed dense Ti-6Al-4V ELI towards biomedical applications., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Multimodal communication in animals is common, and is particularly well studied in signals that include both visual and auditory components. Multimodal signals that combine acoustic and olfactory components are less well known. Multimodal communication plays a crucial role in agonistic interactions in many mammals, but relatively little is known about this type of communication in nocturnal mammals. Here, we used male Great Himalayan leaf-nosed bats Hipposideros armiger to investigate multimodal signal function in acoustic and olfactory aggressive displays. We monitored the physiological responses (heart rate [HR]) when H. armiger was presented with 1 of 3 stimuli: territorial calls, forehead gland odors, and bimodal signals (calls + odors). Results showed that H. armiger rapidly increased their HR when exposed to any of the 3 stimuli. However, the duration of elevated HR and magnitude of change in HR increased significantly more when acoustic stimuli were presented alone compared with the presentation of olfactory stimuli alone. In contrast, the duration of elevated HR and magnitude of change in HR were significantly higher with bimodal stimuli than with olfactory stimuli alone, but no significant differences were found between the HR response to acoustic and bimodal stimuli. Our previous work showed that acoustic and chemical signals provided different types of information; here we describe experiments investigating the responses to those signals. These results suggest that olfactory and acoustic signals are non-redundant signal components, and that the acoustic component is the dominant modality in male H. armiger , at least as it related to HR. This study provides the first evidence that acoustic signals dominate over olfactory signals during agonistic interactions in a nocturnal mammal., (© The Author(s) (2021). Published by Oxford University Press on behalf of Editorial Office, Current Zoology.)

Hierarchically porous-structured materials show tremendous potential for catalytic applications. In this work, a facile method through the combination of three-dimensional (3D) printing and chemical dealloying was employed to synthesize a nanoporous-copper-encapsulating microporous-diamond-cellular-structure (NPC@DCS) catalyst. The developed NPC@DCS catalyst was utilized as a heterogeneous photo-Fenton-like catalyst where its catalytic applications in the remediation of organic wastewater were exemplified. The experimental results demonstrated that the NPC@DCS catalyst possessed a remarkable degradation efficiency in the removal of rhodamine B with a reaction rate of 8.24 × 10 -2 min -1 and displayed attractive stability, durability, mineralization capability, and versatility. This work not only manifests the applicability of the proposed NPC@DCS catalyst for wastewater purification in practical applications but also is anticipated to inspire the incorporation of the 3D printing technology and chemical synthesis to design high-performance metal catalysts with tunable hierarchical micro- and nanopores for functional applications.

Nitrogen fixation to produce ammonia is a vital process since nitrogen is an essential element for the human body. Industrial nitrogen fixation mainly relies on the Haber-Bosch process. However, this process requires huge energy consumption and leads to pollution emission. In this study, the behaviors of intermediates in the nitrogen reduction reaction (NRR) are investigated for fifteen double-atom catalysts (DACs) through density functional theory calculations, revealing that under the synergistic effect of active sites on appropriate DACs, intermediates can be adsorbed through different configurations according to the activity improvement needs. VFe-N-C shows the best catalytic activity for electrochemical NRR with a limiting potential of -0.36 V vs. the reversible hydrogen electrode. The proposed synergistic effect of active sites on DACs for NRR could provide a new method for design of NRR catalysts., (© 2021 Wiley-VCH GmbH.)

Purpose: The purpose of this study was to identify independent prognostic factors for breast cancer patients with T1-2 tumors and 1-3 positive lymph nodes, and discuss the role of postmastectomy radiotherapy(PMRT) in these patients., Methods: Between January 2005 and December 2015, the data on 840 eligible patients with breast cancer were retrospectively reviewed. Of these patients, 368 women received PMRT and 472 did not. The endpoints were locoregional recurrence (LRR) and distant metastasis (DM)., Results: With a median follow-up of 62.0 months, multivariate analysis identified the following independent risk factors for increased LRR: tumor size ≥ 4 cm (HR: 2.994, 95% CI: 1.190-7.535, P = 0.020), ER- and PR-negative tumor (HR: 2.540, 95% CI: 1.165-5.537, P = 0.019), preoperative high neutrophil-to-lymphocyte ratio (NLR) (HR: 4.716, 95% CI: 1.776-12.528, P = 0.002)and low neutrophil-to-monocyte ratio (NMR) (HR: 0.231, 95% CI: 0.084-0.633, P = 0.004). And independent risk factors for increased DM: ER- and PR-negative tumor (HR: 2.540, 95% CI: 1.880-5.625, P = 0.000), high NLR (HR: 2.693, 95% CI: 1.426-5.084, P = 0.002) and low NMR (HR: 0.460, 95% CI: 0.257-0.824, P = 0.009). The high-risk patients (≥ 2 risk factors) had worse LRRFS and DFS than low-risk patients (0-1 risk factor) (all, P < 0.05). In the subgroup analysis, both low- and high-risk patients received PMRT had better LRRFS and DFS than those who without PMRT (all, P < 0.05), and the high-risk patients received PMRT had similar 5-year rates of LRRFS and DFS than low-risk patients who without PMRT (94.5 vs. 94.3%, P = 0.402; 83.4 vs.87.4%, P = 0.877, respectively)., Conclusions: Tumor size, ER/PR status, preoperative NLR and NMR were independent predictors of risk of recurrence. PMRT could improve locoregional control even in low-risk subgroup of breast cancer patients with T1-2 tumors and 1-3 positive lymph nodes significantly.

Laryngeal squamous cell carcinoma (LSCC) is a common head and neck cancer with high mortality in developing countries. A comprehensive understanding of the molecular mechanisms of tumor progression in laryngeal cancer is needed for new treatment strategies. MicroRNA-29a has been emerged as a critical miRNA in various cancers, and shown to regulate multiple oncogenic processes. In this study, we investigated the tumor suppressive role of miRNA-29a in laryngeal squamous cell carcinoma. We performed cell-based functional analysis after overexpression of miR-29a in AMC-HN-8 and HEP2 cells in vitro. It turned out that ectopic expression of miR-29a significantly inhibited cell proliferation, invasion, and migration in two LSCC cell lines (AMC-HN-8 and Hep2) in vitro. Further downstream target analysis by western blotting and luciferease reporter assay showed that overexpression of miR-29a significantly downregulated expression of STAT3, and STAT3 is a putative direct target of miR-29a in LSCC. In addition, the tumor suppressive function of miR-29a can be abrogated by forced expression of STAT3. Taken together, our data suggest that miR-29a functions as a tumor suppressor through targeting STAT3 in laryngeal squamous cell carcinoma., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Objective: To explore the relationship between job stress, job burnout and turnover intention of operating room nurses in a tertiary hospital in Shandong Province. Methods: From January 2016 to January 2019, the operating room nurses with an average daily operation volume of more than 200 operating rooms in a tertiary hospital in Shandong Province were selected as the research objects. The work pressure, job burnout and turnover intention of nurses were investigated with the Chinese nurses' job stressor scale, job burnout table and turnover intention table. Pearson related factors were used to analyze job stress, job burnout and turnover intention Multivariate logistic regression was used to analyze the factors influencing turnover intention. A total of 98 questionnaires were distributed and 98 questionnaires were returned, with a recovery rate of 100%. Results: The average score of job stress, job burnout and turnover intention were 85.49±5.42, 36.17±3.52 and 14.99±3.32, respectively. There were differences in the scores of work stress among different age, working years, education background and establishment groups ( P <0.05) . The scores of job burnout among nurses with different working years, education background, professional title, salary and establishment were different ( P < 0.05) ; the scores of turnover intention of nurses in different age, working years, professional title, salary and establishment group were different ( P <0.05) ; salary, job burnout and occupational pressure were the influencing factors of turnover intention ( P <0.05) . Conclusion: The operating room nurses have high work pressure and job burnout is an important factor leading to high turnover intention.

Recently, accumulating evidences have shown that microRNAs (miRNAs) could play key roles in the development and progression of multiple important human diseases. Nonetheless, due to the shortcoming of being expensive and time-consuming existing in experimental approaches, computational methods are needed for the prediction of potential miRNA-disease associations. In our study, we proposed a computational model named Heterogeneous Network-based MiRNA-Disease Association prediction (HNMDA) for the latent miRNA-disease association prediction by integrating known miRNA-disease associations, miRNA functional similarity, disease semantic similarity and Gaussian interaction profile kernel similarity. The Gaussian interaction profile kernel similarity can make up for the shortages of the traditional similarity calculation methods. Furthermore, we applied a heterogeneous network-based method, in which we first implemented a network diffusion algorithm of random walk with restart, and then we applied a method to find the optimal projection from miRNA space to disease space, which enabled the prediction of new miRNA-disease associations that are not experimentally confirmed so far. In the cross-validation, HNMDA obtained the AUC of 0.8394, which achieved improvement compared with previous methods. In the case studies of breast neoplasms, esophageal neoplasms and kidney neoplasms based on known miRNA-disease associations in the HMDD V2.0 database, there were 82, 76 and 84% of top 50 predicted related miRNAs that were confirmed to have associations with these three diseases, respectively. In the further case studies for new diseases without any known related miRNAs and the case using HMDD V1.0 database as known associations, there were also high ratio of the predicted miRNAs confirmed by experimental reports.

Solid polymer electrolytes, such as polyethylene oxide (PEO) based systems, have the potential to replace liquid electrolytes in secondary lithium batteries with flexible, safe, and mechanically robust designs. Previously reported PEO nanocomposite electrolytes routinely use metal oxide nanoparticles that are often 5-10 nm in diameter or larger. The mechanism of those oxide particle-based polymer nanocomposite electrolytes is under debate and the ion transport performance of these systems is still to be improved. Herein we report a 6-fold ion conductivity enhancement in PEO/lithium bis(trifluoromethanesulfonyl) imide (LiTFSI)-based solid electrolytes upon the addition of fullerene derivatives. The observed conductivity improvement correlates with nanometer-scale fullerene crystallite formation, reduced crystallinities of both the (PEO)6:LiTFSI phase and pure PEO, as well as a significantly larger PEO free volume. This improved performance is further interpreted by enhanced decoupling between ion transport and polymer segmental motion, as well as optimized permittivity and conductivity in bulk and grain boundaries. This study suggests that nanoparticle induced morphological changes, in a system with fullerene nanoparticles and no Lewis acidic sites, play critical roles in their ion conductivity enhancement. The marriage of fullerene derivatives and solid polymer electrolytes opens up significant opportunities in designing next-generation solid polymer electrolytes with improved performance.

Spices have enjoyed a long history and a worldwide application. Of particular interest is the pharmaceutical value of spices in addition to its basic seasoning function in cooking. Concretely, equipped with complex chemical compositions, spices are of significant importance in pharmacologic actions, like antioxidant, antibacterial, antitumor, as well as therapeutical effects in gastrointestinal disorders and cardiovascular disease. Although increasing evidences in support of its distinct role in the medical field has recently reported, little information is available for substantive, thorough and sophisticated researches on its chemical constituents and pharmacological activities, especially mechanism of these actions. Therefore, in popular wave of studies directed at a single spice, this review presents systematic studies on the chemical constituents and pharmacological activities associated with common used spices, together with current typical individual studies on functional mechanism, in order to pave the way for the exploitation and development of new medicines derived from the chemical compounds of spice (such as, piperine, curcumin, geniposide, cinnamaldehyde, cinnamic acid, linalool, estragole, perillaldehyde, syringic acid, crocin).

Here we report the low-temperature dielectric and viscoelastic properties of phosphoric acids in the range of H2O:P2O5 1.5-5. Both dielectric and viscosity measurements allow us to determine the glass-transition temperatures of phosphoric acids. The obtained glass-transition temperatures are in good agreement with previous differential scanning calorimetric measurements. Moreover, our analysis reveals moderate decoupling of ionic conductivity from structural relaxation in the vicinity of the glass transition.

Electrode polarization analysis is frequently used to determine free-ion diffusivity and number density in ionic conductors. In the present study, this approach is critically examined in a wide variety of electrolytes, including aqueous and nonaqueous solutions, polymer electrolytes, and ionic liquids. It is shown that the electrode polarization analysis based on the Macdonald-Trukhan model [J. Chem. Phys. 124, 144903 (2006); J. Non-Cryst. Solids 357, 3064 (2011)] progressively fails to give reasonable values of free-ion diffusivity and number density with increasing salt concentration. This should be expected because the original model of electrode polarization is designed for dilute electrolytes. An empirical correction method which yields ion diffusivities in reasonable agreement with pulsed-field gradient nuclear magnetic resonance measurements is proposed. However, the analysis of free-ion diffusivity and number density from electrode polarization should still be exercised with great caution because there is no solid theoretical justification for the proposed corrections.

The A2A adenosine receptor (A2AR) is a G-protein-coupled receptor. We previously reported that the C terminus of the A2AR binds to translin-associated protein X (TRAX) and modulates nerve growth factor (NGF)-evoked neurite outgrowth in PC12 cells. Herein, we show that neuritogenesis of primary hippocampal neurons requires p53 because blockage of p53 suppressed neurite outgrowth. The impaired neuritogenesis caused by p53 blockage was rescued by activation of the A2AR (designated the A2A rescue effect) in a TRAX-dependent manner. Importantly, suppression of a TRAX-interacting protein (kinesin heavy chain member 2A, KIF2A) inhibited the A2A rescue effect, whereas overexpression of KIF2A caused a rescue effect. Expression of a KIF2A fragment (KIF2A514), which disturbed the interaction between KIF2A and TRAX, blocked the rescue effect. Transient colocalization of TRAX and KIF2A was detected in the nucleus of PC12 cells upon NGF treatment. These data suggest that functional interaction between KIF2A and TRAX is critical for the A2A rescue effect. Moreover, p53 blockage during NGF treatment prevented the redistribution of KIF2A from the nucleus to the cytoplasmic region. Expression of a nuclear-retained KIF2A variant (NLS-KIF2A) did not rescue the impaired neurite outgrowth as did the wild-type KIF2A. Therefore, redistribution of KIF2A to the cytoplasmic fraction is a prerequisite for neurite outgrowth. Collectively, we demonstrate that KIF2A functions downstream of p53 to mediate neuritogenesis of primary hippocampal neurons and PC12 cells. Stimulation of the A2AR rescued neuritogenesis impaired by p53 blockage via an interaction between TRAX and KIF2A., (Copyright (c) 2010 Wiley Periodicals, Inc.)

Blockage of the p53 tumor suppressor has been found to impair nerve growth factor (NGF)-induced neurite outgrowth in PC-12 cells. We report herein that such impairment could be rescued by stimulation of the A(2A) adenosine receptor (A(2A)-R), a G protein-coupled receptor implicated in neuronal plasticity. The A(2A)-R-mediated rescue occurred in the presence of protein kinase C (PKC) inhibitors or protein kinase A (PKA) inhibitors and in a PKA-deficient PC-12 variant. Thus, neither PKA nor PKC was involved. In contrast, expression of a truncated A(2A)-R mutant harboring the seventh transmembrane domain and its C terminus reduced the rescue effect of A(2A)-R. Using the cytoplasmic tail of the A(2A)-R as bait, a novel-A(2A)-R-interacting protein [translin-associated protein X (TRAX)] was identified in a yeast two-hybrid screen. The authenticity of this interaction was verified by pull-down experiments, coimmunoprecipitation, and colocalization of these two molecules in the brain. It is noteworthy that reduction of TRAX using an antisense construct suppressed the rescue effect of A(2A)-R, whereas overexpression of TRAX alone caused the same rescue effect as did A(2A)-R activation. Results of [(3)H]thymidine and bromodeoxyuridine incorporation suggested that A(2A)-R stimulation inhibited cell proliferation in a TRAX-dependent manner. Because the antimitotic activity is crucial for NGF function, the A(2A)-R might exert its rescue effect through a TRAX-mediated antiproliferative signal. This antimitotic activity of the A(2A)-R also enables a mitogenic factor (epidermal growth factor) to induce neurite outgrowth. We demonstrate that the A(2A)-R modulates the differentiation ability of trophic factors through a novel interacting protein, TRAX.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by a CAG trinucleotide expansion in exon 1 of the Huntingtin (Htt) gene. We show herein that in an HD transgenic mouse model (R6/2), daily administration of CGS21680 (CGS), an A(2A) adenosine receptor (A(2A)-R)-selective agonist, delayed the progressive deterioration of motor performance and prevented a reduction in brain weight. 3D-microMRI analysis revealed that CGS reversed the enlarged ventricle-to-brain ratio of R6/2 mice, with particular improvements in the left and right ventricles. (1)H-MRS showed that CGS significantly reduced the increased choline levels in the striatum. Immunohistochemical analyses further demonstrated that CGS reduced the size of ubiquitin-positive neuronal intranuclear inclusions (NIIs) in the striatum of R6/2 mice and ameliorated mutant Htt aggregation in a striatal progenitor cell line overexpressing mutant Htt with expanded polyQ. Moreover, chronic CGS treatment normalized the elevated blood glucose levels and reduced the overactivation of a major metabolic sensor [5'AMP-activated protein kinase (AMPK)] in the striatum of R6/2 mice. Since AMPK is a master switch for energy metabolism, modulation of energy dysfunction caused by the mutant Htt might contribute to the beneficial effects of CGS. Collectively, CGS is a potential drug candidate for the treatment of HD.

We isolated and characterized a 4.8-kb 5' flanking region of the rat A2A adenosine receptor (A2A-R) gene in the present study. Promoter activity was observed with this DNA fragment in PC12 cells and C6 cells which contain endogenous A2A-Rs. A fusion fragment consisting of the 4.8-kb promoter-proximal DNA fragment of the A2A-R gene, and the coding region of lacZ was utilized to produce mice harbouring the fusion gene. In three independent founder lines, proteins and transcripts of the transgene were found in many areas of the central nervous system (CNS), but not in three peripheral tissues examined. Double immunohistochemical analyses revealed that the transgene was coexpressed with endogenous A2A-R and proper neuronal markers in the brain. Specifically, the transgene in the striatum was found in the enkephalin-containing GABAergic neurons and in the cholinergic neurons as was found for the endogenous A2A-R. However, a selectively enriched striatal expression of the transgene was not found as was observed for the endogenous A2A-R. Collectively, the 4.8-kb promoter-proximal DNA fragment of the rat A2A-R gene contains important element(s) to direct its expression in the CNS where functional A2A-R are found, but were not sufficient to confer the highly concentrated expression of the striatal A2A-R. Furthermore, expressions of A2A-R and the transgene were found in both neurons and astrocytes, suggesting that adenosine might mediate its function through A2A-R in both cell types.

By a combination of PCR and DNA walking technique, we isolated a 4.8-kb DNA fragment containing a 4.3 kb 5'-flanking region and a 0.5-kb 5'-untranslated region of the rat A(2A) adenosine receptor (A(2A)-R) gene. Various lengths of the 5'-flanking region of the A(2A)-R gene were inserted into an expression vector and transfected into several different cell lines for promoter analysis. Our results reveal that a consensus NF1 element (designated as A(2A)-R/NF1), located between bases -2846 and -2827 of the A(2A)-R gene, functions as a repressor for A(2A)-R promoters in the rat brain-derived type-2 astrocyte cell line (RBA2), which expresses no A(2A)-R. Electrophoretic gel mobility shift assay (EMSA) revealed that two A(2A)-R/NF1-protein complexes of RBA2 nuclear extract were formed. Supershift experiments using an anti-NF1 antibody suggest that NF1 proteins exist in both A(2A)-R/NF1-protein complexes. Furthermore, mutations in the conserved NF1 binding site of this A(2A)-R/NF1 element disturbed DNA-protein formation. Thus, NF1 proteins appear to mediate this cell line-specific suppression of A(2A)-R promoters in RBA2 cells. The importance of NF1 proteins in regulating A(2A)-R promoters was further confirmed in another cell line (Siha) which expresses no endogenous A(2A)-R. Moreover, addition of the A(2A)-R/NF1element upstream of an irrelevant thymidine kinase (TK) promoter suppressed its promoter activity in Siha cells, but not in RBA2 cells. Thus, the NF1-mediated inhibition of the A(2A)-R promoter was promoter- and cell line-specific. In summary, we have defined a distal negative element (A(2A)-R/NF1) that plays a functional role in modulating the expression of A(2A)-R.

We previously showed that phosphorylation of Ser(10) of the N terminus domain of the type VI adenylyl cyclase (ACVI) partly mediated protein kinase C (PKC)-induced inhibition of ACVI. We now report that phosphorylation of the other two cytosolic domains (C1 and C2), which form the catalytic core complex of ACVI, also contributes to PKC-mediated inhibition. In vitro phosphorylation by PKC of the recombinant C1a and C2 domains, and of the synthetic peptides representing potential PKC phosphorylation sites, suggests that Ser(568) and Ser(674) of the C1 domain and Thr(931) of the C2 domain might act as substrates for PKC. We next created several full-length ACVI mutants in which one or more of the four likely PKC phosphorylation sites (Ser(10), Ser(568), Ser(674), and Thr(931)) were mutated to alanine. Simultaneous mutation of at least two of the three likely residues located in the N and C1 domains (Ser(10), Ser(568), and Ser(674)) was required to render ACVI variants completely insensitive to PKC treatment. In contrast, a single mutation of Thr(931) was sufficient to create a functional ACVI mutant that exhibited no detectable PKC-mediated inhibition, demonstrating the essentiality of Thr(931) to PKC-mediated regulation. Based on these results, we propose that the three cytosolic domains of ACVI might form a regulatory complex. Phosphorylation of this regulatory complex at different sites might induce a fine-tuning of the catalytic core complex and subsequently lead to alternation in the catalytic activity of ACVI.

Four glutathione S-transferase (GST, EC 2.5.1.18) isozymes have been characterized in the larvae of the diamondback moth (DBM), Plutella xylostella L., a cosmopolitan insect pest of crucifiers. This work aimed at cloning and heterologously expressing the cDNA of DBM GST-3, an isozyme involved in this insect resistance to some organophosphorus insecticides, and studying the molecular basis for its increased expression in the resistant strains. Reverse-transcription polymerase chain reaction (RT-PCR) using midgut mRNA from a methyl parathion resistant MPA strain and degenerate primers complimentary to the N-terminal and internal amino acid sequences of GST-3 generated a 128 bp DNA product. A clone of 809 bp, obtained by screening a midgut cDNA library of MPA strain using this PCR product as probe, encoded a protein of 216 amino acids (calculated Mr 24,083 and pI 8.50). This GST of DBM, PxGST3, shared the highest (46.3%) amino acid sequence identity, among insects, to MsGST1 of Manduca sexta. PxGST3 mRNA level was considerably higher in MPA than in susceptible strains, and Southern blots suggested that gene amplification was probably not involved in the increased expression of this GST isozyme. Enzymatically active PxGST3 expressed heterologously in E. coli exhibited similar biochemical and toxicological properties as GST-3 purified from DBM larvae. It is the first cloned GST with a well-defined role in insecticide resistance.

In human spermatide cytoplasmic annulate lamellae and intranuclear lamellae were found. In the cytoplasm, the annulate lamellae may vary from 3 to 11 layers of cisternal structures. There is evidence that the annulate lamellae are formed from the outer nuclear envelope. The intranuclear membranes are often seen directly connected to the inner nuclear membrane. These morphologic findings suggest that annulate lamellae may be involved in the transfer of genetic material from the nucleus to the cytoplasm.

The authors have provided ultrastructural demonstration from human material of a small rod shaped organism that represents the causative agent of Legionnaires' disease. The organisms are found mainly in intracytoplasmic vacuoles of alveolar cells. They have a thin outer cell wall and a more delicate plasma membrane. On the basis of morphology, arguments are presented to classify these unusual organisms as rickettsia-like.

Examination of brain tissue taken at autopsy from a 55-year old male showed the presence of herpes simplex viral antigen by immunofluorescence techniques. A suspension of this tissue was inoculated into cultures of human diploid fibroblasts. After 72 h, viral particles at different stages of development were seen inside the nuclei. Envelopment of the viral particles seemed to occur in association with the nuclear membranes, endoplasmic reticulum, cytoplasmic vacuoles and plasma membranes. Severely altered nuclear membranes, with as many as 4 to 8 layers of nuclear envelope, emphasized the common occurrence of membrane reduplication associated with herpes simplex infection. Unusual membrane-bound, dense, rounded inclusions, 100 to 500 nm in diameter, were present in the nucleus. These inclusions apparently were formed between the inner and outer lamellae of the nuclear membrane.

Extracellular cross-striated banded structures have been found in the connective tissues of a variety of organs. Rarely have they been found in conditions such as: hyperplastic parathyroid, follicular adenoma of the thyroid, metastatic hemangiopericytoma in the lung, sarcoidosis of lymph node, and two biopsies of nerve as reported here. The shape and size of the structure varies, as well as its periodicity, which is usually in the range of 1000 to 15000 A. An unusual exception to this general rule is the banded structure in the follicular adenoma of the thyroid where the periodicity is 270 A. These structures are quite different from native collagen or experimentally reconstituted long-space collagen, both by staining reaction with phosphotungstic acid or uranyl acetate and lead nitrate, and in their ultrastructural morphology.

Electron microscopical study of an oncocytoma of the parotid gland disclosed the presence of two cell types, typical oncocytes and condensed oncocytes. The typical oncocyte contains abundant mitochondria that were tightly packed almost completely filling the entire cytoplasm. Only small areas were left for the remaining cytoplasmic organelles. The nuclei in these typical onocytes appeared oval or spheroid. Condensed oncocytes were scattered singly or in nests in tumorous tissue. These cells were also replete with mitochondria, but differed, however, in that many mitochondria showed evidence of degeneration and fusion. Nuclei of these cells were irregular, dense, and contained inclusions and glycogen granules. The relationship of nuclear envelopes to mitochondria and other cellular components has been described previously, but to our knowledge, this represents an original finding in the case of an oncocytoma.

Annulate lamellae have been found in a primitive neuroectodermal tumor, a metastatic cerebellar tumor, a testicular seminoma, a retinoblastoma and three melanomas. These annulate lamellae are arranged in stacked parallel arrays in the cytoplasm of tumor cells. The number of annulate lamellae observed to comprise a single stack varies from 2--4 in the seminoma tumor to 5--18 in the cerebellar tumor. Although the functional significance of annulate lamallae is still unknown, in many instances they have been found to be continuous with rough-surfaced cisternae of the endoplasmic reticulum and ribosomes have been demonstrated on the surface of annulate lamellae. This may suggest that annulate lamellae participate in protein synthesis.

Many spheric bodies with radiating, needle-like filaments are present in semen of patients with fertility problems. These bodies are most likely degenerated sperm. The intermediate forms and a sequential series of degeneration are discussed.