Search

Background: Although individuals with vision impairment are at greater risk for depression and anxiety, there has been limited study of mental healthcare utilization among this population., Objectives: To address this gap, this pooled cross-sectional study estimates the prevalence of mental healthcare utilization among individuals with vision impairment during the COVID-19 pandemic., Methods: We calculated adjusted relative risk ratios and 95% confidence intervals of depression and/or anxiety symptoms and mental healthcare utilization using multinomial logistic regression, accounting for demographics, social determinants of health, and survey week. The population-based, U.S. Census Bureau Household Pulse Survey was administered April 2021-March 2022. Participants included 800,935 US adults (weighted population: 174,598,530) RESULTS: Adjusting for other factors, adults with vision impairment were more likely to report depression symptoms (RRR: 2.33; 95% CI: 2.03-2.68), anxiety symptoms (RRR: 2.12; 95% CI: 1.94-2.33, and comorbid depression and anxiety symptoms (RRR: 3.77; 95% CI: 3.51-4.04) compared with individuals with no vision impairment. Among individuals reporting anxiety or depression symptoms, individuals with vision impairment (RRR: 1.46; 95% CI: 1.35-1.59) were more likely to lack of mental healthcare utilization compared with individuals with no vision impairment., Conclusion: Findings suggest that individuals with vision impairment are at increased risk for depression and/or anxiety symptoms and report reduced mental healthcare utilization compared with individuals without vision impairment. Additional programs and policies are needed to improve mental healthcare utilization among individuals with vision impairment and depression and/or anxiety symptoms, such as increased telehealth accessibility and coordination of behavioral health and ophthalmology services., Competing Interests: Declaration of competing interest The authors do not have any conflicts of interest to disclose., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Physician-patient discussions regarding lung cancer screening (LCS) are uncommon and its racial and ethnic disparities are under-investigated. We examined the racial and ethnic disparities in the trends and frequency of LCS discussion among the LCS-eligible United States (US) population., Methods: We analyzed data from the Health Information National Trends Survey from 2014 to 2020. LCS-eligible individuals were defined as adults aged 55 to 80 years old who have a current or former smoking history. We estimated the trends and frequency of LCS discussions and adjusted the probability of having an LCS discussion by racial and ethnic groups., Results: Among 2136 LCS-eligible participants (representing 22.7 million US adults), 12.9% (95% CI, 10.9%-15%) reported discussing LCS with their providers in the past year. The frequency of LCS discussion was lowest among non-Hispanic White participants (12.3%, 95% CI, 9.9%-14.7%) compared to other racial and ethnic groups (14.1% in Hispanic to 15.3% in non-Hispanic Black). A significant increase over time was only observed among non-Hispanic Black participants (10.1% in 2014 to 22.1% in 2020; P = .05) and non-Hispanic Whites (8.5% in 2014 to 14% in 2020; P = .02). In adjusted analyses, non-Hispanic Black participants (14.6%, 95% CI, 12.3%-16.7%) had a significantly higher probability of LCS discussion than non-Hispanic Whites (12.1%, 95% CI, 11.4%-12.7%)., Conclusion: Patient-provider LCS discussion was uncommon in the LCS-eligible US population. Non-Hispanic Black individuals were more likely to have LCS discussions than other racial and ethnic groups. There is a need for more research to clarify the discordance between LCS discussions and the actual screening uptake in this population., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Competing Interests: The authors have no disclosures or conflicts of interest.

Background: Smoking remains a major contributor to mortality and morbidity. Dental care professionals are positioned to help patients quit smoking. Results of clinical trials have shown the efficacy of dental care professionals' smoking-cessation advice; however, the evidence of its effectiveness in the general population in the United States is limited. The authors examined the association between smoking-cessation advice from dental care professionals and quitting behaviors of adult smokers in the general population., Methods: The authors used an observational study design with data from the National Health and Nutrition Examination Survey for the years 2015 through 2018. The authors included 1,024 respondents 18 years and older who were current or former smokers who quit smoking within the past 12 months and reported a dental visit within the past 12 months., Results: Among the study sample, 44.6% received smoking-cessation advice from a dental care professional. The authors found no significant association between smoking-cessation advice and any attempt to quit smoking (as a binary outcome; adjusted odds ratio, 1.11; 95% CI, 0.68 to 1.80; P = .677). Although respondents who received smoking-cessation advice reported 18% more quit attempts (on a continuous scale; adjusted rate ratio, 1.18; 95% CI, 1.00 to 1.39, P = .05), smoking-cessation advice was not associated with smoking abstinence beyond 6 months., Conclusions: Receiving smoking-cessation advice from a dental care professional was associated with more attempts to quit smoking but not with abstinence of 6 months or longer., Practical Implications: Additional efforts seem to be needed for smoking cessation in dental practices. Implementing enhanced incentive programs or promoting tobacco-use cessation certification in dental education may be an effective strategy to enhance dental professionals' knowledge and skills in providing additional support to their patients., (Copyright © 2022 American Dental Association. Published by Elsevier Inc. All rights reserved.)

Objectives: Patient-provider discussion about treatment costs has been recognized as a key component of shared clinical decision making in cancer care. This study examined the association of patient-provider cost discussion with out-of-pocket spending among cancer survivors., Methods: Using data from the 2016-2017 Medical Expenditure Panel Survey-Experiences with Cancer Survivorship Supplement, cancer survivors in the United States who reported having a detailed discussion about treatment costs were identified. Multivariable generalized linear model with gamma distribution and log-link was fitted to analyze average total out-of-pocket spending between those who had the discussion and those who did not. We also examined whether having the cost discussion is associated with the likelihood of reporting receipt of all cancer care they believed was necessary using a multivariable logistic regression model. All analyses controlled for patient socioeconomic and health-related characteristics., Results: Among 1525 individuals, representing 14.6 million cancer survivors in the United States (mean age, 65.5 years; 59% women; 80.4% white), only 10.4% (95% confidence interval [CI], 8.7%-12.1%) reported having the detailed cost discussion with their providers during their cancer care. Having a cost discussion was associated with a -33.8% reduction in (95% CI, -38.2% to -29.6%; an absolute difference of -$478) average total out-of-pocket spending. The probability of receiving all necessary patient-reported cancer care was not different between those who had the discussion and those who did not., Conclusion: Detailed patient-provider cost discussions were associated with lower average total out-of-pocket spending. Patients who had detailed cost discussions with providers did not seem to sacrifice the appropriate utilization of necessary cancer treatments., (Copyright © 2020 ISPOR–The Professional Society for Health Economics and Outcomes Research. Published by Elsevier Inc. All rights reserved.)

Objective: To examine how the teach-back, interactive communication loop between patient and provider, is utilized and its role in diabetes care delivery., Methods: This was a cross-sectional analysis of the Medical Expenditure Panel Survey (MEPS) 2011-2016. The study sample included US adults aged 18 or older with diabetes. Survey-design adjusted analyses were used to examine patterns of teach-back utilization across patient socioeconomic/clinical characteristics, patient-provider interactions, and diabetes care education., Results: Analysis of 2901 US adults with diabetes showed that 25.0 % reported patient teach-back experience during their visit to care. Compared with patients without teach-back, those with teach-back experience had higher scores on interaction quality with their providers (composite score: 90.8 vs. 55.8, P < .001). Those with teach-back were also more to receive additional advice on diet and exercise from providers (67.0 % vs. 60.9 %, P = 0.03) and to report that they were confident in diabetes self-care management (75.7 % vs. 70.3 %, P =0.03)., Conclusion: Teach-back communication appears to be effective in patient-provider interaction and diabetes care education, leading to higher confidence in self-care management., Practice Implications: Despite its potential, the utilization of teach-back communication is suboptimal. More effort is needed to promote effective use of teach-back communication in routine diabetes care., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Obesity is a significant risk factor of several cancers that imposes a substantial economic burden on US healthcare that remains to be quantified. We estimated the excess costs and economic burden of obesity-related cancers in the United States., Methods: From the Medical Expenditure Panel Survey (2008-2015) data, we identified 19 405 cancer survivors and 175 498 non-cancer individuals. We estimated annual health expenditures using generalized linear regression with log link and gamma distribution by cancer types (stratified by 11 obesity-related cancers and other cancer types), controlling for sociodemographic and clinical characteristics. All cost estimates were adjusted to 2015 USD value., Results: The average annual total health expenditures were $21 503 (95% CI, $20 946-$22 061) for those with obesity-related cancer and $13 120 (95% CI, $12 920-$13 319) for those with other cancer types. There was a positive association between body mass index and health expenditures among cancer survivors: for each additional 5-unit increase in body mass index, the average predicted expenditures increase by $1503 among those with obesity-related cancer and by $722 among those with other cancers. With adjustments for sociodemographic and clinical characteristics, the mean incremental expenditures of treating obesity-related cancer were 2.1 times higher than those of other cancers ($4492 vs $2139) and more considerable among the non-elderly cancer population. Obesity-related cancers accounted for nearly 43.5% of total direct cancer care expenditures, estimated at $35.9 billion in 2015., Conclusion: The economic burden of obesity-related cancer in the United States is substantial. Our findings suggest a need for the inclusion of comprehensive obesity prevention and treatment in cancer care., (Copyright © 2019. Published by Elsevier Inc.)

Context: Palliative care will play an important role to alleviate disease suffering and improve quality of life for cancer patients and their family caregivers., Objective: We examined the knowledge penetration of palliative care in a nationally representative sample of U.S. adults., Methods: We used the 2018 National Cancer Institute's Health Information National Trends Survey to determine the proportion of respondents who had knowledge of palliative care as well as the depth and sources of their knowledge. We used the Pearson chi-square test and a multivariable logistic regression model to assess the association of respondents' basic demographic characteristics as well as health status and having knowledge of palliative care., Results: We identified 3194 respondents (weighted sample size: 229,591,005) who met the inclusion criteria. About 71% (2097) of all respondents had no knowledge of palliative care and 84.5% of Hispanic respondents had no knowledge of palliative care. Multivariable analyses indicated the middle-aged (50-64 years old, odds ratio, 1.58; 95% CI, 1.15-2.19, P = 0.006) and elder population (65 years or older, odds ratio, 1.70, 95% CI, 1.30-2.22, P < 0.001) have a significantly better knowledge of palliative care than those under age 50. Common misconceptions existed in respondents, even those who had self-reported adequate knowledge of palliative care., Conclusion: The proportion of adults who have knowledge of palliative care is low in the U.S. Greater efforts are needed to promote palliative care and reduce the misconceptions of palliative care in the general population., (Copyright © 2019 American Academy of Hospice and Palliative Medicine. Published by Elsevier Inc. All rights reserved.)

Objectives: Using the Andersen behavioral model, we examined the complex relationships among geographic access to care, financial disadvantage, patient-centered care factors, and access to care outcomes., Study Design: This was a retrospective, cross-sectional study of the US civilian non-institutionalized population., Methods: Our analytic sample included 15,787 US adults aged 18 years or older who had health insurance coverage for a full year in Medical Expenditure Panel Survey 2014-2015. Structural equation modeling was used to determine the associations among usual source of care, travel time to provider, financial disadvantage, patient-centered care factors (perceived interaction with health provider, shared decision-making, and value of health care), and access to care (perceived access to care and unmet need of health services)., Results: Our analysis showed that patient-centered care factors were associated with improved perceived access to care (β = 0.03 to 0.56, P = .002) and reduced unmet needs of health care (β = -0.03 to -0.17, P = .03 to < .001). Although longer travel time to provider and having financial disadvantage of paying medical bills had negative effects on access to care outcomes, these associations were mediated by patient-centered care quality factors., Conclusions: Our findings suggest that better patient-centered care factors are associated with enhanced patient access to care. Efforts that focus on improving patient experience could be an effective approach along with coverage expansion to enhance access to quality care., (Copyright © 2019 The Royal Society for Public Health. Published by Elsevier Ltd. All rights reserved.)

Odorant receptors are the largest subfamily of G protein-coupled receptors and were recently suggested to play critical roles in nonolfactory tissues. However, the expression and function of odorant receptors in astrocytes, the most abundant cells in the brain, are not well known. We demonstrate that Olfr920 is highly expressed and propose that it functions as a short-chain fatty acid sensor in primary cortical astrocytes. The short-chain fatty acid isobutyric acid (IBA) was identified via a luciferase assay as an Olfr920 ligand. We show that IBA activates the G s protein-adenylyl cyclase-cAMP pathway via Olfr920 in primary cortical astrocytes by using cAMP and knockdown analyses. In addition, IBA reduces lipopolysaccharide-induced glial fibrillary acidic protein expression in reactive astrocytes. These results suggest that astrocytic Olfr920 is a potential novel target for increased reactive astrocytes., (Copyright © 2019. Published by Elsevier Inc.)

GSK3β interacting protein (GSKIP) is a naturally occurring negative regulator of GSK3β and retains both the Protein Kinase A Regulatory subunit binding (PKA-RII) domain and GSK3β interacting domain. Of these two domains, we found that PKA-RII is required for forming a working complex comprising PKA/GSKIP/GSK3β/Drp1 to influence phosphorylation of Drp1 Ser637. In this study, bioinformatics and experimental explorations re-analyzing GSKIP's biofunctions suggest that the evolutionarily conserved Domain of Unknown Function (DUF727) is an ancestral prototype of GSKIP in prokaryotes, and acquired the C-terminal GSK3β binding site (tail) in invertebrates except for Saccharomyces spp., after which the N-terminal PKA-RII binding region (head) evolved in vertebrates. These two regions mutually influence each other and modulate GSKIP binding to GSK3β in yeast two-hybrid assays and co-immunoprecipitation. Molecular modeling showed that mammalian GSKIP could form a dimer through the L130 residue (GSK3β binding site) rather than V41/L45 residues. In contrast, V41/L45P mutant facilitated a gain-of-function effect on GSKIP dimerization, further influencing binding behavior to GSK3β compared to GSKIP wild-type (wt). The V41/L45 residues are not only responsible for PKA RII binding that controls GSK3β activity, but also affect dimerization of GSKIP monomer, with net results of gain-of-function in GSKIP-GSK3β interaction. In addition to its reported role in modulating Drp1, Ser637 phosphorylation caused mitochondrial elongation; we postulated that GSKIP might be involved in the Wnt signaling pathway as a scavenger to recruit GSK3β away from the β-catenin destruction complex and as a competitor to compete for GSK3β binding, resulting in accumulation of S675 phosphorylated β-catenin., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Objective: To investigate patient attitudes toward acceptable levels of disability after decompressive surgery for malignant middle cerebral artery infarction, including impact of patient age, to improve process of surgical informed consent., Methods: Patients who had experienced a recent minor stroke were asked to complete a questionnaire containing 2 parts: demographic information, including patient age, sex, years of education, working status, religion, and economic status, and acceptable level of disability based on modified Rankin Scale (mRS) with corresponding illustrations to explain mRS levels., Results: Patient age was identified as an independent determinant of the worst acceptable mRS score with a negative correlation. For nondominant hemispheric malignant infarction, the worst acceptable mRS score was significantly lower (mean ± SD 2.0 ± 1.3) for the oldest patients (>70 years old) compared with patients <60 years old (mean ± SD 3.0 ± 0.6) and 60-70 years old (mean ± SD 3.0 ± 0.8). For dominant hemispheric malignant infarction with language impairment, all age groups showed a significantly lower value for worst acceptable mRS score (mean ± SD 1.8 ± 1.1 for patients <60 years old, 1.8 ± 1.2 for patients 60-70 years old, and 1.0 ± 1.4 for patients >70 years old)., Conclusions: Patients showed different attitudes toward disability according to their age. Patients >70 years old showed the lowest worst acceptable mRS score after surgical treatment of malignant infarction. Language impairment with dominant hemispheric infarction further decreased the worst acceptable mRS score., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Scrub typhus is caused by the obligate intracellular bacterium Orientia tsutsugamushi. Macrophages are host cells for its replication and clearance. Severe complications in patients are mainly caused by a cytokine storm resulting from overproduction of proinflammatory cytokines; nevertheless, the molecular mechanism for the occurrence remains obscure. Herein, we investigate the interactive regulation of cytokines and micro-RNA (miR) in human macrophages infected with low and high doses of O. tsutsugamushi. During low dose infection, macrophages produce high levels of IL-10 through extracellular signal-regulated kinase activation, which inhibits proinflammatory cytokine production and facilitates pathogen replication. Increasing levels of pathogen results in reduced levels of IL-10, and macrophages begin to generate high levels of proinflammatory cytokines through NF-κB activation. However, during a high dose infection, macrophages produce high levels of miR-155 to slow the proinflammatory response. The extracellular signal-regulated kinase/IL-10 axis suppresses the NF-κB/tumor necrosis factor alpha axis via activation of signal transducer and activator of transcription 3. Both IL-10 and miR-155 inhibit the NF-κB signaling pathway. Furthermore, IL-10 is a potent inhibitor of miR-155. Patients susceptible to a cytokine storm, peripheral blood mononuclear cells showed significantly lower IL-10 and miR-155 responses to O. tsutsugamushi challenge. Thus, IL-10 and miR-155 operate inhibitory mechanisms to achieve a proper defense mechanism and prevent a cytokine storm., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Tremella Polysaccharides (TPS) have been reported to play an important role in regulating immune responses. Tregs are widely identified as the critical reason for immune dysfunction during sepsis. However, whether TPS could influence the immunomodulatory activities of Tregs in post-burn sepsis mice remains unclear. In this experiment, we researched the effects of TPS on peripheral blood Tregs in sepsis mouse induced by burn plus Pseudomonas aeruginosa infection. Results showed that TPS reversed the influences of Tregs on CD4⁺T cells proliferation and polarization and declined the level of IL-10 in burn plus P. aeruginosa infection mice. In addition, TPS notably reduced the mortality of post-burn sepsis mice. Therefore, TPS could inhibit the abnormal activities of CD4⁺CD25(high) Tregs in burn with P. aeruginosa infection mice, at least in part via inhibiting IL-10 secretion, and trigger a shift of Th2 to Th1 with activation of CD4⁺T cells in burn with P. aeruginosa infection mice., (Copyright © 2014. Published by Elsevier Inc.)

The dimerization of 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase was studied by interrupting the salt bridge interactions between D249 and R167 in the dimeric interface. Substitution of alanine, lysine and serine for D249 decreased catalytic efficiency 30, 1400 and 1.4-fold, and lowered the melting temperature 6.9, 5.4 and 7.6 degrees C, respectively. The mutated enzymes have the dimeric species but the equilibrium between monomer and dimer for these mutants varies from each other, implying that these residues might contribute differently to the dimer stability. Thermal and urea-induced unfolding profiles for wild-type and mutant enzymes appeared as a two-state transition and three-state transition, respectively. In addition, mutation on D249 breaks the salt bridges and causes different effects on the loss of enzymatic activity for D249A, D249K and D249S mutants in the urea-induced unfolding profiles. Hence, D249 at the dimeric interface in 3alpha-HSD/CR is essential for conformational stability, oligomeric integrity and enzymatic activity.

Epstein-Barr virus (EBV) expresses the immediate-early protein Rta to activate the transcription of EBV lytic genes and the lytic cycle. We show that RanBPM acts as a binding partner of Rta in yeast two-hybrid analysis. The binding was confirmed by glutathione-S-transferase pull-down assay. A coimmunoprecipitation experiment and confocal microscopy revealed that RanBPM and Rta interact in vivo and colocalize in the nucleus. The interaction appears to involve the SPRY domain in RanBPM and the region between amino acid residues 416 to 476 in Rta. The interaction promotes the transactivation activity of Rta in activating the transcription of BMLF1 and p21 in transient transfection assays. Additionally, RanBPM interacts with SUMO-E2 (Ubc9) to promote sumoylation of Rta by SUMO-1. This fact explains why the expression of RanBPM enhances the transactivation activity of Rta. Taken together, the present results indicate a new role of RanBPM in regulating a viral protein that is critical to EBV lytic activation.

Beta-catenin has essential roles in morphogenesis and human cancer, both as a subunit of adhesive complexes in the cell membrane and as a transcriptional coactivator in the Wnt signaling pathway. In addition, beta-catenin also has the ability to transport lymphoid enhancer binding factor-1 into the nucleus. In this study, we examined a constitutive active mutation, beta-catenin (T41A, S45A), for its potential as a nuclear import receptor for T-cell transcription factor 4 in 293 cells. Immunoblot analysis demonstrated that this constitutive active form of beta-catenin increased the amount of Tcf4 in the nucleus about 4-5-fold compared to controls. However, the overall expression of Tcf4 remained the same with or without over-expression of beta-catenin (T41A, S45A). T-cell transcription factor 4 reporter gene and electrophoretic mobility shift assay further indicated that the increase in Tcf4 in the nucleus was consistent with its accrued DNA binding capacity and transcription activity. Microscopic immunofluorescence examination showed that Tcf4 was mainly located in the cytoplasm and transported into the nucleus, without or with over-expression of beta-catenin (T41A, S45A), respectively. Our results suggest that beta-catenin might be a major factor regulating the import of Tcf4 from the cytoplasm into the nucleus, consequently controlling its transcription activity.

A centrosomal-associated protein, ninein is a microtubules minus end capping, centrosome position, and anchoring protein, but the underlying structure and physiological functions are still unknown. To identify the molecules that regulate the function of human ninein in centrosome, we performed yeast two-hybrid screen and isolated the SUMO-conjugating E2 enzyme, Ubc9, and SUMOylation enhancing enzymes, including PIAS1 and PIASxalpha, as binding partners of hNinein. These interactions as well as the interaction between hNinein and SUMO-1 are also confirmed by a glutathione S-transferase (GST) pull-down experiment. Furthermore, the C-terminal region of hNinein can be SUMOylated in vitro and in HeLa cells transfected with a plasmid expressing GFP-hNinein. Our findings firstly place SUMOylation target on the centrosome structure protein, hNinein, which results in the switch localization from centrosome to nucleus, suggesting the importance of the SUMOylation of hNinein and probably other centrosomal proteins may also be involved in the centrosome activity.

To explore more possible roles for GSK3beta function, the yeast two-hybrid screening using GSK3beta as a bait protein was performed. In this study, we demonstrated that two variants of CABYR (281 and 379) interacted with GSK3beta in the yeast two-hybrid and GST pull down assay. Molecular characterization showed that CABYR variants formed a dimer with a proline-rich extensin-like domain, which slightly overlapped with GSK3beta-binding site. In kinase assay, we also showed that CABYR variants act as an ideal substrate for GSK3beta within the extensin-like domain and phosphorylation sites on CABYR were mapped. Interestingly, Northern blot showed that CABYR transcripts were expressed more distinctly in the fetal brain than in the adult brain, suggesting that this protein may play a role during brain development. Moreover, differential expression of CABYR variants may exhibit aberrant expression in brain tumors and cancer cell lines.

The human dynamin-like protein, HdynIV, has recently been cloned and shown to be involved in the formation and trafficking of coated vesicles. In particular, one of the HdynIV variant overexpressions has been suggested to contribute to the pathogenesis of brain tumors. In this paper, we report on the genomic organization of the human HdynIV gene. The gene was found to correspond to 20 exons of genomic sequence on human chromosome 12, distributed over 64kb of genomic DNA. The two exons, numbers 15 and 16, are subjected to differential splicing, generating four different transcripts of a perfect match to our recent report on the four different spliced HdynIV variants [DNA Cell Biol. 19 (2000) 189]. We have also characterized the 5(') regulatory region of the HdynIV gene in order to understand the molecular mechanisms regulating its expression. The transcriptional initiation site was identified by 5(')-RACE. The 5(')-flanking sequence of the HdynIV gene contains three GC boxes that concatenate Ap2- and Sp1-binding motifs, but that does not contain either the TATA or CAAT consensus sequence. A region between -140 and +92 contributed to high promoter activity. Deletion analysis demonstrated that the minimal promoter activity required the region of -110 to -100. Electrophoretic mobility shift assay demonstrated that a putative transcriptional factor bound to the region of -119 to -90. Site-directed mutagenesis analysis of this region revealed that nucleotides at -108 to -100 were essential for transactivation mediated by this transcriptional factor. In conclusion, we have characterized the minimal HdynIV promoter and shown that CTCCCAGCA (-108 to -100) sequence may act as a novel transcriptional element for regulating HdynIV gene expression.

Aims: Rac1 is a member of the Ras superfamily of small GTPase and plays a fundamental role in cytoskeleton reorganization, regulation of gene expression and cell proliferation, and cellular transformation. Though recent studies point to an involvement of rac1 in tumorigenesis, little is known about the alteration of rac1 gene in human brain tumours., Methods: Reverse transcription-polymerase chain reaction (RT-PCR), TA cloning, and DNA sequencing were performed to detect rac1 gene mutations in the surgical specimens of 45 human brain tumours., Results: Twelve of 45 cases had base changes in the rac1 gene. The frequency of rac1 alterations was seven of 18 meningiomas, three of 14 astrocytomas, one of seven pituitary adenomas, and one of four metastatic brain tumours. No mutation was detected in acoustic neurilemomas. The subtypes of seven meningiomas include three meningotheliomatous, two atypical, one transitional and one angioblastic meningioma. Three astrocytomas had rac1 gene mutation, including one grade II, one grade III, and one grade IV astrocytoma. All of single base changes were transitions, five of them being T to C transitions. Sites of rac1 mutation were found in codons 34, 41 (two cases), 42 (two cases), 43, 44, 46 and 58. These mutations are mainly localized in the putative effector-domain of rac1 gene and may enhance the activity of rac1, which increases the survival of brain tumours., Conclusion: Our results suggest that rac1 gene may play a role in some brain tumours of divergent histogenesis and that the alterations of rac1 gene may contribute to tumorigenesis and/or metastasis.

The centrosomal protein ninein has been identified as a microtubules minus end capping, centriole position, and anchoring protein, but the true physiological function remains to be determined. In this report, using immunofluorescence analysis and GFP-fusions we show that coiled-coil II domain (CCII domain, 1303-2096) co-localized with gamma-tubulin and centrin at the centrosome. We further narrow down within 83 amino acids and classify a new centrosomal targeting signal. Interestingly, antibodies raised against CCII domain reveal that ninein protein declines from spindle poles during mitosis, but reaccumulates at centrosomes at the end of cell division. Moreover, the data also suggest that fragment 1783-1866 may be attributed to declined signal of ninein. In kinase assay, we show that CCII domain could readily be phosphorylated by AIK and PKA. Taken together, our results suggest that ninein protein contains two distinct subdomains which are required for targeting and regulating asymmetry centrosomes. Importantly, the decline of ninein during mitosis implies that this centrosomal protein may play a role to regulate the process of chromosome segregation without discrimination. The model we propose here will foster a clearer picture of how two asymmetric centrosomes could direct and ensure the correct segregation of chromosomes during the mitotic stage.

SH3 domains are found in many signal transduction proteins where they mediate protein-protein binding by recognizing specific peptides rich in proline. Based on the analysis of sequence alignment data, the NADPH oxidase p67(phox) C-terminal SH3 domain possesses a typical compact beta-barrel consisting of five beta-strands arranged in two antiparallel beta-sheets of three and two beta-strands. Multiple amino acid substitutions were made at beta e and its flanking residues to determine the role of the boundary sequences in binding activity and conformational specificity of the domain. Analysis of amino acid P55 indicated that all mutants were completely abolished in their binding activities. The substitution of F58 with Y58 showed no effect of the binding, whereas substitution with stop codon abolished activity. Furthermore, when amino acid V59 was substituted with stop codon, activity was also completely abolished. Substitution of E60 with stop codon showed no effect of binding. Moreover, our data show that V59 particularly could not be replaced by Leu. Taken together, these data suggest that V59 may not only contribute the exact boundary site but also play on the specificity for protein-protein interactions in phagocyte NADPH oxidase., (Copyright 2001 Academic Press.)

The centrosome plays a key role in the formation of the mitotic spindle, cell polarity, and cell locomotion. Previously we identified a novel centrosomal associated protein hNinein using GSK-3beta as a bait in the yeast two-hybrid assay. In this report, the hNinein genome was found to correspond to 29 exons of genomic sequence on human chromosome 14q22. Promoter analysis predicts that hNinein contains a TATA, two CCAAT, and three GC boxes. The promoter exhibits the following potential transcription factor binding sites: Sp1, p300, and AP-1. In addition, an alternatively spliced isoform, encoded a 2041-amino-acid protein of 237,900 Da, which was designated hNinein-Lm (GenBank AF302773). The hNinein-Lm genome was found to correspond to 28 exons (2'-29). Amino acid sequence comparison with hNinein showed that hNinein-Lm exhibited an EF-hand Ca2+ binding domain in the N-terminus which similar to mouse ninein. Northern blot showed that this hNinein-Lm isoform was expressed more than hNinein in tissues examined. Differential RT-PCR combining Southern blotting also showed that hNinein-Lm is much more abundant compared to hNinein. Two forms of ninein may also imply the status of ninein associated with a pair of the centrioles in the centrosome structure. Furthermore, molecular characterization shows that human ninein is oligomerized at the C-terminal end which overlapped with GSK-3beta binding site, suggesting that oligomerization of ninein may be regulated by GSK-3beta phosphorylation.

Dynamin-like protein, a large GTP-binding protein, has recently been cloned, and studies have shown that it may be involved in the formation of coated vesicles. In this report, three different alternatively spliced dynamin-like protein variants (DLP1-WT, -11, and -37) from rat brain were identified by reverse transcription/polymerase chain reaction (RT-PCR). One novel rat alternatively spliced variant (DLP1-37), not described previously, was identified. We examined the interaction of these three rat brain dynamin-like protein variants with glycogen synthase kinase 3beta (Gsk-3beta) using the yeast two-hybrid screening, in vitro binding assay, and immunoprecipitation analysis. It was found that all three examined rat brain dynamin-like protein variants can bind to Gsk-3beta. Moreover, in vitro kinase (phosphorylation) assay showed that mammalian dynamin-like protein acts as a substrate for glycogen synthase kinase 3beta. These data suggest that Gsk-3beta may participate in a functional role in dynamin-like proteins in vesicle trafficking., (Copyright 2000 Academic Press.)

The cDNA encoding cobrotoxin was constructed from the cellular RNA isolated from the venom glands of Naja naja atra (Taiwan cobra). The cDNA was subcloned into the expression vector pET20b(+) and transformed into BL21(DE3) Escherichia coli strain. Expressed cobrotoxin was isolated from inclusion bodies of E. coli and subjected to refolding into its folded structure. The refolded cobrotoxin was purified by high-performance liquid chromatography and exhibited a neurotoxicity in inhibiting acetylcholine-induced muscle contractions. Recombinant cobrotoxin showed a tendency to isomerize its disulfide bonds as that observed with native cobrotoxin. An appreciable decrease in the rate of isomerization reaction was observed when Glu-38 was replaced with Gln-38 or Lys-47 was replaced with Glu-47 or Gln-47. These results reflect that the element in controlling the disulfide isomerization of cobrotoxin is closely associated with the charged side chains in the cobrotoxin molecule., (Copyright 1999 Academic Press.)

Members of the dynamin superfamily are implicated in vesicle trafficking. Using human glycogen synthase kinase 3 beta (Gsk-3 beta) as bait in the yeast two-hybrid system, we identified a novel human dynamin-like protein IV (HdynIV). When the full-length cDNA of HdynIV was sequenced, it showed that HdynIV's carboxyl terminal lacks a proline-rich domain that can bind to Gsk-3 beta. By Northern blot analysis and isoform-specific PCR, we found that HdynIV is expressed ubiquitously in all human tissues examined. Two transcripts of 2.4 and 4.4 kb are shown to be more abundant in heart, brain, and skeletal muscle. Interestingly, the 2.4-kb transcript is expressed more distinctly in the fetal liver than in the adult liver, suggesting that this protein might play a role during development. In the present report, we have demonstrated that HdynIV interacts with the Gsk-3 beta through its carboxyl-terminal region, implying than HdynIV may also be involved in cell signaling.

Rac GTPases regulate activation of the phagocyte NADPH oxidase, a multi-component enzyme complex that produces superoxide in response to host infection. GTP-bound Rac binds to the cytosol protein p67-phox enabling it to participate in oxidase assembly. Details of this interaction are poorly understood. Previous studies showed that Rac/p67-phox binding is GTP-dependent and that several Rac1 mutants lost the ability to activate the oxidase even though they still bound p67-phox. Using two hybrid and blot overlay binding methods, we identified a novel binding site in the p67-phox C-terminus that binds Rac1, Rac2, and Cdc42, a related GTPase which does not activate the oxidase. Binding was independent of the GDP/GTP state. We also showed that GTP-Cdc42 binds p67-phox N-terminus similar to GTP-Rac. Therefore, Rac binding to p67-phox is not synonymous with NADPH oxidase activation, and Rac probably participates in other steps of oxidase activation in addition to binding p67-phox., (Copyright 1998 Academic Press.)