Responsiveness of respiratory function in Parkinson's Disease to an integrative exercise programme: A prospective cohort study.

INTRODUCTION: Respiratory disorders are the most common cause of death in Parkinson's Disease (PD). Conflicting data exist on the aetiology of respiratory dysfunction in PD and few studies examine the effects of exercise-based interventions on respiratory measures. This study was conducted to better understand respiratory dysfunction in PD and to identify measures of dysfunction responsive to an integrative exercise programme. OBJECTIVES: The objectives were to compare baseline respiratory measures with matched, published population norms and to examine immediate and longer-term effects of a 12-week integrated exercise programme on these measures. DESIGN: Twenty-three people with mild PD (median Hoehn & Yahr = 2) self-selected to participate in this exploratory prospective cohort study. Evaluation of participants occurred at three time points: at baseline; following the 12-week exercise programme and at 4-month follow-up. OUTCOME MEASURES: Outcome measures included: Forced Vital Capacity (FVC), Forced Expiratory Volume in 1 second (FEV1), FEV1/FVC ratio, Peak Expiratory Flow (PEF), Inspiratory Muscle Strength (MIP), Expiratory Muscle Strength (MEP), Peak Cough Flow (PCF), and Cardiovascular Fitness measures of estimated VO2 max and 6-Minute Walk Test (6MWT). RESULTS: Compared to published norms, participants had impaired cough, reduced respiratory muscle strength, FEV, FVC, PEF and cardiovascular fitness. Post exercise intervention, statistically significant improvements were noted in MEP, cardiovascular fitness, and PEF. However only gains in PEF were maintained at 4-month follow-up. CONCLUSIONS: Significant respiratory dysfunction exists, even in the early stages of PD. Metrics of respiratory muscle strength, peak expiratory flow and cardiovascular fitness appear responsive to an integrative exercise programme.

Benefits following community treatment orders have an inverse relationship with rates of use: meta-analysis and meta-regression.

BACKGROUND: Community treatment order (CTO) use in Australia and New Zealand ranges from less than 40 per 100 000 population in Western Australia and Canterbury to over 100 per 100 000 in Victoria, South Australia and Waitemata. Recent publications on CTO use now permit a meta-regression to investigate whether differences in CTO use by jurisdiction affect either the possible predictors or outcomes of CTOs. AIMS: To assess whether factors associated with CTO placement or subsequent outcomes vary by rates of use. METHOD: A systematic search of PubMed/Medline, Embase, CINAHL, the Cochrane Central Register of Controlled Trials and PsycINFO for any Australian or New Zealand study comparing CTO cases with controls receiving voluntary psychiatric treatment. This study was prospectively registered with PROSPERO (protocol registration number: CRD42022351500). RESULTS: There were 35 articles from 16 studies identified in the search, plus unpublished data from a further study. Of these, 29 publications were included in meta-analyses. Two were from New Zealand. People who were male, single and not engaged in work, study or home duties were significantly more likely to be on CTOs. In addition, those from migrant backgrounds were 47% more likely to be on an order. On meta-regression, cases in jurisdictions with higher CTO rates had higher proportions of females or individuals with diagnoses other than non-affective psychoses. High-use jurisdictions were also less likely to show reductions in readmission rates or bed-days. CONCLUSIONS: There are marked differences in the possible predictors and outcomes of CTO placement between high- and low-use jurisdictions in Australia and New Zealand. These findings may have implications elsewhere and indicate that better-targeted CTO placement might improve outcomes.

A systematic review and meta-analysis of respiratory dysfunction in Parkinson's disease.

INTRODUCTION: Respiratory dysfunction in Parkinson's disease (PD) is common and associated with increased hospital admission and mortality rates. Central and peripheral mechanisms have been proposed in PD. To date no systematic review identifies the extent and type of respiratory impairments in PD compared with healthy controls. METHODS: PubMed, EMBASE, CINAHL, Web of Science, Pedro, MEDLINE, Cochrane Library and OpenGrey were searched from inception to December 2021 to identify case-control studies reporting respiratory measures in PD and matched controls. RESULTS: Thirty-nine studies met inclusion criteria, the majority with low risk of bias across Risk of Bias Assessment tool for Non-randomized Studies (RoBANS) domains. Data permitted pooled analysis for 26 distinct respiratory measures. High-to-moderate certainty evidence of impairment in PD was identified for vital capacity (standardised mean difference [SMD] 0.75; 95% CI 0.45-1.05; p < 0.00001; I2  = 10%), total chest wall volume (SMD 0.38; 95% CI 0.09-0.68; p = 0.01; I2  = 0%), maximum inspiratory pressure (SMD 0.91; 95% CI 0.64-1.19; p < 0.00001; I2  = 43%) and sniff nasal inspiratory pressure (SMD 0.58; 95% CI 0.30-0.87; p < 0.00001; I2  = 0%). Sensitivity analysis provided high-moderate certainty evidence of impairment for forced vital capacity and forced expiratory volume in 1 s during medication ON phases and increased respiratory rate during OFF phases. Lower certainty evidence identified impairments in PD for maximum expiratory pressure, tidal volume, maximum voluntary ventilation and peak cough flow. CONCLUSIONS: Strong evidence supports a restrictive pattern with inspiratory muscle weakness in PD compared with healthy controls. Limited data for central impairment were identified with inconclusive findings.

Treatment strategies for clozapine-induced nocturnal enuresis and urinary incontinence: a systematic review.

BACKGROUND: Clozapine is the most effective medication for treatment-refractory schizophrenia but is associated with significant adverse drug reactions, including nocturnal enuresis and urinary incontinence. This side effect can be burdensome and lead to medication nonadherence and psychotic relapse. Evidence to guide treatment of clozapine-induced nocturnal enuresis and urinary incontinence is sparse. We therefore aimed to synthesize the evidence base to guide management for clinicians, patients, and their carers. METHODS: We systematically searched PubMed, Embase, PsycInfo, CINAHL, and the Cochrane Trial Registry databases from inception to May 2021 for publications on management of clozapine-induced nocturnal enuresis and urinary incontinence using a PROSPERO preregistered search strategy. RESULTS: We identified 22 case reports and case series describing 74 patients. Interventions included clozapine dose reduction, nonpharmacological treatment, and pharmacological treatments. Among pharmacological treatments, desmopressin, oxybutynin, trihexyphenidyl, tolterodine, imipramine, amitriptyline, ephedrine, pseudoephedrine, aripiprazole, and verapamil were associated with complete resolution of nocturnal enuresis and urinary incontinence. Balancing evidence for effectiveness against risk of adverse effects, we developed a management framework for clozapine-induced nocturnal enuresis and urinary incontinence. CONCLUSIONS: Following assessment of urological, psychiatric, pharmacological, and common comorbid medical issues, first-line treatments should be nonpharmacological, including bathroom alarms, voiding before bedtime, and nocturnal fluid restriction. If these interventions do not provide adequate relief, aripiprazole should be trialed. Desmopressin may be considered for severe refractory cases, but monitoring for hyponatremia is essential.

Occurrence, prevention, and management of the psychological effects of emerging virus outbreaks on healthcare workers: rapid review and meta-analysis.

OBJECTIVE: To examine the psychological effects on clinicians of working to manage novel viral outbreaks, and successful measures to manage stress and psychological distress. DESIGN: Rapid review and meta-analysis. DATA SOURCES: Cochrane Central Register of Controlled Trials, PubMed/Medline, PsycInfo, Scopus, Web of Science, Embase, and Google Scholar, searched up to late March 2020. ELIGIBILITY CRITERIA FOR STUDY SELECTION: Any study that described the psychological reactions of healthcare staff working with patients in an outbreak of any emerging virus in any clinical setting, irrespective of any comparison with other clinicians or the general population. RESULTS: 59 papers met the inclusion criteria: 37 were of severe acute respiratory syndrome (SARS), eight of coronavirus disease 2019 (covid-19), seven of Middle East respiratory syndrome (MERS), three each of Ebola virus disease and influenza A virus subtype H1N1, and one of influenza A virus subtype H7N9. Of the 38 studies that compared psychological outcomes of healthcare workers in direct contact with affected patients, 25 contained data that could be combined in a pairwise meta-analysis comparing healthcare workers at high and low risk of exposure. Compared with lower risk controls, staff in contact with affected patients had greater levels of both acute or post-traumatic stress (odds ratio 1.71, 95% confidence interval 1.28 to 2.29) and psychological distress (1.74, 1.50 to 2.03), with similar results for continuous outcomes. These findings were the same as in the other studies not included in the meta-analysis. Risk factors for psychological distress included being younger, being more junior, being the parents of dependent children, or having an infected family member. Longer quarantine, lack of practical support, and stigma also contributed. Clear communication, access to adequate personal protection, adequate rest, and both practical and psychological support were associated with reduced morbidity. CONCLUSIONS: Effective interventions are available to help mitigate the psychological distress experienced by staff caring for patients in an emerging disease outbreak. These interventions were similar despite the wide range of settings and types of outbreaks covered in this review, and thus could be applicable to the current covid-19 outbreak.

Effects of body size on estimation of mammalian area requirements.

Accurately quantifying species' area requirements is a prerequisite for effective area-based conservation. This typically involves collecting tracking data on species of interest and then conducting home-range analyses. Problematically, autocorrelation in tracking data can result in space needs being severely underestimated. Based on the previous work, we hypothesized the magnitude of underestimation varies with body mass, a relationship that could have serious conservation implications. To evaluate this hypothesis for terrestrial mammals, we estimated home-range areas with global positioning system (GPS) locations from 757 individuals across 61 globally distributed mammalian species with body masses ranging from 0.4 to 4000 kg. We then applied block cross-validation to quantify bias in empirical home-range estimates. Area requirements of mammals <10 kg were underestimated by a mean approximately15%, and species weighing approximately100 kg were underestimated by approximately50% on average. Thus, we found area estimation was subject to autocorrelation-induced bias that was worse for large species. Combined with the fact that extinction risk increases as body mass increases, the allometric scaling of bias we observed suggests the most threatened species are also likely to be those with the least accurate home-range estimates. As a correction, we tested whether data thinning or autocorrelation-informed home-range estimation minimized the scaling effect of autocorrelation on area estimates. Data thinning required an approximately93% data loss to achieve statistical independence with 95% confidence and was, therefore, not a viable solution. In contrast, autocorrelation-informed home-range estimation resulted in consistently accurate estimates irrespective of mass. When relating body mass to home range size, we detected that correcting for autocorrelation resulted in a scaling exponent significantly >1, meaning the scaling of the relationship changed substantially at the upper end of the mass spectrum.

Efectos del Tamaño Corporal sobre la Estimación de los Requerimientos de Área de Mamíferos Resumen La cuantificación precisa de los requerimientos de área de una especie es un prerrequisito para que la conservación basada en áreas sea efectiva. Esto comúnmente implica la recolección de datos de rastreo de la especie de interés para después realizar análisis de la distribución local. De manera problemática, la autocorrelación en los datos de rastreo puede resultar en una subestimación grave de las necesidades de espacio. Con base en trabajos previos, formulamos una hipótesis en la que supusimos que la magnitud de la subestimación varía con la masa corporal, una relación que podría tener implicaciones serias para la conservación. Para probar esta hipótesis en mamíferos terrestres, estimamos las áreas de distribución local con las ubicaciones en GPS de 757 individuos de 61 especies de mamíferos distribuidas mundialmente con una masa corporal entre 0.4 y 4,000 kg. Después aplicamos una validación cruzada en bloque para cuantificar el sesgo en estimaciones empíricas de la distribución local. Los requerimientos de área de los mamíferos <10 kg fueron subestimados por una media ∼15% y las especies con una masa ∼100 kg fueron subestimadas en ∼50% en promedio. Por lo tanto, encontramos que la estimación del área estaba sujeta al sesgo inducido por la autocorrelación, el cual era peor para las especies de talla grande. En combinación con el hecho de que el riesgo de extinción incrementa conforme aumenta la masa corporal, el escalamiento alométrico del sesgo que observamos sugiere que la mayoría de las especies amenazadas también tienen la probabilidad de ser aquellas especies con las estimaciones de distribución local menos acertadas. Como corrección, probamos si la reducción de datos o la estimación de la distribución local informada por la autocorrelación minimizan el efecto de escalamiento que tiene la autocorrelación sobre las estimaciones de área. La reducción de datos requirió una pérdida de datos del ∼93% para lograr la independencia estadística con un 95% de confianza y por lo tanto no fue una solución viable. Al contrario, la estimación de la distribución local informada por la autocorrelación resultó en estimaciones constantemente precisas sin importar la masa corporal. Cuando relacionamos la masa corporal con el tamaño de la distribución local, detectamos que la corrección de la autocorrelación resultó en un exponente de escalamiento significativamente >1, lo que significa que el escalamiento de la relación cambió sustancialmente en el extremo superior del espectro de la masa corporal.

Management and 5-year outcome of patients with coronary artery disease in different periods of stent technology.

BACKGROUND: The availability of bare metal stents (BMS) followed by drug-eluting stents of first- (DES1) and second-generation (DES2) progressively increased the rate of the percutaneous revascularizations [percutaneous coronary intervention (PCI)] with unknown impact on the long-term outcome of real-world patients with established coronary artery disease. We sought to investigate treatments applied in patients with coronary artery disease in BMS, DES1 and DES2 eras and their 5-year outcome. METHODS: A total of 3099 consecutive patients with at least one coronary stenosis more than 50% observed in 2002 (BMS era), 2005 (DES1 era) and 2011(DES2 era) were enrolled at 13 hospitals in Veneto region, Italy. RESULTS: Moving from BMS to DES1 and DES2 eras patients became significantly older, had more comorbidities and received more frequently statins, betablockers, renin-angiotensin modulators and antiplatelets (P < 0.0001 for all). The PCI/conservative therapy ratio increased from 1.9 to 2.2 and 2.3, the PCI/coronary artery by-pass surgery ratio from 3.6 to 4.0 and 5.1. The crude 5-year survival was 84.9, 83.4 and 81.4% (P = 0.20) and survival free of myocardial infarction, stroke or further revascularizations was 62.1, 60.2 and 60.1% (P = 0.68), with cardiovascular mortality accounting for 60.9, 55.6 and 43.4% of deaths. At multivariable analysis cardiovascular mortality was significantly lower in patients enrolled in 2011 vs. 2002 (hazard ratio = 0.712, 95% confidence interval 0.508-0.998, P = 0.048). CONCLUSION: From BMS to DES1 and DES2 eras progressive worsening of patients characteristics, improvement of medical treatment standards and increase in PCI/conservative therapy and PCI/coronary artery by-pass surgery ratios were observed. Five-year outcomes remained similar in the three cohorts, but in the DES2 era cardiovascular mortality was reduced.

Activation of vitamin D in the gingival epithelium and its role in gingival inflammation and alveolar bone loss.

BACKGROUND AND OBJECTIVE: Both chronic and aggressive periodontal disease are associated with vitamin D deficiency. The active form of vitamin D, 1,25(OH)2 D3 , induces the expression of the antimicrobial peptide LL-37 and innate immune mediators in cultured human gingival epithelial cells (GECs). The aim of this study was to further delineate the mechanism by which vitamin D enhances the innate defense against the development of periodontal disease (PD). MATERIALS AND METHODS: Wild-type C57Bl/6 mice were made deficient in vitamin D by dietary restriction. Cultured primary and immortalized GEC were stimulated with 1,25(OH)2 D3 , followed by infection with Porphyromonas gingivalis, and viable intracellular bacteria were quantified. Conversion of vitamin D3 to 25(OH)D3 and 1,25(OH)2 D3 was quantified by ELISA. Effect of vitamin D on basal IL-1α expression in mice was determined by topical administration to the gingiva of wild-type mice, followed by qRT-PCR. RESULTS: Dietary restriction of vitamin D led to alveolar bone loss and increased inflammation in the gingiva in the mouse model. In primary human GEC and established human cell lines, treatment of GEC with 1,25(OH)2 D3 inhibited the intracellular growth of P. gingivalis. Cultured GEC expressed two 25-hydroxylases (CYP27A1 and CYP2R1), as well as 1-α hydroxylase, enabling conversion of vitamin D to both 25(OH)D3 and 1,25(OH)2 D3 . Topical application of both vitamin D3 and 1,25(OH)2 D3 to the gingiva of mice led to rapid inhibition of IL-1α expression, a prominent pro-inflammatory cytokine associated with inflammation, which also exhibited more than a 2-fold decrease from basal levels in OKF6/TERT1 cells upon 1,25(OH)2 D3 treatment, as determined by RNA-seq. CONCLUSION: Vitamin D deficiency in mice contributes to PD, recapitulating the association seen in humans, and provides a unique model to study the development of PD. Vitamin D increases the activity of GEC against the invasion of periodontal pathogens and inhibits the inflammatory response, both in vitro and in vivo. GEC can convert inactive vitamin D to the active form in situ, supporting the hypothesis that vitamin D can be applied directly to the gingiva to prevent or treat periodontal disease.

Impact of Exercise Intervention in Parkinson's Disease can be Quantified Using Inertial Sensor Data and Clinical Tests.

Parkinson's Disease (PD) has the second-highest prevalence rate of all neurodegenerative disorders. It effects approximately 1% of the population over the age of 60, with this proportion rising further, in more elderly cohorts. PD manifests as several motor and non-motor disfunctions, which develop progressively over time. Gait and mobility problems are amongst the most debilitating symptoms for people with PD. They severely affect a person's ability to carry out daily activities of living and can lead to a decreased quality of life. However, recent research has shown exercise intervention to be effective in improving gait, and overall functional mobility, in persons with PD. In this paper, we study the effect of an exercise intervention, comprised of three separate methods of exercise - all which have been shown previously to be effective individually - on a cohort with early-to-moderate stage PD. We also examine the ability of the Timed Up and Go (TUG) test - instrumented with inertial sensors (QTUG) - and the Unified Parkinson's Disease Rating Scale (UPDRS) Part III in measuring the response to the exercise intervention. We found that TUG time and the QTUG-derived frailty index - along with many additional parameters derived from QTUG - showed a significant change between baseline and post-intervention, while the UPDRS Part III score did not. The direction of the changes in the QTUG parameters also align with the expected exercise effect from the literature. Our results suggest QTUG may be a more sensitive measure than UPDRS Part III for assessing the effect of exercise intervention on functional mobility in people with early-to-moderate stage PD.

Evaluation of micro-GPS receivers for tracking small-bodied mammals.

GPS telemetry markedly enhances the temporal and spatial resolution of animal location data, and recent advances in micro-GPS receivers permit their deployment on small mammals. One such technological advance, snapshot technology, allows for improved battery life by reducing the time to first fix via postponing recovery of satellite ephemeris (satellite location) data and processing of locations. However, no previous work has employed snapshot technology for small, terrestrial mammals. We evaluated performance of two types of micro-GPS (< 20 g) receivers (traditional and snapshot) on a small, semi-fossorial lagomorph, the pygmy rabbit (Brachylagus idahoensis), to understand how GPS errors might influence fine-scale assessments of space use and habitat selection. During stationary tests, microtopography (i.e., burrows) and satellite geometry had the largest influence on GPS fix success rate (FSR) and location error (LE). There was no difference between FSR while animals wore the GPS collars above ground (determined via light sensors) and FSR generated during stationary, above-ground trials, suggesting that animal behavior other than burrowing did not markedly influence micro-GPS errors. In our study, traditional micro-GPS receivers demonstrated similar FSR and LE to snapshot receivers, however, snapshot receivers operated inconsistently due to battery and software failures. In contrast, the initial traditional receivers deployed on animals experienced some breakages, but a modified collar design consistently functioned as expected. If such problems were resolved, snapshot technology could reduce the tradeoff between fix interval and battery life that occurs with traditional micro-GPS receivers. Our results suggest that micro-GPS receivers are capable of addressing questions about space use and resource selection by small mammals, but that additional techniques might be needed to identify use of habitat structures (e.g., burrows, tree cavities, rock crevices) that could affect micro-GPS performance and bias study results.

Induction of triggering receptor expressed on myeloid cells (TREM-1) in airway epithelial cells by 1,25(OH)2 vitamin D3.

The airway epithelium plays a role in host defense through the binding of innate immune receptors, which leads to the activation of inflammatory mediators, including antimicrobial peptides. The active form of vitamin D, 1,25(OH)(2)D(3), induces the expression of the antimicrobial peptide LL-37 in both myeloid cells and airway epithelial cells (AEC). Here, we demonstrate that mRNA encoding triggering receptor expressed on myeloid cells (TREM)-1 was induced up to 12-fold by 1,25(OH)(2)D(3) in normal human bronchial epithelial (NHBE) cells and in well-differentiated cultures of six airway epithelial cell lines from patients with cystic fibrosis and healthy individuals. TREM-2 and DAP12 were also expressed in airway cultures, but not induced by vitamin D. Induction occurs through a vitamin D response element identified in its proximal promoter region, and was regulated by PU.1 expressed in the AEC. Activation of TREM-1 by a cross-linking antibody led to an induction of both human ß-defensin-2 and TNF-α mRNA, demonstrating its functionality in these cells. Our results expand on the role played by the airway epithelium in innate immunity and suggest that vitamin D can modulate the innate immune defense of the airway epithelium, and could potentially be developed as an adjunctive therapy for airway infections.

Vitamin D-mediated induction of innate immunity in gingival epithelial cells.

Human gingival epithelial cells (GEC) produce peptides, such as ß-defensins and the cathelicidin LL-37, that are both antimicrobial and that modulate the innate immune response. In myeloid and airway epithelial cells, the active form of vitamin D(3) [1,25(OH)(2)D(3)] increases the expression and antibacterial activity of LL-37. To examine the activity of vitamin D on the innate immune defense of the gingival epithelium, cultured epithelial cells were treated with either 10(-8) M 1,25(OH)(2)D(3) or ethanol for up to 24 h. A time-dependent induction of LL-37 mRNA up to 13-fold at 24 h in both standard monolayer and three-dimensional cultures was observed. Induction of the vitamin D receptor and the 1-α-hydroxylase genes was also observed. The hydroxylase was functional, as LL-37 induction was observed in response to stimulation by 25(OH)D(3). Through microarray analysis of other innate immune genes, CD14 expression increased 4-fold, and triggering receptor expressed on myeloid cells-1 (TREM-1) was upregulated 16-fold after 24 h of treatment with 1,25(OH)(2)D(3). TREM-1 is a pivotal amplifier of the innate immune response in macrophages, leading to increased production by inflammatory response genes. Activation of TREM-1 on the GEC led to an increase in interleukin-8 (IL-8) mRNA levels. Incubation of three-dimensional cultures with 1,25(OH)(2)D(3) led to an increase in antibacterial activity against the periodontal pathogen Aggregatibacter actinomycetemcomitans when the bacteria were added to the apical surface. This study is the first to demonstrate the effect of vitamin D on antibacterial defense of oral epithelial cells, suggesting that vitamin D(3) could be utilized to enhance the innate immune defense in the oral cavity.

Measuring antimicrobial peptide activity on epithelial surfaces in cell culture.

To more accurately assess the activity and role of epithelial cell-derived antimicrobial peptides in their native settings, it is essential to perform assays at the surfaces under relevant conditions. In order to carry this out, we utilize three-dimensional cultures of airway and gingival epithelium, which are grown at an air-liquid interface. Under these conditions, the cultures can be subjected to challenge with a variety of factors known to cause an increase in antimicrobial peptide gene expression. The functional relevance of this induction can then be assessed by quantifying antibacterial activity either directly on the surface of the cells or using the fluid secreted onto the apical surface of the cultures. The relative contribution of the peptides can also be measured by pre-incubation of the secreted fluid with specific inhibitory antibodies. Thus, a relatively inexpensive in vitro model can be used to evaluate the role of antimicrobial peptides in mucosal epithelium.

Knockdown of alphaII spectrin in normal human cells by siRNA leads to chromosomal instability and decreased DNA interstrand cross-link repair.

Nonerythroid alpha-spectrin (alphaIISp) is a structural protein involved in repair of DNA interstrand cross-links and is deficient in cells from patients with Fanconi anemia (FA), which are defective in ability to repair cross-links. In order to further demonstrate the importance of the role that alphaIISp plays in normal human cells and in the repair defect in FA, alphaIISp was knocked down in normal cells using siRNA. Depletion of alphaIISp in normal cells by siRNA resulted in chromosomal instability and cellular hypersensitivity to DNA interstrand cross-linking agents. An increased number of chromosomal aberrations were observed and, following treatment with a DNA interstrand cross-linking agent, mitomycin C, cells showed decreased cell growth and survival and decreased formation of damage-induced alphaIISp and XPF nuclear foci. Thus depletion of alphaIISp in normal cells leads to a number of defects observed in FA cells, such as chromosome instability and a deficiency in cross-link repair.

Deficiency in incisions produced by XPF at the site of a DNA interstrand cross-link in Fanconi anemia cells.

Repair of DNA interstrand cross-links is a multistep process, critical to which is production of incisions at the site of the lesion resulting in the unhooking of the cross-link from DNA. We have previously shown that XPF is involved in production of incisions at the site of a psoralen interstrand cross-link and that in Fanconi anemia, complementation group A (FA-A) cells, there is a deficiency in these incisions. We now demonstrate that in FA complementation group B, C, D2, F, and G cells there is also a deficiency in production of these incisions. Involvement of FA proteins in this process is demonstrated by the ability of FA cells, corrected with the appropriate FANC cDNAs, to produce these incisions and by inhibition of these incisions by antibodies against these proteins. This incision deficiency correlates with reduced levels of DNA repair synthesis in these cells and is not due to reduced levels of XPF. FA proteins could be influencing this incision process by interacting either with proteins involved in the unhooking step or with damaged DNA, acting as a damage sensor. The results also demonstrate that FA cells are undergoing apoptosis by 12 h after interstrand cross-link damage. It is thus proposed that the single-strand breaks known to be created in DNA during apoptosis could mask the deficiency in ability of FA cells to incise cross-linked DNA and could account for the reported discrepancy as to whether FA cells are deficient in the incision step of the repair process.

alphaII-Spectrin interacts with five groups of functionally important proteins in the nucleus.

Nonerythroid alpha-spectrin (alphaSpIISigma( *)) is a structural protein that has been identified in the nucleus of mammalian cells and shown to be involved in DNA repair. It is also deficient in cells from the clinically diverse genetic disorder Fanconi anemia (FA). In order to get a clearer understanding of the role of alphaSpIISigma( *) in DNA repair, and whether it may have other important functions in the nucleus, studies were undertaken to identify specific alphaSpIISigma( *) protein binding partners in the nucleus. The results demonstrate that multiple proteins co-immunoprecipitate with alphaSpIISigma( *) from nuclear extracts from normal human lymphoblastoid and HeLa cells. These can be grouped into five categories: structural proteins, proteins involved in DNA repair, chromatin remodeling proteins, FA proteins, and transcription and RNA processing factors. These studies indicate that alphaSpIISigma( *) may play a role in a number of diverse and important processes in the nucleus and that a deficiency in this protein, as occurs in FA, could affect a number of critical cellular pathways.

Partial amino acid sequence and mRNA analysis of cytosolic pyridoxine-beta-D-glucoside hydrolase from porcine intestinal mucosa: proposed derivation from the lactase-phlorizin hydrolase gene.

We have previously identified and purified a novel beta-glucosidase, designated PNGH (pyridoxine-5'-beta-D-glucoside hydrolase), from the cytosolic fraction of pig intestinal mucosal. PNGH catalyses the hydrolysis of PNG (pyridoxine-5'-beta-D-glucoside), a plant derivative of vitamin B6 that exhibits partial nutritional bioavailability in humans and animals. Preliminary amino acid sequence analysis indicated regions of close similarity of PNGH to the precursor form of LPH (lactase-phlorizin hydrolase), the beta-glucosidase localized to the brush-border membrane. We report in the present study amino acid sequence data for PNGH and results of Northern blot analyses, upon which we propose a common genomic origin of PNGH and LPH. Internal Edman sequencing of the PNGH band isolated by SDS/PAGE yielded data for 16 peptides, averaging 10.8 amino acids in length. These peptides from PNGH (approx. 140 kDa) were highly similar to sequences existing over most of the length of the >200 kDa precursor of rabbit LPH; however, we found no PNGH sequences that corresponded to approx. 350 amino acids between positions 463 and 812 of the LPH precursor, a region encoded by exon 7 of the LPH precursor gene (amino acids 568-784), and no sequences that corresponded to regions near the N-terminus. MS analysis of tryptic peptides yielded 25 peptides, averaging 15 amino acids, with masses that matched segments of the rabbit LPH precursor. Northern blot analysis of pig and human small intestinal polyadenylated mRNA using a non-specific LPH cDNA probe showed an expected approx. 6 kb transcript of the LPH precursor, but also an approx. 4 kb transcript that was consistent with the size predicted from the PNGH protein mass. Using a probe specific to the region encoded by exon 7, hybridization occurred only with the 6 kb transcript. Based on these observations, we propose that both PNGH and LPH enzymes have the same genomic origin, but differ in transcriptional and, possibly, post-translational processing.

Two sets of interacting collagens form functionally distinct substructures within a Caenorhabditis elegans extracellular matrix.

A ubiquitous feature of collagens is protein interaction, the trimerization of monomers to form a triple helix followed by higher order interactions during the formation of the mature extracellular matrix. The Caenorhabditis elegans cuticle is a complex extracellular matrix consisting predominantly of cuticle collagens, which are encoded by a family of approximately 154 genes. We identify two discrete interacting sets of collagens and show that they form functionally distinct matrix substructures. We show that mutation in or RNA-mediated interference of a gene encoding a collagen belonging to one interacting set affects the assembly of other members of that set, but not those belonging to the other set. During cuticle synthesis, the collagen genes are expressed in a distinct temporal series, which we hypothesize exists to facilitate partner finding and the formation of appropriate interactions between encoded collagens. Consistent with this hypothesis, we find for the two identified interacting sets that the individual members of each set are temporally coexpressed, whereas the two sets are expressed approximately 2 h apart during matrix synthesis.

Nonerythroid alphaII spectrin is required for recruitment of FANCA and XPF to nuclear foci induced by DNA interstrand cross-links.

The events responsible for repair of DNA interstrand cross-links in mammalian cells, the proteins involved and their interactions with each other are poorly understood. The present study demonstrates that the structural protein nonerythroid alpha spectrin (alphaSpIISigma*), present in normal human cell nuclei, plays an important role in repair of DNA interstrand cross-links. These results show that alphaSpIISigma* relocalizes to nuclear foci after damage of normal human cells with the DNA interstrand cross-linking agent 8-methoxypsoralen plus ultraviolet A (UVA) light and that FANCA and the known DNA repair protein XPF localize to the same nuclear foci. That alphaSpIISigma* is essential for this re-localization is demonstrated by the finding that in cells from patients with Fanconi anemia complementation group A (FA-A), which have decreased ability to repair DNA interstrand cross-links and decreased levels of alphaSpIISigma*, there is a significant reduction in formation of damage-induced XPF as well as alphaSpIISigma* nuclear foci, even though levels of XPF are normal in these cells. In corrected FA-A cells, in which levels of alphaSpIISigma* are restored to normal, numbers of damage-induced nuclear foci are also returned to normal. Co-immunoprecipitation studies show that alphaSpIISigma*, FANCA and XPF co-immunoprecipitate with each other from normal human nuclear proteins. These results demonstrate that alphaSpIISigma*, FANCA and XPF interact with each other in the nucleus and indicate that there is a close functional relationship between these proteins. These studies suggest that an important role for alphaSpIISigma* in the nucleus is to act as a scaffold, aiding in recruitment and alignment of repair proteins at sites of damage.