The influence of clinic care on perceptions and knowledge of non-communicable diseases and physical activity from a low-resourced community: a mixed-method study.

BACKGROUND: Health promotion for the management of risk factors for non-communicable diseases (NCDs) is an integral part of standard care in South Africa. Most persons presenting with NCDs utilise public primary health care centres for disease management. This mixed-methods study aimed at expanding current understanding of the the influence of standard clinic care (usual care) on perceptions and knowledge of risk factors for NCDs and physical activity (PA) among persons from a low-resourced community. Qualitatively the perceptions of women from a low-resourced community about risk factors for NCDs and PA were explored throughout 24-weeks of standard clinic care. Parallel quantitative data was collected to describe changes in risk factors for NCDs and trends in self-reported knowledge about risk factors of NCDs and PA. METHOD: A convergent-parallel mixed-methods research design was used. The study was carried out in a public primary health care setting, in the North West Province, South Africa. From a convenience sample of 100 participants, 77 African women aged between 34 and 79 years were recruited for the study. Data were collected at three time-points including baseline, 12 weeks, and 24 weeks of a standard clinic care health-promotion programme. The qualitative data was collected during focus group discussions, and the quantitative data included questionnaires on knowledge of physical activity and risk factors for NCDs as well as anthropometric and biological measurements. Qualitative and quantitative data were analysed independently for each phase and then consolidated for interpretation. All data was collected in the same setting. RESULTS: Participants' initial understanding and perceptions of NCD risk factors were poor. Qualitative findings showed that participants knew little about the specific physical activity they could engage in and the role of PA in NCD management. Participants preferred low-intensity activities. Heart-disease knowledge improved significantly at 12 weeks intervention compared to baseline MD = -3.655, p < 0.001. There were improvements in PA knowledge at 12 weeks from baseline MD = -0.625 p = 0.02. There were significant weight (MD = 1.420, p = 0.002) and waist circumference reductions (MD = 0.621, p = 0.02) from baseline to 24 weeks. CONCLUSION: Standard clinic care improved knowledge of physical activity and risk factors for NCDs, but perceptions of risk factors for NCDs and PA were unchanged. This study offers insight into the perceptions held by women from a low-resource setting and how future interventions to manage and prevent NCDs should be structured. TRIAL REGISTRATION: PACTR201609001771813 .

The use of absorbable staples for skin closure after tibial plateau leveling osteotomy.

OBJECTIVE: To compare the use of stainless steel staples with absorbable staples for closure of skin incisions in dogs undergoing tibial plateau leveling osteotomy (TPLO). STUDY DESIGN: Prospective study. SAMPLE POPULATION: Client-owned dogs (n = 80). METHODS: With client consent, dogs were randomly assigned a staple type (stainless steel or absorbable) immediately prior to closure of a TPLO skin incision. Incisions were compared for length, staple type and number, and an inflammation-infection score 2 weeks after surgery. RESULTS: Overall, 18.8% of incisions were diagnosed with inflammation or infection. No difference was found between inflammation-infection scores, incision length, number of staples used, or general anesthetic time between the 2 staple groups. However, wound closure was faster with stainless steel staples (22.50 seconds; range, 11-180) by approximately 30 seconds compared with absorbable staples (56.50 seconds; range, 18-190; P < .001). Time taken to close the incision correlated negatively with the number of occasions that absorbable staples were used (P = .01). CONCLUSION: Absorbable skin staples were successfully used to close skin incisions after TPLO and were not associated with an increased level of inflammation or infection in our clinical setting. CLINICAL SIGNIFICANCE: Absorbable staples may be considered to close surgical wounds when subsequent suture removal would be impractical, without specific concerns over inflammation or infection of the wound.

Translational profiling of retinal ganglion cell optic nerve regeneration in Xenopus laevis.

Unlike adult mammals, adult frogs regrow their optic nerve following a crush injury, making Xenopus laevis a compelling model for studying the molecular mechanisms that underlie neuronal regeneration. Using Translational Ribosome Affinity Purification (TRAP), a method to isolate ribosome-associated mRNAs from a target cell population, we have generated a transcriptional profile by RNA-Seq for retinal ganglion cells (RGC) during the period of recovery following an optic nerve injury. Based on bioinformatic analysis using the Xenopus laevis 9.1 genome assembly, our results reveal a profound shift in the composition of ribosome-associated mRNAs during the early stages of RGC regeneration. As factors involved in cell signaling are rapidly down-regulated, those involved in protein biosynthesis are up-regulated alongside key initiators of axon development. Using the new genome assembly, we were also able to analyze gene expression profiles of homeologous gene pairs arising from a whole-genome duplication in the Xenopus lineage. Here we see evidence of divergence in regulatory control among a significant proportion of pairs. Our data should provide a valuable resource for identifying genes involved in the regeneration process to target for future functional studies, in both naturally regenerative and non-regenerative vertebrates.

Evaluating different concentrations of hydrogen peroxide in an automated room disinfection system.

UNLABELLED: A comparative study was made on the efficacy of 5, 10 and 35% weight by weight (w/w) hydrogen peroxide solutions when applied using an automated room disinfection system. Six-log biological indicators of methicillin-resistant Staphylococcus aureus (MRSA) and Geobacillus stearothermophilus were produced on stainless steel coupons and placed within a large, sealed, environmentally controlled enclosure. Five percent hydrogen peroxide was distributed throughout the enclosure using a Bioquell hydrogen peroxide vapour generator (BQ-50) for 40 min and left to reside for a further 200 min. Biological indicators were removed at 10-min intervals throughout the first 120 min of the process. The experiment was repeated for 10 and 35% hydrogen peroxide solutions. Five percent and 10% hydrogen peroxide solutions failed to achieve any reduction of MRSA, but achieved full kill of G. stearothermophilus spores at 70 and 40 min respectively. Thirty-five percent hydrogen peroxide achieved a 6-log reduction of MRSA after 30 min and full kill of G. stearothermophilus at 20 min. The concentration of 5% hydrogen peroxide within the enclosure after the 200-min dwell was measured at 9·0 ppm. This level exceeds the 15-min Short Term Exposure Limit (STEL) for hydrogen peroxide of 2·0 ppm. Users of automated hydrogen peroxide disinfection systems should review system efficacy and room re-entry protocols in light of these results. SIGNIFICANCE AND IMPACT OF THE STUDY: This research allows hospital infection control teams to consider the impact and risks of using low concentrations of hydrogen peroxide for disinfection within their facilities, and to question automated room disinfection system providers on the efficacy claims they make. The evidence that low concentration hydrogen peroxide solutions do not rapidly, autonomously break down, is in contradiction to the claims made by some hydrogen peroxide equipment providers and raises serious health and safety concerns. Facilities using hydrogen peroxide systems that claim autonomous break down of hydrogen peroxide should introduce monitoring procedures to ensure rooms are safe for re-entry and patient occupation.

Integrating population- and individual-level information in a movement model of Yellowstone bison.

Throughout the world, fragmentation of landscapes by human activities has constrained the opportunity for large herbivores to migrate. Conflict between people and wildlife results when migrating animals transmit disease to livestock, damage property, and threaten human safety. Mitigating this conflict requires understanding the forces that shape migration patterns. Bison Bos bison migrating from Yellowstone National Park into the state of Montana during winter and spring concern ranchers on lands surrounding the park because bison can transmit brucellosis (Brucella abortus) to cattle. Migrations have been constrained, with bison being lethally removed or moved back into the park. We developed a state-space model to support decisions on bison management aimed at mitigating conflict with landowners outside the park. The model integrated recent GPS observations with 22 years (1990-2012) of aerial counts to forecast monthly distributions and identify factors driving migration. Wintering areas were located along decreasing elevation gradients, and bison accumulated in wintering areas prior to moving to areas progressively lower in elevation. Bison movements were affected by time since the onset of snowpack, snowpack magnitude, standing crop, and herd size. Migration pathways were increasingly used over time, suggesting that experience or learning influenced movements. To support adaptive management of Yellowstone bison, we forecast future movements to evaluate alternatives. Our approach of developing models capable of making explicit probabilistic forecasts of large herbivore movements and seasonal distributions is applicable to managing the migratory movements of large herbivores worldwide. These forecasts allow managers to develop and refine strategies in advance, and promote sound decision-making that reduces conflict as migratory animals come into contact with people.

Cell type-specific translational profiling in the Xenopus laevis retina.

BACKGROUND: Translating Ribosome Affinity Purification (TRAP), a method recently developed to generate cell type-specific translational profiles, relies on creating transgenic lines of animals in which a tagged ribosomal protein is placed under regulatory control of a cell type-specific promoter. An antibody is then used to affinity purify the tagged ribosomes so that cell type-specific mRNAs can be isolated from whole tissue lysates. RESULTS: Here, cell type-specific transgenic lines were generated to enable TRAP studies for retinal ganglion cells and rod photoreceptors in the Xenopus laevis retina. Using real time quantitative PCR for assessing expression levels of cell type-specific mRNAs, the TRAP method was shown to selectively isolate mRNAs expressed in the targeted cell and was efficient at purifying mRNAs expressed at both high and low levels. Statistical measures used to distinguish cell type-specific RNAs from low level background and non-specific RNAs showed TRAP to be highly effective in Xenopus. CONCLUSIONS: TRAP can be used to purify mRNAs expressed in rod photoreceptors and retinal ganglion cells in X. laevis. The generated transgenic lines will enable numerous studies into the development, disease, and injury of the X. laevis retina.

Modelling hospital disinfectant against multi-drug-resistant dry surface biofilms grown under artificial human sweat.

BACKGROUND: Dry surface biofilms (DSBs) have been found abundantly across hospital surfaces within intensive care units and may explain how nosocomial pathogens can remain virulent and persist on surfaces for extended periods. Testing standards governing the performance of disinfectant products employ planktonic models under routine growth conditions, which are known to be less tolerant than their biofilm counterpart. AIM: To evaluate biofilm models cultured under artificial human sweat (AHS), a source of nutrient expected on touch surfaces, to assess the antimicrobial performance of common cleaning agents, including a quaternary ammonium, hydrogen peroxide and active chlorine. METHODS: Five single-species biofilms, using pathogenic bacteria such as Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus faecalis, were generated on stainless-steel substrates using a sedimentation protocol under both AHS and nutrient-rich conditions for a direct comparison of phenotypic tolerance. The biofilm models were grown over five days followed by desiccation cycles, before being submerged into the disinfectant solutions for up to 25 min. Epifluorescence (EF) microscopy using LIVE/DEAD™ stain was used to visualize microcolony viability. FINDINGS: The results revealed biofilms cultured under AHS exhibited a greater antimicrobial tolerance and reduced speed of kill for all cleaning agents compared with the routine media; an average reduction of 72.4% vs 96.9%, respectively. EF microscopy revealed traces of viable bacteria across all coupons after disinfection indicating a potential opportunity for regrowth and recontamination. CONCLUSION: The notable difference in biocidal performance between the two growth conditions highlights potential pitfalls within current antimicrobial test standards, and the importance of accurate representation of the microbial challenge.

An automated contact model for transmission of dry surface biofilms of Acinetobacter baumannii in healthcare.

BACKGROUND: Dry surface biofilms (DSBs) have been recognized across environmental and equipment surfaces in hospitals and could explain how microbial contamination can survive for an extended period and may play a key role in the transmission of hospital-acquired infections. Despite little being known on how they form and proliferate in clinical settings, DSB models for disinfectant efficacy testing exist. AIM: In this study we develop a novel biofilm model to represent formation within hospitals, by emulating patient to surface interactions. METHODS: The model generates a DSB through the transmission of artificial human sweat (AHS) and clinically relevant pathogens using a synthetic thumb capable of emulating human contact. The DNA, glycoconjugates and protein composition of the model biofilm, along with structural features of the micro-colonies was determined using fluorescent stains visualized by epifluorescence microscopy and compared with published clinical data. RESULTS: Micrographs revealed the heterogeneity of the biofilm across the surface; and reveal protein as the principal component within the matrix, followed by glycoconjugates and DNA. The model repeatably transferred trace amounts of micro-organisms and AHS, every 5 min for up to 120 h on to stainless-steel coupons to generate a biofilm model averaging 1.16 × 103 cfu/cm2 falling within the reported range for clinical DSB (4.20 × 102 to 1.60 × 107 bacteria/cm2). CONCLUSION: Our in vitro DSB model exhibits many phenotypical characteristics and traits to those reported in situ. The model highlights key features often overlooked and the potential for downstream applications such as antibiofilm claims using more realistic microbial challenges.

Evaluating the environmental microbiota across four National Health Service hospitals within England.

Hospital surfaces contaminated with microbial soiling, such as dry surface biofilms (DSBs), can act as a reservoir for pathogenic micro-organisms, and inhibit their detection and removal during routine cleaning. Studies have recognized that such increases in bioburden can hinder the impact of disinfectants and mask the detection of potential pathogens. Cleanliness within healthcare settings is often determined through routine culture-based analysis, whereby surfaces that exhibit >2.5 colony-forming units (CFU) per cm2 pose a risk to patient health; therefore, any underestimation could have detrimental effects. This study quantified microbial growth on high-touch surfaces in four hospitals in England over 19 months. This was achieved using environmental swabs to sample a variety of surfaces within close proximity of the patient, and plating these on to non-specific low nutrient detection agar. The presence of DSBs on surfaces physically removed from the environment was confirmed using real-time imaging through episcopic differential interference contrast microscopy combined with epifluorescence. Approximately two-thirds of surfaces tested exceeded the limit for cleanliness (median 2230 CFU/cm2), whilst 83% of surfaces imaged with BacLight LIVE/DEAD staining confirmed traces of biofilm. Differences in infection control methods, such as choice of surface disinfectants and cleaning personnel, were not reflected in the microbial variation observed and resulting risk to patients. This highlights a potential limitation in the effectiveness of the current standards for all hospital cleaning, and further development using representative clinical data is required to overcome this limitation.

Neurotrophins use the Erk5 pathway to mediate a retrograde survival response.

Growth factors synthesized and released by target tissues promote survival and differentiation of innervating neurons. This retrograde signal begins when growth factors bind receptors at nerve terminals. Activated receptors are then endocytosed and transported through the axon to the cell body. Here we show that the mitogen-activated protein kinase (MAPK) signaling pathways used by neurotrophins during retrograde signaling differ from those used following direct stimulation of the cell soma. During retrograde signaling, endocytosed neurotrophin receptors (Trks) activate the extracellular signal-related protein kinase 5 (Erk5) pathway, leading to nuclear translocation of Erk5, phosphorylation of CREB, and enhanced neuronal survival. In contrast, Erk1/2, which mediates nuclear responses following direct cell body stimulation, does not transmit a retrograde signal. Thus, the Erk5 pathway has a unique function in retrograde signaling. Differential activation of distinct MAPK pathways may enable an individual growth factor to relay information that specifies the location and the nature of stimulation.

Early municipal bacteriology in Brighton, Aberdeen and Bristol: blessing or burden?

In contrast to the idea that bacteriology was introduced as a tool for the diagnosis and management of the individual patient, this review highlights the work of the municipal bacteriological laboratory in the United Kingdom to illustrate how bacteriological laboratories were introduced as means to control community epidemic disease. Using the examples of municipal laboratories in Brighton, Bristol and Aberdeen, it shows how public health considerations of community infectious diseases such as diphtheria and typhoid dominated the early development and workload of the municipal laboratory, rather than examination of patients with pathological states of uncertain aetiology. It argues that this public health focus of the Medical Officer of Health limited the range of diagnostic tests carried out in such laboratories for over two decades. The growing number of pathogenic microbes being discovered in the late 19th century appears to have had little impact on the tests being carried out in the municipal laboratory. Municipal bacteriological facilities in three towns, a central municipal laboratory (in Brighton), a central university pathological department (Aberdeen) or a combination of both (Bristol) all provided the same limited set of tests. This restricted set of bacteriological examinations is likely to have diminished the value and status of bacteriology in what should have been a period of increasing scope.

Modelling vaporised hydrogen peroxide efficacy against mono-species biofilms.

This pilot study investigates a novel approach towards efficacy testing of antimicrobial cleaning agents; focusing primarily on hydrogen peroxide vapour (HPV). Contaminated surfaces are recognised modes of pathogen transmission within healthcare environments and increase the risk of pathogen acquisition in newly admitted patients. Studies have shown these pathogens can survive on surfaces for extended periods of time in spite of cleaning. This resilience is characteristic of biofilm formation and recent publications have identified their presence in hospitals. In this study, biofilm models comprised of multidrug-resistant organisms (MDROs) were generated using a drip flow reactor and exposed to HPV decontamination. The MDROs included Acinetobacter baumannii, Enterococcus faecalis, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus. Upon exposure, samples were periodically removed and enumerated to generate kill curves for each species. Consequently revealing any inherent resistances; such as catalase-producing organisms which expressed reduced susceptibility. Epifluorescence microscopy revealed an abundance of viable and non-viable microcolonies before and after decontamination, respectively. Greater than 6-Log10 reduction was achieved within a 100 minutes exposure time. This pilot study puts forward a potential methodology for testing antimicrobial agents against biofilms and supports the efficacy of HPV.

A review of treatment options for behavioural manifestations of clinical anxiety as a comorbidity in dogs with idiopathic epilepsy.

Psychiatric comorbidities affect a large percentage of people with epilepsy and have a detrimental impact on their quality of life. Recently, behavioural comorbidities, with similar characteristics to human psychiatric diseases, have been identified in dogs with idiopathic epilepsy. In particular, behaviours motivated by the fear-anxiety emotional system have been found to be associated with the occurrence of idiopathic epilepsy in both dogs receiving anti-epileptic drugs, and drug-naïve dogs. There has been little research into the relationship between epilepsy and behavioural signs, and even less into potential treatment protocols. The following article will review available literature from human medicine to describe the current state of knowledge about the bi-directional relationship between anxiety and epilepsy, draw parallels from reported anxiogenic and anxiolytic properties of anti-epileptic drugs and attempt to provide pharmaceutical and behavioural guidance for veterinary patients with epilepsy and comorbid anxiety.

Sequence of the variant thyroxine-binding globulin of Australian aborigines. Only one of two amino acid replacements is responsible for its altered properties.

A form of thyroxine-binding globulin (TBG) with reduced affinity for hormone and increased susceptibility to heat and acid denaturation has been identified in Australian Aborigines (TBG-A). Results of heat denaturation of TBG established that the TBGA allele is X linked and has a frequency of 50.9% in Western Australian Aborigines. The sequence of an isolated TBGA allele differed at two positions from that of the normal TBG allele (TBGC). One substitution was in codon 191, ACA (threonine) rather than GCA (alanine), and the other was in codon 283, TTT (phenylalanine) instead of TTG (leucine). These nucleotide substitutions resulted in the loss of sites for the enzymes Bgl 1 and Tth 111 II, respectively. The nucleotide substitutions in the TBG-A allele was confirmed by digestion of genomic DNA segments amplified using the polymerase chain reaction. The Bgl 1 and Tth 111 II sites were absent in the genes of two Aboriginal men expressing TBG-A and were present in those of three Aboriginal and six Caucasian males expressing TBG-C. The TBG gene of a seventh Caucasian male possessed the Bgl 1 site but had lost the Tth 111 II site; sequencing of this allele revealed only the substitution in codon 283 identical to that in the TBGA allele. As the biochemical properties of TBGPhe-283 expressed by this individual were indistinguishable from normal TBGLeu-283, we believe that the abnormal properties of TBG-A are due to substitution of alanine for threonine at residue 191.

Three centuries of stomach symptoms in Scotland.

BACKGROUND: Stomach pain and discomfort have been reported since antiquity. AIM: To follow the time trends since the 18th century of dyspepsia, gastric ulcer, duodenal ulcer, and benign oesophageal disease to test when dyspepsia started to become a major clinical problem. METHODS: The annual in- and out-patient records of the last three centuries from the Scottish Royal Infirmaries of Edinburgh, Aberdeen, Glasgow and Dundee were analysed. In addition, dispensary attendances, clinicians' casebooks, students' notebooks and medical texts have been scrutinized for historic statistics of upper gastrointestinal disease. RESULTS: Dyspepsia was first recorded in the 1750s and increased markedly subsequently. Such dyspepsia persisted after gastric and duodenal ulcers appeared in the late 19th century and then declined again in the late 20th century. Non-ulcer dyspepsia has remained the commonest diagnosis made after endoscopy for stomach pain in the beginning of the 21st century. CONCLUSION: The current commonest diagnosis of stomach pain, dyspepsia dates from the mid-18th century. Any explanations of its causation need to consider this timing.

Rapid nuclear responses to target-derived neurotrophins require retrograde transport of ligand-receptor complex.

Target-derived neurotrophins initiate signals that begin at nerve terminals and cross long distances to reach the cell bodies and regulate gene expression. Neurotrophin receptors, Trks, themselves serve as retrograde signal carriers. However, it is not yet known whether the retrograde propagation of Trk activation reflects movement of Trk receptors from neurites to cell bodies or reflects serial activation of stationary Trk molecules. Here, we show that neurotrophins selectively applied to distal neurites of sensory neurons rapidly induce phosphorylation of the transcription factor cAMP response element-binding protein (CREB) and also cause a slower increase in Fos protein expression. Both nuclear responses require activation of neurotrophin receptors (Trks) at distal nerve endings and retrograde propagation of Trk activation to the nerve cell bodies. Using photobleach and recovery techniques to follow biologically active, green fluorescent protein (GFP)-tagged BDNF receptors (TrkB-GFP) in live cells during retrograde signaling, we show that TrkB-GFP moves rapidly from neurites to the cell bodies. This rapid movement requires ligand binding, Trk kinase activity, and intact axonal microtubules. When they reach the cell bodies, the activated TrkB receptors are in a complex with ligand. Thus, the retrograde propagation of activated TrkB from neurites to cell bodies, although rapid, reflects microtubule-dependent transport of phosphorylated Trk-ligand complexes. Moreover, the relocation of activated Trk receptors from nerve endings to cell bodies is required for nuclear signaling responses. Together, these data support a model of retrograde signaling whereby rapid vesicular transport of ligand-receptor complex from the neurites to the cell bodies mediates the nuclear responses.

Gene expression in the brain across the hibernation cycle.

The purpose of this study was to characterize changes in gene expression in the brain of a seasonal hibernator, the golden-mantled ground squirrel, Spermophilus lateralis, during the hibernation season. Very little information is available on molecular changes that correlate with hibernation state, and what has been done focused mainly on seasonal changes in peripheral tissues. We produced over 4000 reverse transcription-PCR products from euthermic and hibernating brain and compared them using differential display. Twenty-nine of the most promising were examined by Northern analysis. Although some small differences were observed across hibernation states, none of the 29 had significant changes. However, a more direct approach, investigating expression of putative hibernation-responsive genes by Northern analysis, revealed an increase in expression of transcription factors c-fos, junB, and c-Jun, but not junD, commencing during late torpor and peaking during the arousal phase of individual hibernation bouts. In contrast, prostaglandin D2 synthase declined during late torpor and arousal but returned to a high level on return to euthermia. Other genes that have putative roles in mammalian sleep or specific brain functions, including somatostatin, enkephalin, growth-associated protein 43, glutamate acid decarboxylases 65/67, histidine decarboxylase, and a sleep-related transcript SD464 did not change significantly during individual hibernation bouts. We also observed no decline in total RNA or total mRNA during torpor; such a decline had been previously hypothesized. Therefore, it appears that the dramatic changes in body temperature and other physiological variables that accompany hibernation involve only modest reprogramming of gene expression or steady-state mRNA levels.

The 3' codon context effect on UAG suppressor tRNA is different in Escherichia coli and human cells.

We have compared the effect of 3' context on the efficiency of nonsense suppressor tRNAs in Escherichia coli and human cells. Plasmids containing amber (UAG) termination codons were constructed in the vector pRSV beta gal by oligonucleotide insertion at an N-terminal location in a lacZ fusion. A family of identical vectors was prepared with either A, C, G or U as the first 3' base following the stop codon. These derivatives of pRSV beta gal were expressed in E. coli as stable plasmids, or transiently in human 293 cell tissue culture. Nonsense suppression was monitored using enzyme assays for beta-galactosidase. In E. coli the efficiency of a plasmid-borne bacterial tRNA(trp) UAG suppressor varied A > G > C = U. When the same lacZ reporter vectors were cotransfected with a human tRNA(ser) UAG suppressor plasmid into human cells, context effects of a different nature were detected. Double reciprocal analysis of dose-response experiments were used to show that the efficiency of suppression varied C > G > U = A. The discovery of different codon context effects on nonsense suppression in human cells suggest that the interaction between mammalian tRNAs or release factors and their target codons may have different characteristics from those in bacteria.

X-ray analyses of aspartic proteinases. V. Structure and refinement at 2.0 A resolution of the aspartic proteinase from Mucor pusillus.

The structure of mucor pusillus pepsin (EC 3.4.23.6), the aspartic proteinase from Mucor pusillus, has been refined to a crystallographic R-factor of 16.2% at 2.0 A resolution. The positions of 2638 protein atoms, 221 solvent atoms and a sulphate ion have been determined with an estimated root-mean-square (r.m.s.) error of 0.15 to 0.20 A. In the final model, the r.m.s. deviation from ideality for bond distances is 0.022 A, and for angle distances it is 0.050 A. Comparison of the overall three-dimensional structure with other aspartic proteinases shows that mucor pusillus pepsin is as distant from the other fungal enzymes as it is from those of mammalian origin. Analysis of a rigid body shift of residues 190 to 302 shows that mucor pusillus pepsin displays one of the largest shifts relative to other aspartic proteinases (14.4 degrees relative to endothiapepsin) and that changes have occurred at the interface between the two rigid bodies to accommodate this large shift. A new sequence alignment has been obtained on the basis of the three-dimensional structure, enabling the positions of large insertions to be identified. Analysis of secondary structure shows the beta-sheet to be well conserved whereas alpha-helical elements are more variable. A new alpha-helix hN4 is formed by a six-residue insertion between positions 131 and 132. Most insertions occur in loop regions: -5 to 1 (five residues relative to porcine pepsin): 115 to 116 (six residues); 186 to 187 (four residues); 263 to 264 (seven residues); 278 to 279 (four residues); and 326 to 332 (six residues). The active site residues are highly conserved in mucor pusillus pepsin; r.m.s. difference with rhizopuspepsin is 0.37 A for 25 C alpha atom pairs. However, residue 303, which is generally conserved as an aspartate, is changed to an asparagine in mucor pusillus pepsin, possibly influencing pH optimum. Substantial changes have occurred in the substrate binding cleft in the region of S1 and S3 due to the insertion between 115 and 116 and the rearrangement of loop 9-13. Residue Asn219 necessitates a shift in position of substrate main-chain atoms to maintain hydrogen bonding pattern. Invariant residues Asp11 and Tyr14 have undergone a major change in conformation apparently due to localized changes in molecular structure. Both these residues have been implicated in zymogen stability and activation.

Phospholipases Involved in Activation of the Neutrophil NADPH Oxidase

Stimulation of neutrophils with a variety of stimuli can result in the activation of phospholipases A2, C, or D with the resultant hydrolysis of plasma membrane phospholipids and the formation of important second messenger molecules. In the neutrophil, the activities of these phospholipases have been implicated in the processes of both stimulating and maintaining oxidase activation. In this review, some of the methods currently used to measure the products of phospholipase activation in the neutrophil are described, along with the possible role of their products in reactive oxidant production by the neutrophil NADPH oxidase.