Feline immunodeficiency virus (FIV) infection in domestic pet cats in Australia and New Zealand: Guidelines for diagnosis, prevention and management.

Despite the passage of over 30 years since its discovery, the importance of feline immunodeficiency virus (FIV) on the health and longevity of infected domestic cats is hotly debated amongst feline experts. Notwithstanding the absence of good quality information, Australian and New Zealand (NZ) veterinarians should aim to minimise the exposure of cats to FIV. The most reliable way to achieve this goal is to recommend that all pet cats are kept exclusively indoors, or with secure outdoor access (e.g., cat enclosures, secure gardens), with FIV testing of any in-contact cats. All animal holding facilities should aim to individually house adult cats to limit the spread of FIV infection in groups of animals that are stressed and do not have established social hierarchies. Point-of-care (PoC) FIV antibody tests are available in Australia and NZ that can distinguish FIV-infected and uninfected FIV-vaccinated cats (Witness™ and Anigen Rapid™). Although testing of whole blood, serum or plasma remains the gold standard for FIV diagnosis, PoC testing using saliva may offer a welfare-friendly alternative in the future. PCR testing to detect FIV infection is not recommended as a screening procedure since a negative PCR result does not rule out FIV infection and is only recommended in specific scenarios. Australia and NZ are two of three countries where a dual subtype FIV vaccine (Fel-O-Vax® FIV) is available and offers a further avenue for disease prevention. Since FIV vaccination only has a reported field effectiveness of 56% in Australia, and possibly lower in NZ, FIV-vaccinated cats should undergo annual FIV testing prior to annual FIV re-vaccination using a suitable PoC kit to check infection has not occurred in the preceding year. With FIV-infected cats, clinicians should strive to be even more thorough than usual at detecting early signs of disease. The most effective way to enhance the quality of life and life expectancy of FIV-infected cats is to optimise basic husbandry and to treat any concurrent conditions early in the disease course. Currently, no available drugs are registered for the treatment of FIV infection. Critically, the euthanasia of healthy FIV-infected cats, and sick FIV-infected cats without appropriate clinical investigations, should not occur.

Detection of Brucella spp. during a serosurvey of pig-hunting and regional pet dogs in eastern Australia.

Brucellosis is a zoonotic disease with worldwide distribution. Brucella suis serotype 1 is thought to be maintained in the Australian feral pig population, with disease prevalence higher in Queensland (Qld) than New South Wales (NSW). Pig hunting is a popular recreational activity in rural Qld and NSW, with feral pigs in these states thought to carry B. suis. Brucellosis associated with B. suis has been diagnosed in dogs engaged in pig hunting in some of these areas. A total of 431 dogs from northern Qld and north-west NSW were recruited. Two distinct cohorts of clinically healthy dogs were tested - (1) 96 dogs from central, north and far north Queensland actively engaged in pig-hunting and (2) 335 dogs from rural and remote north-west NSW that were primarily companion (non-pig hunting) animals. Serum samples were tested for antibodies to Brucella spp. using the Rose Bengal test (RBT) test followed by complement fixation testing (CFT) for RBT-positive samples. A subset of samples was retested using RBT and CFT. Seven dogs were considered seropositive for B. suis from Qld and remote NSW, including 4/96 (4.2%; 95% CI 3.5% to 4.3%) from the pig-hunting cohort and 3/335 (0.9%) from the regional pet dog cohort. The use of RBT and CFT in dogs to detect anti-Brucella antibodies requires validation. Veterinarians treating pig-hunting dogs and physicians treating pig hunters in central, north and far north Qld need to be aware of the zoonotic risk posed by B. suis to these groups.

Seroprevalence of Coxiella burnetii in pig-hunting dogs from north Queensland, Australia.

The causative agent of Q fever, Coxiella burnetii, is endemic to Queensland and is one of the most important notifiable zoonotic diseases in Australia. The reservoir species for C. burnetii are classically ruminants, including sheep, cattle and goats. There is increasing evidence of C. burnetii exposure in dogs across eastern and central Australia. The present study aimed to determine if pig-hunting dogs above the Tropic of Capricorn in Queensland had similar rates of C. burnetii exposure to previous serosurveys of companion dogs in rural north-west New South Wales. A total of 104 pig-hunting dogs had serum IgG antibody titres to phase I and phase 2 C. burnetii determined using an indirect immunofluorescence assay test. Almost one in five dogs (18.3%; 19/104; 95% confidence interval 9.6%-35.5%) were seropositive to C. burnetii, with neutered dogs more likely to test positive compared to entire dogs (P = 0.0497). Seropositivity of the sampled pig-hunting dogs was one of the highest recorded in Australia. Thirty-nine owners of the pig-hunting dogs completed a survey, revealing 12.8% (5/39) had been vaccinated against Q fever and 90% (35/39) were aware that both feral pigs and dogs could potentially be sources of C. burnetii. Our findings indicate that pig hunters should be aware of the risk of exposure to Q fever during hunts and the sentinel role their dogs may play in C. burnetii exposure.

Stathmin and EGFR correlates to HPV status and clinical outcome in sinonasal inverted papilloma.

BACKGROUND: Inverted papilloma (IP) is a locally destructive benign tumour of the sinonasal mucosa with a tendency for malignant transformation. Stathmin and epidermal growth factor receptor (EGFR) are important markers in cancer prognosis. Here we investigate if expression of stathmin and EGFR correlate to dysplasia, recurrence and HPV in IP. METHODS: 98 patients with IP diagnosed 2000-2010 were analyzed for stathmin and EGFR by immunohistochemistry (IHC) and HPV by polymerase chain reaction assay (PCR). RESULTS: All IPs expressed stathmin while its expression was absent or weak in normal mucosa. Dysplasia was present in 26,7% of IPs with high stathmin expression while only 7.4% of IPs with low stathmin expression showed dysplasia. Stathmin positive IPs showed a trend towards earlier recurrences. 57.1% of IP expressed EGFR but no significant association was seen between EGFR-positivity and recurrence or dysplasia. EGFR was expressed by 91.7% of the HPV-positive IPs compared to 52,3% of the HPV negative IPs. CONCLUSIONS: EGFR expression is significantly higher in HPV positive IP. Stathmin is expressed by all IP tumour cells. Stathmin was also associated with dysplasia and a trend towards a correlation between stathmin positivity and recurrence was found. Stathmin and EGFR might therefore be considered therapeutic targets.

Diagnosing feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV) infection: an update for clinicians.

With the commercial release in Australia in 2004 of a vaccine against feline immunodeficiency virus (FIV; Fel-O-Vax FIV®), the landscape for FIV diagnostics shifted substantially. Point-of-care (PoC) antibody detection kits, which had been the mainstay for diagnosing FIV infection since the early 1990s, were no longer considered accurate to use in FIV-vaccinated cats, because of the production of vaccine-induced antibodies that were considered indistinguishable from those produced in natural FIV infections. Consequently, attention shifted to alternative diagnostic methods such as nucleic acid detection. However, over the past 5 years we have published a series of studies emphasising that FIV PoC test kits vary in their methodology, resulting in differing accuracy in FIV-vaccinated cats. Importantly, we demonstrated that two commercially available FIV antibody test kits (Witness™ and Anigen Rapid™) were able to accurately distinguish between FIV-vaccinated and FIV-infected cats, concluding that testing with either kit offers an alternative to PCR testing. This review summarises pertinent findings from our work published in a variety of peer-reviewed research journals to inform veterinarians (particularly veterinarians in Australia, New Zealand and Japan, where the FIV vaccine is currently commercially available) about how the approach to the diagnosis of FIV infection has shifted. Included in this review is our work investigating the performance of three commercially available FIV PoC test kits in FIV-vaccinated cats and our recommendations for the diagnosis of FIV infection; the effect of primary FIV vaccination (three FIV vaccines, 4 weeks apart) on PoC test kit performance; our recommendations regarding annual testing of FIV-vaccinated cats to detect 'vaccine breakthroughs'; and the potential off-label use of saliva for the diagnosis of FIV infection using some FIV PoC test kits. We also investigated the accuracy of the same three brands of test kits for feline leukaemia virus (FeLV) diagnosis, using both blood and saliva as diagnostic specimens. Based on these results, we discuss our recommendations for confirmatory testing when veterinarians are presented with a positive FeLV PoC test kit result. Finally, we conclude with our results from the largest and most recent FIV and FeLV seroprevalence study conducted in Australia to date.

The protective rate of the feline immunodeficiency virus vaccine: An Australian field study.

A case-control field study was undertaken to determine the level of protection conferred to client-owned cats in Australia against feline immunodeficiency virus (FIV) using a commercial vaccine. 440 cats with outdoor access from five Australian states/territories underwent testing, comprising 139 potential cases (complete course of primary FIV vaccinations and annual boosters for three or more years), and 301 potential controls (age, sex and postcode matched FIV-unvaccinated cats). FIV status was determined using a combination of antibody testing (using point-of-care test kits) and nucleic acid amplification, as well as virus isolation in cases where results were discordant and in all suspected FIV-vaccinated/FIV-infected cats ('vaccine breakthroughs'). Stringent inclusion criteria were applied to both 'cases' and 'controls'; 89 FIV-vaccinated cats and 212 FIV-unvaccinated cats ultimately satisfied the inclusion criteria. Five vaccine breakthroughs (5/89; 6%), and 25 FIV-infected controls (25/212; 12%) were identified, giving a vaccine protective rate of 56% (95% CI -20 to 84). The difference in FIV prevalence rates between the two groups was not significant (P=0.14). Findings from this study raise doubt concerning the efficacy of Fel-O-Vax FIV® under field conditions. Screening for FIV infection may be prudent before annual FIV re-vaccination and for sick FIV-vaccinated cats. Owners should not rely on vaccination alone to protect cats against the risk of acquiring FIV infection; other measures such as cat curfews, the use of 'modular pet parks' or keeping cats exclusively indoors, are recommended.

Paving the way of systems biology and precision medicine in allergic diseases: the MeDALL success story: Mechanisms of the Development of ALLergy; EU FP7-CP-IP; Project No: 261357; 2010-2015.

MeDALL (Mechanisms of the Development of ALLergy; EU FP7-CP-IP; Project No: 261357; 2010-2015) has proposed an innovative approach to develop early indicators for the prediction, diagnosis, prevention and targets for therapy. MeDALL has linked epidemiological, clinical and basic research using a stepwise, large-scale and integrative approach: MeDALL data of precisely phenotyped children followed in 14 birth cohorts spread across Europe were combined with systems biology (omics, IgE measurement using microarrays) and environmental data. Multimorbidity in the same child is more common than expected by chance alone, suggesting that these diseases share causal mechanisms irrespective of IgE sensitization. IgE sensitization should be considered differently in monosensitized and polysensitized individuals. Allergic multimorbidities and IgE polysensitization are often associated with the persistence or severity of allergic diseases. Environmental exposures are relevant for the development of allergy-related diseases. To complement the population-based studies in children, MeDALL included mechanistic experimental animal studies and in vitro studies in humans. The integration of multimorbidities and polysensitization has resulted in a new classification framework of allergic diseases that could help to improve the understanding of genetic and epigenetic mechanisms of allergy as well as to better manage allergic diseases. Ethics and gender were considered. MeDALL has deployed translational activities within the EU agenda.

Structural activity relationship analysis (SAR) and in vitro testing reveal the anti-ageing potential activity of acetyl aspartic acid.

BACKGROUND: Acetyl aspartic acid (A-A-A) was discovered through gene array analysis with corresponding connectivity mapping (Cmap), aiming for identification of new compounds with anti-ageing properties. OBJECTIVE: The aim of this study was to use structural activity relationship (SAR) analysis to identify a predictive mechanism of action of A-A-A. The findings from SAR will be further characterized by in vitro activity testing. Furthermore, we aimed to investigate the role of polymerized filamentous F-actin in ageing fibroblasts and to evaluate the effect of A-A-A on this model. METHODS: To predict the mode of action of A-A-A, we used the PASS computer program as a SAR model. In vitro, scratch motility tests with immortalized keratinocytes were used as a model for wound healing potential. Matrix metalloproteinase 1-3 (MMP 1-3) was analysed using multiplex protein assays (Luminex), and polymerized actin was detected by phalloidin staining in dermal fibroblasts (HDF). RESULTS: SAR analysis predicted that A-A-A would possess both epidermal and dermal activities with identification of wound healing and MMP inhibition potential. Further in vitro studies confirmed the wound healing potential using keratinocyte scratch motility assays. We were also able to confirm the dermal activities predicted by inhibition of MMP (MMP 1-3) in HDF by A-A-A. In addition, we found a positive relationship between age and F-actin expression. We also discovered that stimulation of HDF with A-A-A for 72 h significantly reduced the polymerized cytoskeletal network as visualized by inhibition of F-actin expression. In fact, A-A-A leveraged the expression of F-actin in middle-aged female fibroblasts (50 years of age) to the level of young female fibroblasts (30 years of age), corresponding to a 40% reduction in F-actin expression. CONCLUSION: Using an in silico and in vitro approach, we were able to demonstrate that A-A-A has the capacity to target different compartments of the skin through keratinocyte regeneration, MMP inhibition and relief in fibroblasts stiffness by reduction of F-actin cytoskeletal network in HDF.

Allergy-related diseases and recurrent abdominal pain during childhood - a birth cohort study.

BACKGROUND: Allergy and immune dysregulation may have a role in the pathophysiology of recurrent abdominal pain of functional origin, but previous studies of allergy-related diseases and abdominal pain have contradictory results. AIM: To examine the association between allergy-related diseases or sensitisation during childhood and abdominal pain at age 12 years. METHODS: In this birth cohort study of 4089 children, parents answered questionnaires regarding asthma, allergic rhinitis, eczema and food hypersensitivity ('allergy-related diseases') at ages 0,1,2,4,8 and 12 years. Blood for analyses of allergen-specific IgE was sampled at 4 and 8 years. At 12 years, the children answered questions regarding abdominal pain. Children with coeliac disease or inflammatory bowel disease were excluded. Associations were examined using multivariable logistic regression. RESULTS: Among 2610 children with complete follow-up, 9% (n = 237) reported abdominal pain at 12 years. All allergy-related diseases were associated with concurrent abdominal pain at 12 years and the risk increased with increasing number of allergy-related diseases (P for trend <0.001). Asthma at 1 and 2 years and food hypersensitivity at 8 years were significantly associated with abdominal pain at 12 years. There was an increased risk of abdominal pain at 12 years in children sensitised to food allergens at 4 or 8 years, but in stratified analyses, this was confined to children whose parents had not reported food hypersensitivity at time of sensitisation. CONCLUSION: Allergy-related diseases as well as sensitisation to food allergens were associated with an elevated risk of abdominal pain, and the risk increased with the number of allergy-related diseases.

Characteristics of persons convicted for offences relating to animal hoarding in New South Wales.

OBJECTIVE: To highlight the characteristics of persons convicted for offences related to animal hoarding in New South Wales, Australia, document the outcomes of cases and compare them with overseas studies. DESIGN: Retrospective case series. METHODS: Records of finalised prosecutions for offences relating to animal hoarding between 2005 and 2011 were examined. Data recorded included: the age of each subject at the first offence, sex, postcode, occupation, living conditions, number of charges, number of prosecutions, title of each charge, number and species of live animals, whether animals needed veterinary attention, the medical conditions that the animals suffered, whether dead animals were on the property, how animals were obtained, veterinary and legal costs accrued and case outcomes. The data were analysed to obtain frequencies and relative frequencies for categorical variables and summary statistics for quantitative variables. Observed frequencies were compared using Chi-square test with the expected frequencies calculated based on the Australian Bureau of Statistics data for NSW. RESULTS: The number of persons included was 29. Most were female (72.4%) and 23 were 40-64 years of age at their first offence. Almost one-third identified themselves as breeders, eight as pensioners and four as unemployed. Most resided in inner regional Australia (45%), 28% lived in major cities and 28% lived in outer regional Australia. Dogs were the species hoarded in 80% of cases. Animals requiring veterinary attention were identified in all cases. Dead animals were found on premises in 41.4% of cases. CONCLUSIONS: Persons prosecuted for charges relating to animal hoarding in NSW have similar characteristics to those of previous studies, although the outcomes may be different. More farm animals and horses were hoarded in NSW and hoarders in NSW were more likely to live in inner regional and outer regional areas (rural areas) than animal hoarders in the USA.

Transcriptional changes in organoculture of full-thickness human skin following topical application of all-trans retinoic acid.

In this study, we developed an organoculture of human skin to investigate the effect of topical applied all-trans retinoic acid using a gene array approach. We could by using this approach confirm previous studies on genes activated by RA in keratinocyte monocultures and also provide new insights on genes that are relevant to RA-activation in human skin. The results in the present study show this model represent a valuable pre-clinical model for studying the effects of retinoids in skin.

The link between parental allergy and offspring allergic and nonallergic rhinitis.

BACKGROUND: Parental allergy-related disease increases the risk for rhinitis, but it remains unknown how different phenotypes of parental allergy affect this risk. The aim of this study was to investigate how parental hay fever, asthma, and eczema affect the risk of allergic rhinitis (AR) and nonallergic rhinitis (NAR) at 8 years of age. METHODS: Information on 2413 children from a population-based birth cohort was used combining questionnaire data and IgE to inhalant allergens. Logistic regression was used to estimate the association between parental allergy-related disease and AR and NAR. In addition, cluster analysis was used to search for latent phenotypes of heredity likely to be associated with AR and NAR. RESULTS: At age 8 years, 13.8% of the children had AR, while 6.4% had NAR. Parental isolated hay fever increased the odds of AR (OR 2.2, 95% CI 1.6-3.2), whereas isolated asthma or eczema did not. The odds of NAR increased when one parent had two or more allergy-related diseases. In the cluster analysis, the highest proportion of AR, 37.5%, was seen in a cluster where both parents had hay fever and pollen allergy and that of NAR, 11.0%, in a cluster where one parent had hay fever, pollen allergy, and eczema. CONCLUSIONS: Parental allergy-related disease may be an important risk factor for NAR as well as AR, and the risk is comparable for maternal and paternal allergy. Parental hay fever seems to be the dominating hereditary risk factor for AR.

Lack of acetylcholine nicotine alpha 7 receptor suppresses development of collagen-induced arthritis and adaptive immunity.

Activation of the alpha7 receptor (α7nAChR) has been shown to be important in inflammation and immune regulation, and is also essential in the neural cholinergic anti-inflammatory pathway. The aim of this study was to investigate the role of α7nAChR in the development of experimental arthritis and immune activation. Mice lacking the α7nAChR were immunized with collagen II and the development of arthritis was assessed. Another group of α7nAChR-deficient mice was immunized with ovalbumin, spleen and lymph node cells were isolated and the proliferative responses to restimulation with ovalbumin or concanavalin A were investigated. We could demonstrate significantly milder arthritis and less cartilage destruction, together with a decrease of T cell content in lymph nodes in mice lacking the α7nAChR compared to wild-type controls. In addition, mice lacking the α7nAChR had a deficient proliferative response to concanavalin A, whereas antigen presentation-dependent proliferation was not affected. These results indicate important roles for α7nAChR in arthritis development as well as in regulation of T cell-dependent immunological mechanisms. In addition, the data implicate α7nAChR as a therapeutic target for modulation of adaptive immune responses.

Cell specific synovial expression of nicotinic alpha 7 acetylcholine receptor in rheumatoid arthritis and psoriatic arthritis.

Neuroimmune interactions are known to influence several chronic inflammatory and rheumatic diseases, but the underlying mechanisms have been insufficiently elucidated. The cholinergic anti-inflammatory pathway is characterized by neural regulation of systemic inflammation, mediated by the vagus nerve and specific cholinergic stimulation of the nicotinic alpha-7 acetylcholine receptor (alpha7nAChR) on immune cells. Moreover, alpha7nAChR has been shown important for immune regulation also in the absence of nerves, but little is known about these mechanisms in chronic joint inflammation. The expression and localization of alpha7nAChR in synovial biopsies from patients with rheumatoid arthritis and psoriatic arthritis was investigated by immunohistochemistry using monoclonal antibody against alpha7nAChR. Surface staining of alpha7nAChR was observed in synovial tissue of all arthritis patients investigated and could also to a lesser extent be detected in the synovium of healthy individuals. alpha7nAChR positive cells were detected in mainly synovial lining cells and vessels. The alpha7nAChR positively stained cells were by double immunofluorescence identified as primarily macrophages and fibroblasts, with the majority of these cells expressing the receptor. These results indicate the importance of alpha7nAChR and cholinergic mechanisms in arthritis pathogenesis and implicate specific cholinergic modulation as a potential anti-inflammatory therapeutic strategy in joint inflammation.

Influence of body weight, age, and weight gain on fertility and prolificacy in four breeds of ewe lambs.

Breeding ewes to lamb at 1 yr of age can improve profitability for some production systems. The first objective of this study was to evaluate the effect of age and weight at breeding and total postweaning weight gain on reproductive performance of ewe lambs. The second objective was to compare the effects of weight and age variables in four major sheep breeds (Columbia, Polypay, Rambouillet, and Targhee). Weights, ages, and the binary traits of fertility (pregnant or nonpregnant) and prolificacy (one lamb born vs. two or more) were collected on 2,055 ewe lambs at the U.S. Sheep Experiment Station, Dubois, ID, from 1984 through 1988. The effects of age and weight at breeding and total weight gain from weaning to breeding on fertility and prolificacy were analyzed with a logit model in a maximum likelihood analyses. Differences (P < 0.001) among breeds for fertility were identified, with a 93% fertility rate for Polypay ewe lambs compared with lower fertility rates in Columbia, Targhee, and Rambouillet ewe lambs (50, 60, and 75%, respectively). The percentage of multiple births (prolificacy rate) also was higher (P < 0.001) in the Polypay (47%) than in Columbia, Targhee, and Rambouillet breeds (1, 13, and 14%, respectively). Averaged across breeds, weight at breeding had a positive effect on fertility and prolificacy (P < 0.001), whereas total weight gain from weaning to breeding had a positive effect only on fertility (P < 0.027). In separate analyses for each breed, increasing age (P < 0.001) and weight at breeding (P < 0.001) increased the probability of pregnancy in Rambouillet ewe lambs. The probability of pregnancy for Targhee ewe lambs increased (P < 0.005) with weight at breeding. Increasing weight at breeding increased (P < 0.004) the probability of multiple births in all breeds. Increasing total postweaning weight gain increased (P < 0.007) the probabilities of multiple births in Rambouillet and Targhee ewe lambs. In conclusion, Polypay ewe lambs were superior in fertility and prolificacy to Columbia, Rambouillet, and Targhee under Western range conditions. Improved reproductive performance of Columbia, Rambouillet, and Targhee ewe lambs may be achieved by increasing age and weight at breeding and postweaning gain.

Expression of microsomal prostaglandin E synthase 1 in rheumatoid arthritis synovium.

OBJECTIVE: Microsomal prostaglandin E synthase 1 (mPGES-1) catalyzes the formation of PGE(2) from cyclooxygenase-derived PGH(2). Microsomal PGES-1 is induced by proinflammatory cytokines and is strongly linked to conditions that result in high PGE(2) biosynthesis. PGE(2) contributes to the pathogenesis of rheumatoid arthritis (RA), acting as a mediator of inflammation and promoting bone destruction. Induction of mPGES-1 in rheumatoid synoviocytes by proinflammatory cytokines has been demonstrated in vitro, indicating an important role in RA pathogenesis. Recent studies using mPGES-1-deficient mice demonstrated the importance of this gene in chronic inflammation. The aim of this study was to investigate the expression and localization of mPGES-1 in synovial biopsy specimens obtained from patients with RA. METHODS: Synovial tissue samples from 24 patients with RA were obtained, and immunohistologic analysis was performed using polyclonal antibodies against mPGES-1. Double immunofluorescence staining was performed with antibodies to CD3, CD19, CD20, CD68, CD163, and prolyl 4-hydroxylase. RESULTS: Intracellular mPGES-1 staining was observed in synovial membranes from all of the RA patients studied. Specifically, strong expression of mPGES-1 was detected in synovial lining cells. In sublining mononuclear and fibroblast-like cells, the extent of mPGES-1 staining was less than that in the synovial lining cells. In some patients, positive staining was observed in endothelial cells. With the double immunofluorescence technique, mPGES-1 production was detected in synovial macrophages and fibroblasts, while mPGES-1 expression was not observed in lymphocytes. CONCLUSION: The demonstration of mPGES-1 expression in synovial tissues from patients with RA suggests a role for mPGES-1 in the RA disease process. Microsomal PGES-1 might be a potential new target for treatment strategies to control PGE(2) synthesis in patients with RA, without the systemic side effects associated with cyclooxygenase inhibitors.

Two active site asparagines are essential for the reaction mechanism of the class III anaerobic ribonucleotide reductase from bacteriophage T4.

Class III ribonucleotide reductase is an anaerobic enzyme that uses a glycyl radical to catalyze the reduction of ribonucleotides to deoxyribonucleotides and formate as ultimate reductant. The reaction mechanism of class III ribonucleotide reductases requires two cysteines within the active site, Cys-79 and Cys-290 in bacteriophage T4 NrdD numbering. Cys-290 is believed to form a transient thiyl radical that initiates the reaction with substrate and Cys-79 to take part as a transient thiyl radical in later steps of the reductive reaction. The recently solved three-dimensional structure of class III ribonucleotide reductase (RNR) from bacteriophage T4 shows that two highly conserved asparagines, Asn-78 and Asn-311, are positioned close to the essential Cys-79. We have investigated the function of Asn-78 and Asn-311 by site-directed mutagenesis and measured enzyme activity and glycyl radical formation in five single (N78(A/C/D) and N311(A/C)) and one double (N78A/N311A) mutant proteins. Our results suggest that both asparagines are important for the catalytic mechanism of class III RNR and that one asparagine can partially compensate for the lack of the other functional group in the single Asn --> Ala mutant proteins. A plausible role for these two asparagines could be in positioning formate in the active site to orient it toward the proposed thiyl radical of Cys-79. This would also control the highly reactive carbon dioxide radical anion form of formate within the active site before it is released as carbon dioxide. A detailed reaction scheme including the function of the two asparagines and two formate molecules is proposed for class III RNRs.

The impact of vacation and job stress on burnout and absenteeism.

Abstract The purpose of the present study was to test the hypothesis that vacation relief decreases psychological and behavioral strains caused by job stressors. We examined the impact of job stress and vacation on strain on 87 blue-collar employees in an industrial enterprise in central Israel. Whereas former respite research focused on the impact of vacation only on psychological strains such as burnout and job and life satisfaction, the current study also examined a behavioral strain, absenteeism. The employees completed questionnaires before and after vacation and again four weeks later. Our findings show that vacation alleviated perceived job stress and bumout as predicted, replicating findings that a respite from work diminishes levels of strain to lower than chronic, on-the-job levels. We found declines in burnout immediately after the vacation and a return to prevacation levels four weeks later, and a similar pattern with regard to absenteeism.

Cysteines involved in radical generation and catalysis of class III anaerobic ribonucleotide reductase. A protein engineering study of bacteriophage T4 NrdD.

Class III ribonucleotide reductase (RNR) is an anaerobic glycyl radical enzyme that catalyzes the reduction of ribonucleotides to deoxyribonucleotides. We have investigated the importance in the reaction mechanism of nine conserved cysteine residues in class III RNR from bacteriophage T4. By using site-directed mutagenesis, we show that two of the cysteines, Cys-79 and Cys-290, are directly involved in the reaction mechanism. Based on the positioning of these two residues in the active site region of the known three-dimensional structure of the phage T4 enzyme, and their structural equivalence to two cysteine residues in the active site region of the aerobic class I RNR, we suggest that Cys-290 participates in the reaction mechanism by forming a transient thiyl radical and that Cys-79 participates in the actual reduction of the substrate. Our results provide strong experimental evidence for a similar radical-based reaction mechanism in all classes of RNR but also identify important differences between class III RNR and the other classes of RNR as regards the reduction per se. We also identify a cluster of four cysteines (Cys-543, Cys-546, Cys-561, and Cys-564) in the C-terminal part of the class III enzyme, which are essential for formation of the glycyl radical. These cysteines make up a CX(2)C-CX(2)C motif in the vicinity of the stable radical at Gly-580. We propose that the four cysteines are involved in radical transfer between Gly-580 and the cofactor S-adenosylmethionine of the activating NrdG enzyme needed for glycyl radical generation.