Identification of the mutation causing progressive retinal atrophy in Old Danish Pointing Dog

Progressive retinal atrophy (PRA) is a common cause of blindness in many dog breeds. It is most often inherited as a simple Mendelian trait, but great genetic heterogeneity has been demonstrated both within and between breeds. In many breeds the genetic cause of the disease is not known, and until now, the Old Danish Pointing Dog (ODP) has been one of those breeds. ODP is one of the oldest dog breeds in Europe. Seventy years ago the breed almost vanished, but today a population still exists, primarily in Denmark but with some dogs in Germany and Sweden. PRA has been diagnosed in ODP since the late 1990s. It resembles late onset PRA in other dog breeds, and it is inherited as an autosomal recessive trait. In the present study, we performed whole‐genome sequencing and identified a single base insertion (c.3149_3150insC) in exon 1 of C17H2orf71. This is the same mutation previously found to cause PRA in Gordon Setters and Irish Setters, and it was later found in Tibetan Terrier, Standard Poodle and the Polski Owczarek Nizinny. The presence of the mutation in such a diverse range of breeds indicates an origin preceding creation of modern dog breeds. Hence, we screened 262 dogs from 44 different breeds plus four crossbred dogs, and can subsequently add Miniature Poodle and another polish sheepdog, the Polski Owczarek Podhalanski, to the list of affected breeds.     

Heterotetrameric forms of human phenylalanine hydroxylase: Co-expression of wild-type and mutant forms in a bicistronic system

Hybrid forms of human phenylalanine hydroxylase (hPAH) mutants have been found to present catalytic activities lower than predicted from the individual recombinant forms, indicating that interallelic complementation could be a major determinant of the metabolic phenotype of compound heterozygous phenylketonuric (PKU) patients. To provide a molecular explanation for interallelic complementation we have here developed a bicistronic expression system and a purification strategy to obtain isolated hPAH heteromeric forms. On co-expression of WT-hPAH (~ 50% tetramer; ~ 10% dimer) and the N- and C-terminally truncated form ΔN102/ΔC24-hPAH (~ 80% dimer) no heterodimers were recovered. Moreover, by co-expression of WT-hPAH and the N-terminally truncated form ΔN102-hPAH (~ 95% tetramer), heterotetramers, as a result of an assembly of two different homodimers, were isolated. The recovered (WT)/(ΔN102)-hPAH heterotetramers revealed a catalytic activity deviating significantly from that calculated by averaging the respective recombinant homotetrameric forms. The heterotetramer assembly also results in conformational changes in the WT-hPAH protomer, as detected by trypsin limited proteolysis. The finding that the presence of two homodimers with different kinetic parameters influences the properties of the resulting heterotetrameric protein indicates that the dimers exhibit interactions which are transmitted across the assembled tetramer. The bicistronic expression system developed here allowed the isolation of hybrid forms that exhibit negative interallelic complementation, and may represent a model system for studying the molecular pathogenic mechanisms of PAH gene mutations in compound heterozygous PKU patients, providing the rationale to understand the observed inconsistencies both in genotype/phenotype correlations and in the response to BH₄ supplementation.     

Human α-synuclein modulates vesicle trafficking through its interaction with prenylated Rab acceptor protein 1

α-Synuclein has been implicated in the pathogenesis of Parkinson’s disease. Although it is highly conserved, its physiological function has not yet been elucidated in detail. In an effort to define the function of α-synuclein, interacting proteins were screened in phage display assays. Prenylated Rab acceptor protein 1 (PRA1) was identified as an interacting partner. A selective interaction between α-synuclein and PRA1 was confirmed by coimmunoprecipitation and GST pull-down assays. PRA1 and α-synuclein were colocalized in N2a neuronal cells. Cotransfection of α-synuclein and PRA1 caused vesicles to accumulate in the periphery of the cytosol in neuronal cells, suggesting that overexpression of α-synuclein hinders proper vesicle trafficking and recycling as a result of the interaction between α-synuclein and PRA1.     

GTRAP3-18 serves as a negative regulator of Rab1 in protein transport and neuronal differentiation

Glutamate transporter associated protein 3–18 (GTRAP3-18) is an endoplasmic reticulum (ER)-localized protein belonging to the prenylated rab-acceptor-family interacting with small Rab GTPases, which regulate intracellular trafficking events. Its impact on secretory trafficking has not been investigated. We report here that GTRAP3-18 has an inhibitory effect on Rab1, which is involved in ER-to-Golg trafficking. The effects on the early secretory pathway in HEK293 cells were: reduction of the rate of ER-to-Golgi transport of the vesicular stomatitis virus glycoprotein (VSVG), slowed accumulation of a Golgi marker plasmid in pre-Golgi structures after Brefeldin A treatment and inhibition of cargo concentration of the neuronal glutamate transporter excitatory amino-acid carrier 1 (EAAC1) into transpor complexes in HEK293 cells, an effect that could be completely reversed in the presence of an excess of Rab1. In accordance with the known role of Rab1 in neurite formation, overexpression of GTRAP3-18 significantly inhibited the length of outgrowing neurites in differentiated CAD cells. The inhibitory effect of GTRAP3-18 on neurite growth was rescued by co-expression with Rab1, supporting the conclusion that GTRAP 3-18 acted by inhibiting Rab1 action. Finally, we hypothesized that expression of GTRAP3-18 in the brain shoul be lower at stages of active synaptogenesis compared to early developmental stages. This was the case as expression of GTRAP3-18 declined from E17 to P0 and adult rat brains. Thus, we propose a model where protein trafficking and neuronal differentiation are directly linked by the interaction of Rab1 and its regulator GTRAP3-18.     

A comparison of mycolic acid analysis for nontuberculous mycobacteria identification by thin-layer chromatography and molecular methods

The development of fast, inexpensive, and reliable tests to identify nontuberculous mycobacteria (NTM) is needed. Studies have indicated that the conventional identification procedures, including biochemical assays, are imprecise. This study evaluated a proposed alternative identification method in which 83 NTM isolates, previously identified by conventional biochemical testing and in-house M. avium IS1245-PCR amplification, were submitted to the following tests: thin-layer chromatography (TLC) of mycolic acids and PCR-restriction enzyme analysis of hsp65(PRA). High-performance liquid chromatography (HPLC) analysis of mycolic acids and Southern blot analysis for M. avium IS1245 were performed on the strains that evidenced discrepancies on either of the above tests. Sixty-eight out of 83 (82%) isolates were concordantly identified by the presence of IS1245 and PRA and by TLC mycolic acid analysis. Discrepant results were found between the phenotypic and molecular tests in 12/83 (14.4%) isolates. Most of these strains were isolated from non-sterile body sites and were most probably colonizing in the host tissue. While TLC patterns suggested the presence of polymycobacterial infection in 3/83 (3.6%) cultures, this was the case in only one HPLC-tested culture and in none of those tested by PRA. The results of this study indicated that, as a phenotypic identification procedure, TLC mycolic acid determination could be considered a relatively simple and cost-effective method for routine screening of NTM isolates in mycobacteriology laboratory practice with a potential for use in developing countries. Further positive evidence was that this method demonstrated general agreement on MAC and M. simiae identification, including in the mixed cultures that predominated in the isolates of the disseminated infections in the AIDS patients under study. In view of the fact that the same treatment regimen is recommended for infections caused by these two species, TLC mycolic acid analysis may be a useful identification tool wherever molecular methods are unaffordable.     

Circadian variations of plasma renin activity (PRA), aldosterone and electrolyte concentrations in plasma in pregnant and non-pregnant goats

The aim of this study was to estimate and analyse circadian variations of the renin-angiotensin-aldosterone system (RAA) activity in blood of goats and the influence of late pregnancy on the circadian variations of RAA system. The study was carried out on a group of 17 non-pregnant and 9 pregnant goats. The animals were kept in uniform environmental conditions, (9 h light/15 h darkness). Blood samples were collected seven times over a period of 24 h, every 4 h. Plasma renin activity (PRA), plasma aldosterone (PA), sodium, potassium and chloride concentrations were determined. PRA and PA of both groups changed during 24 h, with the highest values in the dark phase and with higher RAA system activity (especially during the night) in the pregnant goats. In the non-pregnant goats, no circadian changes in PRA and PA were observed. The circadian changes in PRA and PA found in pregnant goats had acrophases at 06:27 h and 01:13 h, respectively. Plasma electrolyte concentrations in both groups of goats also changed during 24 h. These results suggest that circadian changes of potassium concentration in plasma of goats during late pregnancy may be one of the main factors affecting the RAA system.     

JM4 is a four-transmembrane protein binding to the CCR5 receptor

The CC chemokine receptor 5 (CCR5) is a major co-receptor for human immunodeficiency virus (HIV) and CCR5 mutants lacking the carboxy (C)-terminus interfere with HIV infection. Therefore, we analysed the C-terminus of CCR5 and here describe Jena-Muenchen 4 (JM4), a novel CCR5-interacting protein. JM4 is membrane-associated, co-precipitates with CCR5, and is ubiquitously expressed. It shares about 62% sequence similarity with JWA and glutamate transporter-associated protein 3-18 (GTRAP3-18), a regulator of an amino acid transporter. JWA, like JM4, is a four-transmembrane protein, which binds to the CCR5 receptor. Furthermore, JM4, JWA, and GTRAP3-18 co-localise and heterodimerise indicating a functional relationship. JM4 co-localises with calnexin in the endoplasmic reticulum and with the mannose 6-phosphate receptor in the Golgi. JM4 and GTRAP3-18 harbor a Rab-acceptor motif, indicating a function in vesicle formation at the Golgi complex. In conclusion, we describe a CCR5-interacting protein, which is suggested to function in trafficking and membrane localisation of the receptor, possibly also other receptors or amino acid transporters.     

Direct application of AvaII PCR restriction fragment length polymorphism analysis (AvaII PRA) targeting 644 bp heat shock protein 65 (hsp65) gene to sputum samples

To evaluate the usefulness of the AvaII PRA method targeting 644-bp hsp65 gene for the direct detection of pathogenic mycobacteria from clinical specimens, we applied this method to 40 sputum samples and compared the results to those obtained by IS 6110 PCR. Although this method showed a sensitivity slightly lower than IS 6110 PCR (97.5% vs. 100%), it detected infections of M. avium complex (MAC) in two patients, which was not possible by IS 6110 PCR. We conclude that AvaII PRA is a highly effective method for directly detecting pathogenic mycobacteria in primary clinical specimens.     

Risk assessment for nonindigenous pests: 2. Accounting for interyear climate variability

The paper firstly discusses the importance of accounting for interyear variability when assessing the likelihood of establishment of an alien pest. The potential establishment of Colorado beetle (Leptinotarsa decemlineata) is used as an illustration within the geographical context of England and Wales. An aggregate risk index is introduced as a probabilistic representation of the likelihood that a pest might complete a single generation over a 30‐year period (1961–90). Data for individual years were used to compute, objectively, the interyear distribution of risk across the landscape. The standard deviation in area at risk (26 800 km²) was high relative to the average proportion of the landscape potentially at risk (95 700 km²). In 40% of years, the area at risk was estimated to be higher than ‘average’. Secondly, the paper demonstrates multiple indices of risk that reflect different aspects of pest risk assessment. Viewing risk from a variety of perspectives provides a means of gauging the consistency and therefore reliability of the results. This contrasts with current practice, where a single mapped output is commonly presented to decision makers. Modelling using a daily time step allowed the use of indices to investigate the long‐term probabilities of biotic and abiotic events of short duration. These indices include estimates of pest activity and flight potential.