The role of biomechanical factors in ankylosing spondylitis: the patient's perspective.

Biomechanical factors including occupational joint physical stressing and joint injury have been linked to spondyloarthritis. We explored such factors in ankylosing spondylitis (AS). A retrospective, online survey was developed alongside the UK National Ankylosing Spondylitis Society (NASS). Questions on early entheseal symptoms, potential precipitating trauma, sporting activity, and physiotherapy were asked. A total of 1026 patients responded with 44% recalling an instance of injury or trauma as a potential trigger for their AS. After symptom onset, 55% modified sporting activities and 28% reported that the initial AS recommended exercises exacerbated symptoms. Patients report physical trauma, exercise and physiotherapy as potential triggers for AS symptoms. These findings further support the experimental evidence for the role of biomechanical factors in disease.

Two-dimensional gas chromatography with heart-cutting for isotope ratio mass spectrometry analysis of steroids in doping control.

The accuracy and precision of gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) measurements are highly dependent on analyte purity. Reliable analysis of urinary steroids for doping control therefore requires extensive and time-consuming sample preparation (i.e. liquid chromatography fraction collection) prior to GC-C-IRMS analysis. The use of two-dimensional GC (GC-GC) with heart-cutting (Deans Switch) as a possible approach to reduce the sample purification required for IRMS analysis is described herein. The system uses a low thermal mass oven (LTM) incorporated into an existing GC-C-IRMS system. GC-GC allowed the use of a cyanopropyl/phenyl column in the first dimension to optimize the separation of underivatized steroids, while a phenyl-methylpolysiloxane column in the second dimension focuses the selectively cut analytes into narrower peaks for more sensitive and reliable MS analysis. In addition, to confirm analyte identity, eluent from the second GC was split, with 20 % entering a scanning MS, and 80 % flowing to the IRMS. As a proof concept, the developed method was then used to analyze a single spot urine (5 ml) from an individual receiving T therapy (2 × 50 mg sachets of Testogel(®)). The T delta value (-27.8 ‰, [T] = 38 ng/ml) was clearly distinct from 11-ketoetiocholanolone (-22.5 ‰) (used as an endogenous reference compound (ERC)), indicating T as being of exogenous origin. The simultaneous analysis by the scanning MS yielded a full scan mass spectrum of the same chromatographic peak, thus confirming the peak to be T.

European Network of Forensic Science Institutes (ENFSI): Evaluation of new commercial STR multiplexes that include the European Standard Set (ESS) of markers.

To support and to underpin the European initiative to increase the European set of standard markers (ESS), by the addition of five new loci, a collaborative project was organised by the European Network of Forensic Science Institutes (ENFSI) DNA working group in order to assess the new multiplex kits available. We have prepared allele frequency databases from 26 EU populations. Concordance studies were carried out to verify that genotyping results were consistent between kits. Population genetics studies were conducted and it was estimated that F(ST)<0.001. The results showed that the kits were comparable to each other in terms of performance and major discrepancy issues were highlighted. We provide details of allele frequencies for each of the populations analysed per laboratory.

Effects of storage temperatures and annealing conditions on the structure and properties of potato (Solanum tuberosum) starch.

Starches were extracted from freshly harvested potatoes (12 cultivars, grown in Perthshire) and the properties of the starches of six cultivars were compared with starches extracted from the same samples but stored at 5, 25 or 55 degrees C for 7 days before extraction. The amylose (total) content of the freshly extracted starches from tubers stored at 5, 25 or 55 degrees C was on average 27.9+/-2.3, 28.3+/-1.7, 29.2+/-2.2 and 28.8+/-1.5%, respectively, with corresponding phosphorus representing 60+/-16, 64+/-9, 61+/-5 and 63+/-9 mg 100 g(-1). The unit chain distribution by chromatography of the amylopectin molecules from the starches extracted from the different conditions was very similar with an average degree of polymerisation (DP) of 26+/-2 where the two major fractions (F1 and F2) represented 54+/-2 and 19+/-1, respectively. Peak gelatinisation temperatures (Tp) and enthalpies (DeltaH) for the freshly extracted starches and from tubers stored at 5 or 25 degrees C were very similar (63.3+/-1.5 degrees C and 18.6+/-0.8 J g(-1); 63.1+/-1.0 degrees C and 17.7+/-1.5 J g(-1) and; 62.9+/-0.7 degrees C and 18.7+/-1.1 J g(-1), respectively) although starches stored at 55 degrees C were annealed, where Tp represented 71.1+/-1.1 degrees C and DeltaH 18.1+/-1.4 J g(-1). These in situ-annealed starches were comparable in terms of gelatinisation characteristics to annealed freshly extracted starches where on average, T(p) represented 72.7+/-1.0 degrees C and DeltaH 20.8+/-1.0 J g(-1). Annealing of tubers in situ prior to processing might be beneficial with respect to developing new potato-based products.

The yeast glycerol 3-phosphatases Gpp1p and Gpp2p are required for glycerol biosynthesis and differentially involved in the cellular responses to osmotic, anaerobic, and oxidative stress.

We have characterized the strongly homologous GPP1/RHR2 and GPP2/HOR2 genes, encoding isoforms of glycerol 3-phosphatase. Mutants lacking both GPP1 and GPP2 are devoid of glycerol 3-phosphatase activity and produce only a small amount of glycerol, confirming the essential role for this enzyme in glycerol biosynthesis. Overproduction of Gpp1p and Gpp2p did not significantly enhance glycerol production, indicating that glycerol phosphatase is not rate-limiting for glycerol production. Previous studies have shown that expression of both GPP1 and GPP2 is induced under hyperosmotic stress and that induction partially depends on the HOG (high osmolarity glycerol) pathway. We here show that expression of GPP1 is strongly decreased in strains having low protein kinase A activity, although it is still responsive to osmotic stress. The gpp1Delta/gpp2Delta double mutant is hypersensitive to high osmolarity, whereas the single mutants remain unaffected, indicating GPP1 and GPP2 substitute well for each other. Transfer to anaerobic conditions does not affect expression of GPP2, whereas GPP1 is transiently induced, and mutants lacking GPP1 show poor anaerobic growth. All gpp mutants show increased levels of glycerol 3-phosphate, which is especially pronounced when gpp1Delta and gpp1Delta/gpp2Delta mutants are transferred to anaerobic conditions. The addition of acetaldehyde, a strong oxidizer of NADH, leads to decreased glycerol 3-phosphate levels and restored anaerobic growth of the gpp1Delta/gpp2Delta mutant, indicating that the anaerobic accumulation of NADH causes glycerol 3-phosphate to reach growth-inhibiting levels. We also found the gpp1Delta/gpp2Delta mutant is hypersensitive to the superoxide anion generator, paraquat. Consistent with a role for glycerol 3-phosphatase in protection against oxidative stress, expression of GPP2 is induced in the presence of paraquat. This induction was only marginally affected by the general stress-response transcriptional factors Msn2p/4p or protein kinase A activity. We conclude that glycerol metabolism plays multiple roles in yeast adaptation to altered growth conditions, explaining the complex regulation of glycerol biosynthesis genes.

MIP-ligand binding assays (pseudo-immunoassays).

Molecular imprint sorbent assays (MIAs) have been applied to an increasing number of analytes of medical and environmental interest: the sensitivities and selectivities of these assays are comparable to immunoassays employing biological antibodies. In a number of cases complete analytical procedures starting from raw samples (blood, plasma and urine) have been demonstrated. There have been significant advances in applying MIPs in new formats and in the use of non-radioisotope labels. Progress in the field is reviewed, with particular emphasis on the technical aspects and new innovations. It is demonstrated that many of the perceived drawbacks of molecular imprinted polymers (MIPs) do not hinder their application in competitive binding assays: Many MIAs have been applied in aqueous systems and a heterogenous distribution of binding sites is not problematic, provided the recognition sites which bind the probe most strongly are selective.

Cholesterol oxidase: sources, physical properties and analytical applications.

Since Flegg (H.M. Flegg, An investigation of the determination of serum cholesterol by an enzymatic method, Ann. Clin. Biochem. 10 (1973) 79-84) and Richmond (W. Richmond, The development of an enzymatic technique for the assay of cholesterol in biological fluids, Scand. J. clin. Lab. Invest. 29 (1972) 25; W. Richmond, Preparation and properties of a bacterial cholesterol oxidase from Nocardia sp. and its application to enzyme assay of total cholesterol in serum, Clinical Chemistry 19 (1973) 1350-1356) first illustrated the suitability of cholesterol oxidase (COD) for the analysis of serum cholesterol, COD has risen to become the most widely used enzyme in clinical laboratories with the exception of glucose oxidase (GOD). The use is widespread because assays incorporating the enzyme are extremely simple, specific, and highly sensitive and thus offer distinct advantages over the Liebermann-Burchard analytical methodologies which employ corrosive reagents and can be prone to unreliable results due to interfering substances such as bilirubin. Individuals can now readily determine their own serum cholesterol levels with a simple disposable test kit. This review discusses COD in some detail and includes the topics: (1) The variety of bacterial sources available; (2) The various extraction/purification protocols utilised in order to obtain protein of sufficient clarification (purity) for use in food/clinical analysis; (3) Significant differences in the properties of the individual enzymes; (4) Substrate specificities of the various enzymes; (5) Examples of biological assays which have employed cholesterol oxidase as an integral part of the analysis, and the various assay protocols; (6) New steroidal products of COD. This review is not a comprehensive description of published work, but is intended to provide an account of recent and current research, and should promote further interest in the application of enzymes to analytical selectivity.

Pre-selection of integration sites imparts repeatable transgene expression.

Variable gene expression amongst transgenic lines occurs due to copy number and to random associations of incoming DNA with chromosomal elements at the site of integration. Here we describe a method of identifying sites permissive for transgene expression and their use for efficient introduction of single copy transgenes by homologous recombination. ES clones were selected in HAT medium for expression of a randomly integrated HPRT marker lying 5' to an Oct4/ lacZ transgene. 794 clones were assessed in vitro for appropriate down-regulation of lacZ following differentiation. Two clones were chosen for further analysis which displayed appropriate and inappropriate gene regulation (clones 710 and 91, respectively). Three developmental promoters (thyroglobulin, Hox2.6 and Myf5) were then sequentially introduced into the original insertion sites in each clone (710 and 91) by homologous recombination, to drive expression of lacZ. Transgenic embryos were assessed for their ability to direct lacZ expression to tissues in which the respective promoter sequences are normally active. The site which appropriately down-regulated lacZ in vitro (710) also showed appropriate in vivo regulation of lacZ from the three developmental promoters. Site 91, however, directed an additional pattern of ectopic expression, which was common to all four promoters. Pre-selection of genomic sites for the introduction of transgenes by gene targeting improves the repeatability of transgene expression and provides an efficient means of single copy transgene introduction by homologous recombination.

Surface partitioning studies of N-methylcarbamate-treated post-harvest crops using SFE-HPLC-postcolumn reaction-fluorescence.

The partitioning characteristics of selected carbamate insecticides (carbaryl, aldicarb, bendiocarb and pirimicarb) on five fruit and vegetable types were investigated. Post-harvest samples were surface-saturated with a methanolic-aqueous mixed carbamate spiking solution for a number of time periods. Samples were taken at 3, 7, 10 and 14 d, and extracted using supercritical CO2 at pressure = 300 atm modified with 10% dimethyl sulfoxide. Extracts were analysed by HPLC-postcolumn reaction-fluorescence detection at lambda ex = 330 nm and lambda em = 450 nm for N-methylcarbamates and at lambda ex = 315 nm and lambda em = 380 nm for pirimicarb. The relative partitioning of each insecticide between sample skin and flesh was investigated. This included the determination of both half-life and normalised matrix metabolic rate studies with respect to each carbamate. Multilinear regression (MLR) was applied to a number of insecticide and matrix-based variables to develop regression models for carbamate partitioning for each matrix type studied. Experimentally derived carbamate half-lives ranged from 3.6 d (carbaryl in pear flesh) to 8.0 d (bendiocarb in banana skin). Determinations of normalised metabolic rates were based on calculating the time period from the point of sampling through to the point where carbamate concentration was reduced to 5% of its initial value. These values ranged from 16.2 d (bendiocarb in potato skin) to 34.7 d (bendiocarb in banana skin). Although no practicable MLR partitioning models were obtained, it was found that the models created indicated that carbamate solubility in water (and hence log P) and the number of days in contact with the spiking solution were the most important parameters in model construction.

The effect of iron limitation on glycerol production and expression of the isogenes for NAD(+)-dependent glycerol 3-phosphate dehydrogenase in Saccharomyces cerevisiae.

When deprived of iron, Saccharomyces cerevisiae rearranges its metabolic flux towards increased glycerol production. This work examines the role and regulation of GPD1 and GPD2, encoding two isoforms of glycerol 3-phosphate dehydrogenase, in glycerol production during iron starvation. The two genes respond differently on transfer of cells to iron-limited conditions. Whereas the expression of GPD2 increases about 3-fold, that of GPD1 does not exhibit significant changes. Deletion of either GPD1 or GPD2 alters the capacity for glycerol production during iron-limited as well as iron sufficient conditions. However, loss of function of either gene does not seem to provoke compensatory flux via the other gene product. As judged from the glycerol production, the amount produced by each single mutant adds approximately up to the level produced by the parental strain. In agreement with the pattern of expression of GPD2, this gene product was estimated to account for the bulk of the glycerol production (about 60%) during iron-limited conditions. The strong growth inhibition caused by iron starvation was reversed by the addition of iron also for a gpd1Deltagpd2Delta double deletion mutant, which is unable to produce any detectable glycerol.

Use of molecularly imprinted polymers in a biotransformation process.

Molecularly imprinted polymers are highly stable and can be sterilised, making them ideal for use in biotransformation process. In this communication, we describe a novel application of molecularly imprinted polymers in an enzymatic reaction. The enzymatic condensation of Z-L-aspartic acid with L-phenylalanine methyl ester to give Z-L-Asp-L-Phe-OMe (Z-aspartame) was chosen as a model system to evaluate the applicability of using molecularly imprinted polymers to facilitate product formation. When the product-imprinted polymer is present, a considerable increase (40%) in product yield is obtained. Besides their use to enhance product yields, as demonstrated here, we suggest that imprinted polymers may also find use in the continuous removal of toxic compounds during biochemical reactions.

Synthesis and properties of new coenzyme mimics based on the artificial coenzyme CL4.

We have previously shown that a range of nicotinamide containing 'biomimetic coenzymes' function as active analogues of NAD+ in the oxidation of alcohols by horse liver alcohol dehydrogenase (HLADH), despite their apparently astonishing lack of structural similarity to the natural coenzyme. The simplest structure as yet shown to exhibit activity is the biomimetic coenzyme CL4. To investigate the effect of the structure of this truncated artificial coenzyme on its activity, a range of close structural analogues of CL4 were designed, synthesized and characterized. The electrochemical reduction potentials of the analogues were strongly influenced by the nature of the groups attached to the pyridine ring. All of the analogues could be chemically reduced using sodium borohydride, to give compounds with altered UV-visible absorption and fluorescence properties. An HPLC-based assay suggested that two of the new analogues were coenzymically active in the oxidation of butan-1-ol by HLADH, with one displaying a significantly higher activity than CL4. The results demonstrate which features of the structures of the coenzymes lead to desirable electrochemical and spectroscopic properties, but suggest that the structural requirements for a functional coenzyme are quite stringent. These observations may be used to design an artificial coenzyme which combines the best features of those studied so far.

Insertion of a foreign gene into the beta-casein locus by Cre-mediated site-specific recombination.

The expression of foreign genes in transgenic animals is generally unpredictable as transgenes are integrated at random after pro-nuclear injection into fertilized oocytes. In many cases, transgene expression is inhibited by neighbouring chromatin structures or by the repeated nature of the multiple transgene copies present at the integration site. A strategy involving homologous and site-specific recombination has been devised by which single copies of a foreign gene can be inserted specifically into the locus of a highly expressed gene. As a first step, a loxP recombination target site is introduced by homologous recombination into a predetermined gene locus such that the loxP sequence is placed next to the promoter region and replaces the translational initiation signal. In a subsequent site-specific recombination reaction, a gene of interest can be integrated into the pre-existing loxP site. This biphasic recombination strategy was used to integrate a luciferase reporter gene into the locus of the murine beta-casein gene in embryonic stem cells.

[Securing evidence after sexual offences is an important task for physicians. Increasing severity of crimes and use of DNA analyses necessitate higher quality standards]. / Viktig uppgift för läkare att säkra spår efter sexbrott. Grövre brottslighet och användning av DNA-analyser bakom högre kvalitetskrav.

Forensic DNA analysis of biological specimens such as blood and semen often yields vital evidence in contemporary criminal investigations. Appropriate, correctly performed physical examination in cases of sexual offences is of crucial importance in providing the forensic analyst with genital and extra-genital samples, for example. Despite the availability of standard guidelines for procedures to be followed by the examining physician during sampling, many cases submitted to the National Laboratory of Forensic Science at Linköping lack adequate material for forensic analysis. A recently modernised 'sexual offence kit' is now available, containing the equipment and instructions needed to facilitate standard forensic sampling for the general physician.

Magnetic molecularly imprinted polymer beads for drug radioligand binding assay.

Molecularly imprinted polymer-magnetic iron oxide composite materials which exhibit recognition properties and can be withdrawn from solution by application of a magnetic field were prepared for the first time. Magnetic iron oxide was incorporated using a suspension polymerisation methodology with a perfluorocarbon liquid as the dispersing phase for the preparation of methacrylic acid-1,1,1-trimethylolpropane trimethacrylate copolymer beads molecularly imprinted with the beta-blocker (S)-propranolol. The resulting superparamagnetic imprinted polymer beads were capable of binding [3H]-(S)-propranolol more strongly than a non-imprinted, otherwise identical, polymer. In a competitive radioligand binding assay using a magnet to separate polymer from solution, (R)-propranolol and (R,S)-metoprolol exhibited cross-reactivities of 19 and 0.7%, respectively, compared with (S)-propranolol.

The importance of the glycerol 3-phosphate shuttle during aerobic growth of Saccharomyces cerevisiae.

Maintenance of a cytoplasmic redox balance is a necessity for sustained cellular metabolism. Glycerol formation is the only way by which Saccharomyces cerevisiae can maintain this balance under anaerobic conditions. Aerobically, on the other hand, several different redox adjustment mechanisms exist, one of these being the glycerol 3-phosphate (G3P) shuttle. We have studied the importance of this shuttle under aerobic conditions by comparing growth properties and glycerol formation of a wild-type strain with that of gut2 delta mutants, lacking the FAD-dependent glycerol 3-phosphate dehydrogenase, assuming that the consequent blocking of G3P oxidation is forcing the cells to produce glycerol from G3P. To impose different demands on the redox adjustment capability we used various carbon sources having different degrees of reduction. The results showed that the shuttle was used extensively with reduced substrate such as ethanol, whereas the more oxidized substrates lactate and pyruvate, did not provoke any activity of the shuttle. However, the absence of a functional G3P shuttle did not affect the growth rate or growth yield of the cells, not even during growth on ethanol. Presumably, there must be alternative systems for maintaining a cytoplasmic redox balance, e.g. the so-called external NADH dehydrogenase, located on the outer side of the inner mitochondrial membrane. By comparing the performance of the external NADH dehydrogenase and the G3P shuttle in isolated mitochondria, it was found that the former resulted in high respiratory rates but a comparably low P/O ratio of 1.2, whereas the shuttle gave low rates but a high P/O ratio of 1.7. Our results also demonstrated that of the two isoforms of NAD-dependent glycerol 3-phosphate dehydrogenase, only the enzyme encoded by GPD1 appeared important for the shuttle, since the enhanced glycerol production that occurs in a gut2 delta strain proved dependent on GPD1 but not on GPD2.

The two isoenzymes for yeast NAD+-dependent glycerol 3-phosphate dehydrogenase encoded by GPD1 and GPD2 have distinct roles in osmoadaptation and redox regulation.

The two homologous genes GPD1 and GPD2 encode the isoenzymes of NAD-dependent glycerol 3-phosphate dehydrogenase in the yeast Saccharomyces cerevisiae. Previous studies showed that GPD1 plays a role in osmoadaptation since its expression is induced by osmotic stress and gpd1 delta mutants are osmosensitive. Here we report that GPD2 has an entirely different physiological role. Expression of GPD2 is not affected by changes in external osmolarity, but is stimulated by anoxic conditions. Mutants lacking GPD2 show poor growth under anaerobic conditions. Mutants deleted for both GPD1 and GPD2 do not produce detectable glycerol, are highly osmosensitive and fail to grow under anoxic conditions. This growth inhibition, which is accompanied by a strong intracellular accumulation of NADH, is relieved by external addition of acetaldehyde, an effective oxidizer of NADH. Thus, glycerol formation is strictly required as a redox sink for excess cytosolic NADH during anaerobic metabolism. The anaerobic induction of GPD2 is independent of the HOG pathway which controls the osmotic induction of GPD1. Expression of GPD2 is also unaffected by ROX1 and ROX3, encoding putative regulators of hypoxic and stress-controlled gene expression. In addition, GPD2 is induced under aerobic conditions by the addition of bisulfite which causes NADH accumulation by inhibiting the final, reductive step in ethanol fermentation and this induction is reversed by addition of acetaldehyde. We conclude that expression of GPD2 is controlled by a novel, oxygen-independent, signalling pathway which is required to regulate metabolism under anoxic conditions.

Physiological response to anaerobicity of glycerol-3-phosphate dehydrogenase mutants of Saccharomyces cerevisiae.

Mutants of Saccharomyces cerevisiae, in which one or both of the genes encoding the two isoforms of NAD-dependent glycerol-3-phosphate dehydrogenase had been deleted, were studied in aerobic batch cultures and in aerobic-anaerobic step change experiments. The respirofermentative growth rates under aerobic conditions with semisynthetic medium (20 g of glucose per liter) of two single mutants, gpd1 delta and gpd2 delta, and the parental strain (mu = 0.5 h-1) were almost identical, whereas the growth rate of a double mutant, gpd1 delta gpd2 delta, was approximately half that of the parental strain. Upon a step change from aerobic to anaerobic conditions in the exponential growth phase, the specific carbon dioxide evolution rates (CER) of the wild-type strain and the gpd1 delta strain were almost unchanged. The gpd2 delta mutant showed an immediate, large (> 50%) decrease in CER upon a change to anaerobic conditions. However, after about 45 min the CER increased again, although not to the same level as under aerobic conditions. The gpd1 delta gpd2 delta mutant showed a drastic fermentation rate decrease upon a transition to anaerobic conditions. However, the CER values increased to and even exceeded the aerobic levels after the addition of acetoin. High-pressure liquid chromatographic analyses demonstrated that the added acetoin served as an acceptor of reducing equivalents by being reduced to butanediol. The results clearly show the necessity of glycerol formation as a redox sink for S. cerevisiae under anaerobic conditions.

Selective ablation of differentiated cells permits isolation of embryonic stem cell lines from murine embryos with a non-permissive genetic background.

Embryonic stem (ES) cells enable the engineering of precise modifications to the mouse genome by gene targeting. Although there are reports of cultured cell contributions to chimaeras in golden hamster, rat and pig, definitive ES cell lines which contribute to the germline have not been demonstrated in any species but mouse. Among mouse strains, genetic background strongly affects the efficiency of ES isolation, and almost all ES lines in use are derived from strain 129 (refs 1,4,5) or, less commonly, C57BL/6 (refs 6-8). The CBA strain is refractory to ES isolation and there are no published reports of CBA-derived ES lines. Hence, CBA mice may provide a convenient model of ES isolation in other species. In ES derivation it is critical that the primary explant be cultured for a sufficient time to allow multiplication of ES cell progenitors, yet without allowing extensive differentiation. Thus, differences in ES derivation between mouse strains may reflect differences in the control of ES progenitor cells by other lineages within the embryo. Here we describe a strategy to continuously remove differentiated cells by drug selection, which generates germline competent ES lines from genotypes that are non-permissive in the absence of selection.