The adenylate kinase family in yeast: identification of URA6 as a multicopy suppressor of deficiency in major AMP kinase.

The gene URA6 encoding uridylate kinase (UK) from Saccharomyces cerevisiae was isolated as a multicopy suppressor of the respiratory-deficient phenotype of an S. cerevisiae mutant defective in the gene AKY2 encoding AMP kinase (AK). The URA6 gene also restored temperature resistance to two different temperature-sensitive mutations in the gene encoding Escherichia coli AK. By contrast, the gene encoding UK of Dictyostelium discoideum on a multicopy yeast shuttle plasmid, expressed under control of the constitutive yeast AKY2 promoter, failed to complement the deficiency in yeast, although such transformants expressed high UK activity. We show that yeast UK exerts significant AK activity which is responsible for the complementation and is absent in the analogous enzyme from D. discoideum. Since UK also significantly phosphorylates CMP (but not GMP), it must be considered an unspecific short-form nucleoside monophosphate kinase. Wild-type mitochondria lack UK activity, but import AKY2. Since multicopy transformation with URA6 heals the Pet- phenotype of AKY2 disruption mutants, the presence of AKY2 in the mitochondrial intermembrane space is not required to maintain respiratory competence. However, furnishing UK with the bipartite intermembrane space-targeting presequence of cytochrome c1 improves the growth rates of AKY2 mutants with nonfermentable substrates, suggesting that AK activity in mitochondria is helpful, though not essential for oxidative growth.     

Characterization of plasminogen activator produced by an established cell line from human ovary

An established cell line (OC-1) was obtained from human ovarian tissue, which yielded a high concentration of plasminogen activator (PA) in the culture medium. The PA (OC-1-PA) produced by the cell line was purified and compared with urokinase (UK), proform of UK (pro-UK), and tissue-type PA (t-PA) purified from human melanoma cells (Bowes). OC-1-PA was purified by Zn chelate-Sepharose affinity chromatography followed by high-performance liquid chromatography with a Zn chelate-5PW column and with a p-amino-benzamidine-5PW column, giving a yield of 58.3% and a purification factor of 15,439. This purified material revealed a single band of Mr 55,000 on sodium dodecylsulfate polyacrylamide gel electrophoresis in the presence or absence of reducing agents. Electrophoretic enzymography demonstrated that the Mr 55,000 protein band had a plasminogen-dependent fibrinolytic activity. Treatment with plasmin did not change the Mr even in the presence of reducing agents. These results suggest that OC-1-PA has a single-chain structure protected from protease degradation, which is completely different from UK. The activator had higher affinities for lysine and fibrin than those of UK or pro-UK. An immunological study demonstrated that OC-1-PA cross-reacted with anti-UK IgG but not with anti-t-PA IgG. All these findings indicate that OC-1-PA belongs immunologically to the UK type, but its structure differs from that of UK.     

国产人尿激酶原的药效学、药理学和毒理学研究

通过对重组人尿激酶原（rhRhpro-UK）不同剂量（8万IU/kg和16万IU/kg）对家兔纤溶功能的影响并与尿激酶（UK）（8万IU/kg）比较实验显示,rhpro-UK和UK都能明显缩短兔优球蛋白溶解时间,rhpro-UK对纤溶参数影响比UK小.rhpro-UK对体外血栓的溶解作用随剂量增加而增加,与同剂量UK比较有显著差别.rhpro-UK和UK对猪冠脉血栓都有明显的溶栓作用,对猪的纤溶指标纤维蛋白原（FG）、纤溶酶原（PLG）和α2抗纤溶酶（α2-AP）无明显影响.rhpro-UK对出血时间、凝血时间、单位时间内出血量均明显低于UK.表明rhpro-UK的副作用明显低于UK.rhpro-UK对小鼠、大鼠、犬和豚鼠回肠的一般药理学实验表明,rhpro-UK对受试动物的一般行为、状态及中枢神经系统、心血管系统、呼吸系统、消化系统等均无明显影响.仅发现犬实验中手术创面有渗血现象,对全身血液有类似肝素化状态.rhpro-UK的毒理实验表明,rhpro-UK的半数致死量为97.5mg/kg;Beagle犬接受2,8,28mg/kgrhpro-UK后,除8mg和28mg组有一过性牙龈充血,凝血时间明显延长,Tchol,TP和Alb含量有升高趋势外,未观察到明显毒性反应,病理学检查也未观察到药物造成直接的脏器损伤,2mg/kg组未观察到任何毒副反应.特殊毒性实验表明,rhpro-UK没有致突变和致畸作用.     

Reversible interactions between plasminogen activators and plasminogen activator inhibitor-1.

We have shown that the urokinase (UK) kringle domain contains a high-affinity plasminogen activator inhibitor-1 (PAI-1) binding site, responsible for the 10-fold faster complex formation between UK and PAI-1 than between PAI-1 and low-molecular-weight urokinase (LMWUK). Complex formation between UK and PAI-1, but not between LMWUK and PAI-1, was suppressed 10-fold in the presence of peptide U-107 derived from the UK kringle domain. Peptide U-373 derived from the UK catalytic domain slowed complex formation between UK and PAI-1 and also LMWUK and PAI-1. Inactivation of tissue-type plasminogen activator (tPA) by PAI-1 was slowed 10-fold in the presence of peptides derived from the tPA finger and kringle-2 domains. DFP-inactivated (DIP) UK and both forms of DIP-tPA inhibited PAI-1 binding to U-107 and to U-373 whereas single-chain urokinase-type PA (scuPA) was unable to compete with either peptide for PAI-1 binding. These data suggest that the reversible PAI-1 binding site in the UK A-chain plays a role in the rapid association with PAI-1 as important as those that reside in the tPA A-chain and that reversible PAI-1 binding sites are expressed on the surface of UK upon conversion from scuPA, in contrast to tPA.     

Genetic isolation of a now extinct population of bottlenose dolphins (Tursiops truncatus)

A number of dolphin species, though highly mobile, show genetic structure among parapatric and sometimes sympatric populations. However, little is known about the temporal patterns of population structure for these species. Here, we apply Bayesian inference and data from ancient DNA to assess the structure and dynamics of bottlenose dolphin (Tursiops truncatus) populations in the coastal waters of the UK. We show that regional population structure in UK waters is consistent with earlier studies suggesting local habitat dependence for this species in the Mediterranean Sea and North Atlantic. One genetically differentiated UK population went extinct at least 100 years ago and has not been replaced. The data indicate that this was a local extinction, and not a case of historical range shift or contraction. One possible interpretation is a declining metapopulation and conservation need for this species in the UK.     

Identification of two types of protein immunochemically related to urinary urokinase occurring in human plasma

Plasma urokinase, a plasminogen activator immunochemically related to urinary urokinase (UK), was removed from human plasma (3.5 ng/ml) by immuno-depletion with antibodies raised against UK. The remaining plasminogen activator activity of the depleted plasma could not be inhibited by anti-UK antibodies and a sensitive ELISA for UK did not detect any UK levels that were higher than the background of the assay (0.1 ng/ml). However, when the depleted plasma was subjected to SDS-PAGE, substantial amounts of protein were found hereafter around 110 and 46 kD which now gave a positive reaction in the ELISA (35-350 ng/ml plasma). From these observations it is concluded that in human plasma two types of UK-related protein occur: Type I, among which the plasma urokinase, has antigenic determinants which are directly accessible to the anti-UK antibodies, Type II has determinants in a latent form. The function of the 110 kD type-II protein is that of a plasminogen activator; that of the 46 kD protein is not yet clear.     

Assessment of UK hops for the occurrence of hop latent and hop stunt viroids

The occurrences and distributions of hop stunt (HSVd) and hop latent (HLVd) viroids were assessed by a nucleic acid hybridisation assay, using samples from 476 commercial hop plantings in the UK. These samples represented about half of the UK production.
HLVd was detected in c. 17% of the samples, with infection in different cultivars ranging from 0% to 89%. This viroid was found in all cultivars sensitive to Verticillium wilt except cv. Sunshine, an old cultivar grown on only one farm in the UK. Two minor wilt-tolerant cultivars were also found to be infected at low frequencies, but the main commercially-important wilt-tolerant cultivars were all uninfected. A high proportion of the nuclear stock mother plants in the "A +" house at the Institute of Horticultural Research Dept of Hops Research, Wye College were infected. Circumstantial evidence, based on the planting dates of infected gardens, suggests that infection became established in the hop propagation system during the late 1970s and that there was a major increase in the prevalence of HLVd as a result. Whether this contamination of propagating material arose because of spread from long-standing infections or because the viroid was newly introduced into the UK, is not known.
All samples were also tested for HSVd but this viroid was not detected in any UK hop material.     

Molecular evidence for a recent founder event in the UK populations of the Adonis blue butterfly (<Emphasis Type="Italic">Polyommatus bellargus</Emphasis>)

Contrary to accepted theories of post-glacial colonisation of the UK approximately 10,000 year BP (yBP), historical population data for Polyommatus bellargus suggests the butterfly was either extremely rare or not present before 1775. We examined the phylogeography of the species by sequencing the ‘hypervariable’ mitochondrial control region of UK and French butterflies. Overall, 22 polymorphic nucleotide sites were identified within the control region. French specimens were highly variable, with 17 polymorphic sites, whereas most UK specimens were monomorphic. Average nucleotide diversity was 0.026 (SD 0.016, n = 8) in France, whilst the UK values ranged from 0.00 (n = 6) (for every UK population outside Dorset, n = 43) to 0.01 (SD 0.008, n = 7) (Dorset). The mean number of pairwise differences among the French samples was 7.42, whilst the UK values ranged from 0.00 (all populations except Dorset) to 0.295 (Dorset). One French haplotype differed from the predominant UK version by just a single nucleotide substitution. It seems implausible that the species can have been resident in the UK for 10,000 years without accumulating variation at this mitochondrial region. Thus, the results suggest that either a severe genetic bottleneck or founder event has occurred recently in the UK.     

Possible effects of ingested lead gunshot on populations of ducks wintering in the UK

Although the use of lead ammunition for shooting wildfowl and/or over listed wetlands in the UK has been banned, c. 70% of ducks shot in England (the only UK country with compliance monitoring) are still shot with lead and the proportion of ducks found dead with signs of lead poisoning from ingested gunshot has not declined significantly since the ban. However, there is little quantitative evidence of the impacts of additional mortality from lead poisoning on duck populations. For the eight duck species that winter in freshwater habitats in the UK, we found that inter‐specific variation in mean population growth rate during the period 1990/1991 to 2013/2014 was significantly negatively correlated with two independent measures of the prevalence of ingested lead gunshot in the UK and Europe. This relationship was found for a wide range of different periods over which population growth was estimated, and also for annual growth rates in the period 1966/1967 to 2013/2014, derived from smoothed population trajectories. These findings support the hypothesis that ingested lead gunshot might affect population trend. An alternative hypothesis, that migratory short‐stopping driven by climate change affected trends in numbers of ducks wintering in the UK, was not supported by simple or partial correlation results. The possible impact of ingested lead gunshot on the Common Pochard Aythya ferina, a species listed as globally threatened, is of special concern.     

Analysis of museum specimens suggests extreme genetic drift in the adonis blue butterfly (Polyommatus bellargus)

We amplified microsatellite DNA from museum specimens over 100 years old of the adonis blue butterfly, Polyommatus bellargus. These results were compared with butterfly samples taken from the same site near Folkestone in southern UK in 1998/9, 200 generations later, and with samples from other extant UK populations. Dramatic changes in allele frequencies have occurred over time, which is indicative of substantial genetic drift or extinction/recolonization. Patterns of heterozygosity in the 1998/9 sample are indicative of a past bottleneck, and one was known to have occurred in the late 1970s in this and many other UK populations. One allele present at high frequency in 1896 was not detected in any extant UK population, suggesting that it may have been lost from the UK (a 'ghost' allele), although the allele may well persist elsewhere within the range of the species. Although the present study is relatively small in scale (20 museum specimens from one site), it serves to reinforce the enormous potential of museum specimens in well represented taxa such as butterflies for examining the effects of demographic events spanning many years.  © 2006 The Linnean Society of London, Biological Journal of the Linnean Society , 2006, 88 , 447–452.     

Increased CCR5 affinity and reduced CCR5/CD4 dependence of a neurovirulent primary human immunodeficiency virus type 1 isolate

Most human immunodeficiency virus type 1 (HIV-1) viruses in the brain use CCR5 as the principal coreceptor for entry into a cell. However, additional phenotypic characteristics are necessary for HIV-1 neurotropism. Furthermore, neurotropic strains are not necessarily neurovirulent. To better understand the determinants of HIV-1 neurovirulence, we isolated viruses from brain tissue samples from three AIDS patients with dementia and HIV-1 encephalitis and analyzed their ability to induce syncytia in monocyte-derived macrophages (MDM) and neuronal apoptosis in primary brain cultures. Two R5X4 viruses (MACS1-br and MACS1-spln) were highly fusogenic in MDM and induced neuronal apoptosis. The R5 viruses UK1-br and MACS2-br are both neurotropic. However, only UK1-br induced high levels of fusion in MDM and neuronal apoptosis. Full-length Env clones from UK1-br required lower CCR5 and CD4 levels than Env clones from MACS2-br to function efficiently in cell-to-cell fusion and single-round infection assays. UK1-br Envs also had a greater affinity for CCR5 than MACS2-br Envs in binding assays. Relatively high levels of UK1-br and MACS2-br Envs bound to CCR5 in the absence of soluble CD4. However, these Envs could not mediate CD4-independent infection, and MACS2-br Envs were unable to mediate fusion or infection in cells expressing low levels of CD4. The UK1-br virus was more resistant than MACS2-br to inhibition by the CCR5-targeted inhibitors TAK-779 and Sch-C. UK1-br was more sensitive than MACS2-br to neutralization by monoclonal antibodies (2F5 and immunoglobulin G1b12 [IgG1b12]) and CD4-IgG2. These results predict the presence of HIV-1 variants with increased CCR5 affinity and reduced dependence on CCR5 and CD4 in the brains of some AIDS patients with central nervous system disease and suggest that R5 variants with increased CCR5 affinity may represent a pathogenic viral phenotype contributing to the neurodegenerative manifestations of AIDS.     

Plasma fibrinolysis after intraduodenal administration of urokinase in rats

Plasma fibrinolysis in rats rose above the level of physiological fluctuations in a curve with two peaks at 1 and 6 h, following intraduodenal administration of high-molecular-weight urokinase (HMW-UK; MW 53,000; 124,000 IU/mg protein). Activation of plasma fibrinolysis was also confirmed with insolubilized enzyme (glass-coupled UK), but lacked the first activity peak. Plasma fibrinolytic enzyme isolated by affinity chromatography revealed strong fibrinolytic (1,120 IU/dl), pyro-Glu-Gly-Arg-pNA amidolytic (3,200 nmol/dl) and Glu-plasminogen activating (24.5 IU/dl) activities. Using specific UK antibody, it appeared that the first peak originated from the administered UK, while the second one derived from endogenous plasminogen activator. Dose response of UK was not observed, and the maximal effect was at about 5,000 IU/kg body weight.     

Tissue-type plasminogen activator inhibits aggregation of platelets in vitro

The effects of tissue-type plasminogen activator (t-PA) on the platelet aggregation were studied using citrated whole blood and platelet-rich plasma (PRP) obtained from human donors. t-PA suppressed adenosine 5'-diphosphate (ADP)- or collagen-induced platelet aggregation in a dose-dependent manner. The 50% inhibitory concentration (IC50) for t-PA was lower by one order of magnitude than that for urokinase (UK) in whole blood and PRP. The suppression of platelet aggregation was not completely inhibited by alpha-2-antiplasmin. t-PA did not cause the degradation of fibrinogen or fibrin in PRP, whereas UK caused the reduction of fibrinogen and fibrin, and the increase of fibrinogen- and fibrin-degradation products (FDP). These results suggest that the mode of action of t-PA in inhibiting platelet aggregation may be different from that of UK.     

International stem cell collaboration: how disparate policies between the United States and the United Kingdom impact research

As the scientific community globalizes, it is increasingly important to understand the effects of international collaboration on the quality and quantity of research produced. While it is generally assumed that international collaboration enhances the quality of research, this phenomenon is not well examined. Stem cell research is unique in that it is both politically charged and a research area that often generates international collaborations, making it an ideal case through which to examine international collaborations. Furthermore, with promising medical applications, the research area is dynamic and responsive to a globalizing science environment. Thus, studying international collaborations in stem cell research elucidates the role of existing international networks in promoting quality research, as well as the effects that disparate national policies might have on research. This study examined the impact of collaboration on publication significance in the United States and the United Kingdom, world leaders in stem cell research with disparate policies. We reviewed publications by US and UK authors from 2008, along with their citation rates and the political factors that may have contributed to the number of international collaborations. The data demonstrated that international collaborations significantly increased an article's impact for UK and US investigators. While this applied to UK authors whether they were corresponding or secondary, this effect was most significant for US authors who were corresponding authors. While the UK exhibited a higher proportion of international publications than the US, this difference was consistent with overall trends in international scientific collaboration. The findings suggested that national stem cell policy differences and regulatory mechanisms driving international stem cell research in the US and UK did not affect the frequency of international collaborations, or even the countries with which the US and UK most often collaborated. Geographical and traditional collaborative relationships were the predominate considerations in establishing international collaborations.     

Gene polymorphisms of cellular senescence marker p21 and disease progression in non-alcohol-related fatty liver disease

Non-alcohol-related fatty liver disease (NAFLD) encompasses a wide spectrum, ranging from steatosis alone to steatohepatitis and fibrosis. Presence of steatohepatitis and fibrosis are key hallmarks of disease progression. Previous studies have demonstrated an association between hepatocyte p21 expression and fibrosis stage in NAFLD. The aim of this study is to investigate the association between the variants of CDKN1A, which encodes p21, and disease progression in NAFLD. To this end, the relation between CDKN1A polymorphism and liver fibrosis was studied in 2 cohorts of biopsy-proven NAFLD patients from UK (n = 323) and Finland (n = 123). Genotyping was performed using DNA isolated from lymphocytes collected at the time of liver biopsy. The findings of the UK cohort were tested in the Finnish cohort. Both the UK and Finnish cohorts were significantly different from each other in basic demographics. In the UK cohort, rs762623, of the 6 SNPs across CDKN1A tested, was significantly associated with disease progression in NAFLD. This association was confirmed in the Finnish cohort. Despite the influence on fibrosis development, SNPs across CDKN1A did not affect the progression of liver fibrosis. In conclusion, CDKN1A variant rs762623 is associated with the development but not the propagation of progressive liver disease in NAFLD.     

A preliminary assessment of the consequences for inhabitants of the UK of the Chernobyl accident

The accident with the nuclear power reactor at Chernobyl in the USSR resulted in the release of substantial quantities of radioactive material and subsequent increases in radioactivity in the environment in many countries. In this paper the situation in the UK is considered and, from the preliminary monitoring measurements, the major routes of exposure of the population are identified and quantified. For the most part exposures in the UK are within variations in levels of natural background radiation to be found in Europe. An exception is the dose likely to have been received by the thyroids of young people in the north of the UK. From reported measurements of I-131 in milk it is predicted that thyroid doses up to 10-20 times the annual doses received from 'normal' natural background radiation might have affected young children drinking fresh cows' milk. The ways in which this component of exposure could have been reduced and the criteria that govern decisions as to whether or not to implement counter-measures are discussed. The importance of I-131 in milk as a route of exposure of the population to radioactivity is a feature that the Chernobyl accident has in common with the Windscale accident in the UK in 1957, and underlines the importance of milk-producing regions in relation to reactor-sitting policy.     

Frequencies of PrP gene haplotypes in British sheep flocks and the implications for breeding programmes

AIMS: To analyse the frequencies of prion (PrP) gene haplotypes in UK sheep flocks and evaluate their relevance to transmissible spongiform encephalopathies (TSEs) and TSE resistance breeding programmes in sheep. METHODS AND RESULTS: Genomic DNA isolated from sheep blood was PCR amplified for the coding region of the PrP gene and then sequenced. This study has analysed the sequence of PrP between codons 110 and 245 in 6287 ARQ haplotypes revealing a total of eight variant sequences, which represent a higher than expected 41% of all ARQ haplotypes. The additional PrP gene dimorphisms were M112T, L141F, M137T, H143R, H151C, P168L, Q175E and P241S. CONCLUSION: The results do not suggest a correlation between the occurrence of a specific ARQ haplotype and the scrapie disease status of a flock. The ARQ haplotype variability appears to be different in the UK sheep flocks compared with sheep flocks from outside the UK. SIGNIFICANCE AND IMPACT OF THE STUDY: Additional PrP dimorphisms may impact on the methodologies used for standard PrP genotyping in sheep breeding programmes. Some of these polymorphisms were found with significant frequencies in the UK sheep flocks and should therefore be considered in breeding programmes.     

Mitochondrial genetic diversity and structure of the European otter (Lutra lutra) in Britain

The European otter (Lutra lutra) is a focus for conservation efforts throughout Europe due to a population decline in recent decades and because of its importance as a biological indicator of the health of rivers and waterways. The aim of this study was to aid the conservation of this species by adding genetic information from samples originating in the United Kingdom (UK), to help build up a picture of the phylogeographic structure of the European otter throughout Europe. This was done by a comparison of 299 base pairs of the mitochondrial DNA control region. Four haplotypes were identified in the UK, one of which has not been found outside the west of the UK in the wild, and one of which was unique. Populations in the UK, and in particular the west were shown to have a higher haplotype diversity than previously found for the European otter in Europe (h = 0.7338 for the 58 UK otters sampled in this study) and an overall nucleotide diversity of π = 0.003. The western UK population was shown to have a high level of genetic distinctiveness. We discuss possible contributory population processes, the importance of the western UK population for the future conservation of the species and comment on future conservation strategies.     

Dietary factors in the fall in coronary heart disease mortality.

The role of dietary change in the fall in heart disease mortality has been hotly debated. Three countries, Australia, USA and UK with equal 'care' and sophistication of surgical techniques have shown different timing in the beginning of the decline of this 'epidemic'; around the mid 1960s in the first two countries, but not until the late 1970s for the UK. The cause of this difference may be the changing food habits of their populations. Using food disappearance data, apparent consumption of butter and margarine show opposite trends (butter down and margarine up) predating the decline in mortality in both the USA and Australia by at least 7 years and also in the UK, but at a later time, (about 1970). Changes in adipose tissue linoleate, a marker for polyunsaturated fat intake, support this indirect evidence, with depot levels rising in the USA from the 1960s and 10 years later in the UK. Other evidence support the view of decreasing saturated fat intake and increasing polyunsaturated intake prior to 1960 in the USA. Although many factors must contribute to the decline in mortality from CHD, change in dietary P/S ratio would seem to be the major dietary contributor.