Induction of Swine Dysentery with a Pure Culture of Treponema Hyodysenteriae in Vitamin E and Selenium Deficient Pigs

Several successful attempts have been made to induce swine dysentery in pigs using pure cultures of Treponema hyodysen-teriae (Taylor & Alexander 1971, Harris et al. 1972, Akkermans & Pomper 1973, Hughes et al. 1975). In these studies, either conventional or specific-pathogen-free pigs were used. In the present study, 2 approximately 8 weeks old conventional pigs (Nos. 1 and 2) were purchased and fed the same basic ration as used by Teige et al. (1977). In addition, 10 % cod liver oil was incorporated in the diet at each feeding. After a feeding period of 25 days rectal swabs were applied and examined for the presence of spirochaetes. The pigs were then fed a 3 days old primary and pure culture of T. hyodysenteriae on TSA-S400 medium (Songer et al. 1976). The culture originated from the colon of a pig with swine dysentery (Pig No. 4, Teige et al. 1977). Each pig received the agar contents of 5 petri dishes which were mixed with the food.     

d-Ribulose-5-Phosphate 3-Epimerase: Cloning and Heterologous Expression of the Spinach Gene, and Purification and Characterization of the Recombinant Enzyme

We have achieved, to our knowledge, the first high-level heterologous expression of the gene encoding d-ribulose-5-phosphate 3-epimerase from any source, thereby permitting isolation and characterization of the epimerase as found in photosynthetic organisms. The extremely labile recombinant spinach (Spinacia oleracea L.) enzyme was stabilized by dl-α-glycerophosphate or ethanol and destabilized by d-ribulose-5-phosphate or 2-mercaptoethanol. Despite this lability, the unprecedentedly high specific activity of the purified material indicates that the structural integrity of the enzyme is maintained throughout isolation. Ethylenediaminetetraacetate and divalent metal cations did not affect epimerase activity, thereby excluding a requirement for the latter in catalysis. As deduced from the sequence of the cloned spinach gene and the electrophoretic mobility under denaturing conditions of the purified recombinant enzyme, its 25-kD subunit size was about the same as that of the corresponding epimerases of yeast and mammals. However, in contrast to these other species, the recombinant spinach enzyme was octameric rather than dimeric, as assessed by gel filtration and polyacrylamide gel electrophoresis under nondenaturing conditions. Western-blot analyses with antibodies to the purified recombinant enzyme confirmed that the epimerase extracted from spinach leaves is also octameric.As a participant in the oxidative pentose phosphate pathway, Ru5P epimerase (EC 5.1.3.1), which catalyzes the interconversion of Ru5P and Xu5P, is widely distributed throughout nature. Beyond its catabolic role, the epimerase is also vital anabolically to photosynthetic organisms in the regenerative phase of the reductive pentose phosphate pathway (the Calvin cycle). In this capacity, Ru5P epimerase directs Xu5P, formed in two distinct transketolase reactions of the cycle, to Ru5P. Phosphorylation of the latter regenerates d-ribulose-1,5-bisphosphate, the substrate for net CO₂ fixation. Because both the oxidative and reductive pentose phosphate pathways coexist in chloroplasts (Schnarrenberger et al., 1995), Ru5P epimerase and R5P isomerase facilitate partitioning of pentose phosphates between the two pathways, as dictated by the metabolic needs and redox status of the cell.Scant structural and mechanistic information about Ru5P epimerase is available despite its inherent importance and dual metabolic roles. This neglect may in part reflect the low natural abundance of the enzyme. For example, achievement of electrophoretic homogeneity required a 2000-fold purification from yeast (Bär et al., 1996) and spinach (Spinacia oleracea L.) chloroplasts (Teige et al., 1998) and 9000-fold purification from beef liver (Terada et al., 1985). Although low overall recoveries (<10%) further limited the availability of pure material, molecular sieving and denaturing electrophoresis established that the epimerases from mammals (Wood, 1979; Karmali et al., 1983; Terada et al., 1985) and yeast (Bär et al., 1996) are homodimers of approximately 23-kD subunits, whereas the enzyme from spinach chloroplasts may be an octamer of 23-kD subunits (Teige et al., 1998). DNA-deduced amino acid sequences of Ru5P epimerases from both photosynthetic and nonphotosynthetic sources, which confirm this estimated subunit size, show greater than 50% similarities among the most evolutionarily distant species examined (Kusian et al., 1992; Blattner et al., 1993; Falcone and Tabita, 1993; Lyngstadaas et al., 1995; Nowitzki et al., 1995; Teige et al., 1995).Although Ru5P epimerase has very recently been purified from a photosynthetic organism (spinach) for the first time (Teige et al., 1998), the low recovery (100 μg from 3.8 g of soluble chloroplast protein, representing an overall yield of 5%) imposes severe constraints on the directions of future experiments. Furthermore, despite successful cloning of cDNA fragments encoding Ru5P epimerase of several photosynthetic organisms (Kusian et al., 1992; Nowitzki et al., 1995; Teige et al., 1995), to our knowledge high-level heterologous expression and purification of enzymically active recombinant enzyme have not been achieved. Because of our interest in the regulation of photosynthetic carbon assimilation and the requisite need for ample supplies of the participant enzymes for use in mechanistic studies, we have attempted to optimize the heterologous expression of the spinach gene for Ru5P epimerase. In this paper we report cDNA clones that encode the mature chloroplastic enzyme or its cytoplasmic precursor. We also describe an efficient isolation procedure for the mature spinach enzyme synthesized in Escherichia coli and some of the properties of the purified enzyme. Contrasting features of the plant Ru5P epimerase, relative to the animal and yeast counterparts, include an octameric rather than a dimeric structure (also see Teige et al., 1998) and striking instability under routine laboratory conditions.     

Linkage and association studies of QTL for nuclear families by mixed models

The transmission disequilibrium test (TDT) has been utilized to test the linkage and association between a genetic trait locus and a marker. Spielman et al. (1993) introduced TDT to test linkage between a qualitative trait and a marker in the presence of association. In the presence of linkage, TDT can be applied to test for association for fine mapping (Martin et al., 1997; Spielman and Ewens, 1996). In recent years, extensive research has been carried out on the TDT between a quantitative trait and a marker locus (Allison, 1997; Fan et al., 2002; George et al., 1999; Rabinowitz, 1997; Xiong et al., 1998; Zhu and Elston, 2000, 2001). The original TDT for both qualitative and quantitative traits requires unrelated offspring of heterozygous parents for analysis, and much research has been carried out to extend it to fit for different settings. For nuclear families with multiple offspring, one approach is to treat each child independently for analysis. Obviously, this may not be a valid method since offspring of one family are related to each other. Another approach is to select one offspring randomly from each family for analysis. However, with this method much information may be lost. Martin et al. (1997, 2000) constructed useful statistical tests to analyse the data for qualitative traits. In this paper, we propose to use mixed models to analyse sample data of nuclear families with multiple offspring for quantitative traits according to the models in Amos (1994). The method uses data of all offspring by taking into account their trait mean and variance-covariance structures, which contain all the effects of major gene locus, polygenic loci and environment. A test statistic based on mixed models is shown to be more powerful than the test statistic proposed by George et al. (1999) under moderate disequilibrium for nuclear families. Moreover, it has higher power than the TDT statistic which is constructed by randomly choosing a single offspring from each nuclear family.     

Regional Ileitis in Pigs Isolation of Campylobacter from Affected Ileal Mucosa

In the last few years special interest has been focused on enteric diseases localized to the lower alimentary tract, especially the ileum of weaned pigs. An increasing frequency of disorders of unknown aetiology described as regional ileitis (Emsbo 1951), necrotic enteritis (Jubb & Kennedy 1970), and intestinal adenomatosis (Rowland et al. 1973) has been reported. The changes include thickening of the ileal mucosa with hypertrophy of the muscular wall. The normal structure of villi is replaced by a proliferation of crypt cells. Necrosis of the mucosa and granulation-tissue proliferation in the submucosa occur in later stages. Regional ileitis in man (Crohn et al. 1932) which is described as a chronic enteric disease with granulomatous inflammatory changes localized to segments of the ileum is also attracting increasing attention in medical research (Liljefors 1972). The lesions are also found in the colon, and the presence of a transmissible agent involved in the aetiology of Crohn's disease has been discussed on the basis of animal experiments (Cave et al. 1973). The disease in pigs is accompanied by haematological changes, including decreased concentration of total serum protein, albumin, alkaline phosphatase, and zinc (Martinsson et al. 1974, 1976).     

Multiple Forms and Distribution of Calcium/Calmodulin-Stimulated Protein Kinase II in Brain

In recent years, the enzyme Ca²⁺/calmodulin-stimulated protein kinase II¹ (CaM-PK II) as attracted a great deal of interest. CaM-PK II is the most abundant calmodulin-stimulated protein kinase in brain, where it is particularly enriched in neurons (Ouimet et al., 1984; Erondu and Kennedy, 1985; Lin et al., 1987; Scholz et al., 1988). Neuronal CaM-PK II has been suggested to be involved in several phenomena associated with synaptic plasticity (Lisman and Goldring, 1988; Kelly, 1992), including long-term potentiation (Malinow et al., 1988; Malenka et al.,1989), neurotransmission (Nichols et al., 1990; Siekevitz, 1991), and learning (for review, see Rostas, 1991). This enzyme has also been postulated to be selectively vulnerable in several pathological condition, including epilepsy/kindling (Bronstein et al.,1990; Wu et al., 1990), cerebral ischemia (Taft et al., 1988), and organophosphorus toxicity (Abou-Donia and Lapadula, 1990).     

Non-random association of human acrocentric chromosomes

Summary The association of human acrocentric chromosomes was found to be nonrandom using fluorescence technic.The occurrence of chromosomes D13-15 in Robertsonian rearrangement has been shown to be non-random (Hecht et al., 1968). Ohno et al. (1961) have postulated these rearrangements might be related to acrocentric association. In two studies (Nakagame, 1969; Shaw et al., 1969), however, a random distribution of the D group chromosomes in association was found by autoradiography in lymphocytes. The recently described fluorescence technic (Caspersson et al., 1971) permits a reliable identification of each of the 23 pairs and we would like to report the results of our re-examination of acrocentric association by this method.

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Zusammenfassung Die Assoziation menschlicher akrozentrischer Chromosomen hat sich als nicht zufällig erwiesen. Es wurde die Fluorescenztechnik benutzt.

     

Isolation of DNA markers linked to a beet cyst nematode resistance locus in Beta patellaris and Beta procumbens.

Summary In cultivated beet no useful level of resistance of the beet cyst nematode (BCN) Heterodera schachtii Schm. has been found, unlike the situation in wild species of the section Procumbentes. Stable introgression of resistance genes from the wild species into Beta vulgaris has not been achieved, but resistant monosomic additions (2n =18 + 1), diploids of B. vulgaris with an extra alien chromosome carrying the resistance locus, have been obtained. Here we describe a new series of resistant monosomic fragment addition material of B. patellaris chromosome 1 (pat-1). We further describe the cloning of a single-copy DNA marker that specifically hybridizes with a monosomic addition fragment of approximately 8 Mb (AN5-90) carrying the BCN resistance locus. This marker and another fragment-specific, single-copy DNA marker probably flank the BCN locus on the addition fragment present in the AN5-203 material, which is approximately 19 Mb in size. Furthermore, several specific repetitive DNA markers have been isolated, one of which hybridizes to AN5-90 and also to DNA from a smaller DNA segment of Beta procumbens, present in line B883, carrying a BCN resistance locus introgressed into the B. vulgaris genome. This suggests that the specific repetitive marker is closely linked to the BCN locus.     

Linkage and association mapping of the LRP5 locus on chromosome 11q13 in type 1 diabetes

Linkage of chromosome 11q13 to type 1 diabetes (T1D) was first reported from genome scans (Davies et al. 1994; Hashimoto et al. 1994) resulting in P <2.2 x 10(-5) (Luo et al. 1996) and designated IDDM4 ( insulin dependent diabetes mellitus 4). Association mapping under the linkage peak using 12 polymorphic microsatellite markers suggested some evidence of association with a two-marker haplotype, D11S1917*03-H0570POLYA*02, which was under-transmitted to affected siblings and over-transmitted to unaffected siblings ( P=1.5 x 10(-6)) (Nakagawa et al. 1998). Others have reported evidence for T1D association of the microsatellite marker D11S987, which is approximately 100 kb proximal to D11S1917 (Eckenrode et al. 2000). We have sequenced a 400-kb interval surrounding these loci and identified four genes, including the low-density lipoprotein receptor related protein (LRP5) gene, which has been considered as a functional candidate gene for T1D (Hey et al. 1998; Twells et al. 2001). Consequently, we have developed a comprehensive SNP map of the LRP5 gene region, and identified 95 SNPs encompassing 269 kb of genomic DNA, characterised the LD in the region and haplotypes (Twells et al. 2003). Here, we present our refined linkage curve of the IDDM4 region, comprising 32 microsatellite markers and 12 SNPs, providing a peak MLS=2.58, P=5 x 10(-4), at LRP5 g.17646G>T. The disease association data, largely focused in the LRP5 region with 1,106 T1D families, provided no further evidence for disease association at LRP5 or at D11S987. A second dataset, comprising 1,569 families from Finland, failed to replicate our previous findings at LRP5. The continued search for the variants of the putative IDDM4 locus will greatly benefit from the future development of a haplotype map of the genome.     

Prevalence of Antibodies to Neospora caninum and Toxoplasma gondii in Swedish Dogs

Neospora caninum is a newly described coccidian parasite which has been found in various species such as the dog, cattle, horse, sheep and goat. Morphologically it resembles Toxoplasma gondii with which it is related (Holmdahl et al. 1994), and with which it has earlier been confused. The life cycle of N caninum is only partially known. Tachyzoites and tissue cysts are the only known stages of the parasite, and transplacental transmission is the only known route of infection. Subclini-cally infected dams can transmit the parasite to their fetuses and successive offspring from the same mother might be born infected (Dubey et al. 1990b). Clinical neosporosis is mostly seen in pups or young dogs, and the majority or all pups in a litter are often affected. The disease is characterized by ascending paralysis of the legs, with the hind legs more severely affected than the front legs, paralysis of the jaw, difficulty in swallowing and muscle flaccidity and atrophy (Dubey 1992, Dubey & Lindsay 1993). Fatal infections with N caninum in dogs have been reported from many countries, e.g. Norway (Bjerkäs & Presthus 1988), USA (Dubey et al. 1988), Sweden (Uggla et al. 1989a,b) and the United Kingdom (Dubey et al. 1990a). Serological surveys for antibodies to N. caninum in dogs from Kansas, USA and England have shown a prevalence of 2 and 13%, respectively (Lindsay et al. 1990, Trees et al. 1993).     

Carbon and water fluxes in ecologically vulnerable areas in China

Ecologic vulnerable areas (EVAs) are the regions where ecosystems are fragile and vulnerable to suffer from degradation with external disturbances, e.g. environmental changes and human activities (Feng et al. 2022; Wang et al. 2019). EVAs in China are widely distributed and account for more than 55% China’s land area (Ministry of Ecology and Environment of the People’s Republic of China 2008). The ecosystem in EVAs, chartered with low stability, weak resistance and high vulnerability, has been experiencing significant degradation owing to the impacts of global climate change and human activities (Bai et al. 2018; Chen et al. 2021; Yu et al. 2022). The EVAs in China are not only the most serious areas of environmental degradation, but also the most poverty-stricken regions (Wang et al. 2019). Harsh environmental condition (drought, low temperature and strong radiation) and limited resource supply (water, soil nutrients, etc.) constrain the vegetation productivity and ecosystem services of EVAs (Li et al. 2021). Climate change adds new challenges with warmer temperatures, changing rainfall regime and increasing frequency of extreme events (drought, heat wave, storms, etc.), which make it is more difficult to predict the changes of ecosystem processes and functions in future scenarios (Piao et al. 2020; Reid et al. 2014). Carbon and water fluxes are the core ecosystem processes, which is linked to diverse ecosystem services (Lian et al. 2021). Therefore, clarifying the variations and controls of ecosystem carbon and water fluxes is an effective approach to clarifying how ecosystem respond to global change in EVAs (Baldocchi 2020). As the only technique can directly measure the carbon, water and energy fluxes between vegetation and atmosphere, eddy covariance technique has been considered as a standard method for flux observations (Chen et al. 2020). By integrating long-term, eddy covariance measurements over time and space, researches are able to assess ecosystem metabolism at different time scales (hours to decades) (Forzieri et al. 2020; Han et al. 2020; Jung et al. 2017). Eddy covariance measurements also produce information on how ecosystem respond to the changes in climate, which is useful for assessing ecosystem carbon sequestration (Hu et al. 2018), water and energy balance (Forzieri et al. 2020), resource use efficiency (Liu et al. 2019) and ecosystem feedback to climate change (Huang et al. 2019; Piao et al. 2020; Yue et al. 2020). Long-term flux measurements are also vital for detecting the responses of ecosystem functions to extreme events, optimizing and validating models on regional and global scales (Baldocchi 2020). Combining with remote sensing and ecosystem modeling techniques, scientists can upscale and evaluate the functional relations between carbon and water fluxes with environmental variables at high resolution and across diverse spatial/temporal scales (Niu et al. 2017; Xia et al. 2020).     

Cellular Redistribution of Protein Tyrosine Phosphatases LAR and PTPσ by Inducible Proteolytic Processing

Most receptor-like protein tyrosine phosphatases (PTPases) display a high degree of homology with cell adhesion molecules in their extracellular domains. We studied the functional significance of processing for the receptor-like PTPases LAR and PTPσ. PTPσ biosynthesis and intracellular processing resembled that of the related PTPase LAR and was expressed on the cell surface as a two-subunit complex. Both LAR and PTPσ underwent further proteolytical processing upon treatment of cells with either calcium ionophore A23187 or phorbol ester TPA. Induction of LAR processing by TPA in 293 cells did require overexpression of PKCα. Induced proteolysis resulted in shedding of the extracellular domains of both PTPases. This was in agreement with the identification of a specific PTPσ cleavage site between amino acids Pro₈₂₁ and Ile₈₂₂. Confocal microscopy studies identified adherens junctions and desmosomes as the preferential subcellular localization for both PTPases matching that of plakoglobin. Consistent with this observation, we found direct association of plakoglobin and β-catenin with the intracellular domain of LAR in vitro. Taken together, these data suggested an involvement of LAR and PTPσ in the regulation of cell contacts in concert with cell adhesion molecules of the cadherin/catenin family. After processing and shedding of the extracellular domain, the catalytically active intracellular portions of both PTPases were internalized and redistributed away from the sites of cell–cell contact, suggesting a mechanism that regulates the activity and target specificity of these PTPases. Calcium withdrawal, which led to cell contact disruption, also resulted in internalization but was not associated with prior proteolytic cleavage and shedding of the extracellular domain. We conclude that the subcellular localization of LAR and PTPσ is regulated by at least two independent mechanisms, one of which requires the presence of their extracellular domains and one of which involves the presence of intact cell–cell contacts. A key element in the regulation of cell–cell and cell– matrix contacts is the tyrosine phosphorylation of proteins that are localized in focal adhesions and at intercellular junctions (for reviews see Kemler, 1993; Clark and Brugge, 1995). While much is known about the protein tyrosine kinases involved in the phosphorylation of cell adhesion components, very little information exists about the identity of protein tyrosine phosphatases (PTPases),¹ which are responsible for the dephosphorylation and thereby regulation of these structural complexes. Probable candidates are those receptor-like PTPases that contain cell adhesion molecule-like extracellular domains and could therefore regulate their intrinsic phosphatase activity in response to cell contact. Recent reports suggest that some PTPases do, in fact, possess properties that resemble those of classical cell adhesion molecules (for review see Brady-Kalnay and Tonks, 1995). A direct involvement in cell–cell contact has so far been demonstrated for PTPμ (Brady-Kalnay et al., 1993; Gebbink et al., 1993) and PTPκ (Sap et al., 1994), for which a homophilic interaction between their extracellular domains was found. The localization of PTPμ (Brady-Kalnay et al., 1995; Gebbink et al., 1995), PTPκ (Fuchs et al., 1996), and PCP-2 (Wang et al., 1996) was restricted to sites of cell–cell contact and surface expression of PTPμ (Gebbink et al., 1995), and PTPκ (Fuchs et al., 1996) was increased in a cell density-dependent manner. Moreover, a direct association of PTPκ (Fuchs et al., 1996) and PTPμ (Brady-Kalnay et al., 1995) with members of the cadherin/catenin family suggests that proteins of the cell adhesion complex represent physiological substrates for these PTPases. A possible regulatory function in cell–matrix adhesion has been proposed for LAR, another receptor-like PTPase, which associated with focal cell–substratum adhesions via the newly identified LAR interacting protein 1, LIP-1 (Serra-Pages et al., 1995).PTPμ (Gebbink et al., 1991), PTPκ (Jiang et al., 1993; Fuchs et al., 1996), PTPδ (Krueger et al., 1990; Mizuno et al., 1993, Pulido et al., 1995a), PCP-2 (Wang et al., 1996), and LAR (Streuli et al., 1988, Pot et al., 1991) are members of the so-called type II receptor-like PTPases. The extracellular domains of these PTPases contain a variable number of Ig-like and fibronectin type III-like (FNIII) domains (for review see Charbonneau and Tonks, 1992). With the exception of PCP-2 (Wang et al., 1996), these PTPases also share characteristics in their biosynthesis. They all underwent proteolytic processing by a furin-like endoprotease and were expressed at the cell surface in two subunits which were not covalently linked (Streuli et al., 1992; Yu et al., 1992; Jiang et al., 1993; Brady-Kalnay and Tonks, 1994; Gebbink et al., 1995; Pulido et al., 1995a; Fuchs et al., 1996). It was shown for LAR that the E subunit, which contains the cell adhesion molecule-like extracellular domain, was shed from the cell surface when cells were grown to a high density (Streuli et al., 1992). This shedding of the E subunit of LAR was the result of an additional proteolytic processing step that could also be induced by treatment of the cells with the phorbol ester TPA (Serra-Pages et al., 1995). An accumulation of E subunits in the supernatant of cells was also observed for PTPμ (Gebbink et al., 1995) and PTPδ (Pulido et al., 1995a), and this suggests a common mechanism in the regulation of type II PTPases. However, the effect of proteolytic processing on either the catalytic activity, the substrate specificity, or the cellular localization of these PTPases has not yet been determined. We report here that PTPσ, a recently identified new member of the family of receptor-like type II PTPases (Pan et al., 1993; Walton et al., 1993; Yan et al., 1993; Ogata et al., 1994; Zhang et al., 1994), underwent biosynthesis and proteolytic processing in a manner that resembled that of the most closely related PTPase LAR. Moreover, further proteolytic processing of PTPσ as well as of LAR could be induced by treatment of the cells with TPA or the calcium ionophore A23187. Transient expression studies indicated that TPA-induced processing of LAR, but not PTPσ, was dependent on the coexpression of PKCα. Inducible processing of both PTPases took place in the extracellular segment of the P subunit in a juxtamembrane position and led to the shedding of the E subunit. Both LAR and PTPσ were predominantly localized in regions of cell–cell contact and accumulated in dot-like structures that could be identified as adherens junctions and desmosomes by colocalization with plakoglobin (Cowin et al., 1986). Moreover, plakoglobin and β-catenin, another component of E-cadherin–containing cell adhesion complexes in adherens junctions, associated directly with the intracellular domain of LAR in vitro. The inducible shedding of the E subunit of LAR and PTPσ was followed by a redistribution of the PTPases within the cell membrane and by an internalization of the cleaved P subunits. It therefore represents a mechanism through which the phosphatase activity of these PTPases could be regulated in response to cell–cell contact. The cell adhesion molecule-like character of LAR and PTPσ was further supported by the fact that the internalization of LAR and PTPσ occurred independently of the proteolytic processing if cells were grown in calcium-depleted growth medium. The analogies in specific localization as well as internalization behavior of PTPσ and LAR, with molecules of the cadherin/catenin family, strongly suggest a direct involvement of PTPσ and LAR in the formation or maintenance of intercellular contacts.     

Role of nonstructural proteins 3A and 3B in host range and pathogenicity of foot-and-mouth disease virus

The genome of foot-and-mouth disease virus (FMDV) differs from that of other picornaviruses in that it encodes a larger 3A protein (>50% longer than poliovirus 3A), as well as three copies of protein 3B (also known as VPg). Previous studies have shown that a deletion of amino acids 93 to 102 of the 153-codon 3A protein is associated with an inability of a Taiwanese strain of FMDV (O/TAW/97) to cause disease in bovines. Recently, an Asian virus with a second 3A deletion (amino acids 133 to 143) has also been detected (N. J. Knowles et al., J. Virol. 75:1551-1556, 2001). Genetically engineered viruses harboring the amino acids 93 to 102 or 133 to 143 grew well in porcine cells but replicated poorly in bovine cells, whereas a genetically engineered derivative of the O/TAW/97 virus expressing a full-length 3A (strain A12) grew well in both cell types. Interestingly, a virus with a deletion spanning amino acid 93 to 144 also grew well in porcine cells and caused disease in swine. Further, genetically engineered viruses containing only a single copy of VPg were readily recovered with the full-length 3A, the deleted 3A (amino acids 93 to 102), or the "super" deleted forms of 3A (missing amino acids 93 to 144). All of the single-VPg viruses were attenuated in porcine cells and replicated poorly in bovine cells. The single-VPg viruses produced a mild disease in swine, indicating that the VPg copy number is an important determinant of host range and virulence. The association of VPg copy number with increased virulence in vivo may help to explain why all naturally occurring FMDVs have retained three copies of VPg.     

Genetic insights into age-related macular degeneration: controversies addressing risk, causality, and therapeutics

Age-related macular degeneration (AMD) is a common condition among the elderly population that leads to the progressive central vision loss and serious compromise of quality of life for its sufferers. It is also one of the few disorders for whom the investigation of its genetics has yielded rich insights into its diversity and causality and holds the promise of enabling clinicians to provide better risk assessments for individuals as well as to develop and selectively deploy new therapeutics to either prevent or slow the development of disease and lessen the threat of vision loss. The genetics of AMD began initially with the appreciation of familial aggregation and increase risk and expanded with the initial association of APOE variants with the disease. The first major breakthroughs came with family-based linkage studies of affected (and discordant) sibs, which identified a number of genetic loci and led to the targeted search of the 1q31 and 10q26 loci for associated variants. Three of the initial four reports for the CFH variant, Y402H, were based on regional candidate searches, as were the two initial reports of the ARMS2/HTRA1 locus variants. Case-control association studies initially also played a role in discovering the major genetic variants for AMD, and the success of those early studies have been used to fuel enthusiasm for the methodology for a number of diseases. Until 2010, all of the subsequent genetic variants associated with AMD came from candidate gene testing based on the complement factor pathway. In 2010, several large-scale genome-wide association studies (GWAS) identified genes that had not been previously identified. Much of this historical information is available in a number of recent reviews (Chen et al., 2010b; Deangelis et al., 2011; Fafowora and Gorin, 2012b; Francis and Klein, 2011; Kokotas et al., 2011). Large meta analysis of AMD GWAS has added new loci and variants to this collection (Chen et al., 2010a; Kopplin et al., 2010; Yu et al., 2011). This paper will focus on the ongoing controversies that are confronting AMD genetics at this time, rather than attempting to summarize this field, which has exploded in the past 5 years.     

Tilorone confers robust in vitro and in vivo antiviral effects against severe fever with thrombocytopenia syndrome virus

Severe fever with thrombocytopenia syndrome virus (SFTSV), an emerging pathogen, is a tick-borne bunyavirus belonging to the genus Bandavirus in the family Phenuiviridae (Kuhn et al., 2020). This pathogen was first identified in China during the heightened surveillance of acute febrile illness in 2009, and has been reported to cause several outbreaks in eastern Asia areas, including China, Japan, and Korea (Yu et al., 2011). Besides, Vietnam has also reported several confirmed SFTS cases (Tran et al., 2019). The mortality rate in hospitalised patients with SFTSV infection is up to 10%–30%. Moreover, SFTSV has been reported to possibly transmitted by the contact of body fluids from person-to-person, and extensive SFTSV contamination was detected in the patient rooms (Kim et al., 2015). These reports suggest that more stringent isolation measures are needed for the prevention of massive SFTSV outbreak.     

Ubiquitin Binding in Endocytosis – How Tight Should It Be and Where Does It Happen?

Ubiquitin is an important tag in membrane transport. From studies in yeast, monoubiquitin has been considered sufficient to elicit uptake of cell surface transporters and receptors into endosomes. Two articles in the current issue of Traffic (Hawryluk et al. and Barriere et al.) indicate that stronger binding is required to retain and concentrate cargo in endocytic microdomains of the plasma membrane. High avidity interactions can be obtained by tandemly arrayed ubiquitin interaction motifs (UIM), in proteins such as the endocytic adaptors epsin and Eps15, interacting with polyubiquitin or by UIM-containing proteins binding several ubiquitins brought together through oligomerization of receptors. A controversial issue has been where such interactions take place. One view is that the association of epsin with ubiquitinated cargo is negatively regulated by its interaction with clathrin (Chen H and De Camilli P. Proc Natl Acad Sci USA 2005;102:2766-2771). This contention is now challenged by the articles of Hawryluk et al. and Barriere et al. Hawryluk et al. demonstrate that epsin and Eps15 consistently co-localize with clathrin but never with caveolin.     

内蒙古西部额济纳旗晚奥陶世生物地理和奥陶-志留系分界

通过最近对内蒙古额济纳旗东南珠斯楞海尔罕一带(阿拉善地块)奥陶—志留系界线地层和生物群的野外调查和室内研究,首次在上奥陶统上部巴丹吉林组中发现阿什极尔中期(mid Ashgill)腕足动物化石巨大全嘴贝(Holorhymchus giganteus Kiaer),这是该属在我国奥陶系中的首次记录。根据未成年个体的切片所发现的腹壳顶端发育中隔板构造,修订该属定义,并证明研究小个体对识别物种特征、探讨个体发育和系统演化有重要意义。分析了全嘴贝属的群落生态、生物地理和演化意义后,发现它在奥陶纪末大灭绝过程中,因居群规模和分布范围极大地缩减,至今未发现其化石记录;但在志留纪初环境好转后继续存活,可暂视其为复活属。根据在拐子湖组近底部发现志留系最下部Akidograptus ascensus带的重要分子Normalograptus lubricus Chen et Lin,确定本区的奥陶—志留系分界。阿拉善地块因不发育Hirnantia动物群,表明奥陶纪末期它与扬子区、西藏、滇西等富含该动物群的地区属于不同的生物地理区系;在阿什极尔中期可能位于热带海域,与祁连山、中亚、乌拉尔等地有重要联系。文中描记H．giganteus种,评述正常笔石类(normalograptid)的系统分类位置并描记N．lubricus种。     

Porcine ileal polypeptide causes an increase in cytoplasmic Ca2+ in both parietal and chief cells resulting in acid and pepsinogen secretion

Porcine ileal polypeptide, an enterooxyntin isolated from distal small intestinal mucosal epithelium, has been observed to stimulate gastric acid secretion in vivo as well as in vitro (Wider, M.D. et al. (1984) Endocrinology 115, 1484-1491, Wider M.D. et al. (1986) Endocrinology 118, 1546-1550). We report here that porcine ileal polypeptide stimulates both acid (aminopyrine accumulation) and pepsinogen secretion in isolated, enriched populations of guinea pig parietal and chief cells in a dose-dependent manner. Further, 10(-9) M porcine ileal polypeptide caused an increase in cytoplasmic Ca2+ concentration in both parietal and chief cells similar in magnitude to that observed with gastrin-17 (10(-8) M) (as measured by both fura-2 and aequorin) and cholecystokinin octapeptide (CCK-OP) (10(-8) M), respectively. Porcine ileal polypeptide has been observed to cause no stimulation of cAMP production in gastric glands from guinea pigs (Gespach, C., personal communication) nor is there any effect of medium Ca2+ depletion on acid production observed with guinea pig gastric mucosal sections. It is concluded that porcine ileal polypeptide, at concentrations similar to circulating levels observed in plasma of normal pigs (5 x 10(-9) M), acts directly on the parietal and chief cells to cause the mobilization of intracellular Ca2+ from the stores resulting in acid and pepsinogen secretion. These experiments demonstrate that this peptide is a potent enterooxyntin and chief cell secretagogue which acts via the same signal transduction mechanisms as gastrin and cholecystokinin.     

Polymorphisms on SSC15q21-q26 Containing QTL for reproduction in Swine and its association with litter size

Several quantitative trait loci (QTL) for important reproductive traits (ovulation rate) have been identified on the porcine chromosome 15 (SSC15). To assist in the selection of positional candidate swine genes for these QTL on SSC15, twenty-one genes had already been assigned to SSC15 in a previous study in our lab, by using the radiation hybrid panel IMpRH. Further polymorphism studies were carried out on these positional candidate genes with four breeds of pigs (Duroc, Erhualian, Dahuabai and Landrace) harboring significant differences in reproduction traits. A total of nineteen polymorphisms were found in 21 genes. Among these, seven in six genes were used for association studies, whereby NRP2 polymorphism was found to be significantly (p < 0.05) associated with litter-size traits. NRP2 might be a candidate gene for pig-litter size based on its chromosome location (Du et al., 2006), significant association with litter-size traits and relationships with Sema and the VEGF super families.     

Association of a pH-sensitive peptide with membrane vesicles: role of amino acid sequence

The solution properties and bilayer association of two synthetic 30 amino acid peptides, GALA and LAGA, have been investigated at pH 5 and 7.5. These peptides have the same amino acid composition and differ only in the positioning of glutamic acid and leucine residues which together compose 47% of each peptide. Both peptides undergo a similar coil to helix transition as the pH is lowered from 7.5 to 5.0. However, GALA forms an amphipathic alpha-helix whereas LAGA does not. As a result, GALA partitions into membranes to a greater extent than LAGA and can initiate leakage of vesicle contents and membrane fusion which LAGA cannot (Subbarao et al., 1987; Parente et al., 1988). Membrane association of the peptides has been studied in detail with large phosphatidylcholine vesicles. Direct binding measurements show a strong association of the peptide GALA to vesicles at pH 5 with an apparent Ka around 10(6). The single tryptophan residue in each peptide can be exploited to probe peptide motion and positioning within lipid bilayers. Anisotropy changes and the quenching of tryptophan fluorescence by brominated lipids in the presence of vesicles also indicate that GALA can interact with uncharged vesicles in a pH-dependent manner. By comparison to the peptide LAGA, the membrane association of GALA is shown to be due to the amphipathic nature of its alpha-helical conformation at pH 5.