Diminished Susceptibility to Cefoperazone/Sulbactam and Piperacillin/Tazobactam in Enterobacteriaceae Due to Narrow-Spectrum β-Lactamases as Well as Omp Mutation

Cefoperazone/sulbactam (CSL) and piperacillin/tazobactam (TZP) are commonly used in clinical practice in China because of their excellent antimicrobial activity. CSL and TZP-nonsusceptible Enterobacteriaceae are typically resistant to extended-spectrum cephalosporins such as ceftriaxone (CRO). However, 11 nonrepetitive Enterobacteriaceae strains, which were resistant to CSL and TZP yet susceptible to CRO, were collected from January to December 2020. Antibiotic susceptibility tests and whole-genome sequencing were conducted to elucidate the mechanism for this rare phenotype. Antibiotic susceptibility tests showed that all isolates were amoxicillin/clavulanic-acid resistant and sensitive to ceftazidime, cefepime, cefepime/tazobactam, cefepime/zidebactam, ceftazidime/avibactam, and ceftolozane/tazobactam. Whole-genome sequencing revealed three of seven Klebsiella pneumoniae strains harbored bla_SHV-1 only, and four harbored bla_SHV-1 and bla_TEM-1B. Two Escherichia coli strains carried bla_TEM-1B only, while two Klebsiella oxytoca isolates harbored bla_OXY-1-3 and bla_OXY-1-1, respectively. No mutation in the β-lactamase gene and promoter sequence was found. Outer membrane protein (Omp) gene detection revealed that numerous missense mutations of OmpK36 and OmpK37 were found in all strains of K. pneumoniae. Numerous missense mutations of OmpK36 and OmpK35 and OmpK37 deficiency were found in one K. oxytoca strain, and no OmpK gene was found in the other. No Omp mutations were found in E. coli isolates. These results indicated that narrow spectrum β-lactamases, TEM-1, SHV-1, and OXY-1, alone or in combination with Omp mutation, contributed to the resistance to CSL and TZP in CRO-susceptible Enterobacteriaceae.Antibiotic susceptibility tests

Aluminum induced proteome changes in tomato cotyledons

Cotyledons of tomato seedlings that germinated in a 20 µM AlK(SO₄)₂ solution remained chlorotic while those germinated in an aluminum free medium were normal (green) in color. Previously, we have reported the effect of aluminum toxicity on root proteome in tomato seedlings (Zhou et al.¹). Two dimensional DIGE protein analysis demonstrated that Al stress affected three major processes in the chlorotic cotyledons: antioxidant and detoxification metabolism (induced), glyoxylate and glycolytic processes (enhanced), and the photosynthetic and carbon fixation machinery (suppressed).Key words: aluminum, cotyledons, proteome, tomatoDifferent biochemical processes occur depending on the developmental stages of cotyledons. During early seed germination, before the greening of the cotyledons, glyoxysomes enzymes are very active. Fatty acids are converted to glucose via the gluconeogenesis pathway.^2,3 In greening cotyledons, chloroplast proteins for photosynthesis and leaf peroxisomal enzymes in the glycolate pathway for photorespiration are metabolized.^2–4 Enzymes involved in regulatory mechanisms such as protein kinases, protein phosphatases, and mitochondrial enzymes are highly expressed.^3,5,6The chlorotic cotyledons are similar to other chlorotic counterparts in that both contains lower levels of chlorophyll, thus the photosynthetic activities are not as active. In order to understand the impact of Al on tomato cotyledon development, a comparative proteome analysis was performed using 2D-DIGE following the as previously described procedure.¹ Some proteins accumulated differentially in Al-treated (chlorotic) and untreated cotyledons (Fig. 1). Mass spectrometry of tryptic digestion fragments of the proteins followed by database search has identified some of the differentially expressed proteins (Open in a separate windowFigure 1Image of protein spots generated by Samspot analysis of Al treated and untreated tomato cotyledons proteomes separated on 2D-DIGE.

Table 1

Proteins identified from tomato cotyledons of seeds germinating in Al-solution

Stress-induced flowering

Many plant species can be induced to flower by responding to stress factors. The short-day plants Pharbitis nil and Perilla frutescens var. crispa flower under long days in response to the stress of poor nutrition or low-intensity light. Grafting experiments using two varieties of P. nil revealed that a transmissible flowering stimulus is involved in stress-induced flowering. The P. nil and P. frutescens plants that were induced to flower by stress reached anthesis, fruited and produced seeds. These seeds germinated, and the progeny of the stressed plants developed normally. Phenylalanine ammonialyase inhibitors inhibited this stress-induced flowering, and the inhibition was overcome by salicylic acid (SA), suggesting that there is an involvement of SA in stress-induced flowering. PnFT2, a P. nil ortholog of the flowering gene FLOWERING LOCUS T (FT) of Arabidopsis thaliana, was expressed when the P. nil plants were induced to flower under poor-nutrition stress conditions, but expression of PnFT1, another ortholog of FT, was not induced, suggesting that PnFT2 is involved in stress-induced flowering.Key words: flowering, stress, phenylalanine ammonia-lyase, salicylic acid, FLOWERING LOCUS T, Pharbitis nil, Perilla frutescensFlowering in many plant species is regulated by environmental factors, such as night-length in photoperiodic flowering and temperature in vernalization. On the other hand, a short-day (SD) plant such as Pharbitis nil (synonym Ipomoea nil) can be induced to flower under long days (LD) when grown under poor-nutrition, low-temperature or high-intensity light conditions.^1–9 The flowering induced by these conditions is accompanied by an increase in phenylalanine ammonia-lyase (PAL) activity.¹⁰ Taken together, these facts suggest that the flowering induced by these conditions might be regulated by a common mechanism. Poor nutrition, low temperature and high-intensity light can be regarded as stress factors, and PAL activity increases under these stress conditions.¹¹ Accordingly, we assumed that such LD flowering in P. nil might be induced by stress. Non-photoperiodic flowering has also been sporadically reported in several plant species other than P. nil, and a review of these studies suggested that most of the factors responsible for flowering could be regarded as stress. Some examples of these factors are summarized in 12^–14

Table 1

Some cases of stress-induced flowering

Escherichia coli O157:H7 and Other E. coli Strains Share Physiological Properties Associated with Intestinal Colonization

Escherichia coli isolates (72 commensal and 10 O157:H7 isolates) were compared with regard to physiological and growth parameters related to their ability to survive and persist in the gastrointestinal tract and found to be similar. We propose that nonhuman hosts in E. coli O157:H7 strains function similarly to other E. coli strains in regard to attributes relevant to gastrointestinal colonization.Escherichia coli is well known for its ecological versatility (15). A life cycle which includes both gastrointestinal and environmental stages has been stressed by both Savageau (15) and Adamowicz et al. (1). The gastrointestinal stage would be subjected to acid and detergent stress. The environmental stage is implicit in E. coli having transport systems for fungal siderophores (4) as well as pyrroloquinoline quinone-dependent periplasmic glucose utilization (1) because their presence indicates evolution in a location containing fungal siderophores and pyrroloquinoline quinone (1).Since its recognition as a food-borne pathogen, there have been numerous outbreaks of food-borne infection due to E. coli O157:H7, in both ground beef and vegetable crops (6, 13). Cattle are widely considered to be the primary reservoir of E. coli O157:H7 (14), but E. coli O157:H7 does not appear to cause disease in cattle. To what extent is E. coli O157:H7 physiologically unique compared to the other naturally occurring E. coli strains? We feel that the uniqueness of E. coli O157:H7 should be evaluated against a backdrop of other wild-type E. coli strains, and in this regard, we chose the 72-strain ECOR reference collection originally described by Ochman and Selander (10). These strains were chosen from a collection of 2,600 E. coli isolates to provide diversity with regard to host species, geographical distribution, and electromorph profiles at 11 enzyme loci (10).In our study we compared the 72 strains of the ECOR collection against 10 strains of E. coli O157:H7 and six strains of E. coli which had been in laboratory use for many years (Table ​(Table1).1). The in vitro comparisons were made with regard to factors potentially relevant to the bacteria''s ability to colonize animal guts, i.e., acid tolerance, detergent tolerance, and the presence of the Entner-Doudoroff (ED) pathway (Table ​(Table2).2). Our longstanding interest in the ED pathway (11) derives in part from work by Paul Cohen''s group (16, 17) showing that the ED pathway is important for E. coli colonization of the mouse large intestine. Growth was assessed by replica plating 88 strains of E. coli under 40 conditions (Table ​(Table2).2). These included two LB controls (aerobic and anaerobic), 14 for detergent stress (sodium dodecyl sulfate [SDS], hexadecyltrimethylammonium bromide [CTAB], and benzalkonium chloride, both aerobic and anaerobic), 16 for acid stress (pH 6.5, 6.0, 5.0, 4.6, 4.3, 4.2, 4.1, and 4.0), four for the ability to grow in a defined minimal medium (M63 glucose salts with and without thiamine), and four for the presence or absence of a functional ED pathway (M63 with gluconate or glucuronate). All tests were done with duplicate plates in two or three separate trials. The data are available in Tables S1 to S14 in the supplemental material, and they are summarized in Table ​Table22.

TABLE 1.

E. coli strains used in this study

Use of methotrexate therapy is not associated with decreased prevalence of metabolic syndrome

With great interest, we read the article by Toms and colleagues [1] in the previous issue of Arthritis Research & Therapy, in which they assessed prevalences of metabolic syndrome (MetS) in rheumatoid arthritis (RA) patients. Moreover, they identified demographic and clinical factors that may be associated with MetS. Toms and colleagues found prevalences of up to 45% of MetS and demonstrated older age and health status (health assessment questionnaire) to be associated with MetS irrespectively of the definition used. Of most interest, an association between methotrexate (MTX) use and decreased presence of MetS was observed in patients more than 60 years of age. The investigators hypothesized that this may be attributed to a drug-specific effect (and not to an anti-inflammatory effect) either by changing levels of adenosine, which is known to interact with glucose and lipid metabolism, or by an indirect effect mediated through concomitant folic acid administration, thereby decreasing homocysteine levels.Recently, we also examined the prevalence of MetS in (a subgroup of) RA patients in the CARRÉ investigation, a prospective cohort study on prevalent and incident cardiovascular disease and its underlying cardiovascular risk factors [2]. The findings of Toms and colleagues stimulated us to perform additional analyses in our total study population (n = 353).The prevalences of MetS were 35% and 25% (Table ​(Table1)1) according to criteria of National Cholesterol Education Program (NCEP) 2004 and NCEP 2001, respectively. In multivariate backward regression analyses, we found significant associations between body mass index, pulse rate, creatinine levels, hypothyroidism and diabetes mellitus and the presence of MetS independently of the criteria used (Table ​(Table2).2). However, an independent association between single use of MTX or use of MTX in combination with other disease-modifying antirheumatic drugs, on the one hand, and a decreased prevalence of MetS, on the other hand, could not be demonstrated (even in the subgroup of patients over the age of 60).

Table 1

Characteristics of the study population

Immunoreactivity for alpha-smooth muscle actin characterizes a potentially aggressive subgroup of little basal cell carcinomas

Basal cell carcinoma (BCC) is a very common malignant skin tumor that rarely metastatizes, but is often locally aggressive. Several factors, like large size (more than 3 cm), exposure to ultraviolet rays, histological variants, level of infiltration and perineural or perivascular invasion, are associated with a more aggressive clinical course. These morphological features seem to be more determinant in mideface localized BCC, which frequently show a significantly higher recurrence rate. An immunohistochemical profile, characterized by reactivity of tumor cells for p53, Ki67 and alpha-SMA has been associated with a more aggressive behaviour in large BCCs. The aim of this study was to verify if also little (<3 cm) basal cell carcinomas can express immunohistochemical markers typical for an aggressive behaviour.Basal cell carcinoma (BCC) is a very common malignant skin tumor that rarely metastatizes, even If Is often locally aggressive. Several factors, like large size (more than 3 cm), face localization, exposure to ultraviolet rays, histological variants, infiltration level and perineural or perivascular invasion, are associated with a more aggressive clinical course. In particular, the incidence of metastasis and/or death correlates with tumors greater than 3 cm in diameter in which setting patients are said to have 1–2 % risk of metastases that increases to 20–25% in lesions greater than 5 cm and to 50% in lesions greater than 10 cm in diameter (Snow et al., 1994). Histologically morpheiform, keratotic types and infiltrative growth of BCC are also considered features of the most aggressive course (Crowson, 2006). This can be explained by the fact that both the superficial and nodular variants of BCC are surrounded by a continuous basement membrane zone comprising collagens type IV and V admixed with laminin, while the aggressive growth variants (i.e. morpheiform, metatypical, and infiltrative growth subtypes) manifest the absence of basement membrane (Barsky et al., 1987).The molecular markers which characterize aggressive BCC include: increased expression of stromolysin (MMP-3) and collagenase-1 (MMP-1) (Cribier et al., 2001), decreased expression of syndecan-1 proteoglycan (Bayer-Garner et al., 2000) and of anti-apoptotic protein bcl-2 (Ramdial et al., 2000; Staibano et al., 2001).C-ras , c-fos (Urabe et al., 1994; Van der Schroeff et al., 1990) and p53 tumor supressor gene mutations (Auepemikiate et al., 2002) are indicative of an aggressive course.Focusing upon bcl-2 and p53 expression in BCC, there have been numerous studies documenting the utility of bcl-2 as a marker of favourable clinical behaviour while p53 expression may be a feature of a more aggressive outcome (Ramdial et al., 2000; Staibano et al., 2001; Bozdogan et al., 2002).An increased expression of cytoskeletal microfilaments like α–smooth muscle actin, frequently found in invasive BCC subtypes (Jones JCR et al., 1989), may explain an enhanced tumor mobility and deep tissue invasion through the stroma. (Cristian et al., 2001; Law et al., 2003). The aim of this preliminary study was to verify if also little (<3 cm) basal cell carcinomas may express aggressive immunohistochemical markers like p53, Ki67 and alpha-SMA. We used 31 excisional BCCs with tumor size less than 2 cm (ranging from 2 up to 20 mm) and with different skin localization (19 in the face, 6 in the trunk and 6 in the body extremities). All cases were immunostained for p53, BCL2, Ki67 and alpha-smooth muscle actin (α-SMA) (

Correlation of Fragile Histidine Triad (Fhit) Protein Structural Features with Effector Interactions and Biological Functions

We have previously shown that Fhit tumor suppressor protein interacts with Hsp60 chaperone machinery and ferredoxin reductase (Fdxr) protein. Fhit-effector interactions are associated with a Fhit-dependent increase in Fdxr stability, followed by generation of reactive oxygen species and apoptosis induction under conditions of oxidative stress. To define Fhit structural features that affect interactions, downstream signaling, and biological outcomes, we used cancer cells expressing Fhit mutants with amino acid substitutions that alter enzymatic activity, enzyme substrate binding, or phosphorylation at tyrosine 114. Gastric cancer cell clones stably expressing mutants that do not bind substrate or cannot be phosphorylated showed decreased binding to Hsp60 and Fdxr and reduced mitochondrial localization. Expression of Fhit or mutants that bind interactor proteins results in oxidative damage and accumulation of cells in G₂/M or sub-G₁ fractions after peroxide treatment; noninteracting mutants are defective in these biological effects. Gastric cancer clones expressing noncomplexing Fhit mutants show reduction of Fhit tumor suppressor activity, confirming that substrate binding, interaction with heat shock proteins, mitochondrial localization, and interaction with Fdxr are important for Fhit tumor suppressor function.Fhit protein is a powerful tumor suppressor that is frequently lost or reduced in cancer cells because of rearrangement of the exquisitely DNA damage-sensitive fragile FHIT gene. Restoration of Fhit expression suppresses tumorigenicity of cancer cells of various types, and the ability to induce apoptosis in cancer cells in vitro is reduced by specific Fhit mutations (1, 2).Through studies of signal pathways affected by Fhit expression, by searches for Fhit protein effectors, and by in vitro analyses of Fhit activity, we and others have defined Fhit enzymatic activity in vitro (3), apoptotic activity in cells and tumors (4–6), and most recently identification of a Fhit protein complex that affects Fhit stability, mitochondrial localization, and interaction with ferredoxin reductase (Fdxr)⁵ (7). The complex includes Hsp60 and Hsp10 that mediate Fhit stability and may affect import into mitochondria, where Fhit interacts with Fdxr, which is responsible for transferring electrons from NADPH to cytochrome P450 via ferredoxin. Virally mediated Fhit restoration in Fhit-deficient cancer cells increases production of intracellular reactive oxygen species (ROS), followed by increased apoptosis of cancer cells under oxidative stress conditions; conversely, Fhit-negative cells escape apoptosis, likely carrying oxidative DNA damage that contributes to accumulation of mutations.The Fhit protein sequence, showing high homology to the histidine triad (HIT) family of proteins, suggested that the protein product would hydrolyze diadenosine tetraphosphate or diadenosine triphosphate (Ap₃A) (8), and in vitro studies showed that Ap₃A was cleaved into ADP and AMP by Fhit. The catalytic histidine triad within Fhit was essential for catalytic activity (3), and a Fhit mutant that substituted Asn for His at the central histidine (H96N mutant) was catalytically inactive, although it bound substrate well (3). Early tumor suppression studies showed that cancer cells stably transfected with wild type (WT) or H96N mutant Fhit were suppressed for tumor growth in nude mice. This suggested the hypothesis that the Fhit-substrate complex sends the tumor suppression signal (9, 10). To test this hypothesis, a series of FHIT alleles was designed to reduce substrate-binding and/or hydrolytic rates and was characterized by quantitative cell-death assays on cancer cells virally infected with each allele. The allele series covered defects as great as 100,000-fold in k_cat and increases as large as 30-fold in K_m. Mutants with 2–7-fold increases in K_m had significantly reduced apoptotic indices and the mutant with a 30-fold increase in K_m retained little apoptotic function. Thus, the proapoptotic function of Fhit, which is likely associated with tumor suppressor function, is limited by substrate binding and is unrelated to substrate hydrolysis (11).Fhit, a homodimeric protein of 147 amino acids, is a target of tyrosine phosphorylation by the Src family protein kinases, which can phosphorylate Tyr-114 of Fhit in vitro and in vivo (12). After co-expression of Fhit with the Elk tyrosine kinase in Escherichia coli to generate phosphorylated forms of Fhit, unphosphorylated, mono-, and diphosphorylated Fhit were purified, and enzyme kinetics studies showed that monophosphorylated Fhit exhibited monophasic kinetics with K_m and k_cat values ∼2- and ∼7-fold lower, respectively, than for unphosphorylated Fhit. Diphosphorylated Fhit exhibited biphasic kinetics; one site had K_m and k_cat values ∼2- and ∼140-fold lower, respectively, than for unphosphorylated Fhit; the second site had a K_m ∼60-fold higher and a k_cat ∼6-fold lower than for unphosphorylated Fhit (13). Thus, it was possible that the alterations in K_m and k_cat values for phosphorylated forms of Fhit might favor formation and lifetime of the Fhit-Ap₃A complex and enhance tumor suppressor activity (see Fhit forms

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Kinetic parameters

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% Sub-G₁

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Direct binding

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Subcellular location

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Co-IP in vivo

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8-OHdG

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Apoptosis

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Tumor suppressor

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K_m (mm)k_cat (s^–1)A549MKN74Hsp60FdxrHsp60Fdxr Fhit WT 1.6 +/– 0.19 2.7 +/– 0.95 43 24 Yes Yes Cyt & mito Yes Yes Yes Yes Yes Catalyt mutants    H96D Up 2-fold Down >2 × 10⁴ 29 NT NT NT Cyt & mito Yes Yes NT Yes NT    H96N Up 2-fold Down >5 × 10⁵ 31 14.4 NT NT Cyt & mito Yes Yes Yes Yes Yes Loop mutants    Y114A Up 23-fold Down 2-fold 3.7 NT NT NT Cyt +/– +/– +/– No No    Y114D NT NT 2.9 6 NT NT Cyt +/– +/– – No –/+    Y114E NT NT NT NT NT NT Cyt & mito –/+ –/+ – No NT    Y114F Up 5-fold Up 1.1-fold 11.5 3 NT NT Cyt & mito –/+ –/+ – No No    Y114W Up 5-fold Up 1.4-fold NT NT NT NT Cyt & mito –/+ – – NT NT    del113–117 Up 10-fold Down 38-fold 5 NT NT NT NT NT NT – No NT Other mutants    L25W Up 7-fold Down 4-fold 15 NT NT NT Cyt – – – NT –/+    I10W,L25W Up 32-fold Down 6-fold 11 NT NT NT NT NT NT NT NT NT    F5W Up 3.3 fold NT NT 5 NT NT NT NT NT +/– No NT Purified pFhit    pFhit Down 0.4-fold Down 7-fold NA NA –/+ Yes NA NA NA NA NA NA    ppFhit Down 0.4-fold Down > 100-fold NA NA –/+ Yes NA NA NA NA NA NA Up 60-fold Down 6-fold Open in a separate windowTo explore the in vivo importance of the Tyr-114 phosphorylation site and define Fhit-mediated signaling events, Semba et al. (14) compared the differential biological effects of Ad-FHIT WT and Ad-FHIT Tyr-114 mutant expression in human lung cancer cells. Caspase-dependent apoptosis was effectively induced only by WT Fhit protein. However, the biological significance of phosphorylation at Tyr-114 has been difficult to study because the endogenous phosphorylated forms have very short half-lives; activation of epidermal growth facto receptor family members induces Fhit phosphorylation by Src and proteasome degradation of phosphorylated Fhit (15).Although there are possible connections among the various pathways known to be altered in Fhit-deficient cells, apoptosis, DNA damage-response checkpoint activation, ROS production, and related biological effects of Fhit loss or overexpression, details of the pathway(s) leading from Fhit overexpression to cell death and tumor suppression have not been delineated. Now that a Fhit signaling complex has been identified, we set out to examine which structural features of Fhit protein might participate in individual steps of the pathway leading from Fhit overexpression through complex formation, subcellular localization, interaction with mitochondrial Fdxr, DNA damage induction, cell cycle changes, apoptosis, and ultimately tumor suppression. The underlying hypotheses were as follows: substrate-binding mutants would behave similarly to WT; nonsubstrate-binding mutants would be defective in some step of the pathway, perhaps complexing with heat shock proteins or Fdxr or perhaps induction of DNA damage; and Tyr-114 mutants, which also affect formation or stability of the enzyme-substrate complex, would also be defective in executing some step of the Fhit overexpression pathway to cell death. One goal was to identify specific mutants that exhibited deficiency in specific steps of the pathway, so that such mutants could be used to dissect each step in more detail. Using in vitro Fhit and Fhit-effector protein interactions, we aimed to determine the following: 1) which proteins of the complex interact directly with Fhit, and 2) the biological role of these interactions in vivo. Using cancer cells expressing exogenous WT and mutant Fhit proteins, we were able to examine the structural features of Fhit that affect the direct interaction with its effectors, participate in ROS production, and are necessary for tumor suppression activity.     

Allelic frequency and genotypes of prion protein at codon 136 and 171 in Iranian Ghezel sheep breeds

PrP genotypes at codons 136 and 171 in 120 Iranian Ghezel sheep breeds were studied using allele-specific PCR amplification and compared with the well-known sheep breeds in North America, the United States and Europe. The frequency of V allele and VV genotype at codon 136 of Ghezel sheep breed was significantly lower than AA and AV. At codon 171, the frequency of allele H was significantly lower than Q and R. Despite the similarities of PrP genotypes at codons 136 and 171 between Iranian Ghezel sheep breeds and some of the studied breeds, significant differences were found with others. Planning of effective breeding control and successful eradication of susceptible genotypes in Iranian Ghezel sheep breeds will not be possible unless the susceptibility of various genotypes in Ghezel sheep breeds to natural or experimental scrapie has been elucidated.Key words: scrapie, Ghezel sheep breed, PrP genotyping, allele specific amplification, codon 136, codon 171Scrapie was first described in England in 1732,¹ and it is an infectious neurodegenerative fatal disease of sheep and goats belonging to the group of transmissible subacute spongiform encephalopathies (TSEs), along with bovine spongiform encephalopathy (BSE), chronic wasting disease and Creutzfeldt-Jakob disease.^2,3 The term prion, proteinaceous infectious particles, coined by Stanley B. Prusiner, was introduced, and he presents the idea that the causal agent is a protein.⁴ Prion proteins are discovered in two forms, the wild-type form (PrP^c) and the mutant form (PrP^Sc).⁵ Although scrapie is an infectious disease, the susceptibility of sheep is influenced by genotypes of the prion protein (PrP) gene.^2,6 Researchers have found that the PrP allelic variant alanine/arginine/arginine (ARR) at codons 136, 154 and 171 is associated with resistance to scrapie in several breeds.^7–14 Most of the sheep populations in the Near East and North African Region (84% of the total population of 255 million) are raised in Iran, Turkey, Pakistan, Sudan, Algeria, Morocco, Afghanistan, Syria and Somalia.¹⁵ In 2003, the Iranian sheep population was estimated at 54,000,000 head. The Ghezel sheep breed, which also is known as Kizil-Karaman, Mor-Karaman, Dugli, Erzurum, Chacra, Chagra, Chakra, Gesel, Gezel, Kazil, Khezel, Khizel, Kizil, Qezel, Qizil and Turkish Brown, originated in northwestern Iran and northeastern Turkey. By considering sheep breeds as one of the main sources of meat, dairy products and related products, a global screening attempt is started in different areas. In compliance with European Union Decision 2003/100/EC, each member state has introduced a breeding program to select for resistance to TSEs in sheep populations to increase the frequency of the ARR allele. A similar breeding program is established in United States and Canada. The Near East and North African Region still needs additional programs to help the global plan of eradication of scrapie-susceptible genotypes. The current study was the first to assess the geographical and molecular variation of codons 136 and 171 polymorphism between Iranian Ghezel sheep breed and well-known sheep breeds.Polymorphism at codon 136 is associated with susceptibility to scrapie in both experimental and natural models.^10,11,13,16 17 and Austrian Carynthian sheep.¹⁸ Swiss White Alpine showed higher frequency of allele V at position 136 than Swiss Oxford Down, Swiss Black-Brown Mountain and Valais Blacknose.¹⁹ Comparison of polymorphism at codon 136 in the current study with some of other breeds (20 some flock of Hampshire sheep²¹ with current study, but the frequency of it is higher than that of some other breeds.

Table 1

Comparison of PrP allelic and genotype frequencies at codon 136 in different breeds

Genome-wide analysis of thioredoxin fold superfamily peroxiredoxins in Arabidopsis and rice

A broad range of peroxides generated in subcellular compartments, including chloroplasts, are detoxified with peroxidases called peroxiredoxins (Prx). The Prx are ubiquitously distributed in all organisms including bacteria, fungi, animals and also in cyanobacteria and plants. Recently, the Prx have emerged as new molecules in antioxidant defense in plants. Here, the members which belong to Prx gene family in Arabidopsis and rice are been identified. Overall, the Prx members constitute a small family with 10 and 11 genes in Arabidopsis and rice respectively. The prx genes from rice are assigned to their functional groups based on homology search against Arabidopsis protein database. Deciphering the Prx functions in rice will add novel information to the mechanism of antioxidant defense in plants. Further, the Prx also forms the part of redox signaling cascade. Here, the Prx gene family has been described for rice.Key words: antioxidant defense, chloroplast, gene family, oxidative stress, reactive oxygen speciesThe formation of free radicals and reactive oxygen species (ROS) occur in several enzymatic and non-enzymatic reactions during cellular metabolism. The accumulation of these reactive and deleterious intermediates is suppressed by antioxidant defense mechanism comprised of low molecular weight antioxidants and enzymes. In photosynthetic organisms, the defense against the damage from free radicals and oxidative stress is crucial. For instance, the ROS production occurs in photosystem II with generation of singlet oxygen (¹O₂) and hydrogen peroxide (H₂O₂),^1,2 photosystem I from superoxide anion radicals (O₂⁻),³ and during photorespiration with generation of H₂O₂.⁴ ROS production may exceed under environmental stress conditions like excess light, low temperature and drought.⁵The antioxidant defense mechanism is activated by antioxidant metabolities and enzymes which detoxify ROS and lipid peroxides. The detoxification of ROS can occur in various cellular compartments such as chloroplasts, mitochondria, peroxisomes and cytosol.⁶ The enzymes like ascorbate peroxidase, catalase, glutathione peroxidase and superoxide dismutase are prominent antioxidant enzymes.⁶ The peroxiredoxins (Prx) emerged as new components in the antioxidant defense network of barley.^7,8 Later, Prx were studied in other plants.^9–14Prx can be classified into four different functional groups, PrxQ, 1-Cys Prx, 2-Cys Prx and Type-2 Prx.^15,16 They are members of the thioredoxin fold superfamily.^17,18 In this study, the prx genes found in Arabidopsis and rice genomes are been identified. The Arabidopsis genome encodes 10 prx genes classified into four functional categories, 1-Cys Prx, 2-Cys Prx, PrxQ and Type-2 Prx.¹³ Of these, one each of 1-Cys Prx and PrxQ, two of 2-Cys Prx (2-Cys PrxA and 2-Cys PrxB) and six Type-2 Prx (PrxA–F) are identified¹³ (LocusAnnotationSynonymA^*B^*C^*AT1G481301-Cysteine peroxiredoxin 1 (ATPER1)1-Cys Prx21624081.36.603AT1G60740Peroxiredoxin type 2Type-2 PrxD16217471.95.2297AT1G65970Thioredoxin-dependent peroxidase 2 (TPX2)Type-2 PrxC16217413.95.2297AT1G65980Thioredoxin-dependent peroxidase 1 (TPX1)Type-2 PrxB16217427.84.9977AT1G65990Type 2 peroxiredoxin-relatedType-2 PrxA55362653.66.4368AT3G06050Peroxiredoxin IIF (PRXIIF)Type-2 PrxF20121445.29.3905AT3G116302-Cys Peroxiredoxin A (2CPA, 2-Cys PrxA)2-Cys PrxA26629091.77.5686AT3G26060ATPRX Q, periredoxin QPrxQ21623677.810.0565AT3G52960Peroxiredoxin type 2Type-2 PrxE23424684.09.572AT5G062902-Cysteine Peroxiredoxin B (2CPB, 2-Cys PrxB)2-Cys PrxB27329779.55.414Open in a separate window^*A, amino acids; B, molecular weight; C, isoelectric point.In rice (rice.plantbiology.msu.edu/), there are 11 genomic loci which encode for Prx proteins (​and33). Interestingly, a new prx gene (LOC_Os07g15670) annotated as “peroxiredoxin, putative, expressed” is identified making the tally of prx genes to eleven in rice as compared to ten in Arabidopsis (​and22). The BLAST search has identified its counterpart in Arabidopsis which has been annotated as “antioxidant/oxidoreductase” (AT1G21350) in the TAIR database (www.arabidopsis.org). The rice LOC_Os07g15670 and Arabidopsis AT1G21350 share protein homology %68/78 for 236 amino acids (ChromosomeLocus IdPutative function/AnnotationA^*B^*C^*1LOC_Os01g16152peroxiredoxin, putative, expressed19920873.68.22091LOC_Os01g24740peroxiredoxin-2E-1, chloroplast precursor, putative10711591.56.79061LOC_Os01g48420peroxiredoxin, putative, expressed16317290.85.68282LOC_Os02g09940peroxiredoxin, putative, expressed22623179.56.5352LOC_Os02g33450peroxiredoxin, putative, expressed26228096.95.77094LOC_Os04g339702-Cys peroxiredoxin BAS1, chloroplast precursor, putative, expressed12213410.24.37056LOC_Os06g09610peroxiredoxin, putative, expressed2662892610.50976LOC_Os06g42000peroxiredoxin, putative, expressed23323688.39.20597LOC_Os07g15670peroxiredoxin, putative, expressed25327684.69.85457LOC_Os07g44440peroxiredoxin, putative, expressed22124232.65.36187LOC_Os07g44430peroxiredoxin, putative25627785.36.8544Open in a separate window^*A, amino acids; B, molecular weight; C, isoelectric point.

Table 3

Identification of rice homologs of peroxiredoxins in A. thaliana

Association of Campylobacter jejuni Cj0859c Gene (fspA) Variants with Different C. jejuni Multilocus Sequence Types

Cj0859c variants fspA1 and fspA2 from 669 human, poultry, and bovine Campylobacter jejuni strains were associated with certain hosts and multilocus sequence typing (MLST) types. Among the human and poultry strains, fspA1 was significantly (P < 0.001) more common than fspA2. FspA2 amino acid sequences were the most diverse and were often truncated.Campylobacter jejuni is the leading cause of bacterial gastroenteritis worldwide and responsible for more than 90% of Campylobacter infections (7). Case-control studies have identified consumption or handling of raw and undercooked poultry meat, drinking unpasteurized milk, and swimming in natural water sources as risk factors for acquiring domestic campylobacteriosis in Finland (7, 9). Multilocus sequence typing (MLST) has been employed to study the molecular epidemiology of Campylobacter (4) and can contribute to virulotyping when combined with known virulence factors (5). FspA proteins are small, acidic, flagellum-secreted nonflagellar proteins of C. jejuni that are encoded by Cj0859c, which is expressed by a σ²⁸ promoter (8). Both FspA1 and FspA2 were shown to be immunogenic in mice and protected against disease after challenge with a homologous strain (1). However, FspA1 also protected against illness after challenge with a heterologous strain, whereas FspA2 failed to do the same at a significant level. Neither FspA1 nor FspA2 protected against colonization (1). On the other hand, FspA2 has been shown to induce apoptosis in INT407 cells, a feature not exhibited by FspA1 (8). Therefore, our aim was to study the distributions of fspA1 and fspA2 among MLST types of Finnish human, chicken, and bovine strains.In total, 367 human isolates, 183 chicken isolates, and 119 bovine isolates (n = 669) were included in the analyses (3). PCR primers for Cj0859c were used as described previously (8). Primer pgo6.13 (5′-TTGTTGCAGTTCCAGCATCGGT-3′) was designed to sequence fspA1. Fisher''s exact test or a chi-square test was used to assess the associations between sequence types (STs) and Cj0859c. The SignalP 3.0 server was used for prediction of signal peptides (2).The fspA1 and fspA2 variants were found in 62.6% and 37.4% of the strains, respectively. In 0.3% of the strains, neither isoform was found. Among the human and chicken strains, fspA1 was significantly more common, whereas fspA2 was significantly more frequent among the bovine isolates (Table ​(Table1).1). Among the MLST clonal complexes (CCs), fspA1 was associated with the ST-22, ST-45, ST-283, and ST-677 CCs and fspA2 was associated with the ST-21, ST-52, ST-61, ST-206, ST-692, and ST-1332 CCs and ST-58, ST-475, and ST-4001. Although strong CC associations of fspA1 and fspA2 were found, the ST-48 complex showed a heterogeneous distribution of fspA1 and fspA2. Most isolates carried fspA2, and ST-475 was associated with fspA2. On the contrary, ST-48 commonly carried fspA1 (Table ​(Table1).1). In our previous studies, ST-48 was found in human isolates only (6), while ST-475 was found in both human and bovine isolates (3, 6). The strict host associations and striking difference between fspA variants in human ST-48 isolates and human/bovine ST-475 isolates suggest that fspA could be important in host adaptation.

TABLE 1.

Percent distributions of fspA1 and fspA2 variants among 669 human, poultry, and bovine Campylobacter jejuni strains and their associations with hosts, STs, and CCs

Single Nucleotide Polymorphism-Based Diagnostic System for Crop-Associated Sclerotinia Species

A molecular diagnostic system using single nucleotide polymorphisms (SNPs) was developed to identify four Sclerotinia species: S. sclerotiorum (Lib.) de Bary, S. minor Jagger, S. trifoliorum Erikss., and the undescribed species Sclerotinia species 1. DNAs of samples are hybridized with each of five 15-bp oligonucleotide probes containing an SNP site midsequence unique to each species. For additional verification, hybridizations were performed using diagnostic single nucleotide substitutions at a 17-bp sequence of the calmodulin locus. The accuracy of these procedures was compared to that of a restriction fragment length polymorphism (RFLP) method based on Southern hybridizations of EcoRI-digested genomic DNA probed with the ribosomal DNA-containing plasmid probe pMF2, previously shown to differentiate S. sclerotiorum, S. minor, and S. trifoliorum. The efficiency of the SNP-based assay as a diagnostic test was evaluated in a blind screening of 48 Sclerotinia isolates from agricultural and wild hosts. One isolate of Botrytis cinerea was used as a negative control. The SNP-based assay accurately identified 96% of Sclerotinia isolates and could be performed faster than RFLP profiling using pMF2. This method shows promise for accurate, high-throughput species identification.Sclerotinia is distinguished morphologically from other genera in the Sclerotiniaceae (Ascomycota, Pezizomycotina, Leotiomycetes) by the production of tuberoid sclerotia that do not incorporate host tissue, by the production of microconidia that function as spermatia but not as a disseminative asexual state, and by the development of a layer of textura globulosa composing the outer tissue of apothecia (8). Two hundred forty-six species of Sclerotinia have been reported, most distinguished morphotaxonomically (Index Fungorum [www.indexfungorum.org]). These include the four species of agricultural importance now recognized plus many that are imperfectly known, seldom collected, or apparently endemic to relatively small geographic areas (2, 5, 6, 7, 8, 9, 17).The main species of phythopathological interest in the genus Sclerotinia are S. sclerotiorum (Lib.) de Bary, S. minor Jagger, S. trifoliorum Erikss., and the undescribed species Sclerotinia species 1. Sclerotinia species 1 is an important cause of disease in vegetables in Alaska (16) and has been found in association with wild Taraxacum sp., Caltha palustris, and Aconitum septentrionalis in Norway (7). It is morphologically indistinguishable from S. sclerotiorum, but it was shown to be a distinct species based on distinctive polymorphisms in sequences from internal transcribed spacer 2 (ITS2) of the nuclear ribosomal repeat (7). The other three species have been delimited using morphological, cytological, biochemical, and molecular characters (3, 8, 9, 10, 12, 15). Interestingly, given that the ITS is sufficiently polymorphic in many fungal genera to resolve species, in Sclerotinia, only species 1 and S. trifoliorum are distinguished by characteristic ITS sequence polymorphisms; S. sclerotiorum and S. minor cannot be distinguished based on ITS sequence (2, 7).Sclerotinia sclerotiorum is a necrotrophic pathogen with a broad host range (1). S. minor has a more restricted host range but causes disease in a variety of important crops such as lettuce, peanut, and sunflower crops (11). S. trifoliorum has a much narrower host range, limited to the Fabaceae (3, 8, 9). Sclerotial and ascospore characteristics also serve to differentiate among the three species. Sclerotinia minor has small sclerotia that develop throughout the colony in vitro and aggregate to form crusts on the host, while the sclerotia of S. sclerotiorum and S. trifoliorum are large and form at the colony periphery in vitro, remaining separate on the host (8, 9). The failure of an isolate to produce sclerotia or apothecia in vitro is not unusual, especially after serial cultivation (8). The presence of dimorphic, tetranucleate ascospores characterizes S. trifoliorum, while S. sclerotiorum and S. minor both have uniformly sized ascospores that are binucleate and tetranucleate, respectively (9, 14).With the apparent exception of Sclerotinia species 1, morphological characteristics are sufficient to delimit Sclerotinia species given that workers have all manifestations of the life cycle in hand. In cultures freshly isolated from infected plants, investigators usually have mycelia and sclerotia but not apothecia. Restriction fragment length polymorphisms (RFLPs) in ribosomal DNA (rDNA) are diagnostic for Sclerotinia species (3, 10), but the assay requires cloned probes (usually accessed from other laboratories) hybridized to Southern blots from vertical gels, an impractical procedure for large samples. We have analyzed sequence data from previous phylogenetic studies (2) and have identified diagnostic variation for the rapid identification of the four Sclerotinia species. The single nucleotide polymorphism (SNP) assay that we report here is amenable to a high throughput of samples and requires only PCR amplification with a standard set of primers and oligonucleotide hybridizations to Southern blots in a dot format.The SNP assay was performed using two independent sets of species-specific oligonucleotide probes, all with SNP sites shown to differentiate the four Sclerotinia species (Fig. ​(Fig.1).1). A panel of 49 anonymously coded isolates (Table ​(Table1)1) was screened using these species-specific SNP probes, as outlined in Fig. ​Fig.1.1. The assay was validated by comparison to Southern hybridizations of EcoRI-digested genomic DNA hybridized with pMF2, a plasmid probe containing the portion of the rDNA repeat with the 18S, 5.8S, and 26S rRNA cistrons of Neurospora crassa (4, 10).Open in a separate windowFIG. 1.Protocol for the SNP-based identification of Sclerotinia species, with diagnostic SNP sites underlined and in boldface type for each hybridization probe.

TABLE 1.

Isolates and hybridization results for all SNP-based oligonucleotide probes^f