R1 Strains Research Articles

Characterization of causal agents of bacterial canker on apricot plantations and risk mapping using GIS in Aras Basin (Türkiye)

AbstractBacterial canker of stone fruits caused by Pseudomonas syringae pv. syringae (Pss) and Pseudomonas syringae pv. morsprunorum (Psm race‐1/Psm race‐2) may lead to significant yield and crop losses in apricot (Prunus armeniaca L.) cultivation areas in Türkiye. Strains pathogenic to apricot were isolated from trees with symptoms (mainly necrotized buds and dieback) of bacterial canker in orchards in Aras Basin. Pathogens were characterized using pathogenicity tests, phenotypic assays, end‐point PCR and multilocus sequence analysis (MLSA). Fifteen Pseudomonas syringae strains were isolated from 205 plant samples collected from apricot orchards showing symptoms of bacterial canker. As a consequence of the diagnostic tests, all isolates were identified as P. syringae pv. syringae. In this study, Pss, Psm R1 and Psm R2 strains in stone fruits were separated into different phylogroups (Pg‐2, Pg‐3, sPg‐1b) based on MLSA. Turkish strains obtained from stone fruits, particularly apricot, showed genetic heterogeneity, and clustered in different sub‐phylogroups (sPg‐2b, sPg‐2c, sPg‐2d). All these strains except strain K258 are also clustered in the same sub‐phylogroups (sPg‐2b and sPg‐2d) with other strains from different countries especially Iran, Lebanon, etc. Strain K258 isolated from apricot was clustered in sPg‐2c with Pss strain 642 (USA). The risk of bacterial canker disease in apricot growing areas is considered using GIS in this study. It was determined that a significant part of the Iğdır Plain, the biggest agricultural area in the Aras Basin, is at very high risk.

Lipopolysaccharide-Linked Enterobacterial Common Antigen (ECALPS) Occurs in Rough Strains of Escherichia coli R1, R2, and R4.

Enterobacterial common antigen (ECA) is a conserved surface antigen characteristic for Enterobacteriaceae. It is consisting of trisaccharide repeating unit, →3)-α-d-Fucp4NAc-(1→4)-β-d-ManpNAcA-(1→4)-α-d-GlcpNAc-(1→, where prevailing forms include ECA linked to phosphatidylglycerol (ECAPG) and cyclic ECA (ECACYC). Lipopolysaccharide (LPS)-associated form (ECALPS) has been proved to date only for rough Shigella sonnei phase II. Depending on the structure organization, ECA constitutes surface antigen (ECAPG and ECALPS) or maintains the outer membrane permeability barrier (ECACYC). The existence of LPS was hypothesized in the 1960–80s on the basis of serological observations. Only a few Escherichia coli strains (i.e., R1, R2, R3, R4, and K-12) have led to the generation of anti-ECA antibodies upon immunization, excluding ECAPG as an immunogen and conjecturing ECALPS as the only immunogenic form. Here, we presented a structural survey of ECALPS in E. coli R1, R2, R3, and R4 to correlate previous serological observations with the presence of ECALPS. The low yields of ECALPS were identified in the R1, R2, and R4 strains, where ECA occupied outer core residues of LPS that used to be substituted by O-specific polysaccharide in the case of smooth LPS. Previously published observations and hypotheses regarding the immunogenicity and biosynthesis of ECALPS were discussed and correlated with presented herein structural data.

Genomic insights into plant growth promoting rhizobia capable of enhancing soybean germination under drought stress

BackgroundThe role of soil microorganisms in plant growth, nutrient utilization, drought tolerance as well as biocontrol activity cannot be over-emphasized, especially in this era when food crisis is a global challenge. This research was therefore designed to gain genomic insights into plant growth promoting (PGP) Rhizobium species capable of enhancing soybean (Glycine max L.) seeds germination under drought condition.ResultsRhizobium sp. strain R1, Rhizobium tropici strain R2, Rhizobium cellulosilyticum strain R3, Rhizobium taibaishanense strain R4 and Ensifer meliloti strain R5 were found to possess the entire PGP traits tested. Specifically, these rhizobial strains were able to solubilize phosphate, produce exopolysaccharide (EPS), 1-aminocyclopropane-1-carboxylate (ACC), siderophore and indole-acetic-acid (IAA). These strains also survived and grew at a temperature of 45 °C and in an acidic condition with a pH 4. Consequently, all the Rhizobium strains enhanced the germination of soybean seeds (PAN 1532 R) under drought condition imposed by 4% poly-ethylene glycol (PEG); nevertheless, Rhizobium sp. strain R1 and R. cellulosilyticum strain R3 inoculations were able to improve seeds germination more than R2, R4 and R5 strains. Thus, genomic insights into Rhizobium sp. strain R1 and R. cellulosilyticum strain R3 revealed the presence of some genes with their respective proteins involved in symbiotic establishment, nitrogen fixation, drought tolerance and plant growth promotion. In particular, exoX, htrA, Nif, nodA, eptA, IAA and siderophore-producing genes were found in the two rhizobial strains.ConclusionsTherefore, the availability of the whole genome sequences of R1 and R3 strains may further be exploited to comprehend the interaction of drought tolerant rhizobia with soybean and other legumes and the PGP ability of these rhizobial strains can also be harnessed for biotechnological application in the field especially in semiarid and arid regions of the globe.

Engineered Deinococcus radiodurans R1 with NiCoT genes for bioremoval of trace cobalt from spent decontamination solutions of nuclear power reactors.

The aim of the present work was to engineer bacteria for the removal of Co in contaminated effluents. Radioactive cobalt ((60)Co) is known as a major contributor for person-sievert budgetary because of its long half-life and high γ-energy values. Some bacterial Ni/Co transporter (NiCoT) genes were described to have preferential uptake for cobalt. In this study, the NiCoT genes nxiA and nvoA from Rhodopseudomonas palustris CGA009 (RP) and Novosphingobium aromaticivorans F-199 (NA), respectively, were cloned under the control of the groESL promoter. These genes were expressed in Deinococcus radiodurans in reason of its high resistance to radiation as compared to other bacterial strains. Using qualitative real time-PCR, we showed that the expression of NiCoT-RP and NiCoT-NA is induced by cobalt and nickel. The functional expression of these genes in bioengineered D. radiodurans R1 strains resulted in >60 % removal of (60)Co (≥5.1 nM) within 90 min from simulated spent decontamination solution containing 8.5 nM of Co, even in the presence of >10 mM of Fe, Cr, and Ni. D. radiodurans R1 (DR-RP and DR-NA) showed superior survival to recombinant E. coli (ARY023) expressing NiCoT-RP and NA and efficiency in Co remediation up to 6.4 kGy. Thus, the present study reports a remarkable reduction in biomass requirements (2 kg) compared to previous studies using wild-type bacteria (50 kg) or ion-exchanger resins (8000 kg) for treatment of ~10(5)-l spent decontamination solutions (SDS).

Adaptive mutation related to cellulose producibility in Komagataeibacter medellinensis (Gluconacetobacter xylinus) NBRC 3288.

Gluconacetobacter xylinus (formerly Acetobacter xylinum and presently Komagataeibacter medellinensis) is known to produce cellulose as a stable pellicle. However, it is also well known to lose this ability very easily. We investigated the on and off mechanisms of cellulose producibility in two independent cellulose-producing strains, R1 and R2. Both these strains were isolated through a repetitive static culture of a non-cellulose-producing K. medellinensis NBRC 3288 parental strain. Two cellulose synthase operons, types I and II, of this strain are truncated by the frameshift mutation in the bcsBI gene and transposon insertion in the bcsCII gene, respectively. The draft genome sequencing of R1 and R2 strains revealed that in both strains the bcsBI gene was restored by deletion of a nucleotide in its C-rich region. This result suggests that the mutations in the bcsBI gene are responsible for the on and off mechanism of cellulose producibility. When we looked at the genomic DNA sequences of other Komagataeibacter species, several non-cellulose-producing strains were found to contain similar defects in the type I and/or type II cellulose synthase operons. Furthermore, the phylogenetic relationship among cellulose synthase genes conserved in other bacterial species was analyzed. We observed that the cellulose genes in the Komagataeibacter shared sequence similarities with the γ-proteobacterial species but not with the α-proteobacteria and that the type I and type II operons could be diverged from a same ancestor in Komagataeibacter.

Searching for genomic region of high-fat diet-induced type 2 diabetes in mouse chromosome 2 by analysis of congenic strains.

SMXA-5 mice are a high-fat diet-induced type 2 diabetes animal model established from non-diabetic SM/J and A/J mice. By using F2 intercross mice between SMXA-5 and SM/J mice under feeding with a high-fat diet, we previously mapped a major diabetogenic QTL (T2dm2sa) on chromosome 2. We then produced the congenic strain (SM.A-T2dm2sa (R0), 20.8–163.0 Mb) and demonstrated that the A/J allele of T2dm2sa impaired glucose tolerance and increased body weight and body mass index in the congenic strain compared to SM/J mice. We also showed that the combination of T2dm2sa and other diabetogenic loci was needed to develop the high-fat diet-induced type 2 diabetes. In this study, to narrow the potential genomic region containing the gene(s) responsible for T2dm2sa, we constructed R1 and R2 congenic strains. Both R1 (69.6–163.0 Mb) and R2 (20.8–128.2 Mb) congenic mice exhibited increases in body weight and abdominal fat weight and impaired glucose tolerance compared to SM/J mice. The R1 and R2 congenic analyses strongly suggested that the responsible genes existed in the overlapping genomic interval (69.6–128.2 Mb) between R1 and R2. In addition, studies using the newly established R1A congenic strain showed that the narrowed genomic region (69.6–75.4 Mb) affected not only obesity but also glucose tolerance. To search for candidate genes within the R1A genomic region, we performed exome sequencing analysis between SM/J and A/J mice and extracted 4 genes (Itga6, Zak, Gpr155, and Mtx2) with non-synonymous coding SNPs. These four genes might be candidate genes for type 2 diabetes caused by gene-gene interactions. This study indicated that one of the genes responsible for high-fat diet-induced diabetes exists in the 5.8 Mb genomic interval on mouse chromosome 2.

Differences in saccharin preference and genetic alterations of the Tas1r3 gene among senescence-accelerated mouse strains and their parental AKR/J strain

The senescence-accelerated mouse (SAM) is used as an animal model of senescence acceleration and age-associated disorders. SAM is derived from unexpected crosses between the AKR/J and unknown mouse strains. There are nine senescence-prone (SAMP) strains and three senescence-resistant (SAMR) strains. Although SAMP strains exhibit strain-specific and age-related pathological changes, the genes responsible for the pathologic changes in SAMP strains have not been comprehensively identified. In the present study, we evaluated sweet taste perception using the two-bottle test. We compared genotypes of the taste related gene, Tas1r3, using SAM strains and the parental AKR/J strain. The two-bottle test revealed that SAMR1 (R1), SAMP6 (P6), SAMP8 (P8), and SAMP10 (P10) mice were saccharin-preferring strains, whereas AKR/J did not prefer saccharin. All genotypes of the R1, P6, P8, and P10 strains at the polymorphic sites in Tas1r3, which is known to influence saccharin preference, were identical to those of C57BL6/J, a well-known saccharin-preferring strain, and were completely different from those of the parental AKR/J strain. These genetic alterations in SAM strains appear to arise from an unknown strain that is thought to have been crossed with AKR/J initially.

Whole Genome Mapping and Re-Organization of the Nuclear and Mitochondrial Genomes of Babesia microti Isolates

Babesia microti is the primary causative agent of human babesiosis, an emerging pathogen that causes a malaria-like illness with possible fatal outcome in immunocompromised patients. The genome sequence of the B. microti R1 strain was reported in 2012 and revealed a distinct evolutionary path for this pathogen relative to that of other apicomplexa. Lacking from the first genome assembly and initial molecular analyses was information about the terminal ends of each chromosome, and both the exact number of chromosomes in the nuclear genome and the organization of the mitochondrial genome remained ambiguous. We have now performed various molecular analyses to characterize the nuclear and mitochondrial genomes of the B. microti R1 and Gray strains and generated high-resolution Whole Genome maps. These analyses show that the genome of B. microti consists of four nuclear chromosomes and a linear mitochondrial genome present in four different structural types. Furthermore, Whole Genome mapping allowed resolution of the chromosomal ends, identification of areas of misassembly in the R1 genome, and genomic differences between the R1 and Gray strains, which occur primarily in the telomeric regions. These studies set the stage for a better understanding of the evolution and diversity of this important human pathogen.

Safety and intestinal microbiota modulation by the exopolysaccharide-producing strains Bifidobacterium animalis IPLA R1 and Bifidobacterium longum IPLA E44 orally administered to Wistar rats

Bifidobacterium animalis subsp. lactis IPLA R1 and Bifidobacterium longum IPLA E44 strains were tested for their safety and ability to modulate the intestinal microbiota in vivo. Chemically simulated gastrointestinal digestion showed considerably lower survival of E44 than R1 strain, the first microorganism also being more sensitive to refrigerated storage in 10% skimmed milk at 4°C. Harmful glycosidic activities were absent, or at low levels, in the strains R1 and E44. Both strains were sensitive to most antibiotics and resistant to aminoglycosides, a common feature in bifidobacteria. Similar to several other bifidobacteria strains, B. animalis subsp. lactis IPLA R1 displayed a moderate resistance against tetracycline which correlated with the presence of tet(W) gene in its genome. The general parameters indicating well-being status, as well as translocation to different organs and histological examination of the gut tissues, revealed no changes induced by the administration of bifidobacteria to rats. Twelve-week-old male Wistar rats were distributed into three groups, eight rats in each. Two groups were administered daily over 108cfu of the corresponding strain suspended in 10% skimmed milk for 24days, whereas rats in the placebo group received skimmed milk without microorganisms added. The microbiota and short chain fatty acids (SCFA) were monitored in faeces at different time points during treatment and in caecum content at the end of the assay. Quantitative PCR (qPCR) showed that faecal and caecal Bifidobacterium levels were higher in bifidobacteria-fed rats than in the placebo rats at the end of the intervention, whereas total anaerobic plate counts did not show significant differences. Quantification of B. animalis and B. longum by qPCR showed that, independent of the microorganism administered, treatment with bifidobacteria resulted in higher levels of B. animalis in the caecum. PCR-DGGE analysis of microbial populations revealed a higher diversity of bands in caecum content of rats fed B. animalis IPLA R1 than in the placebo group and rats fed B. longum IPLA E44. Remarkably, although no variations in the proportion of acetate, propionate and butyrate were found, at the end of the assay the total SCFA concentration in the faeces of rats fed bifidobacteria was significantly higher and those in caecum content significantly lower, than that of the placebo group. This suggests a displacement of the SCFA production to parts of the colon beyond the caecum in rats receiving bifidobacteria. Therefore, the oral administration of B. animalis IPLA R1 and B. longum E44 can be considered safe, these microorganisms having the ability to modulate the intestinal microbiota of rats by influencing SCFA and the bifidobacterial population levels.

Quorum sensing is necessary for the virulence of Pseudomonas aeruginosa during urinary tract infection

Quorum sensing is a signaling pathway used by bacteria to monitor their population density by detecting small diffusible molecules. To understand the role of quorum sensing in pathogenesis of urinary tract infections, wild type Pseudomonas aeruginosa, having both functional las and rhl quorum sensing systems, and its isogenic single and double mutants were used in a mouse model of ascending urinary tract infection. Mice were evaluated on the basis of bacteriological and histopathological examinations, detection of acyl homoserine lactone, and the pathologic index factors of renal and bladder tissue. Single as well as double mutant strains were able to reach renal tissue, but they were not able to multiply and cause tissue damage. Reduced rates of infection, mild histopathologic lesions, and decreased production of pathologic index factors like myeloperoxidase, malondialdehyde, and reactive nitrogen intermediates were found in these groups of mice as compared to mice infected with a standard strain (PAO1) possessing both functional sensing systems. Loss of any one of the quorum-sensing components significantly reduced the in vivo virulence of P. aeruginosa in this mouse model of ascending urinary tract infection. Our results suggests that quorum-sensing signals may act as virulence factors and are essential for P. aeruginosa multiplication and virulence during the course of urinary tract infection.

Change in gene expression of mouse embryonic stem cells derived from parthenogenetic activation

We previously established parthenogenetic mouse embryonic stem cells (ESCs) and this study was subsequently conducted for elucidating the influence of oocyte parthenogenesis on gene expression profile of ESCs. Gene expression of parthenogenetic ESC (pESC)-1 or pESC-2 was separately compared with that of two normally fertilized ESC (nfESC) lines (B6D2F1 and R1 strains), and quantification of mRNA expression was conducted for validating microarray data. In two sets of comparison, reaction of 11 347 and 15 454 gene probes were altered by parthenogenesis, while strain difference changed the expression of 15 750 and 14 944 probes. Level of correlation coefficient was higher in the comparisons between normal fertilization and parthenogenesis (0.974-0.985) than in the comparisons between strains of nfESCs (0.97-0.971). Overall, the expression of 3276-3329 genes was changed after parthenogenesis, and 88% (96/109) of major functional genes differentially (P < 0.01) expressed in one comparison set showed the same change in the other. When we monitored imprinted genes, expression of nine paternal and eight maternal genes were altered after parthenogenesis and 88% (14/16) of these was confirmed by mRNA quantification. The change in gene expression after parthenogenesis was similar to, or less than, the change induced by a strain difference under a certain genetic background. These results may suggest the clinical feasibility of parthenogenesis-derived, pluripotent cells.

Deinococcus radiodurans PprI Switches on DNA Damage Response and Cellular Survival Networks after Radiation Damage

Preliminary findings indicate that PprI is a regulatory protein that stimulates transcription and translation of recA and other DNA repair genes in response to DNA damage in the extremely radioresistant bacterium Deinococcus radiodurans. To define the repertoire of proteins regulated by PprI and investigate the in vivo regulatory mechanism of PprI in response to gamma radiation, we performed comparative proteomics analyses on wild type (R1) and a pprI knock-out strain (YR1) under conditions of ionizing irradiation. Results of two-dimensional electrophoresis and MALDI-TOF MS or MALDI-TOF/TOF MS indicated that in response to low dose gamma ray exposure 31 proteins were significantly up-regulated in the presence of PprI. Among them, RecA and PprA are well known for their roles in DNA replication and repair. Others are involved in six different pathways, including stress response, energy metabolism, transcriptional regulation, signal transduction, protein turnover, and chaperoning. The last group consists of many proteins with uncharacterized functions. Expression of an additional four proteins, most of which act in metabolic pathways, was down-regulated in irradiated R1. Additionally phosphorylation of two proteins was under the control of PprI in response to irradiation. The different functional roles of representative PprI-regulated genes in extreme radioresistance were validated by gene knock-out analysis. These results suggest a role, either directly or indirectly, for PprI as a general switch to efficiently enhance the DNA repair capability and extreme radioresistance of D. radiodurans via regulation of a series of pathways.

Oxidative stress on the astrocytes in culture derived from a senescence accelerated mouse strain

Astrocytes are one of the predominant glial cell types in the adult central nervous system functioning as both supportive and metabolic cells for the brain. Our objective in this experiment is to study the direct effects of hydrogen peroxide induced oxidative stress on astrocytes in culture. These astrocytes were derived from both an aged mouse strain (P8) and a matched control strain (R1). The astrocytes for both the P8 and R1 strains were treated with increasing concentrations of hydrogen peroxide. Our results showed that the oxidative stress had a similar effect in both strains of astrocytes; decreases in 3-(4,5-dimethylthiazol-2-yl)-2,2-diphenyltetrazolium bromide (MTT) and glial fibrillary acidic protein (GFAP) levels, and increases in terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) staining, lactate dehydrogenase (LDH) staining, and superoxide dismutase (SOD), caspase-3 and B-cell lymphoma 2-associated protein X (bax) levels. At a hydrogen peroxide concentration of 400μM, the differences of the above parameters between P8 cultures and R1 cultures were statistically significant (p<0.05). This strongly suggested that astrocytes derived from P8 and R1 strains reacted to oxidative stress with similar mechanisms and consequences. However, the mechanisms were not able to compensate for the oxidative stress in the P8 strain at a hydrogen peroxide concentration of 400μM. The inability of the P8 astrocytes to counteract the oxidative stress might lead to inadequate protection from neuronal loss possibly resulting in significantly more astrocytic death. Our results suggested that the changes of astrocytes in peroxide detoxification may play a role in aging of the central nervous system, and further aging studies should examine the oxidative status of the samples.

Protective ability of subcellular extracts from Salmonella Enteritidis and from a rough isogenic mutant against salmonellosis in mice

We evaluated the efficacy of surface components enriched hot saline extracts (HE) from parental and two isogenic rough mutant strains of Salmonella Enteritidis as subcellular vaccine candidates. By a randomized mutagenesis approach from a clinical isolate of S. Enteritidis there were selected two rough mutants defective in LPS synthesis (R1 and R2 mutants). The mutations mapped to the wcaI gene and gmd gene, respectively, of the O-antigen gene cluster involved in O-antigen synthesis. BALB/c mice received intraperitoneally one single dose of 30 μg of HE from parental and mutant strains, and the protection against a lethal infection with S. Enteritidis was determined. In contrast to the wild type extract, immunization with rough extracts did not induce any distress symptoms in the mice. HE extract from wild type and R1 strains induced the highest immunogenic response with respect IFN-γ eliciting splenic cells, in contrast with HE-R2. These results correlated with the obtained levels of protection. Thus, at day 63 post-infection, HE from parental strain rendered an 80% level of protection; HE-R1 conferred a 60% level of protection, whereas HE-R2 did not protect the mice. Any of the antigenic extracts elicited systemic IgG1 and IgG2a responses, although these antibodies did not, however, correlate with protection. These results put forward the importance of cellular immune response mediated by IFN-γ in protection against salmonellosis. The significantly different protective capacity between HE extracts from both rough mutants suggest that other factors independent of the O-chain, like outer membrane proteins and fimbrial antigens, may be involved in protection. In summary, the HE is a good candidate acellular extract for evaluation of its protective ability against salmonellosis following vaccination in poultry.

Age-related changes in the oxidation–reduction characteristics and the 8-OHdG accumulation in liver, lung, brain of SAMP1 and SAM R1

Age-related changes of endogenous generation of reactive free radicals (reactive oxygen species, ROS) and anti-oxidative defense systems were studied in the tissues of 5- and 9-month-old scenescence-acceralated mice (SAM) P1 and R1 strains. With advancing age, TBARS reflecting the production of ROS and their scavenger glutathione (GSH) in extra-hepatic tissues such as lung, brain, and aorta of the two strains were reduced suggesting a decline in the metabolism in general, whereas anti-oxidative enzymes such as GSH-dependent (GSH-pex) and non-dependent (POD) peroxidases showed increased activities. In addition to significantly increased activities of GSH-pex and POD in liver, a 42% increase with age in hepatic GSH may be a compensatory response for supplying GSH to its reduced extra-hepatic contents. In the nucleus, the significant increase of 8-hydroxydeoxyguanosine (8-OHdG), hydroxy free radical adduct of DNA, with age was observed in liver, lung, and brain of two strains. Despite of a 29% age-related increase in the activity of hepatic 8-oxo-7,8-dihydro-2′-deoxyguanosine 5′-triphosphatase (8-OXOdGTPase) which hydrolyze and eliminate 8-oxo-dGTP in SAMP1, there was a two-fold increase in 8-OHdG in the liver. These results suggest that the defect in protective systems in SAMP1 against ROS produced through normal cellular metabolism may cause extensive damage to DNA and may contribute to accelerated aging and to degenerative diseases of aging.

Age-related decrease in spontaneity observed in senescence-accelerated mice (SAMP10) and the involvement of the dopaminergic system in behavioral disorders

We studied age-related behavioral changes in senescence-accelerated P10/Ta mice (SAMP10) reared under specific pathogen-free (SPF) conditions. Unlike SAMP8/Ta (SAMP8), the SAMP10 did not exhibit low anxiety-like behavior in the plus-maze test and the impairment of the passive avoidance task of SAMP10 was milder than that of SAMP8. SAMP10 showed more age-related abnormal behavior than did SAMP8, such as a decrease in shock sensitivity, increases in the duration of immobility during the tail suspension and the forced swimming tests and a decrease in swimming speed in a water maze task. SAMP10 showed a significant decrease in dopamine content in the anterior cortex. Furthermore, the behavioral depression observed in SAMP10 was ameliorated by dopaminergic stimulants such as apomorphine and a sustained release formulation of thyrotropin-releasing hormone (TRH-SR), at doses having no significant effects on spontaneous motor activity. These findings suggest that the dopaminergic function may contribute to behavioral depression. The present results indicate that SAMP10 shows a greater age-related decrease in the responsiveness to averse or stressful stimuli than do the R1 and P8 strains, which suggests that SAMP10 may be a useful animal model of age-related behavioral depression or decrease in spontaneity.

Mechanisms of resistance to fungicides in field strains of Botrytis cinerea.

Field strains of Botrytis cinerea Pers ex Fr, the causal agent of grey mould diseases, were collected from French vineyards between 1993 and 2000. Several phenotypes have been characterized according to the inhibitory effects of fungicides towards germ-tube elongation and mycelial growth. Two types of benzimidazole-resistant strains (Ben R1 and Ben R2) could be detected; negative cross-resistance to phenylcarbamates (e.g. diethofencarb) was only found in Ben R1. Benzimidazole resistance was related to point mutations at codon 198 (Ben R1) or 200 (Ben R2) of the beta-tubulin gene. Most dicarboximide-resistant strains were also weakly resistant to aromatic hydrocarbon fungicides (e.g. dicloran) but remained sensitive to phenylpyrroles (e.g. fludioxonil). These resistant field strains (Imi R1) contained a single base pair mutation at position 365 in a two-component histidine kinase gene, probably involved in the fungal osmoregulation. Three anilinopyrimidine-resistant phenotypes have been identified. In the most resistant one (Ani R1), resistance was restricted to anilinopyrimidines, but no differences were observed in the amino-acid sequences of cystathionine beta-lyase (the potential target site of these fungicides) from Ani R1 or wild-type strains. In the two other phenotypes (Ani R2 and Ani R3), resistance extended to various other groups of fungicide, including dicarboximides, phenylpyrroles and sterol biosynthesis inhibitors. This multi-drug resistance was probably determined by over-production of ATP-binding cassette transporters. The hydroxyanilide fenhexamid is a novel botryticide whose primary target site is the 3-keto reductase involved in sterol C-4 demethylations. Apart from the multi-drug-resistant strain Ani R3, three other fenhexamid-resistant phenotypes have been recognized. For two of them (Hyd R1 and Hyd R2) fenhexamid-resistance seemed to result from P450-mediated detoxification. Reduced sensitivity of the target site could be the putative resistance mechanism operating in the third resistant phenotype (Hyd R3). Increased sensitivity to inhibitors of sterol 14 alpha-demethylase recorded in Hyd R1 strains was related to two amino-acid changes at positions 15 and 105 of this enzyme.

Remarkable decrease in d-amino acid oxidase activity in the kidney of both prone and resistant strains of senescence-accelerated mouse

d-Amino acid oxidase (DAO) activity was remarkably low in the kidney of both prone P1 and resistant R1 strains of the senescence-accelerated mouse (SAM) compared with that of AKR/J mouse, in spite of the presence of a nearly equal amount of protein as demonstrated by Western blot analysis. A nucleotide sequence analysis of DAO-cDNA from SAMP1/Yag kidney indicated that the 541st guanine was replaced by adenine, resulting in the change of the 181st amino acid from glycine to arginine. The results of reverse transcription-polymerase chain reaction-restriction fragment length polymorphism analysis indicate the presence of this mutation in all SAM strains examined (P1, P2, P3 and R5).

Experimental myelitis in BALB/cN and C57BL/6N mice caused by herpes simplex virus type 1 compared with herpes simplex virus type 2

Intraperitoneal and footpad inoculations of herpes simplex virus type 1 (HSV) into BALB/cN (HSV-susceptible) and C57BL/6N (HSV-resistant) mice were carried out to induce experimental myelitis. Standard laboratory strains (McIntyre, F, RK, and recently Okinawa strain R1) were inoculated in mice. As a control, the HSV 2 standard laboratory strain SAV was also inoculated. The McIntyre strain was the most virulent, while the F strain was the least. RK and R1 were both moderately virulent. Myelitis was induced in BALB/cN mice after intraperitoneal and footpad inoculations of low to high doses of the McIntyre strain, and intraperitoneal inoculation of moderate and high doses of the RK and R1 strains. Symptoms of paraplegia of the hind legs and rectal and urinary incontinence were observed, but not until 3-5 hours before death. The symptoms caused by footpad inoculation were slightly different from those following intraperitoneal inoculation; rectal incontinence, in particular, was inconspicuous in the former. In the case of footpad inoculation of RK and R1, only one mouse inoculated with R1 showed symptoms and histology of myelitis. The F strain caused no symptoms. In the case of C57BL/6N mice, high dose intraperitoneal and footpad inoculations of the McIntyre strain also caused myelitis, and the symptoms were observed about 6-7 hours before death. In only one C57BL/6N mouse intraperitoneally inoculated with a high dose of R1 did symptoms appear about 6 hours before death. The same symptoms caused by intraperitoneal and footpad inoculations of HSV 2 (SAV) were observed more clearly and for a longer period (half to one day) than those caused by HSV 1 inoculation. Spinal cord necrosis was noted with McIntyre, RK and R1 inoculations, but it was not marked with randomly located foci, when compared with that caused by SAV. Further, the foci of necrosis in C57BL/6N mice were smaller than in BALB/cN mice, even when high dose McIntyre strain was used. Nuclear pyknosis and edema of the brain in the dead mice following HSV 1 inoculation were more marked than in those killed by SAV.

Genetic Control of the Immune Response to Collagen by the Major Histocompatibility Complex in the Rat: I. Humoral Responses to Bovine and Chick Type II Collagen

A number of congenic and recombinant rat strains with different RT1 haplotypes on a PVG/c background were immunised with native bovine or chick type II collagen. Only strains with the “u” haplotype were good responders to bovine type II collagen whilst all strains responded to chick type II collagen. However, the “u” haplotype was again associated with the production of the highest amounts of antibody. All the strains responded to a non-collagenous antigen, keyhole limpet haemocyanin, showing that this effect was not a general one relating to all antigens.When antibodies were produced to the immunising collagen, binding of antibodies to native rat type II collagen was also seen. This association, of high amounts of antibody with the “u” haplotype, was inherited in a dominant fashion when F1 and F2 hybrids between low responder (RT1a)and high reponder (RT1u) were examined.The recombinant r1 (class I av1, class II c) and r8 (class I av1 and class II u) strains showed that “u” haplotype at class II rather than at class I was associated with the strong response to type II collagen.The absence of arthritis in the “u” haplotype rats, suggests that the “u” haplotype alone is insufficient for arthritis induction after immunisation with type II collagen. It suggests that background genes outside the RT1 complex influence arthritis production as Wistar rats with the RT1u haplotype (but different minor genes outside RT1 complex) give an arthritis incidence of 50% under identical immunisation protocols.