Distinct and opposite roles for SH2 and SH3 domains of <Emphasis Type="Italic">v-src</Emphasis> in embryo survival and hemangiosarcoma formation

The cellular proto-oncogene c-src is thought to be involved in formation, progression, and metastasis of a variety of tumor cell types, although its exact role during tumor cell genesis is not well defined. v-src, the viral oncogene counterpart of c-src, causes metastatic sarcomas, hemorrhagic disease, and hemangiosarcomas in chicken embryos and, thus, can be used as a constitutively activated form of src for experimentally-induced tumorigenesis. Here, we used retroviral vectors to express wild-type v-src or SH2 or SH3 domain-deleted forms (ΔSH2 or ΔSH3) to determine if different pathogenic effects resulted. Vectors were injected into early chick embryo midbrain ventricles and embryos were sacrificed at various ages up to embryonic day (E) 18. Retroviral expression of all forms of v-src resulted in transformation of pial connective tissue cells into large, rounded abnormal-appearing cells. Surprisingly, all forms of v-src were lethal. The v-src retrovirus was lethal and killed most embryos by E15 with the development of hemangiosarcomas over the injection site between E10–E12. The ΔSH3 retrovirus was the most deadly, killing most embryos by E12, however, it never resulted in hemangiosarcoma formation. The ΔSH2 retrovirus injected embryos survived longer than v-src or ΔSH3 embryos, and some of these embryos also developed large hemangiosarcomas over the injection site between E13 and E18. These results demonstrate that the src SH2 domain is required to be fully lethal, whereas the presence of the SH3 domain attenuated lethality. Furthermore, the formation of hemangiosarcomas absolutely required the presence of the src SH3 domain and to some extent required the SH2 domain. This implicates distinct and opposite roles for SH2 and SH3 domains of src and their cellular binding partners in tumorigenesis and hemorrhagic disease.     

Comparison of the chicken and zebra finch Z chromosomes shows evolutionary rearrangements

Using fluorescent in-situ hybridization (FISH) of zebra finch (Taeniopygia guttata) bacterial artificial chromosome (BAC) clones, we determined the chromosomal localizations of 14 zebra finch genes that are Z-linked in chickens: ATP5A1, CHD1, NR2F1, DMRT1, PAM, GHR, HSD17B4, NIPBL, ACO1, HINT1, SMAD2, SPIN, NTRK2 and UBE2R2. All 14 genes also map to the zebra finch Z chromosome, indicating substantial conservation of gene content on the Z chromosome in the two avian lineages. However, the physical order of these genes on the zebra finch Z chromosome differed from that of the chicken, in a pattern that would have required several inversions since the two lineages diverged. Eight of 14 zebra finch BAC DNA showed cross-hybridization to the W chromosome, usually to the entire W chromosome, suggesting that repetitive sequences are shared by the W and Z chromosomes. These repetitive sequences likely evolved in the finch lineage after it diverged from the Galliform lineage.     

The role of the O-antigen lipopolysaccharide on the colonizationin vivoof the germfree chicken gut byAeromonas hydrophilaserogroup O:34

We compared the ability of differentAeromonas hydrophilastrains from serogroup O:34 grown at different temperatures to colonizein vivothe germfree chicken gut. We found a good colonization when the strains were grown at 20°C but not when they were grown at 37°C. We previously described that these strains were able to form the O-antigen lipopolysaccharide (LPS) when they grow at low temperature but not at high temperature. We also obtained by transposon mutagenesis mutants only devoid of the O-antigen LPS (rfbmutants), and showed that they were unable to colonize the germfree chicken gut. All these results prompted us to conclude that the O-antigen LPS, in these strains, is a main factor for colonization in this animal model system.     

Studies on the association of BF1/BF2 gene expression patterns with traits of genetic resistance to Marek's disease in chickens

The aim was try to clarify the association of BF1 and BF2 gene expression patterns with the traits of genetic resistance to Marek's disease (MD) in chickens. An MD-resistant line and a common line of Xiayan chickens were used in the experiment. The productions of mRNA of BF1 and BF2 and Marek's disease virus (MDV) gene, meq, were quantified by quantitative real-time PCR on different days post-infection (dpi) with virulent MDV in the peripheral blood leukocytes (PBLs). Results indicated that the MDV loads were lower in the MD-resistant line compared to the common line. The mRNA levels of BF1 were significantly lower and levels of BF2 higher in the MD-resistant line whereas levels of BF1 were significantly higher and levels of BF2 lower in the common line. The results demonstrated that the expression modes of BF1 and BF2 genes might be related to traits of genetic resistance to MD in the chicken.     

Functional annotation of the T‐cell immunoglobulin mucin family in birds

T‐cell immunoglobulin and mucin (TIM) family molecules are cell membrane proteins, preferentially expressed on various immune cells and implicated in recognition and clearance of apoptotic cells. Little is known of their function outside human and mouse, and nothing outside mammals. We identified only two TIM genes (chTIM) in the chicken genome, putative orthologues of mammalian TIM1 and TIM4, and cloned the respective cDNAs. Like mammalian TIM1, chTIM1 expression was restricted to lymphoid tissues and immune cells. The gene chTIM4 encodes at least five splice variants with distinct expression profiles that also varied between strains of chicken. Expression of chTIM4 was detected in myeloid antigen‐presenting cells, and in γδ T cells, whereas mammalian TIM4 is not expressed in T cells. Like the mammalian proteins, chTIM1 and chTIM4 fusion proteins bind to phosphatidylserine, and are thereby implicated in recognition of apoptotic cells. The chTIM4–immunoglobulin fusion protein also had co‐stimulatory activity on chicken T cells, suggesting a function in antigen presentation.     

Validation of a quantitative Eimeria spp. PCR for fresh droppings of broiler chickens

A quantitative Polymerase Chain Reaction (qPCR) for the seven chicken Eimeria spp. was modified and validated for direct use on fresh droppings. The analytical specificity of the qPCR on droppings was 100%. Its analytical sensitivity (non-sporulated oocysts/g droppings) was 41 for E. acervulina, ≤2900 for E. brunetti, 710 for E. praecox, 1500 for E. necatrix, 190 for E. tenella, 640 for E. maxima, and 1100 for E. mitis. Field validation of the qPCR was done using droppings with non-sporulated oocysts from 19 broiler flocks. To reduce the number of qPCR tests five grams of each pooled sample (consisting of ten fresh droppings) per time point were blended into one mixed sample. Comparison of the oocysts per gram (OPG)-counting method with the qPCR using pooled samples (n?=?1180) yielded a Pearson’s correlation coefficient of 0.78 (95% CI: 0.76–0.80) and a Pearson’s correlation coefficient of 0.76 (95% CI: 0.70–0.81) using mixed samples (n?=?236). Comparison of the average of the OPG-counts of the five pooled samples with the mixed sample per time point (n?=?236) showed a Pearson’s correlation coefficient (R) of 0.94 (95% CI: 0.92–0.95) for the OPG-counting method and 0.87 (95% CI: 0.84–0.90) for the qPCR. This indicates that mixed samples are practically equivalent to the mean of five pooled samples. The good correlation between the OPG-counting method and the qPCR was further confirmed by the visual agreement between the total oocyst/g shedding patterns measured with both techniques in the 19 broiler flocks using the mixed samples.     

Generation of antibody and development of an enzyme-linked immunosorbant assay for the feed additive roxarsone

Roxarsone is used in the poultry and swine industries as a feed additive to treat coccidiosis and other intestinal disorders as well as to improve feed efficiencies and weight gain. 3-Amino-4-hydroxyphenylarsonic acid (AHPA) was conjugated with keyhole limpet hemocyanin in the presence of bis-sulfosuccinimidyl-suberate for the immunogen in rabbits. The antibody has minor cross-reactivity towards 2-aminophenylarsonic acid (7.3%, n=3), arsanilic acid (5.3%, n=3), AHPA (4%, n=3), and phenylarsonic acid (3%, n=3). The mean IC₅₀s for PBST, muscle 1:10, and muscle 1:20 were 11.7±1.50, 13.1±2.53 and 12.0±2.45 ng/mL (n=6). Using a standard curve prepared in chicken muscle extract and diluted with PBST at 1:20, muscle samples spiked with roxarsone at 2.5, 5, 10 and 25 ng/mL had recoveries of 117%, 98%, 80% and 65% having coefficients of variation of 36, 9.2, 10.2 and 11.5 respectively (n=6).     

The complete DNA sequence of the mitochondrial genome of the dermatophyte fungus Epidermophyton floccosum

We report here the complete nucleotide sequence of the 30.9-kb mitochondrial genome of the dermatophyte fungus Epidermophyton floccosum. All genes are encoded on the same DNA strand and include seven subunits of the reduced nicotinamide adenine dinucleotide ubiquinone oxireductase (nad1, nad2, nad3, nad4, nad4L, nad5, and nad6), three subunits of cytochrome oxidase (cox1, cox2, and cox3), apocytochrome b (cob), three subunits of ATP synthase (atp6, atp8, and atp9), the small and large ribosomal RNAs (rns and rnl), and 25 tRNAs. A ribosomal protein gene (rps5) is present as an intronic ORF in the large ribosomal subunit. The genes coding for cob and cox1 carry one intron and nad5 carries two introns with ORFs. The mtDNA of E. floccosum has the same gene order as Trichophyton rubrum mtDNA, with the exception of some tRNA genes. Maximum likelihood phylogenetic analysis confirms T. rubrum as a close relative of E. floccosum. This is the first complete mitochondrial sequence of a species of the order Onygenales. This sequence is available under GenBank accession number AY916130.     

Molecular and cytogenetic organization of the 5S ribosomal DNA array in chicken (Gallus gallus)

The 5S ribosomal (r) RNA genes encode a small (120-bp) highly-conserved component of the large ribosomal subunit. The objective of the present research was to study the molecular and cytogenetic organization of the chicken 5S rDNA. A predominant 2.2-kb gene (5S) consisting of a coding and intergenic spacer (IGS) region was identified in ten research and commercial populations. A variant gene repeat of 0.6kb (5S) was observed in some of the populations. Genetic linkage analysis and cytogenetic localization by fluorescence in-situ hybridization assigned the 5S rDNA to chromosome 9. The 5S rDNA array was determined to be 80.2±7.0kb upon electrophoretic sizing following EcoRV digestion. Sequence analysis of 5S IGS regions revealed considerable conservation between chicken subspecies (98.4% identity) as well as homology with vertebrate Pol III promoter and regulatory sequence motifs. Minor intraindividual sequence variation within 1000bp of IGS was observed in four cloned Red Jungle Fowl (Gallus gallus gallus) 5S repeats (95.5% identity in this region). Sequence comparisons between IGS regions of 5S and 5S genes indicated two short continuous (>20bp) and many short non-continuous homologous regions as well as other conserved features such as promoter and termination motifs.     

Ameliorative Effect of Grape Seed Proanthocyanidin Extract on Cadmium‐Induced Meiosis Inhibition During Oogenesis in Chicken Embryos

Cadmium (Cd) is an environmental endocrine disruptor that has toxic effects on the female reproductive system. Here the ameliorative effect of grape seed proanthocyanidin extract (GSPE) on Cd‐induced meiosis inhibition during oogenesis was explored. As compared with controls, chicken embryos exposed to Cd (3 µg/egg) displayed a changed oocyte morphology, decreased number of meiotic germ cells, and decreased expression of the meiotic marker protein γH2AX. Real time RT‐PCR also revealed a significant down‐regulation in the mRNA expressions of various meiosis‐specific markers (Stra8, Spo11, Scp3, and Dmc1) together with those of Raldh2, a retinoic acid (RA) synthetase, and of the receptors (RARα and RARβ). In addition, exposure to Cd increased the production of H₂O₂ and malondialdehyde in the ovaries and caused a corresponding reduction in glutathione and superoxide dismutase. Simultaneous supplementation of GSPE (150 µg/egg) markedly alleviated the aforementioned Cd‐induced embryotoxic effects by upregulating meiosis‐related proteins and gene expressions and restoring the antioxidative level. Collectively, the findings provided novel insights into the underlying mechanism of Cd‐induced meiosis inhibition and indicated that GSPE might potentially ameliorate related reproductive disorders. Anat Rec, 299:450–460, 2016. © 2016 Wiley Periodicals, Inc.     

Non-invasive imaging of optical parameters of biological tissues

The normalised back-scattered intensity (NBI) profiles at various locations on the forearms of ten human subjects were obtained by moving the multi-probe of a laser reflectometer. The statistical analysis of the NBI data showed that the variation in the NBI was significantly higher at the ulnar region compared with that at other regions. For determination of the scattering (μ _s ) and absorption (μ _a ) coefficients and the anisotropy parameter g at each location on the forearm, these profiles were matched with the NBI profiles simulated by a Monte Carlo procedure (χ _0.99 ² ). For the reconstruction of images of variation of these parameters, the averaged values ofμ _a ,μ _s and g at all locations on the forearms of the subjects were determined. The absorption coefficient had a minimum (1.92 cm⁻¹) and maximum (2.21 cm⁻¹) at the wrist and the lateral region of the forearm, respectively. The scattering coefficient had a maximum (194 cm⁻¹) at the medial side and near the elbow, and a minimum (186 cm⁻¹) at the lateral side of the forearm. Similar changes in the anisotropy parameter were also observed. By interpolation of the data of each parameter on a 100×100 image matrix and after median filtering, colour-coded images of the variation in the optical parameters were constructed. These images could be useful for diagnostic and therapeutic applications of lasers.     

Age dependence of skin autofluorescence

The nontryptophane intrinsic fluorescence of finger pad skin was studied in men aged 20–70 years in order to evaluate the possibility of using this parameter as a biomarker of aging. Linear correlation coefficients (r) between the fluorescence intensity and age varied from 0.50 to 0.66 for various skin sites. Age dependence of the mean value of fluorescence (F) measured on 4 fingers can be approximated by a second-degree polynomial:F=2.82−0.083T+0.0014T ² (r ₁=0.64,r ₂=0.71), whereT is chronological age in years. The proposed measurement of skin autofluorescence is a simple, noninvasive, rapid test for evaluating the aging of the skin in subjects over 40 with a high age-related determination (r ²=0.5). Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 127, No. 3, pp. 351–353, March, 1999     

Seven new microsporidian parasites of springtails (Collembola) in the Federal Republic of Germany

Three new species ofNosema (N. lepidocyrti, N. onychiurus andN. petrosa), oneEncephalitozoon (E. flavescens) two species ofThelohania (T. bomboschi andT. collembolae) and a new genusAuraspora n.g. withA. canningae were described from Collembola in soil samples of Lower Saxony, Federal Republic of Germany.

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Sieben neue Mikrosporidien-Arten der Springschwänze (Collembola) aus der Bundesrepublik Deutschland

Zusammenfassung Obwohl die Springschwänze (Collembola) als wichtige Streuzersetzer seit langem Gegenstand bodenbiologischer Forschung sind, gab es bisher keine Hinweise auf das Vorkommen pathogener Protozoen als Parasiten dieser Insekten. Im Rahmen einer vergleichenden Bodentieruntersuchung in verschiedenen Waldstandorten Niedersachsens wurden 1320 Collembolen auf Parasitierung durch pathogene Protozoen geprüft. Dabei konnten erstmals Mikrosporidien (Microsporida, Protozoa) als Krankheitserreger von Collembolen festgestellt werden. Sie werden neu beschrieben:Nosema lepidocyrti sp.n. inLepidocyrtus lignorum Tullberg (Tomoceridae),Nosema onychiurus sp.n. inOnychiurus quadriocellatus Gisin (Onychiuridae),Nosema petrosa sp.n. inLepidocyrtus cyaneus Tullberg (Tomoceridae),Thelohania bomboschi sp.n. inTomocerus flavescens Tullberg (Tomoceridae),Thelohania collembolae sp.n. inL. lignorum undT. flavescens, Encephalitozoon flavescens sp.n. inT. flavescens.Eine Art ist keinem bekannten Genus zuzuordnen:Auraspora gen.n.canningae sp.n. inL. lignorum.Der nachgewiesene Mikrosporidienbefall betrug beiO. quadriocellatus 3% (n=220),L. lignorum 7% (n=130),T. flavescens 8%(n=193) andL. cyaneus 6% (n=63).

     

Noninvasive assessment of cardiac contractility by using (dP/dt)/P of carotid artery pulses during exercise

In earlier studies we have shown that both the pressure (P) of the carotid artery pulse (CAP) and its first derivative (CAP dP/dt) could be recorded during moderate exercise. To establish that the CAP (dP/dt)/P is a noninvasive substitute for the left ventricular (LV) value, LV (dP/dt)/P, an index of cardiac contractility, we studied CAP (dP/dt)/P under various states of activity in the autonomic nervous system in 12 healthy male subjects. Increased sympathetic nerve activities yielded by passive tilting, emotional load, or cold stress increased CAP (dP/dt)/P significantly (P< 0.05). Increased parasympathetic nerve activity by ocular compression, however, did not significantly affect the value. Moderate exercise at a heart rate of approximately 150 beats·min^–1 increased it significantly from 16.7 to 25.2·s^–1 in a supine position (P<0.001) and from 16.6 to 24.8·s^–1 in an upright position (P<0.001). It increased monotonically as heart rate increased, but the slope was steeper when the heart rate was greater than approximately 100 beats·min^–1 than it was when the rate was less than 100 beats·min^–1. In conclusion, the present study indicated that CAP (dP/dt)/P can be used as a noninvasive index of cardiac contractility even in moderate exercise.     

Cooperative effect of two amino acid mutations in the coat protein of Pepper mild mottle virus overcomes L3-mediated resistance in Capsicum plants

We found that an L ³ resistance-breaking field isolate of Pepper mild mottle virus (PMMoV), designated PMMoV-Is, had two amino acid changes in its coat protein (CP), namely leucine to phenylalanine at position 13 (L13F) and glycine to valine at position 66 (G66V), as compared with PMMoV-J, which induces a resistance response in L ³ -harboring Capsicum plants. The mutations were located to a CP domain corresponding to the outer surface of PMMoV particles in computational molecular modeling. Analyses of PMMoV CP mutants containing either or both of these amino acid changes revealed that both changes were required to efficiently overcome L ³ -mediated resistance with systemic necrosis induction. Although CP mutants containing either L13F or G66V could not efficiently overcome L ³ -mediated resistance, these amino acid changes had different effects on the elicitor activity of PMMoV CP. L13F caused a slight reduction in the elicitor activity, resulting in virus restriction to necrotic local lesions that were apparently larger than those induced by wild-type PMMoV, while G66V rendered wild-type PMMoV the ability to overcome L ³ -mediated resistance, albeit with a lower efficiency than PMMoV with both changes. These results suggest that a cooperative effect of the L13F and G66V mutations on the elicitor activity of CP is responsible for overcoming the L ³ -mediated resistance.     

Embryonic expression of the transforming growth factor beta ligand and receptor genes in chicken

Results: Here we report the embryonic mRNA expression patterns in chicken embryos of the canonical TGFβ ligands (TGFB1, TGFB2, and TGFB3) and receptors (TGFBR1, TGFBR2, TGFBR3), plus the Activin A receptor, type 1 (ACVR1) and co receptor Endoglin (ENG) that also transduce TGFβ signaling. 相似文献   

Real-time multiplex PCR for simultaneous detection of Campylobacter jejuni, Salmonella, Shigella and Yersinia species in fecal samples

Diarrheal diseases due to notifiable bacterial infections require rapid diagnosis of the causative pathogens. To facilitate detection, a real-time multiplex PCR was developed that identifies common diarrhea-causing bacteria in fecal samples. On the basis of published sequence data, sets of primers and probes were designed that were specific for Campylobacter jejuni, Salmonella, Shigella/enteroinvasive Escherichia coli EIEC, and Yersinia species, suitable for use in a one-tube PCR assay. The assay was assessed using a list of 137 well-defined intestinal bacterial strains or isolates. Furthermore, 393 routine clinical stool samples were analyzed, and the results of real-time multiplex PCR were compared with those obtained by established microbiological methods. The PCR yielded results within 3 h including DNA purification. No false-positive signals or cross-reactions were observed. The analytical sensitivity was 10³ cfu mL⁻¹ for Campylobacter jejuni, 10⁴ cfu mL⁻¹ for Salmonella, and 10⁵ cfu mL⁻¹ for Shigella/EIEC and Yersinia, respectively. Compared with culture, PCR detected 79 out of 81 Campylobacter jejuni (97.5%), 71 out of 74 Salmonella (96%), 8 out of 8 Shigella (100%), and 10 out of 10 Yersinia-positive (100%) clinical samples. In culture-negative samples (n = 192), PCR additionally detected 2 Shigella, 1 Salmonella, and 5 Campylobacter jejuni infections. Thus, the new real-time multiplex PCR provides reliable results within a short time and might be useful as an additional diagnostic tool whenever time is important in the diagnosis of enteropathogenic bacteria.     

Partially Inverted Tandem Repeat Isolated from Pericentric Region of Chicken Chromosome 8

The majority of chicken repetitive sequence is nuclear-membrane-associated sequence (CNM), which resides in a large number of microchromosomes (chromosomes 11–39) and is absent from macrochromosomes 1–5, ZW, and some of the intermediate chromosomes 6–10. Two repetitive families, EcoRI/XhoI, are confined to the female-specific W chromosome. The core repeat units of the three families are 21 bp, containing (A)_3–5 and (T)_3–5 clusters separated by 5–7-bp sequences. In this article, we describe the isolation and initial characterization of a novel repeat family that is related to CNM/EcoRI/XhoI families. The novel family, designated as PIR, consists of multiple types of partially inverted repeat units of about 1.2, 1.4 and 1.6 kb. The PIR sequence is restricted to chicken chromosome 8, and accounts for about 3.8 mb, or 2500 copies of the 1.4-kb units, of the chicken genome. The evolution of PIR and related sequences is discussed.     

Prevalence of resistance phenotypes and genotypes to macrolide, lincosamide and streptogramin antibiotics in Gram-positive cocci isolated in Tunisian Bone Marrow Transplant Center

To investigate the prevalence of resistance to macrolide, lincosamide and streptogramin (MLS) antibiotics in Gram-positive cocci isolated in a Bone Marrow Transplant Center of Tunisia, we tested the antibiotic susceptibility of 172 clinical isolates of Staphylococcus epidermidis, Streptococcus mitis and Enterococcus faecium to macrolide erythromycin and spiramycin, the lincosamide clindamycin and the streptogramin pristinamycin. These three groups of organisms were mostly resistant to macrolides and lincosamide, but were commonly susceptible to pristinamycin. The resistance phenotypes of erythromycin-resistant isolates were determined by the five-disc test with erythromycin, spiramycin, lincomycin, clindamycin and pristinamycin, which showed that most exhibited constitutive MLS resistance. In order to determine the prevalence of the resistance genotypes and the resistance mechanisms, the prevalence of the erythromycin resistance methylase (erm) (A), erm(B), erm(C), msr(A) and macrolide efflux (mef) (A) genes in the erythromycin-resistant isolates was identified by polymerase chain reaction (PCR) analysis. The resistance was due mainly to the presence of ermB in E. faecium (80%), ermC in S. epidermidis (53%) and mefA in S. mitis (65%).