Analysis of gene order data supports vertical inheritance of the leukotoxin operon and genome rearrangements in the 5' flanking region in genus <Emphasis Type="Italic">Mannheimia</Emphasis>

Background  

The Mannheimia subclades belong to the same bacterial genus, but have taken divergent paths toward their distinct lifestyles. For example, M. haemolytica + M. glucosida are potential pathogens of the respiratory tract in the mammalian suborder Ruminantia, whereas M. ruminalis, the supposed sister group, lives as a commensal in the ovine rumen. We have tested the hypothesis that vertical inheritance of the leukotoxin (lktCABD) operon has occurred from the last common ancestor of genus Mannheimia to any ancestor of the diverging subclades by exploring gene order data.     

Differential pattern of spinal sympathetic outflow in response to stimulation of the caudal medullary raphe

In urethan-anesthetized cats, frequency domain analysis was used to explore the mechanisms of differential responses of inferior cardiac (CN), vertebral (VN), and renal (RN) sympathetic nerves to electrical stimulation of a discrete region of the medullary raphe (0-2 mm caudal to the obex). Raphe stimulation in baroreceptor-denervated cats at frequencies (7-12 Hz) that entrained the 10-Hz rhythm in nerve activity decreased CN and RN activities but increased VN activity. The reductions in CN and RN discharges were associated with decreased low-frequency (相似文献   

Recurrent hybridisation events between Primula vulgaris,P. veris and P. elatior (Primulaceae,Ericales) challenge the species boundaries: using molecular markers to re‐evaluate morphological identifications

Three Primula species, Primula vulgaris, P. veris and P. elatior, have been objects of fascination for gardeners and botanists over several centuries. The species are able to hybridise, and where they co‐occur, hybrids are commonly found. In Denmark, Møns Klint on the island of Møn and Købelev Skov on Lolland are examples of localities where all three species occur and where the hybrids P. × digenea, the hybrid between P. vulgaris and P. elatior, and P. × polyantha, the hybrid between P. veris and P. vulgaris, can also be found. To investigate relationships between the species and their hybrids, we sampled 168 specimens from 10 geographical locations and subjected them to genetic analysis. The samples were also identified based on morphological traits: primarily inflorescense structure, the size, shape, colour and markings of corolla and leaf basis, leaf blade texture and hairiness. After identifying species‐specific SNPs in the Internal Transcribed Spacer sequence, these were used to resolve species and hybrid boundaries and status through a cleaved amplified polymorphic sequence assay. Polymorphisms in the chloroplast trnL sequence were used as a high‐throughput marker and used to determine the maternal parent of hybrids. Ten simple sequence repeat markers were applied to obtain further insight into the genetic makeup of the accessions using structure and Introgress, providing information of genetic variability within and between populations. Our results indicated that backcrossing of P. × digenea hybrids with parental species has occurred, and that many of the P. × digenea sampled were later‐generation hybrids rather than F₁s. Analyses of P. × polyantha specimens show mostly the expected pattern for primary hybrids but indications of P. veris ancestry of a P. vulgaris plant was discovered. Our results further indicate that some of the specimens initially identified as P. elatior include P. vulgaris among their progenitors and thus challenge currently accepted species boundaries.     

A synthetic peptide homologous to functional domain of human IL-10 down-regulates expression of MHC class I and Transporter associated with Antigen Processing 1/2 in human melanoma cells

Tumor cells treated with IL-10 were shown to have decreased, but peptide-inducible expression of MHC class I, decreased sensitivity to MHC class I-restricted CTL, and increased NK sensitivity. These findings could be explained, at least partially, by a down-regulation of TAP1/TAP2 expression. In this study, IT9302, a nanomeric peptide (AYMTMKIRN), homologous to the C-terminal of the human IL-10 sequence, was demonstrated to mimic these previously described IL-10 effects on MHC class I-related molecules and functions. We observed a dose-dependent down-regulation of MHC class I at the cell surface of melanoma cells after 24-h treatment with IT9302. The IL-10 homologue peptide also caused a dose-dependent inhibition of the IFN-gamma-mediated surface induction of MHC class I in a melanoma cell line. We demonstrated, using Western blot and flow cytometry, that IT9302 inhibits the expression of TAP1 and TAP2 proteins, but not MHC class I H chain or low molecular protein molecules. Finally, peptide-treated melanoma cells were shown to be more sensitive to lysis by NK cells in a dose-dependent way. Taken together, these results demonstrate that a small synthetic peptide derived from IL-10 can mimic the Ag presentation-related effects mediated by this cytokine in human melanomas and increase tumor sensitivity to NK cells, which can be relevant in the designing of future strategies for cancer immune therapy.     

Limited repair of 8-hydroxy-7,8-dihydroguanine residues in human testicular cells

Oxidative damage in testicular DNA is associated with poor semen quality, reduced fertility and increased risk of stillbirths and birth defects. These DNA lesions are predominantly removed by base excision repair. Cellular extracts from human and rat testicular cells and three enriched populations of rat male germ cells (primary spermatocytes, round spermatids and elongating/elongated spermatids) all showed proficient excision/incision of 5-hydroxycytosine, thymine glycol and 2,6-diamino-4-hydroxy-5-formamidopyrimidine. DNA containing 8-oxo-7,8-dihydroguanine was excised poorly by human testicular cell extracts, although 8-oxoguanine-DNA glycosylase-1 (hOGG1) was present in human testicular cells, at levels that varied markedly between 13 individuals. This excision was as low as with human mononuclear blood cell extracts. The level of endonuclease III homologue-1 (NTH1), which excises oxidised pyrimidines, was higher in testicular than in somatic cells of both species. Cellular repair studies of lesions recognised by formamidopyrimidine-DNA glycosylase (Fpg) or endonuclease III (Nth) were assayed with alkaline elution and the Comet assay. Consistent with the enzymatic activities, human testicular cells showed poor removal of Fpg-sensitive lesions but efficient repair of Nth-sensitive lesions. Rat testicular cells efficiently repaired both Fpg- and Nth-sensitive lesions. In conclusion, human testicular cells have limited capacity to repair important oxidative DNA lesions, which could lead to impaired reproduction and de novo mutations.     

NMR and interval PLS as reliable methods for determination of cholesterol in rodent lipoprotein fractions

Risk of cardiovascular disease is related to cholesterol distribution in different lipoprotein fractions. Lipoproteins in rodent model studies can only reliably be measured by time- and plasma-consuming fractionation. An alternative method to measure cholesterol distribution in the lipoprotein fractions in rat plasma is presented in this paper. Plasma from two rat studies (n = 68) was used in determining the lipoprotein profile by an established ultracentrifugation method and proton nuclear magnetic resonance (NMR) spectra of replicate samples was obtained. From the ultracentrifugation reference data and the NMR spectra, an interval partial least-square (iPLS) regression model to predict the amount of cholesterol in the different lipoprotein fractions was developed. The relative errors of the prediction models were between 12 and 33% and had correlation coefficients (r) between 0.96 and 0.84. The models were tested with an independent test set giving prediction errors between 19 and 46% and r between 0.96 and 0.76. Prediction of High, Low and Very Low Density Lipoprotein (HDL, LDL and VLDL) and total cholesterol was conducted in a study where rats had been supplemented with two doses of air-dried apple-powder. No significant difference in LDL, VLDL and total cholesterol was observed between the groups. The high apple-powder (20%) group had significantly lower HDL cholesterol (11%, P = 0.0452) than the control group. It is concluded that the iPLS approach yielded excellent regression models and thus univocal established chemometric analysis of NMR spectra of rat plasma as a strong and efficient way to quantify lipoprotein fractions in rat studies.     

New insights into porcine-human synteny conservation

Eleven genes were mapped to the porcine genome with the aim of improving the human-porcine comparative gene map. Five of these genes were from regions of the human genome painted by porcine chromosomal probes; of these, two mapped to chromosomes not expected from the painting results. Among the six genes from human regions not painted by porcine chromosomal probes, three genes did not map where expected by the principle of parsimony. Several of the gene assignments indicate the existence of small regions of conserved synteny not detected by heterologous chromosome painting, especially in telomeric regions. We have also detected new rearrangements in gene order within the regions of correspondence between human Chromosome (HSA) 15 and porcine Chromosome (SSC) 1 as well as between HSA4 and SSC8. Received: 30 September 1998 / Accepted: 3 December 1998     

Patch Clamp on the Luminal Membrane of Exocrine Gland Acini from Frog Skin (Rana esculenta) Reveals the Presence of Cystic Fibrosis Transmembrane Conductance Regulator–like Cl? Channels Activated by Cyclic AMP

Chloride channels in the luminal membrane of exocrine gland acini from frog skin (Rana esculenta) constituted a single homogeneous population. In cell-attached patches, channels activated upon exposure to isoproterenol, forskolin, or dibutyryl-cAMP and isobutyl-1-methyl-xanthine rectified in the outward direction with a conductance of 10.0 ± 0.4 pS for outgoing currents. Channels in stimulated cells reversed at 0 mV applied potential, whereas channels in unstimulated cells reversed at depolarized potentials (28.1 ± 6.7 mV), indicating that Cl⁻ was above electrochemical equilibrium in unstimulated, but not in stimulated, cells. In excised inside-out patches with 25 mM Cl⁻ on the inside, activity of small (8-pS) linear Cl⁻-selective channels was dependent upon bath ATP (1.5 mM) and increased upon exposure to cAMP-dependent protein kinase. The channels displayed a single substate, located just below 2/3 of the full channel amplitude. Halide selectivity was identified as P_Br > P_I > P_Cl from the Goldman equation; however, the conductance sequence when either halide was permeating the channel was G_Cl > G_Br >> G_I. In inside-out patches, the channels were blocked reversibly by 5-nitro-2-(3-phenylpropylamino)benzoic acid, glibenclamide, and diphenylamine-2-carboxylic acid, whereas 4,4-diisothiocyanatostilbene-2,2-disulfonic acid blocked channel activity completely and irreversibly. Single-channel kinetics revealed one open state (mean lifetime = 158 ± 72 ms) and two closed states (lifetimes: 12 ± 4 and 224 ± 31 ms, respectively). Power density spectra had a double-Lorentzian form with corner frequencies 0.85 ± 0.11 and 27.9 ± 2.9 Hz, respectively. These channels are considered homologous to the cystic fibrosis transmembrane conductance regulator Cl⁻ channel, which has been localized to the submucosal skin glands in Xenopus by immunohistochemistry (Engelhardt, J.F., S.S. Smith, E. Allen, J.R. Yankaskas, D.C. Dawson, and J.M. Wilson. 1994. Am. J. Physiol. 267: C491–C500) and, when stimulated by cAMP-dependent phosphorylation, are suggested to function in chloride secretion.     

Effect of aluminum on cytoplasmic Ca2+ homeostasis in root hairs of Arabidopsis thaliana (L.)

Aluminum inhibition of root growth is a major world agricultural problem where the cause of toxicity has been linked to changes in cellular calcium homeostasis. Therefore, the effect of aluminum ions (Al) on changes in cytoplasmic free calcium concentration ([Ca²⁺]_c) was followed in root hairs of wild-type, Al-sensitive and Al-resistant mutants of Arabidopsis thaliana (L.) Heynh. Generally, Al exposure resulted in prolonged elevations in tip-localized [Ca²⁺]_c in both wild-type and Al-sensitive root hairs. However, these Al-induced increases in [Ca²⁺]_c were not tightly correlated with growth inhibition, occurring up to 15 min after Al had induced growth to stop. Also, in 32% of root hairs examined growth stopped without a detectable change in [Ca²⁺]_c. In contrast, Al-resistant mutants showed little growth inhibition in response to AlCl₃ exposure and in no case was a change in [Ca²⁺]_c observed. Of the other externally applied stresses tested (oxidative and mechanical stress), both were found to inhibit root hair growth, but only oxidative stress (H₂O₂, 10 μM) caused a prolonged rise in [Ca²⁺]_c similar to that induced by Al. Again this increase occurred after growth had been inhibited. The lack of a tight correlation between Al exposure, growth inhibition and altered [Ca²⁺]_c dynamics suggests that although exposure of root hairs to toxic levels of Al causes an alteration in cellular Ca²⁺ homeostasis, this may not be a required event for Al toxicity. The elevation in [Ca²⁺]_c induced by Al also strongly suggests that the phytotoxic action of Al in root hairs is not through blockage of Ca²⁺-permeable channels required for Ca²⁺ influx into the cytoplasm. Received: 24 October 1997 / Accepted: 6 March 1998