Immunocytochemical evidence for the presence of enzymes synthesizing GABA and GHB in the same neuron

A specific and sensitive immunocytochemical double staining for visualization of glutamate decarboxylase (GAD) and semialdehyde succinate reductase (SSR₂) in the same brain section has been developed. SSR₂ is the enzyme responsible for the transformation of succinic semialdehyde into γ-hydroxybutyrate (GHB). GAD was detected using specific rabbit GAD-antibodies and unlabeled antibody enzyme peroxidase antiperoxidase, and SSR₂ using specific guinea-pig SSR₂ antibodies conjugate to a fluorescein-labeled second antibody. The coexistence of GAD and SSR₂ in the same neuron was demonstrated by a peroxidase reaction superimposed on fluorescent compounds. Cell bodies containing both antigens were observed in the cerebellum, dorso-median hypothalamus and raphe nuclei. GHB is present in most GABA containing neurons. Some neurons contain only SSR₂; these neurons may synthesize GHB by an active uptake of GABA.     

PKA and Epac cooperate to augment bradykinin-induced interleukin-8 release from human airway smooth muscle cells

Background

Platelet-derived growth factor A (PDGF-A) signals solely through PDGF-Rα, and is required for fibroblast proliferation and transdifferentiation (fibroblast to myofibroblast conversion) during alveolar development, because pdgfa-null mice lack both myofibroblasts and alveoli. However, these PDGF-A-mediated mechanisms remain incompletely defined. At postnatal days 4 and 12 (P4 and P12), using mouse lung fibroblasts, we examined (a) how PDGF-Rα correlates with ki67 (proliferation marker) or alpha-smooth muscle actin (αSMA, myofibroblast marker) expression, and (b) whether PDGF-A directly affects αSMA or modifies stimulation by transforming growth factor beta (TGFβ).

Methods

Using flow cytometry we examined PDGF-Rα, αSMA and Ki67 in mice which express green fluorescent protein (GFP) as a marker for PDGF-Rα expression. Using real-time RT-PCR we quantified αSMA mRNA in cultured Mlg neonatal mouse lung fibroblasts after treatment with PDGF-A, and/or TGFβ.

Results

The intensity of GFP-fluorescence enabled us to distinguish three groups of fibroblasts which exhibited absent, lower, or higher levels of PDGF-Rα. At P4, more of the higher than lower PDGF-Rα + fibroblasts contained Ki67 (Ki67+), and Ki67+ fibroblasts predominated in the αSMA + but not the αSMA- population. By P12, Ki67+ fibroblasts comprised a minority in both the PDGF-Rα + and αSMA+ populations. At P4, most Ki67+ fibroblasts were PDGF-Rα + and αSMA- whereas at P12, most Ki67+ fibroblasts were PDGF-Rα- and αSMA-. More of the PDGF-Rα + than - fibroblasts contained αSMA at both P4 and P12. In the lung, proximate αSMA was more abundant around nuclei in cells expressing high than low levels of PDGF-Rα at both P4 and P12. Nuclear SMAD 2/3 declined from P4 to P12 in PDGF-Rα-, but not in PDGF-Rα + cells. In Mlg fibroblasts, αSMA mRNA increased after exposure to TGFβ, but declined after treatment with PDGF-A.

Conclusion

During both septal eruption (P4) and elongation (P12), alveolar PDGF-Rα may enhance the propensity of fibroblasts to transdifferentiate rather than directly stimulate αSMA, which preferentially localizes to non-proliferating fibroblasts. In accordance, PDGF-Rα more dominantly influences fibroblast proliferation at P4 than at P12. In the lung, TGFβ may overshadow the antagonistic effects of PDGF-A/PDGF-Rα signaling, enhancing αSMA-abundance in PDGF-Rα-expressing fibroblasts.     

Paternal Leakage and Heteroplasmy of Mitochondrial Genomes in Silene vulgaris: Evidence From Experimental Crosses

The inheritance of mitochondrial genetic (mtDNA) markers in the gynodioecious plant Silene vulgaris was studied using a series of controlled crosses between parents of known mtDNA genotype followed by quantitative PCR assays of offspring genotype. Overall, ∼2.5% of offspring derived from crosses between individuals that were homoplasmic for different mtDNA marker genotypes showed evidence of paternal leakage. When the source population of the pollen donor was considered, however, population-specific rates of leakage varied significantly around this value, ranging from 10.3% to zero. When leakage did occur, the paternal contribution ranged from 0.5% in some offspring (i.e., biparental inheritance resulting in a low level of heteroplasmy) to 100% in others. Crosses between mothers known to be heteroplasmic for one of the markers and homoplasmic fathers showed that once heteroplasmy enters a maternal lineage it is retained by ∼17% of offspring in the next generation, but lost from the others. The results are discussed with regard to previous studies of heteroplasmy in open-pollinated natural populations of S. vulgaris and with regard to the potential impact of mitochondrial paternal leakage and heteroplasmy on both the evolution of the mitochondrial genome and the evolution of gynodioecy.MATERNAL inheritance of the mitochondrial genome seems to be the usual case in angiosperms, with only occasional reports of paternal leakage (Birky 2001). The mode of inheritance has several interesting consequences for the evolution of the plant mitochondrial genome and plant mating systems. One is that maternal inheritance contributes to homoplasmy, or within-individual genetic homogeneity, in that it precludes the mixing of mitochondrial genomes of differing origin at the time of fertilization. Homoplasmy is further enforced by repeated sampling events associated with the transmission of a finite number of mitochondria from mother to daughter cells during mitotic or meiotic events (Birky 2001). This within-individual genetic drift is sometimes known as vegetative sorting (McCauley and Olson 2008). Paternal leakage would allow the possibility of mitochondrial heteroplasmy (within-individual cytoplasmic genetic diversity) when it leads to some degree of biparental inheritance. With homoplasmy the mitochondrial genome evolves as an effectively asexual lineage. While intra- or intermolecular recombination associated with repeat sequences often found in noncoding regions of plant mitochondrial genomes can result in structural rearrangements (Mackenzie and McIntosh 1999), there is limited opportunity for such events to generate novel genotypic combinations. Heteroplasmy enhances the possibility that recombination can occur between divergent genomes and generate novel genotypes.A second consequence of the mode of inheritance concerns the evolution of gynodioecy or the co-occurrence of female and hermaphrodite individuals. This phenomenon is often ascribed to the interaction between mitochondrial genes that confer cytoplasmic male sterility (CMS) and nuclear genes, known as restorers, that counteract the effects of CMS and restore male function (Frank 1989), a topic that continues to be the object of much study by plant evolutionary biologists (McCauley and Bailey 2009). The evolutionary dynamics of these interactions are usually evaluated on the basis of the assumption of pure maternal inheritance of mitochondrial genes. This maximizes the potential for genetic conflict between a CMS gene and its restorers, since a difference in the mode of inheritance between the mitochondrial and nuclear genomes results in a difference in their respective currency of fitness. With paternal leakage, pollen production is no longer unimportant for the fitness of the mitochondrial genes carried by a hermaphrodite (Wade and McCauley 2005).Recently there has been increased appreciation of the potential role of paternal leakage and heteroplasmy in the evolution of the mitochondrial genomes of a broad array of eukaryotes (Kmiec et al. 2006; White et al. 2008). This includes studies of the plant genus Silene, which have provided evidence of at least occasional paternal transmission of mitochondria in several species, as well as mitochondrial heteroplasmy. Observations supporting the possibility of mitochondrial paternal leakage and heteroplasmy in the genus Silene are especially intriguing given the occurrence of gynodioecy in this genus. Evidence of paternal leakage comes primarily from two types of observation. First are observations of mitochondrial genotypes that most likely result from intra- or intergenic recombination (see studies by Städler and Delph 2002 for S. acaulis and McCauley et al. 2005; Houliston and Olson 2006; and McCauley and Ellis 2008 for S. vulgaris). Second, direct evidence of heteroplasmy in S. vulgaris comes from studies that utilize real time quantitative PCR (q-PCR) to quantify the within-individual diversity of mitochondrial marker genes (Welch et al. 2006; Pearl et al. 2009). The likelihood that heteroplasmy is due to paternal leakage in S. vulgaris was inferred from observations by Pearl et al. (2009) of heteroplasmic offspring of open-pollinated homoplasmic mothers. A second observation by Pearl et al. (2009) bears on the inheritance of heteroplasmy. Heteroplasmic mothers were more likely than homoplasmic mothers to produce heteroplasmic offspring, but this heteroplasmy was also lost between generations in many cases, in keeping with the theory of vegetative sorting.One interesting result from Welch et al. (2006) and Pearl et al. (2009) is that incidents of heteroplasmy and apparent leakage do not seem to be evenly distributed among the natural populations from which samples were taken. Most of the heteroplasmic individuals documented by Welch et al. (2006) were from just one of the three populations studied. Similarly, while the apparent leakage rate observed by Pearl et al. (2009) was ∼8% when all 14 study populations are considered together, if the rate is calculated on a population-by-population basis, it exceeds 10% in 3 of them and is zero in 3 others (see their Supplementary Table 2). Population-to-population variation in the rate of leakage might suggest that variable environmental conditions influence leakage or that any genetic variation that influences the traits that determine mode of inheritance is geographically structured.Much of the current evidence for mitochondrial paternal leakage in Silene is indirect in that it is derived from observations of apparent recombinant genotypes or of heteroplasmy. While this evidence is compelling, alternate explanations, such as mutational hotspots within the genes under study, are at least possible. Even the evidence of leakage presented by Pearl et al. (2009) was based on mother–offspring comparisons of individuals collected from natural populations, in which the pollen donor was unknown. Though some evidence for paternal leakage and heteroplasmy reported in McCauley et al. (2005) comes from controlled crosses of S. vulgaris, those crosses were few in number and any incidents of heteroplasmy were based on qualitative observations rather than the q-PCR method used more recently. Thus, it would be valuable to conduct a large number of controlled crosses between S. vulgaris individuals of known mitochondrial genotype to assay directly the rate and magnitude of paternal leakage and any resulting heteroplasmy and also to assay the degree to which heteroplasmy is transmitted between generations. Taken together, this information would allow one to begin to ask, not only about the origins of mitochondrial heteroplasmy in Silene, but also about the degree to which the frequency of mitochondrial heteroplasmy in natural populations results from gains through paternal leakage vs. loss from vegetative sorting. Furthermore, since the among-population heterogeneity in levels of heteroplasmy and leakage summarized above could be due to either real differences between populations in factors promoting these phenomena or simply ascertainment bias associated with differences between populations in the level of polymorphism of the q-PCR markers, it would be valuable to test for a population effect in an experimental setting.Here we present comparisons of parent and offspring mitochondrial genotypes obtained by q-PCR following three types of controlled crosses in which either (1) the two parents are homoplasmic for different alleles of a marker gene, (2) both parents are homoplasmic for the same allele, or (3) the maternal parent is heteroplasmic. In the first cross type any contribution of the pollen donor to the offspring mitochondrial genotype would be detectable. This quantifies the likelihood of leakage. Knowing the natural population from which the pollen donor and pollen recipient trace their respective ancestry allows investigation of the possibility of a population effect without the confounding effects of varying levels of marker polymorphism present in field studies. In the second cross type, any observed mother–offspring difference would most likely be due to error of some sort (or the unlikely possibility of mutation at the SNP that defines the marker). Thus, these crosses act as a control by estimating the experimental error rate. The third type of cross measures the frequency with which heteroplasmy is transmitted maternally to offspring or is lost. Taken together this study represents what is, to our knowledge, the first attempt to combine experimental crosses and q-PCR methodology to examine mitochondrial genome inheritance and heteroplasmy in a plant species; important information given that it is not yet clear how widespread mitochondrial leakage and heteroplasmy are in the genus Silene, in other gynodioecious species, or in other species of plants in general.     

The Nicotinic Acetylcholine Receptor and the Na,K-ATPase α2 Isoform Interact to Regulate Membrane Electrogenesis in Skeletal Muscle

The nicotinic acetylcholine receptor (nAChR) and the Na,K-ATPase functionally interact in skeletal muscle (Krivoi, I. I., Drabkina, T. M., Kravtsova, V. V., Vasiliev, A. N., Eaton, M. J., Skatchkov, S. N., and Mandel, F. (2006) Pflugers Arch. 452, 756–765; Krivoi, I., Vasiliev, A., Kravtsova, V., Dobretsov, M., and Mandel, F. (2003) Ann. N.Y. Acad. Sci. 986, 639–641). In this interaction, the specific binding of nanomolar concentrations of nicotinic agonists to the nAChR stimulates electrogenic transport by the Na,K-ATPase α2 isozyme, causing membrane hyperpolarization. This study examines the molecular nature and membrane localization of this interaction. Stimulation of Na,K-ATPase activity by the nAChR does not require ion flow through open nAChRs. It can be induced by nAChR desensitization alone, in the absence of nicotinic agonist, and saturates when the nAChR is fully desensitized. It is enhanced by noncompetitive blockers of the nAChR (proadifen, QX-222), which promote non-conducting or desensitized states; and retarded by tetracaine, which stabilizes the resting nAChR conformation. The interaction operates at the neuromuscular junction as well as on extrajunctional sarcolemma. The Na,K-ATPase α2 isozyme is enriched at the postsynaptic neuromuscular junction and co-localizes with nAChRs. The nAChR and Na,K-ATPase α subunits specifically coimmunoprecipitate with each other, phospholemman, and caveolin-3. In a purified membrane preparation from Torpedo californica enriched in nAChRs and the Na,K-ATPase, a ouabain-induced conformational change of the Na,K-ATPase enhances a conformational transition of the nAChR to a desensitized state. These results suggest a mechanism by which the nAChR in a desensitized state with high apparent affinity for agonist interacts with the Na,K-ATPase to stimulate active transport. The interaction utilizes a membrane-delimited complex involving protein-protein interactions, either directly or through additional protein partners. This interaction is expected to enhance neuromuscular transmission and muscle excitation.     

Effect of abrupt weaning at housing on leukocyte distribution,functional activity of neutrophils,and acute phase protein response of beef calves

Background  

Sixteen, spring-born, single suckled, castrated male calves of Limousin × Holstein-Friesian and Simmental × Holstein-Friesian dams respectively, were used to investigate the effect of weaning on total leukocyte and differential counts, neutrophil functional activity, lymphocyte immunophenotypes, and acute phase protein response. Calves grazed with their dams until the end of the grazing season when they were housed in a slatted floor shed. On the day of housing, calves were assigned to a treatment, (i) abruptly weaned (W: n = 8) or (ii) non-weaned (controls) (C: n = 8). Weaned calves were housed in pens without their dams, whereas non-weaned (control) calves were housed with their dams. Blood was collected on day -7, 0 (housing), 2, 7, and 14 to determine total leukocyte and differential counts and concentration of fibrinogen and haptoglobin. Lymphocyte immunophenotypes were characterised using selected surface antigens (CD4⁺, CD8⁺, WC1⁺ (γδ T cells), MHC Class II⁺ lymphocytes), and the functional activities of neutrophils (surface expression of L-selectin (CD62L), phagocytic and oxidative burst activity) were investigated using flow cytometry.     

Incision of trivalent chromium [Cr(III)]-induced DNA damage by Bacillus caldotenax UvrABC endonuclease

Some hexavalent chromium [Cr(VI)]-containing compounds are lung carcinogens. Once within cells, Cr(VI) is reduced to trivalent chromium [Cr(III)] which displays an affinity for both DNA bases and the phosphate backbone. A diverse array of genetic lesions is produced by Cr including Cr-DNA monoadducts, DNA interstrand crosslinks (ICLs), DNA-Cr-protein crosslinks (DPCs), abasic sites, DNA strand breaks and oxidized bases. Despite the large amount of information available on the genotoxicity of Cr, little is known regarding the molecular mechanisms involved in the removal of these lesions from damaged DNA. Recent work indicates that nucleotide excision repair (NER) is involved in the processing of Cr-DNA adducts in human and rodent cells. In order to better understand this process at the molecular level and begin to identify the Cr-DNA adducts processed by NER, the incision of CrCl(3) [Cr(III)]-damaged plasmid DNA was studied using a thermal-resistant UvrABC NER endonuclease from Bacillus caldotenax (Bca). Treatment of plasmid DNA with Cr(III) (as CrCl(3)) increased DNA binding as a function of dose. For example, at a Cr(III) concentration of 1 microM we observed approximately 2 Cr(III)-DNA adducts per plasmid. At this same concentration of Cr(III) we found that approximately 17% of the plasmid DNA contained ICLs ( approximately 0.2 ICLs/plasmid). When plasmid DNA treated with Cr(III) (1 microM) was incubated with Bca UvrABC we observed approximately 0.8 incisions/plasmid. The formation of endonuclease IV-sensitive abasic lesions or Fpg-sensitive oxidized DNA bases was not detected suggesting that the incision of Cr(III)-damaged plasmid DNA by UvrABC was not related to the generation of oxidized DNA damage. Taken together, our data suggest that a sub-fraction of Cr(III)-DNA adducts is recognized and processed by the prokaryotic NER machinery and that ICLs are not necessarily the sole lesions generated by Cr(III) that are substrates for NER.     

A DNA fragment from the human X chromosome short arm which detects a partially homologous sequence on the Y chromosomes long arm.

An X linked human DNA fragment (named DXS31 ) which detects partially homologous sequences on the Y chromosome has been isolated. Regional localisation of the two sex linked sequences was determined using a panel of rodent-human somatic cell hybrids. The X specific sequence is located at the tip of the short arm ( Xp22 .3-pter), i.e. within or close to the region which pairs with the Y chromosome short arm at meiosis. However the Y specific sequence is located in the heterochromatic region of the long arm ( Yq11 -qter) and lies outside from the pairing region. DNAs from several XX male subjects were probed with DXS31 and in all cases a double dose of the X linked fragment was found, and the Y specific fragment was absent. DXS31 detects in chimpanzee a male-female differential pattern identical to that found in man. However results obtained in a more distantly related species, the brown lemur, suggest that the sequences detected by DXS31 in this species might be autosomally coded. The features observed with these X-Y related sequences do not fit with that expected from current hypotheses of homology between the pairing regions of the two sex chromosomes, nor with the pattern observed with other X-Y homologous sequences recently characterized. Our results suggest also that the rule of conservation of X linkage in mammals might not apply to sequences present on the tip of the X chromosome short arm, in bearing with the controversial issue of steroid sulfatase localisation in mouse.     

Actin-like sequences are present on human X and Y chromosomes.

The human genome contains greater than 20 actin-related sequences, six of which at least are expressed as protein. We have shown by blot hybridization the presence of actin-like sequences on both the X and the Y chromosomes. These sequences can be detected in HindIII digests of genomic DNA, using as probe cDNA clones corresponding to human alpha skeletal actin or to a hamster (beta or gamma) cytoskeletal actin; they show more homology to the latter probe. The actin probes also detect a polymorphic DNA fragment showing autosomal inheritance with a frequency for the major allele of 0.55 in the population studied. The X-linked actin sequence has been assigned to a centromeric region between Xp11 and Xq11 by hybridization to DNAs from a panel of human-mouse hybrid cell lines, and thus lies outside the postulated region of homology between the X and Y chromosomes. The Y-linked actin sequence can serve as a marker to analyse anomalies of sex determination or of gametogenesis in man. It was found in all XY males studied but was absent from the genomic DNA of four unrelated 'XX male' subjects and two XX hermaphrodites. This shows that the region of chromosome Y which contains the actin sequence is not translocated onto the X chromosome (or onto autosomes) in these patients.