Cytogenetic studies in Eigenmannia virescens (Sternopygidae, Gymnotiformes) and new inferences on the origin of sex chromosomes in the Eigenmannia genus

Background

Previous studies suggested that multiple domestication events in South and South-East Asia (Yunnan and surrounding areas) and India have led to the genesis of modern domestic chickens. Ha Giang province is a northern Vietnamese region, where local chickens, such as the H'mong breed, and wild junglefowl coexist. The assumption was made that hybridisation between wild junglefowl and Ha Giang chickens may have occurred and led to the high genetic diversity previously observed. The objectives of this study were i) to clarify the genetic structure of the chicken population within the Ha Giang province and ii) to give evidence of admixture with G. gallus. A large survey of the molecular polymorphism for 18 microsatellite markers was conducted on 1082 chickens from 30 communes of the Ha Giang province (HG chickens). This dataset was combined with a previous dataset of Asian breeds, commercial lines and samples of Red junglefowl from Thailand and Vietnam (Ha Noï). Measurements of genetic diversity were estimated both within-population and between populations, and a step-by-step Bayesian approach was performed on the global data set.

Results

The highest value for expected heterozygosity (> 0.60) was found in HG chickens and in the wild junglefowl populations from Thailand. HG chickens exhibited the highest allelic richness (mean A = 2.9). No significant genetic subdivisions of the chicken population within the Ha Giang province were found. As compared to other breeds, HG chickens clustered with wild populations. Furthermore, the neighbornet tree and the Bayesian clustering analysis showed that chickens from 4 communes were closely related to the wild ones and showed an admixture pattern.

Conclusion

In the absence of any population structuring within the province, the H'mong chicken, identified from its black phenotype, shared a common gene pool with other chickens from the Ha Giang population. The large number of alleles shared exclusively between Ha Giang chickens and junglefowl, as well as the results of a Bayesian clustering analysis, suggest that gene flow has been taking place from junglefowl to Ha Giang chickens.     

Trade-off of energy metabolites as well as body color phenotypes for starvation and desiccation resistance in montane populations of Drosophila melanogaster

Storage of energy metabolites has been investigated in different sets of laboratory selected desiccation or starvation resistant lines but few studies have examined such changes in wild-caught populations of Drosophila melanogaster. In contrast to parallel selection of desiccation and starvation tolerance under laboratory selection experiments, opposite clines were observed in wild populations of D. melanogaster. If resistance to desiccation and starvation occurs in opposite directions under field conditions, we may expect a trade-off for energy metabolites but such correlated changes are largely unknown. We tested whether there is a trade-off for storage as well as actual utilization of carbohydrates (trehalose and glycogen), lipids and proteins in D. melanogaster populations collected from different altitudes (512-2500 m). For desiccation resistance, darker flies (> 50% body melanization) store more body water content and endure greater loss of water (higher dehydration tolerance) as compared to lighter flies (< 30% body melanization). Based on within population analysis, we found evidence for coadapted phenotypes i.e. darker flies store and actually utilize more carbohydrates to confer greater desiccation resistance. In contrast, higher starvation resistance in lighter flies is associated with storage and actual utilization of greater lipid amount. However, darker and lighter flies did not vary in the rate of utilization of carbohydrates under desiccation stress; and of lipids under starvation stress. Thus, we did not find support for the hypothesis that a lower rate of utilization of energy metabolites may contribute to greater stress resistance. Further, for increased desiccation resistance of darker flies, about two-third of total energy budget is provided by carbohydrates. By contrast, lighter flies derive about 66% of total energy content from lipids which sustain higher starvation tolerance. Our results support evolutionary trade-off for storage as well as utilization of energy metabolites for desiccation versus starvation resistance in D. melanogaster.     

Proper Restoration of Excitation-Contraction Coupling in the Dihydropyridine Receptor ��1-null Zebrafish Relaxed Is an Exclusive Function of the ��1a Subunit

The paralyzed zebrafish strain relaxed carries a null mutation for the skeletal muscle dihydropyridine receptor (DHPR) β_1a subunit. Lack of β_1a results in (i) reduced membrane expression of the pore forming DHPR α_1S subunit, (ii) elimination of α_1S charge movement, and (iii) impediment of arrangement of the DHPRs in groups of four (tetrads) opposing the ryanodine receptor (RyR1), a structural prerequisite for skeletal muscle-type excitation-contraction (EC) coupling. In this study we used relaxed larvae and isolated myotubes as expression systems to discriminate specific functions of β_1a from rather general functions of β isoforms. Zebrafish and mammalian β_1a subunits quantitatively restored α_1S triad targeting and charge movement as well as intracellular Ca²⁺ release, allowed arrangement of DHPRs in tetrads, and most strikingly recovered a fully motile phenotype in relaxed larvae. Interestingly, the cardiac/neuronal β_2a as the phylogenetically closest, and the ancestral housefly β_M as the most distant isoform to β_1a also completely recovered α_1S triad expression and charge movement. However, both revealed drastically impaired intracellular Ca²⁺ transients and very limited tetrad formation compared with β_1a. Consequently, larval motility was either only partially restored (β_2a-injected larvae) or not restored at all (β_M). Thus, our results indicate that triad expression and facilitation of 1,4-dihydropyridine receptor (DHPR) charge movement are common features of all tested β subunits, whereas the efficient arrangement of DHPRs in tetrads and thus intact DHPR-RyR1 coupling is only promoted by the β_1a isoform. Consequently, we postulate a model that presents β_1a as an allosteric modifier of α_1S conformation enabling skeletal muscle-type EC coupling.Excitation-contraction (EC)³ coupling in skeletal muscle is critically dependent on the close interaction of two distinct Ca²⁺ channels. Membrane depolarizations of the myotube are sensed by the voltage-dependent 1,4-dihydropyridine receptor (DHPR) in the sarcolemma, leading to a rearrangement of charged amino acids (charge movement) in the transmembrane segments S4 of the pore-forming DHPR α_1S subunit (1, 2). This conformational change induces via protein-protein interaction (3, 4) the opening of the sarcoplasmic type-1 ryanodine receptor (RyR1) without need of Ca²⁺ influx through the DHPR (5). The release of Ca²⁺ from the sarcoplasmic reticulum via RyR1 consequently induces muscle contraction. The protein-protein interaction mechanism between DHPR and RyR1 requires correct ultrastructural targeting of both channels. In Ca²⁺ release units (triads and peripheral couplings) of the skeletal muscle, groups of four DHPRs (tetrads) are coupled to every other RyR1 and hence are geometrically arranged following the RyR-specific orthogonal arrays (6).The skeletal muscle DHPR is a heteromultimeric protein complex, composed of the voltage-sensing and pore-forming α_1S subunit and auxiliary subunits β_1a, α₂δ-1, and γ₁ (7). While gene knock-out of the DHPR γ₁ subunit (8, 9) and small interfering RNA knockdown of the DHPR α₂δ-1 subunit (10-12) have indicated that neither subunit is essential for coupling of the DHPR with RyR1, the lack of the α_1S or of the intracellular β_1a subunit is incompatible with EC coupling and accordingly null model mice die perinatally due to asphyxia (13, 14). β subunits of voltage-gated Ca²⁺ channels were repeatedly shown to be responsible for the facilitation of α₁ membrane insertion and to be potent modulators of α₁ current kinetics and voltage dependence (15, 16). Whether the loss of EC coupling in β₁-null mice was caused by decreased DHPR membrane expression or by the lack of a putative specific contribution of the β subunit to the skeletal muscle EC coupling apparatus (17, 18) was not clearly resolved. Recently, other β-functions were identified in skeletal muscle using the β₁-null mutant zebrafish relaxed (19, 20). Like the β₁-knock-out mouse (14) zebrafish relaxed is characterized by complete paralysis of skeletal muscle (21, 22). While β₁-knock-out mouse pups die immediately after birth due to respiratory paralysis (14), larvae of relaxed are able to survive for several days because of oxygen and metabolite diffusion via the skin (23). Using highly differentiated myotubes that are easy to isolate from these larvae, the lack of EC coupling could be described by quantitative immunocytochemistry as a moderate ∼50% reduction of α_1S membrane expression although α_1S charge movement was nearly absent, and, most strikingly, as the complete lack of the arrangement of DHPRs in tetrads (19). Thus, in skeletal muscle the β subunit enables EC coupling by (i) enhancing α_1S membrane targeting, (ii) facilitating α_1S charge movement, and (iii) enabling the ultrastructural arrangement of DHPRs in tetrads.The question arises, which of these functions are specific for the skeletal muscle β_1a and which ones are rather general properties of Ca²⁺ channel β subunits. Previous reconstitution studies made in the β₁-null mouse system (24, 25) using different β subunit constructs (26) did not allow differentiation between β-induced enhancement of non-functional α_1S membrane expression and the facilitation of α_1S charge movement, due to the lack of information on α_1S triad expression levels. Furthermore, the β-induced arrangement of DHPRs in tetrads was not detected as no ultrastructural information was obtained.In the present study, we established zebrafish mutant relaxed as an expression system to test different β subunits for their ability to restore skeletal muscle EC coupling. Using isolated myotubes for in vitro experiments (19, 27) and complete larvae for in vivo expression studies (28-31) and freeze-fracture electron microscopy, a clear differentiation between the major functional roles of β subunits was feasible in the zebrafish system. The cloned zebrafish β_1a and a mammalian (rabbit) β_1a were shown to completely restore all parameters of EC coupling when expressed in relaxed myotubes and larvae. However, the phylogenetically closest β subunit to β_1a, the cardiac/neuronal isoform β_2a from rat, as well as the ancestral β_M isoform from the housefly (Musca domestica), could recover functional α_1S membrane insertion, but led to very restricted tetrad formation when compared with β_1a, and thus to impaired DHPR-RyR1 coupling. This impairment caused drastic changes in skeletal muscle function.The present study shows that the enhancement of functional α_1S membrane expression is a common function of all the tested β subunits, from β_1a to even the most distant β_M, whereas the effective formation of tetrads and thus proper skeletal muscle EC coupling is an exclusive function of the skeletal muscle β_1a subunit. In context with previous studies, our results suggest a model according to which β_1a acts as an allosteric modifier of α_1S conformation. Only in the presence of β_1a, the α_1S subunit is properly folded to allow RyR1 anchoring and thus skeletal muscle-type EC coupling.     

Up-regulation of early growth response gene-1 via the CXCR3 receptor induces reactive oxygen species and inhibits Na+/K+-ATPase activity in an immortalized human proximal tubule cell line

The CXCR3 chemokine receptor, a member of the CXCR family, has been linked to a pathological role in autoimmune disease, inflammatory disease, allograft rejection, and ischemia. In the kidney, expression of the CXCR3 receptor and its ligands is up-regulated in states of glomerulonephritis and in allograft rejection, but little is known about the expression and functional role the CXCR3 receptor might play. Here, we study the function of the CXCR3 chemokine receptor in an immortalized human proximal tubular cell line (IHKE-1). Stimulation of the CXCR3 receptor by its selective agonist monokine induced by IFN-gamma leads via a Ca(2+)-dependent mechanism to an up-regulation of early growth response gene (EGR)-1. Overexpression of EGR-1 induces down-regulation of copper-zinc superoxide dismutase and manganese superoxide dismutase and stimulates the generation of reactive oxygen species (ROS) via the NADH/NADPH-oxidase system. EGR-1 overexpression or treatment with monokine induced by IFN-gamma resulted in a ROS-dependent inhibition of basolateral Na(+)/K(+)-ATPase activity, compromising sodium transport in these cells. Thus, activation of the CXCR3 receptor in proximal tubular cells might disturb natriuresis during inflammatory and ischemic kidney disease via EGR-1-mediated imbalance of ROS.     

Use of positive ion fast atom bombardment mass spectrometry for rapid identification of a bile alcohol glucuronide isolated from cerebrotendinous xanthomatosis patients

The identification of a major biliary and plasma bile alcohol glucuronide, 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 25-tetrol-3-0-beta-D-glucuronide, present in cerebrotendinous xanthomatosis (CTX) patients, was investigated by positive ion fast atom bombardment mass spectrometry (FAB-MS). The spectrum was characterized by abundant ions formed by attachment of a proton, [M + H]+, or of alkali ions, [M + Na]+ and [M + 39K]+, to the glucuronide salt. These ions allowed an unambiguous deduction of the molecular weight of the sample. It is suggested that FAB-MS could be used in the rapid diagnosis of CTX.     

Synthesis of biological precursors of cholic acid

This paper describes a new and convenient procedure for the synthesis of 5β-cholestane-3α,7α,12α,24-tetrol(24R and 24 S) and 5β-cholestane-3α, 7α, 12α, 26-tetrol starting from 5β-cholestane-3α,7α,12α,25-tetrol. Dehydration of the 25-hydroxytetrol with glacial acetic acid and acetic anhydride yielded a mixture of 5β-cholest-24-ene-3α,7α,12α-triol and the corresponding Δ²⁵ compound. Hydroboration and oxidation of the mixture of Δ²⁴ and Δ²⁵ unsaturated bile alcohols resulted in the formation of 5β-cholestane-3α,7α,12α,24ξ-tetrol and 5β-cholestane-3α,7α,12α,26-tetrol. In addition, smaller amounts of 5β-cholestane-3α, 7α, 12α, 23ξ-tetrol and 5β-cholestane-3α, 7α, 12α-triol were also obtained.The bile alcohols epimeric at C-24 were resolved by analytical and preparative TLC, characterized by gas-liquid chromatography and mass-spectrometry. Tentative assignments of the 24R and 24S configuration was made on the basis of molecular rotation differences. These compounds will be useful for biological studies of cholic acid biosynthesis.     

Cytochrome P450 1A1 (CYP1A1) in blood lymphocytes evidence for catalytic activity and mRNA expression.

Studies initiated to characterise the catalytic activity and expression of CYP1A1 in rat blood lymphocytes revealed significant activity of 7-ethoxyresorufin-O-deethylase (EROD) in rat blood lymphocytes. Pretreatment with 3-methylcholanthrene (MC) and beta-naphthoflavone (NF) resulted in significant induction in the activity of lymphocyte EROD suggesting that like the liver enzyme, EROD activity in lymphocytes is inducible and is mediated by the MC inducible isoenzymes of P450. The increase in the activity of EROD was associated with a significant increase in the apparent Vmax and affinity of the substrate towards EROD. That this increase in the activity of EROD could be primarily due to the increase in the expression of CYP1A1 isoenzymes was demonstrated by RT-PCR and western immunoblotting studies indicating an increase in the expression of CYP1A1 in blood lymphocytes after MC pretreatment. Significant inhibition in the EROD activity of MC induced lymphocyte by anti-CYP1A1/1A2 and alpha-naphthoflavone further provided evidence that the CYP1A1/1A2 isoenzymes are involved in the activity of EROD in blood lymphocytes. The data indicating similarities in the regulation of CYP1A1 in blood lymphocytes with the liver isoenzyme suggests that factors which may affect expression of CYP1A1 in liver may also affect expression in blood lymphocytes and that blood lymphocytes could be used as a surrogates for studying hepatic expression of the xenobiotic metabolising enzymes.