Development of cross-tolerance to 5-hydroxytryptamine in organotypic cultures of mouse spinal cord-ganglia during chronic exposure to morphine

Exposure of organotypic explants of mouse spinal cord with attached dorsal root ganglia (DRGs) to low concentrations (～10nM) of 5-hydroxytryptamine (5-HT) markedly depressed sensory-evoked dorsal-horn network responses, resembling the acute effects of opioids in these cultures. Attenuation of cord responses by 5-HT was not prevented by exposure to the 5-HT antagonists, methysergide and cyproheptadiene, nor to the opiate antagonist, naloxone. Explants that had become tolerant to morphine after chronic exposure (1 μM) for > 2 days often developed cross-tolerance to 5-HT. Acute exposure of morphine-tolerant explants to naloxone (1 μM) further attenuated the effects of 5-HT so that the minimum depressant levels of 5-HT were often increased up to 30-fold. Increasing the extra-cellular Ca⁺⁺ concentration (to 5 mM) and/or introduction of 4-aminopyridine markedly antagonized the depressant effects of 5-HT on DRG-evoked cord responses, so that 5-HT levels comparable to those used on morphine-tolerant explants were required to depress naive explants. These depressant effects of 5-HT on cord-DRG explants are consonant with antinociceptive actions of 5-HT administered to dorsal cord $\text{in situ}$. Our data suggest that 5-HT may block neuronal components of dorsal horn networks at similar regions to those that are depressed by opiates, e.g. presynaptic DRG nerve terminals where abundant opiate receptors are located. The marked attenuation of the depressant effects of both 5-HT and opiates on cord-DRG explants by high Ca⁺⁺ raises the possibility that cross-tolerance to 5-HT in morphine-tolerant explants may result from the same neuronal alterations that render dorsal-horn networks tolerant to opiates. Furthermore, the increased degree of cross-tolerance to 5-HT after acute introduction of naloxone in morphine-tolerant cultures may be an expression of opiate dependence.     

Bile acid synthesis precursors in familial combined hyperlipidemia: The oxysterols 24S-hydroxycholesterol and 27-hydroxycholesterol

Familial combined hyperlipidemia (FCHL), the most common inherited disorder of lipid metabolism is characterized by increasing cholesterol synthesis precursors due to hepatic overproduction of cholesterol. The bile acids synthesis pathway has not been previously studied in FCHL. The aim of this work was to study the oxysterol levels which are involved in the bile acids synthesis from cholesterol in FCHL. Clinical parameters and subclinical atherosclerosis were studied in a total of 107 FCHL patients and 126 normolipidemic controls. Non cholesterol sterols (desmosterol and lanosterol) and oxysterols (27-hydroxycholesterol and 24S-hydroxycholesterol) were measured by high performance liquid chromatography tandem mass spectrometry. Desmosterol and lanosterol, markers of cholesterol synthesis, had a positive correlation with BMI and apo B. However, no correlation was found for 24S-hydroxycholesterol and 27-hydroxycholesterol, precursors of bile acids, with these clinical parameters. Only 27-hydroxycholesterol had a positive correlation with apo B, ρ = 0.204 (P = 0.037). All oxysterol levels were higher in FHCL as compared to normal controls. A total of 59 FCHL subjects (59%) presented values of 24S-hydroxycholesterol above the 95th percentile of this oxysterol in the control population. All oxysterols showed no association with fat mass in contrast with non-cholesterol sterols. FCHL subjects with oxysterol overproduction had less carotid intima media thickness (cIMT), which suggests less atherosclerosis in these subjects. In summary, our data indicate that high oxysterol levels might be good markers of FCHL, unrelated to fat mass, and may exert a protective mechanism for cholesterol accumulation.     

Phreatophytic vegetation response to climatic and abstraction-induced groundwater drawdown: Examples of long-term spatial and temporal variability in community response

The influence of climatic drought and groundwater abstraction on phreatophytic vegetation dynamics was investigated in the southwest of Western Australia. Two contrasting examples of long-term phreatophytic plant community response to reduced water availability are presented. Multivariate analysis of vegetation and hydrological parameters determined depth to watertable as the dominant biophysical driver of floristic spatial and temporal patterns. Under lower rates of watertable drawdown (9 cm year^?1), a progressive change in floristic composition was observed over a 33-year period. The abundance of species with a preference for wetter sites was significantly reduced, whereas that of more drought-tolerant species increased. Higher rates of drawdown (50 cm year^?1) where groundwater abstraction exacerbated climatic drought resulted in a threshold response in vegetation and 33% dissimilarity to pre-abstraction floristics in 12 years. In the context of an ecohydrological state and transition conceptual model, it is suggested higher rates of groundwater drawdown result in a threshold breach and subsequent transition to an alternative ecohydrological state, whilst lower rates result in a progressive floristic transition.     

Fucosylation of glycoproteins in rat spermatocytes and spermatids

Glycoprotein synthesis in pachytene spermatocytes and round spermatids, isolated from rat testes, was studied by analysis of the incorporation of (³H)-fucose. The isolated germ cells were capable of incorporating (³H)-fucose into cell-bound, acid-precipitable components for an incubation period of at least 23 hours (at 32°C). In young spermatids, engaged in the formation of the acrosome, (³H)-fucose was incorporated into more than 16 different glycoproteins within the molecular weight range of 20.000–100,000. A qualitatively similar set of glycoproteins was found to be labeled in spermatocytes. Radioautography showed that after 4 hr most of the incorporated radioactivity was present at one pole in the perinuclear zone of spermatocytes and spermatids, which could reflect incorporation of fucose in the Golgi apparatus. The newly fucosylated glycoproteins were associated with a particulate subcellular fraction (membrane fraction). Trypsin treatment of whole cells after 25 hours of incubation with (³H)-fucose, however, did not cause significant lysis of tritiated glycoproteins. From the results it was concluded that the majority of the newly fucosylated glycoproteins in spermatocytes and spermatids remained associated with an intracellular membrane system, presumably the Golgi apparatus and the vesicles that arise from this structure, to be deposited subsequently in proacrosomic granules and the acrosome. The results also suggest that initiation of the synthesis of spermatidal glycoproteins occurs during the prophase of meiosis in spermatocytes.     

Role of gonadal and adrenal steroids and thyroid hormones in the regulation of molting in domestic goose

Plasma levels of testosterone (T), 17-β-estradiol (E2), progesterone (P4), dehydroepiandrosterone (DHEA), corticosterone (B), thyroxine (T4) and triiodothyronine (T3) were monitored during postnuptial and the prenuptial molt in domestic goose (Anser anser domesticus) in both sexes. 1. At the beginning of postnuptial molt (when the old, worn dawny-, and cover feathers' loss starts) in ganders, the levels of T, E2, P4 decrease while DHEA and B significantly increase. The elevated levels of T4 and low T3 concentrations characteristic of the last phase of the reproduction, remain unchanged. In layers, similar changes were observed, however, B decreases. 2. In the early phase of outgrowth of wing and cover feathers, plasma levels of T, E2 and P4 are low. Elevated B, DHEA and T4 concentrations decrease in ganders, while in layers DHEA increases and B and T4 levels remain unchanged. T3 increases in both sexes. 3. The subsequent intensive outgrowth period of wing- and cover feathers both in ganders and in layers is characterized by very low levels of T, E2, DHEA and T4, but P4 increased, and T3 concentration remain high. 4. At the end of postnuptial molt - when the outgrowth of dawny, cover-, and wing feathers stops - very low T, E2, P4, DHEA and T4 levels and and high T3 plasma levels were found in both sexes. Fast increase of plasma B was detected in ganders, while in geese, B concentration remain high. 5. During prenuptial molting (outgrowth of contour and tail feathers) low E2, P4 and T4, increasing T and DHEA, but very high T3 and B plasma concentration were measured in ganders. In layers, very low T, E2, P4, DHEA and T4 levels, and very high B and T3 levels were found. 6. At the beginning of the fall-winter sexual repose (postmolting stage) T, E2, P4, DHEA and T4 levels increase, T3 and B declines in both sexes. 7. In the subsequent phase of fall-winter period (preparatory stage) there is a further increase in T, P4 and T4, a fast increase of B and a decrease of E2, DHEA and T3 in ganders. In layers, T, P4 and DHEA decrease, B increases and the T4 and T3 do not change. 8. At the beginning of reproduction high T level, unchanged DHEA, slightly declined P4, and decreased E2, T4, T3 and a strong decline of B concentrations occur in ganders. In layers, T is further increased, E2 and P4 shows high levels, and, at the same time DHEA and T3 remain unchanged, while B and T4 decrease.     

Reconstructing the Origins of the Somatostatin and Allatostatin-C Signaling Systems Using the Accelerated Evolution of Biodiverse Cone Snail Toxins

Somatostatin and its related peptides (SSRPs) form an important family of hormones with diverse physiological roles. The ubiquitous presence of SSRPs in vertebrates and several invertebrate deuterostomes suggests an ancient origin of the SSRP signaling system. However, the existence of SSRP genes outside of deuterostomes has not been established, and the evolutionary history of this signaling system remains poorly understood. Our recent discovery of SSRP-like toxins (consomatins) in venomous marine cone snails (Conus) suggested the presence of a related signaling system in mollusks and potentially other protostomes. Here, we identify the molluscan SSRP-like signaling gene that gave rise to the consomatin family. Following recruitment into venom, consomatin genes experienced strong positive selection and repeated gene duplications resulting in the formation of a hyperdiverse family of venom peptides. Intriguingly, the largest number of consomatins was found in worm-hunting species (>400 sequences), indicating a homologous system in annelids, another large protostome phylum. Consistent with this, comprehensive sequence mining enabled the identification of SSRP-like sequences (and their corresponding orphan receptor) in annelids and several other protostome phyla. These results established the existence of SSRP-like peptides in many major branches of bilaterians and challenge the prevailing hypothesis that deuterostome SSRPs and protostome allatostatin-C are orthologous peptide families. Finally, having a large set of predator–prey SSRP sequences available, we show that although the cone snail’s signaling SSRP-like genes are under purifying selection, the venom consomatin genes experience rapid directional selection to target receptors in a changing mix of prey.     

Refuge areas and suture zones in the Pyrenean and Cantabrian regions: geographic variation of the female MPI sex-linked alleles among oviparous populations of the lizard Lacerta (Zootoca) vivipara

The viviparous lizard Lacerta (Zootoca) vivipara exhibits several alleles of the mannose-6-phosphate isomerase (MPI) enzyme that are carried exclusively on the female W sex-chromosome. Previous studies showed that both the oviparous and viviparous forms of L. (Zootoca) vivipara have these female sex-linked alleles. We document the existence of geographic variation of these alleles among the oviparous populations of southwestern France and northwestern Spain. Two oviparous subgroups were identified: all females from the eastern and central Pyrenees and most females from Aquitaine and from the northern slope of the western Pyrenees exhibited the fast migrating alleles MPI¹¹⁰ or MPI¹²⁰, whereas all females from the Cantabric mountains, Spanish Basque country, and from the south slope of the western Pyrenees exhibited the slow migrating allele MPI⁹⁰. Populations with both fast and slow migrating alleles occurred at some stations in the upper Ossau valley (western Pyrenees) and also at a lowland station of south Aquitaine. The hypothesis that several oviparous forms could have retreated to different places of the Pyreneo-Iberian refugia during the Quaternary glaciations could explain the conservation or the evolution of the polymorphism of the MPI alleles, and that is consistent with the phylogeographic scenario previously proposed to account for the reproductive and cytogenetical variation observed in this species.     

Comparative Analysis of Dynamic Cell Viability,Migration and Invasion Assessments by Novel Real-Time Technology and Classic Endpoint Assays

Background

Cell viability and motility comprise ubiquitous mechanisms involved in a variety of (patho)biological processes including cancer. We report a technical comparative analysis of the novel impedance-based xCELLigence Real-Time Cell Analysis detection platform, with conventional label-based endpoint methods, hereby indicating performance characteristics and correlating dynamic observations of cell proliferation, cytotoxicity, migration and invasion on cancer cells in highly standardized experimental conditions.

Methodology/Principal Findings

Dynamic high-resolution assessments of proliferation, cytotoxicity and migration were performed using xCELLigence technology on the MDA-MB-231 (breast cancer) and A549 (lung cancer) cell lines. Proliferation kinetics were compared with the Sulforhodamine B (SRB) assay in a series of four cell concentrations, yielding fair to good correlations (Spearman''s Rho 0.688 to 0.964). Cytotoxic action by paclitaxel (0–100 nM) correlated well with SRB (Rho>0.95) with similar IC₅₀ values. Reference cell migration experiments were performed using Transwell plates and correlated by pixel area calculation of crystal violet-stained membranes (Rho 0.90) and optical density (OD) measurement of extracted dye (Rho>0.95). Invasion was observed on MDA-MB-231 cells alone using Matrigel-coated Transwells as standard reference method and correlated by OD reading for two Matrigel densities (Rho>0.95). Variance component analysis revealed increased variances associated with impedance-based detection of migration and invasion, potentially caused by the sensitive nature of this method.

Conclusions/Significance

The xCELLigence RTCA technology provides an accurate platform for non-invasive detection of cell viability and motility. The strong correlations with conventional methods imply a similar observation of cell behavior and interchangeability with other systems, illustrated by the highly correlating kinetic invasion profiles on different platforms applying only adapted matrix surface densities. The increased sensitivity however implies standardized experimental conditions to minimize technical-induced variance.