Sexing river buffalo (Bubalus bubalis L.), sheep (Ovis aries L.), goat (Capra hircus L.), and cattle spermatozoa by double color FISH using bovine (Bos taurus L.) X- and Y-painting probes

River buffalo, sheep, and goat spermatozoa were cross-hybridized using double color fluorescence in situ hybridization (FISH) with bovine Xcen- and Y-chromosome painting probes, prepared by DOP-PCR of laser-microdissected-catapulted chromosomes, to investigate the possibility of using bovine probes for sexing sperm of other members of the family Bovidae. Before sperm analysis, the probes were hybridized on metaphase chromosomes of each species, as control. Frozen-thawed spermatozoa of cattle, river buffalo, sheep, and goat were decondensed in suspension with 5 mM DTT. Sperm samples obtained from three individuals of each species were investigated, more than 1,000 spermatozoa were scored in each animal. FISH analysis of more than 12,000 sperm revealed high level of sperm with X- or Y-signals in all of the species investigated, indicating FISH efficiency over 99%. Significant interspecific differences were detected in the frequency of aberrant spermatozoa (aneuploid and diploid) between goat (0.393%) and sheep (0.033%) (P < 0.01), goat and cattle (0.096%) (P < 0.5), as well as between river buffalo (0.224%) and sheep (P < 0.5). There was no significant difference between river buffalo and cattle. The present study demonstrated that it is possible to use bovine X-Y painting probes for sexing and analyzing sperm of other species of the family, thus facilitating future studies on the incidence of chromosome abnormalities in sperm as well as on sex predetermination of embryos for the livestock industry. Mol. Reprod. Dev. 67: 108-115, 2004.     

How can river-estuarine planktonic copepods survive river floods?

Distribution of brackish-water zooplankton was surveyed in the Chikugo River estuary, Japan, at 3-h intervals for 24h, including several hours when the river was heavily flooded because of rainfall and discharge from an upstream dam. The populations of the dominant copepods Pseudodiaptomus inopinus and Sinocalanus sinensis were concentrated in the mixing zone of the salt wedge before river flooding. During the period of flooding, they were almost completely swept out from the river, but examination of water trapped by a sediment grab sampler revealed that adults of Pseudodiaptomus inopinus were aggregated immediately above the bottom of the submerged channel outside the river mouth. After the flood, aggregations of both species appeared again in the river together with the salt wedge, and the proportion of adults in the aggregations became significantly higher than before. These results suggest that immature copepods suffer population losses through river flooding, but that the copepods can survive such flooding by means of both behavioral and reproductive mechanisms; that is, adult copepods maintain their position on the bottom of submerged channels outside the river mouth during a river flood and replace the population loss through reproduction after the flood. Channels outside river mouths are thus regarded as important topographic refuges for river-estuarine zooplankton to survive a flood.     

Wood ash treatment affects seasonal N fluctuations in needles of adult<Emphasis Type="Italic"> Picea abies</Emphasis> trees: a <Superscript>15</Superscript>N-tracer study

A ¹⁵N-tracer experiment was carried out in a stand of adult spruce trees [Picea abies (L.) Karst.] located on the Swiss Plateau in order to investigate the effects of wood ash treatment on seasonal nitrogen fluctuations in fine roots and needles. Treatments included irrigation (W), liquid fertilization (LF) and wood ash (A) application. ¹⁵N fluctuation in fine roots and current to 3-year-old needles was studied after one ¹⁵N pulse for 2 consecutive years (1999, 2000). ¹⁵N tracer was rapidly incorporated into the fine roots of adult trees, and ¹⁵N values reached similar levels in all treatments 2 months after the pulse. In the needles, the largest increase in ¹⁵N was observed in those of the current year. Following the initial peak during spring growth, ¹⁵N values in needles of control trees showed an oscillating pattern through the season. This oscillation is attributed to the increased use of internal N sources, as soon as the roots can no longer meet the increased N demand during the sprouting phase. However, W-, LF- and A-treated trees no longer showed the oscillation in ¹⁵N. Additional water (W and LF) as well as fertilizer (A and LF) may have induced shifts in the microbial flora, thus increasing the unlabelled N release from the soil. The strongest dampening was observed for the A treatment, indicating sufficient N availability from the soil, and making intensive use of the internal N sources unnecessary. Treatment with wood ash thus resulted in a similar fertilizer response to liquid fertilization.     

Modelling faecal indicator concentrations in large rural catchments using land use and topographic data

AIMS: To characterize and model spatial variations in faecal-indicator organism concentrations in watercourses draining a large rural catchment during the bathing season. METHODS AND RESULTS: Presumptive coliform (PC), presumptive Eschericia coli (PE) and presumptive enterococci (PEnt) concentrations were determined under base- and high-flow conditions at 24 monitoring points in the Rheidol/Ystwyth catchment, Wales. Relationships with land use and topographic variables within their 'subcatchments' were investigated. Geometric mean (GM) concentrations typically increase more than 10-fold at high flow. The proportion of improved pasture within subcatchments explains 81.6, 73.0 and 85.8%, respectively, of the variance in high-flow GM PC, PE and PEnt concentrations. Distributed modelling suggests that organisms derived from more distant parts of subcatchments may be lost through die-off and sedimentation along watercourses at base flow, although not at high flow. Reductions in concentrations are also evident downstream of reservoir impoundments. CONCLUSIONS: Large rural catchments may contribute substantially to faecal indicator loadings in coastal waters. Indicator concentrations can be modelled successfully using land use and topographic data. SIGNIFICANCE AND IMPACT OF THE STUDY: Development of a modelling approach that provides valuable insight into faecal indicator sources and the transport and survival of these organisms within large catchments.     

Ribozyme Relationships: The Hammerhead, Hepatitis Delta, and Hairpin Ribozymes Have a Common Origin

The hammerhead and the hairpin ribozymes of plant viroids/virusoids and the hepatitis delta ribozyme were generally considered to be unrelated to one another. Here we report notable sequence interrelationships and some structural ones connecting all three. Received: 8 September 1999 / Accepted: 31 March 2000     

Expression and properties of recombinant HbA2 (alpha2delta2) and hybrids containing delta-beta sequences

Hemoglobin A₂ (₂₂), which is present at low concentration (1–2%) in the circulating red cells of normal individuals, has two important features that merit its study, i.e., it inhibits polymerization of sickle HbS and its elevated concentration in some thalassemias is a useful clinical diagnostic. However, reports on its functional properties regarding O₂ binding are conflicting. We have attempted to resolve these discrepancies by expressing, for the first time, recombinant hemoglobin A₂ and systematically studying its functional properties. The construct expressing HbA₂ contains only and genes so that the extensive purification required to isolate natural HbA₂ is circumvented. Although natural hemoglobin A₂ is expressed at low levels in vivo, the amount of recombinant ₂₂ expressed in yeast is similar to that found for adult hemoglobin A and for fetal hemoglobin F when the + or the + genes, respectively, are present on the construct. Recombinant HbA₂ is stable, i.e., not easily oxidized, and it is a cooperative functional hemoglobin with tetramer-dimer dissociation properties like those of adult HbA. However, its intrinsic oxygen affinity and response to the allosteric regulators chloride and 2,3-diphosphoglycerate are lower than the corresponding properties for adult hemoglobin. Molecular modeling studies which attempt to understand these properties of HbA₂ are described.     

Across the Southern Alps by river capture? Freshwater fish phylogeography in South Island,New Zealand

We used DNA analysis of galaxiid fish to test a hypothesis of localized headwater capture in South Island, New Zealand. The restricted western, but widespread eastern, distributions of three nonmigratory freshwater fish species suggest that part of the east-flowing Waiau River has been captured by the west-flowing Buller River. However, mitochondrial control region (Kimura 2-parameter distance = 4.1-5.4%) and microsatellite flanking sequences do not support a relationship between Waiau (N = 4 fish sequences) and western populations (N = 8) of Galaxias vulgaris. Instead, the point of capture is probably to the north-east, perhaps the Nelson lakes region. Phylogenetic analysis indicates that western populations, along with populations in the north-east (N = 18), represent a previously unidentified monophyletic Evolutionarily Significant Unit, possibly a cryptic species. We suggest a general caveat for zoogeographic conclusions based on distributional data alone.     

Variation in material transport and water chemistry along a large ephemeral river in the Namib Desert

• 1 The chemical characteristics of floodwaters in ephemeral rivers are little known, particularly with regard to their organic loads. These rivers typically exhibit a pronounced downstream hydrological decay but few studies have documented its effect on chemical characteristics and material transport. To develop a better understanding of the dynamics of floods and associated material transport in large ephemeral rivers, floods of the ephemeral Kuiseb River in south‐western Africa were tracked and repeatedly sampled at multiple points along the river's lower 220 km.
• 2 We quantified the composition and transport of solute and sediment loads in relation to longitudinal hydrological patterns associated with downstream hydrological decay. Source and sink areas for transported materials were identified, and the composition and transport dynamics of the organic matter load were compared to those described from more mesic systems.
• 3 Concentrations of sediments and solutes transported by floods in the Kuiseb River tended to increase downstream in association with pronounced hydrological decay. The contribution of particulate organic matter to total organic load is among the highest recorded, despite our observation of unusually high levels of dissolved organic matter. Hydrological decay resulted in deposition of all transported material within the lower Kuiseb River, with no discharge of water or materials to the Atlantic Ocean.
• 4 Our results suggest that longitudinal variation in surface flow and associated patterns of material transport renders the lower Kuiseb River a sink for materials transported from upstream. The downstream transport and deposition of large amounts of labile organic matter provides an important carbon supplement to heterotrophic communities within the river's lower reaches.

     

Regulation of PI3K/Akt/GSK-3 pathway by cannabinoids in the brain

Delta9-tetrahydrocannabinol (THC), the main psychoactive component in Cannabis sativa preparations, exerts its central effects mainly through the G-protein coupled receptor CB1, a component of the endocannabinoid system. Several in vitro and in vivo studies have reported neuroprotective effects of cannabinoids in excitotoxicity and neurodegeneration models. However, the intraneuronal signaling pathways activated in vivo by THC underlying its central effects remain poorly understood. We report that THC acute administration (10 mg/kg, i.p.) increases the phosphorylation of Akt in mouse hippocampus, striatum, and cerebellum. This phosphorylation was mediated by CB1 receptors as it was blocked by the selective CB1 antagonist rimonabant. Moreover, PI3K inhibition by wortmannin abrogated THC-induced phosphorylation of Akt, but blockade of extracellular signal-regulated protein kinases by SL327 did not modify this activation/phosphorylation of Akt. Moreover, administration of the dopaminergic D1 (SCH 23390) and D2 (raclopride) receptor antagonists did not block the activation of PI3K/Akt pathway induced in the striatum by cannabinoid receptor stimulation, suggesting that this effect is independent of the dopaminergic system. In addition, THC increased the phosphorylation of glycogen synthase kinase 3 beta. Therefore, activation of the PI3K/Akt/GSK-3 signaling pathway may be related to the in vivo neuroprotective properties attributed to cannabinoids.