Immunohistochemical localization of gonadotropin-releasing hormone (GnRH) in the brain and infundibulum of the sheep

Summary To determine how neural influences control the function of the pineal gland, morphological and biochemical relationships after pharmacological treatment have been studied in rat pineal cells in monolayer cultures. Norepinephrine (NE) and dibutyryl cyclic 3,5-adenosine monophosphate (dBcAMP) treatment of cells that had been in culture for 5 and 21 days produced a stimulation in the enzyme activity of serotonin N-acetyl transferase, an enzyme important in indole synthesis. NE and dBcAMP also produced morphological changes which were dependent on the time of cells in culture. When 5 day-cultures were treated with NE and dBcAMP, light and dark cells were noted and endoplasmic reticulum increased and became more organized. Only dBcAMP treatment at 5 days produced an increase in dense granules and an elongation of cytoplasmic processes. Treatment of 21 day-cultures with dBcAMP also produced an increase in cytoplasmic processes while treatment with NE produced an increase in the synaptic ribbons and clear vesicles within the processes.     

Light effects upon dry lettuce seeds

Germination of certain dry seeds (achenes) of Lactuca sativa L. cv. Grand Rapids was increased to ca. 75% after irradiation with 665 nm red light (R; 1x10³ J m^-2); this response was eliminated by far-red light (FR) following the R. The response of dry seeds required an order of magnitude more light than that of wetted seeds, and was not maximal until 48 h after irradiation. Other seeds, which could not be stimulated by R in dry state, showed a partial response after 10 min hydration. Irradiation of dry seeds (or seeds wetted 1 h) with FR (1x10³ J m^-2) reduced dark germination from 26% to 2%. Seeds dehydrated in an oven (60°C, 90 min) showed a decrease in germination if irradiated with R (1x10⁵ J m^-2) before wetting. The results show that phytochrome is present in dry lettuce seeds (and functional in some seed lots) prior to wetting; and that in other seed lots the molecule becomes functional within minutes after wetting the seeds. Transformation of the FR absorbing from of phytochrome (P_FR) to the inactive from (P_R) occurs at lower seed moisture content than the reverse reaction. It appears that dormancy in seeds ripened in sunlight might be assured during seed drying and maturation by the more effective transformation of P_FR to P_R than vice versa as phytochrome is dehydrated.Abbreviations FR far-red - R red - CAL seeds from California - NC seeds from North Carolina (see text)     

Management of diabetes mellitus in children.

In many respects the management of diabetes mellitus in children differs from the management in adults. Guidelines for the short- and long-term management of the child with diabetes mellitus, particularly as it relates to clinical presentation, treatment of ketoacidosis, long-term therapy and psychological counseling, are presented. The specific aspects of diabetes management that are unique to the child are best met by an increased understanding of the problems by the child and the family and an integrated approach by the physician and allied health personnel.     

As old as the hills: Pliocene palaeogeographical processes influence patterns of genetic structure in the widespread,common shrub Banksia sessilis

The impact of Quaternary glaciation on the development of phylogeographic structure in plant species is well documented. In unglaciated landscapes, phylogeographic patterns tend to reflect processes relating to persistence and stochasticity, yet other factors, associated with the palaeogeographical history of the landscape, including geomorphological events, can also have a significant influence. The unglaciated landscape of south‐western Western Australia is an ideal location to observe these ancient drivers of lineage diversification, with tectonic activity associated with the Darling Fault in the late Pliocene attributed to patterns of deep phylogeographic divergence in a widespread tree from this region. Interestingly, other species within this region have not shown this pattern and this palaeogeographical boundary therefore presents an opportunity to examine age and historical distribution of plant species endemic to this region. In this study, we assess patterns of genetic diversity and structure across 28 populations of the widespread shrub Banksia sessilis using three cpDNA markers and nine nuclear microsatellite markers. Sixteen cpDNA haplotypes were identified, comprising two major chloroplast DNA lineages that are estimated to have diverged in the Pliocene, approximately 3.3 million years ago. This timing coincides with major geomorphological processes in the landscape, including the separation of the Darling Plateau from the adjacent Swan Coastal Plain, as well as eustatic changes on the Swan Coastal Plain that are likely to have resulted in the physical isolation of historical plant lineages. Chloroplast lineages were broadly aligned with populations associated with older lateritic soils of the Darling Plateau and Geraldton sandplains or the younger sandy soils associated with the Swan Coastal Plain and Southern Coastline. This structural pattern of lateritic versus non‐lateritic division was not observed in the nuclear microsatellite data that identified three genetic clades that roughly corresponded to populations in the North, South, and Central portions of the distributions.     

Hexazonium pararosaniline as a fixative for animal tissues

Hexazonium pararosaniline is a valuable reagent that has been used in enzyme activity histochemistry for 50 years. It is an aqueous solution containing the tris-diazonium ion derived from pararosaniline, an aminotriarylmethane dye, and it contains an excess of nitrous acid that was not consumed in the diazotization reaction. Other investigators have found that immersion for 2 min in an acidic (pH 3.5) 0.0015 M hexazonium pararosaniline solution can protect cryostat sections of unfixed animal tissues from the deleterious effects of aqueous reagents such as buffered solutions used in immunohistochemistry, while preserving specific affinities for antibodies. In the present investigation hexazonium pararosaniline protected lymphoid tissue and striated muscle against the damaging effects of water or saline. The same protection was conferred on unfixed sections treated with dilute nitrous or hydrochloric acid in concentrations similar to those in hexazonium pararosaniline solutions. Model tissues (solutions, gels or films containing gelatin and/or bovine albumin) responded predictably to well known cross-linking (formaldehyde) or coagulant (mercuric chloride) fixatives. Hexazonium pararosaniline solutions prevented the dissolution of protein gels in water only after 9 or more days of contact, during which time considerable swelling occurred. It is concluded that there is no evidence for a “fixative” action of hexazonium pararosaniline. The protective effect on frozen sections of unfixed tissue is attributable probably to the low pH of the solution.     

Next-Generation Site-Directed Transgenesis in the Malaria Vector Mosquito Anopheles gambiae: Self-Docking Strains Expressing Germline-Specific phiC31 Integrase

Diseases transmitted by mosquitoes have a devastating impact on global health and the situation is complicated due to difficulties with both existing control measures and the impact of climate change. Genetically modified mosquitoes that are refractory to disease transmission are seen as having great potential in the delivery of novel control strategies. The Streptomyces phage phiC31 integrase system has been successfully adapted for site-directed transgene integration in a range of insects, thus overcoming many limitations due to size constraints and random integration associated with transposon-mediated transformation. Using this technology, we previously published the first site-directed transformation of Anopheles gambiae, the principal vector of human malaria. Mosquitoes were initially engineered to incorporate the phiC31 docking site at a defined genomic location. A second phase of genetic modification then achieved site-directed integration of an anti-malarial effector gene. In the current publication we report improved efficiency and utility of the phiC31 integrase system following the generation of Anopheles gambiae self-docking strains. Four independent strains, with docking sites at known locations on three different chromosome arms, were engineered to express integrase under control of the regulatory regions of the nanos gene from Anopheles gambiae. The resulting protein accumulates in the posterior oocyte to provide integrase activity at the site of germline development. Two self-docking strains, exhibiting significantly different levels of integrase expression, were assessed for site-directed transgene integration and found to demonstrate greatly improved survival and efficiency of transformation. In the fight against malaria, it is imperative to establish a broad repertoire of both anti-malarial effector genes and tissue-specific promoters to regulate their expression, enabling those offering maximum effect with minimum fitness cost to be identified. The improved technology we describe here will facilitate comparative studies of effector transgenes, allowing informed choices to be made that potentially lead to transmission blockade.     

CDC25B Overexpression Stabilises Centrin 2 and Promotes the Formation of Excess Centriolar Foci

CDK-cyclin complexes regulate centriole duplication and microtubule nucleation at specific cell cycle stages, although their exact roles in these processes remain unclear. As the activities of CDK-cyclins are themselves positively regulated by CDC25 phosphatases, we investigated the role of centrosomal CDC25B during interphase. We report that overexpression of CDC25B, as is commonly found in human cancer, results in a significant increase in centrin 2 at the centrosomes of interphase cells. Conversely, CDC25B depletion causes a loss of centrin 2 from the centrosome, which can be rescued by treatment with the proteasome inhibitor MG132. CDC25B overexpression also promotes the formation of excess centrin 2 “foci”. These foci can accumulate other centrosome proteins, including γ-tubulin and PCM-1, and can function as microtubule organising centres, indicating that these represent functional centrosomes. Formation of centrin 2 foci can be blocked by specific inhibition of CDK2 but not CDK1. CDK2-mediated phosphorylation of Monopolar spindle 1 (Mps1) at the G1/S transition is essential for the initiation of centrosome duplication, and Mps1 is reported to phosphorylate centrin 2. Overexpression of wild-type or non-degradable Mps1 exacerbated the formation of excess centrin 2 foci induced by CDC25B overexpression, while kinase-dead Mps1 has a protective effect. Together, our data suggest that CDC25B, through activation of a centrosomal pool of CDK2, stabilises the local pool of Mps1 which in turn regulates the level of centrin 2 at the centrosome. Overexpression of CDC25B may therefore contribute to tumourigenesis by perturbing the natural turnover of centrosome proteins such as Mps1 and centrin 2, thus resulting in the de novo assembly of extra-numerary centrosomes and potentiating chromosome instability.     

Economic evaluation of water loss saving due to the biological control of water hyacinth at New Year’s Dam,Eastern Cape province,South Africa

Water hyacinth Eichhornia crassipes is considered the most damaging aquatic weed in the world. However, few studies have quantified the impact of this weed economically and ecologically, and even fewer studies have quantified the benefits of its control. This paper focuses on water loss saving as the benefit derived from biological control of this plant between 1990 and 2013 at New Year’s Dam, Alicedale, Eastern Cape, South Africa. Estimates of water loss due to evapotranspiration from water hyacinth vary significantly; therefore, the study used three different rates, high, medium and low. A conservative raw agriculture value of R 0.26 per m³ was used to calculate the benefits derived by the water saved. The present benefit and cost values were determined using 10% and 5% discount rates. The benefit/cost ratio at the low evapotranspiration rate was less than one, implying that biological control was not economically viable but, at the higher evapotranspiration rates, the return justified the costs of biological control. However, at the marginal value product of water, the inclusion of the costs of damage to infrastructure, or the adverse effects of water hyacinth on biodiversity, would justify the use of biological control, even at the low transpiration rate.     

The Tau/A152T mutation,a risk factor for frontotemporal‐spectrum disorders,leads to NR2B receptor‐mediated excitotoxicity

We report on a novel transgenic mouse model expressing human full‐length Tau with the Tau mutation A152T (hTau^AT), a risk factor for FTD‐spectrum disorders including PSP and CBD. Brain neurons reveal pathological Tau conformation, hyperphosphorylation, mis‐sorting, aggregation, neuronal degeneration, and progressive loss, most prominently in area CA3 of the hippocampus. The mossy fiber pathway shows enhanced basal synaptic transmission without changes in short‐ or long‐term plasticity. In organotypic hippocampal slices, extracellular glutamate increases early above control levels, followed by a rise in neurotoxicity. These changes are normalized by inhibiting neurotransmitter release or by blocking voltage‐gated sodium channels. CA3 neurons show elevated intracellular calcium during rest and after activity induction which is sensitive to NR2B antagonizing drugs, demonstrating a pivotal role of extrasynaptic NMDA receptors. Slices show pronounced epileptiform activity and axonal sprouting of mossy fibers. Excitotoxic neuronal death is ameliorated by ceftriaxone, which stimulates astrocytic glutamate uptake via the transporter EAAT2/GLT1. In summary, hTau^AT causes excitotoxicity mediated by NR2B‐containing NMDA receptors due to enhanced extracellular glutamate.