Endogenous opiates inhibit gastric acid secretion induced by central administration of Thyrotropin-Releasing Hormone (TRH)

Thyrotropin-releasing hormone (TRH) administered intraventricularly (ICV) to rats causes a dose-dependent increase in gastric acid secretion over a range of 0.01 μg to 10 μg in the pyloris ligated rat. The maximum increase in gastric acid secretion occurs in the first hour. This effect of TRH is not mediated by its metabolites, histidyl-proline diketopiperazine or pyroglutamyl-histidyl-proline (acid TRH). β-endorphin, D-alanine-methionine-enkephalin and the leucine-enkephalin precursor, dynorphin, all inhibit TRH-induced gastric acid secretion. Bombesin, which reduces basal gastric acid secretion had no effect on TRH-induced secretion.     

The influence of coal ash and thermal discharges upon the distribution and bioaccumulation of aquatic invertebrates

The distribution, density and uptake of twenty elements by aquatic invertebrates inhabiting a drainage system, that received excessive coal ash effluent (275 JTU of turbidity) at one end and thermal loading (44.5°C) at the other end, was studied for 15 months. The ash settling basin filled during the first eight months of sampling which resulted in the release of ash effluent directly into the receiving system. Density of invertebrates was lowest in the 300 m stream between the ash basin and swamp and highest 1200 m beyond the stream-swamp confluence where ash influence was minimal. Invertebrate density was lowest in the stations where turbidity from ash effluent was greatest. The most tolerant invertebrates to coal ash stress were odonates (Libellula sp. and Enallagma sp.), crayfish (Procambarus sp.), amphipods (Gammarus sp.) and gastropods (Physa. sp.), and midges (Chironomidae) when the basin was filling. During the period of ash overflow, all groups were either reduced in numbers or absent. In the thermally stressed station, Libellula sp. was the predominant invertebrate sampled when water temperature ranged from 25.5–45.5°C (257-1=28.7°C) all aquatic invertebrates were limited in numbers and density when temperature exceeded the lower and upper ranges of 10.0–38.0°C.This research was supported by AEC Contract AT (38-1-824)This research was supported by AEC Contract AT (38-1-824)     

Minimization of a sodium dithionite-derived interference in nitrate reductase-methyl viologen reactions

The use of sodium dithionite as the reducing agent to produce the radical cation of methyl viologen cofactor for the nitrate reductase-catalyzed reduction of nitrate to nitrite causes a negative interference in the diazonium salt azo dye determination of nitrite. The interference results from bisulfite, a reaction product of dithionite, reacting with the diazonium salt formed from nitrite and sulfanilamide. Addition of formaldehyde before the diazotization reaction masks this interference but at low acidity introduces a nonlinearity in the nitrite calibration curve. This nonlinearity is due to a competing reaction of formaldehyde with sulfanilamide to yield a Schiff base and can be eliminated by increasing the HCL content of the sulfanilamide reagent.     

Constrained evolution of overlapping genes in viral host adaptation: Acquisition of glycosylation motifs in hepadnaviral precore/core genes

Hepadnaviruses use extensively overlapping genes to expand their coding capacity, especially the precore/core genes encode the precore and core proteins with mostly identical sequences but distinct functions. The precore protein of the woodchuck hepatitis virus (WHV) is N-glycosylated, in contrast to the precore of the human hepatitis B virus (HBV) that lacks N-glycosylation. To explore the roles of the N-linked glycosylation sites in precore and core functions, we substituted T77 and T92 in the WHV precore/core N-glycosylation motifs (⁷⁵NIT⁷⁷ and ⁹⁰NDT⁹²) with the corresponding HBV residues (E77 and N92) to eliminate the sequons. Conversely, these N-glycosylation sequons were introduced into the HBV precore/core gene by E77T and N92T substitutions. We found that N-glycosylation increased the levels of secreted precore gene products from both HBV and WHV. However, the HBV core (HBc) protein carrying the E77T substitution was defective in supporting virion secretion, and during infection, the HBc E77T and N92T substitutions impaired the formation of the covalently closed circular DNA (cccDNA), the critical viral DNA molecule responsible for establishing and maintaining infection. In cross-species complementation assays, both HBc and WHV core (WHc) proteins supported all steps of intracellular replication of the heterologous virus while WHc, with or without the N-glycosylation sequons, failed to interact with HBV envelope proteins for virion secretion. Interestingly, WHc supported more efficiently intracellular cccDNA amplification than HBc in the context of either HBV or WHV. These findings reveal novel determinants of precore secretion and core functions and illustrate strong constraints during viral host adaptation resulting from their compact genome and extensive use of overlapping genes.     

ALT-FISH quantifies alternative lengthening of telomeres activity by imaging of single-stranded repeats

Alternative lengthening of telomeres (ALT) occurs in ∼10% of cancer entities. However, little is known about the heterogeneity of ALT activity since robust ALT detection assays with high-throughput in situ readouts are lacking. Here, we introduce ALT-FISH, a method to quantitate ALT activity in single cells from the accumulation of single-stranded telomeric DNA and RNA. It involves a one-step fluorescent in situ hybridization approach followed by fluorescence microscopy imaging. Our method reliably identified ALT in cancer cell lines from different tumor entities and was validated in three established models of ALT induction and suppression. Furthermore, we successfully applied ALT-FISH to spatially resolve ALT activity in primary tissue sections from leiomyosarcoma and neuroblastoma tumors. Thus, our assay provides insights into the heterogeneity of ALT tumors and is suited for high-throughput applications, which will facilitate screening for ALT-specific drugs.     

Proposed megakaryocytic regulon of p53: the genes engaged to control cell cycle and apoptosis during megakaryocytic differentiation

During endomitosis, megakaryocytes undergo several rounds of DNA synthesis without division leading to polyploidization. In primary megakaryocytes and in the megakaryocytic cell line CHRF, loss or knock-down of p53 enhances cell cycling and inhibits apoptosis, leading to increased polyploidization. To support the hypothesis that p53 suppresses megakaryocytic polyploidization, we show that stable expression of wild-type p53 in K562 cells (a p53-null cell line) attenuates the cells' ability to undergo polyploidization during megakaryocytic differentiation due to diminished DNA synthesis and greater apoptosis. This suggested that p53's effects during megakaryopoiesis are mediated through cell cycle- and apoptosis-related target genes, possibly by arresting DNA synthesis and promoting apoptosis. To identify candidate genes through which p53 mediates these effects, gene expression was compared between p53 knock-down (p53-KD) and control CHRF cells induced to undergo terminal megakaryocytic differentiation using microarray analysis. Among substantially downregulated p53 targets in p53-KD megakaryocytes were cell cycle regulators CDKN1A (p21) and PLK2, proapoptotic FAS, TNFRSF10B, CASP8, NOTCH1, TP53INP1, TP53I3, DRAM1, ZMAT3 and PHLDA3, DNA-damage-related RRM2B and SESN1, and actin component ACTA2, while antiapoptotic CKS1B, BCL2, GTSE1, and p53 family member TP63 were upregulated in p53-KD cells. Additionally, a number of cell cycle-related, proapoptotic, and cytoskeleton-related genes with known functions in megakaryocytes but not known to carry p53-responsive elements were differentially expressed between p53-KD and control CHRF cells. Our data support a model whereby p53 expression during megakaryopoiesis serves to control polyploidization and the transition from endomitosis to apoptosis by impeding cell cycling and promoting apoptosis. Furthermore, we identify a putative p53 regulon that is proposed to orchestrate these effects.     

Citric acid production from sucrose using a recombinant strain of the yeast Yarrowia lipolytica

The yeast Yarrowia lipolytica is able to secrete high amounts of several organic acids under conditions of growth limitation and carbon source excess. Here we report the production of citric acid (CA) in a fed-batch cultivation process on sucrose using the recombinant Y. lipolytica strain H222-S4(p67ICL1) T5, harbouring the invertase encoding ScSUC2 gene of Saccharomyces cerevisiae under the inducible XPR2 promoter control and multiple ICL1 copies (10–15). The pH-dependent expression of invertase was low at pH 5.0 and was identified as limiting factor of the CA-production bioprocess. The invertase expression was sufficiently enhanced at pH 6.0–6.8 and resulted in production of 127–140 g l⁻¹ CA with a yield Y _CA of 0.75–0.82 g g⁻¹, whereas at pH 5.0, 87 g l ⁻¹ with a yield Y _CA of 0.51 gg⁻¹ were produced. The CA-productivity Q _CA increased from 0.40 g l ⁻¹ h⁻¹ at pH 5.0 up to 0.73 g l ⁻¹ h⁻¹ at pH 6.8. Accumulation of glucose and fructose at high invertase expression level at pH 6.8 indicated a limitation of CA production by sugar uptake. The strain H222-S4(p67ICL1) T5 also exhibited a gene–dose-dependent high isocitrate lyase expression resulting in strong reduction (<5%) of isocitric acid, a by-product during CA production.     

Abundance,distribution, and territory areas of rock-dwelling Lake Tanganyika cichlid fish species

Lake Tanganyika, the second-oldest and second-deepest lake in the world, harbors an impressive cichlid fish fauna counting about 250 endemic species that are characterized by a great level of ecological, morphological, and behavioral specialization. This study describes and compares cichlid fish communities at two rocky shores with differential human impact in the south of Lake Tanganyika. Species inventories and depth-dependent abundances were elaborated. About 41 and 46 sympatric cichlid species were recorded in the two study sites, respectively. Variabilichromis moorii was the most abundant species (29–60% of total number of fishes), followed by Aulonocranus dewindti (3–19%), Tropheus moorii (12%), Ophthalmotilapia ventralis (4–10%), Eretmodus cyanostictus (6–11%), and Cyathopharynx furcifer (0.01–9%). All other species had abundances below 5%. It further emerged that large cichlids such as Petrochromis species, Cyathopharynx furcifer, and Lobochilotes labiatus were very rare at one location, with frequencies of 0.55% or less. Territorial sizes of three particularly abundant species, Variabilichromis moorii, Aulonocranus dewindti, and Tropheus moorii, were assessed by behavioral observations. We distinguished between territorial core areas and total defended area, yielding average core areas between 0.4 (V. moorii) and 1.6 m² (T. moorii), and total defended areas averaging for each species between 1.6 (V. moorii) and 5.0 m² (A. dewindti) with no significant differences between the two study sites. The data on individual densities are also relevant for evolutionary studies, in that they allow more accurate calculations of effective population sizes.     

Assessing anthropogenic impact on boreal lakes with historical fish species distribution data and hydrogeochemical modeling

Quantifying the effects of human activity on the natural environment is dependent on credible estimates of reference conditions to define the state of the environment before the onset of adverse human impacts. In Europe, emission controls that aimed at restoring ecological status were based on hindcasts from process‐based models or paleolimnological reconstructions. For instance, 1860 is used in Europe as the target for restoration from acidification concerning biological and chemical parameters. A more practical problem is that the historical states of ecosystems and their function cannot be observed directly. Therefore, we (i) compare estimates of acidification based on long‐term observations of roach (Rutilus rutilus) populations with hindcast pH from the hydrogeochemical model MAGIC; (ii) discuss policy implications and possible scope for use of long‐term archival data for assessing human impacts on the natural environment and (iii) present a novel conceptual model for interpreting the importance of physico‐chemical and ecological deviations from reference conditions. Of the 85 lakes studied, 78 were coherently classified by both methods. In 1980, 28 lakes were classified as acidified with the MAGIC model, however, roach was present in 14 of these. In 2010, MAGIC predicted chemical recovery in 50% of the lakes, however roach only recolonized in five lakes after 1990, showing a lag between chemical and biological recovery. Our study is the first study of its kind to use long‐term archival biological data in concert with hydrogeochemical modeling for regional assessments of anthropogenic acidification. Based on our results, we show how the conceptual model can be used to understand and prioritize management of physico‐chemical and ecological effects of anthropogenic stressors on surface water quality.