Mechanisms for gonadotropin-releasing hormone potentiation of growth hormone rebound following norepinephrine inhibition in goldfish pituitary cells

In the goldfish, norepinephrine (NE) inhibits growth hormone (GH) secretion through activation of pituitary alpha(2)-adrenergic receptors. Interestingly, a GH rebound is observed after NE withdrawal, which can be markedly enhanced by prior exposure to gonadotropin-releasing hormone (GnRH). Here we examined the mechanisms responsible for GnRH potentiation of this "postinhibition" GH rebound. In goldfish pituitary cells, alpha(2)-adrenergic stimulation suppressed both basal and GnRH-induced GH mRNA expression, suggesting that a rise in GH synthesis induced by GnRH did not contribute to its potentiating effect. Using a column perifusion approach, GnRH given during NE treatment consistently enhanced the GH rebound following NE withdrawal. This potentiating effect was mimicked by activation of PKC and adenylate cyclase (AC) but not by induction of Ca(2+) entry through voltage-sensitive Ca(2+) channels (VSCC). Furthermore, GnRH-potentiated GH rebound could be alleviated by inactivation of PKC, removal of extracellular Ca(2+), blockade of VSCC, and inhibition of Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII). Inactivation of AC and PKA, however, was not effective in this regard. These results, as a whole, suggest that GnRH potentiation of GH rebound following NE inhibition is mediated by PKC coupled to Ca(2+) entry through VSCC and subsequent activation of CaMKII. Apparently, the Ca(2+)-dependent cascades are involved in GH secretion during the rebound phase but are not essential for the initiation of GnRH potentiation. Since GnRH has been previously shown to have no effects on cAMP synthesis in goldfish pituitary cells, the involvement of cAMP-dependent mechanisms in GnRH potentiation is rather unlikely.     

The time effect of pressure on tissue viability: investigation using an experimental rat model

An experimental rat model was used to investigate the time-pressure effect on tissue viability. External loading equivalent to 13.3 kPa (100 mm Hg) of pressure was applied to the greater trochanter and tibialis area of Sprague-Dawley rats using pneumatic indentors for duration of 6 hrs each day for 1 to 4 days. It was observed that postocclusive hyperemic responses were gradually increased at the trochanter throughout the 4 days of loading, whereas for the tibia there was a significant increase (P = 0.04) in postocclusive hyperemic flow between Days 2 and 3. In histologic evaluations, cutaneous tissue damage was observed at the trochanter area but not at the tibialis area after 2 consecutive days of load application. In contrast, degeneration of muscle cells characterized by numerous increases of nuclei occupying the central of the muscle fibers was observed after 2 days of load application at the tibialis. The situation was found to progress with time (P = 0.17). The presence of other histologic signs, including the internalization of peripherally located nuclei, replacement of muscle cells by fibrosis and adipose tissues, and the presence of pyknotic nuclei as well as karyorrhexis, confirmed that the affected tissues were damaged. These findings suggest that postocclusive hyperemia and the distress of tissues under loading could be closely related.     

Post‐release dispersal and spawning movements of a translocated lake sturgeon (Acipenser fulvescens,Rafinesque 1817) population in the Mattagami River,Ontario

Species translocations are increasingly being used as a management tool to mitigate population losses due to such factors as habitat degradation and fragmentation, but post‐introduction follow‐up is relatively sparse. Post‐translocation telemetry can assess success by identifying activity, emigrations, survival, habitat usage, and reproductive events, aiding in the continued management of translocated populations and informing future efforts. This study assessed movement of translocated adult lake sturgeon (Acipenser fulvescens) immediately post‐release and a decade later, and tested for associations between environmental variables and spawning movements. Prior to their translocation in 2002, 13 of 51 adult lake sturgeon were surgically implanted with radio telemetry tags and tracked for 1 year. In 2011 and 2013, eight additional adults were captured within the reintroduction site and implanted with radio‐tags. Six of the 13 sturgeon tagged in 2002 dispersed downstream over a dam during the early post‐release period. In spring 2014, tagged adults were tracked to the spillway at the release area's inflow, and spawning was confirmed by larval captures. Movement data for tagged adults differed between the two tracking periods, showing marked differences in behaviour over time. Water velocity was correlated with upstream and downstream spawning movements, with water temperature also correlated with downstream movement. Research regarding post‐translocation movement and dispersal provides insight on behavioural responses following translocation, and may improve outcomes by informing future efforts.     

Identification and Characterization of PERK Activators by Phenotypic Screening and Their Effects on NRF2 Activation

Endoplasmic reticulum stress plays a critical role to restore the homeostasis of protein production in eukaryotic cells. This vital process is hence involved in many types of diseases including COPD. PERK, one branch in the ER stress signaling pathways, has been reported to activate NRF2 signaling pathway, a known protective response to COPD. Based on this scientific rationale, we aimed to identify PERK activators as a mechanism to achieve NRF2 activation. In this report, we describe a phenotypic screening assay to identify PERK activators. This assay measures phosphorylation of GFP-tagged eIF2α upon PERK activation via a cell-based LanthaScreen technology. To obtain a robust assay with sufficient signal to background and low variation, multiple parameters were optimized including GFP-tagged eIF2α BacMam concentration, cell density and serum concentration. The assay was validated by a tool compound, Thapsigargin, which induces phosphorylation of eIF2α. In our assay, this compound showed maximal signal window of approximately 2.5-fold with a pEC₅₀ of 8.0, consistent with literature reports. To identify novel PERK activators through phosphorylation of eIF2α, a focused set of 8,400 compounds was screened in this assay at 10 µM. A number of hits were identified and validated. The molecular mechanisms for several selected hits were further characterized in terms of PERK activation and effects on PERK downstream components. Specificity of these compounds in activating PERK was demonstrated with a PERK specific inhibitor and in PERK knockout mouse embryonic fibroblast (MEF) cells. In addition, these hits showed NRF2-dependent anti-oxidant gene induction. In summary, our phenotypic screening assay is demonstrated to be able to identify PERK specific activators. The identified PERK activators could potentially be used as chemical probes to further investigate this pathway as well as the link between PERK activation and NRF2 pathway activation.     

Cryptococcus neoformans capsule regrowth experiments reveal dynamics of enlargement and architecture

The polysaccharide capsule of fungal pathogen Cryptococcus neoformans is a critical virulence factor that has historically evaded complete characterization. Cryptococcal polysaccharides are known to either remain attached to the cell as capsular polysaccharides (CPSs) or to be shed into the extracellular space as exopolysaccharides (EPSs). While many studies have examined the properties of EPS, far less is known about CPS. In this work, we detail the development of new physical and enzymatic methods for the isolation of CPS which can be used to explore the architecture of the capsule and isolated capsular material. We show that sonication or Glucanex enzyme cocktail digestion yields soluble CPS preparations, while use of a French pressure cell press or Glucanex digestion followed by cell disruption removed the capsule and produced cell wall–associated polysaccharide aggregates that we call “capsule ghosts”, implying an inherent organization that allows the CPS to exist independent of the cell wall surface. Since sonication and Glucanex digestion were noncytotoxic, it was also possible to observe the cryptococcal cells rebuilding their capsule, revealing the presence of reducing end glycans throughout the capsule. Finally, analysis of dimethyl sulfoxide-extracted and sonicated CPS preparations revealed the conservation of previously identified glucuronoxylomannan motifs only in the sonicated CPS. Together, these observations provide new insights into capsule architecture and synthesis, consistent with a model in which the capsule is assembled from the cell wall outward using smaller polymers, which are then compiled into larger ones.     

Metabolite changes associated with heat shocked avian fibroblast mitochondira

A previous report from our laboratory (Collier et al 1993) showed that the elongated tubules of mitochondria in the cytoplasm of cultured chicken embryo fibroblasts collpased to irregularly shaped structures surrounding the nuclear membrane after a 1 h heat shock treatment. The normal mitochondiral morphology reappeared upon removal of then thermal stress. We have now determined that several changes occured in mitochondrial-related metabolites under these same heat shock and recovery conditions. Among these were significant decreases in the levels of fumarate and malate and increases in the amounts of aspartate and glutamate. In contrast, other intermediates of the tri-carboxylic acid cycle were unaltered as were levels of ATP and phosphocreatine. The changes observed might result from heat shock-induced changes in enzyme activities of the mitochondria or from alterations in the membrane-embedded specialized carrier proteins that transport metabolites between cytosol and mitochondria, from a disorganization of the electron-transport system normally coupled to oxidative metabolism. The rapid recovery, however, suggested that these changes were transient and readily reversible.     

LEVELS OF CEREBRAL CORTICAL GLYCOLYTIC AND CITRIC ACID CYCLE METABOLITES DURING HYPOGLYCEMIC STUPOR AND ITS REVERSAL

Abstract— Blood glucose, cerebral cortical glucose, and eight metabolites of the glycolytic pathway and citric acid cycle were measured during insulin hypoglycemic stupor and during the first 100s after glucose administration. In hypoglycemic mice that had lost righting ability, blood and brain glucose were decreased 89% and 96% respectively, but glucose-6-phosphate fell only 23%. Other glycolytic and citric acid cycle intermediates were decreased 31–77%. Fructose bisphosphate, 3-phosphoglycerate and phosphopyruvate fell more than glucose-6-phosphate, but less than pyruvate and lactate. Citrate fell less than a-ketoglutarate and malate. These results suggest that in severe hypoglycemia there is a decrease in brain glucose utilization, mediated by phosphofructokinase, but probably caused by decreased neuronal activity. An intravenous injection of glucose restored brain glucose to 75% of normal within 10s and caused return of righting ability within 60s. Glucose-6-phosphate, fructose bisphosphate, 3-phosphoglycerate, and phosphopyruvate rose to normal or near normal levels within 60s, whereas pyruvate, lactate, citrate, ã-ketoglutarate, and malate changed little in this period. This suggests that although glucose given to hypoglycemic animals rapidly enters the glycolytic pathway in brain (and behavior is almost normal), total neuronal activity, and hence overall glucose metabolism, remains subnormal for several minutes.     

Recombinational exchanges at the capsular polysaccharide biosynthetic locus lead to frequent serotype changes among natural isolates of Streptococcus pneumoniae

Serotype 19F variants of the major Spanish multiresistant serotype 23F clone of Streptococcus pneumoniae have been proposed to have arisen by recombinational exchanges at the capsular biosynthetic locus. Members of the Spanish multiresistant serotype 23F clone and the serotype 19F variants were confirmed to be essentially identical in overall genotype, as they were indistinguishable by REP-PCR, and had identical sequences at three polymorphic housekeeping genes. Eight serotype 19F variants were studied and all had large recombinational replacements at the capsular biosynthetic locus. In all cases, one of the recombinational cross-over points appeared to be upstream of dexB which flanks one end of the capsular locus, and in six of the variants the other cross-over point was downstream of aliA , which flanks the other end of the locus. In two strains a recombinational cross-over point between the introduced serotype 19F capsular region and that of the Spanish serotype 23F clone could be clearly identified, within cpsN in one strain and within cpsM in the other. The differences in the recombinational junctions and sequence polymorphisms within the introduced capsular genes, suggested that the eight serotype 19F variants emerged on at least four separate occasions. Changes in capsular type by recombination may therefore be relatively frequent in pneumococci and this has implications for the long-term efficacy of conjugate pneumococcal vaccines that will protect against only a limited number of serotypes.