Ionotropic glutamate receptor AMPA 1 is associated with ovulation rate

Ionotropic glutamate receptors mediate most excitatory neurotransmission in the central nervous system by opening ion channels upon the binding of glutamate. Despite the essential roles of glutamate in the control of reproduction and anterior pituitary hormone secretion, there is a limited understanding of how glutamate receptors control ovulation. Here we reveal the function of the ionotropic glutamate receptor AMPA-1 (GRIA1) in ovulation. Based on a genome-wide association study in Bos taurus, we found that ovulation rate is influenced by a variation in the N-terminal leucine/isoleucine/valine-binding protein (LIVBP) domain of GRIA1, in which serine is replaced by asparagine. GRIA1(Asn) has a weaker affinity to glutamate than GRIA1(Ser), both in Xenopus oocytes and in the membrane fraction of bovine brain. This single amino acid substitution leads to the decreased release of gonadotropin-releasing hormone (GnRH) in immortalized hypothalamic GT1-7 cells. Cows with GRIA1(Asn) have a slower luteinizing hormone (LH) surge than cows with GRIA1(Ser). In addition, cows with GRIA1(Asn) possess fewer immature ovarian follicles before superovulation and have a lower response to hormone treatment than cows with GRIA1(Ser). Our work identified that GRIA1 is a critical mediator of ovulation and that GRIA1 might be a useful target for reproductive therapy.     

An Optimized Pentaplex PCR for Detecting DNA Mismatch Repair-Deficient Colorectal Cancers

Purpose

Microsatellite instability (MSI) is used to screen colorectal cancers (CRC) for Lynch Syndrome, and to predict outcome and response to treatment. The current technique for measuring MSI requires DNA from normal and neoplastic tissues, and fails to identify tumors with specific DNA mismatch repair (MMR) defects. We tested a panel of five quasi-monomorphic mononucleotide repeat markers amplified in a single multiplex PCR reaction (pentaplex PCR) to detect MSI.

Experimental Design

We investigated a cohort of 213 CRC patients, comprised of 114 MMR-deficient and 99 MMR-proficient tumors. Immunohistochemical (IHC) analysis evaluated the expression of MLH1, MSH2, PMS2 and MSH6. MSI status was defined by differences in the quasi-monomorphic variation range (QMVR) from a pool of normal DNA samples, and measuring differences in allele lengths in tumor DNA.

Results

Amplification of 426 normal alleles allowed optimization of the QMVR at each marker, and eliminated the requirement for matched reference DNA to define MSI in each sample. Using ≥2/5 unstable markers as the criteria for MSI resulted in a sensitivity of 95.6% (95% CI = 90.1–98.1%) and a positive predictive value of 100% (95% CI = 96.6%–100%). Detection of MSH6-deficiency was limited using all techniques. Data analysis with a three-marker panel (BAT26, NR21 and NR27) was comparable in sensitivity (97.4%) and positive predictive value (96.5%) to the five marker panel. Both approaches were superior to the standard approach to measuring MSI.

Conclusions

An optimized pentaplex (or triplex) PCR offers a facile, robust, very inexpensive, highly sensitive, and specific assay for the identification of MSI in CRC.     

Effects of elevated seawater temperature and phosphate enrichment on the crustose coralline alga Porolithon onkodes (Rhodophyta)

Both global and local environmental changes threaten coral reef ecosystems. To evaluate the effects of high seawater temperature and phosphate enrichment on reef‐building crustose coralline algae, fragments of Porolithon onkodes were cultured for 1 month under laboratory conditions. The calcification rate of the coralline algae was not affected at 30°C, but it decreased to the negatives at 32°C in comparison to the control treatment of 27°C, indicating that the temperature threshold for maintaining positive production of calcium carbonate lies between 30 and 32°C. Phosphate enrichment of 1–2 μmol L ^?1 did not affect the calcification rate. The net oxygen production rate was enhanced by phosphate enrichment, suggesting that the photosynthetic rate was limited by the availability of phosphate. It was concluded that moderate phosphate enrichment does not directly deteriorate algal calcification but instead ameliorates the negative effects of high seawater temperature on algal photosynthesis.     

Recovery of blood gases and haematological parameters upon anaesthesia with benzocaine,MS-222 or Aqui-S in the air-breathing catfish <Emphasis Type="Italic">Pangasianodon hypophthalmus</Emphasis>

Fish anaesthesia is used to minimize handling stress and damage during harvesting, transportation, and surgical procedures. Through depression of cardiovascular and respiratory functions, it causes significant changes in blood gases and pH. Here, we present the effects of benzocaine (100 mg l^?1), MS-222 (100 mg l^?1), and Aqui-S (30 mg l^?1) on blood gases and haematological parameters of commercial-sized (≈1 kg) striped catfish (Pangasianodon hypophthalmus) and the time course of recovery. Blood was taken through a dorsal aorta catheter immediately after catheterization, and regularly during the following 72 h recovery in aerated water. All anaesthetics caused increases in PCO₂ and lactate resulting in a decrease in pH_e, closely mirrored by RBC pH_i, as well as a marked rise in Hct, associated with elevated [cortisol] and [glucose] and increased RBC counts but no change in RBC volume, as confirmed by the lack of an adrenergic response of RBC in vitro. All anaesthetics showed similar efficacy and blood parameters were normalized within 24 to 48 h.     

Underwater observations of the rare deep-sea fish <Emphasis Type="Italic">Triodon macropterus</Emphasis> (Actinopterygii,Tetraodontiformes, Triodontidae), with comments on the fine structure of the scales

Underwater observations of two living individuals and an examination of a freshly dead individual of the rare deep-sea fish Triodon macropterus revealed that they usually have the large ventral flap completely uplifted and seamlessly retracted into the abdominal region of the trunk: one individual was collected at a depth of 280 m by hook and line east of Tonaki-jima Island, Ryukyu Islands, and kept in a tank at the Okinawa Churaumi Aquarium for six years; the second living individual was observed by the ROV Hakuyo 2000 at a depth of 275 m east of Ishigaki-jima Island, Ryukyu Islands; and another individual collected at depths of 250?350 m by hook and line off Funchu Point, Yoron-jima Island (27°00.15’N, 128°26.06’E), Ryukyu Islands, provided an opportunity to demonstrate that the ventral flap could be manually uplifted by rotating the pelvis. When the ventral flap was uplifted in all of these specimens, whether naturally or manually, there were no scaleless linear bands or streaks and gaps evident in the skin between the retracted ventral flap and the abdominal region. The fine structure of the scales of the body and the ventral flap was observed by light microscopy, scanning electron microscopy, and X-ray CT scanning. The scales of the ventral flap are arranged in rows radiating from the antero-dorsal part of the ventral flap where the anterior part of the pelvis articulates with and rotates around the pectoral girdles. The orientation of the rows on the ventral flap changes from a posterior direction dorsally to a posteroventral direction more ventrally. A ridge with many sharp serrations is raised and bent from the edge of each scale on the body and ventral flap. The raised ridges on the lower scales on the ventral flap become packed together with the upper scales to form a seamless surface when the ventral flap is uplifted into the abdominal region.     

Lipophagy maintains energy homeostasis in the kidney proximal tubule during prolonged starvation

Macroautophagy/autophagy is a self-degradation process that combats starvation. Lipids are the main energy source in kidney proximal tubular cells (PTCs). During starvation, PTCs increase fatty acid (FA) uptake, form intracellular lipid droplets (LDs), and hydrolyze them for use. The involvement of autophagy in lipid metabolism in the kidney remains largely unknown. Here, we investigated the autophagy-mediated regulation of renal lipid metabolism during prolonged starvation using PTC-specific Atg5-deficient (atg5-TSKO) mice and an in vitro serum starvation model. Twenty-four h of starvation comparably induced LD formation in the PTCs of control and atg5-TSKO mice; however, additional 24 h of starvation reduced the number of LDs in control mice, whereas increases were observed in atg5-TSKO mice. Autophagic degradation of LDs (lipophagy) in PTCs was demonstrated by electron microscopic observation and biochemical analysis. In vitro pulse-chase assays demonstrated that lipophagy mobilizes FAs from LDs to mitochondria during starvation, whereas impaired LD degradation in autophagy-deficient PTCs led to decreased ATP production and subsequent cell death. In contrast to the in vitro assay, despite impaired LD degradation, kidney ATP content was preserved in 48-h starved atg5-TSKO mice, probably due to increased utilization of ketone bodies. This compensatory mechanism was accompanied by a higher plasma FGF21 (fibroblast growth factor 21) level and its expression in the PTCs; however, this was not essential for the production of ketone bodies in the liver during prolonged starvation. In conclusion, lipophagy combats prolonged starvation in PTCs to avoid cellular energy depletion.     

Effects of magnesium deficiency on magnesium uptake activity of rice root,evaluated using <Superscript>28</Superscript> Mg as a tracer

Aims

The mechanisms underlying magnesium (Mg) uptake by plant roots remain to be fully elucidated. In particular, there is little information about the effects of Mg deficiency on Mg uptake activity. A Mg uptake kinetic study is essential for better understanding the Mg uptake system.

Methods

We performed a Mg uptake tracer experiment in rice plants using ²⁸?Mg.

Results

Mg uptake was mediated by high- and low-affinity transport systems. The K _m value of the high-affinity transport system was approximately 70 μM under Mg-deficient conditions. The Mg uptake activity was promoted by Mg deficiency, which in turn fell to the basal level after 5- min of Mg resupply. The induced uptake rate was inhibited by ionophore treatment, suggesting that an energy-dependent uptake system is enhanced by Mg deficiency.

Conclusions

The Mg uptake changes rapidly with Mg conditions in rice, as revealed by a ²⁸?Mg tracer experiment. This technique is expected to be applicable for Mg uptake analyses, particularly in mutants or other lines.