Choline Transport Activity Regulates Phosphatidylcholine Synthesis through Choline Transporter Hnm1 Stability

Choline is a precursor for the synthesis of phosphatidylcholine through the CDP-choline pathway. Saccharomyces cerevisiae expresses a single high affinity choline transporter at the plasma membrane, encoded by the HNM1 gene. We show that exposing cells to increasing levels of choline results in two different regulatory mechanisms impacting Hnm1 activity. Initial exposure to choline results in a rapid decrease in Hnm1-mediated transport at the level of transporter activity, whereas chronic exposure results in Hnm1 degradation through an endocytic mechanism that depends on the ubiquitin ligase Rsp5 and the casein kinase 1 redundant pair Yck1/Yck2. We present details of how the choline transporter is a major regulator of phosphatidylcholine synthesis.     

Thermoregulation in leopard tortoises in the Nama-Karoo: The importance of behaviour and core body temperatures

Despite being ectotherms, reptiles have an ability to thermoregulate which is enhanced by adopting a variety of behavioural mechanisms. Different behavioural postures, the use of retreat sites and selection of microhabitats enable reptiles to maintain their core body temperatures (T_b) above that of ambient temperatures (T_a) in winter or below T_a maximum in summer. This study describes the daily activity patterns of leopard tortoises (Stigmochelys pardalis) in relation to T_b and T_a, and the extent to which leopard tortoises can manipulate their T_b in response to seasonal changes in T_a. Ten and nine leopard tortoises were radio-tracked in 2002 and 2003, respectively and cloacal T_b and behaviours observed. Core T_bs were measured using Thermocron iButtons^™ surgically implanted into the body cavities of 4 and 5 adult telemetered tortoises for summer and winter 2003, respectively. There were seasonal differences in the extent to which certain behaviours were practiced and the time of day that these occurred. Leopard tortoises generally had unimodal activity patterns in winter and bimodal ones in summer. In winter tortoises were active at lower T_bs, and at lower T_a, than in summer. Tortoises maintained their core T_b well below T_a minimum profiles in summer and well above these in winter. Core T_b closely followed the increase in T_a minimum profiles in the mornings, however tortoises exhibited an extended thermal lag when T_a minimum profiles cooled overnight. By using different behavioural mechanisms in summer and winter, leopard tortoises maintained their core T_b at different levels compared with T_a minimum and maximum profiles. Consequently although they are ectotherms, they maintained their core T_b independent of T_a.     

Multiple, distributed interactions of μ-conotoxin PIIIA associated with broad targeting among voltage-gated sodium channels

The first μ-conotoxin studied, μCTX GIIIA, preferentially blocked voltage-gated skeletal muscle sodium channels, Na(v)1.4, while μCTX PIIIA was the first to show significant blocking action against neuronal voltage-gated sodium channels. PIIIA shares >60% sequence identity with the well-studied GIIIA, and both toxins preferentially block the skeletal muscle sodium channel isoform. Two important features of blocking by wild-type GIIIA are the toxin's high binding affinity and the completeness of block of a single channel by a bound toxin molecule. With GIIIA, neutral replacement of the critical residue, Arg-13, allows a residual single-channel current (~30% of the unblocked, unitary amplitude) when the mutant toxin is bound to the channel and reduces the binding affinity of the toxin for Na(v)1.4 (~100-fold) [Becker, S., et al. (1992) Biochemistry 31, 8229-8238]. The homologous residue in PIIIA, Arg-14, is also essential for completeness of block but less important in the toxin's binding affinity (~55% residual current and ~11-fold decrease in affinity when substituted with alanine or glutamine). The weakened dominance of this key arginine in PIIIA is also seen in the fact that there is not just one (R13 in GIIIA) but three basic residues (R12, R14, and K17) for which individual neutral replacement enables a substantial residual current through the bound channel. We suggest that, despite a high degree of sequence conservation between GIIIA and PIIIA, the weaker dependence of PIIIA's action on its key arginine and the presence of a nonconserved histidine near the C-terminus may contribute to the greater promiscuity of its interactions with different sodium channel isoforms.     

GROα regulates human embryonic stem cell self-renewal or adoption of a neuronal fate

Previously we reported that feeders formed from human placental fibroblasts (hPFs) support derivation and long-term self-renewal of human embryonic stem cells (hESCs) under serum-free conditions. Here, we show, using antibody array and ELISA platforms, that hPFs secrete ～6-fold higher amounts of the CXC-type chemokine, GROα, than IMR 90, a human lung fibroblast line, which does not support hESC growth. Furthermore, immunocytochemistry and immunoblot approaches revealed that hESCs express CXCR, a GROα receptor. We used this information to develop defined culture medium for feeder-free propagation of hESCs in an undifferentiated state. Cells passaged as small aggregates and maintained in the GROα-containing medium had a normal karyotype, expressed pluripotency markers, and exhibited apical-basal polarity, i.e., had the defining features of pluripotent hESCs. They also differentiated into the three primary (embryonic) germ layers and formed teratomas in immunocompromised mice. hESCs cultured as single cells in the GROα-containing medium also had a normal karyotype, but they downregulated markers of pluripotency, lost apical-basal polarity, and expressed markers that are indicative of the early stages of neuronal differentiation-βIII tubulin, vimentin, radial glial protein, and nestin. These data support our hypothesis that establishing and maintaining cell polarity is essential for the long-term propagation of hESCs in an undifferentiated state and that disruption of cell-cell contacts can trigger adoption of a neuronal fate.     

Methicillin-resistant Staphylococcus aureus (MRSA) pyruvate kinase as a target for bis-indole alkaloids with antibacterial activities

Novel classes of antimicrobials are needed to address the emergence of multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA). We have recently identified pyruvate kinase (PK) as a potential novel drug target based upon it being an essential hub in the MRSA interactome (Cherkasov, A., Hsing, M., Zoraghi, R., Foster, L. J., See, R. H., Stoynov, N., Jiang, J., Kaur, S., Lian, T., Jackson, L., Gong, H., Swayze, R., Amandoron, E., Hormozdiari, F., Dao, P., Sahinalp, C., Santos-Filho, O., Axerio-Cilies, P., Byler, K., McMaster, W. R., Brunham, R. C., Finlay, B. B., and Reiner, N. E. (2011) J. Proteome Res. 10, 1139-1150; Zoraghi, R., See, R. H., Axerio-Cilies, P., Kumar, N. S., Gong, H., Moreau, A., Hsing, M., Kaur, S., Swayze, R. D., Worrall, L., Amandoron, E., Lian, T., Jackson, L., Jiang, J., Thorson, L., Labriere, C., Foster, L., Brunham, R. C., McMaster, W. R., Finlay, B. B., Strynadka, N. C., Cherkasov, A., Young, R. N., and Reiner, N. E. (2011) Antimicrob. Agents Chemother. 55, 2042-2053). Screening of an extract library of marine invertebrates against MRSA PK resulted in the identification of bis-indole alkaloids of the spongotine (A), topsentin (B, D), and hamacanthin (C) classes isolated from the Topsentia pachastrelloides as novel bacterial PK inhibitors. These compounds potently and selectively inhibited both MRSA PK enzymatic activity and S. aureus growth in vitro. The most active compounds, cis-3,4-dihyrohyrohamacanthin B (C) and bromodeoxytopsentin (D), were identified as highly potent MRSA PK inhibitors (IC(50) values of 16-60 nM) with at least 166-fold selectivity over human PK isoforms. These novel anti-PK natural compounds exhibited significant antibacterial activities against S. aureus, including MRSA (minimal inhibitory concentrations (MIC) of 12.5 and 6.25 μg/ml, respectively) with selectivity indices (CC(50)/MIC) >4. We also report the discrete structural features of the MRSA PK tetramer as determined by x-ray crystallography, which is suitable for selective targeting of the bacterial enzyme. The co-crystal structure of compound C with MRSA PK confirms that the latter is a target for bis-indole alkaloids. It elucidates the essential structural requirements for PK inhibitors in "small" interfaces that provide for tetramer rigidity and efficient catalytic activity. Our results identified a series of natural products as novel MRSA PK inhibitors, providing the basis for further development of potential novel antimicrobials.     

Effects of Piper hispidinervum on spermatogenesis and histochemistry of ovarioles of Spodoptera frugiperda

The fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), not only damages crops, but controlling its population also requires synthetic insecticides, which leads to selection of resistant populations and environmental contamination. Essential oils are an alternative for controlling this insect. There are few studies of the effects of these oils on the insect's reproductive system. We evaluated the effects of the long pepper, Piper hispidinervum, essential oil on the gonads of the armyworm and tested its possible influence on the fertility of this insect. Dosages of 30 and 50 mg/ml were tested in 3^rd instar caterpillars using the leaf immersion method. Testes and ovarioles were collected, fixed with 10% formalin and embedded in Historesin. The sections were stained with toluidine blue and Mallory trichrome to detect connective tissue, periodic acid-Schiff to detect neutral carbohydrates, and bromophenol blue to detect proteins. We found that the long pepper essential oil affected negatively the spermatogenesis and altered the histochemistry of the ovarioles of S. frugiperda. The effects of long pepper oil suggest that it is a promising tool for controlling the armyworm pest.     

Ascending Aortic Constriction in Rats for Creation of Pressure Overload Cardiac Hypertrophy Model

Ascending aortic constriction is the most common and successful surgical model for creating pressure overload induced cardiac hypertrophy and heart failure. Here, we describe a detailed surgical procedure for creating pressure overload and cardiac hypertrophy in rats by constriction of the ascending aorta using a small metallic clip. After anesthesia, the trachea is intubated by inserting a cannula through a half way incision made between two cartilage rings of trachea. Then a skin incision is made at the level of the second intercostal space on the left chest wall and muscle layers are cleared to locate the ascending portion of aorta. The ascending aorta is constricted to 50–60% of its original diameter by application of a small sized titanium clip. Following aortic constriction, the second and third ribs are approximated with prolene sutures. The tracheal cannula is removed once spontaneous breathing was re-established. The animal is allowed to recover on the heating pad by gradually lowering anesthesia. The intensity of pressure overload created by constriction of the ascending aorta is determined by recording the pressure gradient using trans-thoracic two dimensional Doppler-echocardiography. Overall this protocol is useful to study the remodeling events and contractile properties of the heart during the gradual onset and progression from compensated cardiac hypertrophy to heart failure stage.