PA,a stress-induced short cut to switch-on ethylene signalling by switching-off CTR1?

Constitutive triple response 1 (CTR1) is a protein kinase that represses plant responses to ethylene. Recently, we have shown that CTR1 function is negatively regulated by the lipid second messenger phosphatidic acid (PA) in vitro.¹ PA was shown to inhibit (1) CTR1''s protein kinase activity, (2) the intramolecular interaction between N-terminus and kinase domain, and (3) the interaction of CTR1 with the ethylene receptor ETR1. PA typically accumulates within minutes in response to biotic or abiotic stresses, which are known to induce ethylene formation. Although long-term treatment with ethephon does stimulate PA accumulation, our results show no fast increase in PA in response to ethylene. A speculative model is presented which explains how stress-induced PA formation could switch on downstream ethylene responses via interaction of the lipid with CTR1.Key words: lipid signaling, phosphatidic acid, ethylene, constitutive triple response 1, plant stress signaling, protein kinase, phospholipase D     

The utility of the morphological variation of pollen for resolving the evolutionary history of Billia (subfam. Hippocastanoideae,Sapindaceae)

In this study, we examined the utility of pollen morphology for resolving questions about the evolutionary history of Billia, which is a poorly known genus of Neotropical trees. Billia has been traditionally circumscribed with two species and treated as sister to Aesculus L. However, the number of species in Billia is uncertain, because the genus exhibits abundant morphological diversity but little discontinuous variation. Therefore, Billia may be monotypic and highly polymorphic, or it may have two species with blurred boundaries due to incipient speciation and/or hybridization. Moreover, one recent molecular phylogenetic study shows Billia nested withinAesculus. Our work sought to address the following questions: (i) Are there discontinuities in the pollen of Billia that may suggest species boundaries? (ii) Does the pollen of Billia show evidence for inter-specific hybridization? (iii) Do the exine morphology and size of pollen in Billia differ from those in Aesculus? Our results from scanning electron microscopy showed that pollen exine morphology is not taxonomically informative in Billia but that there are significant differences in pollen size between red- and white-flowered individuals. Thus, our pollen data support the utility of flower color in Billia for species delimitation. Our assessments of pollen viability do not support hybridization in the genus, but cannot be used to rule it out. Finally, pollen exine morphology may lend some support to an evolutionary origin ofBillia within eastern North American Aesculus. In contrast, data on pollen size suggest that Billia may belong in a topological position outside of Aesculus.     

Molecular Evolution of Broadly Neutralizing Llama Antibodies to the CD4-Binding Site of HIV-1

To date, no immunization of humans or animals has elicited broadly neutralizing sera able to prevent HIV-1 transmission; however, elicitation of broad and potent heavy chain only antibodies (HCAb) has previously been reported in llamas. In this study, the anti-HIV immune responses in immunized llamas were studied via deep sequencing analysis using broadly neutralizing monoclonal HCAbs as a guides. Distinct neutralizing antibody lineages were identified in each animal, including two defined by novel antibodies (as variable regions called VHH) identified by robotic screening of over 6000 clones. The combined application of five VHH against viruses from clades A, B, C and CRF_AG resulted in neutralization as potent as any of the VHH individually and a predicted 100% coverage with a median IC50 of 0.17 µg/ml for the panel of 60 viruses tested. Molecular analysis of the VHH repertoires of two sets of immunized animals showed that each neutralizing lineage was only observed following immunization, demonstrating that they were elicited de novo. Our results show that immunization can induce potent and broadly neutralizing antibodies in llamas with features similar to human antibodies and provide a framework to analyze the effectiveness of immunization protocols.     

Autonomic Substrates of the Response to Pups in Male Prairie Voles

Caregiving by nonparents (alloparenting) and fathers is a defining aspect of human social behavior, yet this phenomenon is rare among mammals. Male prairie voles (Microtus ochrogaster) spontaneously exhibit high levels of alloparental care, even in the absence of reproductive experience. In previous studies, exposure to a pup was selectively associated with increased activity in oxytocin and vasopressin neurons along with decreased plasma corticosterone. In the present study, physiological, pharmacological and neuroanatomical methods were used to explore the autonomic and behavioral consequences of exposing male prairie voles to a pup. Reproductively naïve, adult male prairie voles were implanted with radiotransmitters used for recording ECG, temperature and activity. Males responded with a sustained increase in heart-rate during pup exposure. This prolonged increase in heart rate was not explained by novelty, locomotion or thermoregulation. Although heart rate was elevated during pup exposure, respiratory sinus arrhythmia (RSA) did not differ between these males and males exposed to control stimuli indicating that vagal inhibition of the heart was maintained. Blockade of beta-adrenergic receptors with atenolol abolished the pup-induced heart rate increase, implicating sympathetic activity in the pup-induced increase in heart rate. Blockade of vagal input to the heart delayed the males’ approach to the pup. Increased activity in brainstem autonomic regulatory nuclei was also observed in males exposed to pups. Together, these findings suggest that exposure to a pup activates both vagal and sympathetic systems. This unique physiological state (i.e. increased sympathetic excitation of the heart, while maintaining some vagal cardiac tone) associated with male caregiving behavior may allow males to both nurture and protect infants.     

A high-throughput microscale method to release N-linked oligosaccharides from glycoproteins for matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis

This report describes a convenient method for the rapid and efficient release of N-linked oligosaccharides from low microgram amounts of glycoproteins. A 96-well MultiScreen assay system containing a polyvinylidene difluoride (PVDF) membrane is employed to immobilize glycoproteins for subsequent enzymatic deglycosylation. Recombinant tissue-type plasminogen activator (rt-PA) is used to demonstrate the deglycosylation of 0.1-50 micrograms of a glycoprotein. This method enabled the recovery of a sufficient amount of N-linked oligosaccharides released enzymatically with peptide N-glycosidase F (PNGaseF) from as little as 0.5 microgram rt-PA for subsequent analysis by matrix-assisted laser desorption/ionization time-of-flight (MALDI- TOF) mass spectrometry. The immobilization of rt-PA to the PVDF membrane did not sterically inhibit the PNGaseF-mediated release of oligosaccharides from rt-PA as determined by tryptic mapping experiments. Comparison of the oligosaccharides released from 50 micrograms of rt-PA by either the 96-well plate method or by a standard solution digestion procedure showed no significant differences in the profiles obtained by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Both neutral and sialylated oligosaccharide standards spiked into wells were recovered equally as determined by HPAEC-PAD. One advantage of this approach is that reduction and alkylation can be performed on submicrogram amounts of glycoproteins with easy removal of reagents prior to PNGaseF digestion. In addition, this method allows 60 glycoprotein samples to be deglycosylated in 1 day with MALDI-TOF or HPAEC-PAD analysis being performed on the following day.      

Irradiation induces G2/M cell cycle arrest and apoptosis in p53-deficient lymphoblastic leukemia cells without affecting Bcl-2 and Bax expression

The tumor suppressor p53 has been implicated in gamma irradiation-induced apoptosis. To investigate possible consequences of wild-type p53 loss in leukemia, we studied the effect of a single dose of gamma irradiation upon p53-deficient human T-ALL (acute lymphoblastic leukemia) CCRF - CEM cells. Exposure to 3 - 96 Gy caused p53-independent cell death in a dose and time-dependent fashion. By electron microscopic and other criteria, this cell death was classified as apoptosis. At low to intermediate levels of irradiation, apoptosis was preceded by accumulation of cells in the G2/M phase of the cell division cycle. Expression of Bcl-2 and Bax were not detectably altered after irradiation. Expression of the temperature sensitive mouse p53 V135 mutant induced apoptosis on its own but only slightly increased the sensitivity of CCRF - CEM cells to gamma irradiation. Thus, in these, and perhaps other leukemia cells, a p53- and Bcl-2/Bax-independent mechanism is operative that efficiently senses irradiation effects and translates this signal into arrest in the G2/M phase of the cell cycle and subsequent apoptosis.     

Retinoic acid receptor belongs to a subclass of nuclear receptors that do not form "docking" complexes with hsp90.

We have recently reported that, in contrast to the glucocorticoid receptor, the thyroid hormone receptor does not bind to hsp90 when the receptor is translated in rabbit reticulocyte lysate [Dalman, F. C., Koenig, R. J., Perdew, G. H., Massa, E., & Pratt, W. B. (1990) J. Biol. Chem. 265, 3615-3618]. All of the steroid receptors that are known to bind hsp90 are recovered in the cytosolic fraction when hormone-free cells are ruptured in hypotonic buffer. In contrast, unliganded thyroid hormone receptors and retinoic acid receptors are tightly associated with nuclear components. In this paper, we translated the human estrogen receptor and the human retinoic acid receptor in reticulocyte lysate and then immunoadsorbed the [35S]methionine-labeled translation products with the 8D3 monoclonal antibody against hsp90. The estrogen receptor is bound to hsp90, as indicated by coimmunoadsorption, but the retinoic acid receptor is not. Translation and immunoadsorption of chimeric proteins containing the DNA binding domain of one receptor and the N-terminal and COOH-terminal segments of the other show that the DNA binding finger region of the estrogen receptor is neither necessary nor sufficient for hsp90 binding. These observations suggest that there are two classes within the steroid receptor family. In one class (e.g., glucocorticoid, mineralocorticoid, sex hormone, and dioxin receptors), the receptors bind to hsp90 and remain in some kind of inactive "docking" mode until hormone-triggered release of hsp90 occurs. In the retinoic acid/thyroid hormone class, the unligated receptors do not bind to hsp90, and the receptors appear to proceed directly to their high-affinity nuclear acceptor sites without entering the "docking" state.     

Rapid translocation of hepatic glucokinase in response to intraduodenal glucose infusion and changes in plasma glucose and insulin in conscious rats

The rate of liver glucokinase (GK) translocation from the nucleus to the cytoplasm in response to intraduodenal glucose infusion and the effect of physiological rises of plasma glucose and/or insulin on GK translocation were examined in 6-h-fasted conscious rats. Intraduodenal glucose infusion (28 mg.kg(-1).min(-1) after a priming dose at 500 mg/kg) elevated blood glucose levels (mg/dl) in the artery and portal vein from 90 +/- 3 and 87 +/- 3 to 154 +/- 4 and 185 +/- 4, respectively, at 10 min. At 120 min, the levels had decreased to 133 +/- 6 and 156 +/- 5, respectively. Plasma insulin levels (ng/ml) in the artery and the portal vein rose from 0.7 +/- 0.1 and 1.8 +/- 0.3 to 11.8 +/- 1.5 and 20.2 +/- 2.0 at 10 min, respectively, and 12.4 +/- 3.1 and 18.0 +/- 4.8 at 30 min, respectively. GK was rapidly exported from the nucleus as determined by measuring the ratio of the nuclear to the cytoplasmic immunofluorescence (N/C) of GK (2.9 +/- 0.3 at 0 min to 1.7 +/- 0.2 at 10 min, 1.5 +/- 0.1 at 20 min, 1.3 +/- 0.1 at 30 min, and 1.3 +/- 0.1 at 120 min). When plasma glucose (arterial; mg/dl) and insulin (arterial; ng/ml) levels were clamped for 30 min at 93 +/- 7 and 0.7 +/- 0.1, 81 +/- 5 and 8.9 +/- 1.3, 175 +/- 5 and 0.7 +/- 0.1, or 162 +/- 5 and 9.2 +/- 1.5, the N/C of GK was 3.0 +/- 0.5, 1.8 +/- 0.1, 1.5 +/- 0.1, and 1.2 +/- 0.1, respectively. The N/C of GK regulatory protein (GKRP) did not change in response to the intraduodenal glucose infusion or the rise in plasma glucose and/or insulin levels. The results suggest that GK but not GKRP translocates rapidly in a manner that corresponds with changes in the hepatic glucose balance in response to glucose ingestion in vivo. Additionally, the translocation of GK is induced by the postprandial rise in plasma glucose and insulin.