Thermotolerant guard cell protoplasts of tree tobacco do not require exogenous hormones to survive in culture and are blocked from reentering the cell cycle at the G1-to-S transition

When guard cell protoplasts (GCPs) of tree tobacco [Nicotiana glauca (Graham)] are cultured at 32 degrees C with an auxin (1-napthaleneacetic acid) and a cytokinin (6-benzylaminopurine), they reenter the cell cycle, dedifferentiate, and divide. GCPs cultured similarly but at 38 degrees C and with 0.1 micro M +/- -cis,trans-abscisic acid (ABA) remain differentiated. GCPs cultured at 38 degrees C without ABA dedifferentiate partially but do not divide. Cell survival after 1 week is 70% to 80% under all of these conditions. In this study, we show that GCPs cultured for 12 to 24 h at 38 degrees C accumulate heat shock protein 70 and develop a thermotolerance that, upon transfer of cells to 32 degrees C, enhances cell survival but inhibits cell cycle reentry, dedifferentiation, and division. GCPs dedifferentiating at 32 degrees C require both 1-napthaleneacetic acid and 6-benzylaminopurine to survive, but thermotolerant GCPs cultured at 38 degrees C +/- ABA do not require either hormone for survival. Pulse-labeling experiments using 5-bromo-2-deoxyuridine indicate that culture at 38 degrees C +/- ABA prevents dedifferentiation of GCPs by blocking cell cycle reentry at G1/S. Cell cycle reentry at 32 degrees C is accompanied by loss of a 41-kD polypeptide that cross-reacts with antibodies to rat (Rattus norvegicus) extracellular signal-regulated kinase 1; thermotolerant GCPs retain this polypeptide. A number of polypeptides unique to thermotolerant cells have been uncovered by Boolean analysis of two-dimensional gels and are targets for further analysis. GCPs of tree tobacco can be isolated in sufficient numbers and with the purity required to study plant cell thermotolerance and its relationship to plant cell survival, growth, dedifferentiation, and division in vitro.     

Actions or hand-object interactions? Human inferior frontal cortex and action observation

Cells in macaque ventral premotor cortex (area F5c) respond to observation or production of specific hand-object interactions. Studies in humans associate the left inferior frontal gyrus, including putative F5 homolog pars opercularis, with observing hand actions. Are these responses related to the realized goal of a prehensile action or to the observation of dynamic hand movements? Rapid, event-related fMRI was used to address this question. Subjects watched static pictures of the same objects being grasped or touched while performing a 1-back orienting task. In all 17 subjects, bilateral inferior frontal cortex was differentially activated in response to realized goals of observed prehensile actions. Bilaterally, precentral gyrus was most frequently activated (82%) followed by pars triangularis (73%) and pars opercularis (65%).     

Structural basis for substrate recognition in the salicylic acid carboxyl methyltransferase family

Recently, a novel family of methyltransferases was identified in plants. Some members of this newly discovered and recently characterized methyltransferase family catalyze the formation of small-molecule methyl esters using S-adenosyl-L-Met (SAM) as a methyl donor and carboxylic acid-bearing substrates as methyl acceptors. These enzymes include SAMT (SAM:salicylic acid carboxyl methyltransferase), BAMT (SAM:benzoic acid carboxyl methyltransferase), and JMT (SAM:jasmonic acid carboxyl methyltransferase). Moreover, other members of this family of plant methyltransferases have been found to catalyze the N-methylation of caffeine precursors. The 3.0-A crystal structure of Clarkia breweri SAMT in complex with the substrate salicylic acid and the demethylated product S-adenosyl-L-homocysteine reveals a protein structure that possesses a helical active site capping domain and a unique dimerization interface. In addition, the chemical determinants responsible for the selection of salicylic acid demonstrate the structural basis for facile variations of substrate selectivity among functionally characterized plant carboxyl-directed and nitrogen-directed methyltransferases and a growing set of related proteins that have yet to be examined biochemically. Using the three-dimensional structure of SAMT as a guide, we examined the substrate specificity of SAMT by site-directed mutagenesis and activity assays against 12 carboxyl-containing small molecules. Moreover, the utility of structural information for the functional characterization of this large family of plant methyltransferases was demonstrated by the discovery of an Arabidopsis methyltransferase that is specific for the carboxyl-bearing phytohormone indole-3-acetic acid.     

Structural basis for the modulation of lignin monomer methylation by caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase

Caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase (COMT) from alfalfa is an S-adenosyl-L-Met-dependent O-methyltransferase involved in lignin biosynthesis. COMT methylates caffeoyl- and 5-hydroxyferuloyl-containing acids, aldehydes, and alcohols in vitro while displaying a kinetic preference for the alcohols and aldehydes over the free acids. The 2.2-A crystal structure of COMT in complex with S-adenosyl-L-homocysteine (SAH) and ferulic acid (ferulate form), as well as the 2.4-A crystal structure of COMT in complex with SAH and 5-hydroxyconiferaldehyde, provide a structural understanding of the observed substrate preferences. These crystal structures identify residues lining the active site surface that contact the substrates. Structurally guided site-directed mutagenesis of active site residues was performed with the goal of altering the kinetic preferences for physiological substrates. The kinetic parameters of the COMT mutants versus wild-type enzyme are presented, and coupled with the high-resolution crystal structures, they will serve as a starting point for the in vivo manipulation of lignin monomers in transgenic plants. Ultimately, this structurally based approach to metabolic engineering will allow the further alteration of the lignin biosynthetic pathway in agronomically important plants. This approach will lead to a better understanding of the in vivo operation of the potential metabolic grid for monolignol biosynthesis.     

Thermal ecology of the Australian agamid Pogona barbata

This study compares the thermal ecology of male bearded dragon lizards (Pogona barbata) from south-east Queensland across two seasons: summer (1994–1995) and autumn (1995). Seasonal patterns of body temperature (T _b) were explored in terms of changes in the physical properties of the thermal environment and thermoregulatory effort. To quantify thermoregulatory effort, we compared behavioral and physiological variables recorded for observed lizards with those estimated for a thermoconforming lizard. The study lizards' field T _bs varied seasonally (summer: grand daily mean (GDM) 34.6 ± 0.6°C, autumn: GDM 27.5 ± 0.3°C) as did maximum and minimum available operative temperatures (summer: GDM T _max 42.1 ± 1.7°C, T _min 32.2 ± 1.0°C, autumn: GDM T _max 31.7 ± 1.2°C, T _min 26.4 ± 0.5°C). Interestingly, the range of temperatures that lizards selected in a gradient (selected range) did not change seasonally. However, P. barbata thermoregulated more extensively and more accurately in summer than in autumn; lizards generally displayed behaviors affecting heat load nonrandomly in summer and randomly in autumn, leading to the GDM of the mean deviations of lizards' field T _bs from their selected ranges being only 2.1 ± 0.5°C in summer, compared to 4.4 ± 0.5°C in autumn. This seasonal difference was not a consequence of different heat availability in the two seasons, because the seasonally available ranges of operative temperatures rarely precluded lizards from attaining field T _bs within their selected range, should that have been the goal. Rather, thermal microhabitat distribution and social behavior appear to have had an important influence on seasonal levels of thermoregulatory effort. Received: 28 April 1997 / Accepted: 29 December 1997     

A molluscan model system in the search for the engram.

A 3-neuron central pattern generator, whose sufficiency and necessity has been directly demonstrated, mediates aerial respiratory behaviour in the pond snail, Lymnaea stagnalis. This behaviour can be operantly conditioned, and this associative learning is consolidated into long-lasting memory. Depending on the operant conditioning training procedure used the learning can be consolidated into intermediate term (ITM) or long-term memory (LTM). ITM persists for only 2-3 h, whilst LTM persists for days to weeks. LTM is dependent on both altered gene activity and new protein synthesis while ITM is only dependent on new protein synthesis. We have now directly established that one of the 3-CPG neurons, RPeD1, is a site of LTM formation and storage. We did this by ablating the soma of RPeD1 and leaving behind a functional primary neurite capable of mediating the necessary synaptic interactions to drive aerial respiratory behaviour by the 3-neuron CPG. However, following soma ablation the neuronal circuit is only capable of mediating learning and ITM. LTM can no longer be demonstrated. However, if RPeD1's soma is ablated after LTM consolidation memory is still present. Thus the soma is not needed for the retention of LTM. Using a similar strategy it may be possible to block forgetting.     

Palmitic acid conjugation enhances potency of tricyclo-DNA splice switching oligonucleotides

Tricyclo-DNA (tcDNA) is a conformationally constrained oligonucleotide analog that has demonstrated great therapeutic potential as antisense oligonucleotide (ASO) for several diseases. Like most ASOs in clinical development, tcDNA were modified with phosphorothioate (PS) backbone for therapeutic purposes in order to improve their biodistribution by enhancing association with plasma and cell protein. Despite the advantageous protein binding properties, systemic delivery of PS-ASO remains limited and PS modifications can result in dose limiting toxicities in the clinic. Improving extra-hepatic delivery of ASO is highly desirable for the treatment of a variety of diseases including neuromuscular disorders such as Duchenne muscular dystrophy. We hypothesized that conjugation of palmitic acid to tcDNA could facilitate the delivery of the ASO from the bloodstream to the interstitium of the muscle tissues. We demonstrate here that palmitic acid conjugation enhances the potency of tcDNA-ASO in skeletal and cardiac muscles, leading to functional improvement in dystrophic mice with significantly reduced dose of administered ASO. Interestingly, palmitic acid-conjugated tcDNA with a full phosphodiester backbone proved effective with a particularly encouraging safety profile, offering new perspectives for the clinical development of PS-free tcDNA-ASO for neuromuscular diseases.     

HLA-Cw*04 and hepatitis C virus persistence

In studies of acute hepatitis C virus (HCV) infection, the early host immune response is one of the determinants of viral persistence. The class I human leukocyte antigens (HLA), which present foreign antigen to cytolytic T cells, are integral components of this response. We hypothesized that the highly polymorphic HLA genes affect the outcome of an HCV infection. To test this hypothesis, we molecularly typed 231 persons with well-documented clearance of an HCV infection and 444 matched persistently infected persons. HLA-A*1101 (odds ratio [OR], 0.49; 95% confidence interval [95% CI], 0.27 to 0.89), HLA-B*57 (OR, 0.62; 95% CI, 0.39 to 1.00), and HLA-Cw*0102 (OR, 0.43; 95% CI, 0.21 to 0.89) were associated with viral clearance, whereas HLA-A*2301 (OR, 1.78; 95% CI, 1.01 to 3.11) and HLA-Cw*04 (OR, 1.78; 95% CI, 1.21 to 2.59) were associated with viral persistence. HLA-Cw*04 is in strong linkage disequilibrium with HLA-B*53 and HLA-B*35, but only HLA-B*53 (OR, 1.70; 95% CI, 0.95 to 3.06) and the Cw*04-B*53 haplotype (OR, 1.76; 95% CI, 0.94 to 3.26) were weakly associated with viral persistence. HLA-B*53 has similar, but not necessarily identical, binding specificity to some HLA-B*35 subtypes (B*35-Px group). The association with the B*35-Px group was less strong than with HLA-B*53 alone. The association of HLA-Cw*04 with HCV persistence was codominant (two copies of the gene were more strongly associated with persistence than one copy). However, HLA-Cw*04 was not associated with HCV RNA levels among the persistently infected individuals. Since Cw*04 is a ligand for the killer immunoglobulin-like receptors on natural killer cells, these cells may be involved in recovery from HCV infection. Further investigation is needed to understand the relationship between class I alleles and HCV clearance.