Rhes,a Physiologic Regulator of Sumoylation,Enhances Cross-sumoylation between the Basic Sumoylation Enzymes E1 and Ubc9

We recently reported that the small G-protein Rhes has the properties of a SUMO-E3 ligase and mediates mutant huntingtin (mHtt) cytotoxicity. We now demonstrate that Rhes is a physiologic regulator of sumoylation, which is markedly reduced in the corpus striatum of Rhes-deleted mice. Sumoylation involves activation and transfer of small ubiquitin-like modifier (SUMO) from the thioester of E1 to the thioester of Ubc9 (E2) and final transfer to lysines on target proteins, which is enhanced by E3s. We show that E1 transfers SUMO from its thioester directly to lysine residues on Ubc9, forming isopeptide linkages. Conversely, sumoylation on E1 requires transfer of SUMO from the thioester of Ubc9. Thus, the process regarded as “autosumoylation” reflects intermolecular transfer between E1 and Ubc9, which we designate “cross-sumoylation.” Rhes binds directly to both E1 and Ubc9, enhancing cross-sumoylation as well as thioester transfer from E1 to Ubc9.     

Bibersteinia trehalosi Inhibits the Growth of Mannheimia haemolytica by a Proximity-Dependent Mechanism

Mannheimia (Pasteurella) haemolytica is the only pathogen that consistently causes severe bronchopneumonia and rapid death of bighorn sheep (BHS; Ovis canadensis) under experimental conditions. Paradoxically, Bibersteinia (Pasteurella) trehalosi and Pasteurella multocida have been isolated from BHS pneumonic lungs much more frequently than M. haemolytica. These observations suggest that there may be an interaction between these bacteria, and we hypothesized that B. trehalosi overgrows or otherwise inhibits the growth of M. haemolytica. Growth curves (monoculture) demonstrated that B. trehalosi has a shorter doubling time (∼10 min versus ∼27 min) and consistently achieves 3-log higher cell density (CFU/ml) compared to M. haemolytica. During coculture M. haemolytica growth was inhibited when B. trehalosi entered stationary phase (6 h) resulting in a final cell density for M. haemolytica that was 6 to 9 logs lower than expected with growth in the absence of B. trehalosi. Coculture supernatant failed to inhibit M. haemolytica growth on agar or in broth, indicating no obvious involvement of lytic phages, bacteriocins, or quorum-sensing systems. This observation was confirmed by limited growth inhibition of M. haemolytica when both pathogens were cultured in the same media but separated by a filter (0.4-μm pore size) that limited contact between the two bacterial populations. There was significant growth inhibition of M. haemolytica when the populations were separated by membranes with a pore size of 8 μm that allowed free contact. These observations demonstrate that B. trehalosi can both outgrow and inhibit M. haemolytica growth with the latter related to a proximity- or contact-dependent mechanism.The bighorn sheep (BHS; Ovis canadensis) population in North America has declined from an estimated two million at the beginning of the 19th century to fewer than 70,000 today (7, 30). The decline of BHS populations is presumably due to loss of habitat, competition for forage with domestic livestock, predation, and disease (9, 19). The most important disease that has limited the growth of BHS populations is pneumonia (13, 14, 19, 31). Bacteria associated with BHS pneumonia are members of the genera Mannheimia and Pasteurella, particularly, the species Mannheimia (Pasteurella) haemolytica, Bibersteinia (Pasteurella) trehalosi, and Pasteurella multocida (6-9, 15, 20, 25, 31). Several independent studies have revealed that M. haemolytica is a major cause of BHS pneumonia. In fact, M. haemolytica is the only pathogen that has been shown to consistently cause severe bronchopneumonia and rapid death of BHS under experimental conditions (10, 14, 23). B. trehalosi has been isolated more often than M. haemolytica from the upper respiratory tract of healthy BHS (10, 12-14, 26, 31). Large numbers of B. trehalosi have also been isolated from the pneumonic lungs of BHS experimentally inoculated with M. haemolytica alone (10). Furthermore, our recent studies with M. haemolytica wild type and leukotoxin deletion mutants in BHS have revealed that the leukotoxin deletion mutant does not cause the death of BHS but instead induces only mild lung lesions, confirming the finding in cattle that leukotoxin is the most important virulence factor of M. haemolytica (10, 24, 29). Our recently concluded BHS inoculation study revealed that only leukotoxin producing strains of B. trehalosi can cause pneumonia, indicating that leukotoxin is the most important virulence determinant in B. trehalosi as well. More than 85% of the B. trehalosi isolates obtained from BHS, however, do not produce leukotoxin (28, 32). Therefore, this observation, together with the results from the animal experiments, indicates that B. trehalosi is unlikely to be the major cause of pneumonia outbreaks in BHS.These observations prompted us to hypothesize that B. trehalosi outgrows or otherwise inhibits the growth of M. haemolytica. The objectives of the present study were to (i) characterize in vitro growth kinetics of M. haemolytica and B. trehalosi; (ii) develop M. haemolytica-specific and B. trehalosi-specific PCR assays to detect either species in mixed cultures; and (iii) determine whether B. trehalosi inhibits the growth of M. haemolytica in vitro and, if it does, characterize the mechanism of inhibition.     

A genome-wide Drosophila screen for heat nociception identifies α2δ3 as an evolutionarily conserved pain gene

Worldwide, acute, and chronic pain affects 20% of the adult population and represents an enormous financial and emotional burden. Using genome-wide neuronal-specific RNAi knockdown in Drosophila, we report a global screen for an innate behavior and identify hundreds of genes implicated in heat nociception, including the α2δ family calcium channel subunit straightjacket (stj). Mice mutant for the stj ortholog CACNA2D3 (α2δ3) also exhibit impaired behavioral heat pain sensitivity. In addition, in humans, α2δ3 SNP variants associate with reduced sensitivity to acute noxious heat and chronic back pain. Functional imaging in α2δ3 mutant mice revealed impaired transmission of thermal pain-evoked signals from the thalamus to higher-order pain centers. Intriguingly, in α2δ3 mutant mice, thermal pain and tactile stimulation triggered strong cross-activation, or synesthesia, of brain regions involved in vision, olfaction, and hearing.     

Reconstruction of Pathways Associated with Amino Acid Metabolism in Human Mitochondria

We have used a bioinformatics approach for the identification and reconstruction of metabolic pathways associated with amino acid metabolism in human mitochon- dria. Human mitochondrial proteins determined by experimental and computa- tional methods have been superposed on the reference pathways from the KEGG database to identify mitochondrial pathways. Enzymes at the entry and exit points for each reconstructed pathway were identified, and mitochondrial solute carrier proteins were determined where applicable. Intermediate enzymes in the mito- chondrial pathways were identified based on the annotations available from public databases, evidence in current literature, or our MITOPRED program, which pre- dicts the mitochondrial localization of proteins. Through integration of the data derived from experimental, bibliographical, and computational sources, we recon- structed the amino acid metabolic pathways in human mitochondria, which could help better understand the mitochondrial metabolism and its role in human health.     

Evolution of bioengineering at UCSD: opening new vistas

Before this, the field of bioengineering refers to biomedical engineering of prosthetic devices in physiology. In addition to exciting applications of engineering principles, UCSD Department of Bioengineering began to extend the notion of engineering models of physiological systems to physiological processes. This led to a conceptual shift in the discipline and contributed to the areas of tissue and physiological process engineering. In 1988, Dr. Shu Chien and Richard Skalak joined UCSD to begin research and education on cellular and molecular bioengineering, especially, mechanobiology. Dr. Fung and Dr. Skalak initiated the new field of tissue engineering. These two decades of evolution of bioengineering and its growth across the country was spearheaded by the Whitaker Foundation, whose leitmotif was the building of bioand biomedical engineering across the country. We have garnered other accomplishments in the following fields: regenerative medicine; bioinspired artificial extracellular matrices; flexible bioelectronics and tatoos; cells show how to synchronize biological clocks; and systems medicine.     

Assessment of Therapeutic Potential of Amantadine in Methamphetamine Induced Neurotoxicity

Methamphetamine epidemic has a broad impact on world’s health care system. Its abusive potential and neurotoxic effects remain a challenge for the anti-addiction therapies. In addition to oxidative stress, mitochondrial dysfunction and apoptosis, excitotoxicity is also involved in methamphetamine induced neurotoxicity. The N-methyl-D-aspartate (NMDA) type of glutamate receptor is thought to be one of the predominant mediators of excitotoxicity. There is growing evidence that NMDA receptor antagonists could be one of the therapeutic options to manage excitotoxicity. Amantadine, a well-tolerated and modestly effective antiparkinsonian agent, was found to possess NMDA antagonistic properties and has shown to release dopamine from the nerve terminals. The current study aimed to evaluate the effect of amantadine pre-treatment against methamphetamine induced neurotoxicity. Results showed that methamphetamine treatment had depleted striatal dopamine, generated of reactive oxygen species and decreased activity of complex I in the mitochondria. Interestingly, amantadine, at high dose (10 mg/kg), did not prevent dopamine depletion moreover it exacerbated the behavioral manifestations of methamphetamine toxicity such as akinesia and catalepsy. Only lower dose of amantadine (1 mg/kg) produced significant scavenging of the reactive oxygen species induced by methamphetamine. Overall results from the present study suggest that amantadine should not be used concomitantly with methamphetamine as it may results in excessive neurotoxicity.     

Continuous wave two-photon scanning near-field optical microscopy.

We have implemented continuous-wave two-photon excitation of near-UV absorbing fluorophores in a scanning near-field optical microscope (SNOM). The 647-nm emission of an Ar-Kr mixed gas laser was used to excite the UV-absorbing DNA dyes DAPI, the bisbenzimidazole Hoechst 33342, and ethidium bromide in a shared aperture SNOM with uncoated fiber tips. Polytene chromosomes of Drosophila melanogaster and the nuclei of 3T3 Balb/c cells labeled with these dyes were readily imaged. The fluorescence intensity showed the expected nonlinear (second order) dependence on the excitation power in the range of 8-180 mW. We measured the fluorescence intensity as a function of the tip-sample displacement in the direction normal to the sample surface in the single- and two-photon excitation modes (SPE, TPE). The fluorescence intensity decayed faster in TPE than in SPE.     

Combinatorial interactions between positive and negative cis-acting elements control spatial patterns of 4CL-1 expression in transgenic tobacco

The phenylpropanoid enzyme 4-coumarate:coenzyme A ligase (4CL) plays a key role in linking general phenylpropanoid metabolism to end-product specific biosynthetic pathways. During vascular system and floral organ differentiation, the parsley 4CL-1 gene is expressed in a restricted set of tissues and cell types where 4CL activity is required to supply precursors for the synthesis of diverse phenylpropanoid-derived products such as lignin and flavonoids. In order to localize cis -acting elements which specify complex patterns of 4CL-1 expression, we analyzed the expression of internally deleted promoter fragment— GUS fusions in tobacco plants and parsley protoplasts. Elements located between −244 and −78 were required for most aspects of developmentally regulated expression. Within this region, three separate promoter domains containing partially redundant cis -elements directed vascular-specific expression when combined with a TATA-proximal domain. A negative cis -acting element which represses phloem expression was revealed in one of the domains and appears to be responsible for restricting vascular expression to the xylem. Distinct but overlapping promoter domain combinations were required for expression in floral organs, suggesting that different combinations of cis -acting elements may direct expression in different organs. Gel retardation assays were used to demonstrate the formation of DNA-protein complexes between factors present in nuclear extracts of parsley tissue culture cells and various tobacco organs and a 4CL-1 promoter fragment. Competition experiments showed that complex formation required the presence of a 42 bp promoter domain shown to be critical for 4CL-1 expression in vascular and floral tissues. The results are discussed in light of the coordinate expression of 4CL and other phenylpropanoid genes.