Characterization of cardiac innervation in the nudibranch,Archidoris montereyensis

Summary The heart of the nudibranch mollusc Archidoris montereyensis is regulated by a small number of powerful effector neurons located in the right pleural and visceral ganglia. Two identifiable neurons in the pleural ganglion, a heart excitor (pl_HE) and a heart inhibitor (Pl_HI), are especially important regulators of cardiac function in that low levels of spontaneous activity in either cell significantly alters the amplitude and rate of heart contractions. These neurons have extensive dendritic arbors within the right pleural ganglion and branching axonal processes within the visceral ganglion. The visceral ganglion also contains a heart excitor neuron (V_HE) and at least two heart inhibitor neurons (V_HI cells), but their influence on cardiac activity is weaker than that of the pleural ganglion cells. All of these heart effector cells appear to be motor neurons with axons that terminate predominately in the atrio-ventricular valve region of the heart via the pericardial nerve. The simplicity and strength of these neuronal connections to the heart of Archidoris make this a favorable preparation for studies of cardiac regulation.Abbreviations Pl _HE pleural ganglion heart excitor neuron - Pl _HI pleural heart inhibitor neuron - V _HE visceral ganglion heart excitor neuron - V _HI cells, visceral heart inhibitor neurons - V _K visceral kidney excitor neuron - V _G visceral gill excitor neuron     

Absolute Quantification of E1, Ubiquitin-Like Proteins and Nedd8–MLN4924 Adduct by Mass Spectrometry

Ubiquitin (Ub) and ubiquitin-like (Ubl) proteins regulate a variety of important cellular processes by forming covalent conjugates with target proteins or lipids. Ubl conjugation is catalyzed by a cascade of proteins including activating enzymes (E1), conjugating enzymes (E2), and in many cases ligation enzymes (E3). The discovery of MLN4924 (Brownell et al., Mol Cell 37: 102–111, 1), an investigational small molecule that is a mechanism-based inhibitor of NEDD8-activating enzyme (NAE), reveals a promising strategy of targeting E1/Ubl pathway for therapeutic purposes. In order to better understand, the biochemical dynamics of Ubl conjugation in cells and tissues, we have developed a mass spectrometry-based method to quantify E1 and Ubls using isotope-labeled proteins as internal standards. Furthermore, we have used the described method to quantify levels of the covalent Nedd8-inhibitor adduct formed in MLN4924 treated cells and tissues. The Nedd8–MLN4924 adduct is a tight-binding inhibitor of NAE, and its cellular concentration represents an indirect pharmacodynamic readout of NAE/Nedd8 pathway inhibition.     

Urocortin-deficient mice display normal stress-induced anxiety behavior and autonomic control but an impaired acoustic startle response

Corticotropin-releasing hormone (Crh) plays an important role in modulating physiological and behavioral responses to stress. Its actions are mediated through two receptors, Crhr1 and Crhr2. Urocortin (Ucn), a Crh-related neuropeptide and the postulated endogenous ligand for Crhr2, is a potential mediator of stress responses. We generated Ucn-deficient mice using embryonic stem cell technology to determine its role in stress-induced behavioral and autonomic responses. Unlike Crhr1- or Crhr2-deficient mice, Ucn-deficient mice exhibit normal anxiety-like behavior as well as autonomic regulation in response to stress. However, the mutant mice display an impaired acoustic startle response that is not due to an obvious hearing defect. Thus, our results suggest that Ucn does not play an essential role in stress-induced behavioral and autonomic responses. Ucn may modulate the acoustic startle response through the Ucn-expressing neuron projections from the region of the Edinger-Westphal nucleus.     

Therapeutic effects of systemic administration of chaperone αB-crystallin associated with binding proinflammatory plasma proteins

The therapeutic benefit of the small heat shock protein αB-crystallin (HspB5) in animal models of multiple sclerosis and ischemia is proposed to arise from its increased capacity to bind proinflammatory proteins at the elevated temperatures within inflammatory foci. By mass spectral analysis, a common set of ～70 ligands was precipitated by HspB5 from plasma from patients with multiple sclerosis, rheumatoid arthritis, and amyloidosis and mice with experimental allergic encephalomyelitis. These proteins were distinguished from other precipitated molecules because they were enriched in the precipitate as compared with their plasma concentrations, and they exhibited temperature-dependent binding. More than half of these ligands were acute phase proteins or members of the complement or coagulation cascades. Consistent with this proposal, plasma levels of HspB5 were increased in patients with multiple sclerosis as compared with normal individuals. The combination of the thermal sensitivity of the HspB5 combined with the high local concentration of these ligands at the site of inflammation is proposed to explain the paradox of how a protein believed to exhibit nonspecific binding can bind with some relative apparent selectivity to proinflammatory proteins and thereby modulate inflammation.     

Nedd8 modification of cul-1 activates SCF(beta(TrCP))-dependent ubiquitination of IkappaBalpha

Regulation of NF-kappaB occurs through phosphorylation-dependent ubiquitination of IkappaBalpha, which is degraded by the 26S proteasome. Recent studies have shown that ubiquitination of IkappaBalpha is carried out by a ubiquitin-ligase enzyme complex called SCF(beta(TrCP)). Here we show that Nedd8 modification of the Cul-1 component of SCF(beta(TrCP)) is important for function of SCF(beta(TrCP)) in ubiquitination of IkappaBalpha. In cells, Nedd8-conjugated Cul-1 was complexed with two substrates of SCF(beta(TrCP)), phosphorylated IkappaBalpha and beta-catenin, indicating that Nedd8-Cul-1 conjugates are part of SCF(beta(TrCP)) in vivo. Although only a minute fraction of total cellular Cul-1 is modified by Nedd8, the Cul-1 associated with ectopically expressed betaTrCP was highly enriched for the Nedd8-conjugated form. Moreover, optimal ubiquitination of IkappaBalpha required Nedd8 and the Nedd8-conjugating enzyme, Ubc12. The site of Nedd8 ligation to Cul-1 is essential, as SCF(beta(TrCP)) containing a K720R mutant of Cul-1 only weakly supported IkappaBalpha ubiquitination compared to SCF(beta(TrCP)) containing WT Cul-1, suggesting that the Nedd8 ligation of Cul-1 affects the ubiquitination activity of SCF(beta(TrCP)). These observations provide a functional link between the highly related ubiquitin and Nedd8 pathways of protein modification and show how they operate together to selectively target the signal-dependent degradation of IkappaBalpha.     

Inactivation of single-celled Ascaris suum eggs by low-pressure UV radiation

Intact and decorticated single-celled Ascaris suum eggs were exposed to UV radiation from low-pressure, germicidal lamps at fluences (doses) ranging from 0 to 8,000 J/m2 for intact eggs and from 0 to 500 J/m2 for decorticated eggs. With a UV fluence of 500 J/m2, 0.44-+/-0.20-log inactivation (mean+/-95% confidence interval) (63.7%) of intact eggs was observed, while a fluence of 4,000 J/m2 resulted in 2.23-+/-0.49-log inactivation (99.4%). (The maximum quantifiable inactivation was 2.5 log units.) Thus, according to the methods used here, Ascaris eggs are the most UV-resistant water-related pathogen identified to date. For the range of fluences recommended for disinfecting drinking water and wastewater (200 to 2,000 J/m2), from 0- to 1.5-log inactivation can be expected, although at typical fluences (less than 1,000 J/m2), the inactivation may be less than 1 log. When the eggs were decorticated (the outer egg shell layers were removed with sodium hypochlorite, leaving only the lipoprotein ascaroside layer) before exposure to UV, 1.80-+/-0.32-log reduction (98.4%) was achieved with a fluence of 500 J/m2, suggesting that the outer eggshell layers protected A. suum eggs from inactivation by UV radiation. This protection may have been due to UV absorption by proteins in the outer layers of the 3- to 4-microm-thick eggshell. Stirring alone (without UV exposure) also inactivated some of the Ascaris eggs (approximately 20% after 75 min), which complicated determination of the inactivation caused by UV radiation alone.     

Steam-explosion pretreatment of wood: Effect of chip size, acid, moisture content and pressure drop

Material balances for pentosan, lignin, and hexosan, during steam-explosion pretreatment of aspenwood, showed almost quantitative recovery of cellulose in the water-insoluble fraction. Dilute acid impregnation resulted in more selective hydrolysis of pentosan relative to undesirable pyrolysis, and gave a more accessible substrate for enzymatic hydrolysis. Thermocouple probes, located inside simulated aspenwood chips heated in 240 degrees C-saturated steam, showed rapid heating of air-dry wood, whereas green or impregnated wood heated slowly. Small chips, 3.2 mm in the fiber direction, whether green or airdry gave approximately equal rates of pentosan destruction and solubilization, and similar yields of glucose and of total reducing sugars on enzymatic hydrolysis with Trichoderma harzianum. Partial pyrolysis, destroying one third of the pentosan of aspenwood at atmospheric pressure by dry steam at 276 degrees C, gave little increase in yield of reducing sugars on enzymatic hydrolysis. Treatment with saturated steam at 240 degrees C gave essentially the same yields of glucose and of total reducing sugars, and the same yields of butanediol and ethanol on fermentation with Klebsiella pneumoniae, whether or not 80% of the steam was bled off before explosion and even if the chips remained intact, showing that explosion was unnecessary.     

Genophore homologies among compatible nocardiae

Deoxyribonucleic acid (DNA) reassociation analyses were employed to determine the molecular relationships between recombinable nocardiae. Analysis of the compatibility system of Nocardia erythropolis Mat-Ce and Mat-cE mating strains demonstrated the existence of extensive homology under both exacting and nonexacting conditions. Labeled N. erythropolis Mat-cE DNA reassociated equally as well with the Mat-Ce test DNA as with its own filter-bound DNA. However, the Mat-cE DNA bound only ca. 60% of the Mat-Ce DNA, when the latter was the reference. The existence of unique nucleotide sequences is postulated on the basis of these results as well as of aberrant segregation patterns which have been observed in certain class types of recombinants. Reassociation data reveal that recombinants representing the inheritance of different portions of each of the parental genomes have inherited the unique portion from the Mat-Ce parent. N. restrictus AY-B-226 exhibited little relatedness (11 to 32%), and N. globerula ATCC 9356 only slightly more (21 to 42%), to either of these mating strains at either exacting or nonexacting temperatures of incubation.     

Carbovir: A Carbocyclic Nucleoside with Potent and Selective Activity Against Human Immunodeficiency Virus (HIV) in Vitro

Abstract

Carbocyclic 2′, 3′-didehydro-2′,3′-dideoxyquanosine (carbovir), a novel nucleoside analog, emerged as a potent and selective anti-HIV agent from a primary screen of a large number of carbocyclic nucleosides.¹ Carbovir inhibited the infectivity and replication of HIV in T-cells at concentrations 200 to 400-fold below toxicity to host cells. Carbovir was also evaluated for its Inhibitory effects on the expression of viral antigen in HIV-infected CEM cells. Production of p 24 core antigen at optimal inhibitory concentrations of the antiviral agents indicated comparable results for AZT, ddA and carbovir.