From malate dehydrogenase to phenyllactate dehydrogenase. Incorporation of unnatural amino acids to generate an improved enzyme-catalyzed activity

Malate dehydrogenase (MDH) from Escherichia coli is highly specific for its keto acid substrate. The placement of the active site-binding groups in MDH effectively discriminates against both the shorter and the longer keto dicarboxylic acids that could potentially serve as alternative substrates. A notable exception to this specificity is the alternative substrate phenylpyruvate. This aromatic keto acid can be reduced by MDH, albeit at a somewhat slower rate and with greatly diminished affinity, despite the presence of several substrate-binding arginyl residues and the absence of a hydrophobic pocket in the active site. The specificity of MDH for phenylpyruvate has now been enhanced, and that for the physiological substrate oxaloacetate has been diminished, through the replacement of one of the binding arginyl residues with several unnatural alkyl and aryl amino acid analogs. This approach, called site-specific modulation, incorporates systematic structural variations at a site of interest. Molecular modeling studies have suggested a structural basis for the affinity of native MDH for phenylpyruvate and a rationale for the improved catalytic activity that is observed with these new, modified phenyllactate dehydrogenases.     

N-pyridin-3-yl- and N-quinolin-3-yl-benzamides: modulators of human vanilloid receptor 1 (TRPV1)

High throughput screening of our compound library revealed a series of N-pyridyl-3-benzamides as low micromolar agonists of the human TRPV1 receptor. Synthesis of analogs in this series led to the discovery of a series of N-quinolin-3-yl-benzamides as low nanomolar antagonists of human TRPV1.     

Bioengineered bugs expressing oligosaccharide receptor mimics: toxin-binding probiotics for treatment and prevention of enteric infections

Many microbial pathogens recognize oligosaccharides displayed on the surface of host cells as receptors for toxins and adhesins. These ligand-receptor interactions are critical for disease pathogenesis, making them promising targets for novel anti-infectives. One strategy with particular utility against enteric infections involves expression of molecular mimics of host oligosaccharides on the surface of harmless bacteria capable of surviving in the gut. This can be achieved in Gram-negative bacteria by manipulating the outer core region of the lipopolysaccharide (LPS) through expression of cloned heterologous glycosyltransferases. The resultant chimeric LPS molecules are incorporated into the outer membrane by the normal assembly route and presented as a closely packed 2-D array of receptor mimics. Several such "designer probiotics" have been constructed, and these bind bacterial toxins in the gut lumen with very high avidity, blocking their uptake by host cells and thereby preventing disease.     

REGIONAL DISTRIBUTION OF HOMOCARNOSINE, HOMOCARNOSINE-CARNOSINE SYNTHETASE AND HOMOCARNOSINASE IN HUMAN BRAIN

Homocarnosine (HCarn) content varied over a 6-fold range in different regions of autopsied human brain, being highest in the dentate nucleus and the inferior olive, and lowest in the caudate nucleus and mesolimbic system. HCarn content was similar in biopsied and autopsied frontal cortex. Very little if any carnosine (Carn) was present in human brain, except for the olfactory bulb, where Carn may have comprised 20% of the imidazole dipeptides present. Only HCarn was present in human CSF. HCarn-Carn synthetase enzyme activity in biopsy specimens of human frontal and temporal cortex was approx 10 times greater than has been reported for rat cerebral cortex. The enzyme synthesized Carn 3–5 times as rapidly as HCarn, when β-alanine (β-Ala) or GABA substrate concentrations were 10 MM. The synthetase was found to have an apparent K_m of 1.8 mM for β-Ala, and 8.8 mM for GABA. HCarn-Carn synthetase activity decreases rapidly after brain death, and was not detectable in autopsied brain specimens frozen more than 6 h after patients’deaths. Homocarnosinase activity was determined in brain, using L-[γaminobutyryl-1-¹⁴C]HCarn as substrate, and measuring radioactive GABA produced by hydrolysis of HCarn at pH 7.2 in the presence of Co²⁺ ions. Homocarnosinase activity was similar in biopsied and autopsied human cerebral cortex, and appeared to be stable for at least 10 h after death in unfrozen brain. Differences in the regional distribution of HCarn-Carn synthetase and homocarnosinase activities, as well as regional differences in GABA content in human brain, do not readily account for regional differences in HCarn content, nor do they suggest a physiological role for HCarn.     

Chemical Defense and the Persistence of Pioneer Plant Seeds in the Soil of a Tropical Cloud Forest

We present evidence that differences in soil seedbank persistence among pioneer plants in the cloud forest of Monteverde, Costa Rica, are influenced by differences in seed chemical defense. We used extracted seed chemicals from Bocconia frutescens (Papaveraceae), Guettarda poasana (Rubiaceae) , Phytolacca rivinoides (Phytolaccaceae) , Urera elata (Urticaceae) , Cecropia polyphlebia (Cecropiaceae), and Witheringia meiantha (Solanaceae) to assess seed chemical defense in two ways: (1) a plant pathogen inhibition assay using Pythium irregulare ; and (2) a brine shrimp toxicity assay using Artemia salina . The combined performance of each species in the two assays positively correlated with seedbank persistence. In the pathogen assay, mycelium growth was reduced when Pythium was cultured on media containing seed extracts from the three species with the greatest seed longevity in the soil ( i.e., Bocconia , Guettarda, and Phytolacca ). Bocconia , the most persistent species, was the only species that contained chemicals toxic to brine shrimp , an indication of defense against arthropods. We focused on Bocconia defense by isolating the chemicals toxic to brine shrimp and identified them as dihydrosanguinarine, dihydrochelirubine, and dihydrochelerthrine. We found these alkaloids in Bocconia seeds at much higher concentrations (∼50 mg/g seed material) than in leaves. These chemicals are likely responsible for the exceptional longevity of Bocconia seeds in the soil. Phytolacca and Guettarda seeds also remain viable in the soil for long periods probably due to antipathogen chemicals detected in our analyses. In contrast, the species that do not persist ( i.e., Urera , Cecropia , and Witheringia ) lacked seed defensive chemicals in our assays.
Abstract in Spanish is available at http://www.blackwell-synergy.com/loi/btp .     

Relationship between evolutionary age and hydroxyproline-containing macromolecules in unicellular algae

For 11 species of unicellular marine algae there is an inverse relationship between evolutionary age and hydroxyproline content of the high MW fraction     

The genus Nomuraea Maublanc

The history of Spicaria rileyi (Farlow) Charles and Spicaria prasina (Maub.) Sawada is discussed. The genus Nomuraea is resurrected and Spicaria rileyi is transferred to that genus. Spicaria prasina is placed in synonomy with Nomuraea rileyi (Farlow) Samson.