Effect of potential amine prodrugs of selective neuronal nitric oxide synthase inhibitors on blood–brain barrier penetration

Several prodrug approaches were taken to mask amino groups in two potent and selective neuronal nitric oxide synthase (nNOS) inhibitors containing either a primary or secondary amino group to lower the charge and improve blood–brain barrier (BBB) penetration. The primary amine was masked as an azide and the secondary amine as an amide or carbamate. The azide was not reduced to the amine under a variety of in vitro and ex vivo conditions. Despite the decrease in charge of the amino group as an amide and as carbamates, BBB penetration did not increase. It appears that the uses of azides as prodrugs for primary amines or amides and carbamates as prodrugs for secondary amines are not universally effective for CNS applications.     

Effect of breath-hold time on DLCO(SB) in patients with airway obstruction

The single-breath diffusing capacity of the lung for CO [DLCO(SB)] is considered a measure of the conductance of CO across the alveolar-capillary membrane and its binding with hemoglobin. Although incomplete mixing of inspired gas with alveolar gas could theoretically influence overall diffusion, conventional calculations of DLCO(SB) spuriously overestimate DLCO(SB) during short breath-holding periods when incomplete mixing of gas within the lung might have the greatest effect. Using the three-equation method to calculate DLCO(SB) which analytically accounts for changes in breath-hold time, we found that DLCO(SB) did not change with breath-hold time in control subjects but increased with increasing breath-hold time in both patients with asthma and patients with emphysema. The increase in DLCO(SB) with increasing breath-hold time correlated with the phase III slope of the single-breath N2 washout curve. We suggest that in patients with ventilation maldistribution, DLCO(SB) may be decreased for the shorter breath-hold maneuvers because overall diffusion is limited by the reduced transport of CO from the inspired gas through the alveolar gas prior to alveolar-capillary gas exchange.     

Excitable behavior can explain the "ping-pong" mode of communication between cells using the same chemoattractant

Here we elucidate a paradox: how a single chemoattractant-receptor system in two individuals is used for communication despite the seeming inevitability of self-excitation. In the filamentous fungus Neurospora crassa, genetically identical cells that produce the same chemoattractant fuse via the homing of individual cell protrusions toward each other. This is achieved via a recently described "ping-pong" pulsatile communication. Using a generic activator-inhibitor model of excitable behavior, we demonstrate that the pulse exchange can be fully understood in terms of two excitable systems locked into a stable oscillatory pattern of mutual excitation. The most puzzling properties of this communication are the sudden onset of oscillations with final amplitude, and the absence of seemingly inevitable self-excitation. We show that these properties result directly from both the excitability threshold and refractory period characteristic of excitable systems. Our model suggests possible molecular mechanisms for the ping-pong communication.     

Wound-induced proteinase inhibitors from tomato leaves. II. The cDNA-deduced primary structure of pre-inhibitor II

A cDNA containing the complete amino acid-coding region of wound-induced tomato Inhibitor II was constructed in the plasmid pUC9. The open reading frame codes for 148 amino acids including a 25-amino acid signal sequence preceding the N-terminal lysine of the mature Inhibitor II. The Inhibitor II sequence exhibits two domains, one domain having a trypsin inhibitory site and the other a chymotrypsin inhibitory site, apparently evolved from a smaller gene by a process of gene duplication and elongation. The amino acid sequence of tomato leaf Inhibitor II exhibits homology with two small proteinase inhibitors isolated from potato tuber and an inhibitor from eggplant. The small potato tuber inhibitors are homologous with 33 amino acids of the N-terminal domain and 19 amino acids from the C-terminal domain. Two identical nucleotide sequences of Inhibitor II cDNA in the 3' noncoding region were present that were also found in an Inhibitor I cDNA. These include an atypical polyadenylation signal, AATAAG, and a 10-base palindromic sequence, CATTATAATG, for which no function is yet known.     

Determination of the pH dependence,substrate specificity,and turnovers of alternative substrates for human ornithine aminotransferase

Hepatocellular carcinoma (HCC) is the most common primary cancer of the liver and occurs predominantly in patients with underlying chronic liver diseases. Over the past decade, human ornithine aminotransferase (hOAT), which is an enzyme that catalyzes the metabolic conversion of ornithine into an intermediate for proline or glutamate synthesis, has been found to be overexpressed in HCC cells. hOAT has since emerged as a promising target for novel anticancer therapies, especially for the ongoing rational design effort to discover mechanism-based inactivators (MBIs). Despite the significance of hOAT in human metabolism and its clinical potential as a drug target against HCC, there are significant knowledge deficits with regard to its catalytic mechanism and structural characteristics. Ongoing MBI design efforts require in-depth knowledge of the enzyme active site, in particular, pKa values of potential nucleophiles and residues necessary for the molecular recognition of ligands. Here, we conducted a study detailing the fundamental active-site properties of hOAT using stopped-flow spectrophotometry and X-ray crystallography. Our results quantitatively revealed the pH dependence of the multistep reaction mechanism and illuminated the roles of ornithine α-amino and δ-amino groups in substrate recognition and in facilitating catalytic turnover. These findings provided insights of the catalytic mechanism that could benefit the rational design of MBIs against hOAT. In addition, substrate recognition and turnover of several fragment-sized alternative substrates of hOATs, which could serve as structural templates for MBI design, were also elucidated.     

The evolutionary dynamics of the lion Panthera leo revealed by host and viral population genomics

The lion Panthera leo is one of the world's most charismatic carnivores and is one of Africa's key predators. Here, we used a large dataset from 357 lions comprehending 1.13 megabases of sequence data and genotypes from 22 microsatellite loci to characterize its recent evolutionary history. Patterns of molecular genetic variation in multiple maternal (mtDNA), paternal (Y-chromosome), and biparental nuclear (nDNA) genetic markers were compared with patterns of sequence and subtype variation of the lion feline immunodeficiency virus (FIV(Ple)), a lentivirus analogous to human immunodeficiency virus (HIV). In spite of the ability of lions to disperse long distances, patterns of lion genetic diversity suggest substantial population subdivision (mtDNA Phi(ST) = 0.92; nDNA F(ST) = 0.18), and reduced gene flow, which, along with large differences in sero-prevalence of six distinct FIV(Ple) subtypes among lion populations, refute the hypothesis that African lions consist of a single panmictic population. Our results suggest that extant lion populations derive from several Pleistocene refugia in East and Southern Africa ( approximately 324,000-169,000 years ago), which expanded during the Late Pleistocene ( approximately 100,000 years ago) into Central and North Africa and into Asia. During the Pleistocene/Holocene transition ( approximately 14,000-7,000 years), another expansion occurred from southern refugia northwards towards East Africa, causing population interbreeding. In particular, lion and FIV(Ple) variation affirms that the large, well-studied lion population occupying the greater Serengeti Ecosystem is derived from three distinct populations that admixed recently.     

The auxilin-like phosphoprotein Swa2p is required for clathrin function in yeast

BACKGROUND: In eukaryotic cells, clathrin-coated vesicles transport specific cargo from the plasma membrane and trans-Golgi network to the endosomal system. Removal of the clathrin coat in vitro requires the uncoating ATPase Hsc70 and its DnaJ cofactor auxilin. To date, a requirement for auxilin and Hsc70 in clathrin function in vivo has not been demonstrated. RESULTS: The Saccharomyces cerevisiae SWA2 gene, previously identified in a synthetic lethal screen with arf1, was cloned and found to encode a protein with a carboxy-terminal DnaJ domain which is homologous to that of auxilin. Like auxilin, Swa2p has a clathrin-binding domain and is able to stimulate the ATPase activity of Hsc70. The swa2-1 allele recovered from the original screen carries a point mutation in its tetratricopeptide repeat (TPR) domain, a motif not found in auxilin but known in other proteins to mediate interaction with heat-shock proteins. Swa2p fractionates in the cytosol and appears to be heavily phosphorylated. Disruption of SWA2 causes slow growth and several phenotypes that are very similar to those exhibited by clathrin mutants. Furthermore, the swa2Delta mutant exhibits a significant increase in membrane- associated or -assembled clathrin relative to a wild-type strain. CONCLUSIONS: These results indicate that Swa2p is a clathrin-binding protein required for normal clathrin function in vivo. They suggest that Swa2p is the yeast ortholog of auxilin and has a role in disassembling clathrin, not only in uncoating clathrin-coated vesicles but perhaps in preventing unproductive clathrin assembly in vivo.