Design and synthesis of a series of potent and orally bioavailable noncovalent thrombin inhibitors that utilize nonbasic groups in the P1 position

As part of an ongoing effort to prepare therapeutically useful orally active thrombin inhibitors, we have synthesized a series of compounds that utilize nonbasic groups in the P1 position. The work is based on our previously reported lead structure, compound 1, which was discovered via a resin-based approach to varying P1. By minimizing the size and lipophilicity of the P3 group and by incorporating hydrogen-bonding groups on the N-terminus or on the 2-position of the P1 aromatic ring, we have prepared a number of derivatives in this series that exhibit subnanomolar enzyme potency combined with good in vivo antithrombotic and bioavailability profiles. The oxyacetic amide compound 14b exhibited the best overall profile of in vitro and in vivo activity, and crystallographic studies indicate a unique mode of binding in the thrombin active site.     

Direction determination in the minus-end-directed kinesin motor ncd

Motor proteins of the kinesin superfamily transport intracellular cargo along microtubules. Although different kinesin proteins share 30-50% amino-acid identity in their motor catalytic cores, some move to the plus end of microtubules whereas others travel in the opposite direction. Crystal structures of the catalytic cores of conventional kinesin (a plus-end-directed motor involved in organelle transport) and ncd (a minus-end-directed motor involved in chromosome segregation) are nearly identical; therefore, the structural basis for their opposite directions of movement is unknown. Here we show that the ncd 'neck' made up of 13 class-specific residues next to the superfamily-conserved catalytic core, is essential for minus-end-directed motility, as mutagenesis of these neck residues reverses the direction of ncd motion. By solving the 2.5 A structure of a functional ncd dimer, we show that the ncd neck (a coiled-coil) differs from the corresponding region in the kinesin neck (an interrupted beta-strand), although both necks interact with similar elements in the catalytic cores. The distinct neck architectures also confer different symmetries to the ncd and kinesin dimers and position these motors with appropriate directional bias on the microtubule.     

5-HT1C receptor-mediated phosphoinositide hydrolysis in the rat choroid plexus after chronic treatment with clozapine

Chronic treatment with clozapine (14 days; 10 and 25 mg/kg/day) decreases 5-HT1C receptor density but not affinity in rat choroid plexus measured with [3H]mesulergine. We now report the effects of the same clozapine treatment regimens on the function of 5-HT1C receptors (measured by maximal stimulation of 5-HT1C receptor-mediated phosphoinositide hydrolysis) in relation to receptor changes in rat choroid plexus. Quantitative 5-HT1C receptor autoradiography indicated that chronic clozapine treatment decreased, in a dose-related manner, 5-HT1C receptor binding sites labeled by antagonist ([3H]mesulergine) and agonist ([125I](+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane, [125I]DOI) radioligands. However, only the higher dose of clozapine decreased statistically significantly the maximal 5-HT1C receptor-mediated phosphoinositide hydrolysis response. Chronic administration of haloperidol (0.5 mg/kg/day) did not change any of the 5-HT1C receptor parameters. In conclusion, chronic clozapine treatment is able to modulate the function of 5-HT1C receptors. This further strengthens the possibility that 5-HT1C receptors may contribute to some of the atypical effects of clozapine.     

Molecular characterisation of a distinct South African cassava infecting geminivirus

African cassava mosaic virus (ACMV) and East African cassava mosaic virus (EACMV) are whitefly-transmitted geminiviruses (WTGs) which are widespread in cassava in Africa and cause serious yield losses. Recently, a new geminivirus affecting cassava in South Africa (SACMV) has been reported. In this work SACMV was found to have DNA-A and DNA-B components. Comparisons of amino acid sequences of the putative coat protein, and nucleotide sequences of the common region and a 687-bp DNA B fragment of SACMV with other WTGs, showed that SACMV clustered with the Old World subgroup of the Begomovirus genus of geminiviruses. Despite its bipartite nature, SACMV was most closely related to monopartite TYLCVs, but was sufficiently different to justify designating it as a distinct virus. In serological studies, SACMV grouped biologically with EACMV isolates.     

Effect of protein content on low temperature inactivation of the extracellular proteinase from Pseudomonas fluorescens 22F

The formation of cholic acid and chenodeoxycholic acid through cleavage of the side chains of CoA esters of 3alpha,7alpha,12alpha-trihydroxy-5beta-choles tan-26-oic acid and 3alpha,7alpha-dihydroxy-5beta-cholestan-26-oic acid is believed to occur in peroxisomes. Recently, we found a new peroxisomal enzyme, D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein, and suggested that this bifunctional protein is responsible for the conversion of 3alpha,7alpha,12alpha-trihydroxy-5beta-cholest-2 4-en-26-oyl-CoA and 3alpha,7alpha-dihydroxy-5beta-cholest-24-en-26-oyl-CoA to their 24-oxo-forms. In the present study, the products of this bifunctional protein reaction were analyzed by gas chromatography-mass spectrometry, and the formation of 24-oxo-27-nor-cholestanes was confirmed. Previously, we found a new thiolase in Caenorhabditis elegans, P-44, and suggested that P-44 and sterol carrier protein x, a peroxisomal protein, constitute a second group of 3-oxoacyl-CoA thiolases. The production of cholic acid and chenodeoxycholic acid from the precursors on incubation with the bifunctional protein and sterol carrier protein x or P-44 was confirmed by gas chromatography.     

Right-sided sciatalgia complicating Crohn's disease

Neurological complications of Crohn's disease due to involvement of the extradural space are extremely rare. A 40-yr-old woman with Crohn's disease affecting the terminal ileum presented with a right-sided sciatalgia. The patient did not complain of diarrhea or constipation. The serum fibrinogen and the C-reactive protein were elevated. Magnetic resonance imaging and computed tomography scan of the abdomen and pelvis demonstrated a mass in front of the sacrum up to but not including the first sacral vertebra. Surgical intervention, with resection of 15 cm of terminal ileum, led to the complete resolution of symptoms. In this case, the underlying cause of the neurological symptoms was most likely an infiltration of the right lumbosacral nerve caused by edema and inflammation of the terminal ileum in the vicinity of the presacral space. Unexplained lumbosacral neurological symptoms in a patient with Crohn's disease necessitate a magnetic resonance imaging or computed tomography scan to detect potential neurological compression.     

Chemical denaturation and modification of ovalbumin alters its dependence on ubiquitin conjugation for class I antigen presentation

Class I presentation of microinjected native OVA by a temperature-sensitive ubiquitin conjugation mutant, ts85, but not wild-type murine cells, was markedly inhibited following incubation at a nonpermissive temperature. In contrast, the nonpermissive temperature did not affect class I presentation of a minimal OVA peptide expressed in the cytosol. Therefore, these results provide a second example in which a temperature sensitive mutation in the ubiquitin conjugation pathway inhibits MHC class I presentation of native OVA. Surprisingly, incubation at the nonpermissive temperature did not inhibit class I presentation of chemically denatured and alkylated OVA microinjected into the cytosol of mutant cells. Similarly, the presentation of endogenously synthesized OVA (which is expressed from a recombinant vaccinia virus and, presumably, is misfolded in the cytosol) was also not inhibited in both mutant cell lines. Methylation of the lysine groups in denatured OVA, which blocks ubiquitin conjugation, reduced but did not eliminate the presentation of denatured OVA, providing evidence for both ubiquitin-dependent and ubiquitin-independent pathways for class I presentation. In contrast, a proteasome inhibitor blocked class I presentation of all forms of OVA, while a control peptide aldehyde was not inhibitory. These results indicate that modification of the structure of a protein can influence its requirements for ubiquitin conjugation for efficient class I presentation, with the key alteration possibly being the loss of proper conformation. However, regardless of the form of the Ag, the proteasome appears to be required for generating peptides from both endogenously synthesized and microinjected OVA for class I presentation.     

Alar fold resection in horses: 24 cases (1979-1992)

Between 1979 and 1992, the alar folds were resected bilaterally in 22 horses and unilaterally in 2 horses. Abnormal respiratory tract noise and exercise intolerance were the primary complaints prior to surgery. Significantly (P = 0.01) more Standardbreds underwent resection of the alar folds, compared with the number of Standardbreds in the hospital population during the same period. The alar folds palpated abnormally thick in 13 horses and normal in 11 horses. Temporary dilatation of the nares with mattress sutures or clips lessened the respiratory tract noise and improved exercise tolerance in all 8 horses in which the diagnostic test was performed. Manual elevation of the alar folds reduced respiratory noise in the 11 horses evaluated. Long-term follow-up evaluation by telephone was available for 14 horses. All surgical incisions had healed cosmetically. Respiratory tract noise was decreased, and exercise tolerance improved in 10 of 14 (71%) horses. Complete charted racing information was obtained for 16 horses. Fourteen horses started their first race a mean of 118 days (range, 13 to 321 days) after surgery. The mean number of starts after surgery was 51, with 14 of 16 (88%) horses starting more than 6 times after surgery. Of the 16 horses, 8 horses raced at least 3 times before and after surgery; 4 had improved racing performance, 2 had similar performance, and 2 had decreased performance. Five Standardbreds never raced, and 1 Standardbred raced once before surgery.(ABSTRACT TRUNCATED AT 250 WORDS)