Allosteric interactions of quaternary strychnine and brucine derivatives with muscarinic acetylcholine receptors

The affinity and allosteric properties of 22 quaternary derivatives of strychnine and brucine at the m1-m4 subtypes of muscarinic receptors have been analyzed and compared. The subtype selectivity, in terms of affinity, was in general m2 > m4 > m1 > m3. The highest affinities were found for N-benzyl, N-2-naphthylmethyl, and N-4-biphenylylmethyl strychnine (13, 14, and 18, respectively). All the strychnine and brucine derivatives were positively cooperative with the antagonist, N-methylscopolamine, at m2 receptors and, in the case of the strychnine analogues, were positively cooperative with N-methylscopolamine at least at one other subtype. The strychnine analogues were negatively cooperative with the neurotransmitter, acetylcholine, at all subtypes whereas brucine and five of the six derivatives examined were positively cooperative with acetylcholine at one or more subtypes (m1-m5) and exhibited different patterns of subtype selectivity. The ability to generate subtype-selective allosteric enhancers of acetylcholine binding and function may be of use in the development of drugs for the treatment of Alzheimer's disease.     

The only active mutant of thymidylate synthase D169, a residue far from the site of methyl transfer, demonstrates the exquisite nature of enzyme specificity

Cysteine is the only variant of D169, a cofactor-binding residuein thymidylate synthase, that shows in vivo activity. The 2.4Å crystal structure of Escherichia coli thymidylate synthaseD169C in a complex with dUMP and the antifolate CB3717 showsit to be an asymmetric dimer, with only one active site covalentlybonded to dUMP. At the active site with covalently bound substrate,C169 S     

Gallium-arsenide buffer store components for Gbit/s optical-fibertransmission systems

In many cases long-haul optical-fiber transmission systems require the use of buffer stores to interface to a larger network, for example, to reduce the effects of jitter accumulation. A set of gallium-arsenide (GaAs) buffer-store components designed and manufactured using a 1-μm process is described. Operation at data rates of at least 2-Gb/s has been achieved     

Physical Forces Modulate Oxidative Status and Stress Defense Meditated Metabolic Adaptation of Yeast Colonies: Spaceflight and Microgravity Simulations

Baker’s yeast (Saccharomyces cerevisiae) has broad genetic homology to human cells. Although typically grown as 1-2mm diameter colonies under certain conditions yeast can form very large (10 + mm in diameter) or ‘giant’ colonies on agar. Giant yeast colonies have been used to study diverse biomedical processes such as cell survival, aging, and the response to cancer pharmacogenomics. Such colonies evolve dynamically into complex stratified structures that respond differentially to environmental cues. Ammonia production, gravity driven ammonia convection, and shear defense responses are key differentiation signals for cell death and reactive oxygen system pathways in these colonies. The response to these signals can be modulated by experimental interventions such as agar composition, gene deletion and application of pharmaceuticals. In this study we used physical factors including colony rotation and microgravity to modify ammonia convection and shear stress as environmental cues and observed differences in the responses of both ammonia dependent and stress response dependent pathways We found that the effects of random positioning are distinct from rotation. Furthermore, both true and simulated microgravity exacerbated both cellular redox responses and apoptosis. These changes were largely shear-response dependent but each model had a unique response signature as measured by shear stress genes and the promoter set which regulates them These physical techniques permitted a graded manipulation of both convection and ammonia signaling and are primed to substantially contribute to our understanding of the mechanisms of drug action, cell aging, and colony differentiation.     

Localization of Jacobsen syndrome breakpoints on a 40-Mb physical map of distal chromosome 11q

-The aim of the present study was to assess the effects of high heart rate on mortality in different subgroups in a French population according to age, gender, and blood pressure levels. We studied 19 386 subjects (12 123 men, 7263 women), aged 40 to 69 years, who had a routine health examination at the Centre d'Investigations Préventives et Cliniques (IPC) between 1974 and 1977. Heart rate (HR) measured by ECG was classified into 4 groups: HR1, <60; HR2, 60 to 80; HR3, 81 to 100; and HR4, >100 bpm. Mortality data were recorded for the period of 1974 through 1994. In both sexes, HR was a significant predictor of noncardiovascular mortality. In men, the relative risk (95% confidence interval) for cardiovascular death after adjustment for age and other risk factors in the HR2, HR3, and HR4 groups was 1.35 (1.01 to 1.80), 1.44 (1.04 to 2.00), and 2.18 (1.37 to 3.47), respectively, when compared with HR1. In women, HR did not influence cardiovascular mortality. The association of HR with cardiovascular mortality in men was (1) related to a strong association with coronary but not cerebrovascular mortality, (2) independent of age and hypertension, and (3) influenced by the level of pulse pressure; in patients with high pulse pressure (>65 mm Hg), accelerated HR was not associated with increased cardiovascular mortality. In conclusion, in a large French population, accelerated resting HR represents an independent predictor of noncardiovascular mortality in both genders, and of cardiovascular mortality in men, independent of age and the presence of hypertension. Further investigations are needed to explain the complex interactions between HR, pulse pressure, and cardiovascular complications.     

Transendothelial migration of lymphocytes from HIV-1-infected donors: a mechanism for extravascular dissemination of HIV-1

To identify factors that cause HIV-1 to establish perivascular foci of infected cells, we studied the transendothelial migration of blood mononuclear leukocytes (MNL) from 76 HIV+ patients and 41 controls. The fraction of patients' lymphocytes that migrated across endothelial cell monolayers in vitro was significantly increased (p < or = 0.03) compared with that of control donors. Migration of patients' CD4+ T cells was particularly enhanced, whereas the migration of monocytes did not differ between patients and controls. Lymphocyte migration correlated with expression of CD11a/CD18 and CD49d/CD29 and with the quantity of TNF-alpha produced as MNLs migrated through the endothelium. Measurement of HIV-1 proviral DNA copies in the patients' MNLs (n = 26) suggested that in half the cases virus-infected cells accumulated preferentially amidst the migratory leukocytes. We observed the same behavior with normal donor MNLs infected, in vitro, with each of 4 strains of HIV-1. The number of HIV-1 proviral DNA copies per million MNLs was 40 to 178 times higher in the migratory population than in the original population added to the endothelium. To test whether only certain strains of HIV-1 stimulate transendothelial migration of infected cells, we used single strand conformation polymorphism analysis to identify quasispecies of HIV-1 in the MNLs. If all strains of HIV-1 were equal in their ability to stimulate transendothelial migration, we expected to find no differences in the quasispecies present in the original and migratory cell populations. In fact the quasispecies differed in 14 of 19 paired samples, suggesting that only certain HIV-1 quasispecies promote transendothelial migration of infected cells.     

Advances in modeling and simulation of vacuum electronic devices

Recent advances in the modeling and simulation of vacuum electronic devices are reviewed. Design of these devices makes use of a variety of physical models and numerical code types. Progress in the development of these models and codes is outlined and illustrated with specific examples. The state of the art in device simulation is evolving to the point such that devices can be designed on the computer, thereby eliminating many trial and error fabrication and test steps. The role of numerical simulation in the design places can be expected to grow further in the future     

Pharmacological characterization of guanine nucleotide exchange reactions in membranes from CHO cells stably transfected with human muscarinic receptors m1-m4

We have studied muscarinic agonist stimulated [35S]GTP gamma S binding and [gamma 32P]GTP hydrolysis (GTPase) in membranes from CHO cells stably transfected with human muscarinic m1-m4 receptors. 'Full' agonists were at least 10-fold more potent at m2 & m4 receptors than at m1 & m3. This pattern was less marked with 'partial' agonists, which had a greater maximal effect at m2 & m4 than at m1 & m3. McN-A343 uniquely was more potent and efficacious at m4 than at m2 receptors. Antagonist affinity constants were estimated by fitting the data from inhibition curves directly to the Schild model. Antagonist affinity estimates were very similar to those measured earlier in binding studies using animal tissues, and confirmed a small degree of m4 selectivity for tropicamide and secoverine. The receptor subtypes activated more than one G-protein subtype; m2 & m4 receptors activated only pertussis (PTX) sensitive G-proteins, while m1 & m3 coupled to both PTX sensitive and insensitive G-proteins. Acetylcholine (ACh) was more potent in stimulating guanine nucleotide exchange in PTX-treated m1 cells than in controls.     

The solution structure of the complex of Lactobacillus casei dihydrofolate reductase with methotrexate

We have determined the three-dimensional solution structure of the complex of Lactobacillus casei dihydrofolate reductase (18.3 kDa, 162 amino acid residues) formed with the anticancer drug methotrexate using 2531 distance, 361 dihedral angle and 48 hydrogen bond restraints obtained from analysis of multidimensional NMR spectra. Simulated annealing calculations produced a family of 21 structures fully consistent with the constraints. The structure has four alpha-helices and eight beta-strands with two other regions, comprising residues 11 to 14 and 126 to 127, also interacting with each other in a beta-sheet manner. The methotrexate binding site is very well defined and the structure around its glutamate moiety was improved by including restraints reflecting the previously determined specific interactions between the glutamate alpha-carboxylate group with Arg57 and the gamma-carboxylate group with His28. The overall fold of the binary complex in solution is very similar to that observed in the X-ray studies of the ternary complex of L. casei dihydrofolate reductase formed with methotrexate and NADPH (the structures of the binary and ternary complexes have a root-mean-square difference over the backbone atoms of 0.97 A). Thus no major conformational change takes place when NADPH binds to the binary complex. In the binary complex, the loop comprising residues 9 to 23 which forms part of the active site has been shown to be in the "closed" conformation as defined by M. R. Sawaya & J. Kraut, who considered the corresponding loops in crystal structures of complexes of dihydrofolate reductases from several organisms. Thus the absence of the NADPH does not result in the "occluded" form of the loop as seen in crystal studies of some other dihydrofolate reductases in the absence of coenzyme. Some regions of the structure in the binary complex which form interaction sites for NADPH are less well defined than other regions. However, in general terms, the NADPH binding site appears to be essentially pre-formed in the binary complex. This may contribute to the tighter binding of coenzyme in the presence of methotrexate.