Crystal structure of cod liver class I alcohol dehydrogenase: substrate pocket and structurally variable segments.

The structural framework of cod liver alcohol dehydrogenase is similar to that of horse and human alcohol dehydrogenases. In contrast, the substrate pocket differs significantly, and main differences are located in three loops. Nevertheless, the substrate pocket is hydrophobic like that of the mammalian class I enzymes and has a similar topography in spite of many main-chain and side-chain differences. The structural framework of alcohol dehydrogenase is also present in a number of related enzymes like glucose dehydrogenase and quinone oxidoreductase. These enzymes have completely different substrate specificity, but also for these enzymes, the corresponding loops of the substrate pocket have significantly different structures. The domains of the two subunits in the crystals of the cod enzyme further differ by a rotation of the catalytic domains by about 6 degrees. In one subunit, they close around the coenzyme similarly as in coenzyme complexes of the horse enzyme, but form a more open cleft in the other subunit, similar to the situation in coenzyme-free structures of the horse enzyme. The proton relay system differs from the mammalian class I alcohol dehydrogenases. His 51, which has been implicated in mammalian enzymes to be important for proton transfer from the buried active site to the surface is not present in the cod enzyme. A tyrosine in the corresponding position is turned into the substrate pocket and a water molecule occupies the same position in space as the His side chain, forming a shorter proton relay system.     

An Object-Oriented Modeling and Simulation Environment for Reactive Systems Development

An environment to support the modeling, analysis, simulation, and development of state transition models, SMOOCHES (State Machines for Object-Oriented Concurrent Hierarchical Engineering Specifications), is presented. SMOOCHES allows the hierarchical construction, analysis, and simulation of state transition models in an object-oriented distributed environment. Statecharts (see Harel 1987b), a powerful mechanism for state transition specification, are fundamental to the development of SMOOCHES. To assist in the specification of hierarchical state transition models for distributed and reactive systems, statecharts are extended by introducing the concept of exit-safe states. SMOOCHES allows the specification of objects in the system with hierarchical state transition models and the derivation of new classes of objects through inheritance. A graphical monitoring system has been developed to represent and simulate the object state life cycles and monitor event generations. The example presented illustrates the modeling and simulation of different state life cycles of an assembly robot.     

Transport of carnosine by mouse intestinal brush-border membrane vesicles

The characteristics of carnosine (beta-alanyl-L-histidine) transport have been studied using purified brush-border membrane vesicles from mouse small intestine. Uptake curves did not exhibit any overshoot phenomena, and were similar under Na+, K+ or choline+ gradient conditions (extravesicular greater than intravesicular). However, uptake of histidine showed an overshoot phenomenon in the presence of a Na+-gradient. There was no detectable hydrolysis of carnosine during 15 min of incubation with membrane vesicles under conditions used for transport experiments. Analysis of intravesicular contents further showed the complete absence of the constituent free amino acids of carnosine, and indicates that intact carnosine is transported. Studies on the effect of concentration on peptide uptake revealed that transport occurred by a saturable process conforming to Michaelis-Menten kinetics with a Km of 9.6 +/- 1.4 mM and a Vmax of 2.9 +/- 0.2 nmol/mg protein per 0.4 min. Uptake of carnosine was inhibited by both di- and tripeptides with a maximum inhibition of 68% by glycyl-L-leucyltyrosine. These results clearly demonstrate that carnosine is transported intact by a carrier-mediated, Na+-independent process.     

Nuclear magnetic resonance study of the impact of ribose 2′-O-methylation on the aqueous solution conformation of cytidylyl-(3′ → 5′)-cytidine

In order to obtain information about the conformational characteristics at the nearestneighbor level in the 2′-O-methylated region of t-RNA, as well as in the bizarre 5′-terminus of eucaryotic mRNA, a detailed nuclear magnetic resonance study of 2′-O-methyl-cytidylyl-(3′ → 5′)-cytidine (CmpC) was conducted. Proton spectra were recorded at 270 MHz in the Fourier mode in D₂O solutions, 0.01M, pD 7.3 in the temperature range 5–80°C. Complete accurate sets of nmr parameters were derived for each of the nucleotidyl units by a combination of homo-nuclear decouplings and simulation iteration methods. The data were translated into conformational parameters using procedures developed in earlier studies from these laboratories. It is shown that the ribofuranose ring exists at a ²E ? ³E equilibrium with clear preference [(75–80)%] for the ³E mode. The C(4′)-C(5′) and C(5′)-O(5′) bonds form a stable conformational network with outspoken preference for conformers in which Ψ₁, Ψ₂ ? 60° and ?₂ ? 180°. The orientation of the 3′-phosphate and 2′-O-methyl groups is such that ?₁′ ? 210° and ?″ ? 60°. The phosphodiester bonds are flexible and shift trends for base, H(1′), and H(5″) suggest the existence of a conformational blend of right-handed stack (g^?g^?), left-handed stack (g⁺g⁺), and unstacked arrays (tg^? and tg⁺). Elevation of temperature perturbs the ²E ? ³E equilibrium accompanied with modest depopulation of ψ₁, ψ₂ ? 60° and ?₂ ? 180° conformers. The major effect of elevation of temperature is in the increase of unstacked arrays at the expense of g^?g^? and g⁺g⁺ conformers. The shift trend of Cmp-H(3′) with temperature shows that torsional variation about O(3′)-P is facilitated by increase in temperature and the preferred rotamer about O(3′)-P in the unstacked form is t (ω₁′ = 180°). A detailed comparison of the aqueous solution conformations of CpC and CmpC reveals that 2′-O-methylation causes: (i) a reduction in the magnitude of _χ1; (ii) an increase in the population of ³E pucker at the 3′-nucleotidyl unit; and (iii) modest perturbations in the O(3′)-P and P-O(5′) bond conformations. Comparison of the aqueous solution conformations of AmpA and CmpC makes clear that the conformational properties of pyrimidine-pyrimidine and purine-purine dimers which carry a 2′-O-methylated 3′-nucleotidyl unit are significantly different.     

Pathology of pulmonary parasitic migration: morphological and bronchoalveolar cellular responses following Nippostrongylus brasiliensis infection in rats

Nippostrongylus brasiliensis has an obligatory migratory phase through the lungs during its development in rats. This migration is associated with marked tissue damage and pronounced cellular reaction. Given that cells from the lower respiratory tract, especially alveolar macrophages, can adhere to and kill larvae of N. brasiliensis in vitro, we studied the time course of morphological changes associated with parasitic migration. Compared to a primary infection, a secondary infection resulted in significant changes in the pulmonary tissue characterized by an early acute inflammation leading to granulomatous reaction in the parenchyma and a leucocytosis in the bronchoalveolar lavage fluids with an anamnestic increase in absolute numbers of neutrophils, alveolar macrophages, eosinophils, and lymphocytes. Scanning electron microscopy showed that inflammatory cells, especially alveolar macrophages, granulocytes, lymphocytes, erythrocytes, and platelets, adhered to the larvae following secondary infection and this adhesion was associated with disruption of cuticular surface in some larvae. Secondary infection also resulted in retention of larvae in granulomatous lesions in the lungs even up to 21 days postinfection. There was mast cell and type II pneumocyte hyperplasia and these cells appeared to be activated. Thus, the histopathological changes in lungs correlated with the bronchoalveolar cellular responses and further document the inflammatory and immunological reactions during the migration of N. brasiliensis larvae.     

Flight responses in a wind tunnel of the parasitoid Microplitis croceipes to three spring and three summer host plants of Heliothis spp.

Flight responses of the parasitoid, Microplitis croceipes (Cresson) (Hymenoptera: Braconidae) to two phenological stages of spring host plants (crimson clover, Trifolium incarnatum L., hairy vetch, Vicia villosa Corbiere, and cutleaf geranium, Geranium dissectum L.) and summer host plants (cotton, Gossypium hirsutum L., sorghum, Sorghum bicolor L., and soybean Glycine max (L.) Merr.) of Heliothis virescens (F.), were measured in a dual choice situation in a wind tunnel. Preflowering clover was more attractive than preflowering geranium, followed by preflowering vetch. Clover was preferred over geranium in flowering stages as well. No within species differences were detected for the two phenological stages. Preflowering and flowering cotton and soybean were preferred over preflowering and flowering sorghum. The findings could be important in pre-determining release sites for M. croceipes in the field.     

Studies on the transport of aliphatic glucosides by hamster small intestine in vitro

It has been found (1) that glucosides with a long alkyl chain (2–18 carbon atoms) as the aglycone can be transported by carrier-mediated processes in the hamster small intestine in vitro, (2) that these glucosides interact with the glucose carrier, and (3) that they compete with glucose and analogs for the binding to the carrier. There are Na⁺- and phlorizin-insensitive components of uptake for the long chain alkyl glucosides which suggest additional interactions or uptake processes.     

A glycosidic germacradienolide from Eupatorium altissimum

The isolation and structure determination of a glycosidic germacradienolide from Eupatorium altissimum are reported. Eupatorin and 5-hydroxy-3′,4′,6,7-tetramethoxyflavone were also found.