Catalysis by dienelactone hydrolase: A variation on the protease mechanism

Dienelactone hydrolase (DLH), an enzyme from the β-ketoadipate pathway, catalyzes the hydrolysis of dienelactone to maleylacetate. Our inhibitor binding studies suggest that its substrate, dienelactone, is held in the active site by hydrophobic interactions around the lactone ring and by the ion pairs between its carboxylate and Arg-81 and Arg-206. Like the cysteine/serine proteases, DLH has a catalytic triad (Cys-123, His-202, Asp-171) and its mechanism probably involves the formation of covalently bound acyl intermediate via a tetrahedral intermediate. Unlike the proteases, DLH seems to protonate the incipient leaving group only after the collapse of the first tetrahedral intermediate, rendering DLH incapable of hydrolyzing amide analogues of its ester substrate. In addition, the triad His probably does not protonate the leaving group (enolate) or deprotonate the water for deacylation; rather, the enolate anion abstracts a proton from water and, in doing so, supplies the hydroxyl for deacylation. © 1993 Wiley-Liss, Inc.     

Amazonian biodiversity: assessing conservation priorities with taxonomic data

Data from 3991 records of museum collections representing 421 species of plants, arthropods, amphibians, fish, and primates were analyzed with GIS to identify areas of high species diversity and endemism in Amazonia. Of the 472 1 × 1° grid cells in Amazonia, only nine cells are included in the highest species diversity category (43–67 total species) and nine in the highest endemic species diversity category (4–13 endemic species). Over one quarter of the grid cells have no museum records of any of the organisms in our study. Little correspondence exists between the centers of species diversity identified by our collections-based data and those areas recommended for conservation in an earlier qualitative study of Amazonian biodiversity. Museum collections can play a vital role in identifying species-rich areas for potential conservation in Amazonia, but a concerted and structured effort to increase the number and distribution of collections is needed to take maximum advantage of the information they contain.     

Apical membrane potentials and intracellular potassium and sodium activities in the gallbladder of the freshwater turtle Mauremys caspica

Open-tip and liquid ion-exchanger microelectrodes were used to measure transapical membrane potential (Va), fractional voltage ratio (fa) and intracellular sodium and potassium activities (aiNa, aiK) in Mauremys caspica gallbladder under open circuit conditions. The average values (+/- SEM) for Va and fa were -32 +/- 3 and 0.20 +/- 0.03 mV respectively. aiNa and aiK were, respectively, 17 +/- 4 and 82 +/- 7 mM. These results suggest that the mechanisms of Na+ and K+ transport in this tissue are essentially similar to those observed in leaky epithelia in general.     

Carbachol and KCl-induced changes in intracellular free calcium concentration in isolated, fura-2 loaded smooth-muscle cells from the anterior byssus retractor muscle of Mytilus edulis

Intracellular free calcium concentration [( Ca2+]1) was measured in suspensions of fura-2 loaded smooth-muscle cells isolated from the anterior byssus retractor muscle of Mytilus edulis. Successive application of 5mM carbachol (CCh) and 100mM KCl to the cells transiently elevated [Ca2+]1 from the resting value of 124 +/- 4.5nM (mean +/- S.E., n = 14) to 295 +/- 15.3 and 383 +/- 20.5 nM, respectively. The response to CCh was concentration-dependent with an ED50 of 10(-5) M. Under the microscope, 67 +/- 3.0 and 83 +/- 1.3 % of fura-2 loaded cells contracted on the addition of 5mM CCh and 100mM KCl, respectively. In Ca2+ -free sea water, the CCh induced change in [Ca2+]1 was partially suppressed whereas that induced by KCl was completely abolished, suggesting an agonist-evoked release of stored Ca2+.     

Amyloid β-Mediated Zn2+ Influx into Dentate Granule Cells Transiently Induces a Short-Term Cognitive Deficit

We examined an idea that short-term cognition is transiently affected by a state of confusion in Zn²⁺ transport system due to a local increase in amyloid-β (Aβ) concentration. A single injection of Aβ (25 pmol) into the dentate gyrus affected dentate gyrus long-term potentiation (LTP) 1 h after the injection, but not 4 h after the injection. Simultaneously, 1-h memory of object recognition was affected when the training was performed 1 h after the injection, but not 4 h after the injection. Aβ-mediated impairments of LTP and memory were rescued in the presence of zinc chelators, suggesting that Zn²⁺ is involved in Aβ action. When Aβ was injected into the dentate gyrus, intracellular Zn²⁺ levels were increased only in the injected area in the dentate gyrus, suggesting that Aβ induces the influx of Zn²⁺ into cells in the injected area. When Aβ was added to hippocampal slices, Aβ did not increase intracellular Zn²⁺ levels in the dentate granule cell layer in ACSF without Zn²⁺, but in ACSF containing Zn²⁺. The increase in intracellular Zn²⁺ levels was inhibited in the presence of CaEDTA, an extracellular zinc chelator, but not in the presence of CNQX, an AMPA receptor antagonist. The present study indicates that Aβ-mediated Zn²⁺ influx into dentate granule cells, which may occur without AMPA receptor activation, transiently induces a short-term cognitive deficit. Extracellular Zn²⁺ may play a key role for transiently Aβ-induced cognition deficits.     

Resistant Starch Alters the Microbiota-Gut Brain Axis: Implications for Dietary Modulation of Behavior

The increasing recognition that the gut microbiota plays a central role in behavior and cognition suggests that the manipulation of microbial taxa through diet may provide a means by which behavior may be altered in a reproducible and consistent manner in order to achieve a beneficial outcome for the host. Resistant starch continues to receive attention as a dietary intervention that can benefit the host through mechanisms that include altering the intestinal microbiota. Given the interest in dietary approaches to improve health, the aim of this study was to investigate whether the use of dietary resistant starch in mice to alter the gut microbiota also results in a change in behavior. Forty-eight 6 week-old male Swiss-Webster mice were randomly assigned to 3 treatment groups (n = 16 per group) and fed either a normal corn starch diet (NCS) or diets rich in resistant starches HA7 diet (HA7) or octenyl-succinate HA7 diet (OS-HA7) for 6 week and monitored for weight, behavior and fecal microbiota composition. Animals fed an HA7 diet displayed comparable weight gain over the feeding period to that recorded for NCS-fed animals while OS-HA7 displayed a lower weight gain as compared to either NCS or HA7 animals (ANOVA p = 0.0001; NCS:HA7 p = 0.244; HA7:OS-HA7 p<0.0001; NCS:OS-HA7 p<0.0001). Analysis of fecal microbiota using 16s rRNA gene taxonomic profiling revealed that each diet corresponded with a unique gut microbiota. The distribution of taxonomic classes was dynamic over the 6 week feeding period for each of the diets. At the end of the feeding periods, the distribution of taxa included statistically significant increases in members of the phylum Proteobacteria in OS-HA7 fed mice, while the Verrucomicrobia increased in HA7 fed mice over that of mice fed OS-HA7. At the class level, members of the class Bacilli decreased in the OS-HA7 fed group, and Actinobacteria, which includes the genus Bifidobacteria, was enriched in the HA7 fed group compared to the control diet. Behavioral analysis revealed that animals demonstrated profound anxiety-like behavior as observed by performance on the elevated-plus maze with time spent by the mice in the open arm (ANOVA p = 0.000; NCS:HA7 p = 0.004; NCS:OS-HA7 p = 1.000; HA7:OS-HA7 p = 0.0001) as well as entries in the open arm (ANOVA p = 0.039; NCS:HA7 p = 0.041; HA7:OS-HA7 p = 0.221; NCS:OS-HA7 p = 1.000). Open-field behavior, a measure of general locomotion and exploration, revealed statistically significant differences between groups in locomotion as a measure of transitions across quadrant boundaries. Additionally, the open-field assay revealed decreased exploration as well as decreased rearing in HA7 and OS-HA7 fed mice demonstrating a consistent pattern of increased anxiety-like behavior among these groups. Critically, behavior was not correlated with weight. These results indicate that diets based on resistant starch can be utilized to produce quantifiable changes in the gut microbiota and should be useful to “dial-in” a specific microbiome that is unique to a particular starch composition. However, undesirable effects can also be associated with resistant starch, including lack of weight gain and increased anxiety-like behaviors. These observations warrant careful consideration when developing diets rich in resistant starch in humans and animal models.