Enzyme models: design and selection

There are two approaches to the discovery of enzyme mimics, that is identifying molecules that are able to bind substrate(s) and then catalyze reactions. The first approach, often inspired by enzymes themselves, utilises chemical knowledge and experience to design the catalyst. The other approach is to create a library and select the best host of a transition state analogue of the required reaction.     

Coregulation of beta-galactoside uptake and hydrolysis by the hyperthermophilic bacterium Thermotoga neapolitana

Regulation of the beta-galactoside transport system in response to growth substrates in the extremely thermophilic anaerobic bacterium Thermotoga neapolitana was studied with the nonmetabolizable analog methyl-beta-D-thiogalactopyranoside (TMG) as the transport substrate. T. neapolitana cells grown on galactose or lactose accumulated TMG against a concentration gradient in an intracellular free sugar pool that was exchangeable with external galactose or lactose and showed induced levels of beta-galactosidase. Cells grown on glucose, maltose, or galactose plus glucose showed no capacity to accumulate TMG, though these cells carried out active transport of the nonmetabolizable glucose analog 2-deoxy-D-glucose. Glucose neither inhibited TMG uptake nor caused efflux of preaccumulated TMG; rather, glucose promoted TMG uptake by supplying metabolic energy. These data show that beta-D-galactosides are taken up by T. neapolitana via an active transport system that can be induced by galactose or lactose and repressed by glucose but which is not inhibited by glucose. Thus, the phenomenon of catabolite repression is present in T. neapolitana with respect to systems catalyzing both the transport and hydrolysis of beta-D-galactosides, but inducer exclusion and inducer expulsion, mechanisms that regulate permease activity, are not present. Regulation is manifest at the level of synthesis of the beta-galactoside transport system but not in the activity of the system.     

Transforming growth factor-beta inhibits Leydig cell steroidogenesis in primary culture

The effects of transforming growth factor (TGF) on Leydig cell steroidogenesis in primary culture were investigated. Basal testosterone levels were 3.7 +/- 0.54 ng/ml (mean +/- SE, N = 7). In the presence of hCG (10 ng/ml), testosterone levels increased to 22.77 +/- 3.05 ng/ml. TGF-beta caused a dose dependent inhibition of hCG-stimulated testosterone formation but without effects on basal levels. TGF-beta also inhibited 8-bromo cyclic AMP-induced testosterone formation and hCG-stimulated cyclic AMP formation. In contrast, TGF-alpha had no effect on either basal or hCG-stimulated testosterone formation and did not modify the inhibitory effect of TGF-beta. Present study indicates that TGF-beta can modulate Leydig cell steroidogenesis.     

Induction of Freezing Tolerance in Spinach during Cold Acclimation

Spinach (Spinacia oleracea L.) seedlings, grown in soil or on an agar medium in vitro, became cold acclimated when exposed to a constant 5°C. Plants subjected to cold acclimation, beginning 1 week postgermination, attained freezing tolerance levels similar to that achieved by seedlings that were cold acclimated beginning 3 weeks after sowing. Seedlings at 1 week of age had only cotyledonary leaves, while 3-week-old seedlings had developed true leaves. Plants grown in vitro were able to increase in freezing tolerance, but were slightly less hardy than soil-grown plants. These results suggest that spinach, a cool-season crop that begins growth in early spring when subzero temperatures are likely, can undergo cold acclimation at the earliest stages of development following germination. Axenic seedlings, grown in vitro, were used to develop a noninjurious radiolabeling technique. Leaf proteins were radiolabeled to specific activities of 10⁵ counts per minute per microgram at 25°C or 5 × 10⁴ counts per minute per microgram at 5°C over a 24 hour period. The ability to radiolabel leaf proteins of in vitro grown plants to high specific activities at low temperature, without injury or microbial contamination, will facilitate studies of cold acclimation.     

Form birefringence of muscle.

We investigate the sensitivity of measurements of muscle birefringence to cross-bridge dynamics in the resting, active, and rigor states. The theory of form birefringence is reviewed, and an optical model is constructed for the form birefringence of muscle. Values for the parameters in the model are selected or deduced from the literature. As an illustration of the use of the model, plausible distributions for the orientations of cross-bridges in the resting, active, and rigor states are constructed using a model for cross-bridge dynamics suggested by Huxley and Kress (1985). The general magnitude of the predictions of our model is comparable with that of published measurements of muscle birefringence. However, the precise values of the predicted birefringence for the resting, active, and rigor states are sensitive to the assumed orientations of cross-bridges. We also investigate the dependence of muscle birefringence on sarcomere length and on disorder in the orientation of the myofilament array. We conclude that measurements of muscle birefringence can play a useful role in distinguishing between proposed models of cross-bridge dynamics.     

Effects of Varying Epoch Lengths,Wear Time Algorithms,and Activity Cut-Points on Estimates of Child Sedentary Behavior and Physical Activity from Accelerometer Data

Objective

To examine the effects of accelerometer epoch lengths, wear time (WT) algorithms, and activity cut-points on estimates of WT, sedentary behavior (SB), and physical activity (PA).

Methods

268 7–11 year-olds with BMI ≥ 85^th percentile for age and sex wore accelerometers on their right hips for 4–7 days. Data were processed and analyzed at epoch lengths of 1-, 5-, 10-, 15-, 30-, and 60-seconds. For each epoch length, WT minutes/day was determined using three common WT algorithms, and minutes/day and percent time spent in SB, light (LPA), moderate (MPA), and vigorous (VPA) PA were determined using five common activity cut-points. ANOVA tested differences in WT, SB, LPA, MPA, VPA, and MVPA when using the different epoch lengths, WT algorithms, and activity cut-points.

Results

WT minutes/day varied significantly by epoch length when using the NHANES WT algorithm (p < .0001), but did not vary significantly by epoch length when using the ≥ 20 minute consecutive zero or Choi WT algorithms. Minutes/day and percent time spent in SB, LPA, MPA, VPA, and MVPA varied significantly by epoch length for all sets of activity cut-points tested with all three WT algorithms (all p < .0001). Across all epoch lengths, minutes/day and percent time spent in SB, LPA, MPA, VPA, and MVPA also varied significantly across all sets of activity cut-points with all three WT algorithms (all p < .0001).

Conclusions

The common practice of converting WT algorithms and activity cut-point definitions to match different epoch lengths may introduce significant errors. Estimates of SB and PA from studies that process and analyze data using different epoch lengths, WT algorithms, and/or activity cut-points are not comparable, potentially leading to very different results, interpretations, and conclusions, misleading research and public policy.     

Phosphotyrosine (p-Tyr)-Dependent and -Independent Mechanisms of p190 RhoGAP-p120 RasGAP Interaction: Tyr 1105 of p190, a Substrate for c-Src, Is the Sole p-Tyr Mediator of Complex Formation

p190 RhoGAP is a 190-kDa protein that stably associates with p120 RasGAP and regulates actin dynamics through members of the Rho family of small GTPases. Previous studies have indicated a direct relationship between levels of p190 tyrosine phosphorylation, the extent and kinetics of epidermal growth factor (EGF)-induced actin rearrangements, and EGF-induced cell cycle progression, suggesting that p190 links Ras-mediated mitogenic signaling with signaling through the actin cytoskeleton. Determining which tyrosine residues in p190 are phosphorylated, what factors regulate phosphorylation of these sites, and what effect tyrosine phosphorylation has on p190 function is key to understanding the role(s) that p190 may play in these processes. To begin investigating these questions, we used biochemical approaches to characterize the number and relative levels of in vivo-phosphorylated tyrosine residues on endogenous p190 from C3H10T1/2 murine fibroblasts. Only two tryptic phosphopeptides containing phosphotyrosine (p-Tyr), a major site, identified as Y1105, and a minor, unidentified site, were detected. Phosphorylation of Y1105, but not the minor site, was modulated in vivo to a greater extent by overexpression of c-Src than by the EGF receptor and was efficiently catalyzed by c-Src in vitro, indicating that Y1105 is a selective and preferential target of c-Src both in vitro and in vivo. In vitro and in vivo coprecipitation analysis using glutathione S-transferase (GST) fusion proteins containing wild-type and Y1105F variants of the p190 middle domain, variants of full-length p190 ectopically expressed in COS-7 cells, and endogenous p190 and p120 in C3H10T1/2 cells revealed that p190 could bind to p120 in the presence and absence of p190 tyrosine phosphorylation. p-Tyr-independent complexes comprised 10 to 20% of the complexes formed in the presence of p-Tyr. Mutation of Y1105 from Tyr to Phe resulted in complete loss of p-Tyr-dependent complex formation, indicating that p-Y1105 was the sole p-Tyr residue mediating binding to p120. These studies describe a specific mechanism by which c-Src can regulate p190-p120 association and also document a significant role for p-Tyr-independent means of p190-p120 binding.     

Structure of the dnaA region of the endosymbiont, Buchnera aphidicola, of aphid Schizaphis graminum

Buchnera aphidicola is an intracellular prokaryote (endosymbiont)that lives in the body cavity of the aphid. Phylogenetic studiesindicated that it is closely related to Escherichia coli andmembers of Enterobacteria. The gene order of the region containingthe dnaA gene is well conserved in many bacteria. Seven genesof the endosymbiont of the aphid Schizaphis graminum, gyrB,dnaN, dnaA, rpmH, rnpA, yidD, and 60K, were found to be homologousin sequence and relative location to those of E. coli. We havefurther sequenced the region downstream of the 60K gene to elucidatethe boundary of the conserved region, and found that one moregene, thdF , is conserved. The comparison of gene organizationsof the dnaA region of the related bacteria supported the closephylogenetic relationship of B. aphidicola to E. coli. In addition,we have identified groES and groEL genesnext to the thdF gene.GroEL protein was reported to be expressed at an elevated levelin the endosymbionts of aphids, and is considered to play animportant role in their association with the aphid host. Comparisonof the structure of the groE operon with that of the endosymbiontof the aphid Acyrthosiphon pisum revealed the conservation ofa sequence resembling the E. coli consensus heat shock promoter,and this sequence may be responsible for the high expressionof the groEL gene in aphid endosymbionts.     

STRUCTURE AND HISTOGENESIS OF THE EMBRYO OF ACER SACCHARINUM. I. EMBRYO SAC AND PROEMBRYO

Structure of the embryo sac and development of the proembryo of Acer saccharinum L. are described from paraffin sections. The embryo sac is monosporic and identical to the 8-nucleate Polygonum type in all respects. Cell, nuclear, and nucleolar sizes are constant within a narrow range and sharply distinctive for all components of the mature sac. Polar nuclei fuse before double fertilization. The longitudinal axis of symmetry of the egg, zygote, and proembryo is variously oriented with respect to the longitudinal axis of the embryo sac and is determined by the point of attachment of the presumptive egg cell to the sac wall. Subsequent development of the young embryo is responsive to aligning factors within the embryo sac and is collateral with the longitudinal axis of the sac. The first segmentation is transverse to the longitudinal axis of the zygote; the second and third are transverse in the basal cell and longitudinal in the apical cell. Descendants of ci form a short irregular suspensor; ca and m give rise to the apical and basal halves respectively of the embryo proper. The contribution of the proembryonic tiers to the older embryo differs in embryos of different initial orientation. Distribution and orientation of mitosis in the proembryo are shown in two accumulation maps.