Involvement of the MAP kinase pathways in induction of GADD45 following UV radiation

Indian hedgehog (Ihh) is highly expressed in prehypertrophic chondrocytes in vivo and has been proposed to regulate the proliferation and maturation of chondrocytes and bone collar formation in the growth plate. In high-density cultures of rabbit growth-plate chondrocytes, Ihh mRNA was also expressed at the highest level in the prehypertrophic stage. To explore endogenous factors that regulate Ihh expression in chondrocytes, we examined the effects of various growth factors on Ihh mRNA expression in this system. Retinoic acid (RA) and bone morphogenetic protein-2 enhanced Ihh mRNA expression, whereas PTH/PTH-related peptide (PTHrP) markedly suppressed Ihh expression. RA at more than 10(-8) M induced the expression of Ihh and Patched 1 (Ptc1) within 3 h, before it increased the type X collagen mRNA level at 6-24 h. Cycloheximide blocked the up-regulation of Ihh by RA, indicating the requirement of de novo protein synthesis for this stimulation. These findings suggest that RA is involved in the up-regulation of Ihh during endochondral bone formation. In contrast to RA, PTH (1-84) at 10(-7) M abolished the mRNA expression of Ihh and Ptc1 within 2-4 h, before it suppressed the expression of type X collagen at 12-24 h. The inhibition of Ihh expression by PTH (1-84) did not require de novo protein synthesis. PTH (1-34), PTHrP (1-34), and (Bu)(2)cAMP also suppressed Ihh expression. On the other hand, Ihh has been reported to induce PTHrP synthesis in the perichondrium. Consequently, the direct inhibitory action of PTH/PTHrP on Ihh appears to be a negative feedback mechanism that prevents excess PTHrP accumulation in cartilage.     

Four loops of the catalytic domain of factor viia mediate the effect of the first EGF-like domain substitution on factor viia catalytic activity

The presence of tissue factor is essential for factor VIIa (FVIIa) to reach its full catalytic potential. The previous work in this laboratory demonstrated that substitution of the EGF1 domain of factor VIIa with that of factor IX (FVII((IXegf1))a) results in a substantial decrease in TF-binding affinity and catalytic activity. Supporting simulations of the solution structures of Ca(2+)-bound factor VIIa and FVII((IXegf1))a with tissue factor are provided. Mutants are generated, based on the simulation model, to study the effect of EGF1 substitution on catalytic activity. The simulations show larger Gla-EGF1 and EGF1-EGF2 inter-domain motions for FVII((IXegf1))a than for factor VIIa. The catalytic domain of the chimeric factor VIIa has been disturbed and several surface loops in the catalytic domain of FVII((IXegf1))a (Loop 170s (170-182), Loop 1 (185-188) and Loop 2 (221A-225)) manifest larger position fluctuations than wild-type. The position of Loop 140s (142-152) of FVII((IXegf1))a, near the N terminus insertion site of the catalytic domain, shifts relative to factor VIIa, resulting in a slight alteration of the active site. The results suggest that these four loops mediate the effect of the EGF1 domain substitution on the S1 site and catalytic residues. To test the model, we prepared mutations of these surface loops, including four FVII mutants, D186A, K188A, L144A and R147A, a FVII mutant with multiple mutations (MM3: L144A+R147A+D186A) and a FVII mutant with Loop 170s partially deleted, Loop 170s(del). The catalytic activities towards a small peptidyl substrate decreased 2.4, 4.5 and 9-fold for Loop 170s(del)a (a, activated), L144Aa and D186Aa, respectively, while MM3a lost almost all catalytic activity. The combined results of the simulations and mutants provide insight into the mechanism by which tissue factor enhances factor VIIa catalytic activity.     

gamma-Aminobutyric acid(A) receptor subunit expression predicts functional changes in hippocampal dentate granule cells during postnatal development

Profound alterations in the function of GABA occur over the course of postnatal development. Changes in GABA(A) receptor expression are thought to contribute to these differences in GABAergic function, but how subunit changes correlate with receptor function in individual developing neurons has not been defined precisely. In the current study, we correlate expression of 14 different GABA(A) receptor subunit mRNAs with changes in the pharmacological properties of the receptor in individual hippocampal dentate granule cells over the course of postnatal development in rat. We demonstrate significant developmental differences in GABA(A) receptor subunit mRNA expression, including greater than two-fold lower expression of alpha1-, alpha4- and gamma2-subunit mRNAs and 10-fold higher expression of alpha5-mRNA in immature compared with adult neurons. These differences correlate both with regional changes in subunit protein level and with alterations in GABA(A) receptor function in immature dentate granule cells, including two-fold higher blockade by zinc and three-fold lower augmentation by type-I benzodiazepine site modulators. Further, we find an inverse correlation between changes in GABA(A) receptor zinc sensitivity and abundance of vesicular zinc in dentate gyrus during postnatal development. These findings suggest that developmental differences in subunit expression contribute to alterations in GABA(A) receptor function during postnatal development.     

Spatial variability in oviposition damage by periodical cicadas in a fragmented landscape

Effects of the periodical cicada (Magicicada spp.) on forest dynamics are poorly documented. A 1998 emergence of M. cassini in eastern Kansas led to colonization of a fragmented experimental landscape undergoing secondary succession. We hypothesized that per-tree rates of oviposition damage by cicadas would reflect: (1) distance from the source of the emergence, (2) patch size, and (3) local tree density. Ovipositing females displayed clear preferences for host species and damage incidence showed predictable spatial patterns. Two species (smooth sumac, Rhus glabra, and eastern red cedar, Juniperus virginiana) were rarely attacked, whereas others (rough-leaved dogwood, Cornus drummondii; slippery elm, Ulmus rubra; box elder, Acer negundo, and honey locust, Gleditsia triacanthos) were strongly attacked. The dominant early successional tree, dogwood, received on average the most attacks. As predicted, attacks per stem declined strongly with distance from the emergence source, and with local stem density (a "dilution" effect). Contrary to expectations, there were more attacks per stem on larger patches. Because ovipositing cicadas cut damaging slits in host tree branches, potentially affecting tree growth rate, competitive ability, and capacity to reproduce, cicada damage could potentially influence spatial variation in secondary succession.     

Solution structure of potassium channel-inhibiting scorpion toxin Lq2

Lq2 is a unique scorpion toxin. Acting from the extracellular side, Lq2 blocks the ion conduction pore in not only the voltage- and Ca2+ -activated channels, but also the inward-rectifier K+ channels. This finding argues that the three-dimensional structures of the pores in these K+ channels are similar. However, the amino acid sequences that form the external part of the pore are minimally conserved among the various classes of K+ channels. Because Lq2 can bind to all the three classes of K+ channels, we can use Lq2 as a structural probe to examine how the non-conserved pore-forming sequences are arranged in space to form similar pore structures. In the present study, we determined the three-dimensional structure of Lq2 using nuclear magnetic resonance (NMR) techniques. Lq2 consists of an alpha-helix (residues S10 to L20) and a beta-sheet, connected by an alphabeta3 loop (residues N22 to N24). The beta-sheet has two well-defined anti-parallel strands (residues G26 to M29 and residues K32 to C35), which are connected by a type I' beta-turn centered between residues N30 and K31. The N-terminal segment (residues Z1 to T8) appears to form a quasi-third strand of the beta-sheet.     

The influence of geometry on hydrodynamic and mass transfer characteristics in an external airlift reactor for the cultivation of filamentous fungi

The influences of geometric configuration, mycelial broth rheology and superficial gas velocity (U_sg) were investigated with respect to the following hydrodynamic parameters: gas holdup (), oxygen transfer coefficient (K_La) and mixing time (t_m). Increases in U_sg and height of gas separator (H_t) caused an increase in and K_La, and a decrease in t_m. Consequently, a diameter ratio (D_d/D_r) of 0.71 and H_t 0.20 m were found to be the best geometry and operation parameters to achieve high aeration and mixing efficiency for the high viscous broth system in the cultivation of filamentous fungi. An external airlift reactor (EALR) was developed and designed for the cultivation of filamentous fungi. The EALR with two spargers excels in reliability and high aeration and mass transfer coefficiency, resulting in a fast mycelial growth and high biomass productivity in the cultivation of the fungus Rhizopus oryzae.