Structural Insight of a Trimodular Halophilic Cellulase with a Family 46 Carbohydrate-Binding Module

Cellulases are the key enzymes used in the biofuel industry. A typical cellulase contains a catalytic domain connected to a carbohydrate-binding module (CBM) through a flexible linker. Here we report the structure of an atypical trimodular cellulase which harbors a catalytic domain, a CBM46 domain and a rigid CBM_X domain between them. The catalytic domain shows the features of GH5 family, while the CBM46 domain has a sandwich-like structure. The catalytic domain and the CBM46 domain form an extended substrate binding cleft, within which several tryptophan residues are well exposed. Mutagenesis assays indicate that these residues are essential for the enzymatic activities. Gel affinity electrophoresis shows that these tryptophan residues are involved in the polysaccharide substrate binding. Also, electrostatic potential analysis indicates that almost the entire solvent accessible surface of CelB is negatively charged, which is consistent with the halophilic nature of this enzyme.     

Production, crystallization, and preliminary X-ray analysis of rabbit skeletal muscle troponin complex consisting of troponin C and fragment (1-47) of troponin I.

Troponin is a ternary protein complex consisting of subunits TnC. TnI, and TnT, and plays a key role in calcium regulation of the skeletal and cardiac muscle contraction. In the present study, a partial complex (CI47) was prepared from Escherichia coli-expressed rabbit skeletal muscle TnC and fragment 1-47 of TnI, which is obtained by chemical cleavage of an E. coli-expressed mutant of rabbit skeletal muscle TnI. Within the ternary troponin complex, CI47 is thought to form a core that is resistant to proteolytic digestion, and the interaction within CI47 likely maintains the integrity of the troponin complex. Complex CI47 was crystallized in the presence of sodium citrate. The addition of trehalose improved the diffraction pattern of the crystals substantially. The crystal lattice belongs to the space group P3(1)(2)21, with unit cell dimensions a = b = 48.2 A, c = 162 A. The asymmetric unit presumably contains one CI47 complex. Soaking with p-chloromercuribenzenesulfonate (PCMBS) resulted in loss of isomorphism, but enhanced the quality of the crystals. The crystals diffracted up to 2.3 A resolution, with completeness of 91% and R(merge) = 6.4%. The crystals of PCMBS-derivative should be suitable for X-ray studies using the multiple-wavelength anomalous diffraction technique. This is the first step for elucidating the structure of the full troponin complex.     

Activation of AMPK by Bitter Melon Triterpenoids Involves CaMKKβ

We recently showed that bitter melon-derived triterpenoids (BMTs) activate AMPK and increase GLUT4 translocation to the plasma membrane in vitro, and improve glucose disposal in insulin resistant models in vivo. Here we interrogated the mechanism by which these novel compounds activate AMPK, a leading anti-diabetic drug target. BMTs did not activate AMPK directly in an allosteric manner as AMP or the Abbott compound (A-769662) does, nor did they activate AMPK by inhibiting cellular respiration like many commonly used anti-diabetic medications. BMTs increased AMPK activity in both L6 myotubes and LKB1-deficient HeLa cells by 20–35%. Incubation with the CaMKKβ inhibitor, STO-609, completely attenuated this effect suggesting a key role for CaMKKβ in this activation. Incubation of L6 myotubes with the calcium chelator EGTA-AM did not alter this activation suggesting that the BMT-dependent activation was Ca²⁺-independent. We therefore propose that CaMKKβ is a key upstream kinase for BMT-induced activation of AMPK.     

Regional distribution of DNA and RNA in rat brain: A sensitive determination using high-performance liquid chromatography with electrochemical detection

DNA and RNA contents in 20 brain regions or nuclei of the rat were determined by a highly sensitive method using high-performance liquid chromatography with electrochemical detection. The high DNA and RNA contents were found in the hypothalamic nuclei, especially the median eminence-arcuate nucleus. These results may be available for the preparation of nucleic acids as the regional control.     

Differential effects of transforming growth factor-beta on the synthesis of extracellular matrix proteins by normal fetal rat calvarial bone cell populations

To determine the effects of transforming growth factor-beta (TGF-beta) on the different cell types that exist in bone, cell populations (I-IV), progressively enriched in osteoblastic cells relative to fibroblastic cells, were prepared from fetal rat calvaria using timed collagenase digestions. TGF-beta did not induce anchorage-independent growth of these cells, nor was anchorage-dependent growth stimulated in most populations studied, despite a two- to threefold increase in the synthesis of cellular proteins. In all populations the synthesis of secreted proteins increased 2-3.5-fold. In particular, collagen, fibronectin, and plasminogen activator inhibitor synthesis was stimulated. However, different degrees of stimulation of individual proteins were observed both within and between cell populations. A marked preferential stimulation of plasminogen activator inhibitor was observed in each population, together with a slight preferential stimulation of collagen; the effect on collagen expression being directed primarily at type I collagen. In contrast, the synthesis of SPARC (secreted protein acidic rich in cysteine/osteonectin was stimulated approximately two-fold by TGF-beta, but only in fibroblastic populations. Collectively, these results demonstrate that TGF-beta stimulates matrix production by bone cells and, through differential effects on individual matrix components, may also influence the nature of the matrix formed by different bone cell populations. In the presence of TGF-beta, osteoblastic cells lost their polygonal morphology and alkaline phosphatase activity was decreased, reflecting a suppression of osteoblastic features. The differential effects of TGF-beta on bone cell populations are likely to be important in bone remodeling and fracture repair.     

猫体感皮层神经元对同时刺激隐神经的A类和C类纤维的反应型式

记录了麻痹猫的体感皮层(SI)神经元的自发和隐神经的A类和C类纤维传入诱发放电(A-ED和C-ED)。用NCCVF分析神经元放电。结果表明,SI区神经元对同时刺激隐神经的A类和C类纤维的反应呈多种型式:(1)A-ED和C-ED共存,包括Ⅰ.A-ED和C-ED始终相互伴随出现;Ⅱ.在刺激之初,只出现A-ED,但是,当阻断A类纤维传入并由C类纤维传入诱发神经元放电后,再同时刺激A类和C类纤维时,A-ED和C-ED便同时出现。(2)A-ED制约C-ED,特点是,只要A-ED存在,C/ED就不出现。只有阻断A类纤维传入后,C-ED才产生。(3)单一A-ED,不管在什么刺激条件下,这类神经元都只有A-ED,而不产生C-ED 结论:根据反应型式的不同,可将SI区的神经元分为Ⅰ.A类和C类纤维传入同时驱动的神经元;Ⅱ.A-ED制约C-ED的神经元;Ⅲ.只由A类纤维传入驱动的神经元。     

Ca2+ dependence of positive inotropic responses of guinea pig isolated cardiac preparations to cAMP-generating and cAMP-independent agonists

The effects of procedures which diminish Ca2+ influx into myocardial cells on responses of isolated cardiac preparations to cAMP-independent histamine H1 receptor stimulation and cAMP-generating beta-receptor stimulation were measured. The histamine response of guinea pig left atria, which appears to be primarily mediated by H1 receptors, was depressed to a greater extent than was the response of this preparation to isoproterenol by decreasing the extracellular Ca2+ concentration, and by the Ca2+ influx blocker D-600. Similarly, while the H1 agonist 2-pyridylethylamine dihydrochloride (PEA) produced increases in tension of a similar magnitude as the partial beta-agonist salbutamol in both left atria and in papillary muscles, responses of both preparations to PEA were depressed to a significantly greater extent by decreasing the extracellular Ca2+ concentration than were responses to salbutamol. Overall, both the basal developed force of papillary muscles and the responses of these preparations to H1 and beta-receptor stimulation appeared to be less depressed by decreasing the extracellular Ca2+ concentration than were those of left atria. These results indicate that responses mediated via cAMP-independent H1 receptors, like those arising from alpha-receptor stimulation, are more sensitive to procedures which diminish Ca2+ influx than are responses arising from stimulation of cAMP-generating beta-receptors. This may reflect differences in the mechanisms by which stimulation of H1, alpha-, and beta-receptors give rise to positive inotropic responses. In addition, left atria may be more dependent than papillary muscles on extracellular Ca2+ for the support of contraction.     

T-cell-mediated regression of "spontaneous" and of Epstein-Barr virus-induced B-cell transformation in vitro: studies with cyclosporin A

The regression of Epstein-Barr (EB) virus-transformed B-cell outgrowth which is seen in experimentally-infected cultures of blood mononuclear (UM) cells from healthy seropositive donors can be abolished in medium containing the T-cell-suppressive agent cyclosporin A (CSA) at concentrations of 0.05 microgram/ml and above. CSA mediates its effect within the first 4 days post-infection of the UM cells and this prevents subsequent in vitro generation of the EB virus-specific cytotoxic-T-cell response which normally brings about regression. Regression can be fully restored by supplementing the CSA-treated culture with interleukin 2 (IL-2)-containing culture supernatants or indeed with purified IL-2 itself, suggesting that CSA mediates its effect in this system through inhibiting the endogenous production of IL-2 which is required to amplify the virus-specific cytotoxic response. "Spontaneous transformation" to EB virus genome-positive lymphoblastoid cell lines in noninfected cultures of UM cells from healthy seropositive donors, though rare in normal medium, is enhanced to such a degree in the presence of CSA that, for many donors, the phenomenon becomes titratable against input cell dose across the 2.0 X 10(6)-2.5 X 10(5) cells/culture range. Cell mixing experiments suggest that the spontaneously transformed cell lines which arise with such efficiency under these conditions do so not by direct in vitro outgrowth of progenitor cells transformed by the virus in vivo, but by a two-step mechanism involving virus release and secondary infection in vitro.