Time-irreversible Subconductance Gating Associated with Ba2+ Block of Large Conductance Ca2+-activated K+ Channels

Ba²⁺ block of large conductance Ca²⁺-activated K⁺ channels was studied in patches of membrane excised from cultures of rat skeletal muscle using the patch clamp technique. Under conditions in which a blocking Ba²⁺ ion would dissociate to the external solution (150 mM N-methyl-d-glucamine⁺ _o, 500 mM K⁺ _i, 10 μM Ba²⁺ _i, +30 mV, and 100 μM Ca²⁺ _i to fully activate the channel), Ba²⁺ blocks with a mean duration of ∼2 s occurred, on average, once every ∼100 ms of channel open time. Of these Ba²⁺ blocks, 78% terminated with a single step in the current to the fully open level and 22% terminated with a transition to a subconductance level at ∼0.26 of the fully open level (preopening) before stepping to the fully open level. Only one apparent preclosing was observed in ∼10,000 Ba²⁺ blocks. Thus, the preopenings represent Ba²⁺-induced time-irreversible subconductance gating. The fraction of Ba²⁺ blocks terminating with a preopening and the duration of preopenings (exponentially distributed, mean = 0.75 ms) appeared independent of changes in [Ba²⁺]_i or membrane potential. The fractional conductance of the preopenings increased from 0.24 at +10 mV to 0.39 at +90 mV. In contrast, the average subconductance level during normal gating in the absence of Ba²⁺ was independent of membrane potential, suggesting different mechanisms for preopenings and normal subconductance levels. Preopenings were also observed with 10 mM Ba²⁺ _o and no added Ba²⁺ _i. Adding K⁺, Rb⁺, or Na⁺ to the external solution decreased the fraction of Ba²⁺ blocks with preopenings, with K⁺ and Rb⁺ being more effective than Na⁺. These results are consistent with models in which the blocking Ba²⁺ ion either induces a preopening gate, and then dissociates to the external solution, or moves to a site located on the external side of the Ba²⁺ blocking site and acts directly as the preopening gate.     

Protein phosphatase 6 regulates mitotic spindle formation by controlling the T-loop phosphorylation state of Aurora A bound to its activator TPX2

Many protein kinases are activated by a conserved regulatory step involving T-loop phosphorylation. Although there is considerable focus on kinase activator proteins, the importance of specific T-loop phosphatases reversing kinase activation has been underappreciated. We find that the protein phosphatase 6 (PP6) holoenzyme is the major T-loop phosphatase for Aurora A, an essential mitotic kinase. Loss of PP6 function by depletion of catalytic or regulatory subunits interferes with spindle formation and chromosome alignment because of increased Aurora A activity. Aurora A T-loop phosphorylation and the stability of the Aurora A-TPX2 complex are increased in cells depleted of PP6 but not other phosphatases. Furthermore, purified PP6 acts as a T-loop phosphatase for Aurora A-TPX2 complexes in vitro, whereas catalytically inactive mutants cannot dephosphorylate Aurora A or rescue the PPP6C depletion phenotype. These results demonstrate a hitherto unappreciated role for PP6 as the T-loop phosphatase regulating Aurora A activity during spindle formation and suggest the general importance of this form of regulation.     

Cutting edge: TLR9 and TLR2 signaling together account for MyD88-dependent control of parasitemia in Trypanosoma cruzi infection

Activation of innate immune cells by Trypanosoma cruzi-derived molecules such as GPI anchors and DNA induces proinflammatory cytokine production and host defense mechanisms. In this study, we demonstrate that DNA from T. cruzi stimulates cytokine production by APCs in a TLR9-dependent manner and synergizes with parasite-derived GPI anchor, a TLR2 agonist, in the induction of cytokines by macrophages. Compared with wild-type animals, T. cruzi-infected Tlr9(-/-) mice displayed elevated parasitemia and decreased survival. Strikingly, infected Tlr2(-/-)Tlr9(-/-) mice developed a parasitemia equivalent to animals lacking MyD88, an essential signaling molecule for most TLR, but did not show the acute mortality displayed by MyD88(-/-) animals. The enhanced susceptibility of Tlr9(-/-) and Tlr2(-/-)Tlr9(-/-) mice was associated with decreased in vivo IL-12/IFN-gamma responses. Our results reveal that TLR2 and TLR9 cooperate in the control of parasite replication and that TLR9 has a primary role in the MyD88-dependent induction of IL-12/IFN-gamma synthesis during infection with T. cruzi.     

Influence of modulated structural dynamics on the kinetics of alpha-chymotrypsin catalysis. Insights through chemical glycosylation, molecular dynamics and domain motion analysis

Although the chemical nature of the catalytic mechanism of the serine protease alpha-chymotrypsin (alpha-CT) is largely understood, the influence of the enzyme's structural dynamics on its catalysis remains uncertain. Here we investigate whether alpha-CT's structural dynamics directly influence the kinetics of enzyme catalysis. Chemical glycosylation [Solá RJ & Griebenow K (2006) FEBS Lett 580, 1685-1690] was used to generate a series of glycosylated alpha-CT conjugates with reduced structural dynamics, as determined from amide hydrogen/deuterium exchange kinetics (k(HX)). Determination of their catalytic behavior (K(S), k(2), and k(3)) for the hydrolysis of N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide (Suc-Ala-Ala-Pro-Phe-pNA) revealed decreased kinetics for the catalytic steps (k(2) and k(3)) without affecting substrate binding (K(S)) at increasing glycosylation levels. Statistical correlation analysis between the catalytic (DeltaG( not equal)k(i)) and structurally dynamic (DeltaG(HX)) parameters determined revealed that the enzyme acylation and deacylation steps are directly influenced by the changes in protein structural dynamics. Molecular modelling of the alpha-CT glycoconjugates coupled with molecular dynamics simulations and domain motion analysis employing the Gaussian network model revealed structural insights into the relation between the protein's surface glycosylation, the resulting structural dynamic changes, and the influence of these on the enzyme's collective dynamics and catalytic residues. The experimental and theoretical results presented here not only provide fundamental insights concerning the influence of glycosylation on the protein biophysical properties but also support the hypothesis that for alpha-CT the global structural dynamics directly influence the kinetics of enzyme catalysis via mechanochemical coupling between domain motions and active site chemical groups.     

Intravesicular Factors Controlling Exocytosis in Chromaffin Cells

Chromaffin granules are similar organelles to the large dense core vesicles (LDCV) present in many secretory cell types including neurons. LDCV accumulate solutes at high concentrations (catecholamines, 0.5–1 M; ATP, 120–300 mM; or Ca²⁺, 40 mM (Bulenda and Gratzl Biochemistry 24:7760–7765, 1985). Solutes seem to aggregate to a condensed matrix to elude osmotic lysis. The affinity of solutes for LDCV matrix is responsible for the delayed release of catecholamines during exocytosis. The aggregation of solutes occurs due to a specific H⁺ pump denominated V-ATPase that maintains an inner acidic media (pH ≈5.5). This pH gradient against cytosol is also responsible for the vesicular accumulation of amines and Ca²⁺. When this gradient is reduced by modulation of the V-ATPase activity, catecholamines and Ca²⁺ are moved toward the cytosol. In addition, some drugs largely accumulate inside LDCV and not only impair the accumulation of natural solutes, but also act as false neurotransmitters when they are co-released with catecholamines. There is much experimental evidence to conclude that the physiological modulation of vesicle pH and the manipulation of intravesicular media with drugs affect the LDCV cargo and change the kinetics of exocytosis. Here, we will present some experimental data demonstrating the participation of drugs in the kinetics of exocytosis through changes in the composition of vesicular media. We also offer a model to explain the regulation of exocytosis by the intravesicular media that conciliate the experimentally obtained data.     

Hypertrophic cardiomyopathy associated with left ventricular aneurysm and normal coronary arteries: Case study indicating genetic tendencies of cardiomyopathy

A 50-year-old man presented with hypertrophic obstructive cardiomyopathy (HOC) associated with a left ventricular aneurysm and normal coronary arteries. His history revealed no evidence of myocardial infarction or atypical angina. Physical examination disclosed HOC but did not suggest the presence of an aneurysm. Although the patient was treated medically, heart failure ensued, and he died suddenly while working his farm. Subsequent investigation of the patient's family revealed that three of his five children were also affected by cardiomyopathy, which was especially pronounced in the eldest, a 22-year-old man. The possible hemodynamic relationship between HOC and left ventricular aneurysm is discussed, along with probable indications. The role of left ventricular aneurysm is also presented in relation to the natural history of the disease.     

Microtubule-associated nuclear envelope proteins in interphase and mitosis

The LINC (linker of nucleoskeleton and cytoskeleton) complex forms a transcisternal bridge across the NE (nuclear envelope) that connects the cytoskeleton with the nuclear interior. This enables some proteins of the NE to communicate with the centrosome and the microtubule cytoskeleton. The position of the centrosome relative to the NE is of vital importance for many cell functions, such as cell migration and division, and centrosomal dislocation is a frequent phenotype in laminopathic disorders. Also in mitosis, a small group of transmembrane NE proteins associate with microtubules when they concentrate in a specific membrane domain associated with the mitotic spindle. The present review discusses structural and functional aspects of microtubule association with NE proteins and how this association may be maintained over the cell cycle.     

The genome sequence of the gram-positive sugarcane pathogen Leifsonia xyli subsp. xyli

The genome sequence of Leifsonia xyli subsp. xyli, which causes ratoon stunting disease and affects sugarcane worldwide, was determined. The single circular chromosome of Leifsonia xyli subsp. xyli CTCB07 was 2.6 Mb in length with a GC content of 68% and 2,044 predicted open reading frames. The analysis also revealed 307 predicted pseudogenes, which is more than any bacterial plant pathogen sequenced to date. Many of these pseudogenes, if functional, would likely be involved in the degradation of plant heteropolysaccharides, uptake of free sugars, and synthesis of amino acids. Although L. xyli subsp. xyli has only been identified colonizing the xylem vessels of sugarcane, the numbers of predicted regulatory genes and sugar transporters are similar to those in free-living organisms. Some of the predicted pathogenicity genes appear to have been acquired by lateral transfer and include genes for cellulase, pectinase, wilt-inducing protein, lysozyme, and desaturase. The presence of the latter may contribute to stunting, since it is likely involved in the synthesis of abscisic acid, a hormone that arrests growth. Our findings are consistent with the nutritionally fastidious behavior exhibited by L. xyli subsp. xyli and suggest an ongoing adaptation to the restricted ecological niche it inhabits.     

Molecular and symbiotic characterization of exopolysaccharide-deficient mutants of Rhizobium tropici strain CIAT899

We studied the symbiotic behaviour of 20 independent Tn5 mutants of Rhizobium tropici strain CIAT899 that were deficient in exopolysaccharide (EPS) production. The mutants produced non-mucoid colonies, were motile, grew in broth cultures at rates similar to those of the parent, and produced significantly less EPS than did CIAT899 in broth culture. A genomic library of strain CIAT899, constructed in pLA2917, was mobilized into all of the mutants, and cosmids that restored EPS production were identified. EcoRI restriction digests of the cosmids revealed nine unique inserts. Mutant complementation and hybridization analysis showed that the mutations affecting EPS production fell into six functional and physical linkage groups. On bean, the mutants were as efficient in nodulation and as effective in acetylene reduction as strain CIAT899, induced a severe interveinal chlorosis, and all but one were less competitive than CIAT899. On siratro, CIAT899 induced nodules that were ineffective in acetylene reduction, whereas the EPS-deficient mutants induced effective nodules. Microscopic examination of thin sections showed that nodules from both siratro and bean plants inoculated with either CIAT899 or an EPS-deficient mutant contained infected cells. These data indicate that EPS is not required for normal nodulation of bean by R. tropici, that it may contribute to competitiveness of R. tropici on bean, and that the loss of EPS production is accompanied by acquisition of the ability to reduce acetylene on siratro.     

Hormonal induction of ovulation and artificial insemination in suckled beef cows under nutritional stress

The objective was to develop a program for inducing estrus (followed by insemination) of suckled beef cows under nutritional stress (poor body condition). A total of 123 cows, from 60 to 75 days postpartum, were classified according to their body condition score (BCS; range from 1 to 5, in increments of 0.5) and allocated into two groups. On Day 0 (without regard to stage of the estrous cycle), cows (n = 59) in the hormone induction (HI) treatment group were given an intravaginal device (IVD) containing 250 mg of medroxiprogesterone acetate (MAP) and an i.m. injection of 2.5 mg estradiol benzoate (EB). On Day 6, these cows were given 500 IU eCG i.m. and calves were weaned for 96 h. The IVD were removed on Day 7. Cows detected in estrus by 45 h after IVD removal were inseminated 12 h after standing estrus; cows not in estrus by 45 h after IVD removal received an i.m. injection of 100 microg gonadorelin (GnRH) and were inseminated 16-18 h later. In the control group (C), cows (n = 64) only had their calves weaned at Day 6 (for 96 h), with estrus detection and AI from Days 6 to 11. Overall, the BCS ranged from 2.0 to 3.0. In the treatment group, estrus and pregnancy rates in cows with BCS 2.0 (20 and 30%, respectively) was lower (P < 0.05) than those with BCS 3.0 (50 and 66.6%, respectively), but did not differ (P > 0.05) from BCS 2.5 (23.3 and 47.6%). In C group, only 2 of 66 cows were detected in estrus and bred (neither was pregnant). In conclusion, the program for induction of ovulation using MAP, EB, eCG and GnRH increased the pregnancy rate in beef cows in poor body condition, enabling AI to be done in a 63-h interval.