Bastadin 10 stabilizes the open conformation of the ryanodine-sensitive Ca(2+) channel in an FKBP12-dependent manner.

The marine sponge Ianthella basta synthesizes at least 25 tetrameric bromotyrosine structures that possess a stringent structural requirement for modifying the gating behavior of ryanodine-sensitive Ca(2+) channels (ryanodine receptors) (RyR)). Bastadin 5 (B5) was shown to stabilize open and closed channel states with little influence on the sensitivity of the channel to activation by Ca(2+) (Mack, M. M., Molinski, T. F., Buck, E. D., and Pessah, I. N. (1994) J. Biol. Chem. 269, 23236-23249). In the present paper, we utilize single channel analysis and measurements of Ca(2+) flux across the sarcoplasmic reticulum to identify bastadin 10 (B10) as the structural congener responsible for dramatically stabilizing the open conformation of the RyR channel, possibly by reducing the free energy associated with closed to open channel transitions (DeltaG*c --> o). The stability of the channel open state induced by B10 sensitized the channel to activation by Ca(2+) to such an extent that it essentially obviated regulation by physiological concentrations of Ca(2+) and relieved inhibition by physiological Mg(2+). These actions of B10 were produced only on the cytoplasmic face of the channel, were selectively eliminated by pretreatment of channels with FK506 or rapamycin, and were reconstituted by human recombinant FKBP12. The actions of B10 were found to be reversible. A structure-activity model is proposed by which substitutions on the Eastern and Western hemispheres of the bastarane macrocycle may confer specificity toward the RyR1-FKBP12 complex to stabilize either the closed or open channel conformation. These results indicate that RyR1-FKBP12 complexes possesses a novel binding domain for phenoxycatechols and raise the possibility of molecular recognition of an endogenous ligand.     

Practical synthesis of 2,6-dideoxy-d-lyxo-hexose (“2-deoxy-d-fucose”) from d-galactose

2,6-Dideoxy- -lyxo-hexose was prepared efficiently from -galactose in eight steps (25% overall yield). The synthesis is amenable to multigram scale-up.     

Proliferating cell nuclear antigen (PCNA) as a proliferative marker during embryonic and adult zebrafish hematopoiesis

We investigated the expression of proliferative cell nuclear antigen (PCNA) in zebrafish to delineate the proliferative hematopoietic component during adult and embryonic hematopoiesis. Immunostaining for PCNA and enhanced green fluorescence protein (eGFP) was performed in wild-type and fli1-eGFP (endothelial marker) and gata1-eGFP (erythroid cell marker) transgenic fish. Expression of PCNA mRNA was examined in wild-type and chordin morphant embryos. In adult zebrafish kidney, the renal tubules are surrounded by endothelial cells and it is separated into hematopoietic and excretory compartments. PCNA was expressed in hematopoietic progenitor cells but not in mature neutrophils, eosinophils or erythroid cells. Some PCNA+ cells are scattered in the hematopoietic compartment of the kidney while others are closely associated with renal tubular cells. PCNA was also expressed in spermatogonial stem cells and intestine crypts, consistent with its role in cell proliferation and DNA synthesis. In embryos, PCNA is expressed in the brain, spinal cord and intermediate cell mass (ICM) at 24 h-post fertilization. In chordin morphants, PCNA is significantly upregulated in the expanded ICM. Therefore, PCNA can be used to mark cell proliferation in zebrafish hematopoietic tissues and to identify a population of progenitor cells whose significance would have to be further investigated.     

A benefit-finding intervention for family caregivers of persons with Alzheimer disease: study protocol of a randomized controlled trial

Background

Patients with ST-elevation myocardial infarction (STEMI) not treated with primary or rescue percutaneous coronary intervention (PCI) are at risk for recurrent ischemia, especially when viability in the infarct-area is present. Therefore, an invasive strategy with PCI of the infarct-related coronary artery in patients with viability would reduce the occurrence of a composite end point of death, reinfarction, or unstable angina (UA).

Methods

Patients admitted with an (sub)acute myocardial infarction, who were not treated by primary or rescue PCI, and who were stable during the first 48 hours after the acute event, were screened for the study. Eventually, we randomly assigned 216 patients with viability (demonstrated with low-dose dobutamine echocardiography) to an invasive or a conservative strategy. In the invasive strategy stenting of the infarct-related coronary artery was intended with abciximab as adjunct treatment. Seventy-five (75) patients without viability served as registry group. The primary endpoint was the composite of death from any cause, recurrent myocardial infarction (MI) and unstable angina at one year. As secondary endpoint the need for (repeat) revascularization procedures and anginal status were recorded.

Results

The primary combined endpoint of death, recurrent MI and unstable angina was 7.5% (8/106) in the invasive group and 17.3% (19/110) in the conservative group (Hazard ratio 0.42; 95% confidence interval [CI] 0.18-0.96; p = 0.032). During follow up revascularization-procedures were performed in 6.6% (7/106) in the invasive group and 31.8% (35/110) in the conservative group (Hazard ratio 0.18; 95% CI 0.13-0.43; p < 0.0001). A low rate of recurrent ischemia was found in the non-viable group (5.4%) in comparison to the viable-conservative group (14.5%). (Hazard-ratio 0.35; 95% CI 0.17-1.00; p = 0.051).

Conclusion

We demonstrated that after acute MI (treated with thrombolysis or without reperfusion therapy) patients with viability in the infarct-area benefit from a strategy of early in-hospital stenting of the infarct-related coronary artery. This treatment results in a long-term uneventful clinical course. The study confirmed the low risk of recurrent ischemia in patients without viability.

Trial registration

ClinicalTrials.gov: NCT00149591.     

Monoclonal antibodies against chicken type V collagen: production, specificity, and use for immunocytochemical localization in embryonic cornea and other organs

Two monoclonal antibodies have been produced against chick type V collagen and shown to be highly specific for separate, conformational dependent determinants within this molecule. When used for immunocytochemical tissue localization, these antibodies show that a major site for the in situ deposition of type V is within the extracellular matrices of many dense connective tissues. In these, however, it is largely in a form unavailable to the antibodies, thus requiring a specific “unmasking” treatment to obtain successful immunocytochemical staining. The specificity of these two IgG antibodies was determined by inhibition ELISA, in which only type V and no other known collagen shows inhibition. In ELISA, mixtures of the two antibodies give an additive binding reaction to the collagen, suggesting that each is against a different antigenic determinant. That both antigenic determinants are conformational dependent, being either in, or closely associated with, the collagen helix is demonstrated by the loss of antibody binding to molecules that have been thermally denatured. The temperature at which this occurs, as assayed by inhibition ELISA, is very similar to that at which the collagen helix melts, as determined by optical rotation. This gives strong additional evidence that the antibodies are directed against the collagen. The antibodies were used for indirect immunofluorescence analyses of cryostat sections of corneas and other organs from 17 to 18-day-old chick embryos. Of all tissues examined only Bowman’s membrane gave a strong staining reaction with cryostat sections of unfixed material. Staining in other areas of the cornea and in other tissues was very light or nonexistent. When, however, sections were pretreated with pepsin dissolved in dilute HAc or, surprisingly, with the dilute HAc itself dramatic new staining by the antibodies was observed in most tissues examined. The staining, which was specific for the anti-type V collagen antibodies, was largely confined to extracellular matrices of dense connective tissues. Experiments using protease inhibitors suggested that the “unmasking” did not involve proteolysis. We do not yet know the mechanism of this unmasking; however, one possibility is that the dilute acid causes swelling or conformational changes in a type-V collagen-containing supramolecular structure. Further studies should allow us to determine whether this is the case.     

How to build a paraspeckle

Noncoding RNAs have recently been identified as essential components of the nuclear suborganelles called paraspeckles. This finding will facilitate our understanding of the molecular dynamics and physiological role of these enigmatic macromolecular structures.     

Synchronous bond molecular dynamics of conjugated chlorocyclopropyl alk-yn-enes revealed by ECD and UV–vis

Chlorocyclopropanes (CCPs) conjugated to alk-yn-enes occur in a unique family of polyketide natural products from marine sponges. Synthesis of several optically enriched analogs of CCPs and measurement of their UV–vis spectra and electronic circular dichroism (ECD) spectra reveal unusually strong hyperconjugation that constrains and aligns the cyclopropyl C-C bond with the π-plane of the distal ene-bond. This alignment imposes a barrier to rotation of at least 5.0 kcal·mol⁻¹. Comparison of red-shifted Cotton effects in chiral CCPs show the barrier is independent of alkene substituent and establishes an empirical rule for assignment of other CCP-containing natural products.