Rearrangements within human spliceosomes captured after exon ligation

In spliceosomes, dynamic RNA/RNA and RNA/protein interactions position the pre-mRNA substrate for the two chemical steps of splicing. Not all of these interactions have been characterized, in part because it has not been possible to arrest the complex at clearly defined states relative to chemistry. Previously, it was shown in yeast that the DEAD/H-box protein Prp22 requires an extended 3′ exon to promote mRNA release from the spliceosome following second-step chemistry. In line with that observation, we find that shortening the 3′ exon blocks cleaved lariat intron and mRNA release in human splicing extracts, which allowed us to stall human spliceosomes in a new post-catalytic complex (P complex). In comparison to C complex, which is blocked at a point following first-step chemistry, we detect specific differences in RNA substrate interactions near the splice sites. These differences include extended protection across the exon junction and changes in protein crosslinks to specific sites in the 5′ and 3′ exons. Using selective reaction monitoring (SRM) mass spectrometry, we quantitatively compared P and C complex proteins and observed enrichment of SF3b components and loss of the putative RNA-dependent ATPase DHX35. Electron microscopy revealed similar structural features for both complexes. Notably, additional density is present when complexes are chemically fixed, which reconciles our results with previously reported C complex structures. Our ability to compare human spliceosomes before and after second-step chemistry has opened a new window to rearrangements near the active site of spliceosomes, which may play roles in exon ligation and mRNA release.     

Copper transport to mammary gland and milk during lactation in rats

The delivery of copper to mammary gland and milk and the effects of lactation were examined in rats. Traces of (67)Cu/(64)Cu(II) were injected intraperitoneally or intravenously into virgin rats or lactating rats (2-5 days postpartum), and incorporation into blood, milk, and tissues was monitored. In virgin rats, most of the isotope first entered the liver and kidney. In lactating rats, almost 60% went directly to the mammary gland. Uptake rates and copper contents of the mammary gland were 20-fold higher in lactation. (67)Cu/(64)Cu appeared in milk and milk ceruloplasmin as rapidly as in mammary tissue and when there was no (67)Cu/(64)Cu-ceruloplasmin in the maternal plasma. Plasma (125)I-labeled albumin entered milk much more slowly. Milk ceruloplasmin (10 mg/l) had 25% of the (67)Cu/(64)Cu. Milk copper was 3.3 mg/l. Thus lactation markedly enhances the avidity of the mammary gland for copper, diverting most of it from liver and kidney to that tissue. Also, the primary source of milk ceruloplasmin is the mammary gland and not the maternal plasma.     

Nodal signaling is required for closure of the anterior neural tube in zebrafish

Background  

Nodals are secreted signaling proteins with many roles in vertebrate development. Here, we identify a new role for Nodal signaling in regulating closure of the rostral neural tube of zebrafish.     

Determination of the period for establishment of a liver network echogenic pattern in Schistosoma japonicum infection

Schistosomiasis is caused by infection with Schistosoma haematobium, S. mansoni, S. japonicum, or S. mekongi. S. japonicum infection results in liver cirrhosis at the final stage. A "network" (NW) echogenic pattern on hepatic ultrasonography appears to be specific to S. japonicum infection. The principal aim of the present study was to determine the exact year(s) or even month(s) required for the establishment of the liver NW echogenic pattern from the initial infection in young patients with schistosomiasis japonica since there are few data on this important point. We conducted yearly ultrasonographic, serologic, coprologic, and physical examinations of schistosomiasis patients in the Philippines from 1996 up to the present. During that period, the total number of patients examined was approximately 2,000, among whom we selected 2 patients for determination of the duration required for NW establishment, when they were 10 years old. Although the exact time of initial exposure to schistosomes cannot be determined, the duration for the establishment of NW was definitively confirmed in patient no. 1 to be between 19-24 months based on the results of serologic and coprologic examinations. For patient no. 2, the circumstantial evidence suggested that the establishment of a NW might require 5 to 6 years at maximum. To the best of our knowledge, this is the first evidence-based report on the determination of the period required for the establishment of a liver NW echogenic pattern in S. japonicum infection in the Philippines.     

Differences in a conformational equilibrium distinguish catalysis by the endothelial and neuronal nitric-oxide synthase flavoproteins

Nitric oxide (NO) is a physiological mediator synthesized by NO synthases (NOS). Despite their structural similarity, endothelial NOS (eNOS) has a 6-fold lower NO synthesis activity and 6-16-fold lower cytochrome c reductase activity than neuronal NOS (nNOS), implying significantly different electron transfer capacities. We utilized purified reductase domain constructs of either enzyme (bovine eNOSr and rat nNOSr) to investigate the following three mechanisms that may control their electron transfer: (i) the set point and control of a two-state conformational equilibrium of their FMN subdomains; (ii) the flavin midpoint reduction potentials; and (iii) the kinetics of NOSr-NADP+ interactions. Although eNOSr and nNOSr differed in their NADP(H) interaction and flavin thermodynamics, the differences were minor and unlikely to explain their distinct electron transfer activities. In contrast, calmodulin (CaM)-free eNOSr favored the FMN-shielded (electron-accepting) conformation over the FMN-deshielded (electron-donating) conformation to a much greater extent than did CaM-free nNOSr when the bound FMN cofactor was poised in each of its three possible oxidation states. NADPH binding only stabilized the FMN-shielded conformation of nNOSr, whereas CaM shifted both enzymes toward the FMN-deshielded conformation. Analysis of cytochrome c reduction rates measured within the first catalytic turnover revealed that the rate of conformational change to the FMN-deshielded state differed between eNOSr and nNOSr and was rate-limiting for either CaM-free enzyme. We conclude that the set point and regulation of the FMN conformational equilibrium differ markedly in eNOSr and nNOSr and can explain the lower electron transfer activity of eNOSr.     

Spectroscopic properties of the main-form and high-salt peridinin-chlorophyll a proteins from Amphidinium carterae

The main-form (MFPCP) and high-salt (HSPCP) peridinin-chlorophyll a proteins from the dinoflagellate Amphidinium carterae were investigated using absorption, fluorescence, fluorescence excitation, two-photon, and fast-transient optical spectroscopy. Pigment analysis has demonstrated previously that MFPCP contains eight peridinins and two chlorophyll (Chl) a molecules, whereas HSPCP has six peridinins and two Chl a molecules [Sharples, F. P., et al. (1996) Biochim. Biophys. Acta 1276, 117-123]. Absorption spectra of the complexes were recorded at 10 K and analyzed in the 400-600 nm region by summing the individual 10 K spectra of Chl a and peridinin recorded in 2-MTHF. The absorption spectral profiles of the complexes in the Q(y) region between 650 and 700 nm were fit using Gaussian functions. The absorption and fluorescence spectra from both complexes exhibit several distinguishing features that become evident only at cryogenic temperatures. In particular, at low temperatures the Q(y) transitions of the Chls bound in the HSPCP complex are split into two well-resolved bands. Fluorescence excitation spectroscopy has revealed that the peridinin-to-Chl a energy transfer efficiency is high (>95%). Transient absorption spectroscopy has been used to measure the rate of energy transfer between the two bound Chls which is a factor of 2.9 slower in HSPCP than in MFPCP. The kinetic data are interpreted in terms of the F?rster mechanism describing energy transfer between weakly coupled, spatially fixed, donor-acceptor Chl a molecules. The study provides insight into the molecular factors that control energy transfer in this class of light-harvesting pigment-protein complexes.     

Functional redundancy of the DE-1 and alpha A-CRYBP1 regulatory sites of the mouse alpha A-crystallin promoter.

Previous studies have implicated the DE-1 (-111/-106) and alpha A-CRYBP1 (-66/-57) sites for activity of the mouse alpha A-crystallin promoter in transiently transfected lens cells. Here we have used the bacterial chloramphenicol acetyltransferase (CAT) reporter gene to test the functional importance of the putative DE-1 and alpha A-CRYBP1 regulatory elements by site-specific and deletion mutagenesis in stably transformed alpha TN4-1 lens cells and in transgenic mice. FVB/N and C57BL/6 x SJL F2 hybrid transgenic mice were assayed for CAT activity in the lens, heart, lung, kidney, spleen, liver, cerebrum, and muscle. F0, F1, and F2 mice from multiple lines carrying single mutations of the DE-1 or alpha A-CRYBP1 sites showed high levels of CAT activity in the lens, but not in any of the non-lens tissues. By contrast, despite activity of the wild-type promoter, none of the mutant promoter/CAT constructs were active in the transiently transfected and stably transformed lens cells. The mice carrying transgenes with either site-specific mutations in both the DE-1 and alpha A-CRYBP1 sites or a deletion of the entire DE-1 and part of the alpha A-CRYBP1 site (-60/+46) fused to the CAT gene did not exhibit CAT activity above background in any of the tissues examined, including the lens. Our results thus indicate that the DE-1 and alpha A-CRYBP1 sites are functionally redundant in transgenic mice. Moreover, the present data coupled with previous transfection and transgenic mouse experiments suggest that this functional redundancy is confined to lens expression within the mouse and is not evident in transiently transfected and stably transformed lens cells, making the cultured lens cells sensitive indicators of functional elements of crystallin genes.