Identification of plant microRNA homologs

MicroRNAs (miRNAs) are a recently discovered class of non-coding RNAs that regulate gene and protein expression in plants and animals. MiRNAs have so far been identified mostly by specific cloning of small RNA molecules, complemented by computational methods. We present a computational identification approach that is able to identify candidate miRNA homologs in any set of sequences, given a query miRNA. The approach is based on a sequence similarity search step followed by a set of structural filters.     

Combined modulation of the mitochondrial ATP-dependent potassium channel and the permeability transition pore causes prolongation of the biphasic calcium dynamics

The permeability transition pore (PTP) and the ATP-dependent potassium (mtK-ATP) channel of mitochondria are known to play key roles in mitochondrially mediated apoptosis. We investigated how modulation of the permeability transition pore (PTP) and the ATP-dependent potassium (mtK-ATP) channel, either as single elements or in combination, affects the proapoptotic intracellular calcium ([Ca(2+)](i)) transients and the mitochondrial membrane potential (psi(m)). For this purpose a model was established exploring the [Ca(2+)](i) transients in N2A cells using continuous application of ATP that causes a biphasic [Ca(2+)](i) response. This response was sensitive to endoplasmatic reticulum (ER) Ca(2+) depletion and a smooth ER Ca(2+)-ATPase (SERCA) antagonist. PTP inhibition by cyclosporine A (CsA) or its non-immunosuppressive derivative NIM811 caused an amplification of the secondary [Ca(2+)](i) peak and induced a hyperpolarization of psi(m). Both the putative mtK-ATP channel inhibitor 5-hydroxydecanoate (5-HD) and the opener diazoxide ameliorated the ATP-induced secondary [Ca(2+)](i) peak. The effect of diazoxide was accompanied by a depolarization of psi(m) whereas 5-HD had no effect on psi(m). When diazoxide and CsA or NIM811 were applied together the secondary [Ca(2+)](i) rise did not return to baseline and a not significant hyperpolarization of psi(m) was observed. So, simultaneous inhibition of PTP and activation of the mtK-ATP channel prevents the increased slope of the secondary [Ca(2+)](i) peak induced by CsA (or NIM811) and also the depolarization after diazoxide application. Hence, we propose that modulation of one of these channels leads to functional changes of the other channel by means of Delta[Ca(2+)](i) and Deltapsi(m).     

The relation of the X-ray B-factor to protein dynamics: insights from recent dynamic solid-state NMR data

In addressing the potential use of B-factors derived from X-ray scattering data of proteins for the understanding the (functional) dynamics of proteins, we present a comparison of B-factors of five different proteins (SH3 domain, Crh, GB1, ubiquitin and thioredoxin) with data from recent solid-state nuclear magnetic resonance experiments reflecting true (rotational) dynamics on well-defined timescales. Apart from trivial correlations involving mobile loop regions and chain termini, we find no significant correlation of B-factors with the dynamic data on any of the investigated timescales, concluding that there is no unique and general correlation of B-factors with the internal reorientational dynamics of proteins.     

Increased hepatic fibrosis and JNK2-dependent liver injury in mice exhibiting hepatocyte-specific deletion of cFLIP

Chronic liver disease promotes hepatocellular injury involving apoptosis and triggers compensatory regeneration that leads to the activation of quiescent stellate cells in the liver. The deposition of extracellular matrix from activated myofibroblasts promotes hepatic fibrosis and the progression to cirrhosis with deleterious effects on liver physiology. The role of apoptosis signaling pathways in the development of fibrosis remains undefined. The aim of the current study was to determine the involvement of the caspase-8 homologue cellular FLICE-inhibitory protein (cFLIP) during the initiation and progression of fibrosis. Liver injury and fibrosis from carbon tetrachloride (CCl(4)) and thioacetamide (TAA) were examined in mice exhibiting a hepatocyte-specific deletion of cFLIP (flip(-/-)). Acute liver injury from CCl(4) and TAA were enhanced in flip(-/-) mice. This was accompanied by increased activation of caspase-3 and -9, pronounced phosphorylation of JNK, and decreased phosphorylation of Erk. Deletion of the cJun NH(2)-terminal kinase 2 (JNK2) in flip(-/-) mice protected from injury. Hepatic fibrosis was increased at baseline in 12-wk-old flip(-/-) mice, and progression of fibrosis from TAA was accelerated compared with the wild type. In conclusion, deletion of cFLIP in hepatocytes leads to increased fibrosis and accelerated fibrosis progression. This is accompanied by increased injury involving the activation of caspases and JNK2. Thus predisposition to liver injury involving increased hepatocellular apoptosis is a critical mediator of accelerated fibrogenesis, and prevention of liver injury will be a most important measure for patients with chronic liver disease.     

Haploinsufficiency of a spliceosomal GTPase encoded by EFTUD2 causes mandibulofacial dysostosis with microcephaly

Mandibulofacial dysostosis with microcephaly (MFDM) is a rare sporadic syndrome comprising craniofacial malformations, microcephaly, developmental delay, and a recognizable dysmorphic appearance. Major sequelae, including choanal atresia, sensorineural hearing loss, and cleft palate, each occur in a significant proportion of affected individuals. We present detailed clinical findings in 12 unrelated individuals with MFDM; these 12 individuals compose the largest reported cohort to date. To define the etiology of MFDM, we employed whole-exome sequencing of four unrelated affected individuals and identified heterozygous mutations or deletions of EFTUD2 in all four. Validation studies of eight additional individuals with MFDM demonstrated causative EFTUD2 mutations in all affected individuals tested. A range of EFTUD2-mutation types, including null alleles and frameshifts, is seen in MFDM, consistent with haploinsufficiency; segregation is de novo in all cases assessed to date. U5-116kD, the protein encoded by EFTUD2, is a highly conserved spliceosomal GTPase with a central regulatory role in catalytic splicing and post-splicing-complex disassembly. MFDM is the first multiple-malformation syndrome attributed to a defect of the major spliceosome. Our findings significantly extend the range of reported spliceosomal phenotypes in humans and pave the way for further investigation in related conditions such as Treacher Collins syndrome.     

Efficient synthesis and 5-LOX/COX-inhibitory activity of some 3-hydroxybenzo[b]thiophene-2-carboxylic acid derivatives

A series of 3-hydroxybenzo[b]thiophene-2-carboxylic acid derivatives has been prepared and subsequently evaluated with regards to the inhibition of 5-LOX/COX. Structure optimization furnished derivatives with promising in vitro activity as dual 5-LOX/COX inhibitors with submicromolar IC(50) values for inhibition of 5-LOX and COX-1, respectively.     

A helical region in the C terminus of small-conductance Ca2+-activated K+ channels controls assembly with apo-calmodulin.

Small conductance Ca(2+)-activated potassium (SK) channels underlie the afterhyperpolarization that follows the action potential in many types of central neurons. SK channels are voltage-independent and gated solely by intracellular Ca(2+) in the submicromolar range. This high affinity for Ca(2+) results from Ca(2+)-independent association of the SK alpha-subunit with calmodulin (CaM), a property unique among the large family of potassium channels. Here we report the solution structure of the calmodulin binding domain (CaMBD, residues 396-487 in rat SK2) of SK channels using NMR spectroscopy. The CaMBD exhibits a helical region between residues 423-437, whereas the rest of the molecule lacks stable overall folding. Disruption of the helical domain abolishes constitutive association of CaMBD with Ca(2+)-free CaM, and results in SK channels that are no longer gated by Ca(2+). The results show that the Ca(2+)-independent CaM-CaMBD interaction, which is crucial for channel function, is at least in part determined by a region different in sequence and structure from other CaM-interacting proteins.