Identification of two human nuclear proteins that recognise the cytosine-rich strand of human telomeres in vitro

Most studies on the structure of DNA in telomeres have been dedicated to the double-stranded region or the guanosine-rich strand and consequently little is known about the factors that may bind to the telomere cytosine-rich (C-rich) strand. This led us to investigate whether proteins exist that can recognise C-rich sequences. We have isolated several nuclear factors from human cell extracts that specifically bind the C-rich strand of vertebrate telomeres [namely a d(CCCTAA)_n repeat] with high affinity and bind double-stranded telomeric DNA with a 100× reduced affinity. A biochemical assay allowed us to characterise four proteins of apparent molecular weights 66–64, 45 and 35 kDa, respectively. To identify these polypeptides we screened a λgt11-based cDNA expression library, obtained from human HeLa cells using a radiolabelled telomeric oligonucleotide as a probe. Two clones were purified and sequenced: the first corresponded to the hnRNP K protein and the second to the ASF/SF2 splicing factor. Confirmation of the screening results was obtained with recombinant proteins, both of which bind to the human telomeric C-rich strand in vitro.     

The monomeric glutamyl-tRNA synthetase from Bacillus subtilis 168 and its regulatory factor. Their purification, characterization, and the study of their interaction

The glutamyl-tRNA synthetase from Bacillus subtilis has been purified to homogeneity. It is a monomer of Mr = 65,500 whose NH2-terminal sequence is Met-Asn-Glu-Val-Arg-Val-Arg-Tyr-Ser-Pro-Ser-Pro-Thr-Gly-His-Leu. The number of tryptic peptides indicates the absence of a significant amount of sequence duplication. Under certain conditions, this monomeric enzyme is co-purified with a polypeptide beta of Mr = 46,000, which increases the affinity of the enzyme about 10-fold for glutamate and for ATP, and stabilizes it against heat inactivation. gamma-Globulins prepared against the monomeric enzyme can inhibit completely the glutamyl-tRNA synthetase activity of a B. subtilis extract and precipitate from this extract both the monomeric enzyme and the regulatory factor beta. These anti-alpha immunoglobulins do nt precipitate pure beta. These results show that the glutamyl-tRNA synthetase of B. subtilis has a structure similar to that of the Escherichia coli enzyme (Lapointe, J., and S?ll, D. (1972) J. Biol. Chem. 247, 4966-4974) and indicate that the beta factor has a function in the regulation of glutamyl-tRNA biosynthesis in vivo.     

SPOC1 modulates DNA repair by regulating key determinants of chromatin compaction and DNA damage response

Survival time-associated plant homeodomain (PHD) finger protein in Ovarian Cancer 1 (SPOC1, also known as PHF13) is known to modulate chromatin structure and is essential for testicular stem-cell differentiation. Here we show that SPOC1 is recruited to DNA double-strand breaks (DSBs) in an ATM-dependent manner. Moreover, SPOC1 localizes at endogenous repair foci, including OPT domains and accumulates at large DSB repair foci characteristic for delayed repair at heterochromatic sites. SPOC1 depletion enhances the kinetics of ionizing radiation-induced foci (IRIF) formation after γ-irradiation (γ-IR), non-homologous end-joining (NHEJ) repair activity, and cellular radioresistance, but impairs homologous recombination (HR) repair. Conversely, SPOC1 overexpression delays IRIF formation and γH2AX expansion, reduces NHEJ repair activity and enhances cellular radiosensitivity. SPOC1 mediates dose-dependent changes in chromatin association of DNA compaction factors KAP-1, HP1-α and H3K9 methyltransferases (KMT) GLP, G9A and SETDB1. In addition, SPOC1 interacts with KAP-1 and H3K9 KMTs, inhibits KAP-1 phosphorylation and enhances H3K9 trimethylation. These findings provide the first evidence for a function of SPOC1 in DNA damage response (DDR) and repair. SPOC1 acts as a modulator of repair kinetics and choice of pathways. This involves its dose-dependent effects on DNA damage sensors, repair mediators and key regulators of chromatin structure.     

The phosphoinositide kinase PIKfyve/Fab1p regulates terminal lysosome maturation in Caenorhabditis elegans

Membrane dynamics is necessary for cell homeostasis and signal transduction and is in part regulated by phosphoinositides. Pikfyve/Fab1p is a phosphoinositide kinase that phosphorylates phosphatidylinositol 3-monophosphate into phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P2] and is implicated in membrane homeostasis in yeast and in mammalian cells. These two phosphoinositides are substrates of myotubularin phosphatases found mutated in neuromuscular diseases. We studied the roles of phosphatidylinositol phosphate kinase 3 (PPK-3), the orthologue of PIKfyve/Fab1p, in a multicellular organism, Caenorhabditis elegans. Complete loss of ppk-3 function induces developmental defects characterized by embryonic lethality, whereas partial loss of function leads to growth retardation. At the cellular level, ppk-3 mutants display a striking enlargement of vacuoles positive for lysosome-associated membrane protein 1 in different tissues. In the intestine, RAB-7-positive late endosomes are also enlarged. Membranes of the enlarged lysosomes originate at least in part from smaller lysosomes, and functional and genetic analyses show that the terminal maturation of lysosomes is defective. Protein degradation is not affected in the hypomorphic ppk-3 mutant and is thus uncoupled from membrane retrieval. We measured the level of PtdIns(3,5)P2 and showed that its production is impaired in this mutant. This work strongly suggests that the main function of PPK-3 is to mediate membrane retrieval from matured lysosomes through regulation of PtdIns(3,5)P2.     

The stimulation of glycolysis by hypoxia in activated monocytes is mediated by AMP-activated protein kinase and inducible 6-phosphofructo-2-kinase

The activation of monocytes involves a stimulation of glycolysis, release of potent inflammatory mediators, and alterations in gene expression. All of these processes are known to be further increased under hypoxic conditions. The activated monocytes express inducible 6-phosphofructo-2-kinase (iPFK-2), which synthesizes fructose 2,6-bisphosphate, a stimulator of glycolysis. During ischemia, AMP-activated protein kinase (AMPK) activates the homologous heart 6-phosphofructo-2-kinase isoform by phosphorylating its Ser-466. Here, we studied the involvement of AMPK and iPFK-2 in the stimulation of glycolysis in activated monocytes under hypoxia. iPFK-2 was phosphorylated on the homologous serine (Ser-461) and activated by AMPK in vitro. The activation of human monocytes by lipopolysaccharide induced iPFK-2 expression and increased fructose 2,6-bisphosphate content and glycolysis. The incubation of activated monocytes with oligomycin, an inhibitor of oxidative phosphorylation, or under hypoxic conditions activated AMPK and further increased iPFK-2 activity, fructose 2,6-bisphosphate content, and glycolysis. In cultured human embryonic kidney 293 cells, the expression of a dominant-negative AMPK prevented both the activation and phosphorylation of co-transfected iPFK-2 by oligomycin. It is concluded that the stimulation of glycolysis by hypoxia in activated monocytes requires the phosphorylation and activation of iPFK-2 by AMPK.     

Agrin binds to the N-terminal region of Lrp4 protein and stimulates association between Lrp4 and the first immunoglobulin-like domain in muscle-specific kinase (MuSK)

Neuromuscular synapse formation depends upon coordinated interactions between motor neurons and muscle fibers, leading to the formation of a highly specialized postsynaptic membrane and a highly differentiated nerve terminal. Synapse formation begins as motor axons approach muscles that are prepatterned in the prospective synaptic region in a manner that depends upon Lrp4, a member of the LDL receptor family, and muscle-specific kinase (MuSK), a receptor tyrosine kinase. Motor axons supply Agrin, which binds Lrp4 and stimulates further MuSK phosphorylation, stabilizing nascent synapses. How Agrin binds Lrp4 and stimulates MuSK kinase activity is poorly understood. Here, we demonstrate that Agrin binds to the N-terminal region of Lrp4, including a subset of the LDLa repeats and the first of four β-propeller domains, which promotes association between Lrp4 and MuSK and stimulates MuSK kinase activity. In addition, we show that Agrin stimulates the formation of a functional complex between Lrp4 and MuSK on the surface of myotubes in the absence of the transmembrane and intracellular domains of Lrp4. Further, we demonstrate that the first Ig-like domain in MuSK, which shares homology with the NGF-binding region in Tropomyosin Receptor Kinase (TrKA), is required for MuSK to bind Lrp4. These findings suggest that Lrp4 is a cis-acting ligand for MuSK, whereas Agrin functions as an allosteric and paracrine regulator to promote association between Lrp4 and MuSK.     

The effects of a change in gravity on the dynamics of prehension

The objective of this study was to measure the forces applied on an object manipulated in different gravitational fields attained during parabolic flights. Eight subjects participated flights (ES) and four were inexperienced (NES). They had to move continuously an instrumented object up and down in three different gravitational conditions (1 g, 1.8 g, 0 g). In 1 g, the grip force precisely anticipated the fluctuations of load force which was maximum and minimum at the bottom and at the top of the arm trajectory respectively. When the gravity changed (0 g and 1.8 g), the grip-load force coupling persisted for all the subjects from the first parabola. While the ES immediately exerted a grip force appropriate to the gravity, the NES dramatically increased their grip when faced with hyper and microgravity for the first time. Then, they progressively released their grip until a continuous grip-load force relationship with regard to 1 g was established after the fifth parabola. We suggest that each new gravitational field is rapidly incorporated into an internal model within the CNS which can then be reused as required by the occasion.     

Widespread Distribution of <Emphasis Type="Italic">Dehalococcoides mccartyi</Emphasis> in the Houston Ship Channel and Galveston Bay,Texas, Sediments and the Potential for Reductive Dechlorination of PCDD/F in an Estuarine Environment

Sediments in the Houston Ship Channel and upper Galveston Bay, Texas, USA, are polluted with polychlorinated dibenzo-p-dioxins/furans (PCDD/F; ≤46,000 ng/kg dry weight (wt.)) with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the most toxic congener, contributing >50 % of the total toxic equivalents (TEQ) at most locations. We measured PCDD/F concentrations in sediments and evaluated the potential for enhanced in situ biodegradation by surveying for Dehalococcoides mccartyi, an obligate organohalide respiring bacterium. Dehalococcoides spp. (98 % similar to D. mccartyi) and 22 other members of the class Dehalococcoidia were predominant 16S ribosomal RNA (rRNA) phylotypes. Dehalococcoides spp. were also present in the active fraction of the bacterial community. Presence/absence PCR screening detected D. mccartyi in sediment cores and sediment grab samples having at least 1 ng/kg dry wt. TEQ at salinities ranging from 0.6 to 19.5 PSU, indicating that they are widespread in the estuarine environment. Organic carbon-only and organic carbon + sulfate-amended sediment microcosm experiments resulted in ～60 % reduction of ambient 2,3,7,8-TCDD in just 24 months leading to reductions in total TEQs by 38.4 and 45.0 %, respectively, indicating that 2,3,7,8-TCDD degradation is occurring at appreciable rates.     

Semiautomatic extraction of cortical thickness and diaphyseal curvature from CT scans

The understanding of locomotor patterns, activity schemes, and biological variations has been enhanced by the study of the geometrical properties and cortical bone thickness of the long bones measured using CT scan cross‐sections. With the development of scanning procedures, the internal architecture of the long bones can be explored along the entire diaphysis. Recently, several methods that map cortical thickness along the whole femoral diaphysis have been developed. Precise homology is vital for statistical examination of the data; however, the repeatability of these methods is unknown and some do not account for the curvature of the bones. We have designed a semiautomatic workflow that improves the morphometric analysis of cortical thickness, including robust data acquisition with minimal user interaction and considering the bone curvature. The proposed algorithm also performs automatic landmark refinement and rigid registration on the extracted morphometric maps of the cortical thickness. Because our algorithm automatically reslices the diaphysis into 100 cross‐sections along the medial axis and uses an adaptive thresholding method, it is usable on CT scans that contain soft tissues as well as on bones that have not been oriented specifically prior to scanning. Our approach exhibits considerable robustness to error in user‐supplied landmarks, suppresses distortion caused by the curvature of the bones, and calculates the curvature of the medial axis.