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Background: The European Working Group on Sarcopenia in Older People (EWGSOP) recommends SARC‐F as a tool for identifying sarcopenia among older adults. However, the role of SARC‐F among older adults with cancer remains unexplored. We aimed to evaluate the diagnostic utility of SARC‐F to identify those with sarcopenia, or low muscle mass (using skeletal muscle index [SMI]), and myosteatosis (using skeletal muscle density [SMD]) from computed tomography (CT) imaging and the association of SARC‐F with all‐cause mortality. Methods: Older adults (≥60 years) presenting for initial consultation at UAB medical oncology clinic who underwent geriatric assessment were enrolled in a prospective cohort study. We identified study participants who completed SARC‐F screening and had available CT imaging within 60 days of study enrollment. Using single‐slice CT images at the L3 vertebral level, we computed SMI and SMD using published methods. Sarcopenia and myosteatosis were defined using published cutpoints. We calculated the sensitivity and specificity of SARC‐F for detecting low muscle mass and low muscle density using published thresholds. Finally, we computed the impact of SARC‐F and CT measures on overall survival using Kaplan–Meier curves and Cox regression models, after adjusting for age, sex, cancer type, and cancer stage. Results: We identified 212 older adults with a median age of 68.8 years; with 60.8% males, 76.6% whites, and pancreatic cancer (21.2%) being the most common malignancy. In the overall cohort, 30.7% had abnormal SARC‐F using published cutpoints. SARC‐F ≥ 4 had a sensitivity of 35% and a specificity of 76% to identify low muscle mass. SARC‐F ≥ 4 had a sensitivity of 38% and a specificity of 74% to identify low muscle density. Those with SARC‐F ≥ 4 and low SMI/SMD had worse survival compared to those with low SMI/SMD alone. Incorporating SARC‐F improved survival prognostication beyond SMI and SMD (HR = 3.1; p < 0.001; Harrel's C from 0.73 to 0.76). Conclusions: SARC‐F as a screening tool has limited diagnostic utility for identifying older adults with low muscle mass and/or density. However, SARC‐F retains prognostic value independent of CT‐based muscle measures in predicting mortality among older adults with cancer. [ABSTRACT FROM AUTHOR]

Chara braunii is a model for early land plant evolution and terrestrialization. Salt stress has a profound effect on water and ion transport activities, thereby interacting with many other processes, including inorganic carbon acquisition for photosynthesis. In this study, we analyzed the impact of salt stress (5 practical salt units, PSU) on the physiology and gene expression in C. braunii. Photosynthesis was only slightly affected 6 h after salt addition and returned to control levels after 48 h. Several organic compounds such as proline, glutamate, sucrose, and 2‐aminobutyrate accumulated in salt‐treated thalli and might contribute to osmotic potential acclimation, whereas the amount of K+ decreased. We quantified transcript levels for 17,387 genes, of which 95 were up‐regulated and 44 down‐regulated after salt addition. Genes encoding proteins of the functional groups ion/solute transport and cell wall synthesis/modulation were enriched among the up‐regulated genes 24–48 h after salt stress, indicating their role in osmotic acclimation. However, a homolog to land plant ERD4 osmosensors was transiently upregulated after 6 h, and phylogenetic analyses suggested that these sensors evolved in Charophyceae. Down‐regulated genes were mainly related to photosynthesis and carbon metabolism/fixation, consistent with the observed lowered growth after extended cultivation. The changed expression of genes encoding proteins for inorganic carbon acquisition might be related to the impact of salt on ionic relations and inorganic carbon uptake. The results indicate that C. braunii can tolerate enhanced salt concentrations in a defined acclimation process, including distinct gene expression changes to achieve new metabolic homeostasis. [ABSTRACT FROM AUTHOR]

Charophyceae are the most complex streptophyte algae, possessing tissue‐like structures, rhizoids and a cellulose‐pectin‐based cell wall akin to embryophytes. Together with the Zygnematophyceae and the Coleochaetophycae, the Charophyceae form a grade in which the Zygnematophyceae share a last common ancestor with land plants. The availability of genomic data, its short life cycle, and the ease of non‐sterile cultivation in the laboratory have made the species Chara braunii an emerging model system for streptophyte terrestrialization and early land plant evolution. In this study, tissue containing nodal cells was prepared under the stereomicroscope, and an RNA‐seq dataset was generated and compared to transcriptome data from whole plantlets. In both samples, transcript coverage was high for genes encoding ribosomal proteins and a homolog of the putative PAX3‐ and PAX7‐binding protein 1. Gene ontology was used to classify the putative functions of the differently expressed genes. In the nodal cell sample, main upregulated molecular functions were related to protein, nucleic acid, ATP‐ and DNA binding. Looking at specific genes, several signaling‐related genes and genes encoding sugar‐metabolizing enzymes were found to be expressed at a higher level in the nodal cell sample, while photosynthesis‐and chloroplast‐related genes were expressed at a comparatively lower level. We detected the transcription of 21 different genes encoding DUF4360‐containing cysteine‐rich proteins. The data contribute to the growing understanding of Charophyceae developmental biology by providing a first insight into the transcriptome composition of Chara nodal cells. [ABSTRACT FROM AUTHOR]

Methylation of histone H3 at lysine 9 (H3K9) is a hallmark of heterochromatin that plays crucial roles in gene silencing, genome stability, and chromosome segregation. In Schizosaccharomyces pombe, Clr4 mediates both di‐ and tri‐methylation of H3K9. Although H3K9 methylation has been intensely studied in mitotic cells, its role during sexual differentiation remains unclear. Here, we map H3K9 methylation genome‐wide during meiosis and show that constitutive heterochromatin temporarily loses H3K9me2 and becomes H3K9me3 when cells commit to meiosis. Cells lacking the ability to tri‐methylate H3K9 exhibit meiotic chromosome segregation defects. Finally, the H3K9 methylation switch is accompanied by differential phosphorylation of Clr4 by the cyclin‐dependent kinase Cdk1. Our results suggest that a conserved master regulator of the cell cycle controls the specificity of an H3K9 methyltransferase to prevent ectopic H3K9 methylation and to ensure faithful gametogenesis. Synopsis: Upon entry into meiosis, Cdk1 is inhibited, Clr4SUV39H is no longer phosphorylated and its substrate histone H3 transitions from H3K9me2 to H3K9me3, which is critical for faithful chromosome segregation during meiosis. Constitutive heterochromatin temporarily transitions from H3K9me2 to H3K9me3 when fission yeast cells commit to meiosis.The meiotic H3K9 methylation switch is accompanied by differential phosphorylation of Clr4, which depends on the cyclin‐dependent kinase Cdk1.Monopolar spindle attachment of sister chromatids critically depends on H3K9me3 at the first meiotic division. [ABSTRACT FROM AUTHOR]

Mouse embryonic stem cells (mESCs) are biased toward producing embryonic rather than extraembryonic endoderm fates. Here, we identify the mechanism of this barrier and report that the histone deacetylase Hdac3 and the transcriptional corepressor Dax1 cooperatively limit the lineage repertoire of mESCs by silencing an enhancer of the extraembryonic endoderm‐specifying transcription factor Gata6. This restriction is opposed by the pluripotency transcription factors Nr5a2 and Esrrb, which promote cell type conversion. Perturbation of the barrier extends mESC potency and allows formation of 3D spheroids that mimic the spatial segregation of embryonic epiblast and extraembryonic endoderm in early embryos. Overall, this study shows that transcriptional repressors stabilize pluripotency by biasing the equilibrium between embryonic and extraembryonic lineages that is hardwired into the mESC transcriptional network. Synopsis: Embryonic stem cells (ESC) predominantly differentiate into epiblast‐like fate as opposed to extraembryonic endoderm, but the molecular mechanism underlying this early developmental preference remains ill‐defined. Using multi‐layered expression profiling, this study identifies histone deacetylase Hdac3 and transcriptional corepressor Dax1 as key determinants of lineage barriers in mouse ESCs. Depletion of Hdac3 or Dax1 increases GATA6‐dependent primitive endoderm (PrE) formation at metastable serum/LIF conditions.Dax1 inhibits mESC transdifferentiation by controlling nuclear receptor Nr5a2 activity.Hdac3 acts with corepressors Ncor1/2 to target a single enhancer upstream of the PrE‐specifying transcription factor gene Gata6.Hdac3 or Dax1 mutant mESCs give rise to bona fide PrE in blastocysts. [ABSTRACT FROM AUTHOR]

The article examines the question of whether gay districts in North America of today continue to persevere into the twenty-first century and adapt to large numbers of gay and lesbian baby boomers as they age. Topics covered include degree of perceived positive and supportive attributes associated with these districts, the definition of gay districts and the different types of gay neighborhoods common in North America, and three distinct types of gay districts.

Protein O-fucosylation is a post-translational modification found on serine/threonine residues of thrombospondin type 1 repeats (TSR). The fucose transfer is catalysed by the enzyme protein O-fucosyltransferase 2 (POFUT2) and >40 human proteins contain the TSR consensus sequence for POFUT2-dependent fucosylation. To better understand O-fucosylation on TSR, we carried out a structural and functional analysis of human POFUT2 and its TSR substrate. Crystal structures of POFUT2 reveal a variation of the classical GT-B fold and identify sugar donor and TSR acceptor binding sites. Structural findings are correlated with steady-state kinetic measurements of wild-type and mutant POFUT2 and TSR and give insight into the catalytic mechanism and substrate specificity. By using an artificial mini-TSR substrate, we show that specificity is not primarily encoded in the TSR protein sequence but rather in the unusual 3D structure of a small part of the TSR. Our findings uncover that recognition of distinct conserved 3D fold motifs can be used as a mechanism to achieve substrate specificity by enzymes modifying completely folded proteins of very wide sequence diversity and biological function. [ABSTRACT FROM AUTHOR]

Butyrate response factor (BRF1) belongs to the Tis11 family of CCCH zinc-finger proteins, which bind to mRNAs containing an AU-rich element (ARE) in their 3'untranslated region and promote their deadenylation and rapid degradation. Independent signal transduction pathways have been reported to stabilize ARE-containing transcripts by a process thought to involve phosphorylation of ARE-binding proteins. Here we report that protein kinase B (PKB/Akt) stabilizes ARE transcripts by phosphorylating BRF1 at serine 92 (S92). Recombinant BRF1 promoted in vitro decay of ARE-containing mRNA (ARE-mRNA), yet phosphorylation by PKB impaired this activity. S92 phosphorylation of BRF1 did not impair ARE binding, but induced complex formation with the scaffold protein 14-3-3. In vivo and in vitro data support a model where PKB causes ARE-mRNA stabilization by inactivating BRF1 through binding to 14-3-3. [ABSTRACT FROM AUTHOR]

Microtubules are cylindrical cytoskeletal structures found in almost all eukaryotic cell types which are involved in a great variety of cellular processes. Reversible acetylation on the ∊-amino group of α-tubulin Lys40 marks stabilized microtubule structures and may contribute to regulating microtubule dynamics. Yet, the enzymes catalysing this acetylation/deacetylation have remained unidentified until recently. Here we report that β-tubulin interacts with histone deacetylase-6 (HDAC-6) in a yeast two-hybrid assay and in vitro. We find that HDAC-6 is a microtubule-associated protein capable of deacetylating α-tubulin in vivo and in vitro. HDAC-6's microtubule binding and deacetylation functions both depend on the hdac domains. Overexpression of HDAC-6 in mammalian cells leads to tubulin hypoacetylation. In contrast, inhibition of HDAC-6 function by two independent mechanisms-pharmacological (HDAC inhibitors) or genetic (targeted inactivation of HDAC-6 in embryonic stem cells)-leads to hyperacetylation of tubulin and microtubules. Taken together, our data provide evidence that HDAC-6 might act as a dual deacetylase for tubulin and histones, and suggest the possibility that acetylated non-histone proteins might represent novel targets for pharmacological therapy by HDAC inhibitors. [ABSTRACT FROM AUTHOR]

We have characterized a glycosylated, 31 amino-acid peptide of 4932 Da isolated from Drosophila melanogaster males. The mature peptide contains a sugar moiety of 1184 Da at a NDT consensus glycosylation site and a disulfide bond. It is synthesized in the male ejaculatory duct via a 54 amino-acid precursor containing an N-terminal signal peptide and Arg-Lys at the C-terminus which is cleaved off during maturation. The gene contains an intron of 53 bp and is localized in the cytological region 99B of the D. melanogaster genome. The peptide is therefore named DUP99B (for ductus ejaculatorius peptide, cytological localization 99B). The C-terminal parts of mature DUP99B and D. melanogaster sex-peptide (ACP70A) are highly homologous. Injected into virgin females, DUP99B elicits the same postmating responses as sex-peptide (increased oviposition, reduced receptivity). These effects are also induced by de-glycosylated native peptide or synthetic DUP99B lacking the sugar moiety. Presence of the glycosyl group, however, decreases the amount needed to elicit the postmating responses. Homologies in the coding regions of the two exons of DUP99B and sex-peptide, respectively, suggest that the two genes have evolved by gene duplication. Thus, we consider these two genes to be members of the new sex-peptide gene family. [ABSTRACT FROM AUTHOR]

Examines the effects of intact talin and talin tail fragment on actin filament dynamics. Discussion on the ionic strength; Effects of intact talin on actin network; Autocorrelation of functions.

Metabolism of sucrose, a mobile source of energy and carbon, is an absolute requirement for the survival of heterotrophic plant organs. In these organs, different isoforms of invertase with discrete subcellular locations hydrolyze the disaccharide into hexoses and, thereby, feed sucrose into various biochemical pathways. In contrast to the invertases with acidic pH optima and a vacuolar or extracellular location, knowledge about the molecular nature of the cytoplasmic invertases (neutral and alkaline invertases) is still in its infancy. Here we report the cDNA cloning of a neutral invertase from carrot based on a partial peptide sequence from the purified enzyme. The function of the encoded polypeptide was confirmed by expression of the cDNA in Escherichia coli. The recombinant protein cleaved sucrose into glucose and fructose with the highest activity between pH 6.5 and 7.0. Unlike acid invertase, neutral invertase does not have the characteristics of a typical plant β-fructofuranosidase and sucrose appears to be its sole substrate. The deduced amino acid sequence shares no similarity with sequences of acid invertases. The polypeptide is cysteine-rich and homologous sequences were only detected in the genomes of plants and photosynthetic bacteria. Hence, this protein must have evolved independently of other sucrose-cleaving enzymes. Transcripts for neutral invertase were found in all organs at different stages of development with slightly higher levels in developing organs, suggesting a more general and possibly a growth-related function of the enzyme in carrot sucrose metabolism. [ABSTRACT FROM AUTHOR]

Partial metal depletion and 113Cd-NMR studies have suggested that the recombinant Cd-containing metallothionein of the sea urchin Strongylocentrotus purpuratus (Cd7-MTA) binds its metal ions in a four-metal (Cd4Cys,11) and a three-metal (Cd7Cys9) cluster associated with the N-terminal and C-terminal halves of the protein, respectively [Wang, Y., Mackay, E. A., Zerbe, O., Hess, D., Hunziker, R E., Vasak, M. & Kägi. J. H. R. (1995) Biochemistry 34, 7460–7467]. This partitioning has now been confirmed by bisecting native Cd?-MTA with subtilisin into products bearing only a single metal-thiolate cluster. Their separation by reverse-phase HPLC and on-line electrospray mass spectrometry in combination with sequence analysis revealed selective cleavage of the protein into a set of N-terminal polypeptides containing 37–39 residues with four Cd ions and a set of C terminal polypeptides containing 24 and 25 residues with three Cd ions. Thus, sea urchin MTA like its mammalian counterparts is made up of two separate cluster-harboring domains. The fragmentation pattern indicated that the sites of cleavage are located in the peptide loop interspaced between the first two metal bound cysteine residues of the C-terminal domain. Accordingly, with cleavage, one of the putative nine thiolate ligands of the three-metal cluster was lost to the N terminal fragment. The coordinational consequences of this repartition were reflected in massive chiroptical changes accompanying the cleavage process. While the liberated N-terminal domain retained the CD profile of the four-metal cluster in the parent protein and thereby indicated preservation of its structure, the CD features attributable to the intact three-metal cluster were largely lost on cleavage. The vanished features bear strong resemblance to the large biphasic ellipticity signal at 250 nm which dominates the CD spectrum of native Cd7 MTA, and allow us thus to attribute this signal to excitonic coupling interactions of Cd-thiolate chromophores in the three-metal cluster. [ABSTRACT FROM AUTHOR]

The gene coding for the 3-phosphoglycerate kinase (EC 2.7.2.3) of Pyrococcus woesei was cloned and sequenced. The gene sequence comprises 1230 bp coding for a polypeptide with the theoretical Mr of 46195. The deduced protein sequence exhibits a high similarity (46.1% and 46.6% identity) to the other known archaeal 3-phosphoglycerate kinases of Methanobacterium bryantiiand Methanothermus fervidus [Fabry. S., Heppner, P., Dietmier, W. & ensel, R. (1990) Gene91, 19-25]. By comparing the 3-phosphoglycerate kinase sequences of the mesophilic and the two thermophilic Archaea, trends in thermoadaptation were confirmed that could be deduced from comparisons of glyceraldehyde-3-phosphate dehydrogenase sequences from the same organisms [Zwickl, P.,Fabry, S., Bogedain, C., Haas, A. & Hensel, R. (1990) J.Bacteriol. 172, 4329-4338]. With increasing temperature the average hydrophobicity and portion of aromatic residues increases, whereas the chain flexibility as well as the content in chemically labile residues (Asn. Cys) decreases. To study the phenotypic properties of the 3-phosphoglycerate kinases from thermophilic Archaea in more detail, the 3-phosphoglycerate kinase genes from P. woesei and M.ferviduswere expressed in Escherichia coli. Comparisons of kinetic and molecular properties of the enzymes from the original organisms and from E.coli indicate that the proteins expressed in the mesophilic host are folded correctly. Besides their higher thermostability according to their origin form hyperthermophilic organisms, both enzymes differ form their bacterial and eucaryotic homologues mainly in two respects. (a) The 3-phosphoglycerate kinases from P.woesei and M. fervidus are homomeric dimers in their native state contrary to all other known 3-phosphoglycerate kinases, which are monomers including the enzyme from the mesophilic Archaeum M. bryantii. (b) Monovalent cations are essential for the activity of both archaeal enzymes with K+ being significantly more efficient than Na+. For the P. woesei enzyme, non-cooperative K+ binding with an apparent Kd (K+) of 88 mM could be determined by kinetic analysis, whereas for the M. fervidus 3-phosphoglycerate kinase the K+ binding is rather complex; form the fitting of the saturation data, non-cooperative binding sites with low selectivity for K+ and Na+ (apparent Kd =270 mM) and at least three cooperative and highly specific K+binding sites/subunit are deduced. At the optimum growth temperature of P.woesei (100°C) and M.fervidus(83°C), the 3-phosphoglycerate kinases show half-lives of inactivation of only 28 min and 44 min, respectively. Potassium salts of polyvalent anions stabilize both 3-phosphoglycerate kinases, like other enzymes form these organisms. In the case of M.fervidus3-phosphoglycerate kinase, the dominant low-molecular-mass compound cyclic D-glycerate 2,3-bisphosphate found in vivo[Hensel, R. & Konig, H.(1998) FEMS Microbiol. Lett. 19,325-333] is the most effective. [ABSTRACT FROM AUTHOR]

The glycoprotein Ib/IX complex on platelets is responsible for the first stage of haemostasis as an essential component in the primary adhesion of platelets to damaged vessel walls. Glycocalicin is the extracellular part of platelet glycoprotein Ibα and contains the yon Willebrand factor and thrombin binding sites. Disulphide bonds are implicated in the yon Willebrand binding site and studies with peptides point towards a region of glycocalicin with four cysteines as containing the binding sites for both yon Willebrand factor and thrombin. The position and linkage of these two disulphide bonds are now determined to be 209–248 and 211–264 and the relevance of this double-loop structure for glycoprotein Ib/IX function is discussed. [ABSTRACT FROM AUTHOR]

We examined the binding kinetics of intact talin and talin head and tail fragment with F-actin at pH 7.0 and at low ionic strength. We observed by a transient kinetic method a fast followed by a slower binding process for intact talin and talin tail fragment with filamentous actin. The latter can be attributed to F-actin cross-linking and/or bundling, which was observed in cosedimentation assays as well as by low shear viscometry and electron microscopy [Zhang, J., Robson, R. M., Schmidt, J. M. & Stromer, M. H. (1996) Biochem. Biophys. Res. Commun. 218, 530–537]. This finding is supported by dynamic light scattering measurements, indicating changes in internal actin filament dynamics due to cross-linking/ bundling events with intact talin and talin tail fragment. No binding of the talin head fragment with F-actin was detected by either method. [ABSTRACT FROM AUTHOR]

Secretory actin-binding protein (SABP), a glycoprotein from human seminal plasma, was isolated according to Akiyama and Kimura [Akiyama, K. & Kimura, H. (1990) Biochim. Biophys. Acta 1040, 206-210]. The complete amino acid sequence of SABP was determined with the aid of fragments generated by trypsin, Staphylococcus aureus V8 protease and pepsin. The single polypeptide chain of SABP contains 118 amino acids with a calculated Mr of 13506 and pyroglutamic acid as the N-terminal residue. A single N-glycosidic carbohydrate moiety is located at Ash77. The carbohydrate composition shows an unusually high amount of fucose. The arrangement of the two disulfide bonds is Cys37-Cys63 and Cys61-Cys95. Sequence comparison revealed a high degree of similarity with a 14-kDa submandibular gland protein from mouse (45% identity and 64% similarity). SABP is identical with a prolactin-inducible protein and a protein termed gross cystic disease fluid protein 15 (sequences translated from cDNA clones), both from human breast tissues. Although SABP was also detected in saliva, in extracts of the submandibular gland and seminal vesicles, little is known of its function. [ABSTRACT FROM AUTHOR]

Developmental cell fate specification is a unidirectional process that can be reverted in response to injury or experimental reprogramming. Whether differentiation and de‐differentiation trajectories intersect mechanistically is unclear. Here, we performed comparative screening in lineage‐related mouse naïve embryonic stem cells (ESCs) and primed epiblast stem cells (EpiSCs), and identified the constitutively expressed zinc finger transcription factor (TF) Zfp281 as a bidirectional regulator of cell state interconversion. We showed that subtle chromatin binding changes in differentiated cells translate into activation of the histone H3 lysine 9 (H3K9) methyltransferase Ehmt1 and stabilization of the zinc finger TF Zic2 at enhancers and promoters. Genetic gain‐of‐function and loss‐of‐function experiments confirmed a critical role of Ehmt1 and Zic2 downstream of Zfp281 both in driving exit from the ESC state and in restricting reprogramming of EpiSCs. Our study reveals that cell type‐invariant chromatin association of Zfp281 provides an interaction platform for remodeling the cis‐regulatory network underlying cellular plasticity. Synopsis: If reprogramming of somatic cells requires the reversal of developmental mechanisms is unclear. In this study, comparative large‐scale screening identifies zinc finger transcription factor Zfp281 as a bi‐directional cell state transition regulator of mouse naïve pluripotency. Zfp281 controls interconversion of naïve embryonic stem cells and primed epiblast stem cells.Zfp281 occupies cis‐regulatory DNA elements (CREs) independent of the cell state.H3K9 methyltransferase EHMT1 and transcription factor Zic2 interact with Zfp281 preferentially in differentiated cells.Genetic depletion of Ehmt1 and Zic2 recapitulates cell state transition phenotypes caused by absence of ZFP281.Zfp281 activates Ehmt1 and recruits Zic2 to CREs. [ABSTRACT FROM AUTHOR]