Negative Impact of Skeletal Muscle Loss after Systemic Chemotherapy in Patients with Unresectable Colorectal Cancer

Background

Skeletal muscle depletion (sarcopenia) is closely associated with limited physical ability and high mortality. This study evaluated the prognostic significance of skeletal muscle status before and after chemotherapy in patients with unresectable colorectal cancer (CRC).

Methods

We conducted a retrospective analysis of 215 consecutive patients with unresectable CRC who underwent systemic chemotherapy. Skeletal muscle cross-sectional area was measured by computed tomography. We evaluated the prognostic value of skeletal muscle mass before chemotherapy and the rate of skeletal muscle change in cross-sectional area after chemotherapy.

Results

One-hundred-eighty-two patients met our inclusion criteria. There were no significant differences in progression-free survival (PFS) or overall survival (OS) associated with skeletal muscle mass before chemotherapy. However, 22 patients with skeletal muscle loss (>5%) after chemotherapy showed significantly shorter PFS and OS compared with those without skeletal muscle loss (PFS, log-rank p = 0.029; OS, log-rank p = 0.009). Multivariate Cox regression analysis revealed that skeletal muscle loss after chemotherapy (hazard ratio, 2.079; 95% confidence interval, 1.194–3.619; p = 0.010) was independently associated with OS.

Conclusions

Skeletal muscle loss after chemotherapy was an independent, negative prognostic factor in unresectable CRC.     

Structure-based design of P3 moieties in the peptide mimetic factor VIIa inhibitor

Selective factor VIIa-tissue factor complex (FVIIa/TF) inhibition is seen as a promising target for developing new anticoagulant drugs. Structure-based designs of the P3 moiety in the peptide mimetic factor VIIa inhibitor successfully lead to novel inhibitors with selectivity for FVIIa/TF and extrinsic coagulation the same as or even higher than those of previously reported peptide mimetic factor VIIa inhibitors. X-ray crystal structure analysis reveals that one of the novel inhibitors shows improved selectivity by forming interactions between the inhibitor and FVIIa as expected. Another of the novel inhibitors achieves improved selectivity through an unexpected hydrogen bond with Gln217, with a unique bent conformation in FVIIa/TF accompanied by conformational changes of the inhibitor and the protein.     

Phospholipids of membranes of cultured cells and the products of protoplast fusion

The variety and distribution of phospholipids in the cell membranes of cultured cells and their fatty acid composition were analysed. Membranes of suspension cultured cells of Rauwolfia serpentina var. Bentham, Nicotiana tabacum var. Samsun, Atropa belladonna and Bouvardia ternifolia had almost the same composition of phosphatidylcholine, PC (ca 50%); phosphatidylethanolamine, PE (ca 25%); phosphatidylinositol, PI (ca 10%); phosphatidylglycerol, PG (several %); and phosphatidic acid, PA (several %). We determined the distribution of the molecular species of the three major phospholipid fractions (PC, PE and PI) in R.serpentina and N.tabacum. Membranes of both cell-types contained basically similar molecular species, 1–16:0/2–18:2 the main type, particularly in the PC- and PE- fractions; 1–18:2/2–18:2 and 1–18:0/2–18:2 for R. serpentina; and 1–16:0/2–18:3, 1-18:0/2-18:3 and 1-18:2/2-18:2 for N.tabacum. The influence of membrane fluidity on protoplast fusion as effected by the phase transition of the phospholipids, is discussed.     

C-terminal Phenylalanine of Bacteriophage T7 Single-stranded DNA-binding Protein Is Essential for Strand Displacement Synthesis by T7 DNA Polymerase at a Nick in DNA

Single-stranded DNA-binding protein (gp2.5), encoded by gene 2.5 of bacteriophage T7, plays an essential role in DNA replication. Not only does it remove impediments of secondary structure in the DNA, it also modulates the activities of the other replication proteins. The acidic C-terminal tail of gp2.5, bearing a C-terminal phenylalanine, physically and functionally interacts with the helicase and DNA polymerase. Deletion of the phenylalanine or substitution with a nonaromatic amino acid gives rise to a dominant lethal phenotype, and the altered gp2.5 has reduced affinity for T7 DNA polymerase. Suppressors of the dominant lethal phenotype have led to the identification of mutations in gene 5 that encodes the T7 DNA polymerase. The altered residues in the polymerase are solvent-exposed and lie in regions that are adjacent to the bound DNA. gp2.5 lacking the C-terminal phenylalanine has a lower affinity for gp5-thioredoxin relative to the wild-type gp2.5, and this affinity is partially restored by the suppressor mutations in DNA polymerase. gp2.5 enables T7 DNA polymerase to catalyze strand displacement DNA synthesis at a nick in DNA. The resulting 5′-single-stranded DNA tail provides a loading site for T7 DNA helicase. gp2.5 lacking the C-terminal phenylalanine does not support this event with wild-type DNA polymerase but does to a limited extent with T7 DNA polymerase harboring the suppressor mutations.Single-stranded DNA (ssDNA)³-binding proteins have been assigned the role of removing secondary structure in DNA and protecting ssDNA from hydrolysis by nucleases (1). However, in addition to these mundane roles, ssDNA-binding proteins are now recognized as a key component of the replisome where they physically and functionally interact with other replication proteins and with the primer-template (2–4). ssDNA-binding proteins are also engaged in DNA recombination and repair (5). In view of these multiple roles, it has been difficult to identify the specific defect in genetically altered ssDNA-binding proteins that leads to an observed phenotype.The crystal structures of several prokaryotic ssDNA-binding proteins have been determined (6–8). These proteins have a conserved oligosaccharide-oligonucleotide binding fold (OB-fold) that is thought to bind the ssDNA by means of stacking and electrostatic interactions (6). Prokaryotic ssDNA-binding proteins also have an acidic C-terminal tail that is essential for bacterial and phage growth (9–13).The ssDNA-binding protein of bacteriophage T7 is encoded by gene 2.5 (14). The gene 2.5 protein (gp2.5) is a homodimer in solution, a structure that is stabilized by its C-terminal tail (9, 15). The C-terminal tail of one monomer of gp2.5 binds in a trans mode to the ssDNA-binding cleft of the other subunit, thus stabilizing the dimer interface observed in the crystal structure (6). The current model proposes that the positively charged DNA-binding cleft is shielded by the electrostatic charges of the C-terminal tail in the absence of ssDNA, thus facilitating oligomerization of gp2.5. Upon binding ssDNA, the dimer dissociates to allow the C-terminal tail to interact with other replication proteins (16). The tail modulates the affinity for ssDNA and protein-protein interactions by functioning as a two-way switch (6, 17). This mode of function is applicable to other prokaryotic ssDNA-binding proteins, namely Escherichia coli SSB protein and T4 gp32 (10, 13, 15, 18–22).gp2.5 is one of four proteins that include the T7 replisome. The other three proteins are the T7 gene 5 DNA polymerase (gp5), its processivity factor, E. coli thioredoxin (trx), and the multifunctional gene 4 helicase-primase (gp4). gp5 and trx bind with high affinity (K_D of 5 nm), and the two proteins are normally found in complex (gp5/trx) at a stoichiometry of one to one (23). The acidic C-terminal tail of gp2.5 is critical for the interactions of the protein with gp5/trx and gp4 (9, 24). The C-terminal tail binds to a positively charged segment located in the thumb subdomain of the gp5 (25). This fragment, designated the trx binding domain (TBD), is also the site of binding of the processivity factor, E. coli trx, and the C terminus of gp4. The multiple interactions of the C terminus of gp2.5 could thus function to coordinate the dynamic reactions occurring at the replication fork. gp2.5 is known to be critical for establishing coordination during leading and lagging strand DNA synthesis (26, 27).This C-terminal tail of gp2.5 is an acidic 26-amino acid segment with an aromatic phenylalanine as the C-terminal residue. The C-terminal tail is not seen in the crystal structure because gp2.5Δ26, lacking the tail, was used for crystallization; the wild-type protein did not yield crystals that diffracted (6). gp2.5ΔF designates a genetically modified gp2.5 lacking the C-terminal phenylalanine. gp2.5ΔF does not support the growth of T7Δ2.5 phage lacking gene 2.5 (28). Interestingly, T7 gene 4 protein also has an acidic C-terminal tail with a C-terminal phenylalanine (29). Again, the phenylalanine is critical for the interaction of gp4 with gp5/trx (29). Further evidence for overlapping binding sites of the C termini of these two proteins comes from studies with chimeric proteins (28, 29). The C-terminal tails of gp2.5 and gp4 can be exchanged, and the chimeric proteins support the growth of T7 phage lacking the corresponding wild-type protein.We recently designed a screen for suppressors of dominant lethal mutations of gp2.5 (30). The screen identified mutations in gene 5, the structural gene for T7 DNA polymerase (Fig. 1), which suppresses the lethal phenotype of gp2.5 mutant in which the C-terminal phenylalanine was moved to the penultimate position (gp2.5ΔF232InsF231). One of the altered suppressor genes (gp5, gp5-sup1) encodes a gp5 in which where glycine at position 371 is replaced by lysine (G371K). Whereas the other (gp5-sup2) encodes a protein in which threonine 258 and alanine 411 are replaced by methionine and threonine, respectively (T258M and A411T). The suppressor mutations in gp5 are necessary and sufficient to suppress the lethal phenotype of gp2.5ΔF232InsF231. The affected residues map in proximity to aromatic residues and to residues in close proximity to DNA as seen in the crystal structure of gp5/trx in complex with DNA (31). Throughout this study, gp2.5ΔF232InsF231 mutant will be referred to as gp2.5-FD because it effectively switches the positions of the C-terminal phenylalanine and the adjacent aspartic acid. E. coli SSB protein also has a C-terminal phenylalanine, and recent studies have shown that this residue inserts into a hydrophobic region consisting of exonuclease I of E. coli (45, 46).Open in a separate windowFIGURE 1.Amino acid changes in gp5 suppressor mutant polymerase(s). The amino acid changes in gp5 arising from the suppressor mutations in gene 5 are identified in the crystal structure of gp5/trx in complex with a primer-template and a nucleoside triphosphate (31). gp5 (light gray), trx (dark gray), and primer/template (red) are depicted. The suppressor mutation G371K (gp5-sup1) is shown in yellow and T258M and A411T (gp5-sup2) in orange.In this study, we have purified the two suppressor DNA polymerases and characterized them individually and in interaction with the other T7 replication proteins. Whereas wild-type gp5 binds with low affinity to gp2.5-FD, the DNA polymerases harboring the suppressor mutations bind with a higher affinity. An interesting finding is that whereas wild-type gp2.5 enables gp5/trx to catalyze strand displacement synthesis at a nick in DNA, gp2.5-FD does not support this reaction. Strand displacement synthesis is necessary for the initiation of leading strand DNA synthesis at a nick because it creates a 5′-single-stranded DNA tail for loading of the T7 helicase (32).     

Molecular phylogeny of terrestrial holocarpic endoparasitic peronosporomycetes, <Emphasis Type="Italic">Haptoglossa</Emphasis> spp., inferred from 18S rDNA

Phylogenetic relationships of seven isolates of the genus Haptoglossa parasitic on terrestrial nematodes within the Peronosporomycetes were analyzed using 18S rDNA sequence data with 21 peronosporomycetes, 2 marine stramenopilous flagellates, and 2 hyphochytridiomycetes. The marine stramenopilous flagellates and hyphochytridiomycetes were used as the outgroup. All Haptoglossa isolates formed a monophyletic clade and clustered with the marine genus Eurychasma. The clade of Haptoglossa and Eurychasma formed a sister-group to the clade that consisted of all other peronosporomycetes. These results suggest that the genus Haptoglossa and other terrestrial peronosporomycetes included in the two subclasses, the Saprolegniomycetidae and the Peronosporomycetidae, might have originally adapted to the terrestrial environment individually. In the maximum-likelihood (ML) analysis, the Haptoglossa clade was divided into three subclades, one aplanosporic species clade and two zoosporic species clades. Phylogenetic analyses of combined 18S rDNA and cox2 genes among five species of Haptoglossa supported the results of the ML analysis using 18S rDNA and suggested that zoosporic species may be separated into two lineages. This topology of the analysis may suggest that aplanosporic species diverged from zoosporic species.     

Human hair-follicle associated pluripotent (hHAP) stem cells differentiate to cardiac-muscle cells

We have previously demonstrated that nestin-expressing hair follicle-associated-pluripotent (HAP) stem cells are located in the bulge area. HAP stem cells have been previously shown to differentiate to neurons, glial cells, keratinocytes, smooth-muscle cells, melanocytes and cardiac-muscle cells in vitro. Subsequently, we demonstrated that HAP stem cells could effect nerve and spinal cord regeneration in mouse models, differentiating to Schwann cells and neurons. In previous studies, we established an efficient protocol for the differentiation of cardiac-muscle cells from mouse HAP stem cells. In the present study, we isolated the upper part of human hair follicles containing human HAP (hHAP) stem cells. The upper parts of human hair follicles were suspended in DMEM containing 10% FBS where they differentiated to cardiac-muscle cells as well as neurons, glial cells, keratinocytes and smooth-muscle cells. This method is appropriate for future use with human hair follicles to produce hHAP stem cells in sufficient quantities for future heart, nerve and spinal cord regeneration in the clinic.     

Cyanobacterial Blue Color Formation during Lysis under Natural Conditions

Cyanobacteria produce numerous volatile organic compounds (VOCs), such as β-cyclocitral, geosmin, and 2-methylisoborneol, which show lytic activity against cyanobacteria. Among these compounds, only β-cyclocitral causes a characteristic color change from green to blue (blue color formation) in the culture broth during the lysis process. In August 2008 and September 2010, the lysis of cyanobacteria involving blue color formation was observed at Lake Tsukui in northern Kanagawa Prefecture, Japan. We collected lake water containing the cyanobacteria and investigated the VOCs, such as β-cyclocitral, β-ionone, 1-propanol, 3-methyl-1-butanol, and 2-phenylethanol, as well as the number of cyanobacterial cells and their damage and pH changes. As a result, the following results were confirmed: the detection of several VOCs, including β-cyclocitral and its oxidation product, 2,2,6-trimethylcyclohexene-1-carboxylic acid; the identification of phycocyanin based on its visible spectrum; the lower pH (6.7 and 5.4) of the lysed samples; and characteristic morphological change in the damaged cyanobacterial cells. We also encountered the same phenomenon on 6 September 2013 in Lake Sagami in northern Kanagawa Prefecture and obtained almost the same results, such as blue color formation, decreasing pH, damaged cells, and detection of VOCs, including the oxidation products of β-cyclocitral. β-Cyclocitral derived from Microcystis has lytic activity against Microcystis itself but has stronger inhibitory activity against other cyanobacteria and algae, suggesting that the VOCs play an important role in the ecology of aquatic environments.     

Oviductin, the oviductal protease that mediates gamete interaction by affecting the vitelline coat in Bufo japonicus: its molecular cloning and analyses of expression and posttranslational activation

Previous studies indicated that the acquisition of egg fertilizability during transit through the pars recta portion of the oviduct in Bufo japonicus is accompanied by hydrolytic conversion of the vitelline coat 40- to 52-kDa components to 39-kDa components induced by a 66-kDa serine protease, "oviductin." In this study, we cloned a 3028-bp cDNA that contained an open reading frame encoding 974 amino acids with a calculated molecular mass of 107.6 kDa, including two protease domains and three repeats of CUB domains. Sequence analysis indicated that the catalytically active 66-kDa protein comprised an N-terminally located oviductin protease and two CUB domains. The oviductin gene was transcribed as a part of 6-kb mRNA that was expressed specifically in the cells lining the bottom of epithelial folds in the oviductal pars recta, and this expression was highly accelerated when the pars recta fragments were cultured in the presence of hCG. Western blot analyses using antibodies against a protease domain revealed that the catalytically inactive 102-kDa proteins in the pars recta granules yield 66-kDa catalytically active and 82- and 59-kDa inactive molecules. We propose that the oviductin translated as 107.6-kDa precursors are processed both N- and C-terminally to give rise to a 66-kDa active form comprising a serine protease and two CUB domains.     

Dissecting the interactions of SERRATE with RNA and DICER-LIKE 1 in Arabidopsis microRNA precursor processing

Efficient and precise microRNA (miRNA) biogenesis in Arabidopsis is mediated by the RNaseIII-family enzyme DICER-LIKE 1 (DCL1), double-stranded RNA-binding protein HYPONASTIC LEAVES 1 and the zinc-finger (ZnF) domain-containing protein SERRATE (SE). In the present study, we examined primary miRNA precursor (pri-miRNA) processing by highly purified recombinant DCL1 and SE proteins and found that SE is integral to pri-miRNA processing by DCL1. SE stimulates DCL1 cleavage of the pri-miRNA in an ionic strength-dependent manner. SE uses its N-terminal domain to bind to RNA and requires both N-terminal and ZnF domains to bind to DCL1. However, when DCL1 is bound to RNA, the interaction with the ZnF domain of SE becomes indispensible and stimulates the activity of DCL1 without requiring SE binding to RNA. Our results suggest that the interactions among SE, DCL1 and RNA are a potential point for regulating pri-miRNA processing.