Isocitrate dehydrogenase 1 R132C mutation occurs exclusively in microsatellite stable colorectal cancers with the CpG island methylator phenotype

The CpG Island Methylator Phenotype (CIMP) is fundamental to an important subset of colorectal cancer; however, its cause is unknown. CIMP is associated with microsatellite instability but is also found in BRAF mutant microsatellite stable cancers that are associated with poor prognosis. The isocitrate dehydrogenase 1 (IDH1) gene causes CIMP in glioma due to an activating mutation that produces the 2-hydroxyglutarate oncometabolite. We therefore examined IDH1 alteration as a potential cause of CIMP in colorectal cancer. The IDH1 mutational hotspot was screened in 86 CIMP-positive and 80 CIMP-negative cancers. The entire coding sequence was examined in 81 CIMP-positive colorectal cancers. Forty-seven cancers varying by CIMP-status and IDH1 mutation status were examined using Illumina 450K DNA methylation microarrays. The R132C IDH1 mutation was detected in 4/166 cancers. All IDH1 mutations were in CIMP cancers that were BRAF mutant and microsatellite stable (4/45, 8.9%). Unsupervised hierarchical cluster analysis identified an IDH1 mutation-like methylation signature in approximately half of the CIMP-positive cancers. IDH1 mutation appears to cause CIMP in a small proportion of BRAF mutant, microsatellite stable colorectal cancers. This study provides a precedent that a single gene mutation may cause CIMP in colorectal cancer, and that this will be associated with a specific epigenetic signature and clinicopathological features.     

Effect of zonal conditions and posture on pulmonary blood flow distribution to subpleural and interior lung.

Observations made on vessels seen directly beneath the pleura may not accurately reflect what occurs in vessels located deeper in the interior of the lung. We quantified flow to subpleural and deeper, interior regions under zone 1 or 2 conditions in excised (n = 5) and in vivo (n = 6) rabbit lungs, in the head-up or inverted position. After infusion of radiolabeled microspheres, lungs were dried at alveolar pressure of 25 cmH(2)O and sliced in 1-cm sections along the gravitational plane and in three planes in the dorsal-ventral axis. Regions located <1 mm from the pleural surface were dissected away from the remaining tissue. In both zonal conditions, 1) weight-normalized flow to the interior exceeded that found in subpleural regions; and 2) flow followed the gravitational gradient, with the correlation varying with the scale of measurement. We conclude that flow through subpleural vessels is less than that which occurs deeper in the interior, but the regional distributions of flow and the effects of zonal conditions are similar in the two regions.     

Volume infusion produces abdominal distension, lung compression, and chest wall stiffening in pigs.

The effect of severe generalized edema on respiratory system mechanics is not well described. We measured airway pressure, gastric pressure, and four vertical pleural pressures in 13 anesthetized paralyzed pigs ventilated in the upright position. Pressure-volume relationships of the respiratory system, chest wall, and lung were measured on deflation from total lung capacity to residual volume and during tidal breathing both before (control) and 50 min after one of two interventions. In one series of experiments, a volume equal to 15-20% of the pig's body weight was infused intravenously. In a second series, a balloon was placed in the peritoneal space to distend the abdomen to the same gastric pressures as achieved in the first series. Measurements were compared before and after either abdominal balloon inflation or volume infusion. Volume infusion increased the pleural pressure in dependent lung regions, decreased both total lung capacity (34%) and functional residual capacity (62%) (both P less than 0.05), and markedly shifted the respiratory system and chest wall pressure-volume curves to the right, but it only moderately affected the lung deflation curve. Tidal compliances of the respiratory system, chest wall, and lung decreased 36, 31, and 49%, respectively (all P less than 0.05). The effect of abdominal balloon inflation on respiratory system mechanics was similar to that of volume infusion. We conclude that infusing large volumes of fluid markedly alters chest wall mechanics, mainly by causing abdominal distension that prohibits descent of the diaphragm.     

The small-subunit ribosomal RNA gene sequences from the hypotrichous ciliates Oxytricha nova and Stylonychia pustulata

We have determined the complete nucleotide sequence of the small- subunit ribosomal RNA genes for the ciliate protozoans Stylonychia pustulata and Oxytricha nova. The sequences are homologous and sufficiently similar that these organisms must be closely related. In a phylogeny inferred from comparisons of several eukaryotic small-subunit ribosomal RNAs, the divergence of the ciliates from the eukaryotic line of descent is seen to coincide with the radiation of the plants, the animals, and the fungi. This radiation is preceded by the divergence of the slime mold, Dictyostelium discoideum.      

Molecular population genetics of ref(2)P, a locus which confers viral resistance in Drosophila

The ref(2)P locus (2-54.2) is polymorphic for two allelic forms in natural populations of Drosophila melanogaster, ref(2)Po and ref(2)Pp. The latter allele confers resistance to the rhabdovirus sigma infecting wild populations. Previous work, based on a small sample of prescreened restrictive (resistant) and permissive (susceptible) alleles, identified a large number of amino acid replacement changes (7) relative to synonymous changes (1). Such protein variability could be the result of variation-enhancing selection. To further test the selection hypothesis, we have examined the DNA sequences of ten randomly chosen lines of D. melanogaster and one line of D. simulans. Nine of the ten lines are permissive; D. simulans does not harbor the virus. The melanogaster alleles contain 4 synonymous changes, 19 noncoding changes, and 13 amino acid replacement changes, indicating a relatively high level of polymorphism. Three sequenced restrictive alleles have nearly identical sequences, indicating that they are relatively young. Compared to the permissive alleles, they share only a complex deletion at codon 34, CAG-AAT to GGA, which our analysis indicates to be the site conferring the restrictive phenotype. Patterns of polymorphism and divergence differ from neutral predictions by several criteria for the amino terminal region, which contains the complex deletion (codons 1-91), but not the remainder of the protein (codons 92-599). We find a higher rate of evolution on the D. melanogaster lineage than on the D. simulans lineage. The relatively large amount of both replacement and silent polymorphism in the permissive alleles and the lack of divergence between permissive and restrictive alleles suggests that the sigma virus and ref(2)P may be engaged in an evolutionary race in which new restrictive alleles are continually arising but are relatively short-lived.      

Relationships among msx gene structure and function in zebrafish and other vertebrates

The zebrafish genome contains at least five msx homeobox genes, msxA, msxB, msxC, msxD, and the newly isolated msxE. Although these genes share structural features common to all Msx genes, phylogenetic analyses of protein sequences indicate that the msx genes from zebrafish are not orthologous to the Msx1 and Msx2 genes of mammals, birds, and amphibians. The zebrafish msxB and msxC are more closely related to each other and to the mouse Msx3. Similarly, although the combinatorial expression of the zebrafish msx genes in the embryonic dorsal neuroectoderm, visceral arches, fins, and sensory organs suggests functional similarities with the Msx genes of other vertebrates, differences in the expression patterns preclude precise assignment of orthological relationships. Distinct duplication events may have given rise to the msx genes of modern fish and other vertebrate lineages whereas many aspects of msx gene functions during embryonic development have been preserved.      

Regional hypoxic pulmonary vasoconstriction in prone pigs.

Hypoxic pulmonary vasoconstriction (HPV) is known to affect regional pulmonary blood flow distribution. It is unknown whether lungs with well-matched ventilation (V)/perfusion (Q) have regional differences in the HPV response. Five prone pigs were anesthetized and mechanically ventilated (positive end-expiratory pressure = 2 cmH2O). Two hypoxic preconditions [inspired oxygen fraction (FI(O2)) = 0.13] were completed to stabilize the animal's hypoxic response. Regional pulmonary blood Q and V distribution was determined at various FI(O2) (0.21, 0.15, 0.13, 0.11, 0.09) using the fluorescent microsphere technique. Q and V in the lungs were quantified within 2-cm3 lung pieces. Pieces were grouped, or clustered, based on the changes in blood flow when subjected to increasing hypoxia. Unique patterns of Q response to hypoxia were seen within and across animals. The three main patterns (clusters) showed little initial difference in V/Q matching at room air where the mean V/Q range was 0.92-1.06. The clusters were spatially located in cranial, central, and caudal portions of the lung. With decreasing FI(O2), blood flow shifted from the cranial to caudal regions. We determined that pulmonary blood flow changes, caused by HPV, produced distinct response patterns that were seen in similar regions across our prone porcine model.     

The MMS22L–TONSL heterodimer directly promotes RAD51‐dependent recombination upon replication stress

Homologous recombination (HR) is a key pathway that repairs DNA double‐strand breaks (DSBs) and helps to restart stalled or collapsed replication forks. How HR supports replication upon genotoxic stress is not understood. Using in vivo and in vitro approaches, we show that the MMS22L–TONSL heterodimer localizes to replication forks under unperturbed conditions and its recruitment is increased during replication stress in human cells. MMS22L–TONSL associates with replication protein A (RPA)‐coated ssDNA, and the MMS22L subunit directly interacts with the strand exchange protein RAD51. MMS22L is required for proper RAD51 assembly at DNA damage sites in vivo, and HR‐mediated repair of stalled forks is abrogated in cells expressing a MMS22L mutant deficient in RAD51 interaction. Similar to the recombination mediator BRCA2, recombinant MMS22L–TONSL limits the assembly of RAD51 on dsDNA, which stimulates RAD51‐ssDNA nucleoprotein filament formation and RAD51‐dependent strand exchange activity in vitro. Thus, by specifically regulating RAD51 activity at uncoupled replication forks, MMS22L–TONSL stabilizes perturbed replication forks by promoting replication fork reversal and stimulating their HR‐mediated restart in vivo.     

Effects of EDTA treatment upon the protein subunit composition and mechanical properties of mammalian single skeletal muscle fibers

Considerable interest has been focused on the role of myosin light chain LC(2) in the contraction of vertebrate striated muscle. A study was undertaken to further our investigations (Moss, R.L., G.G. Giulian, and M.L. Greaser, 1981, J. Biol. Chem., 257:8588-8591) of the effects of LC(2) removal upon contraction in skinned fibers from rabbit psoas muscles. Isometric tension and maximum velocity of shortening, V(max), were measured in fiber segments prior to LC(2) removal. The segments were then bathed at 30 degrees C for up to 240 min in a buffer solution containing 20 mM EDTA in order to extract up to 60 percent of the LC(2). Troponin C (TnC) was also partially removed by this procedure. Mechanical measurements were done following the EDTA extraction and the readditions of first TnC and then LC(2) to the segments. The protein subunit compositions of the same fiber segments were determined following each of these procedures by SDS PAGE of small pieces of the fiber. V(max) was found to decrease as the LC(2) content of the fiber segments was reduced by increasing the duration of extraction. EDTA treatment also resulted in substantial reductions in tension due mainly to the loss of TnC, though smaller reductions due to the extraction of LC(2) were also observed. Reversal of the order of recombination of LC(2) and TnC indicated that the reduction in V(max) following EDTA treatment was a specific effect of LC(2) removal. These results strongly suggest that LC(2) may have roles in determining the kinetics and extent of interaction between myosin and actin.