Colorectal Cancers Mimic Structural Organization of Normal Colonic Crypts

Colonic crypts are stereotypical structures with distinct stem cell, proliferating, and differentiating compartments. Colorectal cancers derive from colonic crypt epithelia but, in contrast, form morphologically disarrayed glands. In this study, we investigated to which extent colorectal cancers phenocopy colonic crypt architecture and thus preserve structural organization of the normal intestinal epithelium. A subset of colon cancers showed crypt-like compartments with high WNT activity and nuclear β-Catenin at the leading tumor edge, adjacent proliferation, and enhanced Cytokeratin 20 expression in most differentiated tumor epithelia of the tumor center. This architecture strongly depended on growth conditions, and was fully reproducible in mouse xenografts of cultured and primary colon cancer cells. Full crypt-like organization was associated with low tumor grade and was an independent prognostic marker of better survival in a collection of 221 colorectal cancers. Our findings suggest that full activation of preserved intestinal morphogenetic programs in colon cancer requires in vivo growth environments. Furthermore, crypt-like architecture was linked with less aggressive tumor biology, and may be useful to improve current colon cancer grading schemes.     

Murine Cytomegalovirus m02 Gene Family Protects against Natural Killer Cell-Mediated Immune Surveillance

The murine cytomegalovirus m02 gene family encodes putative type I membrane glycoproteins named m02 through m16. A subset of these genes were fused to an epitope tag and cloned into an expression vector. In transfected and murine cytomegalovirus-infected cells, m02, m04, m05, m06, m07, m09, m10, and m12 localized to cytoplasmic structures near the nucleus, whereas m08 and m13 localized to a filamentous structure surrounding the nucleus. Substitution mutants lacking the m02 gene (SMsubm02) or the entire m02 gene family (SMsubm02-16) grew like their wild-type parent in cultured cells. However, whereas SMsubm02 was as pathogenic as the wild-type virus, SMsubm02-16 was markedly less virulent. SMsubm02-16 produced less infectious virus in most organs compared to wild-type virus in BALB/c and C57BL/6J mice, but it replicated to wild-type levels in the organs of immunodeficient gamma(c)/Rag2 mice, lacking multiple cell types including natural killer cells, and in C57BL/6J mice depleted of natural killer cells. These results argue that one or more members of the m02 gene family antagonize natural killer cell-mediated immune surveillance.     

Synthesis of N,N-Dialkylaniline-2′-deoxyuridine Conjugates for DNA-Mediated Electron Transfer Studies

Abstract

Syntheses of two analogs of deoxyuridine with N,N-dialkylaniline chromophores are reported. 5-[3-(N-methylphenylamino)propanoyl]-2′-deoxyuridine (1) and 5-[2-(4-N,N-dimethylaminophenyl)ethyl)]-2′-deoxyuridine (2) are prepared by palladium-mediated coupling. Preparation of 2 was facilitated by in situ transient O⁴-trimethylsilyl protection during alkynylation which suppressed secondary cyclization of the coupling adduct.     

Identification of protein side chains near the membrane-aqueous interface: a site-directed spin labeling study of KcsA.

This study presents an approach to identifying surface residues on membrane proteins that are exposed toward the membrane-aqueous interface. The method employs a lipid Ni(II) chelate that localizes the metal ion to a region near the membrane-aqueous interface. Lateral diffusion of the lipid chelate results in Heisenberg exchange (HE) with nitroxide side chains in the protein only if direct contact occurs between the paramagnetic species during a collision. Thus, HE serves as a signature for residues facing the bilayer in the neighborhood of the membrane-aqueous interface. To evaluate the method, 13 surface residues on the extracellular half of KcsA, a prokaryotic potassium channel of known structure, were examined for HE with the Ni(II) chelate. The HE rate between the two species is found to depend strongly on the vertical position of the nitroxide with respect to the membrane-aqueous interface. Nitroxides introduced near the interface experience relatively high HE rates, whereas nitroxides that are immersed in the bilayer interior or sterically sheltered from collision experience low or undetectable rates. The results indicate that residues near the interface can be identified on the basis of their high rates of collision with the headgroup region of the bilayer.     

Sampling for Explosives-Residues at Fort Greely,Alaska

Fort Greely, Alaska has an extensive complex of weapon training and testing areas located on lands withdrawn from the public domain under the Military Lands Withdrawal Act (PL106-65). The Army has pledged to implement a program to identify possible munitions contamination. Because of the large size (344,165,000 m²) of the high hazard impact areas, characterization of these constituents will be difficult. We used an authoritative sampling design to find locations most likely to contain explosives-residues on three impact areas. We focused our sampling on surface soils and collected multi-increment and discrete samples at locations of known firing events and from areas on the range that had craters, pieces of munitions, targets, or a designation as a firing point. In the two impact areas used primarily by the Army, RDX was the most frequently detected explosive. In the impact area that was also used by the Air Force, TNT was the most frequently detected explosive. Where detected, the explosives concentrations generally were low (<0.05 mg/kg) except in soils near low-order detonations, where the explosive-filler was in contact with the soil surface. These low-order detonations potentially can serve as localized sources for groundwater contamination if positioned in recharge areas.     

Quantifying Biomass Changes of Single CD8+ T Cells during Antigen Specific Cytotoxicity

Existing approaches that quantify cytotoxic T cell responses rely on bulk or surrogate measurements which impede the direct identification of single activated T cells of interest. Single cell microscopy or flow cytometry methodologies typically rely on fluorescent labeling, which limits applicability to primary cells such as human derived T lymphocytes. Here, we introduce a quantitative method to track single T lymphocyte mediated cytotoxic events within a mixed population of cells using live cell interferometry (LCI), a label-free microscopy technique that maintains cell viability. LCI quantifies the mass distribution within individual cells by measuring the phase shift caused by the interaction of light with intracellular biomass. Using LCI, we imaged cytotoxic T cells killing cognate target cells. In addition to a characteristic target cell mass decrease of 20–60% over 1–4 h following attack by a T cell, there was a significant 4-fold increase in T cell mass accumulation rate at the start of the cytotoxic event and a 2–3 fold increase in T cell mass relative to the mass of unresponsive T cells. Direct, label-free measurement of CD8+ T and target cell mass changes provides a kinetic, quantitative assessment of T cell activation and a relatively rapid approach to identify specific, activated patient-derived T cells for applications in cancer immunotherapy.     

Passive irrigation and functional morphology of crustacean burrows in a tropical mangrove swamp

Flushing measurements and a resin cast of a burrow inhabited by Sesarma messa and Alpheus cf macklay were taken from a Rhizophoraspp. forest. The burrow had 9 openings and occupied a swamp surface area of 0.64 m². Passive irrigation of the burrow was investigated by recording change in conductivity of burrow water in a chamber 45 cm below the swamp surface during tidal inundation of the swamp. The chamber was completely flushed within approximately one hour, i.e. by a single tidal event. Burrow morphology was determined by means of resin casting. The investigated burrow was of discrete structure, with an overall depth of 1.2 m and a total volume of 68 l, i.e. ca. 9% of the volume of swamp soil. The below ground surface area of chambers and tunnels was 3.8 m². The mean and maximum chamber/tunnel diameter was 7 cm and 11 cm respectively. The soil in the close vicinity of the burrow was extensively penetrated by roots, and any two parts of the burrow were located no further than 20 cm away from each other. By reducing diffusion distances within the soil and by being well flushed, the burrows provide an efficient mechanism for removal of excess salt accumulated in the soil around mangrove roots due to exclusion.     

Phosphorylation of rat heart glycogen synthase: studies in cardiomyocytes and in vitro phosphorylations with cAMP-dependent kinase and protein phosphatase-1

The phosphorylation of glycogen synthase has been studied in freshly isolated adult rat cardiomyocytes. Six peaks of 32P-labeled tryptic peptides are recovered via C-18 high performance liquid chromatography (HPLC) when synthase is immunoprecipitated from 32P-labeled cardiomyocytes and digested with trypsin. When epinephrine treated cells are used as a source of enzyme, the same HPLC profile is obtained with a dramatic enhancement of 32P recovered in two of the HPLC peaks. In vitro phosphorylation of rat heart synthase by cAMP-dependent protein kinase stimulates the conversion of synthase from the I to the D form and results in the recovery of the same tryptic peptides from the C-18 as is the case for synthase derived from cardiomyocytes. Treatment of cAMP-dependent kinase phosphorylated synthase with protein phosphatase-1 leads to a reactivation of the enzyme and a dephosphorylation of the same tryptic peptides that are selectively phosphorylated in epinephrine treated cardiomyocytes. These results are discussed in relation to hormonal control of glycogen metabolism in cardiac tissue.     

Regulation of cardiac cyclic GMP-dependent protein kinase

An assay method based on the ability of high concentrations of Mg²⁺ to stimulate phosphorylation of histone in the presence of low concentrations of ATP was developed for the measurement of cyclic GMP-dependent protein kinase activity ratios (activity -cyclic GMP/activity + cyclic GMP). In tissues which contain only trace amounts of cyclic GMP-dependent protein kinase, the basal activity ratios were high due to interference from a cyclic nucleotide-independent protein kinase. In order to study the regulation of the cardica cyclic GMP-dependent protein kinase, factors affecting the equilibrium between the active and inactive forms of the enzyme were determined. Since the rate of dissociation of cyclic GMP from its binding site(s) was relatively slow at 0–4°C at pH 7.0, the amount of time required to process tissue samples was the major limiting factor for preserving the equilibrium between active and inactive forms of the enzyme. Dilution of heart tissue extracts at 0–4°C did not significantly alter the activity ratio of the enzyme under conditions of basal or elevated cyclic GMP levels. Experiments using charcoal or exogenous cyclic GMP-dependent protein kinase in the homogenizing medium demonstrated that the release of sequestered cyclic GMP was not responsible for the elevation of the cyclic GMP-dependent protein kinase activity ratios by agents like acetylcholine. Therefore, the assay reflected in part, at least, the retention of kinase-bound cyclic GMP in the tissue extracts. The effects of acetylcholine and sodium nitroprusside on cyclic GMP levels, the cyclic GMP-dependent protein kinase activity ratios, and force of contraction were studied in the perfused rat heart. Both agents produced rapid, dose-dependent increases in cardiac cyclic GMP. Optimal concentrations of acetylcholine produced a 2–3-fold increase in the levels of cyclic GMP and an increase in the cyclic GMP-dependent protein kinase activity ratio. No significant effect of acetylcholine on cyclic nucleotide-independent protein kinase activity was observed. Associated witth the acetylcholine-induced protein kinase, factors affecting the equilibrium between the active and inactive forms of the enzyme were determined. Since the rate of dissociation of cyclic GMP from its binding site(s) was relatively slow at 0–4°C at pH 7.0, the amount of time required to process tissue samples was the major limiting factor for preserving the equilibrium between active and inactive forms of the enzyme. Dilution of heart tissue extracts at 0–4°C did not significantly alter the activity ratio of the enzyme under conditions of basal elevated cyclic GMP levels. Experiments using charcoal or exogenous cyclic GMP-dependent protein kinase in the homogenizing medium demonstrated that the release of sequestered cyclic GMP was not responsible for the elevation of the cyclic GMP-dependent protein kinase activity ratios by agents like acetylcholine. Therefore, the assay reflected in part, at least, the retention of kinase-bound cyclic GMP in the tissue extracts. The effects of acetylcholine and sodium nitroprusside on cyclic GMP levels, the cyclic GMP-dependent protein kinase activity ratios, and force of contraction were studied in the perfused rat heart. Both agents produced rapid, dose-dependent increases in cardiac cyclic GMP. Optimal concentrations of acetylcholine produced a 2–3-fold increase in the levels of cyclic GMP and an increase in the cyclic GMP-dependent protein kinase activity ratio. No significant effect of acetylcholine on cyclic nucleotide-independent protein kinase activity was observed. Associated with the acetylcholine-induced increase in cyclic GMP and the cyclic GMP-dependent protein kinase activity ratio was a reduction in the force of contraction. In contrast, nitroprusside produced little or no increase in the cyclic GMP-dependent protein kinase activity ratio despite increasing the level of cyclic GMP 8–10-fold. Nitroprusside also had no effect on contractile force. In combination, nitroprusside and acetylcholine produced additive effects on cyclic GMP levels, but protein kinase activation and force of contraction were similar to those seen with acetylcholine alone. The results suggest that the cyclic GMP produced by acetylcholine in the rat heart is coupled to activation of the cyclic GMP-dependent protein kinase, while that produced by nitroprusside is not.     

Organic-solvent water pollution and low birth weight in Michigan.

This pilot study explored the association between a measure of water pollution caused by benzene or chlorinated solvents and the incidence of low birth weights for white residents of Michigan counties. A positive relationship between water pollution by these contaminants and the per cent of low-weight births (less than 2,500 grams, or about 5.5 pounds) resulted despite controls for the incidence of teenaged childbearing, infrequent prenatal care, and mean household income from wages or salaries. Water pollution showed an association with the incidence of low birth weight that was as strong as that between low birth weight and low prenatal care. While correlation cannot prove causation, the finding suggests that impure water may impair fetal growth in Michigan.