Microsatellite mutation rates in cancer cell lines deficient or proficient in mismatch repair.

A selectable system has been used to determine mutation rates within a microsatellite sequence in human cancer cell lines with or without defects in mismatch repair. A sequence consisting of 17 repeats of poly (dC-dA).poly(dT-dG) [abbreviated as (Ca)17] was inserted near the 5' end of the bacterial neomycin-resistance gene in a plasmid vector, such that the reading frame of the neo gene is disrupted. This plasmid was introduced into cancer cell lines, where it became integrated into the cellular genome. Clones with insertions or deletions of CA-repeats that restored the normal reading frame of the neo gene were selected in G418, and mutation rates were determined by fluctuation analysis. The rates of reversion in LoVo cells, which are deficient for hMSH2, were about one in a thousand per generation, which is approximately two orders of magnitude higher than in the repair-proficient HT-1080 human fibrosarcoma cell line. The mutation rates in H6 cells, which are derived from the hMLH1-deficient HCT116 line, were more heterogeneous than in LoVo, but all were considerably higher than in the repair-proficient line. Nearly all of the revertants of the repair-deficient lines had deletions of a single CA-repeat from the microsatellite sequence, whereas repair-proficient cells had a broader spectrum of mutations.

Isolation of chicken primordial germ cells using fluorescence-activated cell sorting.

Presently, it is difficult to undertake germ line modification of the chicken with primordial germ cells (PGC) because it has been difficult to efficiently fractionate the PGC from the total somatic cell population. The objective of this study was to develop a method that allows isolation of an enriched population of viable PGC from embryonic blood and embryonic gonadal tissue. Blood was harvested from early chick embryos (stages 13 to 15), and cells were liberated from the gonads of stage 27 chick embryos. Subsequently, viable PGC were labeled with anti-stage-specific embryonic antigen-1 (SSEA-1), which was detected with goat-anti-mouse IgM-fluorescein isothiocyanate. Fluorescently labeled cells were sorted from the unlabeled cells using fluorescence-activated cell sorting (FACS), and the identities of the PGC were confirmed using periodic acid-Schiff (PAS) staining or anti-embryonic mouse antigen-1 (EMA-1) staining followed by microscopic evaluation. Finally, PGC were sorted from somatic cells of sex-identified embryos. Less than 0.1% of the blood cell population was collected as SSEA-1-positive cells. Similarly, approximately 2% of the gonadal cell population were collected as SSEA-1-positive cells. Therefore, fewer (-1,000 to 9,000) PGC were recovered from each isolate. Placing the sorted SSEA-1-positive cells on a glass slide from a microcentrifuge tube resulted in a recovery rate of 53 to 73% relative to the number detected by FACS. Furthermore, the proportions of sorted cells that stained with PAS or anti-EMA-1 following sorting were 92+/-4% PAS positive and 94+/-1% anti-EMA-1 positive. Finally, the sorted SSEA-1-positive cells were maintained in vitro to demonstrate their viability after sorting. It was demonstrated that it is possible to label blood and gonadal chicken PGC with SSEA-1 and subsequently to sort viable SSEA-1-positive PGC from somatic cells.

Ovine fascioliasis following reinfection.

Sheep were reinfected with matacercariae once. Half were treated with anthelmintic one week before reinfection. Compared with a primary infection, the reinfecting flukes in most seemed to migrate faster through the liver although in one treated sheep migration seemed to be longer and more destructive. Migrations extended into areas little affected by the primary infection thus producing more widespread fibrosis. Although a temporary retardation in fluke growth rate occurred, there was no reduction in the numbers of flukes recovered. Flukes killed by anthelmintic formed large granulomata.

Morphology and classification of subdivisions of the intrahepatic vascular and biliary systems in sheep.

Tensol casts and histology were used to demonstrate sub-divisions of the portal and hepatic veins, and arterial and biliary systems. Portal and hepatic venous subdivisions were most readily identified. Based upon size, location, sequence of branching and histological characteristics a nomenclature for the sub-divisions of the portal vascular system was proposed. The names of distributing, primary, terminal, secondary (long and short) and tertiary veins were adopted to identify portal veins branches. The same criteria allowed the identification of three hepatic vein branches, central, sublobular and hepatic; the central hepatic veins and to a much lesser extent the sublobular hepatic veins drained the sinusoids. Casts of the arterial system demonstrated the arterial blood supply to the biliary system, to the sinusoids and portal vein vasa vasorum but they were of little use in identifying subdivisions. Arterial subdivision identification is possible by using accompanying portal veins as morphological markers and designating the subdivision after the vein. This approach is also applicable to the biliary system and provides a more accurate means of subdivision identification than does the broader division into ductules, intermediate and large ducts.

Filaroides hirthi in a British bred beagle dog.

Filaroides hirthi was identified in large numbers in the lungs of a British bred beagle dog that had received 10 monthly intra-articular injections of a corticosteroid. The dog was terminally hyperpnoeic and eosinophil and reticulocyte counts were raised. At autopsy, the lungs failed to collapse and minute black and red foci were found throughout the lung parenchyma. These foci were identified histologically as parasites and focal haemorrhage. Migrating larvae were found in the liver and mesenteric lymph nodes. F hirthi has not previously been definitively identified in British dogs and the principal features of its identification and control are discussed.

Treatment of stormwater runoff from row crop farming in Ruskin, Florida.

A wet-detention pond, constructed to treat agricultural runoff from winter vegetables, was studied to document constituent concentrations, measure hydrology and analyze conditions in the treatment system. The efficiency of the pond to remove pollutants was affected by the unseasonable amount of rainfall induced by the El Niño phenomenon and the succeeding dry La Niña year. During the two years of study (50 rain events), about 90 per cent of all the pollutant loads for potentially toxic metals entered the pond during five El Niño storms; and since higher pollutant loads are often more easily reduced these conditions contributed to the greater per cent reduction of metals during 1998. Another condition which may have enhanced constituent reduction was made possible by the newly evacuated sediments, since clean soils provide more attachment sites for constituent removal. Annual data show pollutant load reductions for 1998 were greater than 90 per cent for most metals, but for 1999 reduction was about 60 per cent. In contrast, inorganic nutrient removal was better in 1999 (> 80 per cent) than during 1998 (to 70 per cent). Organic nitrogen had the poorest removal for both years (20 to 40 per cent). Total phosphorus levels were measured at high median concentrations (1 mg/L at the inflow) compared to other studies for stormwater runoff.

Intrahepatic vascular lesions in experimental and natural ovine fascioliasis.

Using acrylic resin casts prepared from the liver vasculature and histology, subdivisions of the portal and hepatic systems stenosed as a result of experimental and natural infections of F. hepatica were identified as terminal, secondary and tertiary portal veins and central and sublobular hepatic veins; primary portal veins were also involved in the experimentally infected livers. Fewer veins tended to be involved in livers naturally-infected and they were more evenly distributed among liver lobes than in the experimental disease where most were found in the ventral lobe. Casts of both types of infection also demonstrated enlargement and tortuosity of arteries in ventral lobes and those forming the peribiliary arterial plexus, as well as showing that multiple anastomotic channels had formed. The arterial changes and anastomoses were suggested as developing to compensate for the effects of vascular stenosis. Portal vein stenosis induced experimentally was the outcome of replacement of eosinophils and oedema-like fluid present in veins around fluke tracks and of the organisation of fluke tracts impinging upon veins. During the post-migratory period of infection, stenosis became more marked, for which no adequate cause was identified. In livers naturally-infected, in addition to stenosed portal and hepatic veins, vascular channels in collagen septa in sinusoids and a slight convolution of arteries were seen.

Pathology of ovine adenovirus type 4 infection in SPF lambs: pulmonary and hepatic lesions.

Seven lambs were challenged with an aerosol of OA4 virus. Four developed mild pulmonary oedema and a sparse accumulation of mononuclear cells around blood vessels similar to results reported previously. In addition, three had focal hepatic necrosis and one of these three had a non-suppurative occlusive cholangitis and portal tract lymphangitis and thrombosis. Basophilic intranuclear IB's were identified in many necrotic hepatocytes, in lymphatic endothelial cells involved in thrombi but in only one bile duct epithelial cell. Ultrastructurally, virions with the characteristics of adenovirus were seen in hepatocytes. Although only one IB was seen in the affected biliary system there was no evidence that it was caused by an agent other than OA4. Virus was considered to infect the respiratory system directly, be swallowed to infect the alimentary system and carried, possibly within cells, via a haematogeneous and/or lymphogenous route to the liver. The effect of naturally acquired virus on sheep is unknown but it is possible that it may predispose the host to more serious infections.

Effect of hypolipidaemic drugs on hepatomegaly and micro-bodies in the rat. A new approach to the nature of hepatic peroxisomal proliferation.

Etofylline clofibrate, a new hypolipidaemic drug of low toxicity, was evaluated for effects of induction of peroxisomal proliferation and hepatomegaly in male rats by comparison with standard hypolipidaemic drugs (bezafibrate, clofibrate, fenofibrate). The dose schedule was selected according to standard toxicological methods in an attempt to determine a dose-dependent relationship with particular reference to a possible "nil effect" dose. At high dose levels (250 mg/kg/day), all test drugs induced proliferation and hepatomegaly. At 50 mg/kg/day, all reference compounds induced proliferation and hepatomegaly while etofylline clofibrate indicated only moderate proliferation and no hepatomegaly. At very low dose levels only standards induced proliferation while etofylline clofibrate neither induced proliferation nor hepatomegaly. The latter was still present in bezafibrate and fenofibrate-treated groups. Results indicated a dose-dependent effect of etofylline clofibrate on both parameters with a "nil effect" dose between 11 and 50 mg/kg. Neither a dose-dependent nor a "nil effect" dose was found with any of the standard drugs. For the most potent peroxisomal proliferators (bezafibrate and clofibrate), numerous large and mis-shapen peroxisomes were found at all dose levels. Hepatic changes in terms of pharmacological and toxicological criteria are discussed with special reference to a proposed drug related toxic effect on liver function.

Structure of the nucleosome core particle at 7 A resolution.

The crystal structure of the nucleosome core particle has been solved to 7 A resolution. The right-handed B-DNA superhelix on the outside contains several sharp bends and makes numerous interactions with the histone octamer within. The central turn of superhelix and H3 . H4 tetramer have dyad symmetry, but the H2A . H2B dimers show departures due to interparticle associations.

A 145-base pair DNA sequence that positions itself precisely and asymmetrically on the nucleosome core.

A 145-bp DNA sequence, cloned from Escherichia coli, was reconstituted into nucleosome core particles by a number of methods. The behaviour of the resulting complex upon sucrose gradient sedimentation and nucleoprotein gel electrophoresis closely resembled that of control bulk nucleosome core particles. DNase I digestion of the 32P-end-labelled complex revealed the 10-bp periodicity of cleavages expected for DNA bound on a histone surface. The narrow cleavage sites observed (1 bp wide) imply that the sequence occupies a single preferred position on the nucleosome core, accurate to the level of single base pairs. By relating the digestion pattern observed to the pattern of site protection found for random sequence nucleosomes, the DNA position was found to be offset by 17 bp from that in the normal core particle. A number of experiments argue against the involvement of length or end effects and suggest that it is some feature of the DNA sequence itself that determines this precise positioning of DNA on the nucleosome.