Effects of grazing platform stocking rate on productivity and profitability of pasture-based dairying in a fragmented farm scenario.

The area adjacent to the milking parlor, accessible for grazing by lactating dairy cows (i.e., the grazing platform [GP]), can be limited on fragmented pasture-based dairy farms. Such farms, with a moderate overall farm stocking rate, typically have a much higher stocking rate of dairy cows on the GP. This study quantified the effects of farm fragmentation on milk and herbage production and profitability in a whole-farm systems-scale study over 3 yr (2017-2019). Four systems, each with an overall farm stocking rate of 2.5 cows/ha but with different grazing platform stocking rates (GPSR), were examined. The proportions of the overall farm area within the GP were 100%, 83%, 71%, and 63% in each of the 4 systems, respectively. Hence, the 4 systems had a GPSR of 2.5, 3.0, 3.5, and 4.0 cows/ha. The GP was used for grazing and silage (ensiled herbage) production, and the non-GP portion of each GPSR system was used solely for silage production. Concentrate supplementation per cow was the same across all GPSR systems; approximately 10% of the annual feed budget. All systems were compact spring-calving with 24 cows per system. We discovered a lower proportion of grazed herbage in the diet with higher GPSR. All silage produced on the non-GP areas was required to support higher GPSR on each of the systems. Annual herbage production and milk production per cow were not different between GPSR systems, resulting in similar milk production per hectare of the overall system area. The economic implications of different GPSR on fragmented farms were modeled in 2 scenarios: (1) quantifying the cost associated with different levels of farm area fragmentation; (2) investigating the optimum GPSR on fragmented pasture-based dairy farms, depending on variable criteria. A greater level of farm fragmentation lowered the profitability of pasture-based dairy production. Costs of production increased with higher GPSR and longer distances between GP and non-GP areas. At a fixed GP area, it was most profitable to increase GPSR up to 4 cows/ha on the GP when milk price was high, land rental price was low, and shorter distance existed between GP and non-GP areas.

Access time to pasture under wet soil conditions: Effects on productivity and profitability of pasture-based dairying.

A long grazing season improves the profitability of pasture-based dairy production. It can entail grazing under wet soil conditions and the risk of damaging swards. Housing cows either temporarily or completely while soil moisture is high can avoid damaging swards. An experiment with 4 grazing systems was conducted over 3 yr (Sep. 1, 2013, to Aug. 31, 2016). The purpose was to evaluate whether soil moisture measurements are an effective decision support to assess the risk of treading damage and effects on pasture productivity and dairy cow performance during wet soil conditions. Access time to pasture between February and December of each grazing season was dependent on volumetric soil moisture content (VSMC, m3/m3) measured each morning: Control = cows were housed at VSMC >0.5 and otherwise allowed 22 h/d access to pasture; S<7 = cows were housed on days with VSMC >0.7 and otherwise allowed 22 h/d access to pasture; S7-6 = cows were housed at VSMC >0.7 and allowed 8 h/d access to pasture at VSMC between 0.7 and 0.6 and 22 h/d access at VSMC ≤0.6; S7-5 = cows were housed at VSMC >0.7 and allowed 8 h/d access to pasture at VSMC between 0.7 and 0.5 and 22 h/d access at VSMC ≤0.5. Cows with 8-h access per day received no other feeding when housed. All herds were compact spring-calving, with a mean calving date of Feb. 19. Mean stocking rate was 2.57 cows/ha. Measurements of VSMC provided an objective indicator for the risk of treading damage. Less time spent at pasture under wet soil conditions lowered treading damage but had no effect on annual pasture production (mean 14.8 t of organic matter/ha). Annual milk solids production per cow was lowest for the control herd (485 kg) and not different between the other systems (503 kg). Reducing treading damage to swards did not improve productivity or profitability of the grazing systems. Nevertheless, measuring soil moisture was a useful decision support for assessing the risk of treading damage when turning cows out to pasture.

The effect of fertilizer nitrogen input to grass-clover swards and calving date on the productivity of pasture-based dairy production.

The objective of this systems-scale study was to investigate grazing season timeframes on pasture and milk production and on milk processability of dairy systems with compact spring-calving dairy cows grazing white clover (Trifolium repens L.) based grassland. Fifty-four primiparous and multiparous Holstein-Friesian dairy cows were used in a one-factor study with 3 systems (n = 18) and repeated over 2 yr (2008/09 and 2009/10). The 3 systems were: early spring calving with annual fertilizer N input of 100 kg·ha-1 applied in spring (ES100N; 2.1 cows·ha-1; grazing February to November), early spring calving without fertilizer N (ES0N; 1.6 cows·ha-1; grazing February to November) and late spring calving without fertilizer N (LS0N; 1.53 cows·ha-1; grazing April to January). Annual pasture production was affected by an interaction between grazing system and year: Mean annual pasture yields for 2008 and 2009 were ES100N; 10.35 and 9.88, ES0N; 8.88 and 8.63, LS0N; 9.18 and 10.31 t of dry matter (DM)·ha-1 (SEM 0.39). LS0N had higher pasture DM yield in 2009 due to higher clover DM production and biological N fixation compared with the other systems. Clover stolon and root mass in the following February was correlated with stolon and root mass in the previous November with 64% of stolon mass present on LS0N in February (R2 = 0.84). There were no detectable differences in per-lactation milk yield (6,335 kg·cow-1), fat, protein and lactose yields (271, 226, 297 kg·cow-1, respectively), cow liveweight (585 kg) or body condition score (3.02). Although winter grazing favored subsequent clover DM production, biological N fixation and pasture DM production, delaying calving date in spring and extending lactation into the following winter led to inefficient use of this pasture by the grazing herd and lowered the quality of late-lactation milk for processing purposes. Hence, a mean calving date in mid- to late-February is recommended for zero-fertilizer N input clover-based grassland.

Anticitrullinated protein antibodies and rheumatoid factor are associated with increased mortality but with different causes of death in patients with rheumatoid arthritis: a longitudinal study in three European cohorts.

OBJECTIVE: Patients with rheumatoid arthritis (RA)-related autoantibodies have an increased mortality rate. Different autoantibodies are frequently co-occurring and it is unclear which autoantibodies associate with increased mortality. In addition, association with different causes of death is thus far unexplored. Both questions were addressed in three early RA populations. METHODS: 2331 patients with early RA included in Better Anti-Rheumatic Farmaco-Therapy cohort (BARFOT) (n=805), Norfolk Arthritis Register (NOAR) (n=678) and Leiden Early Arthritis Clinic cohort (EAC) (n=848) were studied. The presence of anticitrullinated protein antibodies (ACPA), rheumatoid factor (RF) and anticarbamylated protein (anti-CarP) antibodies was studied in relation to all-cause and cause-specific mortality, obtained from national death registers. Cox proportional hazards regression models (adjusted for age, sex, smoking and inclusion year) were constructed per cohort; data were combined in inverse-weighted meta-analyses. RESULTS: During 26 300 person-years of observation, 29% of BARFOT patients, 30% of NOAR and 18% of EAC patients died, corresponding to mortality rates of 24.9, 21.0 and 20.8 per 1000 person-years. The HR for all-cause mortality (95% CI) was 1.48 (1.22 to 1.79) for ACPA, 1.47 (1.22 to 1.78) for RF and 1.33 (1.11 to 1.60) for anti-CarP. When including all three antibodies in one model, RF was associated with all-cause mortality independent of other autoantibodies, HR 1.30 (1.04 to 1.63). When subsequently stratifying for death cause, ACPA positivity associated with increased cardiovascular death, HR 1.52 (1.04 to 2.21), and RF with increased neoplasm-related death, HR 1.64 (1.02 to 2.62), and respiratory disease-related death, HR 1.71 (1.01 to 2.88). CONCLUSIONS: The presence of RF in patients with RA associates with an increased overall mortality rate. Cause-specific mortality rates differed between autoantibodies: ACPA associates with increased cardiovascular death and RF with death related to neoplasm and respiratory disease.

The effect of target postgrazing height on sward clover content, herbage yield, and dairy production from grass-white clover pasture.

White clover (Trifolium repens) is an important legume for grazed grassland that can increase the profitability and environmental sustainability of milk production. Previous experiments on mown grass-clover plots suggest that low postgrazing heights (PGH) can increase sward clover content and herbage production. However, this has not been tested in actual strip or rotational grazing systems with dairy cows. Furthermore, lowering PGH in grass-only swards (typically perennial ryegrass without white clover) has previously been associated with reduced milk yields per cow. The objective of this experiment was to investigate the effect of PGH by dairy cows on clover content, herbage production, and milk production from strip-grazed grass-white clover swards in Ireland. Three target PGH treatments of 4, 5, and 6 cm were in place for entire grazing seasons (February to November) for 3 consecutive years (2007 to 2009). Each treatment had a mean of 21 Holstein-Friesian dairy cows that strip-grazed a mean annual area of 10.2 ha. Postgrazing height was measured twice a day with a rising plate meter, and cows were moved to the next strip once the target PGH was reached. Annual fertilizer nitrogen input was 90 kg of N/ha for each treatment. The PGH treatment did not significantly affect annual milk yield (6,202 kg/cow), solids-corrected milk yield (6,148 kg/cow), fat, protein, or lactose yields (265, 222, and 289 kg/cow, respectively), cow liveweight (592 kg) or body condition score (3.01). The PGH treatment also had no significant effect on sward white clover content (196 g/kg). However, herbage production of both grass and clover were significantly higher with the 4-cm PGH treatment compared with the 6-cm treatment. Mean annual herbage yields were 11.1, 10.2, and 9.1 t of organic matter (OM)/ha for the 4-, 5-, and 6-cm PGH treatments, respectively. The lower herbage production in the 6-cm PGH treatment resulted in lower annual silage production, greater housing requirements, and a substantially higher net silage deficit (-1,917 kg of OM/cow) compared with the 5- or 4-cm treatments (-868 and -192 kg of OM/cow, respectively). Grazing to a PGH of 4 cm is therefore recommended for grass-white clover swards.

Life cycle assessment of milk production from commercial dairy farms: the influence of management tactics.

Little consideration has been given to how farm management, specifically tactics used to implement the management strategy, may influence the carbon footprint (CF) and land use for milk produced on commercial farms. In this study, the CF and land use of milk production from 18 Irish commercial dairy farms were analyzed based on foreground data from a 12-mo survey capturing management tactics and background data from the literature. Large variation was found in farm attributes and management tactics; for example, up to a 1.5-fold difference in fertilizer nitrogen input was used to support the same stocking density, and up to a 3.5-fold difference in concentrate fed for similar milk output per cow. However, the coefficient of variation for milk CF between farms only varied by 13% and for land use by 18%. The overall CF and overall land use of the milk production from the 18 dairy farms was 1.23±0.04kg of CO2 Eq and 1.22±0.05 m(2) per kilogram of energy-corrected milk. Milk output per cow, economic allocation between exports of milk and liveweight, and on-farm diesel use per ha were found to be influential factors on milk CF, whereas the fertilizer N rate, milk output per cow, and economic allocation between exports of milk and liveweight were influential on land use. Effective sward management of white clover within a few farms appeared to lower the CF but increased on-farm land use. It was concluded that a combination of multiple tactics determines CF and land use for milk production on commercial dairy farms and, although these 2 measures of environmental impact are correlated, a farm with a low CF did not always have low land use and vice versa.

The carbon footprint of pasture-based milk production: can white clover make a difference?

Carbon footprint (CF) calculated by life cycle assessment (LCA) was used to compare greenhouse gas emissions from pasture-based milk production relying mainly on (1) fertilizer N (FN), or (2) white clover (WC). Data were sourced from studies conducted at Solohead Research Farm in Ireland between 2001 and 2006. Ten FN pastures stocked between 2.0 and 2.5 livestock units (LU)/ha with fertilizer N input between 180 and 353 kg/ha were compared with 6 WC pastures stocked between 1.75 and 2.2 LU/ha with fertilizer N input between 80 and 99 kg/ha. The WC-based system had 11 to 23% lower CF compared with FN (average CF was 0.86 to 0.87 and 0.97 to 1.13 kg of CO(2)-eq/kg of energy-corrected milk, respectively, 91% economic allocation). Emissions of both N(2)O and CO(2) were lower in WC, whereas emissions of CH(4) (per kg of energy-corrected milk) were similar in both systems. Ratio sensitivity analysis indicated that the difference was not caused by error due to modeling assumptions. Replacing fertilizer N by biological nitrogen fixation could lower the CF of pasture-based milk production.

Energy demand on dairy farms in Ireland.

Reducing electricity consumption in Irish milk production is a topical issue for 2 reasons. First, the introduction of a dynamic electricity pricing system, with peak and off-peak prices, will be a reality for 80% of electricity consumers by 2020. The proposed pricing schedule intends to discourage energy consumption during peak periods (i.e., when electricity demand on the national grid is high) and to incentivize energy consumption during off-peak periods. If farmers, for example, carry out their evening milking during the peak period, energy costs may increase, which would affect farm profitability. Second, electricity consumption is identified in contributing to about 25% of energy use along the life cycle of pasture-based milk. The objectives of this study, therefore, were to document electricity use per kilogram of milk sold and to identify strategies that reduce its overall use while maximizing its use in off-peak periods (currently from 0000 to 0900 h). We assessed, therefore, average daily and seasonal trends in electricity consumption on 22 Irish dairy farms, through detailed auditing of electricity-consuming processes. To determine the potential of identified strategies to save energy, we also assessed total energy use of Irish milk, which is the sum of the direct (i.e., energy use on farm) and indirect energy use (i.e., energy needed to produce farm inputs). On average, a total of 31.73 MJ was required to produce 1 kg of milk solids, of which 20% was direct and 80% was indirect energy use. Electricity accounted for 60% of the direct energy use, and mainly resulted from milk cooling (31%), water heating (23%), and milking (20%). Analysis of trends in electricity consumption revealed that 62% of daily electricity was used at peak periods. Electricity use on Irish dairy farms, therefore, is substantial and centered around milk harvesting. To improve the competitiveness of milk production in a dynamic electricity pricing environment, therefore, management changes and technologies are required that decouple energy use during milking processes from peak periods.

Examining area-level variation in service organisation and delivery across the breadth of primary healthcare. Usefulness of measures constructed from routine data.

BACKGROUND: Australia has a universal healthcare system, yet organisation and delivery of primary healthcare (PHC) services varies across local areas. Understanding the nature and extent of this variation is essential to improve quality of care and health equity, but this has been hampered by a lack of suitable measures across the breadth of effective PHC systems. Using a suite of measures constructed at the area-level, this study explored their application in assessing area-level variation in PHC organisation and delivery. METHODS: Routinely collected data from New South Wales, Australia were used to construct 13 small area-level measures of PHC service organisation and delivery that best approximated access (availability, affordability, accommodation) comprehensiveness and coordination. Regression analyses and pairwise Pearson's correlations were used to examine variation by area, and by remoteness and area disadvantage. RESULTS: PHC service delivery varied geographically at the small-area level-within cities and more remote locations. Areas in major cities were more accessible (all measures), while in remote areas, services were more comprehensive and coordinated. In disadvantaged areas of major cities, there were fewer GPs (most disadvantaged quintile 0.9[SD 0.1] vs least 1.0[SD 0.2]), services were more affordable (97.4%[1.6] bulk-billed vs 75.7[11.3]), a greater proportion were after-hours (10.3%[3.0] vs 6.2[2.9]) and for chronic disease care (28%[3.4] vs 17.6[8.0]) but fewer for preventive care (50.7%[3.8] had cervical screening vs 62.5[4.9]). Patterns were similar in regional locations, other than disadvantaged areas had less after-hours care (1.3%[0.7] vs 6.1%[3.9]). Measures were positively correlated, except GP supply and affordability in major cities (-0.41, p < .01). IMPLICATIONS: Application of constructed measures revealed inequity in PHC service delivery amenable to policy intervention. Initiatives should consider the maldistribution of GPs not only by remoteness but also by area disadvantage. Avenues for improvement in disadvantaged areas include preventative care across all regions and after-hours care in regional locations.

Symptoms in first-degree relatives of patients with rheumatoid arthritis: evaluation of cross-sectional data from the symptoms in persons at risk of rheumatoid arthritis (SPARRA) questionnaire in the PRe-clinical EValuation of Novel Targets in RA (PREVeNT-RA) Cohort.

BACKGROUND: First-degree relatives (FDRs) of people with rheumatoid arthritis (RA) have a fourfold increased risk of developing RA. The Symptoms in Persons At Risk of Rheumatoid Arthritis (SPARRA) questionnaire was developed to document symptoms in persons at risk of RA. The aims of this study were (1) to describe symptoms in a cohort of FDRs of patients with RA overall and stratified by seropositivity and elevated CRP and (2) to determine if patient characteristics were associated with symptoms suggestive of RA. METHODS: A cross-sectional study of FDRs of patients with RA, in the PREVeNT-RA study, who completed a study questionnaire, provided a blood sample measured for rheumatoid factor, anti-CCP and CRP and completed the SPARRA questionnaire. Moderate/severe symptoms and symmetrical, small and large joint pain were identified and described. Symptoms associated with both seropositivity and elevated CRP were considered suggestive of RA. Logistic regression was used to determine if symptoms suggestive of RA were associated with patient characteristics. RESULTS: Eight hundred seventy participants provided all data, 43 (5%) were seropositive and 122 (14%) had elevated CRP. The most frequently reported symptoms were sleep disturbances (20.3%) and joint pain (17.9%). Symmetrical and small joint pain were 11.3% and 12.8% higher, respectively, in those who were seropositive and 11.5% and 10.7% higher in those with elevated CRP. In the logistic regression model, seropositivity, older age and feeling depressed were associated with increased odds of small and symmetrical joint pain. CONCLUSIONS: This is the first time the SPARRA questionnaire has been applied in FDRs of patients with RA and has demonstrated that the presence of symmetrical and small joint pain in this group may be useful in identifying people at higher risk of developing RA.

Human gut microbial communities dictate efficacy of anti-PD-1 therapy in a humanized microbiome mouse model of glioma.

BACKGROUND: Although immunotherapy works well in glioblastoma (GBM) preclinical mouse models, the therapy has not demonstrated efficacy in humans. To address this anomaly, we developed a novel humanized microbiome (HuM) model to study the response to immunotherapy in a preclinical mouse model of GBM. METHODS: We used 5 healthy human donors for fecal transplantation of gnotobiotic mice. After the transplanted microbiomes stabilized, the mice were bred to generate 5 independent humanized mouse lines (HuM1-HuM5). RESULTS: Analysis of shotgun metagenomic sequencing data from fecal samples revealed a unique microbiome with significant differences in diversity and microbial composition among HuM1-HuM5 lines. All HuM mouse lines were susceptible to GBM transplantation, and exhibited similar median survival ranging from 19 to 26 days. Interestingly, we found that HuM lines responded differently to the immune checkpoint inhibitor anti-PD-1. Specifically, we demonstrate that HuM1, HuM4, and HuM5 mice are nonresponders to anti-PD-1, while HuM2 and HuM3 mice are responsive to anti-PD-1 and displayed significantly increased survival compared to isotype controls. Bray-Curtis cluster analysis of the 5 HuM gut microbial communities revealed that responders HuM2 and HuM3 were closely related, and detailed taxonomic comparison analysis revealed that Bacteroides cellulosilyticus was commonly found in HuM2 and HuM3 with high abundances. CONCLUSIONS: The results of our study establish the utility of humanized microbiome mice as avatars to delineate features of the host interaction with gut microbial communities needed for effective immunotherapy against GBM.

Using geospatial methods to measure the risk of environmental persistence of avian influenza virus in South Carolina.

Avian influenza (AIV) is a highly contagious virus that can infect both wild birds and domestic poultry. This study aimed to define areas within the state of South Carolina (SC) at heightened risk for environmental persistence of AIV using geospatial methods. Environmental factors known to influence AIV survival were identified through the published literature and using a multi-criteria decision analysis with GIS was performed. Risk was defined using five categories following the World Organization for Animal Health Risk Assessment Guidelines. Less than 1% of 1km grid cells in SC showed a high risk of AIV persistence. Approximately 2% - 17% of counties with high or very high environmental risk also had medium to very high numbers of commercial poultry operations. Results can be used to improve surveillance activities and to inform biosecurity practices and emergency preparedness efforts.

Analysis of breeding and pathology helps refine management practices of a large-scale N'-ethyl-N'-nitrosourea mouse mutagenesis programme.

N'-ethyl-N'-nitrosourea (ENU) is a powerful germline mutagen used in conjunction with phenotype-driven screens to generate novel mouse mutants. ENU also induces genetic lesions in somatic cells and dosage requires optimization between maximum germline mutation rate versus induced sterility and tumourigenesis that compromise the welfare and fecundity of the ENU-treated males. Here, we present our experience with BALB/cAnNCrl and C57BL/6J mice in terms of the pathology induced by ENU and its impact on breeding. In both mouse strains, morbidity and mortality rises with ENU dose. In more than 75% of C57BL/6J males, morbidity and mortality were attributable to the development of malignant T-lymphoblastic lymphoma. Approximately 50% of ENU-treated BALB/cAnNCrl males develop early malignant T-lymphoblastic lymphoma, but the cohort that survives develops late-onset lung carcinoma. Within strains, the latency of these clinically important tumour(s) was not dosage-dependent, but the proportion of mice developing tumours and consequently removed from the breeding programme increased with ENU dosage. The median number of offspring per ENU-treated C57BL/6J male in standard matings with C3H/HeH females decreased with increasing dosage. The two most important underlying causes for lower male fecundity were increased infertility in the highest dosage group and reduced numbers of litters born to the remaining fertile C57BL/6J males due to a higher incidence of morbidity. These findings have allowed us to refine breeding strategy. To maximize the number of offspring from each ENU-treated male, we now rotate productive males between two cages to expose them to more females. This optimizes the number of mutation carrying offspring while reducing the number of ENU-treated males that must be generated.

Phosphorus and nitrogen losses from temperate permanent grassland on clay-loam soil after the installation of artificial mole and gravel mole drainage.

Mole (M) and gravel-mole (GM) drainage systems improve the permeability of soils with high clay contents. They collect and carry away infiltrating water during episodic rainfall events. Characterisation of nutrient fluxes (concentration and flows) in overland flow (OF) and in mole drain flow (MF) across sequential rainfall events is important for environmental assessment of such drainage systems. The objective of this study is to assess the impact of drainage systems on soil nutrient losses. Three treatments were imposed on grazed permanent grassland on a clay loam soil in Ireland (52°30'N, 08°12'W) slope 1.48%: undrained control (C), mole drainage (M) and gravel mole drainage (GM). Plots (100 m × 15 m) were arranged in a randomized complete block design with four replicated blocks. Nitrogen (N) and phosphorus (P) concentrations in OF, MF and groundwater (GW) were measured from each plot over 15 consecutive rainfall events. The results showed that M and GM (P < 0.05) deepened the watertable depth and decreased OF. M and GM increased losses of nitrate-N (22%) and ammonium-N (14%) in GW. Nitrate-N concentrations from all the flow pathways (mean and standard error (s.e.): 0.99 s.e. 0.10 mg L-1) were well below the 11.3 mg L-1 threshold for drinking water. Ammonium-N concentrations from all the flow pathways (mean: 0.64 s.e. 0.14 mg L-1) exceeded drinking water quality standards. On the other hand M and GM lowered total P losses (mean annual losses from C, M and GM: 918, 755 and 853 s.e. 14.1 g ha-1 year-1) by enhancing soil P sorption. Hence M and GM can be implemented on farms under similar management to that described in the present study with a minor impact on N (increased concentration on averaged 18% to GW) and P (reduced by on avenged 114 g ha-1 year-1).

N-methyl-N-nitrosourea-induced rat mammary tumors. Hormone responsiveness but lack of spontaneous metastasis.

N-Methyl-N-nitrosourea (MNU) given iv to rats 50-55 days old induced mammary tumors in 70% of F344/N and 91% W/ICRF inbred females with mean latency periods of 149 and 93 days, respectively. Reduction of the MNU dose did not affect tumor incidence in W/ICRF rats. Of the mammary tumors, 98% were classified histologically as adenocarcinomas, which grew progressively. Primary tumors of nonmammary origin were detected at low incidence. Upon histologic examination, no evidence was found for metastases of either the mammary or other primary tumors. No evidence for tumor-induced hypercalcemia was found. Oophorectomy at the time of MNU administration prevented tumor development; oophorectomy when at least 1 tumor/animal was palpable caused growth delay or regression. All MNU-induced and 7,12-dimethylbenz[a]anthracene-induced mammary tumors tested contained cytoplasmic estrogen receptor (ER) at similar concentrations and were indistinguishable histologically. MNU-induced tumors in F344 rats were transplantable and retained ER through three transplantations.

Biochemical and cellular pharmacology of 1843U89, a novel benzoquinazoline inhibitor of thymidylate synthase.

Studies on a series of benzoquinazoline folate analogues as inhibitors of human thymidylate synthase led to the selection of 1843U89 for further evaluation. This compound had a Ki of 90 pM versus human thymidylate synthase and was noncompetitive with (6R,S)-5,10-methylenetetrahydrofolate. It was a good substrate for the addition of the second glutamate by hog liver folylpolyglutamate synthetase, having a Vmax/Km value 7.8-fold higher than (6R,S)-tetrahydrofolate. The data indicate that 1843U89 was transported into cells via the reduced folate carrier. The Kt for 1843U89 in MOLT-4 cells was 0.33 microM, which was 3-fold lower than that for methotrexate and 16-fold lower than that for (6S-5-formyltetrahydrofolate. V/K values were 20.3 for 1843U89 versus 1.2 and 1.9 for methotrexate and (6S)-5-formyltetrahydrofolate, respectively. It was a potent inhibitor of the growth of human cells, having 50% inhibitory concentrations below 1 nM for all cell lines tested. Growth inhibition was reversed by thymidine alone, indicating that thymidylate synthase was the only site of action of this compound. Growth inhibition was not affected by (6R,S-5-formyltetrahydrofolate at concentrations below 5 microM. However, the 50% inhibitory concentration increased when the concentration in the medium was increased to 100 microM, presumably due to competition for transport. Relative to the human cell lines used, murine cell lines were 80-1300-fold less sensitive to 1843U89 and the other benzoquinazolines tested. This decreased sensitivity appeared to be due, at least in part, to decreased transport or accumulation in murine cells. Ki values for inhibition of methotrexate transport for the benzoquinazolines were 5-17-fold higher in L1210 cells than in MOLT-4 cells. 1843U89, the benzoquinazoline which was transported most efficiently and which was the most potent inhibitor of the growth of human cells, exhibited the largest difference between binding to the MOLT-4 human and L1210 murine transporter. The V/K for L1210 transport was 80-fold less than that for MOLT-4. Initial antitumor studies, using the human thymidine kinase-deficient line GC3TK- to circumvent problems associated with murine transport as well as the high circulating thymidine levels in mice, indicated that 1843U89 had marked in vivo antitumor activity.

Sequence organisation in nuclear DNA from Physarum polycephalum. Arrangement of highly-repeated sequences.

Recombinant plasmids containing highly repetitive Physarum DNA segments were identified by colony hybridisation using a radioactively-labelled total Physarum DNA probe. A large number of these clones also hybridised to a foldback DNA probe purified from Physarum nuclear DNA. The foldback DNA probe was characterised by reassociation kinetic analysis. About one-half of this component was shown to consist of highly repeated sequences with a kinetic complexity of 1100 bp and an average repetition frequency of 5200. Direct screening of 67 recombinant plasmids for foldback sequences using the electron microscope revealed that about one-half were located in segments of DNA containing highly repetitive sequences; the remainder were present in clones containing low-copy number repeated elements. Analysis of two DNA clones showed that they contained repetitive elements located in over half of all DNA segments containing highly repetitive DNA and that the foci containing these highly repetitive sequences had different sequence arrangements. The results are consistent with the hypothesis that the most highly repeated DNA sequence families in the Physarum genome are few in number and are clustered together in different arrangements in about one-sixth of the genome. Over one-half of the foldback DNA complement in the Physarum genome is derived from these segments of DNA.