Development of an absolute gas-counting capability for low to medium activities.

Pacific Northwest National Laboratory (PNNL) is developing a capability to measure the absolute activity concentration of gaseous radionuclides using length-compensated proportional-counting. This capability will enable the validation of low-level calibration standards for use in PNNL's new shallow underground laboratory. Two sets of unequal length proportional counters have been fabricated; one set has been fabricated using ultra-low background (ULB) electroformed copper and a second set fabricated from Oxygen-Free High-Conductivity Copper (OFHC).

A new shallow underground gas-proportional counting lab--first results and Ar-37 sensitivity.

A new ultra-low-background proportional counter was recently developed with an internal volume of 100 cm(3) and has been characterized at pressures from 1-10 atm with P-10 (90% Ar, 10% methane) gas. This design, along with a counting system providing event digitization and passive and active shielding, has been developed to complement a new shallow underground laboratory (30 m water-equivalent). Backgrounds and low-level reference materials have been measured, and system sensitivity for (37)Ar has been calculated.

A shallow underground laboratory for low-background radiation measurements and materials development.

Pacific Northwest National Laboratory recently commissioned a new shallow underground laboratory, located at a depth of approximately 30 meters-water-equivalent. This new addition to the small class of radiation measurement laboratories located at modest underground depths houses the latest generation of custom-made, high-efficiency, low-background gamma-ray spectrometers and gas proportional counters. This paper describes the unique capabilities present in the shallow underground laboratory; these include large-scale ultra-pure materials production and a suite of radiation detection systems. Reported data characterize the degree of background reduction achieved through a combination of underground location, graded shielding, and rejection of cosmic-ray events. We conclude by presenting measurement targets and future opportunities.

Toward a biologically based dose-response model for developmental toxicity of 5-fluorouracil in the rat: acquisition of experimental data.

Biologically based dose-response (BBDR) models represent an emerging approach to improving the current practice of human health-risk assessment. The concept of BBDR modeling is to incorporate mechanistic information about a chemical that is relevant to the expression of its toxicity into descriptive mathematical terms, thereby providing a quantitative model that will enhance the ability for low-dose and cross-species extrapolation. Construction of a BBDR model for developmental toxicity is particularly complicated by the multitude of possible mechanisms. Thus, a few model assumptions were made. The current study illustrates the processes involved in selecting the relevant information for BBDR modeling, using an established developmental toxicant, 5-fluorouracil (5-FU), as a prototypic example. The primary BBDR model for 5-FU is based on inhibition of thymidylate synthetase (TS) and resultant changes in nucleotide pools, DNA synthesis, cell-cycle progression, and somatic growth. A single subcutaneous injection of 5-FU at doses ranging from 1 to 40 mg/kg was given to pregnant Sprague-Dawley rats at gestational day 14; controls received saline. 5-FU was absorbed rapidly into the maternal circulation, and AUC estimates were linear with administered doses. We found metabolites of 5-FU directly incorporated into embryonic nucleic acids, although the levels of incorporation were low and lacked correlation with administered doses. On the other hand, 5-FU produced dose-dependent inhibition of thymidylate synthetase in the whole embryo, and recovery from enzyme inhibition was also related to the administered dose. As a consequence of TS inhibition, embryonic dTTP and dGTP were markedly reduced, while dCTP was profoundly elevated, perhaps due to feedback regulation of intracellular nucleotide pools. The total contents of embryonic macromolecules (DNA and protein) were also reduced, most notably at the high doses. Correspondingly, dose-related reductions of fetal weight were seen as early as GD 15, and these deficits persisted for the remainder of gestation. These detailed dose-response parameters involved in the expression of 5-FU developmental toxicity were incorporated into mathematical terms for BBDR modeling. Such quantitative models should be instrumental to the improvement of high-to-low dose and cross-species extrapolation in health-risk assessment.

The effect of follicle stimulating hormone and epidermal growth factor on the developmental capacity of in-vitro matured mouse oocytes.

This study investigates the effects of follicle stimulating hormone (FSH) or epidermal growth factor (EGF) on the development of mouse oocytes matured in vitro. The data show that addition of FSH or EGF does not significantly increase the proportion of oocytes maturing to metaphase II but does increase the ability of these oocytes to cleave to the 2-cell stage after fertilization. After transfer of 2-cell embryos to pseudopregnant recipients, 64-78% of the embryos implanted, which was significantly reduced compared to embryos from in-vivo matured controls (89%). Fewer fetuses at day 14 of gestation were produced from embryos derived from oocytes matured in basal conditions (26%), or in the presence of EGF (32%), compared to oocytes matured in vivo (64%) or in the presence of FSH (58%). Examination of polar bodies and pronuclei of oocytes matured in vitro suggests that an increase in the rate of triploidy may be partly responsible for the increased fetal loss after maturation in the absence of FSH. This study shows that the fertilization rate after in-vitro maturation can be improved by FSH and EGF and that subsequent embryonic development is improved specifically by FSH.

Repetitive sperm-induced Ca2+ transients in mouse oocytes are cell cycle dependent.

Mature mouse oocytes are arrested at metaphase of the second meiotic division. Completion of meiosis and a block to polyspermy is caused by a series of repetitive Ca2+ transients triggered by the sperm at fertilization. These Ca2+ transients have been widely reported to last for a number of hours but when, or why, they cease is not known. Here we show that Ca2+ transients cease during entry into interphase, at the time when pronuclei are forming. In fertilized oocytes arrested at metaphase using colcemid, Ca2+ transients continued for as long as measurements were made, up to 18 hours after fertilization. Therefore sperm is able to induce Ca2+ transients during metaphase but not during interphase. In addition metaphase II oocytes, but not pronuclear stage 1-cell embryos showed highly repetitive Ca2+ oscillations in response to microinjection of inositol trisphosphate. This was explored further by treating in vitro maturing oocytes at metaphase I for 4-5 hours with cycloheximide, which induced nuclear progression to interphase (nucleus formation) and subsequent re-entry to metaphase (nuclear envelope breakdown). Fertilization of cycloheximide-treated oocytes revealed that continuous Ca2+ oscillations in response to sperm were observed after nuclear envelope breakdown but not during interphase. However interphase oocytes were able to generate Ca2+ transients in response to thimerosal. This data suggests that the ability of the sperm to trigger repetitive Ca2+ transients in oocytes is modulated in a cell cycle-dependent manner.

Cryopreservation of immature mouse oocytes.

Mouse oocytes enclosed in cumulus cells were isolated from antral follicles at the germinal vesicle (GV) stage. They were stored in straws at -196 degrees C by a conventional mouse embryo freezing method using dimethylsulphoxide (1.5 M) as the cryoprotectant. Overall survival assessed after removal of the cumulus cells was 93% (299/320). A significantly greater proportion of fresh oocytes remained arrested at the GV stage during culture (11 versus 1%), but the rate of maturation to metaphase II was not significantly different between frozen and fresh oocytes (83 versus 74%). The rate of fertilization in vitro was similar for frozen and fresh oocytes matured in vitro (70 versus 81%) but significantly less than with mature ovulated oocytes (96%). Fertilization of frozen and fresh oocytes arrested after germinal vesicle breakdown was similar (77 versus 95%). No evidence of parthenogenetic activation was found in the different groups after overnight incubation of metaphase II oocytes. Implantation was similar for embryos derived from fresh and frozen GV-stage oocytes matured in vitro and mature ovulated oocytes, but the loss of embryos after implantation was significantly higher in the in-vitro matured groups (frozen, 40% and fresh, 46% versus 24%). The overall survival of oocytes frozen at the GV stage was 27%. This compares favourably with the estimated overall survival of mature oocytes cryopreserved by a similar procedure. We conclude that the increased post-implantation loss is due to suboptimal conditions for maturation in vitro rather than freezing injury.

A comparison of 1- and 2-cell ova production by F2 50% Meishan versus F1 White line gilts.

The objective of this study was to compare recovery of pronuclear and 2-cell ova from F2 50% Meishan (MX) gilts versus F1 White line (L42) gilts. Sexually mature MX and L42 gilts were allocated across 2 treatments: Super (MX:n=9; L42:n=10) and Control (MX:n=6; L42:n=5) in a 2 x 2 factorial experiment. Allyl trenbolone (AT) was used to synchronize estrus in all gilts. Super gilts were given pregnant mare serum gonadotropin (PMSG: 1250 IU) at 24 h after AT withdrawal. Eighty-five hours after PMSG administration, all Super gilts received 750 IU of human chorionic gonadotropin (hCG). Super gilts which exhibited estrus within 24 h of hCG administration (MX-Super: n=6; L42-Super: n=5) and all Control gilts were bred naturally to Line 3 boars at 12 and 24 hours after the onset of estrus. Ova were recovered from Super gilts between 60 and 64 h after hCG and Control gilts at 48 h after the onset of estrus. All 1- and 2-cell ova were centrifuged at 15000 x g and observed using differential interference contrast microscopy. The mean ovulation rate was greater (P<0.05) for both MX-Super and L42-Super gilts in comparison to their respective Control groups. No differences were detected in the mean ovulation rate (P>0.38) or the mean number of 1- and 2-cell ova recovered (P>0.50) between MX-Super and L42-Super gilts. The proportion of 1- and 2-cell ova which exhibited visible pronuclei or nuclei was also similar among MX-SUPER and L42-SUPER gilts. This study demonstrates that MX gilts respond/perform comparably to L42 gilts with respect to estrus synchronization, superovulation, ova yield, and the ease of visibility of pronuclei or nuclei in the ova.

Alpha 2-macroglobulin and proliferative retinopathy in type 1 diabetes.

Serum alpha 2-macroglobulin (alpha 2m) and total glycosylated haemoglobin (HbA1) concentrations were measured in 110 insulin dependent Type 1 diabetics with minimal or no fundoscopic retinopathy, referred to as non-retinopaths, and in 52 proliferative retinopaths. Proteinuria was recorded in 8 (7%) non-retinopaths and 29 (56%) retinopaths and was accompanied by elevated alpha 2m concentrations in both groups of diabetics but only significantly so in the non-retinopaths. Diabetics without proteinuria showed a significant correlation between alpha 2m concentration and duration of diabetes, HbA1 and age (being higher at extremes of age). Alpha 2m concentrations were significantly higher in retinopaths than in non-retinopaths without proteinuria when allowance was made for the influence of age and duration of diabetes on alpha 2m. This difference may be attributed to the higher HbA, levels found in retinopaths than in non-retinopaths and was no longer evident when account was taken of the prevailing HbA1 concentration in individual patients.

Immunological and biochemical characteristics of acid citrate eluates from tumour cells: a major non-immunoglobulin component.

Using competitive double-antibody radioimmunoassays we have shown that immunoglobulin (especially IgA) can be recovered in pH 3.5, 0.12M acid citrate eluates of freshly excised CCH1 tumour-cell suspensions. Studies with 125I-labelled eluates indicate that such preparations exhibit a variable, but appreciable, degree of non-specific binding to unrelated syngeneic tumour and normal tissues. PAGE/SDS gel electrophoresis of the labelled eluates revealed the presence of a major non-immunoglobulin component of 33-36K dalton which could account in part for the non-specific binding observed. This component was also detected in similar eluates from cultured CCH1 tumour and in all other tumour-cell eluates examined to date. In contrast, preliminary data suggest that it is less prevalent in acid citrate eluates from normal tissue, with the exception of peritoneal-exudate cells. The possible origins, nature and significance of this non-immunoglobulin component are discussed.

Serum alpha 2-macroglobulin levels in diabetes.

Serum alpha 2-macroglobulin levels have been determined in diabetic patients by quantitative radial immunodiffusion and compared with those observed in age- and sex-matched controls. In addition, the results in diabetics have been analysed with respect to such variables as the age and sex of the patient, the duration of disease, treatment, control, and the occurrence of retinopathy or nephropathy. The alpha 2-macroglobulin levels in diabetic patients were found to be significantly higher than in age- and sex-matched controls, thus confirming previous observations. However, these differences were most apparent in the more extreme age groups. Multiple regression analysis also revealed that the only variables contributing significantly to the regression apart from age and sex were control and retinopathy.