Long term retention of 237Np in rats

This interim report summarizes the results of observations during the first year after a single injection of 237Np nitrate (0.2 or 1.0 mg/kg body weight) into adult female rats and further preliminary data obtained with young animals. The retention of 237Np was followed by whole body counting and serial sacrifice of groups of animals. The retention data could be fitted to three-component exponential equations which show no major differences between the two 237Np dose levels. The half-times and extrapolated initial fractions calculated from the first two exponential terms indicate that one fraction, representing about 40 per cent of the injected 237Np was excreted within the first 5 days and an additional 15 per cent within the first 5 months, while the rest was excreted with a half-time of about 3.5 years. This final long term component is assumed to indicate the rate of loss of 237Np from the skeletal compartment. In young animals both whole-body and skeletal retention of 237Np during the first 5 months of observation was about 50 per cent higher than in the adults. Several soft tissue tumours, mostly mammary tumours, have appeared to approximately the same extent in both control and 237Np treated adult rats but no osteosarcomas were detected up to 15 months after injection of 237Np.     

Effects of fasting and/or oxidizing and reducing agents on absorption of neptunium from the gastrointestinal tract of mice and adult or neonatal rats

Neptunium-237(V) nitrate was administered by gavage to groups of fed or fasted adult and 5-day-old rats. Some groups also received the oxidants quinhydrone or ferric iron, and others received the reducing agent ferrous iron. Adult mice received ferric or ferrous iron and 235Np. When the adult rats were killed at 7 days after gavage, measurements showed that, compared with rats that were fed, a 24-hr fast caused a fivefold increase in 237Np absorption and retention. Both quinhydrone and ferric iron caused an even greater increase in absorption in both fed and fasted rats. Ferrous iron, on the other hand, decreased absorption in fasted rats to values lower than those obtained in fed rats. Similar results were obtained in mice treated with 235Np and either ferric or ferrous iron. The highest absorption obtained after gavage of ferric iron to fasted rats and mice was about two orders of magnitude higher than the value obtained in animals that were fed before gavage. The effects of ferric and ferrous iron on neptunium absorption by neonatal rats were similar to their effects on adult animals but of lesser magnitude. These results are consistent with the hypothesis that Np(V), when given in small mass quantities to fed animals, is reduced in the gastrointestinal tract to Np(IV), which is less well absorbed than Np(V).     

Analysis of the mitochondrial haplogroups of farm and wild-living raccoon dogs in Poland

Raccoon dogs' mitochondrial DNA genes (MT-CYTB, MT-CO1 and MT-CO2) containing a total of 1.5?kbp exhibited 11 synonymous substitutions in the polymorphism described, with almost 73% found in the gene MT-CO2. The polymorphism was observed in 27.3% of the loci in wild-living animals, and in over 90% of the loci in farm raccoon dogs. Seven mitochondrial haplogroups have been determined, among which three (Np1, Np2 and Np4) were found in the wild-living raccoon dogs and the other four (Np3, Np5, Np6 and Np7) in the farm animals. The occurrence of new haplogroups in the farm animals indicates the appearance of adaptive mutations. Differences were observed between the sequence under study and the reference sequence in MT-CYTB; they involved two non-synonymous substitutions (T304I and F332L). Analyses carried out to determine the deleterious effect of mutations indicated an almost 50% probability that a single amino acid substitution (T304I) in the protein has a negative impact on its function.     

A complex three breakpoint translocation involving chromosomes 2, 4, and 9 identified by meiotic investigations of a human male ascertained for subfertility

Summary Whole mount pachytene spreads were used to investigate the pairing of a supposed balanced reciprocal t(4;9) translocation in a human male ascertained for subfertility. All well spread pachytene spermatocytes analysed by light microscopy and electron microscopy contained a hexavalent instead of the expected quadrivalent this suggesting that a third chromosome was involved. The hexavalent showed a high efficiency of synapsis with the six arms fully paired except for the proximal segments adjacent to the breakpoints. Further meiotic investigations by the air-drying technique and the reassessment of the mitotic karyotype using stretched chromosomes revealed that the rearrangement is indeed a complex three breakpoint translocation t(2;4;9)(p13;q25;p12). There was an indication of a reduced chiasma frequency of the hexavalent but no interchromosomal effect on chiasma pattern could be detected. No selective association between the hexavalent and the XY configuration was found at any stage, and unless the central lack of pairing is of relevance we have no explanation for the subfertility and reduced testicular size. Except for the hexavalent the most impressive feature of the meiosis of this complex translocation was in fact its normality including the end product with repeated spermiograms being indistinguishable from the normal. Karyotyping of individual spermatozoa has, however, not been performed.     

Cellular membrane contrast and contrast differentiation with osmium triazole and tetrazole complexes

Summary Addition of heterocyclic nitrogen compounds to the classical osmium tetroxide postfixation medium, applied after glutaraldehyde fixation, results in enhanced membrane contrast in ultrathin sections of liver tissue. The addition of similar compounds to potassium osmate solutions, results in contrast differences in some cellular membranes. The membranes of the rough endoplasmic reticulum, the nuclear envelope and the plasma membrane acquire contrast, while the mitochondrial membranes do not. The apolar regions of membranes are contrasted when osmium tetroxide is combined with heterocyclic nitrogen compounds, whereas the polar regions are contrasted by combinations of potassium osmate with these compounds. This polar membrane contrast is probably due to the presence of an amino-group in the heterocyclic nitrogen compounds. Compounds without the amino-group do not contrast membranes, although the glycogen is contrasted.X-ray microanalysis served to establish the relative osmium content in contrasted glycogen, and showed that such nitrogen compounds play a role in complexation of cations in aldehyde-fixed tissues. Electron spectroscopy for chemical analysis (ESCA) measurements of isolated muscle glycogen show that after treatment with various osmium tetroxide or potassium osmate solutions, hexavalent and quadrivalent osmium species are present in the glycogen. The presence of (heterocyclic) nitrogen compounds in such solutions stabilizes certain osmium valency species, and this may account for the contrast observed.     

The quantitative analysis of chromosome pairing and chiasma formation based on the relative frequencies of M I configurations

In autotetraploids, chromosome pairing may be in the form of quadrivalents or bivalent pairs. Whether or not the quadrivalents are maintained until first meiotic metaphase depends on the formation of chiasmata. The relative frequencies of M I configurations thus contain information both on pairing and on chiasma formation. With distal chiasma localisation six configurations can be recognised and their relative frequencies determined: ring quadrivalents, chain quadrivalents, trivalents (with univalent), ring bivalents, open (rod) bivalents, univalent pairs. These represent five degrees of freedom permitting five parameters to be estimated: the frequency (f) of quadrivalent pairing; the frequencies of chiasmate association of the two ends (arms in metacentrics), a′, b′, after quadrivalent pairing, and a, b after bivalent pairing. — The appropriate formulae have been derived and applied to observations on Tradescantia virginiana (4n=24) which has pronounced distal chiasma localisation. Slight modifications make the model applicable to autotetraploids with interstitial in addition to distal chiasmata. In T. virginiana, chromosome pairing appeared to be random between homologues (65.8% quadrivalent pairing; 55.4% observed at M I). After quadrivalent pairing chiasmate association is frequent in the “average long” arm (95.0%) and much less so in the other arm (60.5%). This is attributed to partner exchange. After bivalent pairing chiasma frequencies are still different for the two arms (93.8% and 83.5% association respectively) but much less pronounced. Various complications are discussed.     

Structure of the 5' terminus of hen oviduct lysozyme messenger ribonucleic acid

Lysozyme mRNA (mRNAlys) was purified from hen oviduct poly(A)-containing RNA by hybridization, labeled with NaB[3H]4 and digested with RNase T1. This revealed the presence of equal amounts of two major oligonucleotides having structures of m7Gppp(Np)7 and m7Gppp(Np)4 plus minor amounts of m7Gppp(Np)2 and m7GpppNp. The total mRNAlys contained the cap structures m7Gpppm6Am, m7GpppGm, m7GpppAm, m7GpppCm, m7GpppA, and m7GpppG, in decreasing order of abundance. The m7Gppp(Np)7 oligonucleotide contained only A-caps and the m7Gppp(Np)4, only G-caps. 32P-labeled 5'-terminal T1-oligonucleotides were prepared, and at least 12 different types were observed, the most abundant being m7Gppp(Np)7 and m7Gppp(Np)4. Their sequences were determined to be m7Gppp(m6)AmNmUCCCG and m7GpppGmNmAG. Taken together with the findings of Grez et al. (Grez, M., Land, H., Giesecke, K., Schutz, G., Jung, A., and Sippel, A. E. (1981) Cell 25, 743-752), these results indicate that in the genomic sequence AGCTTGCAGTCCCGT, 52% of the mRNAlys molecules begin at the underlined A residue and 38% at the underlined G residue.     

Inhibition of rat brain prostaglandin D synthase by inorganic selenocompounds

Various inorganic selenocompounds dose-dependently inhibited the rat brain prostaglandin (PG) D synthase, both in the purified enzyme preparation and in the crude brain supernatant. All of the quadrivalent selenium compounds tested had a very limited range of IC50 values in the purified enzyme (11-12 microM) and in the brain supernatant (9-15 microM). A divalent selenium compound was also inhibitory, but a hexavalent selenium compound was ineffective. In contrast, organic selenocompounds such as selenomethionine and selenourea had no effect on the PGD synthase activity. Furthermore, sodium sulfate and sodium sulfite up to 10 mM did not inhibit the activity. The inhibition by selenium required the preincubation of the metal with sulfhydryl compounds such as dithiothreitol (DTT), indicating that the formation of selenotrisulfide or some other adduct(s) is essential for the inhibition. Furthermore, the inhibition was reversed by an excess amount of dithiothreitol, suggesting that the selenotrisulfide derivative of DTT binds to the SH group of the PGD synthase. The kinetic analysis revealed the inhibition by selenite to be noncompetitive with a Ki value of 10.1 microM. On the other hand, glutathione-dependent PGD synthase from rat spleen was much less inhibited, and PGF synthase and PGD2 11-ketoreductase activities were not inhibited by the selenium compound.     

Serum-Free and Xenobiotic-Free Preservation of Cultured Human Limbal Epithelial Cells

Aim/Purpose of the Study

To develop a one-week storage method, without serum and xenobiotics, that would maintain cell viability, morphology, and phenotype of cultured human limbal epithelial sheets.

Materials and Methods

Human limbal explants were cultured on intact human amniotic membranes for two weeks. The sheets were stored in a hermetically sealed container at 23°C in either a serum-free medium with selected animal serum-derived compounds (Quantum 286) or a xenobiotic-free medium (Minimal Essential Medium) for 4 and 7 days. Stored and non-stored cultures were analyzed for cell viability, amniotic membrane and epithelial sheet thickness, and a panel of immunohistochemical markers for immature cells (ΔNp63α, p63, Bmi-1, C/EBP∂, ABCG2 and K19), differentiated cells (K3 and Cx43), proliferation (PCNA), and apoptosis (Caspase-3).

Results

The cell viability of the cultures was 98 ± 1% and remained high after storage. Mean central thickness of non-stored limbal epithelial sheets was 23 ± 3 μm, and no substantial loss of cells was observed after storage. The non-stored epithelial sheets expressed a predominantly immature phenotype with ΔNp63α positivity of more than 3% in 9 of 13 cultures. After storage, the expression of ABCG2 and C/EBP∂ was reduced for the 7 day Quantum 286-storage group; (P = 0.04), and Bmi-1 was reduced after 4 day Quantum 286-storage; (P = 0.02). No other markers varied significantly. The expression of differentiation markers was unrelated to the thickness of the epithelia and amniotic membrane, apart from ABCG2, which correlated negatively with thickness of limbal epithelia (R = -0.69, P = 0.01) and ΔNp63α, which correlated negatively with amniotic membrane thickness (R = -0.59, P = 0.03).

Conclusion

Limbal epithelial cells cultured from explants on amniotic membrane can be stored at 23°C in both serum-free and xenobiotic-free media, with sustained cell viability, ultrastructure, and ΔNp63α-positivity after both 4 and 7 days.     

Transfer of Np(V) nitrate from gastrointestinal segments of the adult rat

The transfer of soluble Np(V) nitrate was measured in gastrointestinal segments from adult rats by two procedures: instillation, in segments in which the physico-chemical form of Np might be modified by gastrointestinal factors; and perfusion, in segments in which the luminal state of Np remains constant. These assays allowed accurate measurement of the Np(V) transferred from the intestine to the whole body. The amount measured was proportional to segment length and to the duration of the experiments, which lasted for periods of 0.25 to 2 h. Under these experimental conditions, hourly transfer values were about 2 percent, both per millilitre of Np(V) solution instilled and per 10 cm of jejunum perfused. This flux is very much greater than that which may be deduced from studies in which Np was gavaged into intact rats. Intestinal transfer of Np was constant for Np concentrations ranging from 5 X 10(-12) M to 1 X 10-4) M. Raising the concentration of Np(V) to more than 1 X 10(-4) M reduced its intestinal transfer. Addition of Fe(II) also reduced it. The small intestine was the main site of Np(V) absorption, since the transfer from instilled jejunum was about 20 times that observed from the stomach, and no difference was noted between jejunal and duodenal transfer.     

Life-span of rats as affected by a combination of external (137Cs) and internal (237Np) radiation

A study was made of a change in the mean life of rats exposed to external gamma-radiation (51.6 mC/kg) and 237Np (a polymeric nitrate form) administered intratracheally (0.2-188.0 kBq/kg) delivered separately and in a combination. It was established that the effects of gamma- and alpha-radiation were summated.     

Glucose clearance in aged trained skeletal muscle during maximal insulin with superimposed exercise.

Insulin and muscle contractions are major stimuli for glucose uptake in skeletal muscle and have in young healthy people been shown to be additive. We studied the effect of superimposed exercise during a maximal insulin stimulus on glucose uptake and clearance in trained (T) (1-legged bicycle training, 30 min/day, 6 days/wk for 10 wk at approximately 70% of maximal O(2) uptake) and untrained (UT) legs of healthy men (H) [n = 6, age 60 +/- 2 (SE) yr] and patients with Type 2 diabetes mellitus (DM) (n = 4, age 56 +/- 3 yr) during a hyperinsulinemic ( approximately 16,000 pmol/l), isoglycemic clamp with a final 30 min of superimposed two-legged exercise at 70% of individual maximal heart rate. With superimposed exercise, leg glucose extraction decreased (P < 0.05), and leg blood flow and leg glucose clearance increased (P < 0.05), compared with hyperinsulinemia alone. During exercise, leg blood flow was similar in both groups of subjects and between T and UT legs, whereas glucose extraction was always higher (P < 0.05) in T compared with UT legs (15.8 +/- 1.2 vs. 14.6 +/- 1.8 and 11.9 +/- 0.8 vs. 8.8 +/- 1.8% for H and DM, respectively) and leg glucose clearance was higher in T (H: 73 +/- 8, DM: 70 +/- 10 ml. min(-1). kg leg(-1)) compared with UT (H: 63 +/- 8, DM: 45 +/- 7 ml. min(-1). kg leg(-1)) but not different between groups (P > 0.05). From these results it can be concluded that, in both diabetic and healthy aged muscle, exercise adds to a maximally insulin-stimulated glucose clearance and that glucose extraction and clearance are both enhanced by training.     

Uptake and clearance of plutonium-238 from liver cells transplanted into fat pads of F344 rats

This research is directed toward understanding the role of liver cells and the liver environment in plutonium biokinetics. Animals injected with liver cells and control animals received a single intraperitoneal injection of 37 kBq (1 microCi) 238Pu citrate and were serially sacrificed 1, 5, 10, 15, 30, 60 or 70 days later. Uptake, retention and distribution of Pu in intact liver and in liver cells growing in fat pads were determined. From these measurements, it was observed that the cells of the intact liver took up about twice as much 238Pu as liver cells transplanted into the fat pads of the same animal. The retention half-life was 8.3 days for the total activity in the liver, 20 days using tracks/cell measurements in the liver and 16 days for the tracks/cell measurements in the liver cells translocated to fat pads. When the data on tracks/cell were standardized relative to the amount of Pu present at 5 days after the injection, there was no significant difference between the retention of Pu in liver cells from intact animals and liver cells transplanted into the fat pads. About 20 per cent of the 5-day Pu liver burden in both liver cells and liver cells transplanted into fat pads was retained at 70 days. The smaller retention and clearance for liver cells in different environments indicate that uptake and clearance of Pu from the body is dependent, to a major extent, upon hepatocyte function.     

Kinetic characterization of mass transfer limited biodegradation of a low water soluble gas in batch experiments--necessity for multiresponse fitting

A method was developed to characterize the kinetics of biodegradation of low water soluble gaseous compounds in batch experiments. The degradation of ethene by resting Mycobacterium E3 cells was used as a model system. The batch degradation data were recorded as the progress curve (i.e., the time course of the ethene concentration in the headspace of the batch vessel). The recorded progress curves, however, suffered gas:liquid mass transfer limitation. A new multiresponse fitting method had to be developed to allow unequivocal identification of both the affinity coefficient, K(aff), and the gas:liquid mass transfer coefficient, K(l)a, in the batch vessel from the mass transfer limited data. Simulation showed that the K(aff) estimate obtained is influenced by the dimensionless (volumetric basis) ethene gas:liquid partitioning coefficient (H). In the fitting procedure, Monod, Teissier, and Blackman biokinetics were evaluated for characterization of the ethene biodegradation process. The fits obtained reflected the superiority of the Blackman biokinetic function. Overall, it appears that resting Mycobacterium E3 cells metabolizing ethene at 24 degrees C have, using Blackman biokinetics, a maximum specific degradation rate, v(max), of 10.2 nmol C(2)H(4) mg(-1) CDW min(-1), and an affinity coefficient, K(aff.g), expressed in equilibrium gas concentration units, of 61.9 ppm, when H is assumed equal to 8.309. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 511-519, 1997.     

四季竹对土壤水分胁迫的生理适应

以四季竹2年生竹苗为材料,采用每天补水控制土壤含水量的方法,设置土壤相对含水率为<30%(T1)、40%～50%(T2)、60%～70%(T3)、80%～90%(T4)和竹蔸部完全水淹(T5)5个土壤水分含量水平,研究四季竹叶片在水分胁迫下的生理活性变化,以探讨四季竹对土壤水分的适应能力。结果表明:(1)随处理时间的延长,T1处理叶片离子渗漏率、MDA含量、叶绿素a/b值、SOD和POD活性、脯氨酸和可溶性蛋白含量均迅速升高,叶绿素含量、类胡萝卜素含量和叶绿素/类胡萝卜素比值迅速下降。(2)T5处理14d后叶片各生理指标随处理时间的延长与T1处理表现出相同的变化规律(类胡萝卜素和脯氨酸除外),并且分别在T1处理14d和T5处理28d后四季竹叶片全部干枯脱落。(3)随处理时间的延长,T2、T3、T4处理的四季竹叶片各生理指标经过一段时间的适应后最终稳定在处理前水平,且处理间均无显著差异。研究发现,四季竹在土壤相对含水率小于30%的土壤中生长不良,甚至死亡,在相对含水率40%～90%的土壤中能正常生长;四季竹耐受水淹胁迫的时间阈值是28d。     

Lactogenic effects of hyperinsulinemic-euglycaemic clamp in goats

In the experiment performed on lactating goats, insulin was infused into the jugular vein over during 2 days every day at the rate 2 mg/kg/hour during 6 hour synchronously with glucose at variable rate to maintain euglycaemia; the transport activity (T, in clearance units) was estimated using the equation: T = Q x E/ (1-E), where Q is plasma flow and E is extraction efficiency. At the end of infusion of the 1st and 2nd days, insulin level in the blood was increased by 63 and 82%, mammary plasma flow by 38 and 78%, milk secretion rate by 23.7 and 31.3 %, milk protein yield by 21.4 and 40%, transport activity of glucose by 63 %, and amino acids by 18% (all p < 0.05) compared to control, respectively. The data obtained suggest that productive effect resulted from elevated metabolic activity of secretory cells and increased mammary blood flow.     

Toxicity and mutagenicity of hexavalent chromium on Salmonella typhimurium.

Four hexavalent and two trivalent chromium compounds were tested for toxicity and mutagenicity by means of the Salmonella typhimurium/mammalian-microsome test. All hexavalent compounds yielded a complete inhibition of bacterial growth at doses of 400 to 800 mug/plate, a significant increase of his(+) revertant colonies at doses ranging from 10 to 200 mug, and no effect at doses of less than 10 mug. The distinctive sensitivity of the four Salmonella strains tested (TA1535, TA1537, TA98, and TA100) suggested that hexavalent chromium directly interacts with bacterial deoxyribonucleic acid by causing both frameshift mutations and basepair substitutions. The latter mutations, which are prevalent, are amplified by an error-prone recombinational repair of the damaged deoxyribonucleic acid. On the average, 1 mumol of hexavalent chromium yielded approximately 500 revertants of the TA100 strain, irrespective of the compound tested (sodium dichromate, calcium chromate, potassium chromate, or chromic acid). The mutagenic potency of the hexavalent metal was not enhanced by adding the microsomal fraction of rat hepatocytes, induced either with sodium barbital or with Aroclor 1254. The two trivalent compounds (chromium potassium sulfate and chromic chloride), with or without the microsomal fraction, were neither toxic nor mutagenic for the bacterial tester strains.     

Comparative toxicity of trivalent and hexavalent chromium compounds in fig moth Ephestia cautella (Walker)

Of the trivalent (CrSO4) and hexavalent (K2Cr2O7) chromium compounds, only the hexavalent produced significant deleterious effects on development and fertility of E. cautella, when eggs were reared on laboratory medium supplemented with different concentrations of these salts.     

Orally induced tolerance generates an efferently acting suppressor T cell and an acceptor T cell that together down-regulate contact sensitivity

Intragastric administration of the hapten trinitrochlorobenzene (TNCB) suppresses development of contact sensitivity (CS) to attempted epicutaneous sensitization with TNCB. Suppression induced by feeding TNCB is hapten specific and can be transferred to normal animals with lymphoid cells from fed mice. The lymphoid cells in hapten-fed mice that cause suppression of CS have been identified as Thy-1.2-positive cells in spleen and mesenteric nodes. The suppression with Peyer's patch cells from hapten-fed mice appears to be attributable to cells bearing Thy-1.2 antigen (T cell) and to cells with surface Ig (B cell). Feeding TNCB induces an efferent-acting suppressor T cell (Ts eff), as well as an intermediary acceptor T cell (T acc) with which it interacts to block adoptive transfer of CS with immune cells. Ts eff emanating from hapten-fed mice was identified by its specificity for the hapten, insensitivity to pretreatment with cyclophosphamide (CY), ability to produce soluble suppressor factor (SSF), and requirement for T acc to be functional. The presence of T acc in hapten-fed mice, on the other hand, was confirmed by its sensitivity to treatment with CY, interaction with Ts eff or SSF, and the ability to produce nonspecific inhibitor of TDTH cells. Thus, the suppressor T cells that are induced by administering the hapten intragastrically appear to function much like the cells of the suppressor T cell cascade that are induced by giving hapten via parenteral routes.