Effects of prolonged moderate body deuteration on proliferative activity in major cell renewal systems in mice

To evaluate the effects of prolonged moderate body deuteration on incorporation of tritiated thymidine (³HTdR) into the DNA of major cell renewal systems, young adult mice were given drinking fluid containing 30 % heavy water for 7, 14, 21, 42 and 70 days. Control mice drank tap water. Three hours prior to sacrifice, 925 kBq of ³HTdR were injected intravenously. Following extraction of the bulk of the soluble activity with an aqueous formalin solution, the residual ³H-activity of the organs was assayed by liquid scintillation counting and by autoradiography. The total thymic ³H-activity and the thymic weight, particularly of the cortex, were significantly reduced in deuterated mice early in the course of the experiment. The fraction of labeled thymocytes diminished to less than one half of the control values of day 70. The ³H-activity of the bone marrow in deuterated mice was reduced to about $\text{3}\text{4}$ of control values. In contrast, the total ³H-activity of the small intestine, as well as mean labeling index and mitotic index of small intestinal epithelia, were not markedly altered in deuterated versus control mice. Drinking water containing 30 % of heavy water did thus not result in generalized, profound and progressive disturbance of ³HTdR incorporation in all the major cell renewal systems in the mouse. The thymus and, to a lesser extent, the bone marrow, were unquestionably affected, but the depression of ³HTdR incorporation did not increase markedly in the course of 10 weeks. In these terms, the toxicity of 30 % heavy water in the drinking fluid appears minor. This is of particular interest since exposure to similar concentrations is known to elicit immunodepressive and/or antineoplastic effects.     

The capacity for reactive DNA synthesis of the myocytes in the heart conduction system in experimental and clinical myocardial pathology]

The DNA synthesis has been studied in the conductive system (CS) myocytes, compared to that in atrial and ventricular myocytes: 1) in the left ventricular myocardial infarction induced in two- and three-week-old and adult rats, 2) after isoproterenol injections to adult rats and mice, and 3) in the hypertrophied human heart. The extent of DNA synthesis reactivation was evaluated by the cumulative labeling indices in experiments with multiple 3HTdR injections to rats and mice. In the human cardiac myocyte nuclei, the DNA content was determined by the Feulgen-cytophotometry. The difference between the control and experimental mean values of the labeling indices for CS myocyte nuclei was statistically significant only for atrioventricular part of the CS in the infarcted hearts of adult rats. In the human heart CS the ability of myocytes to polyploidization varies from one cell type to another, the lowest being in nodal cells.     

Growth of human renal cortical tissue on collagen gel

Summary A model system for 3-dimensional “native-state” culture of tissues on collagen gels (Proc. Natl. Acad. Sci. USA 86:2013–2017; 1989) has been applied in this study to histologically normal human renal cortical tissue from 11 patients undergoing nephrectomy for renal cell carcinoma elsewhere in the kidney. Microbial contamination occurred in 12/90 cultures, the rest (78) were studied by visual inspection, histology, immunohistochemical analysis for pankeratin (epithelial cell origin), vimentin (mesenchymal cell origin), andp-glycoprotein (associated with proximal tubules), transmission electron microscopy (EM), incorporation of tritiated thymidine (³HTdR). In the first 10 days, explants showed³HTdR-labeled cells in tubule structures. The surrounding gel was invaded by cells forming tubule structures, sometimes with basement membrane. Some of these cells showed labeling by³HTdR and immunostaining positive for pankeratin andp-glycoprotein. EM showed well-polarized epithelial cells in tubule structures with tight junctions, interdigitating lateral processes, and microvilli characteristic of proximal and distal convoluted tubules.³HTdR-labeled cells in tubule structures were observed even 2 mo. after Passage 1, 6 mo. after the initial explantation. Tubule growth was most active and fibroblast proliferation was negligible from 2 to 4 wk postexplantation. The proliferation of tubulelike cells and formation of tubulelike structures in this system represents an opportunity to study human renal cortical tissue in vitro, under conditions more closely resembling in vivo circumstances than are present in other in vitro systems suitable for long-term study. This model has potential use for in vitro toxicology studies and studies of renal physiology.     

Glycoprotein secretion in the hypothalamo-neurohypophyseal system of the rat

Summary Although the secretory products of the hypothalamoneurohypophyseal system are not glycoproteins, synthesis and migration of these macromolecules occur within its secretory neurons. After being labeled with ³H-fucose in the Golgi apparatus, newly synthesized glycoproteins migrate to secretion granules, lysosomes and the plasma membrane of the secretory neurons, as demonstrated by quantitative electron-microscopic radioautography. Secretion granules bearing newly synthesized glycoproteins migrate to the pars nervosa, the labeling pattern of which was studied in rats killed from 4 h to 14 days after the isotope injection. Most of the silver grains were observed to overly the secretory axons. Labeling of pituicytes was negligible and the number of silver grains over the perivascular spaces was about 10% of the total at certain postinjection intervals. In the secretory axons, most of the silver grains were seen to overly the secretion granules. The proportion of silver grains over the different portions of the secretory axons changed with time. At the longer intervals, the percentage of silver grains increased over the nerve swellings (including Herring bodies) and decreased concomitantly in the undilated portions of the axons and in the nerve endings. This labeling pattern conforms with observations on the secretion products. Water deprivation increased the release of neurosecretion as well as glycoproteins from the pars nervosa. However, glycoproteins inside the Herring bodies were not easily releasible. There was a parallel decrease in the amount of secretion granules and ³H-fucose-labeled glycoproteins indicating that the glycoproteins are predominantly a constituent of the granule content. Some newly synthesized glycoproteins were probably also used in the renewal of the axonal membrane. The labeling of smooth vesicles in nerve endings was discussed. In conclusion, most of the glycoproteins synthesized in the perikarion of the hypothalamic secretory neurons migrate inside secretion granules along the axon to the pars nervosa where they are secreted.     

Localization of the neuropeptide NGIWYamide in the holothurian nervous system and its effects on muscular contraction

NGIWYamide is a peptide recently isolated from the sea cucumber Apostichopus japonicus. It stiffens the connective tissue of the holothurian body wall. Localization of NGIWYamide was investigated by immunohistochemical staining with antiserum raised against NGIWYamide. In holothurian nervous systems NGIWYamide-like immunoreactivity (NGIWYa-LI) was observed in the hyponeural and ectoneural regions of the radial nerve cord, as well as in the circumoral nerve ring, podial nerves, tentacular nerves, the basiepithelial nerve plexus of the intestine and in cellular processes running through the body wall dermis. Labelled nerve fibres from the hyponeural part of the radial nerve running towards the circular muscle and from the podial nerve into the body wall dermis suggest that NGIWYamide controls both muscle and connective tissue. We examined the effect on muscle activity of the sea cucumber. NGIWYamide (10^-7 to 10^-4 M) caused contraction of the longitudinal body wall muscle. Tentacles showed contraction only at a higher dose (10^-4 M). NGIWYamide (10^-4 M) inhibited spontaneous contraction of the intestine.     

Use of anin situ labeling technique for the determination of seasonal14C distribution in Ponderosa pine

A¹⁴C labeling apparatus was developed to permit the labeling of four-year-old Ponderosa pine with¹⁴CO₂ in the field. The labeling system is a completely closed canopy system with¹⁴CO₂ monitored by a GM tube ratemeter apparatus. The level of¹⁴CO₂ corresponding to ambient levels is monitored by a microloggercomputer which controls a¹⁴CO₂ generating system. The generated¹⁴CO₂ is mixed in the canopy by circulating the atmosphere with 12V diaphram pumps. The portable system requires little operator attention. At approximately monthly intervals over a one-year period two four-year-old Ponderosa pine trees were labeled for three to five days using this labeling apparatus. After an assimilate distribution period, one tree was excavated and analyzed for¹⁴C distribution. During late spring and early summer most of the carbon assimilated (>60%) was found in the active growing tips and new needles, with little being allocated to the roots (<10%) or woody material (<20%). During mid to late fall there was an increase in root labeling along with an increase in carbon going to woody material. Over the winter period, most of the fixed carbon (65%) resided in the older leaves. The early spring labeling period showed another pulse of root labeling along with some labeling of woody tissues.     

Effects of vitamin A-deprivation on hamster tracheal epithelium. A quantitative morphologic study

The effects of vitamin A-deprivation on the tracheal epithelium were studied in 35-day old hamsters that had been raised since birth on a vitamin A-deficient diet. Colchicine and 3HTdR were given 6 hours before death and the proliferative activities of basal cells and mucous cells were quantified separately by 3HTdR labeling indices and mitotic rates. Vitamin A-deprivation decreased replication of basal cells and mucous cells in tracheal epithelium which showed minimal morphologic change. The mitotic rates and labeling indices were reduced 3 to 4-fold in basal cells and 14-fold in mucous cells (analyzed as percent of total number of each cell type) compared with controls. Thus, replication of mucous cells was more inhibited by lack of vitamin A, than replication of basal cells. The disparate hypoplasia of basal cells and mucous cells in epithelium showing minimal change, resulted in a relative increase in the proportion of basal cells and a relative decrease in the proportion of mucous cells, which could be erroneously interpreted as "basal cell hyperplasia". Proportions of preciliated and ciliated cells were also decreased compared to controls. At foci of stratification and epidermoid metaplasia, cell replication rates were increased over controls and more than 70% of all mitotic activity was associated with "non-basal" cells. Genesis of these lesions was coincident with cell death and cell loss. The histogenesis of stratification and epidermoid metaplasia was characterized. Morphological evidence indicated that these lesions were closely related histogenetically and were composed, for the most part, of altered mucous cells which expressed dual phenotypes i.e. keratinization and mucus synthesis.     

DNA synthesis in rat heart cells after injury and the regeneration of myocardia

The regenerative responses of the myocardia of post-natal rats of different age groups (1, 2, 3 and 4 weeks old) to an injury made by a clinical electricator were studied. DNA synthesis and the ultrastructural organization of the cardiac myocytes of the injured myocardia were examined for an evaluation of the potential for regeneration of the developing myocardia. The maximum labeling index of cardiac myocytes was observed in 1-week-old rats showing 8% labeled myocytes 3 days after injury as opposed to 3.2, 2.2 and 0.2% indices in 2-, 3- and 4-week-old rats respectively, 3 days after injury. In subsequent days after injury the labeling indices declined considerably in all age group hearts, and attained values less than 1% labeled myocytes 30 days after injury with the lowest labeling index in the oldest age group heart. When DNA synthesis in uninjured myocardial tissue adjacent to the injured tissue was examined, it was found to be significantly lower than it was in the injured tissue. However, both injured and adjacent uninjured tissue attained a peak in the labeling indices 3 days after injury, with the exception of 3- and 4-week-old uninjured tissue. The overall incorporation of ³H-thymidine into the DNA of heart cells as revealed by scintillation counts showed that the rate of incorporation of the isotope in younger hearts was significantly higher than in the older hearts. Non-muscle cells contributed significantly to the rise of scintillation counts in hearts of all age groups.Ultrastructural analyses of 1- to 4-week-old hearts showed that 24 hr after injury, injured areas of myocardia were heavily crowded with macrophages that surrounded damaged myocytes. Later on, fibroblasts and other non-muscle cells predominated the injury sites along with fibrous connective tissue. Scattered regenerating cardiac myocytes were frequently observed in the injury sites of 1- and 2-week-old hearts 3 days after injury. Myocytes were rare in the corresponding regions of 3- and 4-week-old hearts. Instead abundant non-muscle cells and fibrous connective tissue were predominant. In the fourth and final week of this study, the repaired areas of myocardia in 1- and 2-week-old rats contained more myocytes than those of the 3- and 4-week-old rats, and the repaired zone of the 1-week-old heart contained more myocytes than the repaired areas of the other age groups. These findings suggest that the mammalian myocardia possess an age-dependent potential for regeneration that involves the healing of injury sites with contractile and connective tissues.     

A novel approach for assessing protein synthesis in channel catfish, Ictalurus punctatus

A comprehensive understanding of animal growth requires adequate knowledge of protein synthesis (PS), which in fish, has traditionally been determined by the flooding dose method. However, this procedure is limited to short-term assessments and may not accurately describe fish growth over extended periods of time. Since deuterium oxide (²H₂O) has been used to non-invasively quantify PS in mammals over short- and long-term periods, we aimed at determining if ²H₂O could also be used to measure PS in channel catfish. Fish were stocked in a 40-L aquarium with ~ 4% ²H₂O and sampled at 4, 8 and 24 h (n = 6 at each time period) to determine ²H-labeling of body water (plasma), as well as protein-free and protein-bound ²H-labeled alanine. The labeling of body water reflected that of aquarium water and the labeling of protein-free alanine remained constant over 24 h and was ~ 3.8 times greater than that of body water. By measuring ²H-labeled alanine incorporation after 24 h of ²H₂O exposure we were able to calculate a rate of PS: 0.04 ± 0.01% h^− 1. These results demonstrate that PS in fish can be effectively measured using ²H₂O and, because this method yields integrative measures of PS, is relatively inexpensive and accounts for perturbations such as feeding, it is a novel and practical assessment option.     

Tritiated thymidine autoradiographic study on the origin and renewal of argentaffin cells in the pyloric gland of hamsters

Summary The origin and renewal of the argentaffin cells in the pyloric glands of hamsters were studied by flash, cumulative and pulse labelling autoradiography with ³H-thymidine. The argentaffin cells were identified by the Diazo Method using Fast Red B Salt.By flash labelling autoradiography, it was shown that the argentaffin cells located from the middle to the lower level of the pyloric mucosa were not labelled with ³H-thymidine, indicating that this cell type has no proliferative activity. On the 10th and the 20th day of cumulative labelling, 31% and 63% of the argentaffin cells in the gland were found to be labelled, respectively. The labelled argentaffin cells were concentrated in the upper part of the gland (around the region of the isthmus), and no label was found over nuclei of the cells at the lowermost level of the gland. These labelled cells were shown to undergo a downward migration in the days following pulse labelling. They were replaced by unlabelled (and weakly or very weakly labelled) cells which arose at the region of the isthmus. The argentaffin cells in the pyloric gland are thought to arise from epithelial precursor cells at the region of the isthmus.The labelled argentaffin cells in the gland were found to decrease in number almost exponentially after pulse labelling. This indicates that the life span of argentaffin cells is not fixed, but their renewal conforms to the random loss system. The half time of turnover of this cell population was 15 days on average.Supported by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture, Japan     

Distribution of the glio-interstitial system in molluscs

Summary Ten hamsters received repeated injections of ³H-thymidine for 4 days and were allowed to survive for 7, 28, 42 and 100 days. Changes in spatial distribution of the labelled cells and in labelling indices of each cell line in the gastric glands were studied at various days after ³H-thymidine injections, and the fate of the mucous neck cell, the replacement of the chief cell and the mode of cell migration were discussed.After 4 days of repeated injections of ³H-thymidine, the labelled parietal cells and the mucous neck cells were concentrated at the neck area. Starting from the neck area, they migrated an average of 3 micra downwards per day. By 42 days, they reached the middle level of the glands, where the labelled mucous neck cells decreased but the labelled chief cells increased in number. The differentiation of the chief cell then appears to take place at the middle level of the glands through transformation of the migratory mucous neck cells. After 4 days of the labelling, about 1.8% of the chief cells located in the lower part of the glands was found to undergo in situ replication. This indicates that the renewal of this cell type is partly assured by its own mitotic activity.The foveolar cell — the future surface epithelium — seems to migrate upwards along the long axis of the glandular tubule in the pipe line system, which means first produced, first migrates. After migrating out from the neck area, the parietal cell and the mucous neck cell (the future chief cell) take an average of 200 days to reach the lower end of the glands. In the process of migration, however, the cells produced contemporaneously at the neck area became scatteringly spread from the neck towards the bottom of the gland. The time required for the newly-formed cells to reach the lower end of the gland varied between 100 and 300 days. In the gastric glands the cells first produced at the neck area do not first reach the lower end of the glands. This mode of random migration is referred to as the stochastic flow system. As one of the probable factors which disturb the pipe line flow of downward cell migration, cellular movements perpendicular to the long axis of the glandular tubule were suggested to occur at random at an any level of the gastric glands.Supported by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture, Japan     

Vitamin D3 (soltriol) nuclear receptors in abdominal scent gland and skin of Siberian hamster (Phodopus sungorus) localized by autoradiography and immunohistochemistry

In vivo autoradiography with [³H]1,25-dihydroxycholecalciferol (vitamin D, soltriol) and immuno-staining with antibodies to vitamin D receptor were applied to identify specific binding sites in the abdominal scent gland of male Siberian hamster (Phodopus sungorus). Nuclear concentration of radiolabeled hormone and receptor antibodies was observed in the corresponding cell types including basal cells of sebaceous glands, cells of the outer hair sheaths and hair bulbs, and also keratinocytes in the epidermis. Cells of the hair dermal papillae and fibroblasts of the dermis did not show nuclear labeling. There was good correspondence between the autoradiographic and immunohistochemical data. The results indicate the presence of receptors for vitamin D-soltriol and suggest a seasonal regulation of scent gland marking activities by this steroid hormone of sunlight in cooperation with the sex steroid testosterone.     

Softenin,a Novel Protein That Softens the Connective Tissue of Sea Cucumbers through Inhibiting Interaction between Collagen Fibrils

The dermis in the holothurian body wall is a typical catch connective tissue or mutable collagenous tissue that shows rapid changes in stiffness. Some chemical factors that change the stiffness of the tissue were found in previous studies, but the molecular mechanisms of the changes are not yet fully understood. Detection of factors that change the stiffness by working directly on the extracellular matrix was vital to clarify the mechanisms of the change. We isolated from the body wall of the sea cucumber Stichopus chloronotus a novel protein, softenin, that softened the body-wall dermis. The apparent molecular mass was 20 kDa. The N-terminal sequence of 17 amino acids had low homology to that of known proteins. We performed sequential chemical and physical dissections of the dermis and tested the effects of softenin on each dissection stage by dynamic mechanical tests. Softenin softened Triton-treated dermis whose cells had been disrupted by detergent. The Triton-treated dermis was subjected to repetitive freeze-and-thawing to make Triton-Freeze-Thaw (TFT) dermis that was softer than the Triton-treated dermis, implying that some force-bearing structure had been disrupted by this treatment. TFT dermis was stiffened by tensilin, a stiffening protein of sea cucumbers. Softenin softened the tensilin-stiffened TFT dermis while it had no effect on the TFT dermis without tensilin treatment. We isolated collagen from the dermis. When tensilin was applied to the suspending solution of collagen fibrils, they made a large compact aggregate that was dissolved by the application of softenin or by repetitive freeze-and-thawing. These results strongly suggested that softenin decreased dermal stiffness through inhibiting cross-bridge formation between collagen fibrils; the formation was augmented by tensilin and the bridges were broken by the freeze-thaw treatment. Softenin is thus the first softener of catch connective tissue shown to work on the cross-bridges between extracellular materials.     

Nitrogen-15 labeling effectiveness of two tropical legumes

Nitrogen-15 foliar applications for the production of field-labeled plant tissues may achieve more effective labeling of plant shoot and root tissues and minimize directly labeling the soil N fraction as occurs when¹⁵ N is soil applied. Consequently, foliar-labeled plant tissues should be better suited for subsequent ¹⁵N mineralization studies. A field experiment was conducted to determine the effectiveness of ¹⁵N-labeling and the accumulation of ¹⁵N in various plant parts of two tropical legumes. Desmodium ovalifolium Guillemin and Perrottet and Pueraria phaseoloides (Roxb.) Benth., grown in 0.5 m² microplots, were labeled with foliar-applied urea containing 99 atom% ¹⁵N. Plants in each microplot received a total of 0.1698 g ¹⁵N that was applied all at once or split equally into two, three or four applications. Legume shoots and roots and soil were destructively harvested and analyzed for total ¹⁵N content. Averaged over both legumes and foliar application rates, total plant (shoots, flowers, leaf litter, and roots) recovery was approximately 79% of the ¹⁵N applied. The soil contained 3% of the ¹⁵N applied, of which 2.5 and 0.5% were in the inorganic and organic fractions, respectively. Nitrogen-15 recovery in shoots (76%) was sixty-five fold greater than in roots (1%) and about nineteen fold greater than the sum of roots and soil (4.1%), a much greater percent recovery than observed in other foliar labeling studies. Averaged over all four foliar split-application rates, ¹⁵N recovery by Desmodium shoots was greater than Pueraria. Results demonstrate that ¹⁵N foliar application to legumes is an effective method for labeling, resulting in atom% excess ¹⁵N levels and ¹⁵N recoveries comparable to those reported with the more traditional soil-labeling approach. Another advantage of this method is a nondestructive, in situ labeling method that permits separation of shoot and root residual N contribution to subsequent crops in N tracer studies.     

Heavy water labeling method for measuring the effect of genistein on mammary gland carcinogenesis

Heavy water labeling method was applied to measure the effect of genistein on mammary gland carcinogenesis by incoporating ²H from ²H₂O into the deoxyribose (dR) moiety of purine deoxyribonucleotides in dividing cells. In the present study, we followed the study design of Lamartiniere group to evaluate the efficacy of ²H₂O labeling on the measurement of mammary gland carconogenesis. Female Sprague–Dawley rats were fed estrogen-free AIN-93G diet starting 1 week before breeding and continuing through pregnancy and lactation. Female pups were assigned to the following groups on postnatal day 16 and fed AIN-93G diet: vehicle (dimethylsulfoxide) (DMSO), genistein, and estradiol benzoate (EB). On postnatal days 16, 18, and 20, female pups were injected subcutaneously with 500 μg genistein/g body wt, 500 ng EB/g body wt, or an equivalent volume of the vehicle. At day 50 postpartum, half of each group were gavaged with 60 mg dimethylbenz[a]anthracene (DMBA) in perila oil. After 1 week of DMBA treatment, all animals were labeled with ²H₂O by administration of 4% ²H₂O in drinking water after single intraperitonial bolus injection with 99.9% ²H₂O until sacrifice on postnatal day 81. The time-dependent weight gains were observed in all groups throughout the experimental period. The enrichment of body ²H₂O was attained at 1.84–2.47% through oral administration of ²H₂O. Mammary epithelial cell proliferation was measured by enrichment (EM1) of dA from rats. DMBA-treated groups showed higher fractional synthesis than DMBA non-treated groups. The group exposed only to genistein showed significantly lower EM1 (1.46 ± 0.87%) than those of control groups, i.e., the DMBA non-treated group (2.28 ± 0.29%) and the DMBA-treated group (2.32 ± 0.28%). Bromodeoxyuridine (BrdU) immunostaining of mammary tissue revealed that genistein reduced proliferation of the mammary epithelial, and the number of cells stained positive for BrdU both in DMBA-treated groups and DMBA non-treated groups. H&E staining of mammary epithelium also showed that the exposure to genistein decreased proliferation of the mammary epithelium. The epithelium in the rats treated with DMBA showed mostly multiple cell layers, in contrast to the mostly double layer shown in the DMBA non-treated rats. The exposure to genistein in the prepubertal period inhibited mammary epithelial cell proliferation. In conclusion, the ²H₂O labeling results were in good agreement with the results of BrdU incorporation and histomorphometry, which demonstrates that ²H₂O labeling can be used as a tool to measure carcinogenesis.     

Optimization and quality assessment of the post-digestion O labeling based on urea for protein denaturation by HPLC/ESI-TOF mass spectrometry

The post-digestion ¹⁸O labeling method decouples protein digestion and peptide labeling. This method allows labeling conditions to be optimized separately and increases labeling efficiency. A common method for protein denaturation in proteomics is the use of urea. Though some previous studies have used urea-based protein denaturation before post-digestion ¹⁸O labeling, the optimal ¹⁸O labeling conditions in this case have not been yet reported. Present study investigated the effects of urea concentration and pH on the labeling efficiency and obtained an optimized protocol. It was demonstrated that urea inhibited ¹⁸O incorporation depending on concentration. However, a urea concentration between 1 and 2 M had minimal effects on labeling. It was also demonstrated that the use of FA to quench the digestion reaction severely affected the labeling efficiency. This study revealed the reason why previous studies gave different optimal pH for labeling. They neglect the effects of different digestion conditions on the labeling conditions. Excellent labeling quality was obtained at the optimized conditions using urea 1–2 M and pH 4.5, 98.4 ± 1.9% for a standard protein mixture and 97.2 ± 6.2% for a complex biological sample. For a 1:1 mixture analysis of the ¹⁶O- and ¹⁸O-labeled peptides from the same protein sample, the average abundance ratios reached 1.05 ± 0.31, demonstrating a good quantitation quality at the optimized conditions. This work will benefit other researchers who pair urea-based protein denaturation with a post-digestion ¹⁸O labeling method.     

DIURNAL VARIATION IN THE LABELING INDEX OF MOUSE EPIDERMIS: A DOUBLE ISOTOPE AUTORADIOGRAPHIC DEMONSTRATION OF CHANGING FLOW RATES

A diurnal rhythmicity in the labeling index was observed in the epidermis of hairless mice, injected with either ¹⁴C- or ³H-thymidine, at different times during a 24 hr period. A modified autoradiographic technique, using ¹⁴C- and ³H-thymidine and two overlying emulsion layers, makes it possible to clearly differentiate synthesizing cells which are singly labeled with either carbon-14 or tritium, and cells labeled with both isotopes. At various times during a 24 hr period, hairless mice were injected with thymidine-2-¹⁴C and colcemid, followed at 2 or 3 hr by a second injection of ³H-thymidine. The labeling indices were calculated for the ¹⁴C- and ³H-thymidine injection times. These labeling indices were consistent with the control, single isotope, labeling indices and exhibited the same diurnal rhythm. Cells singly labeled with ³H- or ¹⁴C-thymidine have either started or completed DNA synthesis during the interval between the two injections. Flow rates into and out of DNA synthesis, throughout the 24 hr period, can be calculated from these singly labeled cells. The flow rates varied rhythmically throughout the day and paralleled changes in the labeling indices. The influx and efflux flow rates, at all times measured, were not equal. The influx flow rate was reflected in the efflux rate at a time later equal to the duration of S. By means of these flow rates, the per cent of cells in DNA synthesis was calculated for each hour during a 24 hr period. The resulting labeling index curve matches the observed 24 hr diurnal rhythm in labeling indices. By extension of these flow rates through mitosis, the resulting mitotic index curve is comparable to the reported 24 hr diurnal rhythm in mitotic indices.     

13CO2示踪不同化学形态氮素添加对高寒草甸植物光合碳分配的影响

外源氮素(N)输入陆地生态系统后会引起植物和土壤各碳库的变化,但是对不同化学形态氮素的长期输入如何影响光合碳在植物组织、土壤、土壤呼吸中的分配及转运知之甚少,尤其是对于氮输入引起光合碳分配变化进而作用于植物和土壤碳库的机制的认识还非常匮乏。基于在青藏高原矮嵩草草甸开展的不同化学形态氮素添加的长期实验,利用~(13)C示踪方法揭示了光合碳在植物地上、地下组织的分配,及其随时间在土壤中的滞留和随土壤呼吸的释放。研究结果表明,外源氮素添加10年后,与对照未添加氮素处理相比,氨态氮处理下的地上生物量增加了49.5%,氨态氮处理下的地下生物量增加了111.3%。土壤中滞留的~(13)C整体呈下降趋势,氨态氮处理下的土壤碳库显著高于硝态氮处理下的值。不同处理下的土壤呼吸中~(13)C的滞留量随时间呈指数衰减的变化趋势,其中,硝态氮处理下的~(13)C衰减最快。~(13)C同位素标记后第1天测定植物茎和叶内的~(13)C约占刚刚标定完茎和叶内~(13)C的80%,不同处理之间没有显著性差异。直至标记后的第30天,茎和叶内~(13)C的滞留量约占初始量的30%。硝态氮处理下的值在第21天和第30天显著低于对照和氨态氮处理下的值,表明硝态氮处理下,植物光合固定的碳在短期内迅速输入地下组织和土壤中。这些结果从机理上阐明了植物光合碳分配对不同化学形态氮素长期输入的响应,进而影响到土壤呼吸CO_2的释放,以及对土壤碳库动态的贡献。加深了对高寒草甸土壤有机碳库稳定性维持机制的认识,能够为高寒草地的科学管理以及资源的可持续利用提供理论指导。     

131I Induced Hematological Alterations in Rat Blood: Protection by Zinc

The present study was planned to determine the potential of zinc in attenuating the toxicity induced by ¹³¹I in rat blood. Female wistar rats were segregated into four main groups. Animals in Group I served as normal controls; Group II animals were administered a dose of 3.7 Mbq of ¹³¹I (carrier free) intraperitoneally, Group III was supplemented with Zinc in the form of ZnSo₄.7H₂O (227 mg/l drinking water), and Group IV was given a combined treatment of Zinc as well as ¹³¹I, in a similar way as was given to Groups IV and II animals, respectively. The effects of different treatments were studied on various parameters in rat blood including hemoglobin (Hb) levels, % hematocrit, zinc protoporphyrins (ZPP), activities of enzymes which included aminolevulinic acid dehydratase (δ-ALAD) and Na⁺ K⁺ ATPase and uptake of ⁶⁵Zn in blood. The study revealed an increase in the levels of hemoglobin, % hematocrit, activities of δ-ALAD, Na⁺ K⁺ ATPase and uptake of ⁶⁵Zn, 7 days after the ¹³¹I treatment. On the contrary, the levels of ZPP were found to be significantly decreased after ¹³¹I treatment. However, zinc treatment to ¹³¹I-treated animals significantly attenuated the various biochemical and hematological indices. Moreover, zinc treatment to the ¹³¹I-treated animals could significantly decrease the uptake of ⁶⁵Zn, which was increased after ¹³¹I treatment. Based upon these data, the present study suggests that zinc has the potential to attenuate ¹³¹I induced toxicity by restoring the altered hematological indices and biochemical changes.     

Evaluation of isotope discrimination in C-based metabolic flux analysis

In a ¹³C experiment for metabolic flux analysis (¹³C MFA), we examined isotope discrimination by measuring the labeling of glucose, amino acids, and hexose monophosphates via mass spectrometry. When Escherichia coli grew in a mix of 20% fully labeled and 80% naturally labeled glucose medium, the cell metabolism favored light isotopes and the measured isotopic ratios (δ¹³C) were in the range of −35 to −92. Glucose transporters might play an important role in such isotopic fractionation. Flux analysis showed that both isotopic discrimination and isotopic impurities in labeled substrates could affect the solution of ¹³C MFA.