An autoradiographic and morphological study of mouse bone marrow littoral cells during and after treatment with urethane.

This study demonstrates that the labeling index of mouse marrow littoral cells can be markedly altered as a result of treatment with ethyl carbamate (urethane). Young C57/BL mice were given daily intraperitoneal urethane injections for periods up to 6 days. Following treatment each day, as well as daily over a 10 day recovery period, a group of animals was administered tritiated thymidine every 3 hr over a 24 hr period and sacrificed 1/2 hr after the last injection. Marrow was embedded in epon and 0-5 mum sections cut for autoradiographic and light microscopic analysis. A thirty-five-fold increase in the percentage labeled littoral cells was observed after three injections of urethane. The labeling index of littoral cells fluctuated during the treatment period and during the 10 day recovery period. The albeling data are discussed in relation to the possible effects of urethane on the cell cycle of mouse marrow littoral cells; the morphological sequela to urethane treatment and the possibility that littoral cells may act as stem cells.     

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.     

KINETICS OF CELL RENEWAL, CELL MIGRATION AND CELL LOSS IN THE HAIRLESS MOUSE DORSAL EPIDERMIS

Forty hairless mice were given injections of tritiated thymidine every 4th hour during 10 days. At 24 hr intervals groups of four mice were killed. The numbers of labelled basal and differentiating cells were determined by autoradiography with a stripping film technique. To determine the background activity skin sections from uninjected control mice were subjected to the same stripping film procedure. Another group of hairless mice was given one single pulse labelling with tritiated thymidine. The number of labelled mitoses was scored for 12 hr after the injection. At 10, 12 and 15 hr after the injection, the numbers of labelled basal and differentiating cells were also determined. A mathematical model of cell population kinetics in the epidermis has been suggested. The results of different simulations on this model were compared with the observed results. The curve of mean grain counts under continuous labelling increased from day to day with two well-defined plateaux. The percentage of all labelled cells increased rapidly up to the 3rd day, and thereafter the curves gradually flattened off. When basal cells and differentiated cells were considered separately the labelling index of the basal cells increased rapidly for the first 3 days and then flattened off at the 100% level on the 5th day. The labelling index of the differentiating cells was low during the first 3–4 days. Then a steep increase in the percentage of labelled differentiating cells was seen, but the curve flattened off again close to the 100 % level after the 7th day. The labelled mitosis curve had its maximum 5 hr after the thymidine injection. The curve fell again to almost zero at 12 hr. Ten, 12 and 15 hr after the injection, 6, 7 and 7% respectively of the labelled cells were found in the spinous layer. It was concluded that three grains over each nucleus could be used as lower limit for considering a cell as labelled. On this basis, tritiated thymidine injections every 4th hour can be considered as continuous labelling.     

Population dynamics of "null" and Thy1lo lymphocytes in mouse bone marrow: genesis of cells with natural killer cell lineage characteristics.

The population dynamics of "null" small lymphocytes lacking B and T lineage markers in mouse bone marrow have been examined using a combination of immunolabeling and hydroxyurea (HU) deletion techniques. The binding of the B lineage-associated mAb, 14.8, and anti-Thy1.2 to bone marrow cells has been detected radioautographically. Null cells lacking 14.8 and Thy1.2 determinants (14.8- Thy1-) formed a substantial subset (12-14%) of bone marrow small lymphocytes, representing 0.5 x 10(6) cells per femur (2-3% of nucleated cells). HU treatment revealed an exceptionally rapid turnover of the null small lymphocyte population (T1/2, 7.5 hr) compared with 14.8+ cells (T1/2, 20.5 hr) and Thy1+ cells (T1/2, 53 hr). Small lymphocytes bearing low intensities of Thy1 (Thy1lo) were also rapidly renewed (T1/2, 28 hr) whereas those with high intensities of Thy1 (Thy1hi) were renewed only slowly (T1/2, 123 hr). During ontogeny, null small lymphocytes first appeared in the fetal liver by Day 11 and the fetal spleen by Day 16, but increased rapidly in the bone marrow in early postnatal life. Double immunolabeling techniques demonstrated that 10% of null small lymphocytes in the bone marrow expressed NK1.1 antigen, while larger proportions bound to tumor (YAC.1) cells in vitro and displayed Fc receptors. The NK1.1-bearing fraction of null small lymphocytes in bone marrow was depleted by HU treatment only after an initial delay. NK1.1 was also expressed on subsets of Thy1lo cells and Thy1hi cells. The results have revealed the continuous production in mouse bone marrow of null and Thy1lo small lymphocytes, totaling 1-3 x 10(7) cells/day and 1.2 x 10(6) cells/day, respectively. The findings suggest that the large-scale production of null lymphocytes in mouse bone marrow includes the genesis of NK lineage cells which express NK1.1 and Thy1lo during a period of terminal maturation.     

Differentiation of lymphocytes in mouse bone marrow. II. Kinetics of maturation and renewal of antiglobulin-binding cells studied by double labeling

DNA labeling by ³H-thymidine in vitro and antiglobulin-¹³¹I binding in vitro were used to determine the development and turnover of immunoglobulin-bearing lymphocytes in mouse bone marrow.Bone marrow cells from CBA mice previously injected repeatedly with ³H-thymidine for 1–84 hr were exposed to ¹³¹I-labeled rabbit-antimouse globulin for 30 min at 0 °C, and examined radioautographically. The antiglobulin-binding cells in bone marrow were predominantly (97–98%) nondividing small lymphocytes. Some plasmacytoid and monocytoid cells, but not the proliferating large lymphoid cells, also bound antiglobulin. The ³H-thymidine labeling index of the small lymphocyte population showed a rapid exponential increase (50% in 32 hr). The first small lymphocytes to show ³H-thymidine labeling were those lacking antiglobulin-binding capacity, reaching approximately 90% ³H-thymidine labeling after 2 days. Small lymphocytes which bound antiglobulin-¹³¹I at a concentration of 1.0 μg/ml became labeled with ³H-thymidine only after a lag of approximately 1.5 days. More avid antiglobulinbinding cells were delayed a further 12 hr in ³H-thymidine labeling. During in vitro culture the proportion of antiglobulin-binding small lymphocytes increased progressively in bone marrow but decreased in spleen cell suspensions.The results demonstrate a continuous, rapid renewal of immunoglobulin-bearing small lymphocytes in adult mouse bone marrow. Surface immunoglobulin molecules are not detectable when marrow small lymphocytes are first formed, but they appear and increase progressively in density as the cells mature.     

LYMPHOCYTE POPULATIONS IN MOUSE BONE MARROW: QUANTITATIVE KINETIC STUDIES IN YOUNG, PUBERTAL AND ADULT C3H MICE

Continuous ³H-thymidine infusion was used to characterize two kinetic subpopula-tions of small lymphocytes in mouse bone marrow during normal growth and development. Young (4 wk), pubertal (8 wk) and mature (16 wk) C3H mice were infused subcutaneously with ³H-thymidine for periods up to 10 days. Femoral marrow was then examined in radioautographic smears. During the first 3 days the proportion of marrow small lymphocytes labelled by ³H-thymidine showed a rapid exponential increase to 93%, 81% and 72% in 4 wk, 8 wk and 16 wk mice respectively. The rate of appearance of labelled small lymphocytes then declined markedly but remained higher in younger than in older animals. The labelling curves were found to represent the summation of two exponential curves from which the proportions and renewal rate of corresponding cell populations were calculated. Most marrow small lymphocytes comprised a rapidly renewing population but in mice of increasing age the relative incidence of these cells fell (93-3% at 4 wk; 88-0% at 8 wk; 78-5% at 16 wk) and their half-renewal time (T_½) lengthened (14 hr at 4 wk; 18 hr at 8 wk; 24 hr at 16 wk). The remaining small lymphocytes were slowly renewing with mean T_½ of 4, 7 and 14 days in 4, 8 and 16 wk mice, respectively. Some heavily labelled small lymphocytes persisted in the marrow up to 10 wk after fourteen daily ³H-thymidine injections in 10–12 wk mice. The numbers of rapidly renewing cells decreased from 604 times 10³ to 228 times 10³ per mm³ of marrow from 4 wk to 16 wk, respectively, while slowly renewing cells increased from 44 times 10³ to 61 times 10³ per mm³. The total number of nucleated marrow cells per femur increased from 4 wk to 16 wk but the rapidly renewing small lymphocytes per femur fell in numbers by 36% and in renewal rate by 63%. The results demonstrate a selective change in bone marrow small lymphocytes with age; rapidly renewing cells decline in number and renewal rate while the number of slowly renewing cells increases. The concept of bone marrow as a primary lymphoid organ is discussed.     

AUTORADIOGRAPHIC STUDY OF TYPE II-CELL HYPERPLASIA IN LUNGS OF MICE CHRONICALL EXPOSED TO URETHANE

Hyperplasia of pulmonary alveolar epithelium was induced by exposure of adult mice to 0–1% urethane in drinking water, and their lungs were analysed using combined autoradiographic and morphometric methods. The response of pulmonary epithelium showed three phases: an initial one of 3 weeks in which the number of type II cells was consistently low and, after a transient decrease in ³H-thymidine labeling index, the latter steadily increased. Degenerating or dead type II cells were found, consequently this period was considered one in which cell death was disproportionately increased over cell production. Between the 3rd and 6th weeks the number of type II cells doubled, and thereafter increased at a slower rate. The ³H-thymidine labeling index reached its peak at 6 weeks. The third phase was marked by a decline in labeling index which returned to near-normal levels by the 16th week. The number of type II cells declined slowly after the 10th week and was still elevated at the end of the observation period. Tumors consisting of type II cells continued to arise even during the period of declining labeling index. The dissociation between the proliferative response which was reversible, and the neoplastic response which was not, supports the assumption that the major part of the observed population growth or hyperplasia induced by urethane was a form of regeneration repair secondary to cell death and may be unrelated to tumor growth.     

ALTERATION OF NEONATAL RAT PAROTID GLAND ACINAR CELL PROLIFERATION BY GUANETHIDINE-INDUCED SYMPATHECTOMY

Newborn rats were injected with guanethidine-sulfate (20 μg/g body weight) every 48 hr from 12 hr after birth until day 14 (eight injections per animal). The guanethidine treatment resulted in an 86% absolute reduction in cell number in the superior cervical ganglia of 15 day old rats. The cells which remained after guanethidine treatment showed destruction of mitochondria and an extensive decrease in endoplasmic reticulum. Chemical sympathectomy with guanethidine induced a 3.1 hr lengthening of the acinar cell generation cycle time (17.4 hr to 20.5 hr), resulting from a longer G₁ period (6.9 hr in the control group as compared to 10.5 hr in the guanethidine-treated group), as well as a decrease in the mean percentage of [³H]thymidine-labeled acinar cells (22.3 ± 0.5% to 19.3 ± 0.5%) and mean acinar cell mitotic index (2.6 ± 0.2% to 2.1 ± 0.1%). A circadian rhythm was found to exist in parotid gland acinar cell mitotic activity of 15 day old rats and the amplitude of the rhythm was reduced from 26.5% to 14.9% in guanethidine-treated rats. This study indicates that the diminution of sympathetic influence on the developing parotid gland results in a slight, but significant alteration in acinar cell proliferation.     

DEVELOPMENT OF VAGINAL EPITHELIUM SHOWING IRREVERSIBLE PROLIFERATION AND CORNIFICATION IN NEONATALLY ESTROGENIZED MICE: AN ELECTRON MICROSCOPE STUDY *

Irreversible proliferation and cornification of the mouse vaginal epithelium were induced by 10 daily injections of 20μg estradiol-17β starting on the day of birth. Development of the irreversible vaginal epithelium during the period of estrogen injections in early postnatal life was observed under light and electron microscopes. Small electron-dense cells (A-cells) in clusters were present in the columnar vaginal epithelium of newborn mice. A-cells were proliferated by 2 daily estrogen injections. At the sites of A-cell clumps, large electron-dense cells (B-cells) characterized by long winding cytoplasmic processes appeared in mice given 3 daily injections, forming nodules which then fused together to form a layer under the columnar epithelium after 4 daily injections. In mice given 7 daily injections, the primary epithelium was shed by the superficial cornification of the newly formed layer. The B-cell membrane bore fewer desmosomes than in the basal and intermediary cells of the vaginal epithelium of ovariectomized ‘normal’ adult mice after 5 daily injections of 100μg estradiol-17β. Hyperplastic epithelial downgrowths in old ovariectomized mice given neonatal estrogen injections contained another type of cells with reduced density which formed much fewer processes and only a few desmosomes (C-cells).     

Dose-Dependent Cytokinetic Changes Following 1-β-D-Arabinofuranosylcytosine and Hydroxyurea In L1210 and S-180 In Vivo

DNA synthesis inhibition and recovery in L1210 and S-180 ascites tumors following 1-β-D-arabinofuranosylcytosine (Ara-C) and hydroxyurea (HU) were measured autoradiographically as a basis for optimizing drug schedules. Tumor bearing mice, 10⁶ cells day O, were treated on day 4 with 20, 200 or 2000 mg/kg Ara-C or 50, 300 or 1800 mg/kg HU. At various intervals following drug, [³H]thymidine was administered i.p. and mice were killed 1 hr later. Tumor cells were analyzed for labeling index (LI) and grain count (GC) to determine the percentage of cells in S phase and the distribution of DNA synthesis rates among the labeled cells, respectively. Following each dose of HU, DNA synthesis was inhibited completely. Recovery of LI was rapid and approached control values by 6 hr. Following each dose of Ara-C, DNA synthesis was inhibited completely for at least 6 hr. Recovery of LI was first noted 6 hr following 20 mg/kg Ara-C and 9 hr following 200 mg/kg. Following both doses the LI reached 100% of the control value by 26 hr. GC analysis indicated that following Ara-C treatment, DNA synthesis was reinitiated first with cells with low GC from 6 to 12 hr followed by cells with increasing GC from 12 to 20 hr. the labeling intensity reached control values by 20 hr and an ‘overshoot’ occurred by 26 hr. These data suggest that the recovery of DNA synthesis rate is a gradual process. Survival data for mice receiving two doses of Ara-C indicated that the optimal interval for retreatment following the lower dose of Ara-C occurred by 6 hr as compared to 12–16 hr for the higher dose. These times coincided in both instances with recovery of LI to 33–50% of control values. Early recovery of LI may be the best method currently available for estimating the optimal time for retreatment with an S phase specific drug.     

Radioprotective effects of serum thymic factor in mice.

Serum thymic factor (FTS) reduced mortality of mice after total-body irradiation with 7.56 Gy X rays. The radioprotective effect was achieved by daily repeated subcutaneous injections of 3-100 micrograms FTS, while doses higher than 300 micrograms/day/mouse were neither radioprotective nor toxic. Similarly, degeneration of the spleen was moderated by 3-100 micrograms FTS but not by 500 micrograms FTS in sublethally (3.78 Gy) irradiated mice. Histological examination showed that hematopoiesis was enhanced in the spleen by daily injections of 10 micrograms FTS. Spleen cells from the FTS-treated mice incorporated more [3H]thymidine in culture with or without concanavalin A. The treatment with FTS increased the production of colony-stimulating factor in the spleen as well as in peritoneal macrophage-like cells, and caused a significant increase in the number of granulocyte-macrophage colony-forming cells both in the spleen and in the femoral bone marrow. Furthermore, FTS prevented a decrease in circulating neutrophils in the sublethally irradiated mice. Prominent overshoot recovery of myelopoiesis, which occurred occasionally in sublethally irradiated mice, did not occur in the FTS-treated mice. The decrease in blood erythrocytes was also significantly reduced. These observations imply that this thymic hormone has potential as a radioprotector.     

Dose-dependent cytokinetic changes following 1-beta-D-arabinofuranosylcytosine and hydroxyurea in L1210 and S-180 in vivo.

DNA synthesis inhibition and recovery in L1210 and S-180 ascites tumors following 1-beta-D-arabinofuranosylcytosine (Ara-C) and hydroxyurea (HU) were measured autoradiographically as a basis for optimizing drug schedules. Tumor bearing mice, 10(6) cells day 0, were treated on day 4 with 20, 200 or 2000 mg/kg Ara-C or 50, 300 or 1800 mg/kg HU. At various intervals following drug, [3H]thymidine was administered i.p. and mice were killed 1 hr later. Tumor cells were analyzed for labeling index (LI) and grain count (GC) to determine the percentage of cells in S phase and the distribution of DNA synthesis rates among the labeled cells, respectively. Following each dose of HU, DNA synthesis was inhibited completely. Recovery of LI was rapid and approached control values by 6 hr. Following each dose of Ara-C, DNA synthesis was inhibited completely for at least 6 hr. Recovery of LI was first noted 6 hr following 20 mg/kg Ara-C and 9 hr following 200 mg/kg. Following both doses the LI reached 100% of the control value by 26 hr. GC analysis indicated that following Ara-C treatment, DNA synthesis was reinitiated first with cells with low GC from 6 to 12 hr followed by cells with increasing GC from 12 to 20 hr. The labeling intensity reached control values by 20 hr and an 'overshoot' occurred by 26 hr. These data suggest that the recovery of DNA synthesis rate is a gradual process. Survival data for mice receiving two doses of Ara-C indicated that the optimal interval for retreatment following the lower dose of Ara-C occurred by 6 hr as compared to 12--16 hr for the higher dose. These times coincided in both instances with recovery of LI to 33--50% of control values. Early recovery of LI may be the best method currently available for estimating the optimal time for retreatment with an S phase specific drug.     

DYNAMICS OF LEUKOCYTE MIGRATION INTO THE MOUSE ASCITES TUMOR

There is a marked increase in the number of peritoneal leukocytes (lymphocytes, monocytes and granulocytes) during the growth of Ehrlich ascites tumor in mice. No local proliferation (as indicated by a labeling at 1 hr following a single ³H-TdR injection) was observed in the normal peritoneal leukocytes or those in the ascites tumor, except for a very minor labeling of some tumor macrophages. Kinetics of peritoneal leukocytes was studied with a series of twelve injections of ³H-thymidine (20 μCi every 8 hr) in normal mice as well as mice injected with 10⁶ tumor cells i.p. 2 hr after the last ³H-TdR injection. Animals were sacrificed at intervals up to 6 days. Granulocyte labeling in the blood as well as peritoneal space was near 100% in both groups of animals at all the intervals. Temporal changes in the labeling of lymphocytes (from 10% at 0 day to 22% at day 6), and monocytes (from 20% at 0 day to 57% at day 6) were identical in the blood and peritoneal space of normal animals, indicating a free exchange of cells between these compartments. Higher labeling indices than those in the controls were attained in the blood of tumor-bearing hosts (viz 40% for lymphocytes and 80% for monocytes at 6 days) suggesting an increased turnover of these cells in the circulation. In addition, peritoneal mononuclear cells of tumor-bearing mice showed even a higher labeling than those in the blood (viz 65% for lymphocytes and 92% for monocytes at 6 days) indicating a selective migration and/or retention of newly formed cells within the tumor, in contrast to a random migration into the normal peritoneal cavity. Furthermore, an identical labeling of macrophages to that of monocytes within the tumor indicated a short monocyte-macrophage transition. The preferential accumulation of young mononuclear cells into the tumor may be of functional importance.     

HYDROXYUREA EFFECTS IN THE C3H MOUSE II. MAMMARY TUMOR CELL KINETICS

The cellular kinetics of C3H mouse mammary tumors were studied following a single dose (3 mg/g body weight) of hydroxyurea (HU). This dose was large enough to cause a significant perturbation in the growth curves of these tumours. This was accomplished by labeling the cells with tritiated 5-iodo-2'-deoxyuridine and performing detailed autoradiographic analysis. This dose of HU caused a temporary inhibition in growth and completely inhibited DNA synthesis for 4–5 hr. The HU-killed cells (pyknotic and karyorrhectic) reach a maximum around 10–12 hr and are apparently all removed in about 1 day. Tumors from a fast-growing line (S102F) showed some evidence for cell synchrony upon recovery from HU inhibition but desynchronization occurred within one cell cycle. The cell generation time was not decreased during the acute recovery phase, but the growth fraction shifted from 0·6 to 1·0, and the data suggested that the normal flow of cells from the proliferating pool to the degenerate pool was temporarily interrupted. The cellular kinetic parameters have probably returned to normal by 48 hr after the HU injection.     

Flow cytometric analysis of adriamycin-perturbed mouse mammary tumors.

The effects of a single intraperitoneal injection of adriamycin (10 mg/kg) on a fast-growing C3H mouse mammary tumor (S102F) have been analyzed volumetrically, biochemically, autoradiographically and flow cytometrically. Mathematical simulation of the data was also used to aid in the interpretation of the recovery kinetics. This dose of adriamycin did not induce regression in tumor volume but did inhibit the growth rate for 4-5 days. 3H-TdR incorporation was gradually inhibited to reach a low of 20% of control at 24 and 36 hr and then recovered back to control by 96 hr after adriamycin treatment. The flow cytometric analysis also showed a marked reduction in the relative fraction of cells in the S-phase with a minimum of 23% of control at 72 hr; however, in contrast to the 3H-TdR incorporation data, the fraction of cells in the S-phase was only at 39% of control at 96 hr after the adriamycin injection. Since the 3H-TdR incorporation data disagreed with the flow cytometry data, autoradiographic analysis was also done at selected times after the adriamycin injections, and qualitatively, this analysis confirms the flow cytometry data in that the labeling index was 29% of control at 96 hr after adriamycin. The mitotic index also dropped from 8 to 1%, respectively, for controls and at 96 hr posttreatment. The degenerate index was about 1% in control tumors and no increase was observed in treated tumors. Adriamycin-induced cell-cycle delay occurs predominately in G1 and G2 but there is also an apparent minor delay in the transit across the S-phase and some apparent cytotoxicity in G2 and/or M. The long delay in volumetric growth appears to be due to the extended cell-cycle delay rather than extensive cell killing.     

Myelosuppression in the Pig (Sus scrofa): Uteroferrin Reduces the Myelosuppressive Effects of 5-Fluorouracil in Young Pigs

The present study investigated the ability of uteroferrin to modulate the myelosuppressive effects of 5-fluorouracil (5-FU) in young pigs (Sus scrofa). Pigs (28–35 days of age; n = 6 per treatment) were infused with equal amounts of 5-FU on days 0 and 1 of the experimental period (37.5 mg/kg cumulative dose). Uteroferrin (100 μg/kg in 0.9% NaCl) or control (equivalent volume of 0.9% NaCl) was administered to pigs as intramuscular injections twice daily (08:00 and 20:00 hr) on days 1 through 21. Peripheral blood cell number, composition and progenitor cells were determined over 28 days. Treatment of pigs with 5-FU resulted in a rapid dose-dependent (P < 0.05) leukocytopenia. Concurrent treatment of pigs with uteroferrin reduced (P < 0.05) the rate of 5-FU-induced leukocytopenia (44 vs 77 ± 7% decline from baseline on day 3) and enhanced (P < 0.05) the recovery from 5-FU on days 10 and 12 postinfusion. The positive effect of uteroferrin on leukocytes resulted primarily from a protection and/or enhanced recovery of neutrophils and monocytes. In addition, uteroferrin attenuated (P < 0.05) the suppression of red blood cell numbers after 5-FU administration (6.9 vs 6.1 ± 0.2 × 10⁶ cells/μl on day 3), an affect reflected in increased hematocrit and hemoglobin concentrations. The effects of uteroferrin appeared to result from enhancement of the proliferation and/or differentiation of primitive pluripotent stem cells resistant to 5-FU, as concurrent treatment of pigs with uteroferrin resulted in a protection and/or enhanced recovery (P < 0.05) of CFU-GEMM, CFU-GM and BFU-E progenitor cells in the peripheral blood. These results are the first to demonstrate that uteroferrin can reduce the myelosuppressive effects of 5-FU in the pig and suggest that uteroferrin has hematopoietic growth factor activity in vivo.     

Clastogenic activity of urethane in mice

Single intraperitoneal (i.p.) treatment of male and female BDF1 (C57B1 × DBA2) mice with urethane (0.5 or 1.0 g/kg) caused a significant increase in micronucleated polychromatic erythrocytes (MNPCE) in bone marrow after 24 h. The clastogenic effect observed was dose-, sex- and age-dependent, the male and younger (6–8 weeks old) animals being more susceptible than the female and older (6 months of age) mice. 3-week oral treatment of female Balb/c mice with urethane (3 g/l added to the drinking water) caused an up to 4-fold increase in the number of micronucleated normochromatic erythrocytes (MNNCE) in mouse peripheral blood. In a month after the carcinogen treatment was stopped, the number of MNNCE dropped to the control values. In addition, a single i.p. treatment of pregnant BDF1 mice on day 17 of gestation with urethane (1.0 g/kg) caused a 514.3% (p < 0.001) elevation of MNPCE in mouse fetal liver after 24 h as well as a 154.4% (p < 0.05) increase in MNPCE frequency in the fetal peripheral blood. At this time point, the clastogenic response in mouse fetal liver erythroblasts was less pronounced than that detected in the maternal bone marrow cells. Urethane is a strong clastogen in mice when administered either intraperitoneally or orally and the micronucleus test applied to adult and fetal erythroblasts is a convenient method of choice for studying the acute and subchronic clastogenicity of this carcinogen, its transplacental effects as well as the influence of modifying factors on these processes.     

Synchronization of estrus in post-partum mares with progesterone and estradiol 17β

Thirty-six mares which foaled over a 10-day period were given 1 to 10 daily intramuscular injections of a combination of 150 mg. progesterone and 10 mg. estradiol 17β. The first injection was given within 18 hours after parturition. Because individual mares foaled on different dates during the 10 day period, commencement of treatment varied, but treatment for all mares ceased on the same day. Teasing and breeding began seven days after the final treatment. The mares were teased daily for 10 days and artifically inseminated every second day until ovulation occurred. The mean interval from the end of treatment to beginning of estrus was 9.4 days (range 7 to 14) and 33 of 26 mares (94.7%) ovulated 10 to 16 days after the final treatment. Both estrus and ovulation were effectively synchronized, resulting in a first estrus pregnancy rate of 80.6% (29 of 36).     

Rat Macrophages in Experimental IgA Nephropathy

To test whether the peripheral macrophage functions as an early index of oxygen free radical release in association with the development of IgA nephropathy (IgAN), we studied female Lewis rats. IgAN was produced by treatment over 8 weeks with 0.1% bovine gamma globulin (BGG) in drinking water, followed by three daily intravenous injections of BGG, 1 mg/dose. Fifteen rats were divided randomly into three groups: control, IgAN, and IgAN fed vitamin E 100 IU/kg chow. At the end of the treatment period, rats were placed in individual metabolic cages for 24-h urine collections and then anesthetized with Inactin (100 mg/kg BW) for aspiration of peritoneal macrophages. The results (means ± SD) extended our previous data in male rats, confirming that the elevated proteinuria of IgAN (3.62 ± 0.79 mg/day) was significantly reduced with vitamin E treatment (2.59 ± 0.28 mg/day) in female rats (P< 0.002) More importantly, we indicated for the first time that oxygen free radicals' production by peritoneal macrophages in IgAN was significantly reduced by vitamin E: 1.58 ± 0.91 nmol/10⁶cells/15 min in the untreated group vs 3.28 ± 0.54 nmol/10⁶cells/15 min in the vitamin E-treated group (P< 0.05).     

NUCLEOCYTOPLASMIC RATIO REQUIREMENTS FOR THE INITIATION OF DNA REPLICATION AND FISSION IN TETRAHYMENA

Hydroxyurea (10 mM) arrests the exponential growth of Tetrahymena by blocking DNA replication during S-phase. After removal of the hydroxyurea (HU), they have a long recovery period during which they are active in DNA synthesis. ³H-TdR uptake showed that on completion of the recovery period, the cells divide (recovery division) and enter a cell cycle which lacks G₁. The frequency, size and DNA content of the extranuclear chromatin bodies (ECB) formed at this division are all markedly increased (2–4) over the corresponding values obtained from exponential growth phase controls. Microspectrophotometric analysis of macronuclear DNA content (N) coupled with the cytoplasmic dry mass (C) values suggest that specific N to C ratios (N/C) are required for the initiation of DNA replication and fission: during a normal (exponential growth) cell cycle, both N and C double, but asynchronously, so that the N/C of both post-fission-daughter cells and pre-fission cells is identical (standardized to N/C = 1) but late G₁ cells have a low N/C. During a 10 hr exposure to HU, the N remains essentially the same whereas the C increases. When the HU is removed, the N increases by 4× and the C continues to increase until just prior to recovery division when it also reaches a value 4× that of the original daughter cells. Thus, the N/C = 1 is re-established. The enlarged ECB formed during recovery division may function to lower the N/C in the daughter cells, which in turn may in some way stimulate immediate DNA replication, thus eliminating G₁. The elimination of G₁ (and shortening in a few subsequent cell cycles) allows less time for cytoplasmic growth and results in the return of the cells to the generation time and the N and C values observed prior to the HU treatment.