The distribution and kinetics of nuclei in rat osteoclasts

Abstract. Osteoclast development and growth were studied by determining the number of labelled nuclei in osteoclasts of different sizes (based on the number of nuclei per osteoclast, N/O) and the number of osteoclasts with labelled nuclei at various intervals after tritiated thymidine ([³H]TdR) injection in young rats. The osteoclast smears were made from the cellular periosteum of the proximal tibia. The frequency distribution of the N/O osteoclasts types in the smears had profiles similar to that of in situ osteoclasts in whole mounts of proximal tibia, which indicates that the osteoclast population of the smears was representative of that on the bone surface. A vast majority of the osteoclasts had a 1–6 N/O, and a number of the cells had as many as 26 or more nuclei. Furthermore, profiles of N/O frequency distributions were similar over the course of the study. Nuclei with [³H]TdR label were initially observed in osteoclasts between 4 and 12 hr after isotope injection. However, fusion of labelled nuclei to osteoclasts continued for at least 150 hr. In general, the labelled osteoclasts exhibited a significantly larger number of nuclei than the unlabelled osteoclasts. The probability of an osteoclast incorporating one or more labelled nuclei increased with time after injection and with an increase in N/O. Labelling intensity decreased with time post injection and with an increase in N/O. The data suggest that turnover of nuclei is more rapid in osteoclasts with high N/O values.     

Influence of Growth Hormone and Thyroxine On Cell Kinetics In the Proximal Tibial Growth Plate of Snell Dwarf Mice

Abstract. In Snell dwarf mice, the influence of short-term treatment with human growth hormone (hGH) or thyroxine on the proliferative and sulphation activity of the proximal tibial growth plate was studied. By autoradiographic methods, the [³H]methylthymidine incorporation after a single injection was measured, after 2 hr incorporation time. the labelling index was calculated and the number of labelled mitoses was counted. In addition, the distribution of the labelled nuclei over the proliferating and degenerating zones was determined by continuous labelling for 25 and 73 hr.
In untreated dwarf mice after [³H]-methylthymidine administration, the number of labelled nuclei in the growth plate is low. Labelling occurs, as expected, mainly in the cells of the proliferative zones. the number of labelled nuclei in control dwarf mice was similar after 25 and 73 hr continuous labelling. This suggests that many cells are in a resting G_o or prolonged G₁ phase. Both hGH and T₄ treatment induce a significant increase of the number of labelled nuclei per growth plate and of the number of mitoses. Since hormonal treatment induces a small number of mitoses after 2 hr incorporation of the label, the minimal G₂ phase of the cell cycle is less than 2 hr. In addition, treatment with hGH and T₄ stimulates chondrocytes in the zone of proliferative and hypertrophic cells to actively incorporate [³⁵S]-sulphate.     

Influence of growth hormone and thyroxine on cell kinetics in the proximal tibial growth plate of Snell dwarf mice

In Snell dwarf mice, the influence of short-term treatment with human growth hormone (hGH) or thyroxine on the proliferative and sulphation activity of the proximal tibial growth plate was studied. By autoradiographic methods, the [3H]methylthymidine incorporation after a single injection was measured, after 2 hr incorporation time. The labelling index was calculated and the number of labelled mitoses was counted. In addition, the distribution of the labelled nuclei over the proliferating and degenerating zones was determined by continuous labelling for 25 and 73 hr. In untreated dwarf mice after [3H]-methylthymidine administration, the number of labelled nuclei in the growth plate is low. Labelling occurs, as expected, mainly in the cells of the proliferative zones. The number of labelled nuclei in control dwarf mice was similar after 25 and 73 hr continuous labelling. This suggests that many cells are in a resting G0 or prolonged G1 phase. Both hGH and T4 treatment induce a significant increase of the number of labelled nuclei per growth plate and of the number of mitoses. Since hormonal treatment induces a small number of mitoses after 2 hr incorporation of the label, the minimal G2 phase of the cell cycle is less than 2 hr. In addition, treatment with hGH and T4 stimulates chondrocytes in the zone of proliferative and hypertrophic cells to actively incorporate [35S]-sulphate.     

Effects of pulse labelling with tritiated thymidine on the circadian rhythmicity in epidermal cell-cycle distribution in mice

The influence of pulse labelling with 50 microCi tritiated thymidine ( [3H]TdR) (2 microCi/g) on epidermal cell-cycle distribution in mice was investigated. Animals were injected intraperitoneally with the radioactive tracer or with saline at 08.00 hours, and groups of animals were sacrificed at intervals during the following 32 hr. Epidermal basal cells were isolated from the back skin of the animals and prepared for DNA flow cytometry, and the proportions of cells in the S and G2 phases of the cell cycle were estimated from the obtained DNA frequency distributions. The proportions of mitoses among basal cells were determined in histological sections from the same animals, as were the numbers of [3H]TdR-labelled cells per microscopic field by means of autoradiography. The results showed that the [3H]TdR activity did not affect the pattern of circadian rhythms in the proportions of cells in S, G2 and M phase during the first 32 hr after the injection. The number of labelled cells per vision field was approximately doubled between 8 and 12 hr after tracer injection, indicating an unperturbed cell-cycle progression of the labelled cohort. In agreement with previous reports, an increase in the mitotic index was seen during the first 2 hr. These data are in agreement with the assumption that 50 microCi [3H]TdR given as a pulse does not perturb cell-cycle progression in mouse epidermis in a way that invalidates percentage labelled mitosis (PLM) and double-labelling experiments.     

Effects of Pulse Labelling With Tritiated Thymidine On the Circadian Rhythmicity In Epidermal Cell-Cycle Distribution In Mice

The influence of pulse labelling with 50 °Ci tritiated thymidine ([³H]TdR) (2 μCi/g) on epidermal cell-cycle distribution in mice was investigated. Animals were injected intraperitoneally with the radioactive tracer or with saline at 08.00 hours, and groups of animals were sacrificed at intervals during the following 32 hr. Epidermal basal cells were isolated from the back skin of the animals and prepared for DNA flow cytometry, and the proportions of cells in the S and G₂ phases of the cell cycle were estimated from the obtained DNA frequency distributions. the proportions of mitoses among basal cells were determined in histological sections from the same animals, as were the numbers of [³H]TdR-labelled cells per microscopic field by means of autoradiography. The results showed that the [³H]TdR activity did not affect the pattern of circadian rhythms in the proportions of cells in S, G₂ and M phase during the first 32 hr after the injection. the number of labelled cells per vision field was approximately doubled between 8 and 12 hr after tracer injection, indicating an unperturbed cell-cycle progression of the labelled cohort. In agreement with previous reports, an increase in the mitotic index was seen during the first 2 hr. These data are in agreement with the assumption that 50 °Ci [³H]TdR given as a pulse does not perturb cell-cycle progression in mouse epidermis in a way that invalidates percentage labelled mitosis (PLM) and double-labelling experiments.     

Comparison of morphological effects of PGE2 and TGFβ on osteoclastogenesis induced by RANKL in mouse bone marrow cell cultures

RANKL, in the presence of M-CSF, induces the development and fusion of TRAP+ osteoclasts in mouse bone marrow cultures at 3–5 days. Early during culture (day 3), most cells are small (up to six nuclei). At lower cell densities, these osteoclasts exhibit a rounded morphology with cytoplasm extending around the cells but, at higher densities, this changes to a stellate morphology with the cytoplasm being retracted around the nuclei with numerous localised cytoplasmic extensions. Under optimal conditions, osteoclast fusion results in conglomerates of many cells, which become large cytoplasmic masses on day 4. PGE2 and TGFβ have both been shown to increase osteoclast development in this model and their effects on the morphology of osteoclasts during fusion and differentiation have been compared under all these conditions. PGE2 or TGFβ increase osteoclast numbers and size and also the number of nuclei, indicating increased osteoclast development and fusion. TGFβ increases the size of rounded osteoclasts (with respect to the number of nuclei) more than PGE2, suggesting that TGFβ increases cytoplasmic extension. TGFβ increases the size and number of nuclei in stellate cells but particularly increases the number and length of the cytoplasmic extensions, in contrast to PGE2. Fusion of these extensions with other osteoclasts results in large networks of interconnected cells. On day 4, spreading cells develop but these are still interconnected by cytoplasmic links, a phenomenon not seen in control wells or after treatment with PGE2. TGFβ is more effective than PGE2 in increasing fusion in the formation of cell conglomerates and cytoplasmic masses. PGE2 decreases overall cell density resulting in additional indirect effects on osteoclast numbers and morphology. However, PGE2 particularly promotes the formation of large mature spreading osteoclasts later during culture.     

Myosin II antibodies inhibit the resorption activity of isolated rat osteoclasts

We present microinjection data in support of an indirect approach by which cytoplasmic protein interactions important in the processes of bone resorption can be elucidated. Three polyclonal antibodies (M1, M3, M5) raised against myosin II from perfused rat liver differently affected the actin-activated Mg ATPase of myosin II. These antibodies microinjected into isolated rat osteoclasts affected osteoclast morphology and activity in bone resorption. M1, which completely inhibited myosin ATPase activity at a antibody:myosin ratio of 10:1, initially promoted the extension/retraction motility of lamellipodia but eventually reduced the spread area of osteoclasts along the substrate after 20 hr. M3, which inhibited ATPase activity by 70%, had similar effects; however, M5, which weakly inhibited ATPase activity, neither promoted extension/retraction nor reduced spread area of osteoclasts. Immunofluorescence showed that these antibodies removed myosin II from the majority of actin filaments in injected osteoclasts. Because antibodies that did not bind to a myosin II column had little effect on the extension/retraction of lamellipodia or the osteoclast spread area, these data suggest that myosin II participates in the stabilization of osteoclast lamellipodia along the substrate. M1 injection strongly inhibited injected osteoclasts from excavating resorption lacunae in bone slices, compared to control antibody. M3 and M5 were less effective but also inhibited bone resorption. These data show that myosin II is functionally important in bone resorption and that the osteoclast-differentiated activity of bone resorption is a more sensitive assay for myosin activity than lamellipodia motility or cell morphology.     

Protective actions of green tea polyphenols and alfacalcidol on bone microstructure in female rats with chronic inflammation

This study investigated the effects of green tea polyphenols (GTP) and alfacalcidol on bone microstructure and strength along with possible mechanisms in rats with chronic inflammation. A 12-week study using a 2 (no GTP vs. 0.5%, w/v GTP in drinking water)×2 (no alfacalcidol vs. 0.05 μg/kg alfacalcidol orally, 5×/week) factorial design was employed in lipopolysaccharide (LPS)-administered female rats. A group receiving placebo administration was used to compare with a group receiving LPS administration only to evaluate the effect of LPS. Changes in tibial and femoral microarchitecture and strength of femur were evaluated. Difference in expression of tumor necrosis factor-α (TNF-α) in proximal tibia using immunohistochemistry was examined. Compared to the placebo group, the LPS-administered-only group had significantly lower femoral mass, trabecular volume, thickness and number in proximal tibia and femur, and lower periosteal bone formation rate in tibial shafts but had significantly higher trabecular separation and osteoclast number in proximal tibia and eroded surface in endocortical tibial shafts. Both GTP and alfacalcidol reversed these LPS-induced detrimental changes in femur, proximal tibia and endocortical tibial shaft. Both GTP and alfacalcidol also significantly improved femoral strength, while significantly suppressed TNF-α expression in proximal tibia. There were significant interactions in femoral mass and strength, trabecular separation, osteoclast number and TNF-α expression in proximal tibia. A combination of both showed to sustain bone microarchitecture and strength. We conclude that a protective impact of GTP and alfacalcidol in bone microarchitecture during chronic inflammation may be due to a suppression of TNF-α.     

Spermatogonial multiplication in the Chinese hamster. IV. Search for long cycling stem cells

In the Chinese hamster, 17 days, i.e. one cycle of the seminiferous epithelium, after two injections of [3H]TdR given 24 hr apart, labelled cells were found among all types of spermatogonia, including stem cells (As). These labelled As spermatogonia derive from one or more self-renewing divisions of the stem cells that originally incorporated [3H]TdR. In the steady state, half of the divisions of the As will be self-renewing and the other half will give rise to Apr spermatogonia that will ultimately become spermatozoa. Theoretically, the labelling index (LI) after 17 days will be similar to that after 1 hr, and in this study twice as high as for the 1-hr interval since only one injection was given. However, experimental values only half that of the theoretical LI were found after 17 days. The following causes for the loss of labelled stem cells are discussed: (1) dilution of label because of division; (2) influx of unlabelled components of false pairs (i.e. newborn stem cells that still have to migrate away, mostly during G1, from their sister cells and are scored as Apr spermatogonia) between 1 hr and 17 days; (3) the existence of long- and short-cycling stem cells, probably combined with preferential differentiation of the short-cycling elements; (4) selective segregation of DNA at stem cell mitosis; and (5) irradiation death of radiosensitive labelled stem cells. As it is not impossible that factors 1, 2, 4 and 5 together account for the total loss of labelled stem cells, LI results do not provide evidence for the existence of separate classes of short- and long-cycling stem cells. The distributions of the LIs of the As, Apr and Aal spermatogonia over the stages of the epithelial cycle at 17 days are similar to those at 1 hr after injection. Hence the regulatory mechanisms that govern the stimulation and inhibition of proliferation of As that give rise to new As for the next epithelial cycle are similar to those of the As that will divide into Apr spermatogonia during the same epithelial cycle. Grain counts revealed that more [3H]TdR is incorporated into As, Apr and Aal spermatogonia that are in S phase during epithelial stages X-IV than in stages V-IX.     

An immunocytochemical method for studying the kinetics of osteoclast nuclei on intact mouse parietal bone

Summary An immunocytochemical method using an antibody against 5-bromo-2-deoxyuridine has been applied to the study of the kinetics of osteoclast nuclei on intact mouse parietal bones. Osteoclasts containing tartrate-resistant acid phosphatase show nuclei that are positive for the thymidine analogue within 24 hours of injection into four-day old mice. Labelled osteoclast nuclei decline in number with a half-life of 1.3 days, compatible with a random mechanism of cell death rather than a fixed lifespan. This is shorter than has previously been reported and the possible reasons for this are suggested. The main advantages compared with autoradiography are the shortened processing time and the large number of osteoclasts that can be examined per parietal bone.     

Dynamics and ultrastructural characteristics of osteoclast formation

Using electron microscopy and 3H-thymidine autoradiography it has been shown that osteoclasts of rats and rabbits are formed by fusion of cell precursors which become members of the osteoclasts 14 hours following S-phase. Monoblasts and promonocytes are considered as DNA-synthesizing forms. A single injection of parathormone in a dose of 10 units/100 g of body weight leads to an increase in the fractions of DNA-synthesizing cells. Monocytes and macrophages appear to be direct osteoclast precursors in resorption zones. As a result of their fusion there appear "unfixed" polynuclear macrophages (young osteoclasts). The number of osteoclast nuclei increases also at the expense of little-differentiated phagocytes, the inclusion of the latter takes place at different stages of the life span of osteoclasts depending on the intensity of bone resorption processes. The structural characteristics of fusion of cell precursors are considered.     

Negative Effect of Iodide On the Survival of Newly Divided Epithelial Cells In Chronically Stimulated Rat Thyroid

Abstract. The aim of this work was to investigate some aspects of the thyroid epithelial cell kinetics during the iodide-induced involution of a hyperplastic goitre in the rat. Rats were made iodine-deficient for 6 months, and propylthiouracil (PTU) (0.15%) was added to the diet during the last 2 months. Thereafter, rats were refed with iodide and PTU was removed (day 0).
Forty-eight hours previously, all the rats were injected with tritiated thymidine ([³H]TdR) (1 μCi/g). Some animals were killed 1 hr or 24 hr after [³H]TdR injection (i.e. on day -2 and -1, day O corresponding to the restoration of a normal iodine diet); the other animals were killed after different delay periods and following L³H]TdR injection. Autoradiography of thyroid sections, iodine determination of plasma iodide and protein-bound iodine (PBI), and RIA of plasma thyroid stimulatory hormone (TSH) were performed. Plasma TSH concentration was very high on day O of iodide refeeding (3000 ± 330 ng/ml) and remained at this level until day 8. Plasma PBI was very low on day O, remained so until day 4 and greatly increased on day 8. Plasma iodide was also very low on day O, but markedly increased on day 1, then did not vary significantly until day 43 of iodine refeeding. Thyroid weight, elevated on day O, decreased relatively quickly until day 30, then more slowly until day 73.     

Effect of methotrexate on cells in a keratinized epithelium with an active thymidine salvage pathway assessed by autoradiography

In the partially synchronized cell system of the hamster cheek pouch epithelium, the inhibitory effect of a bolus injection of methotrexate (Mtx) (2 g/m2, injected at 1200 hr) was analysed by means of both autoradiography and flow cytometry (FCM) in a 21-hr experiment. For autoradiography [3H]TdR and [3H]UdR were used as tracers for salvage and de novo pathways of thymidylate (TMP) synthesis, respectively. For FCM no tracers were injected. The autoradiographic studies demonstrated an active TdR salvage pathway for DNA synthesis, not affected by the impaired de novo TMP synthesis. The blocked de novo TMP synthesis was partially released 7 hr after Mtx injection, but it had not totally recovered at the end of the experiment. The decrease in the fraction of S-phase cells detected about 10 hr after Mtx injection by autoradiographic labelling with [3H]TdR and by FCM was found to be caused by a decrease in the number of cells entering S phase. However, Mtx did not influence the salvage TMP synthesis rate of cells entering S phase.     

Attenuation of bone mass and increase of osteoclast formation in decoy receptor 3 transgenic mice

Decoy receptor 3 (DcR3), a soluble receptor for FasL, LIGHT, and TL1A, induces osteoclast formation from monocyte, macrophage, and bone stromal marrow cells. However, the function of DcR3 on bone formation remains largely unknown. To understand the function of DcR3 in bone formation in vivo, transgenic mice overexpressing DcR3 were generated. Bone mineral density (BMD) and bone mineral content (BMC) of total body were significantly lower in DcR3 transgenic mice as compared with wild-type controls. The difference in BMD and BMC between DcR3 transgenic and control mice was confirmed by histomorphometric analysis, which showed a 35.7% decrease in trabecular bone volume in DcR3 transgenic mice in comparison with wild-type controls. The number of osteoclasts increased in DcR3 transgenic mice. In addition, local administration of DcR3 (30 microg/ml, 10 microl, once/day) into the metaphysis of the tibia via the implantation of a needle cannula significantly decreased the BMD, BMC, and bone volume of secondary spongiosa in tibia. Local injection of DcR3 also increased osteoclast numbers around trabecular bone in tibia. Furthermore, coadminstration of soluble tumor necrosis factor receptor inhibitor/Fc chimera (TNFRSF1A) but not osteoprotegerin inhibited the action of DcR3. In addition, in an assay of osteoclast activity on substrate plates, DcR3 significantly increased the resorption activity of mature osteoclasts. Treatment with higher concentrations of DcR3 slightly increased nodule formation and alkaline phosphatase activity of primary cultured osteoblasts. These results indicate that DcR3 may play an important role in osteoporosis or other bone diseases.     

Negative effect of iodide on the survival of newly divided epithelial cells in chronically stimulated rat thyroid

The aim of this work was to investigate some aspects of the thyroid epithelial cell kinetics during the iodide-induced involution of a hyperplastic goitre in the rat. Rats were made iodine-deficient for 6 months, and propylthiouracil (PTU) (0.15%) was added to the diet during the last 2 months. Thereafter, rats were refed with iodide and PTU was removed (day 0). Forty-eight hours previously, all the rats were injected with tritiated thymidine ([3H]TdR) (1 microCi/g). Some animals were killed 1 hr or 24 hr after [3H]TdR injection (i.e. on day -2 and -1, day 0 corresponding to the restoration of a normal iodine diet); the other animals were killed after different delay periods and following [3H]TdR injection. Autoradiography of thyroid sections, iodine determination of plasma iodide and protein-bound iodine (PBI), and RIA of plasma thyroid stimulatory hormone (TSH) were performed. Plasma TSH concentration was very high on day 0 of iodide refeeding (3000 +/- 330 ng/ml) and remained at this level until day 8. Plasma PBI was very low on day 0, remained so until day 4 and greatly increased on day 8. Plasma iodide was also very low on day 0, but markedly increased on day 1, then did not vary significantly until day 43 of iodine refeeding. Thyroid weight, elevated on day 0, decreased relatively quickly until day 30, then more slowly until day 73. The [3H]TdR labelling index (LI) of the thyroid epithelial cells (TEC) was high on day 0 (56 +/- 3 labelled cells/10,000 cells), and 24 hr thereafter increased to 104 +/- 3, by division of the labelled cells. On day 1 of iodine refeeding, the LI had abruptly decreased to about half this value and then remained stable for 3 more days. Between day 4 and day 16, a progressive decline in the LI, (by about 3-4 per day), was observed. The LI showed no further modification, up to day 73, the longest period investigated. The decrease in LI occurred without any significant changes in the labelling intensity (grain count) of the remaining labelled cells between day 1 and 16, this indicates that no cell division took place during this period. The data are therefore interpreted as showing a biphasic elimination after iodide refeeding, of cells that were actively proliferating during the goitrous state.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evidence for discrete cell kinetic subpopulations in mouse epidermis based on mathematical analysis

Abstract. Continuous (repeated) labelling studies in mouse epidermis indicate that nearly all cells are labelled after about 100 hr. Percentage labelled mitoses studies ([³H]TdR at 15.00 and 03.00 hours) have a first peak that does not reach 100% and has a half-width of about 10 hr. Small second and third peaks can be detected at about 90 and 180 hr, respectively. The changes with time in the number of labelled cells show a difference dependent on the time of day of [³H]TdR administration. Both curves show an early doubling in labelled cells which then decline, forming a peak of labelled cells. A second peak occurs at about 120 hr. This is followed by a progressive decline with no further peaks until values of about 1% labelling are obtained at 340 hr.
These experiments have been investigated mathematically. A computer programme has been devized that permits all three types of experiments to be analysed simultaneously. More importantly, it can analyse situations with a heterogeneity in cell cycle parameters in all proliferative subpopulations.
Various models for epidermal cell replacement have been considered. The data as a whole can best be explained if the basal layer contains at least two distinct subpopulations of cells and an exponentially decaying post-mitotic population with a half-life of about 30 hr. The proliferative sub-populations must be characterized by near integer differences in the length of cycle, the precursor (stem) compartment having the longer cycle. An inverse relationship is required for the length of S, i.e. the shortest time for the stem cells.
A full range of cell kinetic parameters can be calculated and are tabulated for the most appropriate model system which is one involving three transit proliferating subpopulations.     

Retention of Labelled Dna Precursor By Murine Cells After A Single Administration of Tritium Labelled Thymidine

Abstract. The central zone of the rat lens epithelium, extending half way from the centre to the periphery of a whole mount preparation, normally has less than 1% of the cells in the cell cycle at any given time. Mechanical wounding initiates a burst of proliferation in the central zone. DNA synthesis begins 14 hr after wounding followed by mitosis 10 hr later. When [³H]TdR was applied at 2 hr prior to S phase, some moderately heavy and some light labelling was observed after the onset of S phase. When [³H]TdR was applied 5 hr before S phase (9 hr after wounding), all the cells were lightly labelled. Only small amounts of the label were available to these cells 5 hr after application. It is significant that there was labelling in this group because it indicates the persistence of relatively small intracellular pools of [³H]TdR for several hours after the initial 'pulse' labelling of cells. Determinations of the duration of S phase were based on the assumption that pulse labelling may be affected by the persistence of the pools of [³H]TdR and consequent light labelling of the cells.     

Autoradiographic Investigations on Cell Proliferation in the Digestive Mucosa of Fishes

The labelling index (TLI) of the digestive mucosa of some fish species was determined following a pulse labelling with tritiated thymidine ([³H]TdR) and light microscopic autoradiography. In the oesophageal epithelium, proliferation was observed to occur in non mucus-secreting cells. In the intestine, both undifferentiated and absorptive cells incorporated [³H)TdR within 1 h after injection. Statistically significant differences in [³H]TdR incorporation were observed between the upper intestine region and both the middle and lower parts on the one hand, and between the middle and lower parts on the other hand. Mucus-secreting cells seemed unable to proliferate. In the stomach, significantly fewer labelled nuclei were counted; they were located in the isthmus epithelium. No significant difference was observed between the TLI of these regions in the different species.     

Osteoactivin is a novel osteoclastic protein and plays a key role in osteoclast differentiation and activity

This study presents gene expression, protein expression, and in situ immunohistochemical evidence that osteoclasts express high levels of osteoactivin (OA), which had previously been reported to be an osteoblast-specific protein in bone. OA expression in osteoclasts was up-regulated upon receptor activator of NFkappaB ligand-induced differentiation. Suppression of functional activity of OA with neutralizing antibody reduced cell size, number of nuclei, fusion, and bone resorption activity of osteoclasts. OA was co-immunoprecipitated with integrin beta3 and beta1, indicating that OA co-localizes with integrin beta3 and/or beta1 in a hetero-polymeric complex in osteoclasts. These findings indicate that OA is a novel osteoclastic protein and plays a role in osteoclast differentiation and/or activity.     

Effect of Methotrexate On Cells In A Keratinized Epithelium With an Active Thymidine Salvage Pathway Assessed By Autoradiography*

In the partially synchronized cell system of the hamster cheek pouch epithelium, the inhibitory effect of a bolus injection of methotrexate (Mtx) (2 g/m², injected at 1200 hr) was analysed by means of both autoradiography and flow cytometry (FCM) in a 21-hr experiment. For autoradiography [³H]TdR and [³H]UdR were used as tracers for salvage and de nouo pathways of thymidylate (TMP) synthesis, respectively. For FCM no tracers were injected. the autoradiographic studies demonstrated an active TdR salvage pathway for DNA synthesis, not affected by the impaired de novo TMP synthesis. the blocked de novo TMP synthesis was partially released 7 hr after Mtx injection, but it had not totally recovered at the end of the experiment. the decrease in the fraction of S-phase cells detected about 10 hr after Mtx injection by autoradiographic labelling with [³H]TdR and by FCM was found to be caused by a decrease in the number of cells entering S phase. However, Mtx did not influence the salvage TMP synthesis rate of cells entering S phase.)