TdR Incorporation Research Articles

Weak peptide agonists reveal functional differences in B7-1 and B7-2 costimulation of human T cell clones.

The influence of costimulation on the T cell response to altered peptide ligands that act as either partial or weak agonists for human CD4+ T cell clones was examined. Using stable Chinese hamster ovary (CHO) cell transfectants expressing DR2 (DRB1*1501) and human B7-1 or B7-2 as APC, presentation of native myelin basic protein (MBP) p85-99 peptide Ag or a partial agonist of MBP p85-99 induced equivalent T cell activation as measured by [3H]TdR incorporation and cytokine secretion. In marked contrast, presentation of cross-reactive peptides of MBP p85-99 that act as weak agonists with B7-1, but not B7-2, costimulation resulted in significant T cell activation as measured by [3H]TdR incorporation and cytokine secretion. These data suggest that decreasing the strength of the signal provided to the TCR allows differences in B7-1 and B7-2 signaling to be observed. Thus, the costimulatory environment during T cell activation may be a mechanism of regulating T cell cross-reactivity in the periphery.

Bacterial dynamics in periphyton from different regions of a tropical coastal lagoon

Various aspects of bacterial dynamics in periphyton attached to leaf detritus of Typha domingensis Pers were examined in a coastal lagoon in the state of Rio de Janeiro, Brazil. Senescent leaves were submerged in eutrophic and oligotrophic regions of this lagoon and the biovolume, abundance, biomass, secondary production ([ 3 H]leucine - leu - and [ 3 H]-thymidine - Tdr - incorporation), and turnover times of the bacterial community were evaluated after five and 25 days of decomposition. An additional experiment was carried out to lest the effects of PP 4 3- and NH 4 + on Tdr incorporation. Bacterial abundance and biomass per unit of detritus surface increased faster in the eutrophic region, but eutrophic and oligotrophic regions did not differ when data were expressed per unit of biofilm dry weight. Despite higher bacterial abundance and biomass, biofilms of the eutrophic region had lower bacterial production, on a biofilm dry weight, and higher turnover times after 25 days of decomposition. Addition of PO 4 3- and NH 4 + significantly increased Tdr incorporation by periphytic bacteria growing in the oligotrophic region significantly, which suggests that this community is P and N limited in that region.

Synergy between CD28 and CD9 costimulation for naive T-cell activation

Our previous study demonstrated that CD9 is expressed on most mature naive T-cells and delivers a potent costimulatory signal that functions independently of CD28. Here, we investigated whether this CD9-mediated signal is different from the CD28-mediated signal in the mode of costimulation and whether both signals function synergistically for T-cell activation. Anti-CD9 or anti-CD28 monoclonal antibody (mAb) increased [ 3H]TdR incorporation of naive T-cells in the absence of antigen-presenting cells (APC) when coimmobilized with submitogenic doses of anti-CD3 mAb. The levels of costimulation induced by ligation of CD9 and CD28 were comparable. However, the costimulatory effect differed between soluble anti-CD9 and CD28 mAb. A soluble form of anti-CD28 mAb could costimulate anti-CD3-triggered T-cells, whereas soluble anti-CD9 mAb failed to costimulate. Although anti-CD28 costimulated naive T-cells treated with phorbol myristate acetate (PMA) instead of anti-CD3 mAb, a combination of PMA plus anti-CD9 mAb could not induce T-cell activation. The combined costimulation of anti-CD3-triggered T-cells with anti-CD9 and anti-CD28 mAbs resulted in strikingly enhanced [ 3H]TdR uptake and lymphokine (IL-2 and IFN- γ) production when compared to those induced by each costimulation. These results suggest that CD9 and CD28 induce T-cell costimulation using different signaling pathways, thereby inducing synergy in T-cell activation.

Secondary productivity (3H-leucine and 3H-thymidine incorporation), abundance and biomass of the epiphytic bacteria attached to detritus of Typha domingensis pers. in a tropical coastal lagoon

Senescent, naturally dried leaves of Typha domingensis were incubated inthe littoral region of a coastal lagoon and epiphytic bacterial volume,abundance, biomass and secondary productivity were measured during 127 daysof decomposition. The peak of cell abundance was registered at t =127 days when expressed per leaf surface area (10.07×107cells cm-2; 7.26 µgC cm-2), and at t= 26 days when expressed per biofilm dry mass (38.10 ×107 cells (mgDM biofilm)-1, 30.52 µgC(mgDM biofilm)-1). The highest values of bacterial biovolumesand lower turnover time were usually obtained in the beginning of thecolonization. Leu:Tdr ratios were also higher in the beginning of thecolonization, when bacterial community presented unbalanced metabolism.Consequently, the highest discrepancies between the bacterial secondaryproduction estimated by leu and Tdr incorporation were observed in the first2 days of decomposition. On average, the bacterial secondary productivityestimated by leu incorporation was 2.1 times higher than the valuesestimated by Tdr incorporation when the empirical factor for Tdr wasobtained from the relationship between Tdr and biomass increment. Thisdifference increased to 4.2 when the empirical factor was obtained from therelationship between Tdr and cell numbers increment. An average of bothmethods (0.0037 to 0.1397 µgC cm-2 h-1)produced results that fall within the range reported in the literature forepiphytic bacteria of freshwater ecosystems.

Seropharmalogical effects of Fuzheng Huayu decoction on rat Ito cell morphology and function in culture.

To investigate the mechanisms of anti-liver fibrosis actions of Fuzheng Huayu (FZHY) decoction, which acts to strengthen the body's resistance and promote blood circulation. Ito cells were isolated from rats and cultured. Serum samples were collected from healthy (normal) rats after administration of FZHY decoction and added to the subcultured cells. The effects of FZHY decoction on the Ito cells were investigated by contrast microscopy (to observe cell morphology), [(3)H]Pro incorporation assay (cell viability), [(3)H]TdR incorporation and MTT colorimetric assay (cell proliferation), and [(3)H]Pro incorporation and collagenase digestion (collagen synthesis rate). The rat sera samples from rats treated with FZHY decoction had no influence on Ito cell morphology, but improved cell viability and markedly inhibited cell proliferation and collagen synthesis. The magnitude of these effects showed dependence on treatment dosage and drug concentration in serum. The seropharmalogical method can be efficiently used to investigate the pharmacological mechanism of anti-fibrotic traditional Chinese herbs and formulae. Inhibition of Ito cell proliferation and collagen synthesis may be two of the major mechanisms underlying the anti-fibrosis actions of the FZHY decoction.

低出力レーザー照射による培養細胞への影響 -第2報-

Many clinical observations have suggested that low power lasers effective in wound healing.However the mechanism of action is not clear. This studies have shown that low power diode laser (Ga-Al-As diode laser,wave length: 835nm, continuous wave) in a suitable irradiation condition (1.12mW/cm2, 0.33J/cm2) increases [3H] TdR incorporation and a cell growth proliferation of oral gingival fibroblast (Gin-1 cell) in cell culture. The irradiation under the same condition stimulates the expression of TIMP-1mRNA and the secretion of TIMP-1 protein,but no effect on the expression of TIMP-2 mRNA.

Suramin disrupts insulin-like growth factor-II (IGF-II) mediated autocrine growth in human SH-SY5Y neuroblastoma cells

Suramin, traditionally used in the treatment of trypanosomiasis, is under investigation in the treatment of cancer. One side effect that limits its use is the onset of a sensorimotor polyneuropathy. In order to investigate the mechanism by which suramin induces polyneuropathy, we examined its effects on SH-SY5Y human neuroblastoma cells, an in vitro model of neuronal growth and differentiation. Addition of 50–400 μg/ml suramin to SH-SY5Y cells grown in 0.6% CS inhibited [ 3H]thymidine ([ 3H]TdR) incorporation and cell growth. Upon removal of suramin, [ 3H]TdR incorporation increased, demonstrating that levels of suramin used were cytostatic and not cytotoxic. Analysis of suramin-treated SH-SY5Y cells by flow cytometry revealed growth arrest in the G 1/G 0 phase of the cell cycle. IGF-II-induced SH-SY5Y growth is mediated by the type I IGF receptor (IGF-IR). Therefore, we examined its effect on IGF-IR tyrosine phosphorylation. Suramin prevented IGF-II-stimulated IGF-IR tyrosine phosphorylation. These results indicate that in SH-SY5Y cells, suramin acts as a cytostatic agent and can block IGF-II-dependent cell growth by preventing IGF-IR activation. Thus, suramin toxicity in the peripheral nervous system may be due, in part, to preventing IGFs and other growth factors from activating their receptors.

The c-Kit Ligand Potentiates the Allogeneic Mixed Lymphocyte Reaction

The allogeneic mixed lymphocyte reaction (MLR) is a complex in vitro assay of T-cell recognition and responsiveness in which interleukin-2 (IL-2) plays a central role. We have previously demonstrated that c-kit ligand (KL) can enhance IL-2-induced proliferation in a subset of human natural killer cells expressing the c-kit tyrosine kinase receptor. In the present study, we asked whether KL could enhance IL-2-mediated T-cell proliferation in the allogeneic MLR. We demonstrate that the vast majority of activated human T-cell clones express the c-kit mRNA transcript. Binding studies performed on activated T cells with radioiodinated KL were consistent with the expression of a single class of c-kit receptors. The addition of exogenous KL to the MLR led to an increase in tritiated thymidine (3[H]-TdR) incorporation in the absence of other exogenous cytokines, and did so in a dose-dependent fashion. A reproducible increase in 3[H]-TdR incorporation was noted at concentrations of KL, which approximate those normally found in vivo. Antibody blocking of KL binding to c-kit, T-cell depletion and sorting experiments suggest that the action of KL is mediated at least in part by a direct effect on both CD4+ and CD8+ T-cells. KL's enhancement of the MLR also requires the binding of IL-2 to its high-affinity IL-2 receptor. Given the abundance of KL normally found in human serum, these data suggest that this cytokine may have a role during T-cell activation in vivo.

Tyrosine phosphorylation is crucial for growth signaling by tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2)

[ 3H]Thymidine (TdR) incorporation by human osteosarcoma cell line MG-63 was significantly stimulated at as early as 3 h after the addition of either TIMP-1 or TIMP-2 alone. Maximum stimulation was attained at a concentration of either 20 ng/ml (0.71 nM) TIMP-1 or 1.0 ng/ml (46 pM) TIMP-2. Tyrosine kinase inhibitors such as genistein, erbstatin, and herbimycin A almost completely inhibited the [ 3H]TdR incorporation stimulated by either of the TIMPs. However, essentially no effect was observed with H-89, H-7, bisindolylmaleimide and K-252a. These inhibition studies suggest a crucial role for tyrosine kinase in the signal transduction of TIMPs. Phosphotyrosine-containing proteins were significantly elevated by the treatment with both TIMPs. We also found that either TIMP stimulated an increase in mitogen-activated protein (MAP) kinase activity, suggesting that MAP kinase plays a role in TIMP-dependent growth signaling.

Adhesion of U-937 monocytes induces cytotoxic damage and subsequent proliferation of cultured human mesangial cells

In an in vitro model of monocyte adhesion to glomerular cells, U-937 myelomonocytic leukemia cells irreversibly bind to human mesangial cell monolayers. Adhesion is enhanced in mesangial cells proliferating in response to fetal bovine serum, and in the presence of several cytokines and vasoactive agents. In the present study, co-culture with U-937 followed by removal of non-adherent cells time-dependently decreased viability of mesangial cells, measured either by fluorometry after dual labeling with calcein acetoxymethylester and ethidium homodimer, or by the release of lactate dehydrogenase. The cytotoxic effects of co-culture with U-937 cells were significantly reduced by a combination of free radical scavengers, indicating involvement of reactive oxygen species. U-937 cells also stimulated subsequent proliferation of mesangial cells, assessed by [3H]-TdR incorporation and direct cell counts 24 hours later (from 1,034 +/- 83 to 14,611 +/- 959 and from 2,931 +/- 201 to 19,400 +/- 2,124 cpm/well, quiescent/cycling mesangial cells, respectively, P < 0.01). Controls to rule out TdR incorporation by adherent U-937 cells included selective [3H]-TdR labeling and demecolcine pretreatment. Cell counts at 24 hours confirmed U-937-induced proliferation of quiescent HMC, from 50,575 +/- 3,596 to 143,012 +/- 10,039 cells/cm2 (P < 0.01). Agents that promote U-937 cell adhesion, such as the TxA2 mimetic, U-46619, or angiotensin II, enhanced cytotoxicity while inhibiting the proliferation of both quiescent and cycling mesangial cells, when added during co-culture and the subsequent 24 hours (+1 microM U-46619, 1,875 +/- 131 and 2,546 +/- 125 cpm/well, respectively, 79,793 +/- 5,744 cells/cm2, P < 0.01 vs. U-937 only; +1 microM Ang II, 5066 +/- 560 and 5,784 +/- 306 cpm/well, respectively, 81,068 +/- 4,671 cells/cm2, P < 0.05). Blocking antibodies against the adhesion molecule ICAM-1 and leukocyte counterreceptors (LFA-1, VLA-4) prevented the proliferative response, which could not be duplicated with the conditioned media of U-937 alone or co-cultured with mesangial cells. These findings may reflect the interactions occurring in vivo between infiltrating leukocytes and resident cells during glomerular inflammation.

Pre-Conditioning of Smooth Muscle Cells via Induction of the Heat Shock Response Limits Proliferation Following Mechanical Injury

Arterial smooth muscle cell (SMC) proliferation is a significant component of post-angioplasty restenosis. We evaluated whether pre-conditioning of SMCs, via induction of the heat shock respone prior to actual physical injury, would result in an alteration in cell proliferation following injury. Rat aortic SMCs were pretreated with either chemical or thermal heat shock inducers and then subjected to scrape-wound injuryin vitro.Cell proliferation at 24 hrs was measured via3H-thymidine (Tdr) incorporation and compared with scrape wounded unstressed controls. A significant decline in cell proliferation post scrape-wound injury was observed for both chemical and thermal heat shock pre-conditioned cultures, compared to untreated controls. Increased expression of heat shock protein 72 was confirmed serially throughout the 24 hr study period for both chemical and thermal inducers. Despite reduced proliferation heat shocked cells remained viable as evidenced by fluorescent cell viability assay and preserved migration. Pre-conditioning of SMCs through induction of the heat shock response prior to physical injury may be a useful approach to limit aggressive proliferation observed with mechanical revascularization injury.

Effects of prostaglandins on deoxyribonucleic acid and aggrecan synthesis in the RCJ 3.1C5.18 chondrocyte cell line: role of second messengers.

PGs play an important role in regulating articular chondrocyte function in both normal and pathological states. However, the mechanisms of the effects of PG on chondrocyte function remain undefined. We, therefore, examined the effects of PGE1, PGE2, and PGE2 alpha on second messenger generation in relation to DNA and aggrecan synthesis in the nontransformed rat RCJ 3.1C5.18 (RCJ) chondrocyte cell line. RCJ cells were grown under minimal attachment conditions on a composite collagen-agarose (0.15%/0.8%) gel to maintain a differentiated phenotype. PGE1 and PGE2 (0.001-100 microM) produced a similar dose-related increase in cAMP accumulation, with a maximal 8-fold increase over basal values, whereas PGF2 alpha produced a minimal 1.3-fold increase in cAMP levels only at 100 microM. On the other hand, both PGE2 and PGE2 alpha raised the intracellular free calcium ([Ca2+]i) concentration, derived primarily from extracellular sources, whereas PGE1 was without effect on [Ca2+]i. These three PGs also had divergent effects on DNA synthesis, as measured by [3H]thymidine ([3H]TdR) incorporation. PGF2 alpha (0.001-5 microM) produced a dose-related increase in [3H]TdR incorporation, with a maximal 1.6-fold increase over baseline values at 5 microM and a slight decline to below maximal levels at 10 microM. PGE2 exhibited a contrasting inverse biphasic response, with an initial small suppressive effect that was maximal at 0.1 microM and a secondary stimulatory phase producing a small increase over control values at 5 microM. PGE1 had a uniformly suppressive effect, producing a 30% decrease at 10 microM. Despite the divergent effects of PGE1, PGE2, and PGE2 alpha on second messenger generation and DNA synthesis, all three PGs produced a dose-related stimulation of aggrecan synthesis. PGF2 alpha was the most potent, producing significant stimulation at 0.001 microM and a maximal 104% increase at 5 microM. PGE1 and PGE2 were approximately equipotent and approximately 60% as effective as PGF2 alpha in stimulating aggrecan synthesis. Northern analysis demonstrated that the effects of PG on aggrecan synthesis were not accompanied by changes in aggrecan core protein steady state messenger RNA levels. Thus, the effects of PG on aggrecan production in RCJ cells appear to be regulated at the posttranscriptional level. Forskolin and (Bu)2cAMP mimicked the suppressive effects of PGE1 on [3H]TdR incorporation, as well as the stimulatory effect of PGE1 on aggrecan synthesis. In addition, the phorbol ester 12-O-tetradecanoyl phorbol acetate mimicked PGF2 alpha stimulation of [3H]TdR incorporation and aggrecan synthesis, and the effects of PGE2 alpha on these processes were blocked by protein kinase C inhibitors. Therefore, it appears that in mammalian chondrocytes, PGE1 primarily activates the cAMP-protein kinase A second messenger system, PGE2 alpha affects primarily the Ca2(+)-protein kinase C system, and PGE2 activates both pathways. Moreover, PG posttranscriptional regulation of aggrecan synthesis in chondrocytes involves both the cAMP-protein kinase A and Ca2(+)-protein kinase C second messenger systems.

Effects of prostaglandins on deoxyribonucleic acid and aggrecan synthesis in the RCJ 3.1C5.18 chondrocyte cell line: role of second messengers

PGs play an important role in regulating articular chondrocyte function in both normal and pathological states. However, the mechanisms of the effects of PG on chondrocyte function remain undefined. We, therefore, examined the effects of PGE1, PGE2, and PGE2 alpha on second messenger generation in relation to DNA and aggrecan synthesis in the nontransformed rat RCJ 3.1C5.18 (RCJ) chondrocyte cell line. RCJ cells were grown under minimal attachment conditions on a composite collagen-agarose (0.15%/0.8%) gel to maintain a differentiated phenotype. PGE1 and PGE2 (0.001-100 microM) produced a similar dose-related increase in cAMP accumulation, with a maximal 8-fold increase over basal values, whereas PGF2 alpha produced a minimal 1.3-fold increase in cAMP levels only at 100 microM. On the other hand, both PGE2 and PGE2 alpha raised the intracellular free calcium ([Ca2+]i) concentration, derived primarily from extracellular sources, whereas PGE1 was without effect on [Ca2+]i. These three PGs also had divergent effects on DNA synthesis, as measured by [3H]thymidine ([3H]TdR) incorporation. PGF2 alpha (0.001-5 microM) produced a dose-related increase in [3H]TdR incorporation, with a maximal 1.6-fold increase over baseline values at 5 microM and a slight decline to below maximal levels at 10 microM. PGE2 exhibited a contrasting inverse biphasic response, with an initial small suppressive effect that was maximal at 0.1 microM and a secondary stimulatory phase producing a small increase over control values at 5 microM. PGE1 had a uniformly suppressive effect, producing a 30% decrease at 10 microM. Despite the divergent effects of PGE1, PGE2, and PGE2 alpha on second messenger generation and DNA synthesis, all three PGs produced a dose-related stimulation of aggrecan synthesis. PGF2 alpha was the most potent, producing significant stimulation at 0.001 microM and a maximal 104% increase at 5 microM. PGE1 and PGE2 were approximately equipotent and approximately 60% as effective as PGF2 alpha in stimulating aggrecan synthesis. Northern analysis demonstrated that the effects of PG on aggrecan synthesis were not accompanied by changes in aggrecan core protein steady state messenger RNA levels. Thus, the effects of PG on aggrecan production in RCJ cells appear to be regulated at the posttranscriptional level. Forskolin and (Bu)2cAMP mimicked the suppressive effects of PGE1 on [3H]TdR incorporation, as well as the stimulatory effect of PGE1 on aggrecan synthesis. In addition, the phorbol ester 12-O-tetradecanoyl phorbol acetate mimicked PGF2 alpha stimulation of [3H]TdR incorporation and aggrecan synthesis, and the effects of PGE2 alpha on these processes were blocked by protein kinase C inhibitors. Therefore, it appears that in mammalian chondrocytes, PGE1 primarily activates the cAMP-protein kinase A second messenger system, PGE2 alpha affects primarily the Ca2(+)-protein kinase C system, and PGE2 activates both pathways. Moreover, PG posttranscriptional regulation of aggrecan synthesis in chondrocytes involves both the cAMP-protein kinase A and Ca2(+)-protein kinase C second messenger systems.

In vitro characterization of a novel series of platelet-derived growth factor receptor tyrosine kinase inhibitors

In this report, we describe the discovery and characterization of a novel biarylhydrazone series of platelet-derived growth factor (PDGF) receptor tyrosine kinase inhibitors typified by the prototype WIN 41662 (3-phenyl- N 1-[1-(4-pyridyl)pyrimidine]hydrazone). WIN 41662 inhibited PDGF-stimulated autophosphorylation of PDGF receptors from human vascular smooth muscle cells (hVSMC) with an IC 50 value of 60 nM. The inhibitor appeared to be competitive with respect to substrate (Mn 2+-ATP), having a calculated K i of 15 ± 5 nM. WIN 41662 was approximately 500-fold more potent in inhibiting the PDGF receptor tyrosine kinase than the p56 lck tyrosine kinase. It was inactive against other serine/threonine and tyrosine kinases tested. WIN 41662 produced concentration-dependent inhibition of PDGF-stimulated receptor autophosphorylation in intact hVSMC with an IC 50 < 100 nM. Intracellular Ca 2+ mobilization and cell proliferation were events that occurred in hVSMC subsequent to PDGF receptor activation. WIN 41662 inhibited PDGF-stimulated Ca 2+ mobilization and cell proliferation ([ 3H]TdR incorporation) with IC 50 values of 430 nM and 2.3 μM, respectively. These effects appeared to be specifically related to PDGF receptor tyrosine kinase inhibition since WIN 41662 was not cytotoxic ( in vitro) and since WIN 72039, a close structural analog that does not inhibit PDGF receptor tyrosine kinase, also did not inhibit PDGF-stimulated receptor autophosphorylation, Ca 2+ mobilization, or hVSMC proliferation. Thus, WIN 41662 is representative of a novel class of selective PDGF receptor tyrosine kinase inhibitors that inhibit PDGF-regulated secondary events in intact cells.

Bacterial activity in a forest soil after soil heating and organic amendments measured by the thymidine and leucine incorporation techniques

The effects of soil heating (200°C, 1 h) and organic amendments (straw and poultry manure) on bacterial activity in a forest soil were studied in a laboratory experiment. Measurements were made over a 15-week incubation using the thymidine (TdR) and leucine (Leu) incorporation techniques. Soil heating initially reduced TdR and Leu incorporation rates by 96–98% within 2 weeks of incubation. Thereafter the bacterial activity recovered, but by the end of the incubation the values were still lower than in the corresponding unheated soil. Reinoculation of heated soil with fresh soil improved the reestablishment of bacterial activity after a long lag, since the values were comparable to those observed for the unheated soil only after 13 weeks of incubation. The organic amendments affected bacterial activity in both unheated and heated soils. An enhanced bacterial activity was found within 2 weeks after the addition of straw or poultry manure to the unheated soils, the increase being greater and maintained longer for the latter treatment. In the heated soil no effect of straw addition was detected, while a large increase in bacterial activity following poultry manure treatment was observed after a long lag (up to 8 weeks). The presence of a toxic substance appeared to be the cause of the prolonged reduced bacterial activity in the heated soils, since an inhibitory effect of water extract from heated soil was found on TdR and Leu incorporation rates of bacteria from both unheated and heated soils. Furthermore, the inhibition was less pronounced for bacteria from the heated soil, indicating that a bacterial community tolerant to the toxic substances had developed.

Effects of Misoprostol on Bone Resorption and Formation in Organ Culture.

Prostaglandins are potent stimulators of bone resorption and formation. Because misoprostol is an analog of prostaglandin E(1) (PGE(1)), we have examined its effects on resorption and formation in organ culture. The results were compared with PGE(2) which can stimulate resorption and both stimulate and inhibit bone formation. Resorption, measured as the release of previously incorporated (45)Ca from 5-day cultures of 19-day fetal-rat long bones, was increased by misoprostol 1.5-fold at 10(minus sign6) M and twofold at 10(minus sign5) M. The effect at 10(minus sign6) M was abolished by addition of indomethacin (10(minus sign6) M). PGE(2) was approximately 100 times more potent and was not affected by indomethacin in this system. In 21-day fetal-rat calvariae, cultured for 24 h in the presence of cortisol (10(minus sign7) M), misoprostol produced a dose-related increase in TdR incorporation between 10(minus sign5) and 10(minus sign7) M. PGE(2) appeared to be only 10-fold more potent in this response. The effects of misoprostol on incorporation of [(3)H]proline into collagenase digestible protein (CDP) and noncollagen protein (NCP) were measured in 72-h cultures, either with continuous treatment or 24-h treatment followed by 48 h in control medium. With continuous treatment at 10(minus sign6) M, misoprostol increased labeling of CDP twofold. A similar effect was observed with 24 h of treatment at 10(minus sign5) M, followed by 48 h in control medium. Again, PGE(2) was approximately 10-fold more potent than misoprostol. When calvariae were treated with insulin-like growth factor I, which increases CDP labeling by 2.5-fold, the effects of misoprostol and PGE(2) were inhibitory. We conclude that misoprostol resembles PGE(2) in its effects on bone but is less potent. Moreover, misoprostol may be relatively less effective in stimulating resorption than in stimulating formation. Therefore, an increase in bone turnover, possibly with a net anabolic effect, might occur in vivo with long-term misoprostol treatment. Misoprostol effects on bone turnover in humans could be evaluated using the sensitive biochemical markers for bone resorption and formation which are currently available.

Effects of temperature on the incorporation of leucine and thymidine by bacterioplankton and bacterial isolates

The effect of temperature on the incorporation of 3H-leucine (W-Leu) and 3H-thymidine (3H-TdR) by bacterioplankton and bacterial isolates from marine, estuarine, and freshwater habitats was examlned. There was a significant, positive correlation between in situ temperature and molar Leu:TdR incorporation by bacterioplankton in oceanic and lagoonal environments. Temperature manipulation experiments showed that Q,,, values for Leu incorporation by bacterial assemblages and isolates were significantly higher than corresponding Q, values for TdR incorporation. Evidence indicated that the temperature-dependence of Leu:TdR ratios was not a funct~on of community compositlon, unbalanced growth, non-specific labelling of macromolecules, or substrate limitation. Thus, Leu and TdR incorporation appear to be affected by temperature differently. These experiments indicate that Leu and/or TdR conversion factors, for estimating bacterial production, are not constant with respect to temperature.

Cell cycle inhibition by sodium butyrate in legume root meristems

Previous investigations demonstrated that butyric, propionic and valeric acids reduced the mitotic index in cultured root meristems of peas ( Pisum sativum). These compounds also inhibited DNA synthesis and the progression of cells through the cell cycle. Cultured pea roots, exposed to either 0.1 mM 14C-labelled butyrate or propionate, incorporated the isotope and transported it into the nucleus. Experiments have now been performed to determine if cultured roots of other legume species such as soybean ( Glycine max), pinto bean ( Phaseolus vulgaris) and broad bean ( Vicia faba) also respond to butyrate in a similar manner. After a 24-hr butyrate exposure, the mitotic index in the root meristems was greatly reduced in all three test species, even at butyrate concentrations as low as 0.1 mM. The mitotic index in broad bean roots exposed to either 1 or 5 mM butyrate was reduced as quickly as 6 hr post-exposure. When broad bean roots were exposed to 5 mM butyrate, [ 3H]-TdR incorporation was inhibited by 8 hr, indicating a reduction in DNA synthesis. These experiments demonstrate that other legume species respond to butyrate exposure by reducing their overall mitotic index and halting progression through the cell cycle.

Effect of lovastatin and fluoromevalonate on phosphatidylinositol 3-kinase activity stimulated with PDGF.

1. Phosphatidylinositol 3-kinase (PI3K) appears to have a crucial role in cellular proliferation induced by platelet-derived growth factor (PDGF). However, the mode of activation of the enzyme has been unclear so far. In the present study, we investigated the effects of a cholesterol lowering drug on [3H]-thymidine ([3H]-TdR) incorporation and PI3K activity in cultured vascular smooth muscle cells (VSMC) stimulated with PDGF. 2. PDGF stimulated both [3H]-TdR incorporation and PI3K activity immunoprecipitated with antiphosphotyrosine antibody in a dose dependent manner (ED50 was 4 ng/mL for [3H]-TdR uptake and 3 ng/mL for PI3K activity). Lovastatin inhibited serum-stimulated [3H]-TdR incorporation dose dependently. PI3K activity induced by PDGF was also inhibited in a dose dependent manner; however, its activity was 61% at 10(-6) mol/L, 72% at 10(-5) mol/L and 8% at 10(-4) mol/L of the control value. 3. These inhibitory effects of lovastatin were completely abolished by adding 1 mmol/L mevalonic acid (MVA), suggesting that MVA metabolites had some important role on the PI3K activation and cellular proliferation. 4. Fluoromevalonate (Fmev), a competitive inhibitor of mevalonate diphosphate (MVA-PP) decarboxylase, inhibited [3H]-TdR incorporation at concentrations more than 10(-6) mol/L. Moreover, marked inhibitory effect was observed at concentrations of 10(-7) and 10(-8) mol/L (76% of control). PI3K activity was also reduced by 10(-3) mol/L Fmev (0.2% of control). However, in contrast to [3H]-TdR uptake, there was no inhibitory effect detected at concentrations up to 10(-4) mol/L. 5. These results suggest that PDGF-stimulated PI3K activity as well as cellular proliferation was modified by protein isoprenylation.

Effects of 22-oxacalcitriol on bone metabolism in vitro: comparison with calcitriol—Effects of 22-oxacalcitriol on osteoclast-like cell formation and bone-resorbing activity

22-Oxacalcitriol (OCT), a synthetic vitamin D3 analog, can mimic the ability of calcitriol to differentiate leukemia and skin cells, to enhance the immune response and to suppress parathyroid hormone secretion, but has much less calcemic activity than that of calcitriol. The mechanism of this selective action remains not fully understood, and the actions of OCT on bone metabolism are little known. The present study was, therefore, designed to investigate the effects of OCT and calcitriol on: the proliferation and functions of osteoblastic MC3T3-E1 cells; osteoclast-like cell formation from hemopoietic blast cells in the absence of stromal cells as well as from unfractionated bone cells in the presence of stromal cells; bone resorption; and the proliferation of MC3T3-E1 cells via monocytes. 22-Oxacalcitriol and calcitriol inhibited [3H]thymidine (TdR) incorporation, alkaline phosphatase activity and collagen synthesis of MC3T3-E1 cells to a similar degree. Both OCT (10(-10)-10(-8) mol/l) and calcitriol significantly and similarly stimulated osteoclast-like cell formation from both hemopoietic blast cells and unfractionated bone cells. 22-Oxacalcitriol (10(-10) and 10(-8) mol/l) significantly stimulated bone resorption, although to a slightly lesser degree than did calcitriol. Human monocyte-conditioned medium (CM) significantly stimulated TdR incorporation into MC3T3-E1 cells. On the other hand, CM obtained from monocytes treated with calcitriol (10(-10)-10(-8) mol/l) significantly inhibited TdR incorporation in a dose-related fashion, whereas CM obtained from monocytes treated with OCT (10(-10)-10(-8) mol/l) significantly stimulated TdR incorporation in a dose-related fashion.(ABSTRACT TRUNCATED AT 250 WORDS)