[Surgical treatment and prognosis of ductal carcinoma in situ: 526 cases analysis].

Objective: To evaluate the choice of surgical treatment of ductal carcinoma in situ (DCIS) and its impact on long-term outcomes. Methods: A retrospective analysis of the clinicopathological features and treatment protocol of DCIS patients who underwent surgical treatment in Comprehensive Breast Health Center, Ruijin Hospital, Shanghai Jiaotong University School of Medicine from January 2009 to August 2016 was done. The factors which could affect surgical treatment were analyzed by χ(2) test and Logistic regression. Survival analysis were performed between different surgical approaches. Kaplan-Meier survival curves and Log-rank tests demonstrated the distribution of disease free survival and overall survival. Results: A total of 526 patients were enrolled in this study, 405 cases (77.0%) underwent mastectomy, 121 cases (23.0%) underwent breast-conserving surgery, of which 88 cases received radiotherapy after breast-conserving surgery. It was shown by univariate and multivariate analysis that age>50 years (OR=0.631, 95% CI: 0.413 to 0.965, P=0.034), first symptom of nipple discharge (OR=0.316, 95% CI: 0.120 to 0.834, P=0.020), excision biopsy (OR=1.831, 95% CI: 1.182 to 2.835, P=0.007) and tumor size >3 cm (OR=0.422, 95% CI: 0.206 to 0.864, P=0.018) were significantly correlated with choice of surgical treatment for breast lesions. Axillary lymph node dissection was performed for 118 cases (22.4%), with sentinel lymph node biopsy for 327 cases (62.2%), and none for 81 cases (15.4%). There was significant statistical difference in the choice of axillary lymph node management in patients of different age (χ(2)=8.124, P=0.017), biopsy type (χ(2)=35.567, P=0.000), breast operation type (χ(2)=149.118, P=0.000) and tumor size (χ(2)=13.394, P=0.010). The 5-year disease free survival rates was 95.7%, 89.6% and 100%, respectively, for mastectomy group, breast-conserving surgery group and breast-conserving surgery plus radiotherapy group. And the 5-year overall survival rates for three groups were 99.0%, 100% and 100%. The differences were not statistically significant (P=0.427, 0.777). Conclusions: For DCIS patients, age, first symptom and tumor size are independent predictors of breast surgery. The choice of axillary lymph node surgery is influenced by age, biopsy, operation type, and tumor size. Different surgical treatment options has no significant effect on disease-free survival and overall survival in DCIS patients.

Leaf-based physiological, metabolic, and ultrastructural changes in cultivated cotton cultivars under cadmium stress mediated by glutathione.

Cadmium (Cd) pollution is present in the world over especially in the industrialized parts of the world. To reduce Cd accumulation in various crops especially food crops, alleviating agents such as reduced glutathione (GSH) can be applied, which are capable either to exclude or to sequester Cd contamination. This study investigated the leaf-based spatial distribution of physiological, metabolic, and microstructural changes in two cotton cultivars (Coker 312 and TM-1) under GSH-mediated Cd stress using single levels of Cd (50 µM) and GSH (50 µM) both separately and in mix along with control. Results showed that GSH revived the morphology and physiology of both cotton cultivars alone or in mix with Cd. Cd uptake was enhanced in all segments of leaf and whole leaf upon the addition of GSH. GSH alleviated Cd-induced reduction in the photosynthetic pigment compositions and chlorophyll a fluorescence parameters. Mean data of biomarkers (2,3,5-triphenyltetrazolium (TTC), total soluble protein (TSP), malondialdehyde (MDA), hydrogen peroxide (H2O2)) revealed the adverse effects of Cd stress on leaf segments of both cultivars, which were revived by GSH. The oxidative metabolism induced by Cd stress was profoundly influenced by exogenous GSH application. The microstructural alterations were mainly confined to chloroplastic regions of leaves under Cd-stressed conditions, which were greatly revived upon the GSH addition. As a whole, Cd stress greatly affected TM-1 as compared to Coker 312. These results suggest a positive role of GSH in alleviating Cd-mediated changes in different leaf sections of cotton cultivars.

Determinants of suicidal ideation in gynecological cancer patients.

OBJECTIVE: Gynecological cancer survivors are at increased risk of psychological problems including suicide risk. Suicidal ideation, which was thought to be precursor to suicide attempts, has not been well studied. This study aimed to investigate the prevalence, and determinants of suicidal ideation for women with gynecological cancer, and then to assess the effect of coping style and social support on suicidal ideation. METHODS: Patients with cervical, ovarian and endometrial cancers seen at Hunan Provincial Tumor Hospital from September 2012 to June 2013 were consecutively recruited and were asked to complete the Zung Self-Rating Depression Scale, Suicidal Ideation of Self-rating Scale, Medical Coping Modes Questionnaire and Social Support Rating Scale. Path analysis was used to examine the relationship among coping style, social support, depression symptoms and suicidal ideation. RESULTS: A total of 579 (579/623, 93.0%) gynecological cancer patients were enrolled in this study and completed all investigations between September 2012 and June 2013. Among them, 105 (18.1%) patients reported suicidal ideation, with the highest rate in patients with ovarian cancer (30.16%). Suicidal ideation was associated with depression symptoms, care providers, chemotherapy history and acceptance-resignation. Path analysis showed that the acceptance-resignation affected suicidal ideation directly as well as mediated by social support and depression symptoms, while confrontation and avoidance affected suicidal ideation entirely through social support and depression symptoms. CONCLUSIONS: Suicidal ideation is high among patients with gynecological cancer, especially among ovarian cancer patients. Coping strategies such as confrontation and avoidance, and social support may be helpful for preventing suicidal ideation among them.

Ultrastructural, metabolic and proteomic changes in leaves of upland cotton in response to cadmium stress.

Present study explores physiological, biochemical and proteomic changes in leaves of upland cotton (ZMS-49) using 500 µM cadmium (Cd) along with control. Leaves' biomass and chlorophyll pigments decreased at 500 µM Cd. Cd contents in roots were higher than leaves. Levels of ROS ( [Formula: see text] and H2O2) both in vivo and in vitro and MDA contents were significantly increased. Chlorophyll parameters (F0, Fm, Fm(') and Fv/Fm), total soluble protein contents and APX showed a decline at 500 µM Cd. SOD, CAT and POD and GR activities significantly enhanced. Less ultrastructural alterations in leaves under Cd stress could be observed. Scanning micrographs at 500 µM Cd possessed less number of stomata as well as near absence of closed stomata. Cd could be located in cell wall, vacuoles and intracellular spaces. Important upregulated proteins were methionine synthase, ribulose 1,5-bisphosphate carboxylase, apoplastic anionic guaiacol peroxidase, glyceraldehydes-3-phosphate dehydrogenase (chloroplastic isoform) and ATP synthase D chain, (mitochondrial). Important downregulated proteins were seed storage proteins (vicilin and legumin), molecular chaperones (hsp70, chaperonin-60 alpha subunit; putative protein disulfide isomerase), ATP-dependent Clp protease, ribulose-1,5-bisphophate carboxylase/oxygenase large subunit. Increase in the activities of ROS-scavenging enzymes, less ultrastructural modification, Cd-deposition in dead parts of cells as well as active regulation of different proteins showed Cd-resistant nature of ZMS-49.

In vitro cadmium-induced alterations in growth and oxidative metabolism of upland cotton (Gossypium hirsutum L.).

Cadmium (Cd) is a toxic pollutant, which cause both dose- and time-dependent physiological and biochemical alterations in plants. The present in vitro study was undertaken to explore Cd-induced physiological and biochemical changes in cotton callus culture at 0, 550, 700, 850, and 1000 µM Cd for four different stress periods (7, 14, 21, and 28 days). At 1000 µM Cd, mean growth values were lower than their respective control. The cell protein contents decreased only after 7-day and 14-day stress treatment. At 550 µM Cd, malondialdehyde (MDA) contents decreased after various stress periods except 21-day period. Superoxide dismutase (SOD) activity at 1000 µM Cd improved relative to its respective controls in the first three stress regimes. Almost a decreasing trend in the hydrogen peroxide (H2O2) and peroxidase (POD) activities at all Cd levels after different stress periods was noticed. Ascorbate peroxidase (APX) activity descended over its relevant controls in the first three stress regimes except at 700 µM Cd after 14- and 21-day stress duration. Moreover, catalase (CAT) mean values significantly increased as a whole. From this experiment, it can be concluded that lipid peroxidation as well as reactive oxygen species (ROS) production was relatively higher as has been revealed by higher MDA contents and greater SOD, CAT activities.

Differential physiological, ultramorphological and metabolic responses of cotton cultivars under cadmium stress.

Cadmium (Cd) stress may cause serious physiological, ultramorphological and biochemical anomalies in plants. Cd-induced physiological, subcellular and metabolic alterations in two transgenic cotton cultivars (BR001, GK30) and their parent line (Coker 312) were evaluated using 10, 100 and 1000 µM Cd. Germination, fresh biomass of roots, stems and leaves were significantly inhibited at 1000 µM Cd. Root volume tolerance index significantly increased (124.16%) in Coker 312 at 1000 µM Cd. In non-Cd stressed conditions, electron micrographs showed well-configured root meristem and leaf mesophyll cells. At 1000 µM Cd, greater ultramorphological alterations were observed in BR001 followed by GK30 and Coker 312. These changes were observed in nucleus, vacuoles, mitochondria and chloroplast. Dense precipitates, probably Cd, were seen in vacuoles, which were also attached to the cell walls. A considerable increase in number of nuclei, vacuoles, starch granules and plastoglobuli was observed in the electron micrographs of both roots and leaves at 1000 µM Cd. MDA contents were higher in roots of BR001 at 1000 µM Cd. Mean values of SOD activity in leaves of both BR001 and GK30 at 1000 µM Cd significantly increased as compared to the controls. POD activity in roots of BR001 and Coker 312 was greater at all Cd (10, 100, 1000 µM) levels over the control. Regarding APX, highest percent increase (71.64%) in roots of GK30 at 1000 µM Cd was found. Non-significant differences in CAT activity were observed at all levels of Cd stress in leaves of BR001 and GK30. Both transgenic cotton cultivars and their parental line invariably responded towards Cd stress. However, Coker 312 showed Cd-resistant behavior as compared to its progeny lines (BR001 and GK30).

Cadmium-induced upregulation of lipid peroxidation and reactive oxygen species caused physiological, biochemical, and ultrastructural changes in upland cotton seedlings.

Cadmium (Cd) toxicity was investigated in cotton cultivar (ZMS-49) using physiological, ultrastructural, and biochemical parameters. Biomass-based tolerance index decreased, and water contents increased at 500 µM Cd. Photosynthetic efficiency determined by chlorophyll fluorescence and photosynthetic pigments declined under Cd stress. Cd contents were more in roots than shoots. A significant decrease in nutrient levels was found in roots and stem. A significant decrease in nutrient levels was found in roots and stems. In response to Cd stress, more MDA and ROS contents were produced in leaves than in other parts of the seedlings. Total soluble proteins were reduced in all parts except in roots at 500 µM Cd. Oxidative metabolism was higher in leaves than aerial parts of the plant. There were insignificant alterations in roots and leaves ultrastructures such as a little increase in nucleoli, vacuoles, starch granules, and plastoglobuli in Cd-imposed stressful conditions. Scanning micrographs at 500 µM Cd showed a reduced number of stomata as well as near absence of closed stomata. Cd depositions were located in cell wall, vacuoles, and intracellular spaces using TEM-EDX technology. Upregulation of oxidative metabolism, less ultrastructural modification, and Cd deposition in dead parts of cells show that ZMS-49 has genetic potential to resist Cd stress, which need to be explored.

Clonal status and clinicopathological features of Langerhans cell histiocytosis.

Some studies have demonstrated that Langerhans cell histiocytosis (LCH) is a neoplastic hyperplasia of Langerhans cells, however some researchers consider that clonality should be assessed in more patients with LCH, both at disease presentation and during the disease course. Monoclonality is a major characteristic of most tumours, whereas normal tissue and reactive hyperplasia are polyclonal. To elucidate the nature of Langerhans cells further, the present study investigated the clinicopathological features and clonality of three cases of LCH in female patients using laser microdissection and a clonality assay, based on X-chromosomal inactivation mosaicism in somatic tissues and polymorphism of the androgen receptor gene. The results indicated that LCH was composed of Langerhans cells with a characteristic morphological appearance, eosinophils, giant cells, neutrophils and foamy cells. Immunohistochemically, the Langerhans cells were positive for CD1a, S-100 protein and vimentin. The clonality assay demonstrated that the Langerhans cells formed a monoclonal population, showing that LCH is neoplastic. We conclude that LCH is characterized by clonal proliferation, although additional studies with larger sample sizes are required to prove this conclusively.

Primary intrapulmonary thymomas: case report and review of the literature.

Primary intrapulmonary thymomas (PIT), which are intrapulmonary tumours without an associated mediastinal component, are very rare; only 29 cases of PIT have been described in the literature since 1951. This report presents a case of PIT in a 59-year old Chinese woman with a type A thymoma (including its pathogenesis, clinical pathological characteristics, immunological phenotype, treatment and prognosis), in the context of a review of the current literature. The origin of thymomas in this unusual location remains unknown. In this case, immunohistochemical analysis demonstrated that the epithelial component was strongly positive for cytokeratin and focally reactive for epithelial membrane antigen. It is concluded that PIT should be considered when the histopathological appearance of a lung tumour is not typical of other pulmonary neoplasms. Complete resection appears sufficient in non-malignant tumours, while in cases of partial resection or malignancy, adjuvant radiotherapy should be considered. Long-term regular clinical follow-up is also warranted, due to the risk of late local recurrence.

Failure behavior of coated nickel-based superalloy under thermomechanical fatigue.

Thermomechanical fatigue (TMF) is a major life limiting factor for gas turbine blades. In this study, the failure behavior of NiCrAlY overlay coated nickel-based superalloy M963 was investigated under out of phase (OP) TMF. All tests were carried out under mechanical strain control with a cyclic period of 200 s. Results revealed that the fatigue life of high velocity oxygen fuel spraying (HVOF) coated specimen was longer than that of air plasma spraying (APS) coated one, but shorter than that of bare superalloy M963 at a given strain range. It was found that cracking process in the APS coating was different from that in the HVOF coating, which was shown by the sketches to understand the crack initiation and propagation behavior.

Transplantation of microencapsulated genetically modified xenogeneic cells augments angiogenesis and improves heart function.

Cell-based gene therapy offers an alternative strategy for therapeutic angiogenesis for the management of myocardial infarction (MI). However, immune rejection poses a significant obstacle to the implantation of genetically engineered allogeneic or xenogeneic cells. In the present study, an ex vivo gene therapy approach utilizing cell microencapsulation was employed to deliver vascular endothelial growth factor (VEGF) to ischemic myocardium. Chinese hamster ovary (CHO) cells were genetically modified to secrete VEGF and enveloped into semipermeable microcapsules. In vitro assay indicated that the microencapsulated engineered CHO cells could secrete VEGF as high as 3852 pg ml(-1) per 48 h at day 8 after encapsulation. Then the microencapsulated CHO cells were implanted into the injured myocardium in a rat MI model, while engineered CHO cells, blank microcapsules and serum-free culture media were implanted as controls. The humoral immunity to xenogeneic CHO cells were evaluated and we found that the titer of anti-CHO antibodies was significantly lower in the microencapsulated CHO transplantation group than the group receiving unencapsulated CHO cells at two weeks after implantation. However, 1 week later, there was almost no difference between these groups. Histology and western blotting confirmed that the microencapsulated CHO cells maintained their original structure and VEGF secretion three weeks after implantation. The capillary density in the treatment region was also significantly higher in the microencapsulated CHO cell group than control groups, which was consistent with gross heart functional improvement. These data suggest that microencapsulated xenogeneic cell-based gene therapy might be a novel approach for therapeutic angiogenesis in ischemic heart disease.

Relationship between the presence of unerupted mandibular third molars and fractures of the mandibular condyle.

Previous studies have shown that the presence of unerupted mandibular third molars predisposes the mandible to angle fractures. This study attempted to relate the presence of unerupted mandibular third molars with the incidence of condyle fractures. The authors compared the proportion of fractures in 439 patients who had unerupted third molars or no unerupted third molars. Fractures at the condylar region showed a significantly higher incidence in patients without unerupted third molars than in those patients with unerupted third molars. This study provides solid clinical evidence to suggest that the removal of unerupted mandibular third molars predisposes the mandible to condyle fractures.

Effect of platelet-rich plasma (PRP) concentration on the viability and proliferation of alveolar bone cells: an in vitro study.

Previous studies have shown that a combination of platelet-rich plasma (PRP) and autogenous bone graft can increase the rate of osteogenesis and enhance bone formation qualitatively. However, contradictory results were reported in a recent animal study. In order to clarify this inconsistency, this study examined the influence of the PRP concentrations on the viability and proliferation of alveolar bone cells in vitro. Bone cells obtained from the alveolar bone chips were exposed to various PRP concentrations. After a culture period of 7 days, cellular viability and proliferation were evaluated by counting the number of cells and a MTT assay. The results showed that the viability and proliferation of alveolar bone cells were suppressed by high PRP concentrations, but were stimulated by low PRP concentrations (1-5%). These in vitro results support the view that variations in the PRP concentrations might influence the bone formation within the PRP-treated bone grafts.

Transplantation of cultured bone marrow stromal cells to improve peripheral nerve regeneration.

The role of cultured bone marrow stromal cells (BMSCs) in peripheral nerve regeneration was examined using an established rabbit peroneal nerve regeneration model. A 15-mm peroneal nerve defect was bridged with a vein filled with BMSCs (1 x 10(6)), which had been embedded in collagen gel. On the contralateral side, the defect was bridged with a vein filled with collagen gel alone. When the regenerated tissue was examined 4, 8 and 12 weeks after grafting, the number and diameter of the myelinated fibers in the side with the BMSCs were significantly higher than in the control side without the BMSCs. This demonstrates the potential of using cultured BMSCs in peripheral nerve regeneration.

Management of mandibular angle fractures using the mandibular angle reduction forceps.

The aim of this study was to determine whether or not the use of the mandibular angle reduction forceps decreases the incidence of post-operative complications. Forty-six patients, who presented with mandibular angle fractures with a displacement or dislocation, were randomly divided into two treatment groups. Both groups underwent an open reduction with a single upper border miniplate and screw fixation. For 23 patients, the mandibular angle reduction forceps was used to aid in fracture reduction, and for 23 patients, the reduction of the fracture was achieved using IMF. The post-reduction radiographs showed that the reduction forceps group had a higher proportion of precise anatomic alignment of fracture than those in the IMF group. In addition, the former group showed a lower rate of post-operative complications, as compared to the latter group. This study found that the use of the mandibular angle reduction forceps is an important factor for decreasing the incidence of post-surgical complication.

Microneural anastomosis using cyanoacrylate adhesives.

We have developed a reliable method of microneural anastomosis using cyanoacrylate adhesives. This method involves overlapping the epineuriums of the two nerve ends and then applying two or three microdrops of cyanoacrylate adhesive on the surface of the epineurium just where the epineuriums overlap. The sciatic nerves of Sprague-Dawley rats were transected and repaired either with 10-0 nylon sutures or by using the described method. Histological evaluation showed no significant difference in the outcome of nerve regeneration between the two groups. It was concluded that the cyanoacrylate repair deserves to be considered as an alternative to the conventional suture technique in microneural anastomosis.

Retinoic acids promote the action of aromatase and 17beta-hydroxysteroid dehydrogenase type 1 on the biosynthesis of 17beta-estradiol in placental cells.

The biosynthesis of 17beta-estradiol (E(2)) in human placenta involves the actions of aromatase and 17beta-hydroxysteroid dehydrogenase type 1 (17HSD1). Aromatase, an enzyme complex comprised of P450aromatase (P450arom) and NADH-cytochrome P450 reductase, converts androgens to estrogens, whereas 17HSD1 catalyzes the reduction of estrone to E(2). In the present study, the effects of retinoic acids (RAs) on P450arom and 17HSD1 expression in placental cells were investigated. Treatment with all-trans-RA (at-RA) or 9cis-RA increased E(2) production in JEG-3 choriocarcinoma cells and cytotrophoblast (CTB) cells isolated from normal early placentas. Meanwhile, the activity of aromatase and expression of P450arom mRNA were induced by at-RA in JEG-3 cells. Northern blot analysis showed that the effect on P450arom mRNA expression occurs in a dose- and time-dependent fashion. Similar to at-RA and 9cis-RA, Ro40-6055, the retinoic acid receptor alpha (RARalpha)-selective activator, increased the expression of P450arom and 17HSD1 mRNA in JEG-3 cells. On the other hand, Ro41-5253 (Ro41), the RARalpha-selective antagonist, blocked the stimulatory effect of RAs on P450arom expression. Surprisingly, Ro41 induced the activity and mRNA expression of 17HSD1 in JEG-3 cells, which is in contrast to the expected inhibitory effect and, moreover, remarkably potentiated the induction by at-RA and 9cis-RA. However, reporter gene analysis revealed that the influence of Ro41 on the transcription of the HSD17B1 gene, which encodes 17HSD1, is considerably milder in JEG-3 cells, and it only additively enhanced the effect of at-RA. Finally, it was found that at-RA and 9cis-RA increased the expression of P450arom and 17HSD1 mRNA in CTB cells, but to a lesser extent. The data suggest that RAs may play a role in promoting the biosynthesis of E(2 )in the placenta. In addition, Ro41 has divergent effects on gene expression in JEG-3 cells.

Activation of protein kinase C inhibits uptake, currents and binding associated with the human dopamine transporter expressed in Xenopus oocytes.

Activation of protein kinase C (PKC) regulates the activity of a number of neurotransmitter transporters. When Xenopus oocytes expressing the cloned human dopamine transporter (hDAT) were pretreated with bath-applied phorbol 12-myristate 13-acetate (PMA), a PKC activator, [3H]DA uptake decreased irreversibly in a time- and dose-dependent manner (IC50 = 22 nM; maximal inhibition = 63-85%). The inhibition appeared to be PKC-specific because incubation with the inactive form of phorbol ester 4alpha-phorbol-12,13-didecanoate (400 nM) did not change the uptake activity and PMA (100 nM) inhibition could be partially blocked by the selective PKC inhibitor bisindolylmaleimide I (1 microM). Saturation studies of [3H]DA uptake showed that PMA-induced inhibition was due to a decrease in V(max) with no change in K(T). Similar to uptake, PMA pretreatment inhibited both the hDAT transport-associated and substrate-independent leak currents. PMA also decreased membrane capacitance (C(m)) by 40%, selectively in hDAT-expressing oocytes. In addition, PMA pretreatment resulted in a 77% decrease in B(max) of [3H]mazindol binding to intact oocytes. In contrast, binding to whole homogenates of PMA-pretreated oocytes was not significantly altered. These results suggest that PMA regulates hDAT expressed in Xenopus oocytes by altering cell surface trafficking of hDAT.

Regulation of transformation frequency by exogenous and endogenous growth factors in rat tracheal epithelial cells.

The purpose of our studies was to re-evaluate the rat tracheal epithelial (RTE) transformation system and to identify critical variables that affect the development of enhanced growth variants (EGV). The enhanced growth variant colony, which is a preneoplastic cell variant, is the quantifiable transformation endpoint in RTE cultures. Using a standard protocol the frequency of EGV colony formation was shown to be inversely related to the number of clonogenic cells (CFU) seeded per dish in control cultures as well as in cultures treated with the transforming agent 6-nitrochrysene (6-NC). Experiments showed that the major mechanisms that underlie the CFU density-dependent inhibition of EGV colony formation are depletion of growth factors from and accumulation of autocrine TGF-beta in the media. Thus the cells themselves are creating the selection environment, which allows only the EGVs to survive. The effects of agents such as 6-NC, which increase the frequency of EGV colony formation, are to induce a cellular phenotype that is less susceptible to the selection environment. We showed that TGF-beta-neutralizing antibodies added to the selection media significantly increased EGV colony formation in control cultures but not in 6-NC-exposed cultures. In addition we demonstrated that the development of EGV colonies is much less susceptible to inhibition by (exogenous) TGF-beta in 6-NC-exposed than in control cultures. Thus spontaneous and 6-NC EGV colony formation are distinguishable based on TGF-beta sensitivity. To conduct quantitative cell transformation experiments with RTE cells it is essential that the number of surviving CFU per dish is the same in control and treated cultures. Under the conditions used in the studies described here, 350-500 CFU per culture was found to be the optimum CFU density. Besides 6-NC, agents that have been shown to increase EGV colony frequency under conditions similar to those described here are nitrosamines, NNK, nickel compounds and X-rays.

Intact cell binding properties of cells expressing altered beta-adrenergic receptors.

During the course of equilibrium competition binding assays with intact cells, agonists induce conversion of beta-adrenergic receptors (BARs) from a native form with high affinity for agonists to a form with a markedly lower apparent affinity. The roles of receptor internalization, receptor-Gs coupling, and receptor phosphorylation in this agonist-induced conversion to the low affinity form were investigated. Agonist and antagonist competition for [125I]iodopindolol binding to intact cells was measured in mouse L cells expressing wild-type BARs (C+I+), mutated BARs that do not couple to Gs but do internalize (C-I+), and mutated BARs that do not couple to Gs and do not internalize (C-I-). For C+I+ and C-I+ cells, most of the receptors exhibited apparent affinities for the agonist isoproterenol that were 500-900-fold lower in equilibrium assays with intact cells than in short-time assays with intact cells or in equilibrium assays with isolated membranes, similar to previous results with cells expressing native BARs. The extent of conversion to this lower affinity form for C-I- cells was markedly decreased. Binding properties for the antagonist metoprolol were similar for all three BARs in both short-time and equilibrium assays. Isoproterenol competition in short-time and equilibrium assays also was compared in Chinese hamster fibroblasts expressing wild-type BARs, mutated BARs that lack BAR kinase sites, mutated BARs that lack cAMP-dependent protein kinase sites, and mutated BARs that lack both types of phosphorylation sites. All three BAR phosphorylation mutants showed only small but significant decreases, relative to the wild-type BAR, in the extent of conversion to the low affinity form. These results provide additional evidence that receptor internalization is the major determinant for the conversion of intact cell BARs to the low affinity form. Receptor phosphorylation may play a minor role in conversion to the low affinity form, whereas receptor coupling to Gs is apparently not required.