3D osteoarthritic changes in TMJ condylar morphology correlates with specific systemic and local biomarkers of disease.

OBJECTIVE: To assess 3D morphological variations and local and systemic biomarker profiles in subjects with a diagnosis of temporomandibular joint osteoarthritis (TMJ OA). DESIGN: Twenty-eight patients with long-term TMJ OA (39.9 ± 16 years), 12 patients at initial diagnosis of OA (47.4 ± 16.1 years), and 12 healthy controls (41.8 ± 12.2 years) were recruited. All patients were female and had cone beam CT scans taken. TMJ arthrocentesis and venipuncture were performed on 12 OA and 12 age-matched healthy controls. Serum and synovial fluid levels of 50 biomarkers of arthritic inflammation were quantified by protein microarrays. Shape Analysis MANCOVA tested statistical correlations between biomarker levels and variations in condylar morphology. RESULTS: Compared with healthy controls, the OA average condyle was significantly smaller in all dimensions except its anterior surface, with areas indicative of bone resorption along the articular surface, particularly in the lateral pole. Synovial fluid levels of ANG, GDF15, TIMP-1, CXCL16, MMP-3 and MMP-7 were significantly correlated with bone apposition of the condylar anterior surface. Serum levels of ENA-78, MMP-3, PAI-1, VE-Cadherin, VEGF, GM-CSF, TGFßb1, IFNγg, TNFαa, IL-1αa, and IL-6 were significantly correlated with flattening of the lateral pole. Expression levels of ANG were significantly correlated with the articular morphology in healthy controls. CONCLUSIONS: Bone resorption at the articular surface, particularly at the lateral pole was statistically significant at initial diagnosis of TMJ OA. Synovial fluid levels of ANG, GDF15, TIMP-1, CXCL16, MMP-3 and MMP-7 were correlated with bone apposition. Serum levels of ENA-78, MMP-3, PAI-1, VE-Cadherin, VEGF, GM-CSF, TGFß1, IFNγ, TNFα, IL-1α, and IL-6 were correlated with bone resorption.

Triaging pregnancies of unknown location: the performance of protocols based on single serum progesterone or repeated serum hCG levels.

STUDY QUESTION: How does a protocol based on a single serum progesterone measurement perform as a triage tool in women with pregnancy of unknown location (PUL) in comparison to protocols based on serial hCG measurement? SUMMARY ANSWER: Triage based on the logistic regression model M4 (using initial hCG and hCG ratio (48 h/0 h)) classifies the majority of PUL into low and high risk groups, in contrast to a progesterone protocol based on a serum level threshold of 10 nmol/l. WHAT IS KNOWN ALREADY: Low progesterone has been shown to identify failing pregnancies and those at low risk of complications. A prediction model (M4) based on the initial hCG and the hCG ratio at 0 and 48 h can successfully classify PUL into low and high risk groups. STUDY DESIGN, SIZE AND DURATION: A multi-centre diagnostic accuracy study of 1271 women was performed retrospectively on data from women at St. George's Hospital (SGH, London, UK) between February 2005 and 2006, Queen Charlottes & Chelsea Hospital (QCCH, London, UK) between April 2009 and August 2012, and the Royal Prince Alfred Hospital (RPAH, Sydney, Australia) between February 2008 and October 2011. The end-points were the final observed outcome for each pregnancy as a failed PUL (low risk), intrauterine pregnancy (IUP, low risk), or ectopic pregnancy (EP, high risk), and any interventions or complications for EP during the follow-up period. PARTICIPANTS/MATERIALS, SETTING AND METHODS: Complete data were available for initial progesterone, 0/48 h hCG and final outcome in 431 of 534 women (81%) at SGH, 396/585 (68%) at QCCH and 96/152 (63%) at RPAH. Missing values were handled using multiple imputation. Three diagnostic approaches were used to classify PUL as high risk: a range of serum progesterone levels were evaluated (>10, 16 and 20 nmol/l) for the progesterone protocol, risk of EP given by the M4 model ≥5% for the M4-based protocol, and hCG ratio was between 0.87 and 1.66 for hCG cut-offs as previously published. Results were analysed using random intercept models or stratified analysis to account for variability between centres. MAIN RESULTS AND THE ROLE OF CHANCE: The progesterone protocol based on levels of >10 nmol/l classified 24% (95% confidence interval 20-28%) of failed PUL, 95% (92-97%) of IUP and 76% (67-83%) of EP as high risk. The M4 protocol classified 14% (11-17%) of failed PUL, 37% (31-43%) of IUP and 84% (76-90%) of EP as high risk. The hCG ratio cut-offs classified 10% (8-12%) of failed PUL, 15% (11-20%) of IUP and 63% (53-71%) of EP as high risk. Using complete cases only, 67% of EP treated with methotrexate (n = 48) and 89% surgically managed (n = 37) were correctly classified by the progesterone protocol, 96 and 81% by M4 protocol and 75 and 65% by hCG ratio cut offs, respectively. LIMITATIONS, REASONS FOR CAUTION: Data were incomplete for 103 (19%), 189 (32%) and 56 (37%) patients at SGH, QCCH and RPAH, respectively; however, we are reassured by the minimal differences seen between the results of complete cases and those following imputation of missing values. The variation in the inclusion criteria between the three centres is also a potential limitation of this study; however, it reflects real clinical practice. Furthermore, the hCG ratio cut-offs were not originally developed to optimize triage. WIDER IMPLICATIONS OF THE FINDINGS: The results show that serum progesterone is less efficient for triage than serial hCG measurements assessed using the M4 model, the striking difference being serum progesterone places nearly all IUP in the high-risk category. A two-step strategy combining single-visit and two-visit approaches should be investigated. STUDY FUNDING/COMPETING INTERESTS: Funding was from Research Foundation-Flanders (FWO). There are no competing interests.

The prediction of pouch of Douglas obliteration using offline analysis of the transvaginal ultrasound 'sliding sign' technique: inter- and intra-observer reproducibility.

STUDY QUESTION: What is the inter-/intra-observer agreement and diagnostic accuracy among gynaecological and non-gynaecological ultrasound specialists in the prediction of pouch of Douglas (POD) obliteration (secondary to endometriosis) at offline analysis of two-dimensional videos using the dynamic real-time transvaginal ultrasound (TVS) 'sliding sign' technique? SUMMARY ANSWER: The inter-/intra-observer agreement and diagnostic accuracy for the interpretation of the TVS 'sliding sign' in the prediction of POD obliteration was found to be very acceptable, ranging from substantial to almost perfect agreement for the observers who specialized in gynaecological ultrasound. WHAT IS KNOWN ALREADY: Women with POD obliteration at laparoscopy are at an increased risk of bowel endometriosis; therefore, the pre-operative diagnosis of POD obliteration is important in the surgical planning for these women. Previous studies have used TVS to predict POD obliteration prior to laparoscopy, with a sensitivity of 72-83% and specificity of 97-100%. However, there have not been any reproducibility studies performed to validate the use of TVS in the prediction of POD obliteration pre-operatively. STUDY DESIGN, SIZE, DURATION: This was a reproducibility study which involved the offline viewing of pre-recorded video sets of 30 women presenting with chronic pelvic pain, in order to determine POD obliteration using the TVS 'sliding sign' technique. The videos were selected on real-time representative quality/quantity; they were not obtained from sequential patients. There were a total of six observers, including four gynaecological ultrasound specialists and two fetal medicine specialists. The study was conducted over a period of 1 month (March 2012-April 2012). PARTICIPANTS/MATERIALS, SETTING, METHODS: The four gynaecological ultrasound observers performed daily gynaecological scanning, while the other two observers were primarily fetal medicine sonologists. Each sonologist viewed the TVS 'sliding sign' video in two anatomical locations (retro-cervix and posterior uterine fundus), i.e. 60 videos in total. The POD was deemed not obliterated, if 'sliding sign' was positive in both anatomical locations (i.e. anterior rectum/rectosigmoid glided smoothly across the retro-cervix/posterior fundus, respectively). If the 'sliding sign' was negative (i.e. anterior rectum/rectosigmoid did not glide smoothly over retro-cervix/posterior fundal region, respectively), the POD was deemed obliterated. Diagnostic accuracy and inter-observer agreement among the six sonologists was evaluated. The same sonologist was also asked to reanalyse the same videos, albeit in a different order, at least 7 days later to assess for intra-observer agreement. A separate analysis of the inter- and intra-observer correlation was also performed to determine the agreement among the four observers who specialized in gynaecological ultrasound. Cohen's κ coefficient <0 meant that there was poor agreement, 0.01-0.20 slight agreement, 0.21-0.40 fair agreement, 0.41-0.60 moderate agreement, 0.61-0.80 substantial agreement and 0.81-0.99 almost perfect agreement. MAIN RESULTS AND THE ROLE OF CHANCE: Agreement (Cohen's κ) between all six observers for the interpretation of the 'sliding sign' for both sets of videos in both regions (retro-cervix and fundus) ranged from 0.354 to 0.927 (fair agreement to almost perfect agreement) compared with 0.630-0.927 (substantial agreement to almost perfect agreement) when only the gynaecological sonologists were included. The overall multiple rater agreement for the interpretation of the 'sliding sign' for both video sets and both regions was Fleiss' κ 0.454 (P-value <0.01) for all six observers and 0.646 (P-value <0.01) for the four gynaecological ultrasound specialists. The multiple rater agreement for all six or all four observers was higher for the retro-cervical region versus the fundal region (Fleiss' κ 0.542 versus 0.370 and 0.732 versus 0.560, respectively). The intra-observer agreement among the six observers for the interpretation of the 'sliding sign' and prediction of POD obliteration ranged from Cohen's κ 0.60-0.95 and 0.46-1.0 (P-value <0.01), respectively. After excluding the fetal medicine specialists, the intra-observer agreement for the interpretation of the 'sliding sign' and the prediction of POD obliteration ranged from Cohen's κ 0.71-0.95 and 0.67-1.0, respectively, indicating substantial to almost perfect agreement. When comparing the four gynaecological observers for the prediction of POD obliteration using the TVS 'sliding sign' (after excluding cases with the POD outcome classified as 'unsure' by the observers), the results for accuracy, sensitivity, specificity, positive and negative predictive value were 93.1-100, 92.9-100, 90.9-100, 77.8-100 and 97.7-100%, respectively. LIMITATIONS, REASONS FOR CAUTION: The 'gold standard' for the diagnosis of POD obliteration is laparoscopy; however, laparoscopic data were available only for 24 out of 30 (80%) TVS 'sliding sign' cases included in this study. Although this should not affect the inter- and intra-observer agreement findings, the ability to draw conclusions regarding the diagnostic accuracy of the TVS 'sliding sign' in the prediction of POD obliteration is somewhat limited. In addition, the diagnostic accuracy findings should be interpreted with the caveat that the cases classified as 'unsure' for the prediction of POD obliteration were excluded from the analysis. WIDER IMPLICATIONS OF THE FINDINGS: We have validated the dynamic real-time TVS 'sliding sign' technique for the prediction of POD obliteration, and this simple ultrasound-based test appears to have very acceptable inter-/intra-observer agreement for those who are experienced in gynaecological ultrasound. Given that women with POD obliteration at laparoscopy have an increased risk of bowel endometriosis and requirement for bowel surgery, the TVS 'sliding sign' test should be considered in the pre-operative imaging work-up for all women with suspected endometriosis, to allow for appropriate surgical planning. We believe the TVS 'sliding sign' technique may be easily learned by sonologists/sonographers who are familiar with performing gynaecological ultrasound, and that further studies are required to confirm the diagnostic accuracy of this new ultrasound technique amongst sonologists/sonographers with various levels of experience. STUDY FUNDING/COMPETING INTEREST(S): This study received no specific grant from any funding agency in the public, commercial or not-for-profit sectors and the authors declare no competing interests.

Regulation of human placental development by oxygen tension.

Cytotrophoblasts, specialized placental cells, proliferate early in pregnancy and then differentiate into tumor-like cells that establish blood flow to the placenta by invading the uterus and its vasculature. In this study, cytotrophoblasts cultured under hypoxic conditions (2 percent oxygen), mimicking the environment near the uterine surface before 10 weeks of gestation, continued proliferating and differentiated poorly. When cultured in 20 percent oxygen, mimicking the environment near uterine arterioles, the cells stopped proliferating and differentiated normally. Thus, oxygen tension determines whether cytotrophoblasts proliferate or invade, thereby regulating placental growth and cellular architecture.

Vaginal delivery compared with elective caesarean section: the views of pregnant women and clinicians.

OBJECTIVE: To quantify the risk of morbidity from vaginal delivery (VD) that pregnant women would be prepared to accept before requesting an elective caesarean section and to compare these views with those of clinicians. DESIGN: Cross-sectional survey. SETTING: Major teaching hospital (nulliparas and midwives) and national samples of medical specialists. SAMPLE: Nulliparas (n = 122), midwives (n = 84), obstetricians (n = 166), urogynaecologists (n = 12) and colorectal surgeons (n = 79). METHODS: Face-to-face interviews (nulliparas) and mailed questionnaire (clinicians). MAIN OUTCOME MEASURES: Maximum level of risk participants would be prepared to accept before opting for an elective caesarean section for each of 17 potential complications of VD. Utility scores for each complication were calculated with higher scores (closer to 1) indicating a greater acceptance of risk. RESULTS: Pregnant women were willing to accept higher risks than clinicians for all 17 potential complications. They were least accepting of the risks of severe anal incontinence (mean utility score 0.32), emergency caesarean section (0.51), moderate anal incontinence (0.56), severe urinary incontinence (0.56), fourth-degree tears (0.59) and third-degree tears (0.72). The views of midwives were closest to those of pregnant women. Urogynaecologists and colorectal surgeons were the most risk averse, with 42 and 41%, respectively, stating that they would request an elective caesarean for themselves or their partners. CONCLUSIONS: Pregnant women were willing to accept significantly higher risks of potential complications of VD than clinicians involved in their care. Pregnant women's views were more closely aligned to midwives than to medical specialists.

Effect of fetal adrenalectomy on catecholamine release and physiologic adaptation at birth in sheep.

Plasma catecholamine levels increase dramatically at birth. To determine the contribution of adrenal catecholamine secretion to the surge in catecholamines at birth and the role in newborn adaptation, we performed surgical adrenalectomy or sham operation on near-term ovine fetuses. After recovery in utero, the animals were delivered and supported by mechanical ventilation. Plasma catecholamine levels, heart rate, blood pressure, cardiac output, pulmonary function, surfactant secretion, and release of free fatty acids (FFA) and glucose were compared in control and adrenalectomized animals. Plasma epinephrine increased rapidly at birth in controls but was undetectable in adrenalectomized animals. Norepinephrine levels were not statistically different. Heart rate, blood pressure, cardiac output and contractility increased abruptly after cord cutting in controls but did not increase in adrenalectomized animals. Lung compliance, pulmonary function, surfactant pool size, glucose and FFA levels were significantly decreased in adrenalectomized animals. These results suggest that adrenal epinephrine secretion is vital to many of the adaptive events at birth.

Specific enzymatic dephosphorylation of the retinoblastoma protein.

The retinoblastoma gene product (RB) undergoes cell cycle-dependent phosphorylation and dephosphorylation. Pulse-chase experiments revealed that the change in RB gel electrophoretic migration which occurs near mitosis is due to enzymatic dephosphorylation (J. W. Ludlow, J. Shon, J. M. Pipas, D. M. Livingston, and J. A. DeCaprio, Cell 60:387-396, 1990). To determine the precise timing of RB dephosphorylation and whether a specific phosphatase is active in this process, we have utilized a nocodazole block and release protocol which allows a large population of cells to progress synchronously through mitosis. In such experiments, RB dephosphorylation began during anaphase and continued until complete dephosphorylation was apparent in the ensuing G1 period. In addition, late mitotic cell extracts were capable of dephosphorylating RB in vitro. This RB-specific mitotic phosphatase activity was more active in anaphase extracts than in pro- or metaphase extracts, which is consistent with the results obtained in vivo. Okadaic acid and protein phosphatase inhibitors 1 and 2 inhibited this specific RB phosphatase activity. These results suggest a role for serine and threonine phosphoprotein phosphatase type 1 in the late mitotic dephosphorylation of RB.

Control of junB and extracellular matrix protein expression by transforming growth factor-beta 1 is independent of simian virus 40 T antigen-sensitive growth-sensitive growth-inhibitory events.

Treatment of Mv1Lu mink lung epithelial cells with transforming growth factor-beta 1 (TGF-beta 1) prevents phosphorylation of the retinoblastoma susceptibility gene product, RB, in late G1 phase of the cell cycle, which is thought to retain RB in a growth-suppressive state. This effect is paralleled by cell cycle arrest in late G1 (M. Laiho, J. A. DeCapric, J. W. Ludlow, D. M. Livingston, and J. Massagué, Cell 62:175-185, 1990). Arrest can be prevented by expression of simian virus 40 T antigen, which binds to underphosphorylated RB, presumably blocking its growth-suppressive activity. The response of cells to TGF-beta 1, however, is complex and includes changes in the levels of expression of genes encoding nuclear transcription factors and extracellular matrix components. To define the relationships among various components of the TGF-beta 1 response, we have investigated the effect of TGF-beta 1 on cells whose growth-inhibitory response to this factor is prevented by T antigen. TGF-beta 1 addition to exponentially growing Mv1Lu cells increased the levels of junB mRNA and of three extracellular matrix proteins: plasminogen activator inhibitor-1, fibronectin, and thrombospondin. Kinetically, the effects on junB and plasminogen activator inhibitor-1 expression occurred faster (half-maximal at 1 to 2 h) than the effects on fibronectin and thrombospondin expression (half-maximal at 6 to 10 h). These effects either preceded or overlapped, respectively, the withdrawal of Mv1Lu cells from the cell cycle. Expression of a transfected T-antigen gene in Mv1Lu cells, however, did not prevent any of these responses to TGF-beta 1. The results indcate that TGF-B1-stimulated expression of junB and extracellular matrix proteins in Mv1Lu cells can occur independently of the T-antigen-sensitive events that lead to growth arrest.

Association of p300 and CBP with simian virus 40 large T antigen.

p300 and the CREB-binding protein CBP are two large nuclear phosphoproteins that are structurally highly related. Both function, in part, as transcriptional adapters and are targeted by the adenovirus E1A oncoprotein. We show here that p300 and CBP interact with another transforming protein, the simian virus 40 large T antigen (T). This interaction depends on the integrity of a region of T which is critical for its transforming and mitogenic properties and includes its LXCXE Rb-binding motif. T interferes with normal p300 and CBP function on at least two different levels. The presence of T alters the phosphorylation states of both proteins and inhibits their transcriptional activities on certain promoters. Although E1A and T show little sequence similarity, they interact with the same domain of p300 and CBP, suggesting that this region exhibits considerable flexibility in accommodating diverse protein ligands.

Quantum chaos and fluctuations in isolated nuclear-spin systems.

Using numerical simulations we investigate dynamical quantum chaos in isolated nuclear spin systems. We determine the structure of quantum states, investigate the validity of the Curie law for magnetic susceptibility and find the spectrum of magnetic noise. The spectrum is the same for positive and negative temperatures. The study is motivated by recent interest in condensed-matter experiments for searches of fundamental parity- and time-reversal-invariance violations. In these experiments nuclear spins are cooled down to microkelvin temperatures and are completely decoupled from their surroundings. A limitation on statistical sensitivity of the experiments arises from the magnetic noise.

Hypoxia arrests ovarian carcinoma cell cycle progression, but invasion is unaffected.

Although hypoxic cells are generally resistant to radiation and chemical therapies designed to halt the spread of neoplastic disease, few investigations have been carried out with regard to the molecular mechanisms responsible for this phenomenon. Here, we report of the development of an in vitro model system with which to study the molecular mechanisms involved in the proliferation and invasion of human ovarian carcinoma cells under hypoxia. Results from [(3)]thymidine incorporation experiments indicate that hypoxia triggers cessation of ovarian carcinoma cell DNA synthesis. Flow cytometry analysis of cellular DNA content for hypoxic cultures revealed that cell cycle progression was arrested. This arrest was found to be reversible upon reoxygenation of the cultures. Concomitant with this growth arrest is hypophosphorylation of pRB and a reduction in cyclin A abundance, suggesting that hypoxia induces growth arrest by regulating the activities of these crucial cell cycle-regulatory proteins. In vitro invasion assays revealed that hypoxia has no appreciable effect on the invasive ability of these cells. Immunoblotting established that the detected proteolytic activity was due to the matrix metalloproteinase MMP-2, the M(r) 72,000 type IV collagenase that is most closely associated with the metastatic phenotype in vitro and in vivo. These data support the notion that populations of ovarian carcinoma cells are capable of surviving and invading extracellular matrix during hypoxic conditions and, after a more suitable oxygen environment is reached, giving rise to new cell colonies.

Selective ability of S-phase cell extracts to dephosphorylate SV40 large T antigen in vitro.

Simian virus 40 (SV40) large tumor antigen (T) is an oncoprotein whose biological and biochemical functions appear to be modulated by phosphorylation. Recently, SV40 DNA replication in vitro has been shown to be activated by dephosphorylation involving the activity of a serine/threonine phosphoprotein phosphatase belonging to the type 2A class (PP2A) [Virshup, D.M., Kauffman, M.G. & Kelly, T.J. (1989) EMBO J., 8, 3891-3898]. To address the question of how specificity of PP2A activity towards T is regulated, an in vitro assay to study the process of T dephosphorylation was developed. Unlabeled extracts from cells enriched for various stages of the cell cycle were incubated with 32P-labeled, immunocomplexed T. Extracts from a population of cells enriched for S phase demonstrated a selective ability to dephosphorylate this labeled protein when compared with extracts prepared from G1- and M-phase cells. The time course of release from growth arrest demonstrated that this T-specific phosphatase activity occurred at the onset of host-cell DNA synthesis. In contrast, when using 32P-labeled phosphorylase a as the substrate, phosphatase activity appeared to be present throughout the cell cycle. The data presented here are consistent with the notion that PP2A activity towards T is regulated in a cell cycle-dependent manner.

Characterization of the mitotic phase pRb-directed protein phosphatase activity.

We have previously reported on the M-phase specific dephosphorylation of pRb and identified a type 1 serine/threonine protein phosphatase (PP1) as the enzyme mediating pRb dephosphorylation. In this report, we have characterized the pRb-directed phosphatase activity found in mitotic cells with respect to dose dependence and demonstrate that the pRb isoform conversion detected in vitro mirrors the pRb isoform conversion which occurs during mitosis of intact cells. Cell fractionation and PP1 catalytic subunit isolation studies support the notion that the pRb-directed phosphatase activity involves subpopulations of PP1 catalytic subunits. Coprecipitation studies revealed that PP1 can form a complex with hypophosphorylated pRb which was converted from the hyperphosphorylated form in mitotic cell extracts. Taken together with data from previous reports in the literature, a model for the regulation of PP1 activity towards pRb during mitotic exit is proposed.

Hypoxia-induced pRB hypophosphorylation results from downregulation of CDK and upregulation of PP1 activities.

Exposure of CV-1P cells to hypoxic conditions results in reversible cell cycle arrest concomitant with accumulation of pRB in the hypophosphorylated, growth suppressive form. Similar to cell cycle arrest induced by serum starvation, we show here that hypoxia-induced arrest is accompanied by a decrease in pRB-directed CDK4 and CDK2 activities, lower cyclin D and E protein levels, and by an increase in p27 protein abundance. Immunoprecipitation studies reveal an increase in p27 association with cyclin E-CDK2 complexes. In contrast to cell cycle arrest induced by serum starvation, hypoxia increases PP1-mediated pRB dephosphorylation. These data reveal that synergy between decreased pRB-directed cyclin/CDK activity and increased pRB-directed phosphatase activity contribute towards inducing and maintaining pRB in its hypophosphorylated, growth suppressive state during hypoxia.

Binding of select forms of pRB to protein phosphatase type 1 independent of catalytic activity.

The product of the retinoblastoma susceptibility gene, pRB, is a demonstrated substrate for the type 1 serine/threonine protein phosphatases (PP1). Curiously, there has been a paucity of data supporting the idea that phosphorylated pRB can be found in a complex with PP1. To more fully characterize the association between these two proteins, we utilized a PP1-affinity chromatography approach to increase our ability to capture from mammalian cell lysate populations of pRB capable of binding to PP1. Western blot analysis of the bound proteins indicates that both faster migrating, hypophosphorylated pRB, as well as slower migrating, hyperphosphorylated pRB can bind. Phosphorylated pRB binding was confirmed by immunoprecipitation of eluted 32P-labeled pRB. In addition, Western blotting of eluted proteins with pRB phosphorylated-site-specific antibodies revealed select phosphorylated forms of pRB binding to PP1. Similar binding studies performed with toxin-inhibited PP1 indicate that catalytic activity of PP1 is not required for pRB binding. The significance of this finding with respect to the functional importance of this interaction is discussed.

Site-specific and temporally-regulated retinoblastoma protein dephosphorylation by protein phosphatase type 1.

pRb is dephosphorylated at mitotic exit by the type 1 serine/threonine protein phosphatases (PP1). Here we demonstrate for the first time that mitotic pRb dephosphorylation is a sequential, temporally-regulated event. We also provide evidence that the three mammalian isoforms of PP1, alpha, gamma-1, and delta, differ in their respective preferences for site-specific pRb dephosphorylation and that the mitotic and G(1) PP1-isoform counterparts exhibit differential activities towards mitotic pRb. Finally, the physiological relevance of the striking contrast between the patterns of Thr821 and Thr826 dephosphorylation, sites known to be important for disrupting binding of LXCXE-containing proteins to pRb, is addressed.

Hypoxic stress induces reversible hypophosphorylation of pRB and reduction in cyclin A abundance independent of cell cycle progression.

The effect of hypoxic stress on the phosphorylation state of the product of retinoblastoma susceptibility gene (pRB) and cyclin A abundance was examined in CV-1P monkey kidney cells. Flow cytometric DNA histogram analysis and [3H]-thymidine incorporation assays demonstrated that hypoxia inhibited cell cycle progression and cell division. Within 6-12 h of hypoxia, pRB became hypophosphorylated and cyclin A abundance fell below detection limits. Hypophosphorylation of pRB and loss of cyclin A detection occurred without progression of cells through S-phase. These effects were found to be reversible by reoxygenation of the hypoxic cultures. Cells were shown to resume DNA synthesis within 12-16 h of reoxygenation concomitant with pRB hyperphosphorylation and an increase in cyclin A detection. These data demonstrate that hypoxic stress blocks the progression of these cells through the phases of the cell cycle and suggests that the effect might arise from the down regulation of key cell cycle controlling elements. The data also raise the possibility that maintaining pRB in a hyperphosphorylated state may be crucial for S-phase progression as well as S-phase entry.

Interaction of the retinoblastoma protein (pRb) with the catalytic subunit of DNA polymerase delta (p125).

The retinoblastoma gene product (pRb) interacts with many cellular proteins to function in the control of cell division, differentiation, and apoptosis. Several pRb binding proteins complex with pRb through an amino acid sequence called the LXCXE motif. The catalytic subunit of DNA polymerase delta (p125) contains a LXCXE motif. To further study the biochemical function of this polymerase, we sought to determine if p125 interacts with pRb. Experiments using GST-pRb fusion proteins showed that p125 from breast epithelial (MCF10A) cell extracts associates with pRb. In addition, GST-p125 fusion proteins bound pRb from the same cell extracts. The pRb that associated with GST-p125 was largely unphosphorylated. Coimmunoprecipitation experiments using cell cycle synchronized cells revealed that p125 and pRb form a complex predominantly during G1 phase, the phase during which pRb is mostly unphosphorylated. In vitro phosphorylation of GST-pRb by the cyclin dependent kinases reduced the ability of p125 to associate with GST-pRh. Addition of the LXCXE containing protein SV40 large T antigen to GST-pRb blocks the ability of p125 to associate with pRb, suggesting that it may be through a LXCXE sequence by which p125 interacts with pRb. Finally, in vitro polymerase assays demonstrate that GST-pRb fusion protein stimulates DNA polymerase delta activity.

Effective dose of dental CBCT-a meta analysis of published data and additional data for nine CBCT units.

OBJECTIVES: This article analyses dose measurement and effective dose estimation of dental CBCT examinations. Challenges to accurate calculation of dose are discussed and the use of dose-height product (DHP) as an alternative to dose-area product (DAP) is explored. METHODS: The English literature on effective dose was reviewed. Data from these studies together with additional data for nine CBCT units were analysed. Descriptive statistics, ANOVA and paired analysis are used to characterize the data. RESULTS: PubMed and EMBASE searches yielded 519 and 743 publications, respectively, which were reduced to 20 following review. Reported adult effective doses for any protocol ranged from 46 to 1073 µSv for large fields of view (FOVs), 9-560 µSv for medium FOVs and 5-652 µSv for small FOVs. Child effective doses from any protocol ranged from 13 to 769 µSv for large or medium FOVs and 7-521 µSv for small FOVs. Effective doses from standard or default exposure protocols were available for 167 adult and 52 child exposures. Mean adult effective doses grouped by FOV size were 212 µSv (large), 177 µSv (medium) and 84 µSv (small). Mean child doses were 175 µSv (combined large and medium) and 103 µSv (small). Large differences were seen between different CBCT units. Additional low-dose and high-definition protocols available for many units extend the range of doses. DHP was found to reduce average absolute error for calculation of dose by 45% in comparison with DAP. CONCLUSIONS: Large exposure ranges make CBCT doses difficult to generalize. Use of DHP as a metric for estimating effective dose warrants further investigation.