Forkhead box protein 3 (FoxP3) mRNA as a diagnostic marker in ovarian tumors: a pilot study.

PURPOSE: To assess the diagnostic performance of forkhead box protein 3 (FoxP3) mRNA in ovarian tumors. MATERIALS AND METHODS: Using quantitative real time reverse transcription PCR (q-RT-PCR), FoxP3 mRNA level was measured in fresh frozen ovarian tumors and its diagnostic performance was compared to those of preoperative serum CA 125 and risk of malignancy index (RMI). RESULTS: FoxP3 mRNA was differentially expressed in the malignant (n = 25) and benign (n = 25) groups, yet without statistically significant differences; positivity rate: 15/25 vs. 10/25; p = 0.157, median: 0.429 vs. 0.046; p = 0.684, and mean ± SD: 73.75 ± 234.68 vs. 247.09 ± 792.17; p = 0.30 1. Although it showed much less diagnostic performance (AUC: 0.534), FoxP3 mRNA enhanced the diagnostic sensitivity and specificity of both CA125 and RMI (96% and 100%, for both). CONCLUSION: FoxP3 mRNA may not be good diagnostic marker in ovarian tumors; however it may prove valuable in defining underlying tumor molecular signature.

Durable survival after chemotherapy in a HIV patient with Burkitt's lymphoma presenting with massive upper gastrointestinal bleeding.

Massive upper gastrointestinal bleeding is an uncommon presentation of Burkitt's lymphoma in a patient with HIV/AIDS, and is seldom reported in the literature. A 39-year-old man who has sex with men presented with abdominal pain and massive haematemesis and a rapid drop in haemoglobin level to 4.8 g/dL. Upper gastrointestinal endoscopy showed a large blood clot in the stomach, and an emergency laparotomy was performed because of unstable haemodynamics. This showed active bleeding from multiple tumours in the fundus and body of the stomach. The patient underwent gastrectomy and gastric biopsy confirmed Burkitt's lymphoma. Further tests showed lymphoma involvement in bone marrow and central nervous system. The patient tested positive for HIV, and had a CD4 count of 212 cells/mm(3) and viral load of 18,000 copies/mL at diagnosis. He was commenced on a chemotherapy regimen of CODOX-M/IVAC, and highly active antiretroviral therapy consisting of indinavir, stavudine and lamivudine. The major side effect was peripheral neuropathy. Infective complications during chemotherapy were controlled by broad-spectrum antibiotics and anti-fungal agents. Complete remission of the lymphoma was achieved after the chemotherapy and remission was maintained for more than 14 years.

Common dental features and craniofacial development of three siblings with Ter Haar syndrome.

BACKGROUND: Ter Haar syndrome is a rare genetic syndrome with <30 cases reported worldwide. There is nothing within the published literature regarding the dental development and dental features of these patients. CASE REPORT: This case series examines three patients with Ter Haar syndrome and tracks their dental development and identifies common dental and skeletal features. FOLLOW-UP: All three patients received dental treatment and regular follow-up at Great Ormond Street Hospital Dental Department. CONCLUSION: These patients have many common dental and craniofacial features which poses the question as to whether these features are due to Ter Haar syndrome.

Prolonged vitamin D intoxication: presentation, pathogenesis and progress.

Vitamin D toxicity from unactivated vitamin D (calciferol) therapy is currently a rare cause of hypercalcaemia. However, the frequency of this event may increase as high-dose unactivated vitamin D preparations become available. Prolonged vitamin D toxicity can cause reversible hypercalcaemia and partially reversible renal impairment. Parathyroid hormone may not be suppressed with unactivated vitamin D toxicity, especially if renal disease coexists.

Palate lengthening by buccinator myomucosal flaps for velopharyngeal insufficiency.

OBJECTIVE : To assess the outcome of palate lengthening by myomucosal buccinator flaps for velopharyngeal insufficiency both in terms of speech and changes in palate length. DESIGN : Thirty-two consecutive patients who underwent the buccinator flap procedure were reviewed retrospectively. Palate length and the presence or absence of a velopharyngeal gap were assessed on pre- and postoperative videofluoroscopic recordings using a calibrated image analysis system. Hypernasality, nasal emission, nasal turbulence, and passive cleft type articulation errors were evaluated blindly by a speech-language pathologist external to the team using pre- and postoperative speech recordings. SETTING : Multidisciplinary cleft team based in a tertiary referral center. Results : In 81% of patients, speech outcome was such that no further velopharyngeal surgery was considered necessary at the time of follow-up. The buccinator flap procedure resulted in a mean palate lengthening of 7.5 mm (±5.5 SD). After the operation, there was a complete elimination of the velopharyngeal gap on lateral videofluoroscopy in 77% of patients. There were significant decreases in hypernasality ratings and passive cleft type articulation errors postoperatively. CONCLUSION: Palatal lengthening with myomucosal buccinator flaps in patients with velopharyngeal insufficiency is effective and safe. It has become one of our routinely practiced procedures for velopharyngeal insufficiency.

The effect Akt2 deletion on tumor development in Pten(+/-) mice.

The serine/threonine kinase Akt is frequently activated in human cancers and is considered an attractive therapeutic target. However, the relative contributions of the different Akt isoforms to tumorigenesis, and the effect of their deficiencies on cancer development are not well understood. We had previously shown that Akt1 deficiency is sufficient to markedly reduce the incidence of tumors in Pten(+/-) mice. Particularly, Akt1 deficiency inhibits endometrial carcinoma and prostate neoplasia in Pten(+/-) mice. Here, we analyzed the effect of Akt2 deficiency on the incidence of tumors in Pten(+/-) mice. Relative to Akt1, Akt2 deficiency had little-to-no effect on the incidence of prostate neoplasia, endometrial carcinoma, intestinal polyps and adrenal lesions in Pten(+/-) mice. However, Akt2 deficiency significantly decreased the incidence of thyroid tumors in Pten(+/-), which correlates with the relatively high level of Akt2 expression in the thyroid. Thus, unlike Akt1 deletion, Akt2 deletion is not sufficient to markedly inhibit tumorigenesis in Pten(+/-) mice in most tested tissues. The relatively small effect of Akt2 deletion on the inhibition of tumorigenesis in Pten(+/-) mice could be explained, in part, by an insufficient decrease in total Akt activity, due to the relatively lower Akt2 versus Akt1 expression, and relatively high blood insulin levels in Pten(+/-)Akt2(-/-) mice. The relatively high blood insulin levels in Pten(+/-)Akt2(-/-) mice may elevate the activity of Akt1, and possibly Akt3, thus, limiting the reduction of total Akt activity and preventing this activity from dropping to a threshold level required to inhibit tumorigenesis.

Trans-cranial placement of an amplatzer device to control intractable cardiac failure in an infant with a vein of galen anomaly. A case report.

Neonates presenting with intractable cardiac failure due to vein of Galen aneurysmal malformations (VGAMs) rapidly progress to multisystem organ failure and death if left untreated. Currently the only viable treatment option is endovascular embolization. Although intracranial embolization of a neonate is a high-risk procedure, successful treatment can reverse cardiac failure and prevent neurological complications associated with VGAMs. Embolization via the arterial route is thought to have a better outcome than embolization via the venous system. However, multiple transarterial embolizations in different sessions may well be contraindicated in neonates, because repeat access via the femoral artery, carries a risk of arterial trauma which, in turn, can jeopardize lower limbs. With this case study we show that after repeat failure of arterial embolization, the transcranial placement of an Amplatzer PFO occluder (AGA Medical, Plymouth, USA) in the aneurysm can effectively reduce intrafistular pressure and venous outflow velocity. We also propose a mathematical model that can be used to calculate flow velocity through the aneurysm, which, in turn, could be used to aid clinical decision-making. Unlike some conventional techniques, the placement of an Amplatzer occluder does not pose the risk of completely obstructing venous drainage and therefore does not increase the risk of venous breakthrough hemorrhage. We propose this endovascular technique as a treatment option for high risk neonates in need of emergency embolization of VGAMs, where multiple arterial embolizations failed to control the condition sufficiently.

Mitochondrial hexokinases, novel mediators of the antiapoptotic effects of growth factors and Akt.

Cell survival has been closely linked to both trophic growth factor signaling and cellular metabolism. Such couplings have obvious physiologic and pathophysiologic implications, but their underlying molecular bases remain incompletely defined. As a common mediator of both the metabolic and anti-apoptotic effects of growth factors, the serine/threonine kinase Akt - also known as protein kinase B or PKB - is capable of regulating and coordinating these inter-related processes. The glucose dependence of the antiapoptotic effects of growth factors and Akt plus a strong correlation between Akt-regulated mitochondrial hexokinase association and apoptotic susceptibility suggest a major role for hexokinases in these effects. Mitochondrial hexokinases catalyse the first obligatory step of glucose metabolism and directly couple extramitochondrial glycolysis to intramitochondrial oxidative phosphorylation, and are thus well suited to play this role. The ability of Akt to regulate energy metabolism appears to have evolutionarily preceded the capacity to control cell survival. This suggests that Akt-dependent metabolic regulatory functions may have given rise to glucose-dependent antiapoptotic effects that evolved as an adaptive sensing system involving hexokinases and serve to ensure mitochondrial homeostasis, thereby coupling metabolism to cell survival. We hypothesize that the enlistment of Akt and hexokinase in the control of mammalian cell apoptosis evolved as a response to the recruitment of mitochondria to the apoptotic cascade. The central importance of mitochondrial hexokinases in cell survival also suggests that they may represent viable therapeutic targets in cancer.

Control of laser heating in clusters through variation in temporal pulse shape.

We present results of experiments and simulations of the interaction between a high intensity, femtosecond laser pulse and an atomic cluster that show the temporal profile of the laser pulse plays a crucial role in the expansion dynamics of the cluster. Experiments were conducted in rare gas clusters of Xe, Kr and Ar of radius 30 - 80 A with a laser pulse of duration 70 - 240 fs and a peak intensity of ~1016 Wcm-2. The shape of the laser pulse was a Gaussian with a shoulder of intensity 0.02 times the peak pulse intensity appearing on either the rising or falling edge of the main pulse depending on the sign of chirp applied to the laser pulse. Significant differences (up to a factor of 2) in the energies of the ions obtained from the laser-cluster interaction were measured when the shape of the laser pulse was varied.

The relationship between arch dimensions and the 5-year index in the primary dentition of patients with complete UCLP.

OBJECTIVE: To compare dental arch dimensions of children in the primary dentition with repaired unilateral clefts of the lip and palate (UCLP) to a noncleft group of a similar age and determine how the dimensions of the cleft arches relate to an index of treatment outcome. METHOD: Dental study casts of 44 5- to 6-year-olds with complete UCLP (22 boys and 22 girls) from a single center, whose primary surgery had been carried out by one surgeon, were matched for age, sex, and ethnicity with dental study casts from a longitudinal growth study. Analysis of variance was used to ascertain differences in arch dimensions between the two groups. The cleft group casts were then assessed with an established index of surgical outcome, the 5-year-old index. Spearman's rank correlation coefficient was used to see how the arch dimensions of the cleft group related to the categories of the index. RESULTS AND CONCLUSIONS: Maxillary arch dimensions were significantly smaller in the cleft group than in the noncleft group, irrespective of sex (p < .05). In the mandibular arch, there was no difference between the cleft and noncleft groups (p > .05). Maxillary arch dimensions of the cleft group correlated significantly with the 5-year-old index for arch length and intercanine width (p < .05) but not intermolar width (p = .842). This would suggest that the 5-year-old index is a suitable tool for assessing the outcome of treatment in the primary dentition for anteroposterior and anterior transverse arch dimensions.

Role of the intramolecular phase in high-harmonic generation.

We study numerically the generation of high-order harmonics by two-center molecules for arbitrary angles between the molecular axis and the laser polarization axis. For fixed angle, the harmonic spectrum exhibits a minimum at a frequency which is independent of the laser parameters. The amplitude of each harmonic is strongly angle dependent, and a pronounced minimum is found at the same angle where a sudden jump in the harmonic phase occurs. By calculating the spatial dependence of the harmonic amplitudes and phases, we are able to explain these effects in terms of interfering contributions from various regions within the molecule.

Growth retardation and increased apoptosis in mice with homozygous disruption of the Akt1 gene.

The serine/threonine kinase Akt has been implicated in the control of cell survival and metabolism. Here we report the disruption of the most ubiquitously expressed member of the akt family of genes, akt1, in the mouse. Akt1(-/-) mice are viable but smaller when compared to wild-type littermates. In addition, the life span of Akt1(-/-) mice, upon exposure to genotoxic stress, is shorter. However, Akt1(-/-) mice do not display a diabetic phenotype. Increased spontaneous apoptosis in testes, and attenuation of spermatogenesis is observed in Akt1(-/-) male mice. Increased spontaneous apoptosis is also observed in the thymi of Akt1(-/-) mice, and Akt1(-/-) thymocytes are more sensitive to apoptosis induced by gamma-irradiation and dexamethasone. Finally, Akt1(-/-) mouse embryo fibroblasts (MEFs) are more susceptible to apoptosis induced by TNF, anti-Fas, UV irradiation, and serum withdrawal.

Myc drives apoptosis in PC12 cells in the absence of Max.

A conditionally active chimeric form of the c-Myc protein fused to the ligand-binding domain of the estrogen receptor (MycER) was expressed in PC12 cells. Induction of Myc activity resulted in a threefold increase in apoptosis after 5 days when cells were maintained in 1% serum. The effect of Myc overexpression was dependent on its DNA-binding domain but not on its heterodimeric binding protein Max, which is absent in PC12 cells. Preincubation of the c-Myc overexpressing cells with either NGF or bFGF, but not EGF, prevented the Myc-mediated increase in apoptosis, although the signaling pathways used by NGF and bFGF to block cell death differed. NGF-mediated rescue was mediated by the phosphatidylinositol 3'-OH (P13) kinase/Akt pathway while rescue by bFGF was not affected by P13 kinase inhibitors. These results show that Myc can induce apoptosis in PC12 cells in a Max-independent manner and that alternate signaling pathways exist to mediate cell survival.

Inhibition of early apoptotic events by Akt/PKB is dependent on the first committed step of glycolysis and mitochondrial hexokinase.

The serine/threonine kinase Akt/PKB is a major downstream effector of growth factor-mediated cell survival. Activated Akt, like Bcl-2 and Bcl-xL, prevents closure of a PT pore component, the voltage-dependent anion channel (VDAC); intracellular acidification; mitochondrial hyperpolarization; and the decline in oxidative phosphorylation that precedes cytochrome c release. However, unlike Bcl-2 and Bcl-xL, the ability of activated Akt to preserve mitochondrial integrity, and thereby inhibit apoptosis, requires glucose availability and is coupled to its metabolism. Hexokinases are known to bind to VDAC and directly couple intramitochondrial ATP synthesis to glucose metabolism. We provide evidence that such coupling serves as a downstream effector function for Akt. First, Akt increases mitochondria-associated hexokinase activity. Second, the antiapoptotic activity of Akt requires only the first committed step of glucose metabolism catalyzed by hexokinase. Finally, ectopic hexokinase expression mimics the ability of Akt to inhibit cytochrome c release and apoptosis. We therefore propose that Akt increases coupling of glucose metabolism to oxidative phosphorylation and regulates PT pore opening via the promotion of hexokinase-VDAC interaction at the outer mitochondrial membrane.

A transcriptional activation function of p53 is dispensable for and inhibitory of its apoptotic function.

The tumor suppressor p53 is an inducer of cell cycle arrest and programmed cell death (apoptosis). The ability of p53 to induce cell cycle arrest is linked to its ability to induce transcription of genes such as the cyclin-dependent kinase inhibitor p21. However, the dependence of p53-mediated apoptosis on transcriptional activation remains controversial. Ectopic expression of a temperature-sensitive (ts) p53 allele induced expression of p53 target genes and elicited both G1 and G2/M cell cycle arrest upon shift to the permissive temperature. Ectopic expression of the same ts p53 allele with two additional point mutations (Gln22, Ser23) that abolish p53-transcriptional activation did not induce p53 target genes and G1 nor G2/M cell cycle arrest. In HCT116 colon carcinoma cells ectopic expression of wild type p53 does not elicit apoptosis whereas p53 mutant deficient in trans-activation induces apoptosis. The ability of wild type p53 to induce apoptosis is restored in HCT116 cells that are null for p21. However, the trans-activation deficient mutant of p53 is still more potent mediator of apoptosis than wild type p53 in the p21 null cells. Although the ability of Gln22,Ser23 to trans-activate p53 target genes is diminished, it retains the ability to repress Bcl-2 expression. Thus, we conclude that while ectopic expression of wild type p53 can induce both G1 and G2/M arrest, in a p21 dependent manner, without apoptosis, a p53 mutant defective in trans-activation elicits apoptosis without inducing cell cycle arrest. Further, the anti-apoptotic function of p53 is dependent on trans-activation and is linked to cell cycle arrest. The results strongly suggest that the trans-activation deficient mutant is a more potent inducer of apoptosis because it lost its anti-apoptotic function and retains its ability to repress pro-apoptotic genes such as Bcl-2. Taken together, the results imply that employing a trans-activation deficient p53 in gene therapy approaches or the use of drugs that convert mutant p53 to a trans-activation-independent mediator of apoptosis may be much more efficient therapeutic approaches than current approaches that employ wild type p53.

Myc represses the p21(WAF1/CIP1) promoter and interacts with Sp1/Sp3.

The cyclin-dependent kinase inhibitor p21((WAF1/CIP1)) inhibits proliferation both in vitro and in vivo, and overexpression of p21 in normal and tumor cell lines results in cell cycle arrest. In contrast, ectopic expression of Myc alleviates G(1) cell cycle arrest. Recent studies showed that Myc can repress p21 transcription, thereby overriding a p21-mediated cell cycle checkpoint. We found that activation of a Myc-estrogen receptor fusion protein by 4-hydroxytamoxifen in mouse cells resulted in suppression of endogenous p21 transcription. This effect was observed in the absence of de novo protein synthesis and was independent of histone deacetylase activity. In transient transfection studies, Myc effectively repressed p21 promoter constructs containing only 119 bp of sequence upstream of the transcription start site. This region contains multiple Sp1-binding sites and a potential initiator element, but no canonical Myc DNA-binding sites. Deletion of the potential initiator element does not affect repression of the p21 promoter by c-Myc. Coimmunoprecipitation and glutathione S-transferase pull-down experiments demonstrate that c-Myc may form complexes with Sp1/Sp3. We found that the central region of c-Myc interacts with the zinc finger domain of Sp1. Because Sp1 is required for p21 transcription, it is possible that Myc may down-regulate p21 transcription, at least in part, by sequestering Sp1. Repression of the p21 promoter may contribute to the ability of c-Myc to promote cell proliferation.

Induction of cell cycle progression and acceleration of apoptosis are two separable functions of c-Myc: transrepression correlates with acceleration of apoptosis.

Analysis of amino-terminus mutants of c-Myc has allowed a systematic study of the interrelationship between Myc's ability to regulate transcription and its apoptotic, proliferative, and transforming functions. First, we have found that c-Myc-accelerated apoptosis does not directly correlate with its ability to transactivate transcription using the endogenous ornithine decarboxylase (ODC) gene as readout for transactivation. Furthermore, deletion of the conserved c-Myc box I domain implicated in transactivation does not inhibit apoptosis. Second, the ability of c-Myc to repress transcription, using the gadd45 gene as a readout, correlates with its ability to accelerate apoptosis. A conserved region of c-Myc implicated in mediating transrepression is absolutely required for c-Myc-accelerated apoptosis. Third, a lymphoma-derived Thr58Ala mutation diminishes c-Myc-accelerated apoptosis through a decreased ability to induce the release of cytochrome c from mitochondria. This mutation in a potential phosphorylation site does not affect cell cycle progression, providing genetic evidence that induction of cell cycle progression and acceleration of apoptosis are two separable functions of c-Myc. Finally, we show that the increased ability of Thr58Ala mutant to elicit cellular transformation correlates with its diminished ability to accelerate apoptosis. Bcl-2 overexpression blocked and the lymphoma-associated Thr58Ala mutation decreased c-Myc-accelerated apoptosis, and both led to a significant increase in the ability of Rat1a cells to form colonies in soft agar. This enhanced transformation was greater in soft agar containing a low concentration of serum, suggesting that protection from apoptosis is a mechanism contributing to the increased ability of these cells to proliferate in suspension. Thus, we show here for the first time that, in addition to mutations in complementary antiapoptotic genes, c-Myc itself can acquire mutations that potentiate neoplastic transformation by affecting apoptosis independently of cell cycle progression.

Activation of NF-kappa B by bradykinin through a Galpha(q)- and Gbeta gamma-dependent pathway that involves phosphoinositide 3-kinase and Akt.

Recent work has suggested a role for the serine/threonine kinase Akt and IkappaB kinases (IKKs) in nuclear factor (NF)-kappaB activation. In this study, the involvement of these components in NF-kappaB activation through a G protein-coupled pathway was examined using transfected HeLa cells that express the B2-type bradykinin (BK) receptor. The function of IKK2, and to a lesser extent, IKK1, was suggested by BK-induced activation of their kinase activities and by the ability of their dominant negative mutants to inhibit BK-induced NF-kappaB activation. BK-induced NF-kappaB activation and IKK2 activity were markedly inhibited by RGS3T, a regulator of G protein signaling that inhibits Galpha(q), and by two Gbetagamma scavengers. Co-expression of Galpha(q) potentiated BK-induced NF-kappaB activation, whereas co-expression of either an activated Galpha(q)(Q209L) or Gbeta(1)gamma(2) induced IKK2 activity and NF-kappaB activation without BK stimulation. BK-induced NF-kappaB activation was partially blocked by LY294002 and by a dominant negative mutant of phosphoinositide 3-kinase (PI3K), suggesting that PI3K is a downstream effector of Galpha(q) and Gbeta(1)gamma(2) for NF-kappaB activation. Furthermore, BK could activate the PI3K downstream kinase Akt, whereas a catalytically inactive mutant of Akt inhibited BK-induced NF-kappaB activation. Taken together, these findings suggest that BK utilizes a signaling pathway that involves Galpha(q), Gbeta(1)gamma(2), PI3K, Akt, and IKK for NF-kappaB activation.