Surveillance imaging with FDG-PET/CT in the post-operative follow-up of stage 3 melanoma.

Background: As early detection of recurrent melanoma maximizes treatment options, patients usually undergo post-operative imaging surveillance, increasingly with FDG-PET/CT (PET). To assess this, we evaluated stage 3 melanoma patients who underwent prospectively applied and sub-stage-specific schedules of PET surveillance. Patients and methods: From 2009, patients with stage 3 melanoma routinely underwent PET +/- MRI brain scans via defined schedules based on sub-stage-specific relapse probabilities. Data were collected regarding patient characteristics and outcomes. Contingency analyses were carried out of imaging outcomes. Results: One hundred and seventy patients (stage 3A: 34; 3B: 93; 3C: 43) underwent radiological surveillance. Relapses were identified in 65 (38%) patients, of which 45 (69%) were asymptomatic. False-positive imaging findings occurred in 7%, and 6% had treatable second (non-melanoma) malignancies. Positive predictive values (PPV) of individual scans were 56%-83%. Negative scans had predictive values of 89%-96% for true non-recurrence [negative predictive values (NPV)] until the next scan. A negative PET at 18 months had NPVs of 80%-84% for true non-recurrence at any time in the 47-month (median) follow-up period. Sensitivity and specificity of the overall approach of sub-stage-specific PET surveillance were 70% and 87%, respectively. Of relapsed patients, 33 (52%) underwent potentially curative resection and 10 (16%) remained disease-free after 24 months (median). Conclusions: Application of sub-stage-specific PET in stage 3 melanoma enables asymptomatic detection of most recurrences, has high NPVs that may provide patient reassurance, and is associated with a high rate of detection of resectable and potentially curable disease at relapse.

Parents' Perceptions of Motor Interventions for Infants and Toddlers with Down Syndrome.

The purpose of the study was to analyze parents' perceptions of their participation in a university-directed, parent-implemented, home-based pediatric strength intervention program as (a) one approach to evaluating the effectiveness of a program conducted over a 4-year period with families of infants and toddlers with Down syndrome and (b) a means of deriving guidelines for future early intervention programs. Participants were 22 parents from 11 families of children with Down syndrome; the children ranged in age from 6 to 42 months. Participatory evaluation research, semistructured audio recorded home interviews, and qualitative content analysis were used. The results indicated that the parents (a) perceived themselves as being empowered to implement the program, (b) perceived their expectations about improved motor development of their children had been met, and (c) perceived the program was worthwhile. The parents' perceptions provided meaningful evaluation data that enabled the development of guidelines for future pediatric strength intervention programs.

Rho-dependence of Schizosaccharomyces pombe Pck2.

BACKGROUND: In metazoans, the HR1 domain, a motif found in a number of proteins including the protein kinase C-related PRKs, is responsible for an interaction with Rho-GTPases. The structural similarity between the Schizosaccaromyces pombe Pck proteins and the mammalian Rho-dependent protein kinase C-related family, has led us to investigate the relationship between the function of Rho and that of Pck1/2. RESULTS: Rho1 is shown to interact with the conserved N-terminal HR1 domain of Pck1/2 in vitro and in vivo. Lethal overproduction of Rho1 is neutralized by co-expression of the Pck2 HR1 domain, which by itself compromises growth when overproduced. The Pck2-Rho1 interaction has a profound effect on the steady state expression of Pck2 and this is shown to parallel the immunoprecipitated activity and phosphorylation of Pck2 at its activation loop site. It is further shown that Pck2 becomes localized at the septum, where Rho1 is also located. CONCLUSIONS: The results demonstrate that the Pck proteins are Rho1 effectors in fission yeast and that the HR1 domain is a universal motif for the Rho-GTPase interaction. Furthermore, the evidence supports the contention that the yeast Pck1 and Pck2 proteins are primitive protein kinases, which in vertebrates have evolved into the two distinct PKC and PRK families.

The New Moms Project: educating mothers about sun protection in newborn nurseries.

Sun protection habits should begin early in life and be taught as part of routine preventive health care. Early teaching of parents aims to introduce an easily achieved means of sun protection with the goal of instilling these practices as habits in the parents and their young children. We developed a maternity nurse-led intervention for 187 mothers at newborn nurseries in Falmouth, Massachusetts, combining educational material and personal discussions. One year after the intervention we successfully contacted 73% of the mothers. Nearly 90% recalled the informational program and equal numbers stated that receiving educational materials in the newborn nursery was timely. Nearly two-thirds of mothers reported that this was the only sun protection information received from a provider in the past year.

Osmotic stress activates phosphatidylinositol-3,5-bisphosphate synthesis.

Inositol phospholipids play multiple roles in cell signalling systems. Two widespread eukaryotic phosphoinositide-based signal transduction mechanisms, phosphoinositidase C-catalysed phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) hydrolysis and 3-OH kinase-catalysed PtdIns(4,5)P2 phosphorylation, make the second messengers inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) sn-1,2-diacylglycerol and PtdIns(3,4,5)P3. In addition, PtdIns(4,5)P2 and PtdIns3P have been implicated in exocytosis and membrane trafficking. We now show that when the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe are hyperosmotically stressed, they rapidly synthesize phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P2) by a process that involves activation of a PtdIns3P 5-OH kinase. This PtdIns(3,5)P2 accumulation only occurs in yeasts that have an active vps34-encoded PtdIns 3-OH kinase, showing that this latter kinase makes the PtdIns3P needed for PtdIns(3,5)P2 synthesis and indicating that PtdIns(3,5)P2 may have a role in sorting vesicular proteins. PtdIns(3,5)P2 is also present in mammalian and plant cells: in monkey Cos-7 cells, its labelling is inversely related to the external osmotic pressure. The stimulation of a PtdIns3P 5-OH kinase-catalysed synthesis of PtdIns(3,5)P2, a molecule that might be a new type of phosphoinositide 'second messenger, thus appears to be central to a widespread and previously uncharacterized regulatory pathway.

Intracellular targeting and homotetramer formation of a truncated inositol 1,4,5-trisphosphate receptor-green fluorescent protein chimera in Xenopus laevis oocytes: evidence for the involvement of the transmembrane spanning domain in endoplasmic reticulum targeting and homotetramer complex formation.

In an attempt to define structural regions of the type I inositol 1, 4,5-trisphosphate [Ins(1,4,5)P3] receptor [Ins(1,4,5)P3R] involved in its intracellular targeting to the endoplasmic reticulum (ER), we have employed the use of green fluorescent protein (GFP) to monitor the localization of a truncated Ins(1,4,5)P3R mutant containing just the putative transmembrane spanning domain and the C-terminal cytoplasmic domain [amino acids 2216-2749; termed inositol trisphosphate receptor(ES)]. We expressed a chimeric GFP-Ins(1,4, 5)P3R(ES) fusion protein in Xenopus laevis oocytes, and used fluorescence confocal microscopy to monitor its intracellular localization. Fluorescence confocal microscopy data showed an intense fluorescence in the perinuclear region and in a reticular-network under the animal pole of the oocyte, consistent with the targeting of expressed GFP-Ins(1,4,5)P3R(ES) to perinuclear ER and ER under the animal pole. These findings are consistent with the intracellular localization of the endogenous Xenopus Ins(1,4, 5)P3R shown previously. Furthermore, electron microscopy data indicate that expressed GFP-Ins(1,4,5)P3R(ES) is in fact targeted to the ER. Sodium carbonate extraction of microsomal membranes and cross-linking experiments indicate that the expressed chimeric protein is in fact membrane anchored and able to form a homotetrameric complex. Our data provides evidence that Ins(1,4, 5)P3R(ES) constitutes the membrane spanning domain of the Ins(1,4, 5)P3R and is able to mediate homotetramer formation, without the need for the large N-terminal cytoplasmic domain. Furthermore, the localization of GFP-Ins(1,4,5)P3R(ES) on the ER indicates that an ER retention/targeting signal is contained within the transmembrane spanning domain of the inositol trisphosphate receptor.

Pharmacological modulators of the inositol 1,4,5-trisphosphate receptor.

Elevation of cytosolic calcium concentrations, induced by many neurotransmitters, plays a crucial role in neuronal function. Some neurotransmitters produce the second messenger InsP3 which activates an intracellular calcium channel (InsP3 receptor) usually located in the endoplasmic reticulum. This article undertakes a comprehensive survey of most pharmacological modulators of the InsP3 receptor so far reported. This review discusses in detail competitive antagonists, non-competitive antagonists and thiol reactive reagents, highlighting their modes of action and in some cases indicating drawbacks in their use.

The inhibition of the inositol 1,4,5-trisphosphate receptor from rat cerebellum by spermine and other polyamines.

Inositol 1,4,5-trisphosphate-induced Ca2+ release from rat cerebellar microsomes can be inhibited by polyamines at mM concentrations. Spermine, one of the most abundant naturally occurring polyamines, inhibits InsP3-induced Ca2+ release with an IC50 of 1 mM. However, the antibiotic neomycin proved most efficacious at inhibiting InsP3-induced Ca2+ release (IC50 0.4mM). The order of potency being neomycin > spermine > spermidine > putrescine. Although binding of [3H]InsP3 to cerebellar microsomes is also inhibited by polyamines, this may be due to InsP3 complexing with the polyamines under the binding conditions used. Under Ca2+ release conditions InsP3 binds weakly to spermine and therefore inhibition of InsP3-induced Ca2+ release is consistent with polyamines interacting with the InsP3 receptor.

The effects of tetrahexyl ammonium cations (THA+) on inositol 1,4,5-trisphosphate-induced calcium release from porcine cerebellar microsomes: THA+ can induce calcium release selectively from the InsP3-sensitive calcium stores.

In this study we show that the potassium-channel blocker tetrahexyl ammonium chloride (THA+) is able to inhibit inositol 1,4,5-trisphosphate (InsP3)-induced calcium release in an apparently biphasic fashion with a IC50 of 3 microM. This inhibition was not alleviated by valinomycin and, therefore, is not consistent with the blocking of K+ counter-ion movement, an observation initially made by Palade et al. (Palade, P., Dettbarn, C., Volpe, P., Alderson, B. and Otero, A.S (1989) Mol. Pharmacol. 36, 664-672). THA+ affected quantal calcium release by reducing the amount of calcium released by InsP3, but did not greatly affect the concentration of InsP3 required to cause half-maximal calcium release. THA+ did not affect the metabolism of InsP3 or its binding to porcine cerebellar microsomes. THA+ could also itself induce calcium release. At concentrations below 100 microM, THA+ appears to release Ca2+ selectively from the InsP3-sensitive calcium stores, since prior depletion of these stores with supramaximal doses of InsP3 abolishes this response. At higher THA+ concentrations (above 100 microM) Ca2+ is released non-selectively from all stores. THA+ has no effect on the Ca(2+)-ATPase activity at concentrations below 100 microM, indicating that selective THA(+)-induced Ca2+ release is not due to non-specific inhibition of the microsomal Ca2+ pumps and does not affect Ca2+ leakage. A number of pharmacological modulators of intracellular calcium channels were also tested on THA(+)-induced calcium release with little effect, except for spermidine which reduced this release by up to 50%. Our observations are consistent with the view that THA+, at concentrations below 100 microM, selectively releases calcium from the InsP3-sensitive calcium stores.

The effects of thimerosal on calcium uptake and inositol 1,4,5-trisphosphate-induced calcium release in cerebellar microsomes.

Thimerosal inhibits calcium uptake in skeletal muscle sarcoplasmic reticulum and rat cerebellar microsomes by inhibiting the Ca(2+)-ATPase. In the presence of 5 mM dithiothreitol (DTT), Ca2+ uptake and ATPase activity were not inhibited by thimerosal, indicating that thimerosal modifies cysteine residues of the Ca(2+)-ATPase. Low thimerosal concentrations (2 microM) sensitize the inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ channel, making it open at lower InsP3 concentrations. Higher concentrations of thimerosal, however, cause inhibition of InsP3-induced Ca2+ release. Both sensitization and inhibition of the InsP3 receptor by thimerosal can be prevented by DTT. The binding and metabolism of InsP3 by cerebellar microsomes is not affected by thimerosal. The amount of InsP3-induced Ca2+ release is co-operatively linked to the InsP3 concentration with a Hill coefficient of 2.0 +/- 0.3. This is decreased to 1.0 +/- 0.2 at inhibitory concentrations of thimerosal. Under our experimental conditions, we observed no dependence of quantal Ca2+ release on intraluminal Ca2+ concentration.

The opening of the inositol 1,4,5-trisphosphate-sensitive Ca2+ channel in rat cerebellum is inhibited by caffeine.

Ins(1,4,5)P3(InsP3)-induced Ca2+ release and [3H]InsP3 binding were measured in rat cerebellar microsomes in the presence or absence of caffeine. The quantal Ca2+ release was shown to occur in an apparently co-operative fashion with a Hill coefficient (h) of 2.2. Half-maximal Ca2+ release was observed at 900 nM-InsP3. Addition of caffeine caused changes both to the concentration of InsP3 required to cause half-maximal Ca2+ release (3.9 microM at 50 mM-caffeine) and to the apparent co-operativity (h = 1.0 at 50 mM-caffeine). Under standard conditions for [3H]InsP3 binding, caffeine had no effect, and it had no effect on InsP3 metabolism. Cyclic AMP also had no effect on the quantal release induced by InsP3. These results are consistent with the view that caffeine affects the opening (Ca2+ release) events rather than the ligand-binding events in the operation of the InsP3-sensitive Ca2+ channel.

Melatonin in the maternal and umbilical circulations during human parturition.

The concentration of melatonin in maternal peripheral plasma was measured during late pregnancy, term and pre-term labour. There was a small increase in the mean concentration of melatonin during labour which was significant in term labour. Umbilical arterial and venous plasma, whether obtained at term, after spontaneous vaginal delivery or at Caesarean section, contained significantly greater concentrations of melatonin than maternal plasma. A significant arterio-venous difference was demonstrated for both groups of umbilical samples with raised venous levels after spontaneous vaginal delivery but higher arterial levels at Caesarean section.

Melatonin in amniotic fluid during human parturition.

Concentrations of melatonin have been measured in amniotic fluid obtained during late pregnancy and labour. Samples obtained by amniotomy during labour contained higher concentrations of melatonin than samples taken at amniotomy for the induction of labour. Amniotic fluid obtained by amniocentesis in late pregnancy contained lower concentrations of melatonin than amniotic fluid obtained by amniotomy. The implications of these findings are discussed in relation to pineal influences on parturition.

Comparison of serum oestrogen concentrations in post-menopausal women taking oestrone sulphate and oestradiol.

Mean serum concentrations of oestradiol-17beta, oestrone, and oestrone sulphate in postmenopausal women were the same when measured up to six hours after treatment with either piperazine oestrone sulphate 1.5 mg or oestradiol valerate 2 mg. Maximum concentrations of oestradiol were less than those of oestrone, but oestrone sulphate reached concentrations about 30 times higher than those of oestrone. The rapid conversion of oestradiol valerate to oestrone and oestrone sulphate does not support the suggestion that in menopausal women oestradiol is less likely to be associated with a risk of endometrial carcinoma than oestrone sulphate, since the two preparations appear to become identical after ingestion.

PIP: In 17 postmenopausal women who were taking estrogens for menopausal symptoms, 10 were taking estradiol valerate, 2 mg daily, and 7 were taking piperazine estrone sulphate, 1.5 mg daily. All stopped these treatments 48 hours before the study began. Blood samples were then taken before and at 2, 4, and 6 hours after estradiol 2 mg or estrone sulphate 1.5 mg. Radioimmunoassay techniques were used. There was no significant difference between the 2 drugs in the 3 estrogen serum concentrations. Estradio concentrations remained the same, while estrone and estrone sulphate showed a 4- to 8-fold rise over pretreatment values. Results suggest that these 2 estrogen preparations have identical effects on the serum concentrations of 3 major estrogens. There was wide variation among patients and during phases of the menstrual cycles. Taking estrone sulphate seemed no more likely to increase risks of endometrial carcinoma than ingesting estradiol since the 2 preparations become identical during metabolism.

Placental transfer and metabolism of betamethasone in human pregnancy.

The concentration of betamethasone has been measured in maternal peripheral plasma, umbilical cord artery and vein, and amniotic fluid following maternal administration of betamethasone phosphate and betamethasone acetate on 3 consecutive days. Betamethasone was measured by radioimmunoassay following column chromatography. The findings show that betamethasone is transferred across the human placenta, circulates in the fetus and appears in amniotic fluid. During the 3 days after the start of treatment, levels of the betamethasone were similar in maternal and umbilical cord blood and in amniotic fluid. Thereafter, although levels in the mother were measurable for up to 7 days after the initial injection, the drug was detected in the cord plasma of only one baby. In vitro incubation studies of human placental tissue with 3H betamethasone identified 11-keto betamethasone as the major metabolite of betamethasone.