Domestic cats convert [2H8]-ß-carotene to [2H4]-retinol following a single oral dose.

Many animals convert ß-carotene to retinol to meet their vitamin A (VA) requirement. However, this pathway is inefficient in many carnivores. This study quantified the plasma response to a single oral dose of [(2) H(8)]-ß-carotene in adult domestic cats, including measurement of [(2) H(4)]-retinol derived from the dose. Cats were fed with either a control diet containing adequate VA (n = 5) or a VA-devoid diet (n = 5) for 28 days. An oral dose of either 5 mg/kg body weight (BW) (n = 4) or 10 mg/kg BW (n = 6) of [(2) H(8) ]-ß-carotene was administered on day 28. Plasma samples were collected prior to dosing and at 6, 12, 24, 32, 48, 72, 120, 168 and 216 h post-dose. Plasma retinoids and ß-carotene were measured using HPLC and [(2) H(4)]-retinol by GC-ECNCI-MS (gas chromatography/electron capture negative chemical ionization/mass spectrometry). ß-carotene was undetectable in plasma prior to dosing. Post-dose, mean peak plasma ß-carotene was 0.37 ± 0.06 nmol/ml at 9.0 ± 1.8 h following the dose, while [(2) H(4) ]-retinol peaked at 3.71 ± 0.69 pmol/ml at 55.2 ± 16.3 h. The ratio per cent of total area under the curve for [(2) H(4)]-retinol compared with the ß-carotene response was 4.6 ± 2.6%. There was little effect of diet or dose on the ß-carotene or [(2) H(4)]-retinol responses. The appearance of [(2) H(4)]-retinol in plasma indicates that cats are capable of converting ß-carotene to active VA. Conversion efficiency was not calculated in this study, but it is likely inadequate to meet cats' VA requirement without the inclusion of preformed VA in the diet.

Influence of a high-protein diet on energy balance in obese cats allowed ad libitum access to food.

The influence of a high-protein [HP, 47% of metabolizable energy (ME)] diet on energy balance was evaluated in obese cats allowed ad libitum access to food. Energy intake, body weight, body composition, energy expenditure, and concentrations of hormones and metabolites associated with carbohydrate and lipid metabolism (glucose, insulin, free fatty acids, triglycerides and leptin) were measured in cats after consuming either a moderate protein (MP, 27% of ME) or HP diet for 4 months. Indirect respiration calorimetry showed that resting and total energy expenditure (kJ/day) adjusted for either body weight or lean body mass was increased in cats consuming the HP in relation to MP diets. However, voluntary energy intake also was increased in the HP treatment and, thus, there was no difference in body weight between animals consuming the two diets. Body composition measurements using deuterium oxide dilution showed that dietary protein content did not alter amounts of either lean body mass or fat mass. No significant differences (p > 0.05) were observed between the two treatment groups for blood glucose, free fatty acid or leptin concentrations, although there was a trend (p = 0.054) towards an increase of serum insulin concentrations in the cats eating the HP diet. This study showed that short-term ad libitum feeding of an HP diet did not reduce food intake or promote weight loss in obese cats. However, energy expenditure was increased in the HP diet group and it is possible that this effect of HP might help promote weight loss when energy intake is restricted.

RSK2 is a new Pim2 target with pro-survival functions in FLT3-ITD-positive acute myeloid leukemia.

Acute myeloid leukemia (AML) with the FLT3 internal tandem duplication (FLT3-ITD AML) accounts for 20-30% of AML cases. This subtype usually responds poorly to conventional therapies, and might become resistant to FLT3 tyrosine kinase inhibitors (TKIs) due to molecular bypass mechanisms. New therapeutic strategies focusing on resistance mechanisms are therefore urgently needed. Pim kinases are FLT3-ITD oncogenic targets that have been implicated in FLT3 TKI resistance. However, their precise biological function downstream of FLT3-ITD requires further investigation. We performed high-throughput transcriptomic and proteomic analyses in Pim2-depleted FLT3-ITD AML cells and found that Pim2 predominantly controlled apoptosis through Bax expression and mitochondria disruption. We identified ribosomal protein S6 kinase A3 (RSK2), a 90 kDa serine/threonine kinase involved in the mitogen-activated protein kinase cascade encoded by the RPS6KA3 gene, as a novel Pim2 target. Ectopic expression of an RPS6KA3 allele rescued the viability of Pim2-depleted cells, supporting the involvement of RSK2 in AML cell survival downstream of Pim2. Finally, we showed that RPS6KA3 knockdown reduced the propagation of human AML cells in vivo in mice. Our results point to RSK2 as a novel Pim2 target with translational therapeutic potential in FLT3-ITD AML.

Modelling of peak-flow wall shear stress in major airways of the lung.

Some respiratory diseases result in the inflammation of the lung airway epithelium. An associated chronic cough, as found in many cases of asthma and in long-term smokers, can exacerbate damage to the epithelial layer. It has been proposed that wall shear stresses, created by peak expiratory flow-rates during a coughing episode, are responsible. The work here uses a computational fluid dynamics technique to model peak expiratory flow in the trachea and major lung bronchi. Calculated wall shear stress values are compared to a limited set of published measurements taken from a physical model. The measurements are discussed in the context of a flow study of a complex bronchial network. A more complete picture is achieved by the calculation method, indicating, in some cases, higher maximum wall shear stresses than measured, confirming the original findings of the experimental work. Recommendations are made as to where further work would be beneficial to medical applications.

Developmental stage-specific life-cycle bioassay for assessment of sediment-associated toxicant effects on benthic copepod production.

In chronic bioassays of sediment organic compounds, toxicant exposures often decline through time, such that the beginning of a test yields disproportionately higher exposures than the end. Thus, those life stages initiating a test often are exposed to the highest concentrations, and for rapidly maturing test fauna, this may lead to varying conclusions regarding compound toxicities depending on the initial life stage chosen. This problem can be addressed by comparative full life-cycle tests initiated with different test-organism life stages. Thus, a full life stage-to-life stage toxicity test was developed for the rapidly maturing meiobenthic copepod Amphiascus tenuiremis to assess the importance of developmental stage at the onset of sediment toxicant exposure relative to reproduction, net population growth, and sex and age structure. Tests were conducted with a model spiked-sediment insecticide, chlorpyrifos, for each of the major life stages (P1) of A. tenuiremis (nauplius, copepodite, and adult). Each P1 stage was allowed to mature and reproduce in low chlorpyrifos concentrations (6-33% of stage-specific 96-h LC50s; 4-22 ng chlorpyrifos/g dry sediment) for 26 d. Test endpoints were numbers of surviving adult females, males, eggs per female (clutch), first generation (F1) nauplii, F1 copepodites, F1 total production, and realized F1 production per surviving female. Only the copepodite P1 test showed a significant decline in survival of an adult age class: females declined by 28% at 22 ng/g. Reductions in total production ranged from 33-96% of controls from nauplius to adult. The P1 naupliar stage was most sensitive, with F1 production being 33-47% of that in controls. However, on a realized production per female basis, both the copepodite and naupliar P1 yielded significantly reduced F1s of 23 and 40% of controls at 11 and 22 ng/g.

Pim kinases phosphorylate Chk1 and regulate its functions in acute myeloid leukemia.

Phosphorylation by Akt on Ser 280 was reported to induce cytoplasmic retention and inactivation of CHK1 with consequent genetic instability in PTEN-/- cells. In acute myeloid leukemia cells carrying the FLT3-internal tandem duplication (ITD) mutation, we observed high rates of FLT3-ITD-dependent CHK1 Ser 280 phosphorylation. Pharmacological inhibition and RNA interference identified Pim1/2, not Akt, as effectors of this phosphorylation. Pim1 catalyzed Ser 280 phosphorylation in vitro and ectopic expression of Pim1/2-induced CHK1 phosphorylation. Ser 280 phosphorylation did not modify CHK1 localization, but facilitated its cell cycle and resistance functions in leukemic cells. FLT3, PIM or CHK1 inhibitors synergized with DNA-damaging agents to induce apoptosis, allowing cells to bypass the etoposide-induced G2/M arrest. Consistently, etoposide-induced CHK1-dependent phosphorylations of CDC25C on Ser 216 and histone H3 on Thr11 were decreased upon FLT3 inhibition. Accordingly, ectopic expression of CHK1 improved the resistance of FLT3-ITD cells and maintained histone H3 phosphorylation in response to DNA damage, whereas expression of unphosphorylated Ser 280Ala mutant did not. Finally, FLT3- and Pim-dependent phosphorylation of CHK1 on Ser 280 was confirmed in primary blasts from patients. These results identify a new pathway involved in the resistance of FLT3-ITD leukemic cells to genotoxic agents, and they constitute the first report of CHK1 Ser 280 regulation in myeloid malignancies.

Elevated plasma norepinephrine inhibits insulin secretion, but adrenergic blockade reveals enhanced ß-cell responsiveness in an ovine model of placental insufficiency at 0.7 of gestation.

In pregnancies complicated by placental insufficiency (PI), fetal hypoglycemia and hypoxemia progressively worsen during the third trimester, which increases circulating norepinephrine (NE). Pharmacological adrenergic blockade (ADR-block) at 0.9 gestation revealed that NE inhibits insulin secretion and enhanced ß-cell responsiveness in fetuses with PI-induced intrauterine growth restriction (IUGR). NE concentrations in PI fetuses at 0.7 gestation were threefold greater compared with age-matched controls, but the levels were similar to near-term controls. Therefore, our objective was to determine whether elevations in plasma NE concentrations inhibit insulin secretion and produce compensatory ß-cell responsiveness in PI fetuses at 0.7 gestation. Fetal insulin was measured under basal, glucose-stimulated insulin secretion (GSIS) and glucose-potentiated arginine-stimulated insulin secretion (GPAIS) conditions in the absence and presence of an ADR-block. Placental weights were 38% lower (P < 0.05) in PI fetus than in controls, but fetal weights were not different. PI fetuses had lower (P < 0.05) basal blood oxygen content, plasma glucose, insulin-like growth factor-1 and insulin concentrations and greater plasma NE concentrations (891 ± 211 v. 292 ± 65 pg/ml; P < 0.05) than controls. GSIS was lower in PI fetuses than in controls (0.34 ± 0.03 v. 1.08 ± 0.06 ng/ml; P < 0.05). ADR-block increased GSIS in PI fetuses (1.19 ± 0.11 ng/ml; P < 0.05) but decreased GSIS in controls (0.86 ± 0.02 ng/ml; P < 0.05). Similarly, GPAIS was 44% lower (P < 0.05) in PI fetuses than in controls, and ADR-block increased (P < 0.05) GPAIS in PI fetuses but not in controls. Insulin content per islet was not different between treatments. We conclude that elevations in fetal plasma NE suppress insulin concentrations, and that compensatory ß-cell stimulus-secretion responsiveness is present before IUGR.

The dual mTORC1 and mTORC2 inhibitor AZD8055 has anti-tumor activity in acute myeloid leukemia.

The serine/threonine kinase mammalian target of rapamycin (mTOR) is crucial for cell growth and proliferation, and is constitutively activated in primary acute myeloid leukemia (AML) cells, therefore representing a major target for drug development in this disease. We show here that the specific mTOR kinase inhibitor AZD8055 blocked mTORC1 and mTORC2 signaling in AML. Particularly, AZD8055 fully inhibited multisite eIF4E-binding protein 1 phosphorylation, subsequently blocking protein translation, which was in contrast to the effects of rapamycin. In addition, the mTORC1-dependent PI3K/Akt feedback activation was fully abrogated in AZD8055-treated AML cells. Significantly, AZD8055 decreased AML blast cell proliferation and cell cycle progression, reduced the clonogenic growth of leukemic progenitors and induced caspase-dependent apoptosis in leukemic cells but not in normal immature CD34+ cells. Interestingly, AZD8055 strongly induced autophagy, which may be either protective or cell death inducing, depending on concentration. Finally, AZD8055 markedly increased the survival of AML transplanted mice through a significant reduction of tumor growth, without apparent toxicity. Our current results strongly suggest that AZD8055 should be tested in AML patients in clinical trials.

Catecholamines mediate multiple fetal adaptations during placental insufficiency that contribute to intrauterine growth restriction: lessons from hyperthermic sheep.

Placental insufficiency (PI) prevents adequate delivery of nutrients to the developing fetus and creates a chronic state of hypoxemia and hypoglycemia. In response, the malnourished fetus develops a series of stress hormone-mediated metabolic adaptations to preserve glucose for vital tissues at the expense of somatic growth. Catecholamines suppress insulin secretion to promote glucose sparing for insulin-independent tissues (brain, nerves) over insulin-dependent tissues (skeletal muscle, liver, and adipose). Likewise, premature induction of hepatic gluconeogenesis helps maintain fetal glucose and appears to be stimulated by both norepinephrine and glucagon. Reduced glucose oxidation rate in PI fetuses creates a surplus of glycolysis-derived lactate that serves as substrate for hepatic gluconeogenesis. These adrenergically influenced adaptive responses promote in utero survival but also cause asymmetric intrauterine growth restriction and small-for-gestational-age infants that are at greater risk for serious metabolic disorders throughout postnatal life, including obesity and type II diabetes.

Perspectives on inhibiting mTOR as a future treatment strategy for hematological malignancies.

Mammalian target of rapamycin (mTOR) is a protein kinase implicated in the regulation of various cellular processes, including those required for tumor development, such as the initiation of mRNA translation, cell-cycle progression and cellular proliferation. In a wide range of hematological malignancies, the mTORC1 signaling pathway has been found to be deregulated and has been designed as a major target for tumor therapy. Given that pre-clinical studies have clearly established the therapeutic value of mTORC1 inhibition, numerous clinical trials of rapamycin and its derivates (rapalogs) are ongoing for treatment of these diseases. At this time, although disease stabilization and tumor regression have been observed, objective responses in some tumor types have been modest. Nevertheless, some of the mechanisms underlying cancer-cell resistance to rapamycin have now been described, thereby leading to the development of new strategy to efficiently target mTOR signaling in these diseases. In this review, we discuss the rationale for using mTOR inhibitors as novel therapies for a variety of hematological, malignancies with a focus on promising new perspectives for these approaches.

Life-table evaluation of sediment-associated chlorpyrifos chronic toxicity to the benthic copepod, Amphiascus tenuiremis.

A partial life-cycle experiment was conducted to assess chronic effects of sediment-associated chlorpyrifos, an organophosphate pesticide, on a marine, benthic copepod population. The static-renewal experiment was initiated with 4 treatments including control, 13 replicates per treatment with one female (bearing first clutch of eggs) per replicate. No males were added because one fertilization is sufficient for several clutches. Once weekly, all replicate chamber contents (10-ml culture tubes with 1.5 ml of sediment and 5 ml of seawater) were sieved and enumerated to determine survival and fecundity. Surviving adult females were placed back into chambers with newly spiked sediments. This process was repeated for 7 weeks until all initial females were dead or reproduction had ceased for at least two weeks. Survival and fecundity data were then used to determine population dynamic parameters such as r (intrinsic rate of natural increase) for each treatment. Results revealed a chronic toxicity response with significant population effects (p<0.05) in all pesticide treatments versus the control; concentrations that represent 7-32% of the 96-hr LC50. The control treatment had an r value 26-52% higher than the pesticide treatments. This translated into a control population rate increase of up to twice that of pesticide treatments. In addition, significant reductions in weekly and total fecundity were found in all chlorpyrifos treatments. Based on these results, usage of population parameters with benthic copepods allows for an integrative measurement of population effects from chronic exposure to sediment-associated contaminants.

Instructed heart rate control in a high heart rate population.

Forty college students were selected from a large number of introductory psychology students on the basis of high heart rate during an initial screening session. Subjects were then contacted and participated in two additional sessions during which heart rate, respiration rate, and skin conductance measures were obtained. Each session consisted of a baseline period followed by five trial periods during which subjects attempted to control their heart rate or performed a visual tracking task. Subjects were randomly assigned to one of four groups. One group served as a control and monitored a visual feedback display driven by their own heart rate but received no instructions to decrease their heart rate. In contrast, the three heart rate control groups were instructed to decrease heart rate during the trial periods by utilizing a relaxation procedure, proportional biofeedback, or proportional biofeedback plus criterion information. No group differences were present during the baseline periods. During feedback trials, however, all the training groups differed from the control in heart rate but did not differ from each other. It is suggested that feedback displays may not facilitate heart rate reduction beyond the level achieved by instructing subjects to use a general relaxation procedure.

A bizarre case of vehicular suicide.

The upper torso of a man was discovered under a highway sign next to tire tread marks leading from the highway and continuing beyond the sign. The victim's lower torso and automobile were also found along the same path 31 m (101 ft) and 41 m (133 ft) beyond the sign, respectively. The decedent was initially thought to be a disposed, homicide victim who had been dismembered by his assailant(s). Accident reconstruction revealed that the victim was the driver of the automobile and was transected by the highway sign stanchion as he protruded through the passenger side window of his moving vehicle. Based on the scene findings, autopsy, and psychological autopsy, the manner of death was classified as suicidal. The criteria used by medical examiners for vehicular suicide are also discussed.

Spin-exchange-induced circularly polarized molecular fluorescence.

We have measured the circular polarization of light emitted from both atomic H and molecular H2 after bombarding H2 with longitudinally polarized electrons. For both atomic and molecular fluorescence near threshold we observe a circular polarization as great as 10% of the electron polarization. This represents the first direct observation of spin transfer in electron-molecule collisions.

The design and development of a triaxial wear-testing joint simulator.

Most of the existing wear testers created to wear test total hip replacements, specifically the acetabular component, are designed to exert only an axial force and provide rotation in a close approximation of the actual femoral movement. The Rocky Mountain Joint Simulator was designed to exert three orthogonal forces and provide rotations about the X-, Y- and Z-axes to more closely simulate the physiological forces and motions found in the human gait cycle. The RMJS was also designed with adaptability for other joints, such as knees or canine hips, through the use of hydraulics and a computer-programmable control system. Such adaptability and functionality allows the researcher to more closely model a gait cycle, thereby obtaining wear patterns that resemble those found in retrieved implants more closely than existing simulators. Research is ongoing into the tuning and evaluation of the machine and preliminary acetabular component wear test results will be presented at the conference.