Quantification of concentration changes in neonatal human cerebral oxidized cytochrome oxidase.

The oxygenation of cerebral cytochrome oxidase in vivo was investigated in eight newborn preterm infants. Near-infrared spectroscopy was used to quantify changes in the concentration of oxidized cytochrome oxidase ([CytO2]) observed during alterations in arterial oxygen saturation (SaO2) in the range of 85-99% and of carbon dioxide tension (PaCO2) in the range of 4.3-9.6 kPa. No relation was found between changes in SaO2 and [CytO2]. Alterations in PaCO2 were positively related both to changes in [CytO2] and total cerebral hemoglobin concentration [( Hb]t). The changes in [CytO2] ranged from 0.09 to 0.33 (median 0.21) mumol.l-1.kPa-1. The ratio [CytO2]/[Hb]t ranged from 0.06 to 0.12 (median 0.08). The relation of delta [CytO2] to the change in cerebral blood volume (delta CBV) was calculated: delta [CytO2]/delta CBV ranged from 0.09 to 0.18 (median 0.11) mumol/ml. These results define a fraction of cerebral cytochrome oxidase in the newborn infant that is oxidized after an increase in PaCO2 but demonstrate that a change in SaO2 in the range studied was not sufficient by itself to change [CytO2]. They differ from results of studies in adults; this may reflect significant differences between adult and neonatal brain.     

Blood flow index using near-infrared spectroscopy and indocyanine green as a minimally invasive tool to assess respiratory muscle blood flow in humans

Near-infrared spectroscopy (NIRS) in combination with indocyanine green (ICG) dye has recently been used to measure respiratory muscle blood flow (RMBF) in humans. This method is based on the Fick principle and is determined by measuring ICG in the respiratory muscles using transcutaneous NIRS in relation to the [ICG] in arterial blood as measured using photodensitometry. This method is invasive since it requires arterial cannulation, repeated blood withdrawals, and reinfusions. A less invasive alternative is to calculate a relative measure of blood flow known as the blood flow index (BFI), which is based solely on the NIRS ICG curve, thus negating the need for arterial cannulation. Accordingly, the purpose of this study was to determine whether BFI can be used to measure RMBF at rest and during voluntary isocapnic hyperpnea at 25, 40, 55, and 70% of maximal voluntary ventilation in seven healthy humans. BFI was calculated as the change in maximal [ICG] divided by the rise time of the NIRS-derived ICG curve. Intercostal and sternocleidomastoid muscle BFI were correlated with simultaneously measured work of breathing and electromyography (EMG) data from the same muscles. BFI showed strong relationships with the work of breathing and EMG for both respiratory muscles. The coefficients of determination (R(2)) comparing BFI vs. the work of breathing for the intercostal and sternocleidomastoid muscles were 0.887 (P < 0.001) and 0.863 (P < 0.001), respectively, whereas the R(2) for BFI vs. EMG for the intercostal and sternocleidomastoid muscles were 0.879 (P < 0.001) and 0.930 (P < 0.001), respectively. These data suggest that the BFI closely reflects RMBF in conscious humans across a wide range of ventilations and provides a less invasive and less technically demanding alternative to measuring RMBF.     

Na/K pump current and [Na](i) in rabbit ventricular myocytes: local [Na](i) depletion and Na buffering

Na/K pump current (I(pump)) and intracellular Na concentration ([Na](i)) were measured simultaneously in voltage-clamped rabbit ventricular myocytes, under conditions where [Na](i) is controlled mainly by membrane transport. Upon abrupt pump reactivation (after 10-12 min blockade), I(pump) decays in two phases. Initially, I(pump) declines with little [Na](i) change, whereas the second phase is accompanied by [Na](i) decline. Initial I(pump) sag was still present at external [K] = 15 mM, but prevented by [Na](i) approximately 100 mM. Initial I(pump) sag might be explained by subsarcolemmal [Na](i) ([Na](SL)) depletion produced by rapid Na extrusion and I(pump). Brief episodes of pump blockade allowed [Na](SL) repletion, since peak postblockade I(pump) exceeded I(pump) at the end of previous activation (without appreciably altered global [Na](i)). The apparent K(m) for [Na](i) was higher for continuous I(pump) activation than peak I(pump) (14.1 +/- 0.2 vs. 11.2 +/- 0.2 mM), whereas that based on d[Na](i)/dt matched peak I(pump) (11.6 +/- 0.3 mM). [Na](SL) depletion (vs. [Na](i)) could be as high as 3 mM for [Na](i) approximately 18-20 mM. A simple diffusion model indicates that such [Na](SL) depletion requires a Na diffusion coefficient 10(3)- to 10(4)-fold below that expected in bulk cytoplasm (although this could be subsarcolemmal only). I(pump) integrals and [Na](i) decline were used to estimate intracellular Na buffering, which is slight (1.39 +/- 0.09).     

Blood lactate accumulation and muscle deoxygenation during incremental exercise.

Near-infrared spectroscopy (NIRS) could allow insights into controversial issues related to blood lactate concentration ([La](b)) increases at submaximal workloads (). We combined, on five well-trained subjects [mountain climbers; peak O(2) consumption (VO(2peak)), 51.0 +/- 4.2 (SD) ml. kg(-1). min(-1)] performing incremental exercise on a cycle ergometer (30 W added every 4 min up to voluntary exhaustion), measurements of pulmonary gas exchange and earlobe [La](b) with determinations of concentration changes of oxygenated Hb (Delta[O(2)Hb]) and deoxygenated Hb (Delta[HHb]) in the vastus lateralis muscle, by continuous-wave NIRS. A "point of inflection" of [La](b) vs. was arbitrarily identified at the lowest [La](b) value which was >0.5 mM lower than that obtained at the following. Total Hb volume (Delta[O(2)Hb + HHb]) in the muscle region of interest increased as a function of up to 60-65% of VO(2 peak), after which it remained unchanged. The oxygenation index (Delta[O(2)Hb - HHb]) showed an accelerated decrease from 60- 65% of VO(2 peak). In the presence of a constant total Hb volume, the observed Delta[O(2)Hb - HHb] decrease indicates muscle deoxygenation (i.e., mainly capillary-venular Hb desaturation). The onset of muscle deoxygenation was significantly correlated (r(2) = 0.95; P < 0.01) with the point of inflection of [La](b) vs., i.e., with the onset of blood lactate accumulation. Previous studies showed relatively constant femoral venous PO(2) levels at higher than approximately 60% of maximal O(2) consumption. Thus muscle deoxygenation observed in the present study from 60-65% of VO(2 peak) could be attributed to capillary-venular Hb desaturation in the presence of relatively constant capillary-venular PO(2) levels, as a consequence of a rightward shift of the O(2)Hb dissociation curve determined by the onset of lactic acidosis.     

Effect of hypercapnia on cerebral blood flow and blood volume in pigs studied by positron emission tomography

Pigs are increasingly used as in vivo models in neuroscience, including studies using positron emission tomography. During anesthesia, cerebral blood flow (CBF) and cerebral blood volume (CBV) are mainly regulated by the partial pressure of CO2 (pCO2) in arterial blood. We sought to determine the effects of increased arterial pCO2 (hypercapnia) on CBF and CBV in anesthetized domestic pigs. We anesthetized 4 pigs and manipulated the tidal volume of the ventilator to different pCO2 levels. Baseline pCO2 was on average 6.5 kPa (n = 9 periods) and hypercapnia pCO2 ranged from 11 to 20 kPa, mean 18.5 kPa (n = 9 periods). Series of dynamic PET scans with H(2)15O (CBF measurements) and C15O (CBV measurements) were performed. CBF increased on average 54%, from mean 0.48 ml blood/min/ml brain tissue during normoxia to 0.74 ml blood/min/ml brain tissue during hypercapnia. CBV increased 41% from mean 0.061 ml blood/ml brain tissue (n = 6) during normoxia to 0.086 ml blood/ml brain tissue (n = 6) during hypercapnia. Our observations indicate that pCO2 levels have a major influence on porcine CBF and CBV and should be controlled in studies where a constant level is crucial.     

Quantitation of cerebral blood volume in human infants by near-infrared spectroscopy

Current methods for measuring cerebral blood volume (CBV) in newborn infants are unsatisfactory. A new method is described in which the effect of a small change (5-10%) in arterial oxygen saturation (SaO2) on cerebral oxyhemoglobin [HbO2] and deoxyhemoglobin [Hb] concentration is observed by near-infrared (NIR) spectroscopy. Previous experiments in which the NIR absorption characteristics of HbO2 and Hb and the pathlength of NIR light through the brain were defined allowed changes in [HbO2] and [Hb] to be quantified from the Beer-Lambert law. It is shown here that CBV can then be derived from the expression CBV = (delta[HbO2] - delta[Hb])/(2. delta SaO2.H.R.), where H is the large vessel total hemoglobin concentration and R to the cerebral-to-large vessel hematocrit ratio. Observations on 12 newborn infants with normal brains, born at 25-40 wk of gestation and aged 10-240 h, gave a mean value for CBV of 2.22 +/- 0.40 (SD) ml/100 g, whereas mean CBV was significantly higher 3.00 +/- 1.04 ml/100 g in 10 infants with brain injury born at 24 to 42 wk of gestation and aged 4-168 h (P less than 0.05).     

Regional blood flow during exercise in humans measured by near-infrared spectroscopy and indocyanine green.

Using near-infrared spectroscopy (NIRS) and the tracer indocyanine green (ICG), we quantified blood flow in calf muscle and around the Achilles tendon during plantar flexion (1-9 W). For comparison, blood flow in calf muscle was determined by dye dilution in combination with magnetic resonance imaging measures of muscle volume, and, for the peritendon region, blood flow was measured by (133)Xe washout. From rest to a peak load of 9 W, NIRS-ICG blood flow in calf muscle increased from 2.4+/-0.2 to 74+/-5 ml x 100 ml tissue(-1) x min(-1), similar to that measured by reverse dye (77+/-6 ml x 100 ml tissue(-1) x min(-1)). Achilles peritendon blood flow measured by NIRS-ICG rose with exercise from 2.2+/-0.5 to 15.1+/-0.2 ml x 100 ml(-1) x min(-1), which was similar to that determined by (133)Xe washout (2.0+/-0.6 to 14.6+/-0.3 ml x 100 ml tissue(-1) x min(-1)). This is the first study using NIRS and ICG to quantify regional tissue blood flow during exercise in humans. Due to its high spatial and temporal resolution, the technique may be useful for determining regional blood flow distribution and regulation during exercise in humans.     

Noninvasive evaluation of skeletal muscle mitochondrial capacity with near-infrared spectroscopy: correcting for blood volume changes

Near-infrared spectroscopy (NIRS) is a well-known method used to measure muscle oxygenation and hemodynamics in vivo. The application of arterial occlusions allows for the assessment of muscle oxygen consumption (mVo(2)) using NIRS. The aim of this study was to measure skeletal muscle mitochondrial capacity using blood volume-corrected NIRS signals that represent oxygenated hemoglobin/myoglobin (O(2)Hb) and deoxygenated hemoglobin/myoglobin (HHb). We also assessed the reliability and reproducibility of NIRS measurements of resting oxygen consumption and mitochondrial capacity. Twenty-four subjects, including four with chronic spinal cord injury, were tested using either the vastus lateralis or gastrocnemius muscles. Ten healthy, able-bodied subjects were tested on two occasions within a period of 7 days to assess the reliability and reproducibility. NIRS signals were corrected for blood volume changes using three different methods. Resting oxygen consumption had a mean coefficient of variation (CV) of 2.4% (range 1-32%). The recovery of oxygen consumption (mVo(2)) after electrical stimulation at 4 Hz was fit to an exponential curve, which represents mitochondrial capacity. The time constant for the recovery of mVo(2) was reproducible with a mean CV of 10% (range 1-22%) only when correcting for blood volume changes. We also examined the effects of adipose tissue thickness on measurements of mVo(2). We found the mVo(2) measurements using absolute units to be influenced by adipose tissue thickness (ATT), and this relationship was removed when an ischemic calibration was performed, supporting its use to compare mVo(2) between individuals of varying ATT. In conclusion, in vivo oxidative capacity can be assessed using blood volume-corrected NIRS signals with a high degree of reliability and reproducibility.     

Naloxone-like influence of TRH and ACTH-(4-7) on hypothalamic blood flow autoregulation in the rat

The influence of intracerebroventricularly (ICV) administered thyrotropin-releasing hormone pGlu-His-Pro-NH2 (TRH), pGlu-His-Phe-NH2 (TRH analog, (TRHa)), Met-Glu-His-Phe(ACTH-(4-7)) and His-Phe-Arg-Trp-Gly (ACTH-(6-10)) on autoregulation of cerebral blood flow was studied in anesthetized, ventilated rats. Autoregulatory capacity of the cerebrovascular bed was tested by hypothalamic blood flow (HBF) and total cerebral blood volume (CBV) determinations during consecutive stepwise lowering of the systemic mean arterial pressure to 80, 60 and 40 mmHg, by hemorrhage. None of the peptides caused a change in resting HBF or CBV upon ICV administration (5 micrograms/kg). However, the same dose of TRH, TRHa and ACTH-(4-7) resulted in impairment of autoregulation. ACTH-(6-10) was not effective. Thus, the disturbed autoregulation may be due to the presence of the dipeptide Glu-His which is common to TRH, TRHa and ACTH-(4-7).     

Characterization of the mechanisms of action and nitric oxide species involved in the relaxation induced by the ruthenium complex

Nitric oxide (NO) plays an important role in the control of vascular tone. NO donors have therapeutic use and the most used NO donors, nitroglycerin and sodium nitroprusside have problems in their use. Thus, new NO donors have been synthesized to minimize these undesirable effects. Nytrosil ruthenium complexes have been studied as a new class of NO donors. trans-[RuCl([15]aneN(4))NO](2+), induces vasorelaxation only in presence of reducing agent. In this study, we characterized the mechanisms of vasorelaxation of trans-[RuCl([15]aneN(4))NO](2+) in denuded rat aorta and identified which NO forms are involved in this relaxation. We also evaluated the effect of this NO donor in decreasing the cytosolic Ca(2+) concentration ([Ca(2+)]c) of the vascular smooth muscle cells. Vasorelaxation to trans-[RuCl([15]aneN(4))NO](2+) (E(max): 101.8 +/- 2.3%, pEC(50): 5.03 +/- 0.15) was almost abolished in the presence of the NO* scavenger hydroxocobalamin (E(max): 4.0 +/- 0.4%; P < 0.001) and it was partially inhibited by the NO(-) scavenger L-cysteine (E(max): 79.9 +/- 6.9%, pEC(50): 4.41 +/- 0.06; P < 0.05). The guanylyl cyclase inhibitor ODQ reduced the E(max) (57.7 +/- 4.0%, P < 0.001) and pEC(50) (4.21 +/- 0.42, P < 0.01) and the combination of ODQ and TEA abolished the response to trans-[RuCl([15]aneN(4))NO](2+). The blockade of voltage-dependent (K(v)), ATP-sensitive (K(ATP)), and Ca(2+)-activated (K(Ca) K(+) channels reduced the vasorelaxation induced by trans-[RuCl([15]aneN(4))NO](2+). This compound significantly reduced [Ca(2+)]c (from 100% to 85.9 +/- 3.5%, n = 4). In conclusion, our data demonstrate that this NO donor induces vascular relaxation involving NO* and NO(-) species, that is associated to a decrease in [Ca(2+)]c. The mechanisms of vasorelaxation involve guanylyl cyclase activation, cGMP production and K(+) channels activation.     

ATP regulation in adult rat cardiomyocytes: time-resolved decoding of rapid mitochondrial calcium spiking imaged with targeted photoproteins

The mechanisms that enable the heart to rapidly increase ATP supply in line with increased demand have not been fully elucidated. Here we used an adenoviral system to express the photoproteins luciferase and aequorin, targeted to the mitochondria or cytosol of adult cardiomyocytes, to investigate the interrelationship between ATP and Ca(2+) in these compartments. In neither compartment were changes in free [ATP] observed upon increased workload (addition of isoproterenol) in myocytes that were already beating. However, when myocytes were stimulated to beat rapidly from rest, in the presence of isoproterenol, a significant but transient drop in mitochondrial [ATP] ([ATP](m)) occurred (on average to 10% of the initial signal). Corresponding changes in cytosolic [ATP] ([ATP](c)) were much smaller (<5%), indicating that [ATP](c) was effectively buffered in this compartment. Although mitochondrial [Ca(2+)] ([Ca(2+)](m)) is an important regulator of respiratory chain activity and ATP production in other cells, the kinetics of mitochondrial Ca(2+) transport are controversial. Parallel experiments in cells expressing mitochondrial aequorin showed that the drop in [ATP](m) occurred over the same time scale as average [Ca(2+)](m) was increasing. Conversely, in the absence or presence of isoproterenol, clear beat-to-beat peaks in [Ca(2+)](m) were observed at 0.9 or 1.3 mum, respectively, concentrations similar to those observed in the cytosol. These results suggest that mitochondrial Ca(2+) transients occur during the contractile cycle and are translated into a time-averaged increase in mitochondrial ATP production that keeps pace with increased cytosolic demand.     

Control of cerebral blood velocity with furosemide-induced hypovolemia and upright tilt

The purpose of this study was to test the hypothesis that exacerbated reductions of cerebral blood velocity (CBV) during upright tilt with dehydration are associated with impaired cerebrovascular control. Nine healthy men were tilted head-up (HUT) to 70° for 10 min on two occasions separated by 7 days under euhydration (EUH) and dehydration (DEH; 40 mg of furosemide and water restriction) conditions. Beat-by-beat arterial pressures and CBV were measured during a 5-min supine baseline and during the first (T1) and last (T2) 5 min of HUT. Cerebral autoregulation and arterial baroreflex sensitivity were assessed in the frequency domain with cross-spectral techniques. DEH reduced plasma volume by 10% (P = 0.008) and supine mean CBV (CBV(mean)) by 11% (P = 0.002). Mean arterial pressure (MAP), stroke volume, and baroreflex sensitivity decreased during HUT (P ≤ 0.002), but absolute reductions were similar between hydration conditions, with the exception of stroke volume, which was lower at T1 during DEH than EUH (P = 0.04). CBV(mean) during DEH was lower (7 cm/s) over the course of the entire 10 min of HUT (P ≤ 0.004) than during EUH. Low-frequency oscillations (0.07-0.2 Hz) of MAP and CBV(mean) and MAP-CBV(mean) coherence were higher during DEH than EUH at T1 (P ≤ 0.02), but not at T2. Our results suggest that increased coherence between arterial pressure and CBV with the combination of DEH and HUT are indicative of altered cerebrovascular control. Increased CBV oscillations with DEH may reflect acute protective mechanisms to ensure adequate cerebral perfusion under conditions of reduced central blood volume.     

Cesium toxicity in Arabidopsis

Cesium (Cs) is chemically similar to potassium (K). However, although K is an essential element, Cs is toxic to plants. Two contrasting hypotheses to explain Cs toxicity have been proposed: (1) extracellular Cs+ prevents K+ uptake and, thereby, induces K starvation; and (2) intracellular Cs+ interacts with vital K(+)-binding sites in proteins, either competitively or noncompetitively, impairing their activities. We tested these hypotheses with Arabidopsis (Arabidopsis thaliana). Increasing the Cs concentration in the agar ([Cs](agar)) on which Arabidopsis were grown reduced shoot growth. Increasing the K concentration in the agar ([K](agar)) increased the [Cs](agar) at which Cs toxicity was observed. However, although increasing [Cs](agar) reduced shoot K concentration ([K](shoot)), the decrease in shoot growth appeared unrelated to [K](shoot) per se. Furthermore, the changes in gene expression in Cs-intoxicated plants differed from those of K-starved plants, suggesting that Cs intoxication was not perceived genetically solely as K starvation. In addition to reducing [K](shoot), increasing [Cs](agar) also increased shoot Cs concentration ([Cs](shoot)), but shoot growth appeared unrelated to [Cs](shoot) per se. The relationship between shoot growth and [Cs](shoot)/[K](shoot) suggested that, at a nontoxic [Cs](shoot), growth was determined by [K](shoot) but that the growth of Cs-intoxicated plants was related to the [Cs](shoot)/[K](shoot) quotient. This is consistent with Cs intoxication resulting from competition between K+ and Cs+ for K(+)-binding sites on essential proteins.     

Fiber type-specific determinants of Vmax for insulin-stimulated muscle glucose uptake in vivo

The aim of this study was to determine barriers limiting muscle glucose uptake (MGU) during increased glucose flux created by raising blood glucose in the presence of fixed insulin. The determinants of the maximal velocity (V(max)) of MGU in muscles of different fiber types were defined. Conscious rats were studied during a 4 mU x kg(-1) x min(-1) insulin clamp with plasma glucose at 2.5, 5.5, and 8.5 mM. [U-(14)C]mannitol and 3-O-methyl-[(3)H]glucose ([(3)H]MG) were infused to steady-state levels (t = -180 to 0 min). These isotope infusions were continued from 0 to 40 min with the addition of a 2-deoxy-[(3)H]glucose ([(3)H]DG) infusion. Muscles were excised at t = 40 min. Glucose metabolic index (R(g)) was calculated from muscle-phosphorylated [(3)H]DG. [U-(14)C]mannitol was used to determine extracellular (EC) H(2)O. Glucose at the outer ([G](om)) and inner ([G](im)) sarcolemmal surfaces was determined by the ratio of [(3)H]MG in intracellular to EC H(2)O and muscle glucose. R(g) was comparable at the two higher glucose concentrations, suggesting that rates of uptake near V(max) were reached. In summary, by defining the relationship of arterial glucose to [G](om) and [G](im) in the presence of fixed hyperinsulinemia, it is concluded that 1) V(max) for MGU is limited by extracellular and intracellular barriers in type I fibers, as the sarcolemma is freely permeable to glucose; 2) V(max) is limited in muscles with predominantly type IIb fibers by extracellular resistance and transport resistance; and 3) limits to R(g) are determined by resistance at multiple steps and are better defined by distributed control rather than by a single rate-limiting step.     

Interpretation of concentration-dependence in aggregation kinetics

Aggregation processes are analyzed by two kinetic models, the random polymerization model and the nucleation-dependent polymerization model. A kinetic equation for the random polymerization model can be derived analytically, revealing the relation between the initial monomer concentration ([M]0), the rate constant (k(a)), time (t), the yield of detectable aggregate ([F]), and the critical aggregation number (m). However, time-course curves for the nucleation-dependent polymerization model can be obtained by numerical calculation. It is found that lag time (t(d)) and half-time (t1/2) are proportional to [M](-1) in the random polymerization model, while t(d) and t1/2 are proportional to [M1](-s) (1 < s < n; n is nucleus size) at the lower concentration and are less dependent on [M1] at the higher concentration in the nucleation-dependent polymerization model.     

Plasma acid-base regulation above and below ventilatory threshold in late gestation.

Stewart's physicochemical approach was used to study the effects of pregnancy on acid-base regulation in arterialized blood. Responses of 15 healthy pregnant women (PG; gestational age, 37.1 +/- 0.2 wk) were compared with those of 15 nonpregnant controls (CG) at rest and during cycling at 70 and 110% of the ventilatory threshold (T(vent)). Hydrogen ion concentration ([H(+)]) was lower in the PG vs. CG at rest and during exercise (P < 0.05 at rest and 70% T(vent)). Exercise-induced changes in [H(+)] were similar between groups. Lower resting [H(+)] values in the PG vs. CG resulted from lower values for arterialized PCO(2) (Pa(CO(2))) and total weak acid ([A](tot)), which were partly offset by a lower strong-ion difference ([SID]). Reductions in [A](tot) and [SID] at rest were primarily the result of reductions in albumin [Alb] and sodium [Na(+)], respectively. In the transition from rest to 70% T(vent), small increases in Pa(CO(2)) and [A](tot) contributed to moderate increases in [H(+)] in both groups, however [SID] increased in the PG and decreased in the CG (P < 0.05 between groups). In the transition from rest to 110% T(vent), decreases in [SID] made a significantly greater contribution to changes in [H(+)] in the CG vs. PG. Exercise-induced increases in [H(+)] are similar in the pregnant vs. nonpregnant state, but there is a reduced contribution of [SID] both above and below T(vent) during pregnancy.     

Interactions between selenium and sulphur nutrition in Arabidopsis thaliana

Selenium (Se) is an essential plant micronutrient, but is toxic at high tissue concentrations. It is chemically similar to sulphur (S), an essential plant macronutrient. The interactions between Se and S nutrition were investigated in the model plant Arabidopsis thaliana (L.) Heynh. Arabidopsis plants were grown on agar containing a complete mineral complement and various concentrations of selenate and sulphate. The Se/S concentration ratio in the shoot ([Se](shoot)/[S](shoot)) showed a complex dependence on the ratio of selenate to sulphate concentration in the agar ([Se](agar)/[S](agar)). Increasing [S](agar) increased shoot fresh weight (FW) and [S](shoot), but decreased [Se](shoot). Increasing [Se](agar) increased both [Se](shoot) and [S](shoot), but reduced shoot FW. The reduction in shoot FW in the presence of Se was linearly related to the shoot Se/S concentration ratio. These data suggest (i) that Se and S enter Arabidopsis through multiple transport pathways with contrasting sulphate/selenate selectivities, whose activities vary between plants of contrasting nutritional status, (ii) that rhizosphere sulphate inhibits selenate uptake, (iii) that rhizosphere selenate promotes sulphate uptake, possibly by preventing the reduction in the abundance and/or activity of sulphate transporters by sulphate and/or its metabolites, and (iv) that Se toxicity occurs because Se and S compete for a biochemical process, such as assimilation into amino acids of essential proteins.     

Continuous measurement of cerebral blood volume in rats with the photoelectric technique: effect of morphine and naloxone

The validity of a photoelectric method for continuous cerebral blood volume (CBV) measurement was tested and modified for the rat's brain. A new way of introducing a miniature light source between the two hemispheres and fixing a light sensitive silicone blue cell to the outer surface of the parietal bone was developed. Light extinction factor of the rat's blood was determined experimentally (Eb rat = 1.38 +/- 0.15) in order to calculate absolute CBV value in this species, resulting in a 4.77 +/- 0.13 vol % absolute CBV value. Data obtained in anesthetized, artificially ventilated rats by simultaneous recording of CBV and local cerebral blood flow (H2-gas clearance technique) show that local hypothalamic blood flow decreased significantly after morphine (1.0 mg/kg s.c.), while total CBV remained unchanged. Opiate receptor blockade with naloxone (1.0 mg/kg s.c.) on the contrary, as well as naloxone and morphine administration, caused no change in local hypothalamic blood flow, but resulted in a significant increase of total cerebral blood volume.     

Perfusion MRI Derived Indices of Microvascular Shunting and Flow Control Correlate with Tumor Grade and Outcome in Patients with Cerebral Glioma

Objectives

Deficient microvascular blood flow control is thought to cause tumor hypoxia and increase resistance to therapy. In glioma patients, we tested whether perfusion-weighted MRI (PWI) based indices of microvascular flow control provide more information on tumor grade and patient outcome than does the established PWI angiogenesis marker, cerebral blood volume (CBV).

Material and Methods

Seventy-two glioma patients (sixty high-grade, twelve low-grade gliomas) were included. Capillary transit time heterogeneity (CTH) and the coefficient of variation (COV), its ratio to blood mean transit time, provide indices of microvascular flow control and the extent to which oxygen can be extracted by tumor tissue. The ability of these parameters and CBV to differentiate tumor grade were assessed by receiver operating characteristic curves and logistic regression. Their ability to predict time to progression and overall survival was examined by the Cox proportional-hazards regression model, and by survival curves using log-rank tests.

Results

The best prediction of grade (AUC = 0.876; p < 0.05) was achieved by combining knowledge of CBV and CTH in the enhancing tumor and peri-focal edema, and patients with glioblastoma multiforme were identified best by CTH (AUC = 0.763; p<0.001). CTH outperformed CBV and COV in predicting time to progression and survival in all gliomas and in a subgroup consisting of only high-grade gliomas.

Conclusion

Our study confirms the importance of microvascular flow control in tumor growth by demonstrating that determining CTH improves tumor grading and outcome prediction in glioma patients compared to CBV alone.     

Synthesis of prostaglandin F ethanolamide by prostaglandin F synthase and identification of Bimatoprost as a potent inhibitor of the enzyme: new enzyme assay method using LC/ESI/MS

Prostaglandin (PG) D(2) ethanolamide (prostamide D(2)) was reduced to 9alpha,11beta-PGF(2) ethanolamide (9alpha,11beta-prostamide F(2)) by PGF synthase, which also catalyzes the reduction of PGH(2) and PGD(2) to PGF(2alpha) and 9alpha,11beta-PGF(2), respectively. These enzyme activities were measured by a new method, the liquid chromatographic-electrospray ionization-mass spectrometry (LC/ESI/MS) technique, which could simultaneously detect the substrate and all products. PGF(2alpha), 9alpha,11beta-PGF(2), PGD(2), PGH(2), 9alpha,11beta-prostamide F(2), and prostamide D(2) were separated on a TSKgel ODS 80Ts column, ionized by electrospray, and detected in the negative mode. Selected ion monitoring (SIM) of m/z 353 ([M-H](-)), 353 ([M-H](-)), 351 ([M-H](-)), 333 ([M-H-H(2)O](-)), 456 ([M+59](-)), and m/z 358 ([M-37](-)) was used for quantifying PGF(2alpha), 9alpha,11beta-PGF(2), PGD(2), PGH(2), 9alpha,11beta-prostamide F(2), and prostamide D(2), respectively. The detection limit for PGF(2alpha) and 9alpha,11beta-PGF(2) was 0.01pmol; that for PGH(2) and PGD(2), 0.1pmol; and that for prostamide D(2) and 9alpha,11beta-prostamide F(2), 0.5 and 0.03pmol, respectively. The LC/ESI/MS technique for measuring PGF synthase activity showed higher sensitivity than other methods. Using this method, we found that Bimatoprost, the ethyl amide analog of 17-phenyl-trinor PGF(2alpha) and an anti-glaucoma agent, inhibited all three reductase activities of PGF synthase when used at a low concentration. These results suggest that Bimatoprost also behaves as a potent PGF synthase inhibitor in addition to having prostamide-like activity.