The efflux of biotin from human peripheral blood mononuclear cells

Peripheral blood mononuclear cells (PMBCs) are readily available for sampling and are a useful model for studying biotin metabolism in human cells. To better understand biotin handling by PMBCs, we investigated the mechanism(s) and kinetics of biotin efflux from PMBCs. Human PMBCs were incubated with [(3)H]biotin at 475 pmol/L to load the cells. The [(3)H]biotin-loaded cells were then harvested and incubated in [(3)H]biotin-free media for up to 20 hours. At various intervals, aliquots of the PMBC suspensions were collected and analyzed for intracellular [(3)H]biotin. [(3)H]Biotin efflux from cells at 37 degrees C was fast and triphasic; the half-lives for the three elimination phases were 0.2 +/- 0.02 hours, 1.2 +/- 0.1 hours, and 21.9 +/- 13.6 hours. Such a triphasic [(3)H]biotin efflux could reflect (1) rapid efflux of free biotin, (2) slower release of biotin bound to intracellular molecules, and (3) even slower release from carboxylases in cellular organelles. Incubation at 4 degrees C rather than 37 degrees C increased the [(3)H]biotin retained at 20 hours from 27% to 85%. This observation is consistent with transporter-mediated efflux. When cellular glucose utilization was reduced by 2-deoxy-d-glucose and sodium fluoride, [(3)H]biotin efflux was similar to controls, suggesting that biotin efflux does not directly require metabolic energy. When [(3)H]biotin-loaded cells were incubated in external medium containing unlabeled biotin analogs, [(3)H]biotin efflux was accelerated approximately two times compared with incubation in a biotin-free medium. This observation suggests that biotin efflux is mediated by the same transporter that mediates biotin uptake from the extracellular medium (i.e., classic countertransport).     

Evaluation of primary production in Lake Erie by multiple proxies

Direct measurements of rates of primary production in Lake Erie are few and uncertainties surround rate measurements based on radiocarbon and the light-dark bottle incubation methods. For these reasons, we conducted a series of simultaneous primary productivity measurements in Lake Erie in July and August of 2003, based on incubation with [14C]-NaHCO3, the light-dark bottle method, and incubation with (18)O enriched water. Significant differences in the rates of primary production obtained by incubations with [(18)O]-H2O (0.19-34.60 mmol-O2 m(-3) h(-1)), [14C]-NaHCO3 (0.03-90.50 mmol-C m(-3) h(-1)), and light-dark bottles (0.06-60.78 mmol-O2 m(-3) h(-1)) were evident in six out of nine comparisons. Within the epilimnion, [(18)O]-H2O rates of primary production were significantly different from rates based on [14C]-NaHCO3 and light-dark bottles in all four comparisons and lower rates were obtained in three out of four comparisons. Eutrophic conditions in Sandusky Bay, Lake Erie were evident from the high primary production rates of 20.50-34.60 mmol-O2 m(-3) h(-1) ([(18)O]-H2O), 34.39-90.50 mmol-C m(-3) h(-1) ([14C]-NaHCO3), and 46.66-60.78 mmol-O2 m(-3) h(-1) (light-dark bottle). The photosynthetic quotient (PQ), or ratio of O2 production to CO2 consumption during photosynthesis, averaged 0.64+/-0.33 and 1.93+/-1.93, respectively, based on a comparison of [(18)O]-H2O to [14C]-NaHCO3 rates or light-dark bottle to [14C]-NaHCO3 production rates, respectively, demonstrating that photosynthesis in Lake Erie communities primarily follows expected stochiometric trends. The average of the ratio of production rates based on incubation with [(18)O]-H2O relative to those obtained by the light-dark incubation method was 0.66+/-0.33, indicating a tendency for the [(18)O]-H2O method to provide slightly lower estimates of production in Lake Erie. Lower estimates of primary production based on [(18)O]-H2O incubation relative to the other two approaches is most likely a consequence of consumption of labeled O2 within the cell or dilution of label by the release of O2 from supersaturated cells. This latter effect may be particularly characteristic of eutrophic environments.     

The Relationship between Ribulose Bisphosphate Concentration, Dissolved Inorganic Carbon (DIC) Transport and DIC-Limited Photosynthesis in the Cyanobacterium Synechococcus leopoliensis Grown at Different Concentrations of Inorganic Carbon

To examine the factors which limit photosynthesis and their role in photosynthetic adaptation to growth at low dissolved inorganic carbon (DIC), Synechococcus leopoliensis was grown at three concentrations (as signified by brackets) of DIC, high (1000-1800 micromolar), intermediate (200-300 micromolar), and low (10-20 micromolar). In all cell types photosynthesis varied from being ribulose bisphosphate (RuBP)-saturated at low external [DIC] to RuBP-limited at high external [DIC]. The maximum rate of photosynthesis (P_max) was achieved when the internal concentration of RuBP fell below the active site density of RuBP carboxylase/oxygenase (Rubisco). At rates of photosynthesis below P_max, photosynthetic capacity was limited by the ability of the cell to transport inorganic carbon and to supply CO₂ to Rubisco. Adaptation to low DIC was reflected by a decrease in the [DIC] required to half-saturate photosynthesis. Simultaneous mass-spectrometric measurement of rates of photosynthesis and DIC transport showed that the initial slope of the photosynthesis versus [DIC] curve is identical to the initial slope of the DIC transport versus [DIC] curve. This provided evidence that the enhanced capacity for DIC transport which occurs upon adaptation to low [DIC] was responsible for the increase in the initial slope of the photosynthesis versus [DIC] curve and therefore the decrease in the half saturation constant of photosynthesis with respect to DIC. Levels of RuBP and in vitro Rubisco activity varied only slightly between high and intermediate [DIC] grown cells but fell significantly (65-70%) in low [DIC] grown cells. Maximum rates of photosynthesis followed a similar pattern with P_max only slightly lower in intermediate [DIC] grown cells than in high [DIC] grown cells, but much lower in low [DIC] grown cells. The changing response of photosynthesis to [DIC] during adaptation to low DIC, may be explained by the interaction between DIC-transport limited and [RuBP]-limited photosynthesis.     

On the control of long-chain-fatty acid synthesis in isolated intact spinach (Spinacia oleracea) chloroplasts

1. Chloroplasts isolated from spinach leaves by using the low-ionic-strength buffers of Nakatani & Barber [(1977) Biochim. Biophys. Acta.461, 510-512] had higher rates of HCO(3) (-)-dependent oxygen evolution (up to 369mumol/h per mg of chlorophyll) and higher rates of [1-(14)C]acetate incorporation into long-chain fatty acids (up to 1500nmol/h per mg of chlorophyll) than chloroplasts isolated by using alternative procedures. 2. Acetate appeared to be the preferred substrate for fatty acid synthesis by isolated chloroplasts, although high rates of synthesis were also measured from H(14)CO(3) (-) in assays permitting high rats of photosynthesis. Incorporation of H(14)CO(3) (-) into fatty acids was decreased by relatively low concentrations of unlabelled acetate. Acetyl-CoA synthetase activity was present 3-4 times in excess of that required to account for rates of [1-(14)C]acetate incorporation into fatty acids, but pyruvate dehydrogenase was either absent or present in very low activity in spinach chloroplasts. 3. Rates of long-chain-fatty acid synthesis from [1-(14)C]acetate in the highly active chloroplast preparations, compared with those used previously, were less dependent on added cofactors, but showed a greater response to light. The effects of added CoA plus ATP, Triton X-100 and sn-glycerol 3-phosphate on the products of [1-(14)C]acetate incorporation were similar to those reported for less active chloroplast preparations. 4. Endogenous [(14)C]acetyl-CoA plus [(14)C]malonyl-CoA was maintained at a constant low level even when fatty acid synthesis was limited by low HCO(3) (-) concentrations. Endogenous [(14)C]acyl-(acyl-carrier protein) concentrations increased with increasing HCO(3) (-) concentration and higher rates of fatty acid synthesis, but were slightly lower in the presence of Triton X-100. It is proposed that rates of long-chain-fatty acid synthesis in isolated chloroplasts at saturating [1-(14)C]acetate concentrations and optimal HCO(3) (-) concentrations may be primarily controlled by rates of removal of the products of the fatty acid synthetase.     

The synthesis of polyribonucleotides by cytoplasmic enzymes

1. The possibility that the cell cytoplasm contains enzymes catalysing the biosynthesis of RNA was investigated in fractions obtained by differential centrifugation of homogenates of Landschutz ascites-tumour cells. 2. The microsomal fraction was shown to be most active in incorporating UMP residues from [alpha-(32)P]UTP into polyribonucleotide material. 3. The same fraction also incorporated [(3)H]CTP, [(3)H]ATP and [(3)H]GTP separately and independently of the presence of complementary ribonucleoside 5'-triphosphates. 4. The reaction was promoted by the addition of RNA and showed an absolute requirement for Mg(2+) ions. 5. Analysis of alkaline hydrolysates of the reaction products after the incorporation of [alpha-(32)P]UTP showed that most of the radioactivity was recovered in (2',3')-UMP residues irrespective of whether CTP, ATP and GTP were present in the reaction mixture. 6. Extraction of RNA from the reaction mixtures after the incorporation of [(3)H]ATP, [(3)H]GTP or [(3)H]CTP and analysis by sucrosedensity-gradient centrifugation showed no labelling of the ribosomal RNA. Radioactive material appeared between the 4s region and the meniscus of the sucrose gradient. In agreement with this observation, determinations of the chain length of the product showed that only short sequences of polynucleotides were synthesized. It is concluded that only homopolyribonucleotide synthesis is catalysed by the microsomal fractions and that there is little or no synthesis of RNA-like heteropolymers.     

A comparison of the metabolism of eighteen-carbon 13C-unsaturated fatty acids in healthy women

Altered use of different dietary fatty acids may contribute to several chronic diseases, including obesity, noninsulin-dependent diabetes mellitus, and cardiovascular disease. However, few comparative data are available to support this link, so the goal of the present study was to compare the metabolism of [(13)C]oleate, [(13)C]alpha-linolenate, [(13)C]elaidate, and [(13)C]linoleate through oxidation and incorporation into plasma lipid fractions and adipose tissue. Each tracer was given as a single oral bolus to six healthy women. Samples were collected over 8 days, and (13)C was analyzed using isotope ratio mass spectrometry. At 9 h postdose, cumulative oxidation was similar for [(13)C]elaidate, [(13)C]oleate, and [(13)C]alpha-linolenate (19 +/- 1%, 20 +/- 4%, and 19 +/- 3% dose, respectively). Significantly lower oxidation of [(13)C]linoleate (12 +/- 4% dose; P < 0.05) was accompanied by its higher incorporation into plasma phospholipids and cholesteryl esters. Abdominal adipose tissue was enriched with [(13)C]alpha-linolenate, [(13)C]elaidate, or [(13)C]linoleate within 6 h. The percentage linoleate in plasma phospholipids correlated positively with [(13)C]linoleate and [(13)C]elaidate oxidation, indicating a potential role of background diet. Conversion of [(13)C]linoleate and [(13)C]alpha-linolenate to longer chain polyunsaturates was a quantitatively minor route of utilization.     

An efficient targeted radiotherapy/gene therapy strategy utilising human telomerase promoters and radioastatine and harnessing radiation-mediated bystander effects

BACKGROUND: Targeted radiotherapy achieves malignant cell-specific concentration of radiation dosage by tumour-affinic molecules conjugated to radioactive atoms. Combining gene therapy with targeted radiotherapy is attractive because the associated cross-fire irradiation of the latter induces biological bystander effects upon neighbouring cells overcoming low gene transfer efficiency. METHODS: We sought to maximise the tumour specificity and efficacy of noradrenaline transporter (NAT) gene transfer combined with treatment using the radiopharmaceutical meta-[(131)I]iodobenzylguanidine ([(131)I]MIBG). Cell-kill was achieved by treatment with the beta-decay particle emitter [(131)I]MIBG or the alpha-particle emitter [(211)At]MABG. We utilised our novel transfected mosaic spheroid model (TMS) to determine whether this treatment strategy could result in sterilisation of spheroids containing only a small proportion of NAT-expressing cells. RESULTS: The concentrations of [(131)I]MIBG and [(211)At]MABG required to reduce to 0.1% the survival of clonogens derived from the TMS composed of 100% of NAT gene-transfected cells were 1.5 and 0.004 MBq/ml (RSV promoter), 8.5 and 0.0075 MBq/ml (hTR promoter), and 9.0 and 0.008 MBq/ml (hTERT promoter), respectively. The concentrations of radiopharmaceutical required to reduce to 0.1% the survival of clonogens derived from 5% RSV/NAT and 5% hTERT/NAT TMS were 14 and 23 MBq/ml, respectively, for treatment with [(131)I]MIBG and 0.018 and 0.028 MBq/ml, respectively, for treatment with [(211)At]MABG. CONCLUSIONS: These results indicate that the telomerase promoters have the capacity to drive the expression of the NAT. The potency of [(211)At]MABG is approximately three orders of magnitude greater than that of [(131)I]MIBG. Spheroids composed of only 5% of cells expressing NAT under the control of the RSV or hTERT promoter were sterilised by radiopharmaceutical treatment. This observation is indicative of bystander cell-kill.     

Efficient sequential synthesis of PET Probes of the COX-2 inhibitor [11C]celecoxib and its major metabolite [11C]SC-62807 and in vivo PET evaluation

Synthesis of [(11)C]celecoxib, a selective COX-2 inhibitor, and [(11)C]SC-62807, a major metabolite of celecoxib, were achieved and the potential of these PET probes for assessing the function of drug transporter in biliary excretion was evaluated. The synthesis of [(11)C]celecoxib was achieved in one-pot by reacting [(11)C]methyl iodide with an excess of the corresponding pinacol borate precursor using Pd(2)(dba)(3), P(o-tolyl)(3), and K(2)CO(3) (1:4:9) in DMF. The radiochemical yield of [(11)C]celecoxib was 63±23% (decay-corrected, based on [(11)C]CH(3)I) (n=7) with a specific radioactivity of 83±23GBq/μmol (n=7). The average time of synthesis from end of bombardment including formulation was 30min with >99% radiochemical purity. [(11)C]SC-62807 was synthesized from [(11)C]celecoxib by further rapid oxidation in the presence of excess KMnO(4) with microwave irradiation. The radiochemical yield of [(11)C]SC-62807 was 55±9% (n=3) (decay-corrected, based on [(11)C]celecoxib) with a specific radioactivity of 39±4GBq/μmol (n=3). The average time of synthesis from [(11)C]celecoxib including formulation was 20min and the radiochemical purity was >99%. PET studies in rats and the metabolite analyzes of [(11)C]celecoxib and [(11)C]SC-62807 showed largely different excretion processes, and consequently, [(11)C]SC-62807 was rapidly excreted via hepatobiliary excretion without further metabolism. [(11)C]SC-62807 was shown to have a high potential as a PET probe for evaluating drug transporter function in biliary excretion.     

In situ observation of crystal growth for poly[(S)-lactide] by temperature-controlled atomic force microscopy

The crystallization behavior and crystalline morphologies of poly[(S)-lactide] (P[(S)-LA]) in thin films crystallized isothermally at over 160 degrees C were characterized by transmission electron microscopy and atomic force microscopy (AFM). The dendritic crystal and hexagonal crystal were formed in thin film with thicknesses below 30 nm or over 50 nm, respectively. The crystal structures of dendritic and hexagonal crystals were identical, suggesting that the crystalline morphology of P[(S)-LA] is strongly dependent upon the film thickness. In situ observation of the crystal growth in the P[(S)-LA] thin film at 165 degrees C from the melt was carried out by using temperature-controlled AFM equipped with a heating stage. The initial stage of crystallization and development of lamellae were successfully observed during isothermal crystallization at 165 degrees C. The first forming crystal showed the edge-on orientation, and grew to S-shaped edge-on lamellae. Dendritic flat-on crystals were developed from the S-shaped edge-on lamellae. The growth rates of flat-on and edge-on lamellae were almost identical.     

静注人免疫球蛋白联合万古霉素治疗小儿败血症的疗效及外周血NLR、PCT变化观察

摘要 目的：观察静注人免疫球蛋白联合万古霉素治疗小儿败血症的疗效及外周血中性粒细胞/淋巴细胞比值（NLR）、降钙素原(PCT)变化。方法：选取2011年1月~2020年1月我院收治的败血症患儿80例为研究对象,按数字随机表法分为对照组和观察组各40例,对照组给予万古霉素治疗,观察组在对照组基础上给予静注人免疫球蛋白治疗,比较两组临床疗效、症状改善时间和住院时间、NLR、PCT、超敏C反应蛋白(hs-CRP)、白细胞计数（WBC）、免疫功能及不良反应发生率。结果：观察组治疗有效率高于对照组（87.50%vs65.00%）（P<0.05）。观察组神经系统症状改善时间、体温改善时间、拒奶改善时间和住院时间为（6.22±1.05）d、（3.88±0.25）d、（5.10±0.86）d、（8.71±2.05）d,均短于对照组的（8.76±1.53）d、（6.22±0.64）d、（7.53±1.46）d和（11.24±3.36）d,比较差异有统计学意义（P<0.05）。治疗后观察组外周血NLR、PCT、hs-CRP、WBC水平为（1.35±0.20）、（0.80±0.34）mg/mL、（3.56±0.62）g/L、（9.12±1.80）×10⁹/L,均显著低于对照组的（1.83±0.32）、（2.19±0.73）mg/mL、（9.78±2.64）g/L和（12.26±2.59）×10⁹/L,比较差异有统计学意义（P<0.05）。治疗后观察组CD4⁺、CD3⁺、CD4/CD8为（42.77±11.36）％、（41.27±11.26）％、（1.70±0.33）,均显著高于对照组的（35.80±9.32）％、（35.66±9.40）％和（1.29±0.25）,比较差异有统计学意义（P<0.05）。两组不良反应发生率比较无差异（10.00%vs7.50%）（P>0.05）。结论：静注人免疫球蛋白联合万古霉素治疗小儿败血症的疗效显著,可降低炎症因子,提高免疫功能,且安全性较高。     

培美曲塞联合长春瑞滨治疗复发转移性宫颈癌患者对疾病控制及预后随访研究

摘要 目的：探讨培美曲塞联合长春瑞滨治疗复发转移性宫颈癌对疾病控制及预后的影响。方法：选择2018年5月~2019年6月收治的100例复发转移性宫颈癌患者,按照随机数表法分观察组和对照组,每组50例。对照组采用紫杉醇联合顺铂化疗,观察组则采用培美曲塞联合长春瑞滨治疗。比较2组疾病控制效果,记录患者化疗前后Karnofsky评分变化,统计2组患者毒副反应及生存率、生存期。结果：观察组的疾病控制率为84.00%,明显高于对照组的62.00%,差异显著（P<0.05）;治疗后,2组患者的Karnofsky评分均升高,且观察组[（86.25±3.02）分]明显高于对照组[（81.15±3.11）分],差异显著（P<0.05）; 2组患者胃肠道反应、骨髓抑制、血小板计数减少、恶心呕吐等毒副反应发生率比较,无明显差异（P>0.05）;观察组患者的1年生存率70.00%、2年生存率58.00%、3年生存率26.00%均高于对照组的50.00%、38.00%、10.00%,观察组的生存期[（18.12±3.21）月]长于对照组[（14.18±2.81）月],差异显著（P<0.05）。结论：培美曲塞联合长春瑞滨治疗复发转移性宫颈癌效果显著,可有效提高患者的生存质量及生存率,延长生存期,毒副反应低,患者耐受性好,可在临床推广运用。     

N-[18F]fluoroethyl-4-piperidyl acetate ([18F]FEtP4A): A PET tracer for imaging brain acetylcholinesterase in vivo

N-[(18)F]Fluoroethyl-4-piperidyl acetate ([(18)F]FEtP4A) was synthesized and evaluated as a PET tracer for imaging brain acetylcholinesterase (AchE) in vivo. [(18)F]FEtP4A was previously prepared by reacting 4-piperidyl acetate (P4A) with 2-[(18)F]fluoroethyl bromide ([(18)F]FEtBr) at 130 degrees C for 30 min in 37% radiochemical yield using an automated synthetic system. In this work, [(18)F]FEtP4A was synthesized by reacting P4A with 2-[(18)F]fluoroethyl iodide ([(18)F]FEtI) or 2-[(18)F]fluoroethyl triflate ([(18)F]FEtOTf in improved radiochemical yields, compared with [(18)F]FEtBr under the corresponding condition. Ex vivo autoradiogram of rat brain and PET summation image of monkey brain after iv injection of [(18)F]FEtP4A displayed a high radioactivity in the striatum, a region with the highest AchE activity in the brain. Moreover, the distribution pattern of (18)F radioactivity was consistent with that of AchE in the brain: striatum>frontal cortex>cerebellum. In the rat and monkey plasma, two radioactive metabolites were detected. However, their presence might not preclude the imaging studies for AchE in the brain, because they were too hydrophilic to pass the blood-brain barrier and to enter the brain. In the rat brain, only [(18)F]fluoroethyl-4-piperidinol ([(18)F]FEtP4OH) was detected at 30 min postinjection. The hydrolytic [(18)F]FEtP4OH displayed a slow washout and a long retention in the monkey brain until the PET experiment (120 min). Although [(18)F]FEtP4A is a potential PET tracer for imaging AchE in vivo, its lower hydrolytic rate and lower specificity for AchE than those of [(11)C]MP4A may limit its usefulness for the quantitative measurement for AchE in the primate brain.     

In vivo investigation of the placental transfer of (13)C-labeled fatty acids in humans

Placental fatty acid transfer in humans in vivo was studied using stable isotopes. Four pregnant women undergoing cesarean section received 4 h before delivery an oral dose of [(13)C]palmitic acid (PA), [(13)C]oleic acid (OA), [(13)C]linoleic acid (LA), and [(13)C]docosahexaenoic acid (DHA). Maternal blood samples were collected at -4 h (basal), -3 h, -2 h, -1 h, 0 h, and +1 h relative to time of cesarean section. At the time of birth, venous cord blood and placental tissue were collected. Fatty acid composition was determined by gas-liquid chromatography and isotopic enrichment by gas chromatography-combustion-isotope ratio mass spectrometry. (13)C-enrichment of fatty acids in the nonesterified fatty acids (NEFA) of cord plasma tended to be higher than in NEFA of placenta, with statistically significant differences for the nonesterified OA and DHA ([(13)C]PA, 0.024 +/- 0.011 vs. 0.001 +/- 0.001; [(13)C]OA, 0.042 +/- 0.008 vs. 0.005 +/- 0.003; [(13)C]LA, 0.038 +/- 0.010 vs. 0.008 +/- 0.002; [(13)C]DHA, 0.059 +/- 0.009 vs. 0.010 +/- 0.003). The ratio of tracer fatty acid concentrations of placenta to maternal plasma was significantly higher for [(13)C]DHA than for the other fatty acids ([(13)C]PA, 7.1 +/- 1%; [(13)C]OA, 3.8 +/- 0.4%; [(13)C]LA, 9.2 +/- 1.3%; [(13)C]DHA, 25.9 +/- 3.4%). These results suggest that only a part of the placental NEFA participated in fatty acid transfer, and that the placenta showed a preferential accretion of DHA relative to the other fatty acids.     

Synthesis of [¹⁸F]-labeled (2-(2-fluoroethoxy)ethyl)triphenylphosphonium cation as a potential agent for myocardial imaging using positron emission tomography

[(18)F]-labeled molecular probe for the detection of myocardial perfusion deficit is driving particular interest due to its high clinical applicability. Thus, we synthesized (2-(2-[(18)F]fluoroethoxy)ethyl)triphenylphosphonium salt ([(18)F]3) that specifically accumulates in myocardium according to mitochondrial membrane potential. Here, we evaluated the performance of [(18)F]3 as a mitochondrial voltage sensor in vitro and in vivo. The [(18)F]3 was synthesized with 20～30% radiochemical yield and radiochemical purity was >98% by analytical HPLC. Specific activity was >6.7TBq/μmol. The cellular uptake assay showed preferential uptake of [(18)F]3 in cardiomyocytes. The results of biodistribution and micro-PET imaging studies of [(18)F]3 in mice and rats showed preferential accumulation in the myocardium. The results suggest that this compound would be a promising candidate for myocardial imaging.     

On the State 1-State 2 phenomenon in photosynthesis

The State 1-State 2 phenomenon of photosynthesis was studied in Chlorella by measuring the flash yield (Y) and the modulated rate (v) of oxygen evolution induced by weak modulated 650-nm light. From light intensity curves, intensities of 650 and 710 nm background and preilluminations were chosen to give maximum values of Y and v. Following long preilluminations in 710 nm (State 1) or in 650 nm (State 2), Y and v were measured in background light of chosen wavelength. The resulting plots of v vs Y show a discontinuity between State 1 and State 2. They confirm the predictions of Bonaventura and Myers [(1969) Biochim. Biophys. Acta 189, 366–383] and are consistent with changes in α (fraction of absorbed light captured by System II) as explanation of the State 1–State 2 phenomenon. When the intensity of 710 nm preillumination was too low, the characteristics of State 1 were not fully developed and the results then were similar to those of Delrieu [(1972) Biochim. Biophys. Acta 256, 293–299].     

Biological evaluation of ring- and side-chain-substituted m-iodobenzylguanidine analogues

A number of ring- and side-chain-substituted m-iodobenzylguanidine analogues were evaluated for their lipophilicity, in vitro stability, uptake by SK-N-SH human neuroblastoma cells in vitro, and biodistribution in normal mice. As expected, the lipophilicity of m-iodobenzylguanidine increased when a halogen was introduced onto the ring and decreased with the addition of polar hydroxyl, amino, and nitro substitutents. Most of the derivatives showed reasonable stability up to 24 h in PBS at 37 degrees C. While N(1)-hydroxy-N(3)-3-[(131)I]iodobenzylguanidine and 3,4-dihydroxy-5-[(131)I]iodobenzylguanidine generated a more nonpolar product in addition to the free iodide, 3-[(131)I]iodo-4-nitrobenzylguanidine decomposed to a product more polar than the parent compound. The specific uptake of 4-chloro-3-[(131)I]iodobenzylguanidine, 3-[(131)I]iodo-4-nitrobenzylguanidine, and N(1)-hydroxy-N(3)-3-[(131)I]iodobenzylguanidine by SK-N-SH human neuroblastoma cells in vitro, relative to that of m-[(125)I]iodobenzylguanidine, was 117 +/- 10%, 50 +/- 4%, and 12 +/- 2%, respectively. The specific uptake of the known m-iodobenzylguanidine analogues 4-hydroxy-3-[(131)I]iodobenzylguanidine and 4-amino-3-[(131)I]iodobenzylguanidine was 80 +/- 4% and 66 +/- 4%, respectively. None of the other m-iodobenzylguanidine derivatives showed any significant specific uptake by SK-N-SH cells. Heart uptake of 4-chloro-3-[(131)I]iodobenzylguanidine in normal mice was higher than that of m-[(125)I]iodobenzylguanidine at later time points (11 +/- 1% ID/g versus 3 +/- 1% ID/g at 24 h; p < 0.05) while uptake of 3-[(131)I]iodo-4-nitrobenzylguanidine and of N(1)-hydroxy-N(3)-3-[(131)I]iodobenzylguanidine in the heart was lower than that of m-iodobenzylguanidine at all time points. In accordance with the in vitro results, none of the other novel m-iodobenzylguanidine derivatives showed any significant myocardial or adrenal uptake in vivo.     

Oestradiol inhibits collagen breakdown in the involuting rat uterus

1. The earlier observation (Woessner, 1969) of oestradiol inhibition of collagen breakdown is confirmed and extended. Administration of 100mug of oestradiol-17beta/day to parturient rats strongly inhibits the loss of collagen from the involuting uterus. Three experiments show that this effect is due to an inhibition of collagen degradation rather than to a stimulation of collagen synthesis. 2. Uterine collagen was labelled with hydroxy[(14)C]-proline by the administration of [(14)C]proline near the end of pregnancy. By 3 days post partum, control uteri lost 83% of their collagen and 90% of their hydroxy[(14)C]proline. Uteri from oestradiol-treated rats lost only 50% of both total and labelled hydroxyproline, with no decrease in the specific radioactivity of the hydroxyproline. 3. Incorporation of [(14)C]proline into uterine collagen hydroxyproline in vivo was not affected by oestradiol treatment. 4. Urinary excretion of hydroxyproline was increased in post-partum control rats and decreased in oestradiol-treated rats. 5. An enzyme capable of cleaving 4-phenylazobenzyloxycarbonyl-l-prolyl-l-leucylglycyl- l-prolyl-d-arginine (a substrate for clostridial collagenase) increased in activity in the post-partum uterus and was unaffected by oestradiol treatment. 6. Uterine homogenates digested uterine collagen extensively at pH3.2. This digestion was unaffected by the oestradiol treatment. 7. Lysosomal fractions prepared by density-gradient centrifugation of uterine homogenates contained coincident peaks of cathepsin D activity and peptide-bound hydroxyproline. The cathepsin D and hydroxyproline contents of this peak were unaffected by oestradiol treatment.     

Radiosynthesis of [13N]dantrolene, a positron emission tomography probe for breast cancer resistant protein, using no-carrier-added [13N]ammonia

Dantrolene (1) is a substrate for breast cancer resistant protein, which is widely distributed in the blood-brain-barrier, intestine, gall bladder, and liver. PET study with 1 labeled with a positron emitter can be used to visualize BCRP and to elucidate the effect of BCRP on the pharmacokinetics of drugs. The objective of this study was to label 1 using nitrogen-13 ((13)N, a positron emitter; half-life: 9.9min). Using no-carrier-added [(13)N]NH(3) as the labeling agent, we synthesized [(13)N]dantrolene ([(13)N]1) for the first time. The reaction of carbomyl chloride 2b with [(13)N]NH(3) gave an unsymmetrical urea [(13)N]3, followed by cyclization of [(13)N]3 to afford [(13)N]1. Due to its instability, 2b was prepared in situ by treating amine 5 with triphosgene in a ratio of 4 to 1 and used for subsequent [(13)N]ammonolysis without purification.     

Blood compartmental metabolism of docosahexaenoic acid (DHA) in humans after ingestion of a single dose of [(13)C]DHA in phosphatidylcholine.

The amount and distribution of [(13)C]docosahexaenoic acid (DHA) in plasma, platelet, and erythrocyte lipid classes were followed as a function of time (1 to 72 h) in young adults after ingestion of a single dose of [(13)C]DHA esterified in a phosphatidylcholine (PC), in using gas chromatography combustion;-isotope ratio mass spectrometry. [(13)C]DHA first appeared in plasma non-esterified fatty acids (NEFA) and triglycerides (TG), with a maximal appearance at 6 h and a further decline, then being delayed 3-fold compared to [(13)C]DHA ingested in triglycerides. Lysophosphatidylcholine (LPC) was also enriched in [(13)C]DHA, due mainly to earlier hepatic secretion, and plateaued at 6 h, whereas phosphatidylethanolamine (PE) and phosphatidylcholine (PC) containing [(13)C]DHA plateaued at 9 h. The labeling of erythrocyte and platelet phospholipids exhibited different kinetics, probably involving different metabolic pathways for [(13)C]DHA incorporation in cell membranes. Computation of the relative contribution of LPC and NEFA for delivery of [(13)C]DHA to blood cells showed that the supply to platelets occurred through NEFA. In contrast, [(13)C]DHA was carried by both LPC and NEFA to erythrocytes, which differs from what was previously been observed after intake of triglycerides labeled with [(13)C]DHA where LPC was the only source of [(13)C]DHA for erythrocytes.We conclude that the lipid form of ingested DHA affects markedly its kinetics and partly its metabolic fate.     

Arginine catabolism in the cyanobacterium Synechocystis sp. Strain PCC 6803 involves the urea cycle and arginase pathway

Cells of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 supplemented with micromolar concentrations of L-[(14)C]arginine took up, concentrated, and catabolized this amino acid. Metabolism of L-[(14)C]arginine generated a set of labeled amino acids that included argininosuccinate, citrulline, glutamate, glutamine, ornithine, and proline. Production of [(14)C]ornithine preceded that of [(14)C]citrulline, and the patterns of labeled amino acids were similar in cells incubated with L-[(14)C]ornithine, suggesting that the reaction of arginase, rendering ornithine and urea, is the main initial step in arginine catabolism. Ornithine followed two metabolic pathways: (i) conversion into citrulline, catalyzed by ornithine carbamoyltransferase, and then, with incorporation of aspartate, conversion into argininosuccinate, in a sort of urea cycle, and (ii) a sort of arginase pathway rendering glutamate (and glutamine) via Delta(1)pyrroline-5-carboxylate and proline. Consistently with the proposed metabolic scheme (i) an argF (ornithine carbamoyltransferase) insertional mutant was impaired in the production of [(14)C]citrulline from [(14)C]arginine; (ii) a proC (Delta(1)pyrroline-5-carboxylate reductase) insertional mutant was impaired in the production of [(14)C]proline, [(14)C]glutamate, and [(14)C]glutamine from [(14)C]arginine or [(14)C]ornithine; and (iii) a putA (proline oxidase) insertional mutant did not produce [(14)C]glutamate from L-[(14)C]arginine, L-[(14)C]ornithine, or L-[(14)C]proline. Mutation of two open reading frames (sll0228 and sll1077) putatively encoding proteins homologous to arginase indicated, however, that none of these proteins was responsible for the arginase activity detected in this cyanobacterium, and mutation of argD (N-acetylornithine aminotransferase) suggested that this transaminase is not important in the production of Delta(1)pyrroline-5-carboxylate from ornithine. The metabolic pathways proposed to explain [(14)C]arginine catabolism also provide a rationale for understanding how nitrogen is made available to the cell after mobilization of cyanophycin [multi-L-arginyl-poly(L-aspartic acid)], a reserve material unique to cyanobacteria.