ESEEM and Mössbauer studies of the ferriheme model compound bis(3-aminopyrazole)tetraphenylporphyrinatoiron(III) chloride, [TPPFe(NH2PzH)2]Cl

A model heme complex, bis(3-aminopyrazole)tetraphenylporphinatoiron(III) chloride, [TPPFe (NH₂PzH)₂]Cl, for which the EPR g-values lead to a rhombicity V/Δ=1.2 if g _zz is the largest g-value, have been investigated by electron spin echo envelope modulation (ESEEM) and Mössbauer spectroscopies. The ESEEM studies focus on the proton sum frequency peaks at near twice the proton Larmor frequency. Analysis of the distant proton peak (mainly due to the pyrrole-H) at exactly twice the proton Larmor frequency shows conclusively that g _zz is aligned along the normal to the porphyrin plane, and thus the electron configuration is (d _xy )²(d _xz ,d _yz )³, with g _zz >g _yy >g _xx . This system is thus another violation to Taylor's "proper axis system" rule. The near proton (the α-H and N-H of the axial ligands) peaks provide distance information for those protons from the metal. Magnetic Mössbauer studies of the same complex confirm the (d _xy )²(d _xz ,d _yz )³ ground state and indicate that, as is the case for cytochrome P450_cam, A _xx is the largest magnitude A-value, and is negative in sign. Other low-spin iron(III) porphyrinates also have A _xx of negative sign, but usually the magnitude is only about half that of A _zz , which is always positive in sign.     

An antitumor promoter from Moringa oleifera Lam.

In the course of studies on the isolation of bioactive compounds from Philippine plants, the seeds of Moringa oleifera Lam. were examined and from the ethanol extract were isolated the new O-ethyl-4-(α- -rhamnosyloxy)benzyl carbamate (1) together with seven known compounds, 4(α- -rhamnosyloxy)-benzyl isothiocyanate (2), niazimicin (3), niazirin (4), β-sitosterol (5), glycerol-1-(9-octadecanoate) (6), 3-O-(6′-O-oleoyl-β- -glucopyranosyl)-β-sitosterol (7), and β-sitosterol-3-O-β- -glucopyranoside (8). Four of the isolates (2, 3, 7, and 8), which were obtained in relatively good yields, were tested for their potential antitumor promoting activity using an in vitro assay which tested their inhibitory effects on Epstein–Barr virus-early antigen (EBV-EA) activation in Raji cells induced by the tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA). All the tested compounds showed inhibitory activity against EBV-EA activation, with compounds 2, 3 and 8 having shown very significant activities. Based on the in vitro results, niazimicin (3) was further subjected to in vivo test and found to have potent antitumor promoting activity in the two-stage carcinogenesis in mouse skin using 7,12-dimethylbenz(a)anthracene (DMBA) as initiator and TPA as tumor promoter. From these results, niazimicin (3) is proposed to be a potent chemo-preventive agent in chemical carcinogenesis.     

Human ether-à-go-go-related gene K+ channel gating probed with extracellular ca2+. Evidence for two distinct voltage sensors

Human ether-à-go-go-related gene (HERG) encoded K+ channels were expressed in Chinese hamster ovary (CHO-K1) cells and studied by whole-cell voltage clamp in the presence of varied extracellular Ca2+ concentrations and physiological external K+. Elevation of external Ca2+ from 1.8 to 10 mM resulted in a reduction of whole-cell K+ current amplitude, slowed activation kinetics, and an increased rate of deactivation. The midpoint of the voltage dependence of activation was also shifted +22.3 +/- 2.5 mV to more depolarized potentials. In contrast, the kinetics and voltage dependence of channel inactivation were hardly affected by increased extracellular Ca2+. Neither Ca2+ screening of diffuse membrane surface charges nor open channel block could explain these changes. However, selective changes in the voltage-dependent activation, but not inactivation gating, account for the effects of Ca2+ on Human ether-à-go-go-related gene current amplitude and kinetics. The differential effects of extracellular Ca2+ on the activation and inactivation gating indicate that these processes have distinct voltage-sensing mechanisms. Thus, Ca2+ appears to directly interact with externally accessible channel residues to alter the membrane potential detected by the activation voltage sensor, yet Ca2+ binding to this site is ineffective in modifying the inactivation gating machinery.     

Enzymatic synthesis of lysophosphatidic acid and phosphatidic acid

Immobilised 1,3-specific lipase from Rhizopus arrhizus was used as catalyst for the esterification of -glycero-3-phosphate and fatty acid or fatty acid vinyl ester in a solvent-free system. With lauric acid vinyl ester as acyl donor, a_w<0.53 favored the synthesis of lysophosphatidic acid (1-acyl-rac-glycero-3-phosphate, LPA1) and the spontaneous acyl migration of the fatty acid on the molecule. Subsequent acylation by the enzyme resulted in high phosphatidic acid (1,2-diacyl-rac-glycero-3-phosphate, PA) formation and high total conversions (>95%). With oleic acid, maximum conversions of 55% were obtained at low water activities. Temperatures below melting point of the product favored precipitation and resulted in high final conversion and high product ratio [LPA/(PA+LPA)]. Thus, LPA was the only product with lauric acid vinyl ester as acyl donor at 25°C. Increased substrate ratio ( -glycero-3-phosphate/fatty acid) from 0.05 to 1 resulted in a higher ratio of LPA to PA formed, but a lower total conversion of -glycero-3-phosphate. Increased amounts of enzyme preparation did not result in higher esterification rates, probably due to high mass-transfer limitations.     

Methyl ketone formation during degradation of phenoxybutyric acid by Penicillium canescens SBUG-M 1139

Penicillium canescens SBUG-M 1139 was shown to be able to grow using phenoxybutyric acid as the sole carbon source. The rapid conversion of the phenoxyalkanoic acid resulted in the formation of phenol, which was metabolized completely. These reactions were accompanied by an accumulation of the methyl ketone phenoxypropan-2-one. Furthermore, during the metabolism of phenoxybutyric acid, 4-phenoxy-2,3-dehydrobutyric acid, 4-phenoxy-3-hydroxybutyric acid, phenoxyacetic acid, and phenoxypropan-2-ol accumulated in minor amounts. Clearly, fungi can metabolize phenoxyalkanoic acids to produce methyl ketones in a manner analogous to that used for the conversion of short- or medium-chain fatty acids by fungi. Received: 7 May 1999 / Accepted: 23 August 1999     

Effect of 2,6-Diisopropylphenol and Halogenated Anesthetics on Tetraphenylphosphonium Uptake by Rat Brain Synaptosomes: Determination of Membrane Potential

The effect of 2,6-diisopropylphenol (propofol) in comparison to that of the halogenated anesthetics enflurane, isoflurane, and halothane on tetrapenylphosphonium uptake by rat brain synaptosomes was studied. A direct method to separately measure the synaptosomal and the mitochondrial transmembrane potential by using the tetraphenylphosphonium cation (TPP⁺) was utilized. The latter is a lipophylic charged molecule which distributes between two compartments according to the transmembrane electrical potential in the presence or absence of 60 mM KCl as a synaptosomal membrane depolarizing agent. After previously reporting the damages induced by general anesthetics on isolated mitochondria, the aim of this paper was to study their possible action on the synaptosomal membrane potential and whether or not drugs concentrations damaging isolated mitochondria are also effective on synaptosomal mitochondria. The results indicated that, in the presence of glucose, mitochondria included in synaptosomes were able to maintain a transmembrane potential of 202 ± 8 mV (mean ± SD) while the synaptosomal membrane showed a potential of 78 ± 8 mV (mean ± SD). When anesthetic concentrations (0.6–1 mM propofol, 10–40 M enflurane, 30–50 M isoflurane, 8–15 M halothane) that impair mitochondrial energy metabolism were used, the synaptosomal transmembrane potential was maintained and, in addition, a slight increase of the TPP⁺ taken up was observed as the anesthetic concentration was increased.     

Relationship between plant cover type and soil properties on Syunkunitai coastal sand dune in eastern Hokkaido

A survey of soils and trees was conducted on Syunkunitai coastal sand dune in eastern Hokkaido to clarify the relationships between the soil properties and the plant cover type. A belt transect of 360m in length was established across the dune. Three community types, that is, a Picea glehnii forest, an Abies sachalinensis forest, and a salt marsh were recognized. Soil types at the study area were determined to be sandy immature soil and peat soil. Their horizon sequences were described as A₀–V–C or T–V–C layers (A₀, T, V, and C indicate layers of leaf litter, peat, volcanic deposit, and parent material, respectively). The Abies sachalinensis forest was characterized by a relatively high calcium concentration in the surface soil layer and a tendency for podzolization in the volcanic deposit layer. The Picea glehnii forest was characterized by peat accumulation because of the high ground water table, volcanic deposits in the soil profile, and the strong influence of sea salt on the soil chemistry. The roots in the Picea glehnii forest were distributed more shallowly than those in the Abies sachalinensis forest, thus avoiding the high water table level as well as the influence of seawater in the soil. The salt marsh showed an extremely high sodium concentration and base saturation, indicating that this area was directly affected by seawater. Recently, the periphery of the Picea glehnii forest on Syunkunitai sand dune has been declining because of seawater inundation caused by ground subsidence.     

蓝斑核调制电刺激包钦格复合体引起的吸气抑制效应

实验选用成年健康家兔,用乌拉坦麻醉,以膈神经放电为指标,观察了电刺激和化学刺激脑桥蓝斑核对延髓包钦格复合体吸气抑制效应的影响。结果观察到:(1)长串电刺激蓝斑核后,在一定时间之内电刺激包钦格复合体所导致的膈神经放电抑制效应明显减弱,与对照组(仅刺激包钦格复合体)相比,抑制程度减弱(28.78 ±19.49)%。(2)蓝斑核内微量注射谷氨酸钠后,电刺激包钦格复合体导致的膈神经放电抑制效应明显减弱,与对照组相比,抑制程度减弱(19.18 ±8.06)%,与长串电刺激蓝斑核的效应一致。这些结果提示,蓝斑核对包钦格复合体吸气抑制效应具有调制作用。     

Application of proteomics in the study of tumor metastasis

Tumor metastasis is the dominant cause of death in cancer patients. However, the molecular and cellular mechanisms underlying tumor metastasis are still elusive.The identification of protein molecules with their expressions correlated to the metastatic process would help to understand the metastatic mechanisms and thus facilitate the development of strategies for the therapeutic interventions and clinical management of cancer. Proteomics is a systematic research approach aiming to provide the global characterization of protein expression and function under given conditions. Proteomic technology has been widely used in biomarker discovery and pathogenetic studies including tumor metastasis. This article provides a brief review of the application of proteomics in identifying molecular factors in tumor metastasis process. The combination of proteomics with other experimental approaches in biochemistry, cell biology, molecular genetics and chemistry, together with the development of new technologies and improvements in existing methodologies will continue to extend its application in studying cancer metastasis.