Stereochemical Control of Valence and Its Application to the Reduction of Coordinated NO and N(2)

The electronic structures of transition metal complexes of NO are controlled by the stereochemistry about the metal atom (stereochemical control of valence). The six-coordinate complex, trans-[CoNO(NCS)-(C₆H₄ [As(CH₃)₂]₂)₂]⁺, consists of [Co(III)-(N=O^-)]²⁺ (angle_Co-N-O = 135°), while the pentacoordinate trigonal bipyramidal complex, [CoNO(C₆H₄[As(CH₃)₂]₂)₂] ²⁺, is best formulated as [Co(I)-(NO⁺]²⁺ (angle_Co-N-O = 179°). Evidence indicates that complexes of (NO)⁺ and N₂ are electronically similar. Hence, the principles of stereochemical control of valence may be applied to metal complexes of N₂. In a linearly coordinated Mⁿ˜m(NN) complex, valence electrons can be transferred from the metal to the N₂ ligand producing a bent, protonated, and/or metallated Mⁿ⁺²-(N=N^2-) complex. This reduction of N₂ can be effected by the addition of an appropriate ligand to M or by a change in the coordination geometry about M. Stereo-chemical control of valence leads to the rejection of one of the previously proposed mechanisms for reduction by nitrogenase.     

The conversion of phenylalanine to tyrosine in man. Direct measurement by continuous intravenous tracer infusions of L-[ring-2H5]phenylalanine and L-[1-13C]tyrosine in the postabsorptive state

Steady state phenylalanine and tyrosine turnover and the rate of conversion of phenylalanine to tyrosine in vivo were determined in 6 healthy postabsorptive adult volunteers. Continuous infusions of tracer amounts of L-[ring-²H₅]phenylalanine were administered intravenously for 13–14 hr. After 9–10 hr, a priming dose followed by a continuous infusion of L-[1-¹³C]tyrosine was added and maintained, along with the [²H₅]phenylalanine infusion, for 4 hr. Venous plasma samples were obtained before the initiation of each infusion and every 30 min during the course of the combined [²H₅]phenylalanine and [¹³C]tyrosine infusion for determination of isotopic enrichments of [²H₅]phenylalanine, [¹³C]tyrosine, and [²H₄[tyrosine by gas chromatograph-mass spectrometric analysis of the N-trifluoroacetyl-, methyl ester derivatives of the amino acids. Calculated from the observed enrichments, free phenylalanine and tyrosine turnover rates were 36.1 ± 5.1 μmole · kg^?1 · h^?1 and 39.8 ± 3.5 μmole · kg^?1 · h^?1, respectively. Phenylalanine was converted to tyrosine at the rate of 5.83 ± 0.59 μmole · kg^?1 · h^?1, accounting for approximately 16% of either the phenylalanine or the tyrosine flux. The results indicate that the normal basal steady state phenylalanine hydroxylase activity in vivo in man is lower than that obtained from phenylalanine loading studies. This supports the existence of some type of substrate activation of the enzyme as reflected in the previously reported exponential relationship between phenylalanine concentration and phenylalanine hydroxylase activity in vitro. The use of continuous simultaneous infusions of tracer amounts of stable isotope-labeled phenylalanine and tyrosine provides a direct means for studying physiological regulation of phenylalanine hydroxylase activity in vivo.     

Synthetic analogues of [Fe4S4(Cys)3(His)] in hydrogenases and [Fe4S4(Cys)4] in HiPIP derived from all-ferric [Fe4S4{N(SiMe3)2}4]

The all-ferric [Fe₄S₄]⁴⁺ cluster [Fe₄S₄{N(SiMe₃)₂}₄] 1 and its one-electron reduced form [1]^- serve as convenient precursors for the synthesis of 3∶1-site differentiated [Fe₄S₄] clusters and high-potential iron-sulfur protein (HiPIP) model clusters. The reaction of 1 with four equivalents (equiv) of the bulky thiol HSDmp (Dmp = 2,6-(mesityl)₂C₆H₃, mesityl = 2,4,6-Me₃C₆H₂) followed by treatment with tetrahydrofuran (THF) resulted in the isolation of [Fe₄S₄(SDmp)₃(THF)₃] 2. Cluster 2 contains an octahedral iron atom with three THF ligands, and its Fe(S)₃(O)₃ coordination environment is relevant to that in the active site of substrate-bound aconitase. An analogous reaction of [1]^- with four equiv of HSDmp gave [Fe₄S₄(SDmp)₄]^- 3, which models the oxidized form of HiPIP. The THF ligands in 2 can be replaced by tetramethyl-imidazole (Me₄Im) to give [Fe₄S₄(SDmp)₃(Me₄Im)] 4 modeling the [Fe₄S₄(Cys)₃(His)] cluster in hydrogenases, and its one-electron reduced form [4]^- was synthesized from the reaction of 3 with Me₄Im. The reversible redox couple between 3 and [3]^- was observed at E_1/2 = -820 mV vs. Ag/Ag⁺, and the corresponding reversible couple for 4 and [4]^- is positively shifted by +440 mV. The cyclic voltammogram of 3 also exhibited a reversible oxidation couple, which indicates generation of the all-ferric [Fe₄S₄]⁴⁺ cluster, [Fe₄S₄(SDmp)₄].     

Determination of retinal Schiff base configuration in bacteriorhodopsin

Resonance Raman spectra of the BR₅₆₈, BR₅₄₈, K₆₂₅, and L₅₅₀ intermediates of the bacteriorhodopsin photocycle have been obtained in ¹H₂O and ²H₂O by using native purple membrane as well as purple membrane regenerated with 14,15-¹³C₂ and 12,14-²H₂ isotopic derivatives of retinal. These derivatives were selected to determine the contribution of the C₁₄—C₁₅ stretch to the normal modes in the 1100- to 1400-cm^-1 fingerprint region and to characterize the coupling of the C₁₄—C₁₅ stretch with the NH rock. Normal mode calculations demonstrate that when the retinal Schiff base is in the C[unk]N cis configuration the C₁₄—C₁₅ stretch and the NH rock are strongly coupled, resulting in a large (≈50-cm^-1) upshift of the C₁₄—C₁₅ stretch upon deuteration of the Schiff base nitrogen. In the C[unk]N trans geometry these vibrations are weakly coupled and only a slight (<5-cm^-1) upshift of the C₁₄—C₁₅ stretch is predicted upon N-deuteration. In BR₅₆₈, the insensitivity of the 1201-cm^-1 C₁₄—C₁₅ stretch to N-deuteration demonstrates that its retinal C[unk]N configuration is trans. The C₁₄—C₁₅ stretch in BR₅₄₈, however, shifts up from 1167 cm^-1 in ¹H₂O to 1208 cm^-1 in ²H₂O, indicating that BR₅₄₈ contains a C[unk]N cis chromophore. Thus, the conversion of BR₅₆₈ to BR₅₄₈ (dark adaptation) involves isomerization about the C[unk]N bond in addition to isomerization about the C₁₃[unk]C₁₄ bond. The insensitivity of the native, [14,15-¹³C₂]-, and [12,14-²H₂]K₆₂₅ and L₅₅₀ spectra to N-deuteration argues that these intermediates have a C[unk]N trans configuration. Thus, the primary photochemical step in bacteriorhodopsin (BR₅₆₈ → K₆₂₅) involves isomerization about the C₁₃[unk]C₁₄ bond alone. The significance of these results for the mechanism of proton-pumping by bacteriorhodopsin is discussed.     

Antitumor properties of organometallic metallocene complexes of tin and germanium

Summary The antitumor activity of the four metallocene compounds decaphenylstannocene [⁵-(C₆H₅)₅C₅]₂Sn(II), decabenzylstannocene [⁵-(C₆H₅CH₂)₅C₅]₂Sn(II), decaphenylgermanocene [⁵-(C₆H₅)₅C₅]₂Ge(II), and decabenzylgermanocene [⁵-(C₆H₅CH₂)₅C₅]₂Ge(II), containing the main group IV elements tin or germanium as the central metal atom and two pentasubstituted cyclopentadienyl ring ligands in sandwich arrangement, were tested against Ehrlich ascites tumor in female CF1 mice. The complexes caused cure rates of 40% to 90% of the animals treated over rather broad dose ranges. With both germanocene complexes, no strong dose-activity relationship was manifest. The toxicity of all four metallocenes was low, the LD₁₀ values of both stannocenes being 460 and 500 mg/kg, and those of both germanocenes higher than 700 mg/kg. Regarding the isolated pentasubstituted cyclopentadiene ligands (C₆H₅)₅C₅H and (C₆H₅CH₂)₅C₅H, these also exhibited antitumor activity which was less pronounced than that of the metal-containing sandwich complexes. Decasubstituted stannocene and germanocene compounds represent a new type of non-platinum group metal antitumor agents structurally differing from known inorganic and organometallic cytostatics.     

n-Type Si-based photoelectrochemical cell: New liquid junction photocell using a nonaqueous ferricenium/ferrocene electrolyte

n-Type Si has been shown to serve as a stable photoanode in a cell for the conversion of light to electricity. The other components of the cell are a Pt cathode and an electrolyte consisting of an ethanol solution of [n-Bu₄N]ClO₄ with a redox couple of ferricenium/ferrocene. Data from a two-compartment cell show that ferrocene is oxidized to ferricenium with 100 ± 2% current efficiency at the Si photoanode. Furthermore, prolonged irradiation of the Si in a one-compartment cell yields constant photocurrent and output characteristics. The maximum open-circuit photopotential is ~700 mV, and the short-circuit quantum yield for electron flow at low light intensity exceeds 0.5. Conversion of monochromatic 632.8-nm light to electricity with ~2% power efficiency at an output voltage of ~200 mV has been sustained. These results represent a stable n-type Si-based photoelectrochemical cell.     

Contribution of galactose and fructose to glucose homeostasis

To determine the contributions of galactose and fructose to glucose formation, 6 subjects (26 ± 2 years old; body mass index, 22.4 ± 0.2 kg/m²) (mean ± SE) were studied during fasting conditions. Three subjects received a primed constant intravenous infusion of [6,6-²H₂]glucose for 3 hours followed by oral bolus ingestion of galactose labeled to 2% with [U-¹³C]galactose (0.72 g/kg); the other 3 subjects received a primed constant intravenous infusion of [6,6-²H₂]glucose followed by either a bolus ingestion of fructose alone (0.72 g/kg) (labeled to 2% with [U-¹³C]fructose) or coingestion of fructose (labeled with [U-¹³C]fructose) (0.72 g/kg) and unlabeled glucose (0.72 g/kg). Four hours after ingestion, subjects received 1 mg of glucagon intravenously to stimulate glycogenolysis. When galactose was ingested alone, the area under the curve (AUC) of [¹³C₆]glucose and [¹³C₃]glucose was 7.28 ± 0.39 and 3.52 ± 0.05 mmol/L per 4 hours, respectively. When [U-¹³C]fructose was ingested with unlabeled fructose or unlabeled fructose plus glucose, no [¹³C₆]glucose was detected in plasma. The AUC of [¹³C₃]glucose after fructose and fructose plus glucose ingestion was 20.21 ± 2.41 and 6.25 ± 0.34 mmol/L per 4 hours, respectively. Comparing the AUC for the ¹³C₃ vs ¹³C₆ enrichments, 67% of oral galactose enters the systemic circulation via a direct route and 33% via an indirect route. In contrast, fructose only enters the systemic circulation via the indirect route. Finally, when ingested alone, fructose and galactose contribute little to glycogen synthesis. After the coingestion of fructose and glucose with the resultant insulin response from the glucose, fructose is a significant contributor to glycogen synthesis.     

The occurrence of C<Subscript>6</Subscript>–C<Subscript>10</Subscript>-dicarboxylic acids,ethylmalonic acid, 5-hydroxycaproic acid,butyrylglycine, isovalerylglycine,isobutyrylglycine, 2-methylbutyrylglycine and glutaric acid in the urine of riboflavin deficient rats

Fifteen children, five with phenylketonuria (PKU), five with hyperphenylalaninaemia, and five phenotypically normal but at risk of being carriers for PKU, were given [ring²H₅]phenylalanine orally in amounts ranging from 75 mg/kg to 10 mg/kg. Plama was assayed for [²H₅]phenylalanine and [²H₄]tyrosine at hourly intervals, the amino acids being measured as theN-acetyl, n-propyl esters by gas chromatography-mass spectroscopy. The results obtained were calculated as the log of the ratio [²H₅]phenylalanine: [²H₄]tyrosine in the plasma. The five patients with PKU had ratios of infinity because no [²H₄]tyrosine was measured in their plasma during the experimental period. The patients with hyperphenylalaninaemia had log ratios over 2.00 throughout the assay period. Among the five normal children three are considered to be carriers for PKU as the logarithms of the [²H₅]phenylalanine: [²H₄]tyrosine ratios were 1.77, 1.73, and 1.33 and remained over 1.00 during the assay period. The other children had log ratios of 1.16 and 1.00 at the first hour which dropped below 1.00 subsequently, suggesting normal activity of phenylalanine hydroxylase.     

Changes of extracellular Na+, K+, Ca2+ and H+ of the ischemic myocardium in pigs

Summary In open-chest pigs during severe myocardial ischemia [K⁺]e, [Ca²⁺]e and [H⁺]e increase, [Na⁺]e increases transiently reaching control values after 30 min. Extracellular osmolality of the ischemic area increases due to an H₂O-shift from the ECS to the ICS. The increase of [Na⁺]e and [Ca²⁺]e must be explained by the shrinkage of the ECS due to the H₂O-shift. The increase of [Ca²⁺]e is additionally caused by the decrease of pHe. The increase of [K⁺]e is mainly caused by the release of K⁺ from the ICS. The changes of [K⁺]e and [K⁺]i cause a decrease of the membrane potential to a range in which slow response potentials and re-entry excitations can occur. The increase of [K⁺e therefore seems to be a major factor to cause early post-ischemic arrhythmias.Supported by Deutsche Forschungsgemeinschaft, SFB 68, A 7     

Monitoring tumor cell death in murine tumor models using deuterium magnetic resonance spectroscopy and spectroscopic imaging

²H magnetic resonance spectroscopic imaging has been shown recently to be a viable technique for metabolic imaging in the clinic. We show here that ²H MR spectroscopy and spectroscopic imaging measurements of [2,3-²H₂]malate production from [2,3-²H₂]fumarate can be used to detect tumor cell death in vivo via the production of labeled malate. Production of [2,3-²H₂]malate, following injection of [2,3-²H₂]fumarate (1 g/kg) into tumor-bearing mice, was measured in a murine lymphoma (EL4) treated with etoposide, and in human breast (MDA-MB-231) and colorectal (Colo205) xenografts treated with a TRAILR2 agonist, using surface-coil localized ²H MR spectroscopy at 7 T. Malate production was also imaged in EL4 tumors using a fast ²H chemical shift imaging sequence. The malate/fumarate ratio increased from 0.016 ± 0.02 to 0.16 ± 0.14 in EL4 tumors 48 h after drug treatment (P = 0.0024, n = 3), and from 0.019 ± 0.03 to 0.25 ± 0.23 in MDA-MB-231 tumors (P = 0.0001, n = 5) and from 0.016 ± 0.04 to 0.28 ± 0.26 in Colo205 tumors (P = 0.0002, n = 5) 24 h after drug treatment. These increases were correlated with increased levels of cell death measured in excised tumor sections obtained immediately after imaging. ²H MR measurements of [2,3-²H₂]malate production from [2,3-²H₂]fumarate provide a potentially less expensive and more sensitive method for detecting cell death in vivo than ¹³C MR measurements of hyperpolarized [1,4-¹³C₂]fumarate metabolism, which have been used previously for this purpose.

Currently, the response of solid tumors to treatment is assessed mainly on the basis of changes in tumor size [Response Evaluation Criteria in Solid Tumors (RECIST) (1)]. However, changes in size may take weeks to appear after the initiation of treatment and, in some cases, may not appear at all, for example, in the case of treatments that inhibit tumor growth but do not result in tumor regression (2, 3). Changes in metabolism can give an earlier indication of treatment response, for example, assessment of glycolytic activity using positron emission tomography (PET) measurements of 2-[¹⁸F]-fluoro-2-deoxy D-glucose uptake (FDG-PET). PET Response Criteria in Solid Tumors (PERCIST) was introduced as a potentially more sensitive method of assessing treatment response when compared to assessment based on changes in tumor size alone (4), particularly with therapies that stabilize disease. Imaging with hyperpolarized [1-¹³C]pyruvate, like FDG-PET, can also be used to detect drug target engagement as well as subsequent tumor cell death (5, 6). We have shown recently that imaging hyperpolarized [1-¹³C]pyruvate metabolism can be more sensitive than FDG-PET in detecting reductions in glycolytic flux associated with tumor cell death posttreatment (7).While metabolic imaging may indicate drug target engagement, and in some cases tumor cell death, there is a need for imaging methods that detect tumor cell death more directly posttreatment and that can give an indication of longer-term treatment outcomes (8). Fumarate is hydrated in the reaction catalyzed by the intracellular enzyme fumarase to produce malate. Previous ¹³C magnetic resonance spectroscopic imaging (MRSI) studies with hyperpolarized [1,4-¹³C₂]fumarate in tumor models and in models of myocardial infarction and acute kidney necrosis (9–16) have demonstrated that the production of labeled malate can be used to image cell death in vivo. The increased production of malate was attributed to loss of the plasma membrane permeability barrier in necrotic cells and increased access of hyperpolarized [1,4-¹³C₂]fumarate to fumarase. However, imaging with hyperpolarized ¹³C-labeled substrates is limited both by the transient nature of the hyperpolarization, which restricts its application to relatively fast metabolic processes, and the requirement for relatively large amounts of ¹³C-labeled compounds and access to clinical hyperpolarizers, which are expensive. The recent demonstration by De Feyter et al. that ²H MRSI can be used to image the metabolism of ²H-labeled substrates in vivo, including in human subjects (17), has provided a potentially lower-cost alternative for clinical metabolic imaging. The relatively low sensitivity of ²H detection is compensated by its very short T₁, which means that signal can be acquired rapidly without saturation. The main limitation is the narrow frequency range, which requires the use of relatively high magnetic field strengths. Nevertheless, by collecting a series of rapidly acquired images, the technique is capable of generating quantitative images of metabolic flux (18). We show here that ²H magnetic resonance spectroscopy (MRS) and MRSI measurements of [2,3-²H₂]fumarate conversion to ²H-labeled malate can be used to detect tumor cell death in vivo and that this is potentially a more sensitive method for detecting cell death than ¹³C MRSI with either hyperpolarized [1-¹³C]pyruvate or [1,4-¹³C₂]fumarate.     

The dominant non-gibberellin-responding dwarf mutant (D8) of maize accumulates native gibberellins

The endogenous gibberellins (GAs) were examined from young vegetative shoots of the dominant mutant, Dwarf-8, a GA-nonresponder, and normal maize; GA₄₄, GA₁₇, GA₁₉, GA₂₀, GA₂₉, GA₁, and GA₈, members of the early-13-hydroxylation pathway, were identified from both kinds of shoots by full-scan mass spectra and Kovats retention indices. In addition, we report the identification of 3-epi-GA₁, GA₃, GA₄, GA₅, GA₇, GA₉, GA₁₂, GA₁₅, GA₂₄, GA₃₄, and GA₅₃ by using the same criteria. [1,7,12,18-¹⁴C₄]GA₅₃ and -GA₄₄, [17-²H₂]GA₁₉, and [17-¹³C,³H₂]GA₂₀, -GA₂₉, -GA₁, -GA₈, and -GA₅ were used as internal standards to determine the endogenous levels of these GAs by measurement of isotope dilution, using capillary gas chromatography and selected ion monitoring. Shoots of Dwarf-8 accumulate relatively high levels of GA₂₀, GA₁, and GA₈. The accumulation of GA₁ appears to be related to gene dosage. Since Dwarf-8 contains the same pattern of GAs as normals (including GA₁ and GA₃), the genetic control point probably lies after GA₁ (and GA₃). Thus Dwarf-8 may be a GA receptor mutant or a mutant that controls a product downstream from the binding of the bioactive GA to a receptor.     

Synthetic Analogs of the Active Sites of Iron-Sulfur Proteins. Structure and Properties of Bis[o-xylyldithiolato-μ2-sulfidoferrate(III)], an Analog of the 2Fe-2S Proteins

The synthetic analog approach has been applied to a clarification of the active sites of 2Fe-2S^* proteins. The compound (Et₄N)₂[FeS(SCH₂)₂C₆H₄]₂, derived from o-xylyl-α,α′-dithiol, has been prepared and its structure has been determined by x-ray diffraction. The centrosymmetric anion contains two tetrahedrally coordinated ferric ions bridged by two sulfide ions and separated by 2.70 Å. Comparison of electronic, Mössbauer, and proton magnetic resonance spectra and magnetic susceptibility of the anion with the corresponding properties of the oxidized forms of the proteins reveals significant degrees of similarity. The anion also exhibits the essential redox capacity of the proteins. We conclude that [FeS(SCH₂)₂C₆H₄]₂^2- possesses the same total oxidation level and electronic configuration as the active sites of the oxidized proteins, and that its structure provides a feasible representation of the minimal structure of the active site. [FeS(SCH₂)₂C₆H₄]₂^2- is thus the first well-defined synthetic analog of the active sites of two-iron ferredoxins.     

Platinum(II) Complexes with Bulky Disubstitute Triazolopyrimidines as Promising Materials for Anticancer Agents

Herein, we present dicarboxylate platinum(II) complexes of the general formula [Pt(mal)(DMSO)(L)] and [Pt(CBDC)(DMSO)(L)], where L is dbtp 5,7-ditertbutyl-1,2,4-triazolo[1,5-a]pyrimidine) or ibmtp (7-isobutyl-5-methyl-1,2,4- triazolo[1,5-a]pyrimidine), as prospective prodrugs. The platinum(II) complexes were synthesized in a one-pot reaction between cis-[PtCl₂(DMSO)₂], silver malonate or silver cyclobutane-1,1-dicarboxylate and triazolopyrimidines. All platinum(II) compounds were characterized by FT-IR, and ¹H, ¹³C, ¹⁵N and ¹⁹⁵Pt NMR; and their square planar geometries with one monodentate N(3)-bonded 5,7-disubstituted-1,2,4-triazolo[1,5-a]pyrimidine, one S-bonded molecule of dimethyl sulfoxide and one O,O-chelating malonato (1, 2) or O,O-chelating cyclobutane-1,1-dicarboxylato (3, 4) was determined. Additionally, [Pt(CBDC)(dbtp)(DMSO)] (3) exhibited (i) substantial in vitro cytotoxicity against the lung adenocarcinoma epithelial cell line (A549) (IC₅₀ = 5.00 µM) and the cisplatin-resistant human ductal breast epithelial tumor cell line (T47D) (IC₅₀ = 6.60 µM); and (ii) definitely exhibited low toxicity against normal murine embryonic fibroblast cells (BALB/3T3).     

H(2)O(2)-induced left ventricular dysfunction in isolated working rat hearts is independent of calcium accumulation

Reactive oxygen species (ROS) and intracellular Ca²⁺ overload play key roles in myocardial ischemia-reperfusion (IR) injury but the relationships among ROS, Ca²⁺ overload and LV mechanical dysfunction remain unclear. We tested the hypothesis that H₂O₂ impairs LV function by causing Ca²⁺ overload by increasing late sodium current (I_Na), similar to Sea Anemone Toxin II (ATX-II). Diastolic and systolic Ca²⁺ concentrations (d[Ca²⁺]_i and s[Ca²⁺]_i) were measured by indo-1 fluorescence simultaneously with LV work in isolated working rat hearts. H₂O₂ (100 μM, 30 min) increased d[Ca²⁺]_i and s[Ca²⁺]_i. LV work increased transiently then declined to 32% of baseline before recovering to 70%. ATX-II (12 nM, 30 min) caused greater increases in d[Ca²⁺]_i and s[Ca²⁺]_i. LV work increased transiently before declining gradually to 17%. Ouabain (80 μM) exerted similar effects to ATX-II. Late I_Na inhibitors, lidocaine (10 μM) or R56865 (2 μM), reduced effects of ATX-II on [Ca²⁺]_i and LV function, but did not alter effects of H₂O₂. The antioxidant, N-(2-mercaptopropionyl)glycine (MPG, 1 mM) prevented H₂O₂-induced LV dysfunction, but did not alter [Ca²⁺]_i. Paradoxically, further increases in [Ca²⁺]_i by ATX-II or ouabain, given 10 min after H₂O₂, improved function. The failure of late I_Na inhibitors to prevent H₂O₂-induced LV dysfunction, and the ability of MPG to prevent H₂O₂-induced LV dysfunction independent of changes in [Ca²⁺]_i indicate that impaired contractility is not due to Ca²⁺ overload. The ability of further increases in [Ca²⁺]_i to reverse H₂O₂-induced LV dysfunction suggests that Ca²⁺ desensitization is the predominant mechanism of ROS-induced contractile dysfunction.     

Novel [FeFe]-Hydrogenase Mimics: Unexpected Course of the Reaction of Ferrocenyl α-Thienyl Thioketone with Fe3(CO)12

The influence of the substitution pattern in ferrocenyl α-thienyl thioketone used as a proligand in complexation reactions with Fe₃(CO)₁₂ was investigated. As a result, two new sulfur–iron complexes, considered [FeFe]-hydrogenase mimics, were obtained and characterized by spectroscopic techniques (¹H, ¹³C{¹H} NMR, IR, MS), as well as by elemental analysis and X-ray single crystal diffraction methods. The electrochemical properties of both complexes were studied and compared using cyclic voltammetry in the absence and in presence of acetic acid as a proton source. The performed measurements demonstrated that both complexes can catalyze the reduction of protons to molecular hydrogen H₂. Moreover, the obtained results showed that the presence of the ferrocene moiety at the backbone of the linker of both complexes improved the stability of the reduced species.     

H2O2-induced left ventricular dysfunction in isolated working rat hearts is independent of calcium accumulation

Reactive oxygen species (ROS) and intracellular Ca²⁺ overload play key roles in myocardial ischemia–reperfusion (IR) injury but the relationships among ROS, Ca²⁺ overload and LV mechanical dysfunction remain unclear. We tested the hypothesis that H₂O₂ impairs LV function by causing Ca²⁺ overload by increasing late sodium current (I_Na), similar to Sea Anemone Toxin II (ATX-II). Diastolic and systolic Ca²⁺ concentrations (d[Ca²⁺]_i and s[Ca²⁺]_i) were measured by indo-1 fluorescence simultaneously with LV work in isolated working rat hearts. H₂O₂ (100 μM, 30 min) increased d[Ca²⁺]_i and s[Ca²⁺]_i. LV work increased transiently then declined to 32% of baseline before recovering to 70%. ATX-II (12 nM, 30 min) caused greater increases in d[Ca²⁺]_i and s[Ca²⁺]_i. LV work increased transiently before declining gradually to 17%. Ouabain (80 μM) exerted similar effects to ATX-II. Late I_Na inhibitors, lidocaine (10 μM) or R56865 (2 μM), reduced effects of ATX-II on [Ca²⁺]_i and LV function, but did not alter effects of H₂O₂. The antioxidant, N-(2-mercaptopropionyl)glycine (MPG, 1 mM) prevented H₂O₂-induced LV dysfunction, but did not alter [Ca²⁺]_i. Paradoxically, further increases in [Ca²⁺]_i by ATX-II or ouabain, given 10 min after H₂O₂, improved function. The failure of late I_Na inhibitors to prevent H₂O₂-induced LV dysfunction, and the ability of MPG to prevent H₂O₂-induced LV dysfunction independent of changes in [Ca²⁺]_i indicate that impaired contractility is not due to Ca²⁺ overload. The ability of further increases in [Ca²⁺]_i to reverse H₂O₂-induced LV dysfunction suggests that Ca²⁺ desensitization is the predominant mechanism of ROS-induced contractile dysfunction.     

Anticancer-agent-linked phosphonates with antiosteolytic and antineoplastic properties: a promising perspective in the treatment of bone-related malignancies?

Summary Bisphosphonates are compounds with a high affinity for bone and other calcified tissues. They inhibit tumor-induced bone destruction and the associated hypercalcemia by hindering the activity of the osteoclasts. Owing to a long biological half-life of bisphosphonates in the bone, a treatment using a prophylactic regimen seems possible. This paper summarizes preclinical studies with the bisphosphonate 3-amino-1-hydroxypropylidene-1, 1-diphosphonic acid and two methyl derivatives; S-N,N-dimethylamino-1-hydroxypropylidene-1,1-diphosphonic acid and 4-N,N-dimetyhlamino-1-hydroxybutylidene-1, 1-diphosphonic acid with respect to their bone-protecting activity in therapy as well as in prophylaxis. To find substances that are useful for the treatment of primary tumor, as well as bone metastasis, we synthesized and testedcis-diammine[nitrilotris(methylphosphonato) (2-)-O ¹ ,N¹]platinum(II) andcis-diammine{[bis-(phosphonatomethyl)amino]acetato(2-)-O¹, N¹} platinum(II), which contain both an osteotropic and an antineoplastic moiety. Experiments were carried out: (a) in the intratibial transplanted Walker carcinosarcoma 256B of the rat, which mimics osteolytic bone metastasis, and (b) in the transplantable osteosarcoma of the rat, which shows a histology and metastatic pattern similar to that found in man. These investigations indicate that it is possible to effect adjuvant therapy of bone metastases by combination of two compounds with different properties into one structure without losing the therapeutic characteristics of the parent compounds. They thus provide evidence that it may be possible to design compounds well suited for the therapeutic or prophylactic treatment of bone-related malignancies.Abbreviations NMR nuclear magnetic resonance - WCS Walker carcinosarcoma 256B - APD 3-amino-1-hydroxypropylidene-1,1-diphosphonic acid - 3Me₂APD theN-dimethyl derivative of APD - 4Me₂ABD A-N, N-dimethylamino-1-hydroxybutylidene-1,1-diphosphonic acid - AMDP cis-diammine[nitrilotris(methylphosphonato)(2-)-O ¹,N ¹]platinum(II) - DBP cis-diammine{[bis(phosphonatomethyl)amino]acetato(2-)-O ¹,N ¹}platmum(II)     

Coupled Ca2+/H+ transport by cytoplasmic buffers regulates local Ca2+ and H+ ion signaling

Ca²⁺ signaling regulates cell function. This is subject to modulation by H⁺ ions that are universal end-products of metabolism. Due to slow diffusion and common buffers, changes in cytoplasmic [Ca²⁺] ([Ca²⁺]_i) or [H⁺] ([H⁺]_i) can become compartmentalized, leading potentially to complex spatial Ca²⁺/H⁺ coupling. This was studied by fluorescence imaging of cardiac myocytes. An increase in [H⁺]_i, produced by superfusion of acetate (salt of membrane-permeant weak acid), evoked a [Ca²⁺]_i rise, independent of sarcolemmal Ca²⁺ influx or release from mitochondria, sarcoplasmic reticulum, or acidic stores. Photolytic H⁺ uncaging from 2-nitrobenzaldehyde also raised [Ca²⁺]_i, and the yield was reduced following inhibition of glycolysis or mitochondrial respiration. H⁺ uncaging into buffer mixtures in vitro demonstrated that Ca²⁺ unloading from proteins, histidyl dipeptides (HDPs; e.g., carnosine), and ATP can underlie the H⁺-evoked [Ca²⁺]_i rise. Raising [H⁺]_i tonically at one end of a myocyte evoked a local [Ca²⁺]_i rise in the acidic microdomain, which did not dissipate. The result is consistent with uphill Ca²⁺ transport into the acidic zone via Ca²⁺/H⁺ exchange on diffusible HDPs and ATP molecules, energized by the [H⁺]_i gradient. Ca²⁺ recruitment to a localized acid microdomain was greatly reduced during intracellular Mg²⁺ overload or by ATP depletion, maneuvers that reduce the Ca²⁺-carrying capacity of HDPs. Cytoplasmic HDPs and ATP underlie spatial Ca²⁺/H⁺ coupling in the cardiac myocyte by providing ion exchange and transport on common buffer sites. Given the abundance of cellular HDPs and ATP, spatial Ca²⁺/H⁺ coupling is likely to be of general importance in cell signaling.Most cells are exquisitely responsive to calcium (Ca²⁺) (1) and hydrogen (H⁺) ions (i.e., pH) (2). In cardiac myocytes, Ca²⁺ ions trigger contraction and control growth and development (3), whereas H⁺ ions, which are generated or consumed metabolically, are potent modulators of essentially all biological processes (4). By acting on Ca²⁺-handling proteins directly or via other molecules, H⁺ ions exert both inhibitory and excitatory effects on Ca²⁺ signaling. For example, in the ventricular myocyte, H⁺ ions can reduce Ca²⁺ release from sarcoplasmic reticulum (SR) stores, through inhibition of the SR Ca²⁺ ATPase (SERCA) pump and ryanodine receptor (RyR) Ca²⁺ channels (5, 6). In contrast, H⁺ ions can enhance SR Ca²⁺ release by stimulating sarcolemmal Na⁺/H⁺ exchange (NHE), which raises intracellular [Na⁺] and reduces the driving force for Ca²⁺ extrusion on Na⁺/Ca²⁺ exchange (NCX), leading to cellular retention of Ca²⁺ (7, 8). Ca²⁺ signaling is thus subservient to pH.Cytoplasmic Ca²⁺ and H⁺ ions bind avidly to buffer molecules, such that <1% of all Ca²⁺ ions and <0.001% of all H⁺ ions are free. Some of these buffers bind H⁺ and Ca²⁺ ions competitively, and this has been proposed to be one mechanism underlying cytoplasmic Ca²⁺/H⁺ coupling (9). Reversible binding to buffers greatly reduces the effective mobility of Ca²⁺ and H⁺ ions in cytoplasm (10, 11) and can allow for highly compartmentalized ionic microdomains, and hence a spatially heterogeneous regulation of cell function. In cardiac myocytes under resting (diastolic) conditions, the cytoplasm-averaged concentration of free [Ca²⁺] ([Ca²⁺]_i) and [H⁺] ([H⁺]_i) ions is kept near 10⁻⁷ M by membrane transporter proteins. Thus, [H⁺]_i is regulated by the balance of flux among acid-extruding and acid-loading transporter proteins at the sarcolemma [e.g., NHE and Cl⁻/OH⁻ (CHE) exchangers, respectively] (4). Similarly, the activity of SERCA and NCX proteins returns [Ca²⁺]_i to its diastolic level after evoked signaling events (3, 12). Despite these regulatory mechanisms, cytoplasmic gradients of [H⁺]_i and [Ca²⁺]_i do occur in myocytes and are an important part of their physiology. Gradients arise from local differences in transmembrane fluxes that alter [H⁺]_i or [Ca²⁺]_i. For example, spatial [H⁺]_i gradients are produced when NHE transporters, expressed mainly at the intercalated disk region, are activated (4, 13) or when membrane-permeant weak acids, such as CO₂, are presented locally (14). Similarly, release of Ca²⁺ through a cluster of RyR channels in the SR produces [Ca²⁺]_i nonuniformity in the form of Ca²⁺ sparks (15). Given the propensity of cytoplasm to develop ionic gradients, it is important to understand their underlying mechanism and functional role.The present work demonstrates a distinct form of spatial interaction between Ca²⁺ and H⁺ ions. We show that cytoplasmic [H⁺] gradients can produce stable [Ca²⁺]_i gradients, and vice versa, and that this interaction is mediated by low-molecular-weight (mobile) buffers with affinity for both ions. We demonstrate that the diffusive counterflux of H⁺ and Ca²⁺ bound to these buffers comprises a cytoplasmic Ca²⁺/H⁺ exchanger. This acts like a “pump” without a membrane, which can, for instance, recruit Ca²⁺ to acidic cellular microdomains. Cytoplasmic Ca²⁺/H⁺ exchange adds a spatial paradigm to our understanding of Ca²⁺ and H⁺ ion signaling.     

Differential diagnosis of hydroxydicarboxylic aciduria based on release of3H2O from [9,10-3H]myristic and [9,10-3H]palmitic acids by intact cultured fibroblasts

Summary Intact cultured fibroblasts from patients with deficiency of long-chain 3-hydroxyacyl-CoA dehydrogenase release³H₂O from [9,10-³H]myristic acid and [9,10-³H]palmitic acid more slowly than normal. The ratio of activity (palmitate/myristate) is also low and the expression (rate with palmitate)²/(rate with myristate) gives good differentiation between affected and unaffected cells. In some patients who have shown hydroxydicarboxylic aciduria when unwell there is reduced³H₂O production from [9,10-³H]myristic and [9,10-³H]palmitic acids by intact cultured fibroblasts but normal 3-hydroxyacyl-CoA dehydrogenase activities in disrupted cells. The palmitate/myristate ratio is higher than in long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency. The basic defect in these patients is still unknown but it is suggested that caution be used over the administration of medium-chain triglyceride.     

Molecular mechanism of water oxidation in photosynthesis based on the functioning of manganese in two different environments

We present a model of photosynthetic water oxidation that utilizes the property of higher-valent Mn ions in two different environments and the characteristic function of redox-active ligands to explain all known aspects of electron transfer from H₂O to Z, the electron donor to P680, the photosystem II reaction center chlorophyll a. There are two major features of this model. (i) The four functional Mn atoms are divided into two groups of two Mn each: [Mn] complexes in a hydrophobic cavity in the intrinsic 34-kDa protein; and (Mn) complexes on the hydrophilic surface of the extrinsic 33-kDa protein. The oxidation of H₂O is carried out by two [Mn] complexes, and the protons are transferred from a [Mn] complex to a (Mn) complex along the hydrogen bond between their respective ligand H₂O molecules. (ii) Each of the two [Mn] ions binds one redox-active ligand (RAL), such as a quinone (alternatively, an aromatic amino acid residue). Electron transfer occurs from the reduced RAL to the oxidized Z. When the experimental data concerning atomic structure of the water-oxidizing center (WOC), electron transfer between the WOC and Z, the electronic structure of the WOC, the proton-release pattern, and the effect of Cl^- are compared with the predictions of the model, satisfactory qualitative and, in many instances, quantitative agreements are obtained. In particular, this model clarifies the origin of the observed absorption-difference spectra, which have the same pattern in all S-state transitions, and of the effect of Cl^--depletion on the S states.