Hemoglobin switching in sheep: commitment of erythroid stem cells to expression of the betaC-globin gene and accumulation of betaC-globin mRNA

The switch from HbA (α₂β₂^A) to HbC (α₂β₂^C) synthesis was induced by injection of erythropoietin into a lamb homozygous for HbA. Serial samples of bone marrow were analyzed to detect the initial commitment of erythroid stem cells (CFU-E) to form colonies which made HbC in vitro, and to detect the initial accumulation of β^C-globin mRNA and the onset of HbC synthesis in erythroblasts in vivo. CFU-E-derived erythroid colonies were formed in plasma clot culture at a low erythropoietin concentration, and the relative amounts of β^A- and β^C-globin synthesized were determined after a 24 hr pulse of ³H-leucine, added after 84 hr in culture. RNA was extracted from nuclei and cytoplasm of “early” and “late” populations of bone marrow erythroblasts which had been fractionated by Ficoll-Hypaque density centrifugation. The concentration of β^A- and β^C-globin mRNA was determined by annealing to purified synthetic DNAs (cDNAs) complementary to β^A and β^C mRNA. No β^C-globin was synthesized in erythroblasts or in CFU-E-derived erythroid colonies prior to the injection of erythropoietin. An increase in the concentration of CFU-E in the bone marrow and the appearance of β^C-globin synthesis in CFU-E-derived colonies were detected 12 hr after the erythropoietin injection. In contrast, β^C mRNA was not detected in either “early” or “late” erythroid cells until 36 hr later. The first measurable β^C-globin mRNA was accompanied by the appearance of β^C-globin synthesis in bone marrow erythroblasts. Our results suggest that the accumulation of β^C-globin mRNA is a relatively late event following induction of HbA to HbC switching by erythropoietin. The expansion of the compartment of erythroid stem cells and the commitment of CFU-E to β^C-globin synthesis appear to precede the detectable accumulation of β^C mRNA by 24–36 hr.     

Schistosoma mansoni: circulating antigens and immune complexes in infected mice.

Circulating schistosome antigens (CSA) and circulating immune complexes (CIC) were investigated during the course of Schistosoma mansoni infection in mice. The radioimmunoprecipitation-polyethylene glycol (PEG) assay (RIPEGA) with [¹²⁵I]anti-S. mansoni antibodies or [¹²⁵I] anti-antigen “4” antibodies detected, respectively, total CSA and antigen “4” in serum and in 3% polyethylene glycol-precipitated CIC from infected mice. Complement fixation test and [¹²⁵I] C_1q-binding test revealed, respectively, an anticomplementary activity and the presence of C_1q-binding CIC. All these substances appeared in infected mice at approximately the same period, i.e., between the 40th- and the 55th- day postinfection. No correlation was observed between the detection of anticomplementary active substances and C_1q-binding CIC. In contrast, a close relationship was noticed between CSA and complement-activating material during the course of the infection. This suggests that substances with anticomplementary activity in serum from infected mice could be one or various CSA. A close correlation was also observed between C_1q-binding CIC and free or “complexed” antigen “4.” This observation supports well the possibility that antigen “4” is one of the major complexed circulating antigen present in schistosomiasis. The immunoglobulins G₁, G_2a, M, and A were also characterized in 3% PEG-precipitated CIC from infected mice during the period in which we detected C_1q-binding CIC. The roles played by specific S. mansoni CIC in either schistosomal nephropathy or protective mechanisms to a challenge infection in mice are discussed.     

<Emphasis Type="Italic">Ab initio</Emphasis> investigation of electric and magnetic dipole electronic transitions in the complex of oxygen with benzene

The electric dipole transitions between pure spin and mixed spin electronic states are calculated at the XMC-QDPT2 and MCSCF levels of theory, respectively, for different intermolecular distances of the C₆H₆ and O₂ collisional complex. The magnetic dipole transition moment between the mixed-spin ground (“triplet”) and the first excited (“singlet”) states is calculated by quadratic response at MCSCF level of theory. The obtained results confirm the theory of intensity borrowing and increasing the intensity of electronic transitions in the C₆H₆?+?O₂ collision. The calculation of magnetically induced current density is performed for benzene molecule being in contact with O₂ at the distances from 3.5 to 4.5 Å. The calculation shows that the aromaticity of benzene is rising due to the conjugation of π-MOs of both molecules. The C₆H₆?+?O₂ complex becomes nonaromatic at the short distances (r?<?3.5 Å). The computation of static polarizability in the excited electronic states of the C₆H₆?+?O₂ collisional complex at various distances supports the theory of red solvatochromic shift of the a?→?X band.

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Graphical abstract The C₆H₆+ O₂ collisional complex

     

Conformational Preferences of Modified Nucleoside N(4)-Acetylcytidine, ac4C Occur at “Wobble” 34th Position in the Anticodon Loop of tRNA

Conformational preferences of modified nucleoside, N(4)-acetylcytidine, ac⁴C have been investigated using quantum chemical semi-empirical RM1 method. Automated geometry optimization using PM3 method along with ab initio methods HF SCF (6-31G**), and density functional theory (DFT; B3LYP/6-31G**) have also been made to compare the salient features. The most stable conformation of N(4)-acetyl group of ac⁴C prefers “proximal” orientation. This conformation is stabilized by intramolecular hydrogen bonding between O(7)···HC(5), O(2)···HC2′, and O4′···HC(6). The “proximal” conformation of N(4)-acetyl group has also been observed in another conformational study of anticodon loop of E. coli elongator tRNA^Met. The solvent accessible surface area (SASA) calculations revealed the role of ac⁴C in anticodon loop. The explicit molecular dynamics simulation study also shows the “proximal” orientation of N(4)-acetyl group. The predicted “proximal” conformation would allow ac⁴C to interact with third base of codon AUG/AUA whereas the ‘distal’ orientation of N(4)-acetyl cytidine side-chain prevents such interactions. Single point energy calculation studies of various models of anticodon–codon bases revealed that the models ac⁴C₍₃₄₎(Proximal):G₃, and ac⁴C₍₃₄₎(Proximal):A₃ are energetically more stable as compared to models ac⁴C₍₃₄₎(Distal):G₃, and ac⁴C₍₃₄₎(Distal):A₃, respectively. MEPs calculations showed the unique potential tunnels between the hydrogen bond donor–acceptor atoms of ac⁴C₍₃₄₎(Proximal):G₃/A₃ base pairs suggesting role of ac⁴C in recognition of third letter of codons AUG/AUA. The “distal” conformation of ac⁴C might prevent misreading of AUA codon. Hence, this study could be useful to understand the role of ac⁴C in the tertiary structure folding of tRNA as well as in the proper recognition of codons during protein biosynthesis process.     

1H-nmr study on the preferred conformations of chiral pyridine-dinucleotide models

The synthesis of four chiral NAD⁺ models 1 and their 1,4-dihydro analogs 2 is described. From the temperature dependence of the ¹H-nmr spectra it is concluded that for these compounds two preferred conformations I and II, differing slightly in energy, exist. Both conformations are “folded” with the more or less parallel p-anisyl and pyridine groups mutually gauche, but in I the pyridine group is rotated by about 180° as compared with II, thus leading to a conspicuous difference in orientation of the substituent Z (NH₂CO, C₆H₅NHSO₂, (CH₂)₄NSO₂, or (C₄H₈ON)SO₂) in the pyridine ring toward the anisyl group. The most stable conformation (I) has Z closest to the center of the p-anisyl group. In 360-MHz spectra of the dihydropyridines at low temperature (?10°C), slow interconversion of I and II leads to the observation of an XY pattern for the C-4 methylene protons of the 1,4-dihydropyridine system. The anisochronity in this methylene group is caused mainly by the anisotropy of the neighboring p-anisyl group.     

Calculations of one-, two- and three-bond nuclear spin-spin couplings in a model peptide and correlations with experimental data

Summary We present ab initio calculations of the Fermi contact term and experimental correlations of six coupling constants, ³J_H ^N _H , ¹J_{C H} , ²J_CH , ¹J_{C N}, ²J_{C N} and ¹J_CN, in a peptide as functions of the backbone dihedral angles, and . Given estimates of experimental uncertainties, we find semiquantitative experimental correlations for ³J_H ^N _H , ¹J_{C N} and ²J_{C N}, qualitative correlations for ¹J_{C H} and ²J_CH , but no experimental correlations of practical utility for ¹J_CN, owing to its complex dependence on at least four dihedral angles. Errors in the estimation of dihedral angles from X-ray crystallographic data for proteins, which result from uncertainties in atom-to-atom distances, place substantial limitations on the quantitative reliability of coupling constant calculations fitted to such data. In the accompanying paper [Edison, A.S. et al., J. Biomol. NMR, 4, 543–551] we apply the results of the coupling constant calculations presented here to the estimation of and angles in staphylococcal nuclease from experimental coupling constants.Abbreviations AO atomic orbital - BPTI basic pancreatic trypsin inhibitor (bovine) - CI-2 chymotrypsin inhibitor 2 - E.COSY exclusive correlation spectroscopy (Griesinger et al., 1986) - ⁿJ_AB single bond (n=1), geminal (n=2), or vicinal (n=3) coupling constant between nuclei A and B - LCAO linear combination of atomic orbitals - NBO natural bond orbital - n lone pair orbitals - bonding orbitals - ^* antibonding orbitals - dihedral angle or molecular orbital wave function; r², correlation coefficient - RHF restricted Hartree-Fock; rmsd, root-mean-square deviation - 3-21G and 6-31G^* molecular orbital basis set designations (Hehre et al., 1986)     

Solution three-dimensional structure of surfactin: A cyclic lipopeptide studied by 1H-nmr,distance geometry,and molecular dynamics

The solution three-dimensional structure of the protonated [Leu7]-surfactin, an hepta-peptide extracted from Bacillus subtilis, has been determined from two-dimensional ¹Hnmr performed in ²H₆-dimethylsulfoxide and combined with molecular modeling. Experimental data included 9 coupling constants, 61 nuclear Overhauser effect derived distances, NH temperature coefficients, and ¹³C relaxation times. Two distance geometry (DISMAN) protocols converged toward models of the structure and the best of them were refined by restrained and unrestrained molecular dynamics (GROMOS). Two structures in accord with the set of experimental constraints are presented. Both are characterized by a “horse saddle” topology for ring atoms on which are attached the two polar Glu and Asp side chains showing an orientation clearly opposite to that of the C_11–13 aliphatic chain. Amphipathic and surface properties of surfactin are certainly related to the existence of such minor polar and a major hydrophobic domains. The particular “claw” configuration of acidic residues observed in surfactin gives important clues for the understanding of its cation binding and transporting ability. © 1994 John Wiley & Sons, Inc.     

Synthesis, crystal structure and esterification of a novel iron(III) 18-metallacrown-6 complex

The trianionic heptadentate ligand, (Z)-3-(5′-chlorosalicylhydrazinocarbonyl) propenoic acid, has been synthesized and reacted with FeCl₃·6H₂O, to produce the complex [Fe^III₆(C₁₂H₈N₂O₅Cl)₆(H₂O)₄(CH₃OH)₂]·8H₂O·4CH₃OH. In the self-assembly process the ligand was esterified and transferred into (Z)-methyl 3-(5′-chlorosalicylhydrazinocarbonyl) propenoate. In the crystal structure, the neutral Fe(III) complex contain a 18-membered metallacrown ring consisting of six Fe(III) and six trianionic ligands. The 18-membered metallacrown ring is formed by the succession of six structural moieties of the type [Fe(III)-N-N]. Due to the meridional coordination of the ligands to the Fe³⁺ ions, the ligands enforce the stereochemistry of the Fe³⁺ ions as a propeller configuration with alternating Λ/Δ forms. The metallacrown can be treated with SnCl₂ or Zn powder to obtain purified ester.     

Crystal and molecular structures of cyclomaltoheptaose inclusion complexes with HI and with methanol

β-Cyclodextrin (β-CD; cyclomaltoheptaose; cyclohepta-amylose; C₄₂H₇₀O₃₅) crystallises from aqueous solutions of HI and of MeOH in the form of stout prisms, which are isomorphous to each other with monoclinic space-group P2₁; cell constants for C₄₂H₇₀O₃₅ · 2HI · 8 H₂O: a = 21.25(3), b = 10.28(2), c = 15.30(2) Å, β = 113.25(9)°, and Z = 2; and for C₄₂H₇₀O₃₅ · MeOH · 6.5 H₂O: a = 21.03(3), b = 10.11(2), c = 15.33(2) Å, β = 111.02(9)°, and Z = 2. X-ray counter data were used to determine the structures of both crystals, which belong to the cage type, with β-CD molecules in nearly identical, “round” shapes. In the HI complex, one I^- is located inside, and one outside, the β-CD cavity; in the MeOH complex, the MeOH is within the cavity. The cavity is closed at the O-2,O-3 side by adjacent β-CD molecules, and at the O-6 side by water molecules hydrogen-bonded to the guest and to surrounding β-CD molecules. Interstices between β-CD molecules are filled by water of hydration molecules in distorted co-ordination.     

On the “filterable aggregates and other particles” interpretation of the extraordinary regime of polyelectrolytes

Quasi-elastic light scattering studies on some polyelectrolyte systems exhibit a somewhat “bizarre” behavior in the profile of the apparent diffusion coefficient D_app as a function of the salt concentration C_s. As C_s is decreased, D_app first increases in accordance with polyelectrolyte theories, and then undergoes a precipitous drop in value by over an order of magnitude at a well-defined critical value C_s = C. This “transition” from C_s > C (ordinary) to C_s < C (extraordinary) is referred to as the “ordinary-extraordinary” (o-e) transition. Ghosh, Peitzsch, and Reed [(1992) Biopolymers, Vol. 32, pp. 1105–1122] proposed a “filterable aggregate” (FA) and “other particle” interpretation for the o-e transition and its reversibility in regard to ionic strength changes. The present communication examines in detail the FA model as applied to the o-e transition. It is shown that the FA model fails to account of the established characteristics of the o-e transition. © 1993 John Wiley & Sons, Inc.     

Direct measurement of critical concentrations and assembly rate constants at the two ends of an actin filament

Actin filament bundles isolated from Limulus sperm were used for quantitative electron microscope studies of F-actin assembly. The assembly rate constants were calculated. In addition, the critical concentrations (C_os) for both filament ends were directly determined. In 75 mM KCI and 1–5 mM Mg⁺⁺, the C_os were 0.1 μM and 0.5 μM for the barbed and pointed ends, respectively. Substitution of Ca⁺⁺ (20–200 μM) for Mg⁺⁺ resulted in C_os of 0.4 μM for both filament ends. Consistent with these findings, filament growth occurred only from the barbed ends of Limulus bundles “seeded” into F-actin solutions in KCI and Mg⁺⁺. Finally, filaments originally grown from the pointed filament ends of Limulus bundles were gradually lost as the actin solution reached steady state. These results demonstrate that actin filaments can “treadmill” under physiological conditions, albeit at very slow rates.     

Accurate geometries for “Mountain pass” regions of the Ramachandran plot using quantum chemical calculations

Unusual local arrangements of protein in Ramachandran space are not well represented by standard geometry tools used in either protein structure refinement using simple harmonic geometry restraints or in protein simulations using molecular mechanics force fields. In contrast, quantum chemical computations using small poly‐peptide molecular models can predict accurate geometries for any well‐defined backbone Ramachandran orientation. For conformations along transition regions—ϕ from −60 to 60°—a very good agreement with representative high‐resolution experimental X‐ray (≤1.5 Å) protein structures is obtained for both backbone C⁻¹‐N‐C_α angle and the nonbonded O⁻¹…C distance, while “standard geometry” leads to the “clashing” of O…C atoms and Amber FF99SB predicts distances too large by about 0.15 Å. These results confirm that quantum chemistry computations add valuable support for detailed analysis of local structural arrangements in proteins, providing improved or missing data for less understood high‐energy or unusual regions.     

On the carbon and sulphur metabolism in the meromictic Lake Faro (Sicily)

The processes of photosynthesis, chemosynthesis and sulphate reduction were quantitatively studied in the brackish meromitic lake Faro (Sicily) with the aid of C¹⁴ and S³⁵. The layer of “red water” was situated at the depht of the chemocline (13–14 m), where the average concentration of H₂S was 10 mg/l. The total biomass of bacterioplankton consisted in this layer mostly of a brown Chlorobium which reached a wet weight of 30 g/m³. The production of photosynthesis in this layer was 30–60 µg C/l/day. The microbial population in the “red water” was found adapted to an extremely low light intensity and to show a light optimum at the depth µg 9m where only 2,5% of outside light penetrates. The photoautotrophic microflora is consumed by infusoria found in mass in the “red water” layer. An active H₂S-production was found in the water column in the upper part of the H₂S-zone and in the bottom sediments. The data are discussed from the view point of the trophology of meromitic basins.     

Selectively 13C-enriched DNA: Dynamics of the C1′-H1′ vector in d(CGCAAATTTGCG)2

Summary In order to examine the internal dynamic processes of the dodecamer d(CGCAAATTTGCG)₂, the ¹³C-enriched oligonucleotide has been synthesized. The three central thymines were selectively ¹³C-labeled at the C1′ position and their spin-lattice relaxation parameters R(C_Z), R(C_X,Y), R(H_Z→C_Z), R(2H_ZC_Z), R(2H_ZC_X,Y) and R(H _infZ ^supC ) were measured. Density functions were computed for two models of internal motions. Comparisons of the experimental data were made with the spin-lattice relaxation rates rather than with the density functions, whose values were altered by accumulation of the uncertainties of each relaxation rate measurement. The spin-lattice relaxation rates were computed with respect to the motions of the sugar around the C1′-N1 bond. A two-state jump model between the anti- and syn-conformations with P(anti)/P(syn)=91/9 or a restricted rotation model with Δχ=28° and an internal diffusion coefficient of 30×10⁷ s^-1 gave a good fit with the experimental data. Twist, tilt or roll base motions have little effect on ¹³C1′ NMR relaxation. Simulation of spin-relaxation rates with the data obtained at several temperatures between 7 and 32 °C, where the dodecamer is double stranded, shows that the internal motion amplitude is independent of the temperature within this range, as expected for internal motion. Using the strong correlation which exists in a B-DNA structure between the χ and δ angle, we suggest that the change in the glycosidic angle value should be indicative of a sugar puckering between the C1′-exo and C2′-endo conformations.     

Topographic analysis of the S7 binding subsite of the tachykinin neurokinin-1 receptor

Conformationally and configurationally restricted rotameric probes of phenylalanine have been incorporated in the sequence of substance P (SP)—Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH₂—for analyzing the binding pockets of Phe⁷ (S₇) and Phe⁸ (S₈), in the neurokinin-1 receptor. These analogues of phenylalanine are (2S, 3R)- and (2S, 3S)-indanylglycines, E- and Z-α, β-dehydrophenylalanines, and 2(S)-α, β-cyclopropylphenylalanines [Δ^E Phe, Δ^ZPhe, ▿^E2(S)Phe, and ▿^Z2(S)Phe]. Binding data obtained with either conformationally (Ing diastereoisomers) or configurationally (Δ^EPhe, Δ^ZPhe) probes have unveiled large differences in the binding potencies of these rotameric probes. With the support of nmr data and energy calculations done on these SP-substituted analogues, we attempt to answer questions inherent to such study. First, none of these six probes prevents the formation of bioactive conformation(s) of the backbone of SP. Second, both diastereoisomers (S, S) and (S, R) of indanylglycine preferentially adopt, in the sequence of SP, the gauche (−) and trans side-chain orientations, respectively, as previously postulated from energy calculations with model peptides. However, in solution, the difference in energy between these rotamers included in the sequence of SP, compared to model peptides, is smaller since the other rotamer can be detected in [(2S, 3R) Ing⁷]SP. Finally, from this study we can hypothesize that the large variations observed in the affinities of Phe⁷ substituted analogues of SP must come from steric hindrance in the S₇ binding site, which drastically restricts the space filling around the C_α (SINGLE BOND) C_β bond of residue 7. © 1996 John Wiley & Sons, Inc.     

Efficiency of photosynthetic water oxidation at ambient and depleted levels of inorganic carbon

Over 40 years ago, Joliot et al. (Photochem Photobiol 10:309–329, 1969) designed and employed an elegant and highly sensitive electrochemical technique capable of measuring O₂ evolved by photosystem II (PSII) in response to trains of single turn-over light flashes. The measurement and analysis of flash-induced oxygen evolution patterns (FIOPs) has since proven to be a powerful method for probing the turnover efficiency of PSII. Stemler et al. (Proc Natl Acad Sci USA 71(12):4679–4683, 1974), in Govindjee’s lab, were the first to study the effect of “bicarbonate” on FIOPs by adding the competitive inhibitor acetate. Here, we extend this earlier work by performing FIOPs experiments at various, strictly controlled inorganic carbon (C_i) levels without addition of any inhibitors. For this, we placed a Joliot-type bare platinum electrode inside a N₂-filled glove-box (containing 10–20 ppm CO₂) and reduced the C_i concentration simply by washing the samples in C_i-depleted media. FIOPs of spinach thylakoids were recorded either at 20-times reduced levels of C_i or at ambient C_i conditions (390 ppm CO₂). Numerical analysis of the FIOPs within an extended Kok model reveals that under C_i-depleted conditions the miss probability is discernibly larger (by 2–3 %) than at ambient conditions, and that the addition of 5 mM HCO₃ ^? to the C_i-depleted thylakoids largely restores the original miss parameter. Since a “mild” C_i-depletion procedure was employed, we discuss our data with respect to a possible function of free or weakly bound HCO₃ ^? at the water-splitting side of PSII.     

Autotrophic carbon sources for heterotrophic bacterioplankton in a floodplain lake of central Amazon

The relative contribution of autotrophic carbon sources (aquatic macrophytes, flooded forest, phytoplankton) for heterotrophic bacterioplankton was evaluated in a floodplain lake of the Central Amazon. Stable carbon isotopes (¹³C) were used as tracers. Values of ¹³C of different autotrophic sources were compared to those of dissolved organic carbon (DOC) and those of bacterially produced CO₂.The percentage of carbon derived from C₄ macrophytes for bacterially produced CO₂ was the highest, on average 89%. The average ¹³C value of CO₂ from bacterial respiration was –18.5 ± 3.3. Considering a fractionation of CO₂ of 3 by bacterial respiration, ¹³C value was –15.5, near C₄ macrophyte ¹³C value (–13.1).The average value of total DOC ¹³C was –26.8 ± 2.4. The percentage of C₄ macrophytes carbon for total DOC was on average 17%. Considering that bacteria consume mainly carbon from macrophytes, the dominance of C₃ plants for total DOC probably reflects a faster consumption of the former source, rather than a major contribution of the latter source.Heterotrophic bacterioplankton in the floodplain may be an important link in the aquatic food web, transferring the carbon from C₄ macrophytes to the consumers.     

Carbon isotope composition of intermediates of the starch-malate sequence and level of the crassulacean acid metabolism in leaves of Kalanchoe blossfeldiana Tom Thumb

Isotype analyses were performed on biochemical fractions isolated from leaves of Kalanchoe blossfeldiana Tom Thumb. during aging under long days or short days. Irrespective of the age or photoperiodic conditions, the intermediates of the starch-malate sequence (starch, phosphorylated compounds and organic acids) have a level of ¹³C higher than that of soluble sugars, cellulose and hemicellulose. In short days, the activity of the crassulacean acid metabolism pathway is predominant as compared to that of C₃ pathway: leaves accumulate organic acids, rich in ¹³C. In long days, the activity of the crassulacean acid metabolism pathway increases as the leaves age, remaining, however, relatively low as compared to that of C₃ pathway: leaves accumulate soluble sugars, poor in ¹³C. After photoperiodic change (long daysshort days), isotopic modifications of starch and organic acids suggest evidence for a lag phase in the establishment of the crassulacean acid metabolism pathway specific to short days. The relative proportions of carbon from a C₃-origin (RuBPC acitivity as strong discriminating step, isotope discrimination in vivo=20) or C₄-origin (PEPC activity as weak discriminating step, isotope discrimination in vivo=4) present in the biochemical fractions were calculated from their ¹³C values. Under long days, 30 to 70% versus 80 to 100% under short days, of the carbon of the intermediates linked to the starch-malate sequence, or CAM pathway (starch, phosphorylated compounds and organic acids), have a C₄-origin. Products connected to the C₃ pathway (free sugars, cellulose, hemicellulose) have 0 to 50% of their carbon, arising from reuptake of the C₄ from malate, under long days versus 30 to 70% under short days.Abbreviations CAM crassulacean acid metabolism - CAM pathway pathway with malate accumulation by -carboxylation of PEP, arising from glycolysis of starch (starch-malate sequence) - C₃-metabolism metabolism with primary carbon fixed by the Calvin and Benson pathway (C₃-origin) - C₄-metabolism metabolism with primary carbon fixed by the Hatch and Slack pathway (C₄-origin) - C₃-pathway pathway with RuBPC activity and the Calvin and Benson pathway, irrespective of the CO₂-source, atmospheric or reuptake of the C₄ from malate - ¹³C()=(R_sample-R_PDR)10³/R_PDB where PDB Pee Dee belemnite (belemnite from the Pee Dee formation, South Carolina) and R=¹³C/¹²C - D isotope discrimination - PEP phosphoenolpyruvate - PEPC (EC 4.1.1.31) PEP carboxylase - PGA phosphoglyceric acid - Py.di-PK (EC 2.7.9.1) pyruvate, Pi-dikinase - RuBP ribulose bisphosphate - RuBPC (EC 4.1.1.39) RuBP carboxylase - SD short days - LD long days     

Synthesis of “Reversed” Methylenecyclopropane Analogues of Antiviral Phosphonates

Synthesis of “reversed” methylenecyclopropane analogues of nucleoside phosphonates 6a,7a, 6b, and 7b is described. 1-Bromo-1-bromomethylcyclopropane 8 was converted to the bromocyclopropyl phosphonate 9 by Michaelis-Arbuzov reaction with triisopropyl phosphite. Base-catalyzed β-elimination and deacetylation gave the key Z- and E-hydroxymethylcyclopropyl phosphonates 10 and 11 separated by chromatography. The Mitsunobu type of alkylation of 10 or 11 with adenine or 2-amino-6-chloropurine afforded phosphonates 12a, 12b, 13a, and 13b. Acid hydrolysis furnished the adenine and guanine analogues 6a, 7a, 6b, and 7b. The E and Z configuration was assigned on the basis of NOE experiments with phosphonates 6b and 7b. All Z- and E-isomers were also distinguished by different chemical shifts of CH₂O or CH₂N (H₄ or H_4′). Significant differences of the chemical shifts of the cyclopropane C_3(3’) carbons and coupling constants ³J_P,C2(2’) or ³J_P,C3(3’) selective for the Z- or E-isomers were also noted. Phosphonates 6a, 7a, 6b, and 7b are devoid of significant antiviral activity.     

Synthesis, 95Mo NMR spectra and x-ray crystal structure of MoO2(C14H11N2O)2(C2H5H)1 and Mo2O5(C14H11N2O)2(C2H5OH)1(H2O)1

The crystal structures and ⁹⁵Mo NMR spectra of two complexes formed between 2-α-hydroxybenzyl- benzimidazole (C₆H₅·CHOH·C₇H₅N₂=HOBB), as its sodium salt, and MoO₂Cl₂ are reported. [MoO₂- (OBB)₂]·EtOH (OBB=deprotonated HOBB) crystallizes in space group P2₁/n, with a=12.8441(7), b=15.917(3), c=13.314(2) Å, β=97.163(8)° and Z =4. The structure was determined from 3096 observed reflections and refined to a final R value of 0.030. The complex is a six coordinate cis-dioxo species, the ⁹⁵Mo spectrum of which shows a single sharp peak at 56 ppm in dimethylformamide (DMF). The second complex, [Mo₂O₅(OBB)₂]·EtOH·H₂O, crystallizes in space group Pbca, with a=22.482(4), b=16.442(3), c=18.407(3) Å and Z=8. The structure was determined from 2936 observed reflections and refined to a final R value of 0.061. The complex is a binuclear doubly bridged species in which one metal atom is six coordinate while the other is five coordinate. Its ⁹⁵Mo NMR spectrum in DMF shows a sharp peak at 124 ppm and a second broader much weaker peak at 51 ppm.