Purification and mode of action of two different arabinoxylan arabinofuranohydrolases from Bifidobacterium adolescentis DSM 20083

Two novel arabinofuranohydrolases (AXH-d3 and AXH-m23) were purified from Bifidobacterium adolescentis DSM 20083. Both enzymes were induced upon growth of Bi. adolescentis on xylose and arabinoxylan-derived oligosaccharides. They were only active with arabinoxylans and therefore denoted as arabinoxylan arabinofuranohydrolases. Their optimal activity was at pH 6 and 30–40 °C. They were very specific in their mode of action and were clearly different from AXH-m from Aspergillus awamori. AXH-m23 released only arabinosyl groups, which were linked to the C-2 or C-3 position of singly substituted xylose residues in arabinoxylan oligomers. AXH-d3 hydrolysed C-3-linked arabinofuranosyl residues of doubly substituted xylopyranosyl residues of arabinoxylans and arab- inoxylan-derived oligosaccharides. No activity was observed with C-2-linked arabinofuranosyl residues of these doubly substituted xylopyranosyl residues, or against C-2- and C-3-linked arabinofuranosyl residues of singly substituted xylopyranosyl residues. The combination of AXH-d3 and AXH-m showed low debranching activity with highly substituted glucurono-arabinoxylans. However, arabinoxylan from wheat flour was debranched almost completely. Received: 12 January 1999 / Accepted: 17 January 1999     

A new arabinofuranohydrolase from Bifidobacterium adolescentis able to remove arabinosyl residues from double-substituted xylose units in arabinoxylan

An arabinofuranohydrolase (AXH-d3) was purified from a cell-free extract of Bifidobacterium adolescentis DSM 20083. The enzyme had a molecular mass of approximately 100 kDa as determined by gel filtration. It displayed maximum activity at pH 6 and 30 °C. Using an arabinoxylan-derived oligosaccharide containing double-substituted xylopyranosyl residues established that the enzyme specifically released terminal arabinofuranosyl residues linked to C-3 of double-substituted xylopyranosyl residues. In addition, this arabinofuranohydrolase released arabinosyl groups from wheat flour arabinoxylan polymer but showed no activity towards p-nitrophenyl α-l-arabinofuranoside or towards sugar-beet arabinan, soy arabinogalactan, arabino-oligosaccharides and arabinogalacto-oligosaccharides. Received: 15 July 1996 / Received revision: 18 October 1996 / Accepted: 18 October 1996     

A H NMR study of the specificity of α-l-arabinofuranosidases on natural and unnatural substrates

Background

The detailed characterization of arabinoxylan-active enzymes, such as double-substituted xylan arabinofuranosidase activity, is still a challenging topic. Ad hoc chromogenic substrates are useful tools and can reveal subtle differences in enzymatic behavior. In this study, enzyme selectivity on natural substrates has been compared with enzyme selectivity towards aryl-glycosides. This has proven to be a suitable approach to understand how artificial substrates can be used to characterize arabinoxylan-active α-l-arabinofuranosidases (Abfs).

Methods

Real-time NMR using a range of artificial chromogenic, synthetic pseudo-natural and natural substrates was employed to determine the hydrolytic abilities and specificity of different Abfs.

Results

The way in which synthetic di-arabinofuranosylated substrates are hydrolyzed by Abfs mirrors the behavior of enzymes on natural arabinoxylo-oligosaccharide (AXOS). Family GH43 Abfs that are strictly specific for mono-substituted d-xylosyl moieties (AXH-m) do not hydrolyze synthetic di-arabinofuranosylated substrates, while those specific for di-substituted moieties (AXH-d) remove a single l-arabinofuranosyl (l-Araf) group. GH51 Abfs, which are supposedly AXH-m enzymes, can release l-Araf from disubstituted d-xylosyl moieties, when these are non-reducing terminal groups.

Conclusions and general significance

The present study reveals that although the activity of Abfs on artificial substrates can be quite different from that displayed on natural substrates, enzyme specificity is well conserved. This implies that carefully chosen artificial substrates bearing di-arabinofuranosyl d-xylosyl moieties are convenient tools to probe selectivity in new Abfs. Moreover, this study has further clarified the relative promiscuity of GH51 Abfs, which can apparently hydrolyze terminal disubstitutions in AXOS, albeit less efficiently than mono-substituted motifs.     

Evidence of the presence of 2-O-beta-D-xylopyranosyl-alpha-l-arabinofuranose side chains in barley husk arabinoxylan

(Glucurono)arabinoxylans were extracted from barley husks and degraded with endo-beta-xylanase or subjected to periodate oxidation. The released oligosaccharide fragments were separated and isolated on Biogel-P2, and their structures were determined by NMR spectroscopy. The oligosaccharides identified consisted of beta-d-(1-->4)-linked xylopyranosyl residues, of which some were substituted at O-3 with alpha-l-arabinofuranosyl groups or at O-2 with 4-O-methylglucuronic acid. In addition to these substituents, a disaccharide side chain, 2-O-beta-d-xylopyranosyl-alpha-l-arabinofuranose, attached at position O-3 of the main chain, was proved to exist in arabinoxylan from barley husks. The compound was fully characterized with NMR, and all (1)H and (13)C NMR signals were assigned. The arabinose to xylose ratio was low (approximately 0.2) and no 2,3-disubstitution existed. No blocks of substituted xylose residues could be observed along the main chain.     

Substrate specificity of three recombinant α-L-arabinofuranosidases from Bifidobacterium adolescentis and their divergent action on arabinoxylan and arabinoxylan oligosaccharides

Bifidobacterium adolescentis possesses several arabinofuranosidases able to hydrolyze arabinoxylans (AX) and AX oligosaccharides (AXOS), the latter being bifidogenic carbohydrates with potential prebiotic properties. We characterized two new recombinant arabinofuranosidases, AbfA and AbfB, and AXH-d3, a previously studied arabinofuranosidase from B. adolescentis. AbfA belongs to glycoside hydrolase family (GH) 43 and removed arabinose from the C(O)2 and C(O)3 position of monosubstituted xylose residues. Furthermore, hydrolytic activity of AbfA was much larger towards substrates with a low amount of arabinose substitutions. AbfB from GH 51 only cleaved arabinoses on position C(O)3 of disubstituted xyloses, similar to GH 43 AXH-d3, making it to our knowledge, the first reported enzyme with this specificity in GH 51. AbfA acted synergistically with AbfB and AXH-d3. In combination with AXH-d3, it released 60% of arabinose from wheat AX. Together with recent studies on other AXOS degrading enzymes from B. adolescentis, these findings allowed us to postulate a mechanism for the uptake and hydrolysis of bifidogenic AXOS by this organism.     

Structural analysis of a glycoside hydrolase family 43 arabinoxylan arabinofuranohydrolase in complex with xylotetraose reveals a different binding mechanism compared with other members of the same family

AXHs (arabinoxylan arabinofuranohydrolases) are alpha-L-arabinofuranosidases that specifically hydrolyse the glycosidic bond between arabinofuranosyl substituents and xylopyranosyl backbone residues of arabinoxylan. Bacillus subtilis was recently shown to produce an AXH that cleaves arabinose units from O-2- or O-3-mono-substituted xylose residues: BsAXH-m2,3 (B. subtilis AXH-m2,3). Crystallographic analysis reveals a two-domain structure for this enzyme: a catalytic domain displaying a five-bladed beta-propeller fold characteristic of GH (glycoside hydrolase) family 43 and a CBM (carbohydrate-binding module) with a beta-sandwich fold belonging to CBM family 6. Binding of substrate to BsAXH-m2,3 is largely based on hydrophobic stacking interactions, which probably allow the positional flexibility needed to hydrolyse both arabinose substituents at the O-2 or O-3 position of the xylose unit. Superposition of the BsAXH-m2,3 structure with known structures of the GH family 43 exo-acting enzymes, beta-xylosidase and alpha-L-arabinanase, each in complex with their substrate, reveals a different orientation of the sugar backbone.     

Structure of an l-arabino-d-xylan from the bark of Cinnamomum zeylanicum

An arabinoxylan isolated from the bark of Cinnamomum zeylanicum was composed of l-arabinose and d-xylose in the molar ratio 1.6:1.0. Partial hydrolysis furnished oligosaccharides which were characterised as α-d-Xylp-(1→3)-d-Ara, β-dXylp-(1→4)-d-Xyl, β-d-Xylp-(1→4)-β-d-Xylp-(1→4)-d-Xyl, β-d-Xylp-(1→4)-β-d-Xylp-(1→4)-β-d-Xylp-Xylp-(1→4)-d-Xyl, xylopentaose, and xylohexaose. Mild acid hydrolysis of the arabinoxylan gave a degraded polysaccharide consisting of l-arabinose (8%) and d-xyolse (92%). Methylation analysis indicated the degraded polysaccharide to be a linear (1→4)-linked d-xlan in which some xylopyranosyl residues were substituted at O-2 or O-3 with l-arabinofuranosyl groups. These data together with the results of methylation analysis and periodate oxidation of the arabinoxylan suggested that it contained a (1→4)-linked β-d-xylan backbone in which each xylopyranosyl residue was substituted both at O-2 and O-3 with l-arabinofuranosyl, 3-O-α-d-xylopyranosyl-l-arabinofuranosyl, and 3-O-l-arabinofuranosyl-l-arabinofuranosyl groups.     

Step-wise enzymatic preparation and structural characterization of singly and doubly substituted arabinoxylo-oligosaccharides with non-reducing end terminal branches

Shearzyme (GH10 endo-1,4-β-d-xylanase) and two different α-l-arabinofuranosidases (AXH-m and AXH-d3) were used stepwise to manufacture arabinoxylo-oligosaccharides (AXOS) with α-l-Araf (1→2)-monosubstituted β-d-Xylp residues or α-l-Araf (1→2)- and (1→3) doubly substituted β-d-Xylp residues from wheat arabinoxylan (AX) in a rather straightforward way. Four major AXOS (d-I, d-II, m-I and m-II) were formed in two separate hydrolyses. The AXOS were purified and the structures were confirmed using TLC, HPAEC-PAD, MALDI-TOF-MS and 1D and 2D NMR spectroscopy. The samples were identified as d-I: α-l-Araf-(1→2)-[α-l-Araf-(1→3)]-β-d-Xylp-(1→4)-β-d-Xylp-(1→4)-d-Xylp, d-II: α-l-Araf-(1→2)-[α-l-Araf-(1→3)]-β-d-Xylp-(1→4)-d-Xylp, m-I: α-l-Araf-(1→2)-β-d-Xylp-(1→4)-β-d-Xylp-(1→4)-d-Xylp and m-II: α-l-Araf-(1→2)-β-d-Xylp-(1→4)-d-Xylp. To our knowledge, this is the first report on structural ¹H and ¹³C NMR analysis of xylobiose-derived AXOS d-II and m-II. The latter compound has not been reported previously. The doubly substituted AXOS were produced for the first time in good yields, as d-I and d-II corresponded to 11.8 and 5.6 wt% of AX, respectively. Singly α-l-Araf (1→2)-substituted AXOS could also be prepared in similar yields by treating the doubly substituted AXOS further with AXH-d3.     

Tuning iridium(III) complexes containing 2-benzo[b]thiophen-2-yl-pyridine based ligands in the red region

Synthesis and characterization of four iridium(III) complexes containing 2-benzo[b]thiophen-2-yl-pyridine based ligands are reported. The absorption, emission, electrochemistry, and thermostability of the complexes were systematically investigated. The (btmp)₂Ir(acac) (btmp = 2-benzo[b]thiophen-2-yl-4-methyl-pyridyl, acac = acetyl acetone) was characterized using X-ray crystallography. Calculation on the electronic ground state for (btmp)₂Ir(acac) was carried out using B3LYP density functional theory, HOMO levels are a mixture of Ir and btmp ligand orbitals, while the LUMO is predominantly btmp ligand based. Introduction of substituents (CH₃, CF₃) into pyridyl ring in a typical red emitter (btp)₂Ir(acac) leads to a marked decrease in the sublimation temperature, which is more suitable for OLEDs process. Electrochemical studies showed that (btmp)₂Ir(acac) has a slightly lower oxidation potential, but (btfmp)₂Ir(acac), (btfmp)₂Ir(dbm), and (btfmp)₂Ir(pic) (btfmp = 2-benzo[b]thiophen-2-yl-5-trifluoromethyl-pyridine, dbm = dibenzoylmethane, pic = 2-picolinic acid) containing CF₃ group are much difficult to oxidate than (btp)₂Ir(acac). The emission characteristics of these complexes can be tuned by either changing the substituents and their position on 2-benzo[b]thiophen-2-yl-pyridine or using different monoanionic ligands, showing emission λ_max values from 604 to 638 nm in CH₂Cl₂ solution at room temperature.     

Synthesis, characterization and electrochemistry of bis(3-aryl-6,6-dimethylcyclohexadienyl)ruthenium complexes

Five bis(3-aryl-6,6-dimethylcyclohexadienyl)ruthenium complexes (4a-4e) are prepared by reactions between di-μ-chlorodichlorobis[(1-3η:6-8η)-2,7-dimethyl-octadienyl]diruthenium and the corresponding dienes. The larger aryl substituents increase the barrier to rotation in 4a-4e relative to bis(3-methyl-6,6-cyclohexadienyl)ruthenium (5b). The activation parameters were determined by line-shape analysis for the exchange process in 4a: ΔG^† (183 K), 8.0 ± 0.2 kcal/mol; ΔH^†, 10.3 kcal/mol; and ΔS^†, 13 cal/mol/K. The electronic effect of the aryl substituents on the cyclohexadienyl ligand on the oxidation potential of the complex are compared to the effect of methyl substituents.     

Biotransformation of glycerol to 3-hydroxypropionaldehyde: Improved production by in situ complexation with bisulfite in a fed-batch mode and separation on anion exchanger

3-Hydroxypropionaldehyde (3HPA) is an important C3 chemical that can be produced from renewable glycerol by resting whole cells of Lactobacillus reuteri. However the process efficiency is limited due to substrate inhibition, product-mediated loss of enzyme activity and cell viability, and also formation of by-products. Complex formation of 3HPA with sodium bisulfite and subsequent binding to Amberlite IRA-400 was investigated as a means of in situ product recovery and for overcoming inhibition. The adsorption capacity and -isotherm of the resin were evaluated using the Langmuir model. The resin exhibited maximum capacity of 2.92 mmol complex/g when equilibrated with 45 mL solution containing an equilibrium mixture of 2.74 mmol 3HPA-bisulfite complex and 2.01 mmol free 3HPA. The dynamic binding capacity based on the breakthrough curve of 3HPA and its complex on passing a solution with 2.49 mmol complex and 1.65 mmol free 3HPA was 2.01 mmol/g resin. The bound 3HPA was desorbed from the resin using 0.20 M NaCl with a high purity as a mixture of complexed- and free 3HPA at a ratio of 0.77 mol/mol. Fed-batch biotransformation of glycerol (818.85 mmol) with in situ 3HPA complexation and separation on the bisulfite-functionalized resin resulted in an improved process with consumption of 481.36 mmol glycerol yielding 325.54 mmol 3HPA at a rate of 17.13 mmol/h and a yield of 68 mol%. Also, the cell activity was maintained for at least 28 h.     

Picosecond fluorescence of intact and dissolved PSI-LHCI crystals

Over the past several years, many crystal structures of photosynthetic pigment-protein complexes have been determined, and these have been used extensively to model spectroscopic results obtained on the same proteins in solution. However, the crystal structure is not necessarily identical to the structure of the protein in solution. Here, we studied picosecond fluorescence of photosystem I light-harvesting complex I (PSI-LHCI), a multisubunit pigment-protein complex that catalyzes the first steps of photosynthesis. The ultrafast fluorescence of PSI-LHCI crystals is identical to that of dissolved crystals, but differs considerably from most kinetics presented in the literature. In contrast to most studies, the data presented here can be modeled quantitatively with only two compartments: PSI core and LHCI. This yields the rate of charge separation from an equilibrated core (22.5 ± 2.5 ps) and rates of excitation energy transfer from LHCI to core (k_LC) and vice versa (k_CL). The ratio between these rates, R = k_CL/k_LC, appears to be wavelength-dependent and scales with the ratio of the absorption spectra of LHCI and core, indicating the validity of a detailed balance relation between both compartments. k_LC depends slightly but nonsystematically on detection wavelength, averaging (9.4 ± 4.9 ps)⁻¹. R ranges from 0.5 (<690 nm) to ∼1.3 above 720 nm.     

Synthesis, properties and solution speciation of lanthanide chloride complexes of triphenylphosphine oxide

The reaction of Ph₃PO with LnCl₃ · nH₂O (Ln=La-Lu ≠ Pm) in a 3.5:1 ratio in acetone produces [LnCl₃(Ph₃PO)₃], whilst from a 6:1 ratio in ethanol the products are [LnCl₂(Ph₃PO)₄]Cl · n(solvate). In the presence of [NH₄][PF₆] in ethanol solution, [LnCl₂(Ph₃PO)₄]PF₆ can be isolated. The last complexes are stable in solution but the [LnCl₃(Ph₃PO)₃] and [LnCl₂(Ph₃PO)₄]Cl partially interconvert in non-coordinating solvents, the neutral species being preferred by the lighter lanthanides, the cationic tetrakis complexes becoming more favoured towards the end of the series. The complexes have been characterised in the solid state by analysis and IR spectroscopy and in solution by ³¹P{¹H} NMR spectroscopy and conductance measurements. The crystal structures of trans-[LnCl₂(Ph₃PO)₄]Cl · nEtOH (Ln=Tb or Yb) and mer-[LnCl₃(Ph₃PO)₃] · 0.5Me₂CO (Ln=La or Ce) are reported and discussed.     

Low-energy collision-activated dissociation electrospray ionization tandem mass spectrometric analysis of Sinorhizobial succinoglycan monomers

Succinoglycan monomers (M1, M2, and M3) are octasaccharides with acetyl, pyruvyl, and/or succinyl groups as substituents derived from Sinorhizobium meliloti 1021. The dissociation patterns of the octasaccharides caused by low-energy collision-activated dissociation (CAD) were investigated using triple quadrupole tandem mass spectrometry (MS) equipped with an electrospray ionization (ESI) source with increasing collision energy (CE) in negative ion mode. None of the succinoglycan monomers were fragmented at a CE of −25 eV. When the CE was applied to −50 or −70 eV, the loss of the terminal Gal residue and/or the succinyl group of the monomers was observed in the product ion scan mode. Interestingly, the acetyl and the pyruvyl groups in the succinoglycan monomers were not lost even when a CE of −70 eV was applied, indicating that the substituents are more stable than the succinyl group in the octasaccharides.     

Cryopreservation of human ovarian tissue: Comparison of novel direct cover vitrification and conventional vitrification

Objective

To compare the effect of novel direct cover vitrification (DCV) and conventional vitrification (CV) for human ovarian tissue.

Study design

Ovarian biopsy specimens obtained from 12 patients were randomly allocated into five groups: Fresh, DCV1, DCV2, DCV3 and CV. Three concentrations of cryoprotectants were used in DCV group. The equilibration solution of DCV1, DCV2, DCV3 was 5% EG + 5% DMSO + DPBS, 7.5% EG + 7.5%DMSO + DPBS, 10% EG + 10% DMSO + DPBS, respectively. And the vitrification solution of DCV1, DCV2, DCV3 was 10% EG + 10% DMSO + DPBS, 15%EG+15% DMSO + DPBS, 20% EG + 20% DMSO + DPBS, respectively. The equilibration solution and the vitrification solution of CV group was same as DCV3 group. The effects of cryopreserved procedure on human ovarian tissue were studied by histology, TUNEL assay, transmission electron microscopy (TEM) and heterotopic allograft.

Results

The percentages of morphologically normal and viable follicles of DCV2 were significantly higher than DCV1, DCV3 and CV groups (P < 0.05). TUNEL assay demonstrated that the incidence of apoptotic cell in vitrification ovarian tissue was significantly higher than fresh tissue (P < 0.05), but there were no difference in various groups with cryopreservation. TEM showed that less damage was detected in DCV2 group. After grafting, the follicle density of DCV2 was greater than DCV1, DCV3 and CV groups (P < 0.05).

Conclusions

The novel cover vitrification with optimal concentration of cryoprotectants is superior to conventional vitrification. It is suitable for human ovarian tissue fragments with high efficiency and facility.     

Determination of uric acid in human urine and serum by capillary electrophoresis with chemiluminescence detection

A simple and sensitive method based on capillary electrophoresis (CE) with chemiluminescence (CL) detection has been developed for the determination of uric acid (UA). The sensitive detection was based on the enhancement effect of UA on the CL reaction between luminol and potassium ferricyanide (K₃[Fe(CN)₆]) in alkaline solution. A laboratory-built reaction flow cell and a photon counter were deployed for the CL detection. Experimental conditions for CL detection were studied in detail to achieve a maximum assay sensitivity. Optimal conditions were found to be 1.0 × 10⁻⁴ M luminol added to the CE running buffer and 1.0 × 10⁻⁴ M K₃[Fe(CN)₆] in 0.2 M NaOH solution introduced postcolumn. The proposed CE-CL assay showed good repeatability (relative standard deviation [RSD] = 3.5%, n = 11) and a detection limit of 3.5 × 10⁻⁷ M UA (signal/noise ratio [S/N] = 3). A linear calibration curve ranging from 6.0 × 10⁻⁷ to 3.0 × 10⁻⁵ M UA was obtained. The method was evaluated by quantifying UA in human urine and serum samples with satisfactory assay results.     

Polysaccharides as potential antioxidative compounds for extended-release matrix tablets

The objective of this study was to identify polysaccharides with antioxidant properties for use as potential antioxidative compounds for extended-release matrix tablets. The antioxidant properties of five different polysaccharides, high molecular weight alginate (H-ALG), low molecular weight alginate (L-ALG), high molecular weight chitosan (H-chitosan), low molecular weight chitosan (L-chitosan), and pectic acid (PA) were examined using N-centered radicals from 1,1′-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and reducing power, based on their ability to reduce Cu²⁺. L-chitosan and PA had acceptable scavenging abilities and were good radical scavengers, with good reducing power, but the H-chitosan and alginate derivatives were much less effective. The results suggest that L-chitosan and PA could be useful in combating oxidative stress. A PA and L-chitosan interpolymer complex (IPC) tablet was prepared and evaluated as an extended-release tablet matrix using theophylline (TPH) as a model drug. The release of TPH from the matrix tablet (TPH/PA/L-chitosan = 200 mg:150 mg:50 mg) was slower than that from PA only (TPH/PA/chitosans = 200 mg:200 mg:0 mg) or L-chitosan only (TPH/PA/L-chitosan = 200 mg:0 mg:200 mg) tablet. Turbidity measurements also indicated the optimum complexation ratio for IPC between PA/L-chitosan to be 1/3, indicating an acceptable relationship between the turbidity of the complex and the release ratio of TPH. These results suggest that an L-chitosan/PA complex would be potentially useful in an extended-release IPC tablet with high antioxidant activity.     

Synthesis, characterization and electrode adsorption studies of porphyrins coordinated to ruthenium(II) polypyridyl complexes

Two new porphyrins, meso-tris-3,4-dimethoxyphenyl-mono-(4-pyridyl)porphyrin (H₂MPy3,4DMPP) and meso-tris-3-methoxy-4-hydroxyphenyl-mono-(4-pyridyl)porphyrin (H₂MPy3M4HPP), and their ruthenium analogs obtained by coordination of [Ru(bpy)₂Cl]⁺ groups (where bpy = 2,2′-bipyridine) to the pyridyl nitrogens have been synthesized and studied by electronic absorption spectroscopy, cyclic voltammetry and spectroelectrochemistry. These ruthenated porphyrins couple Ru chromophores to porphyrins containing electroactive meso-substituents. The highest energy electronic absorption for the ruthenated complexes is assigned as a bpy(π) → bpy(π*) intraligand charge transfer while the next lowest energy electronic absorption is assigned as Ru(dπ) → bpy(π*) metal-to-ligand charge transfer (MLCT) transition. The Ru^III/II couples occur at approximately 0.95 V versus the SHE reference electrode in acetonitrile solutions. The first oxidation of the porphyrin is localized on the 3,4-dimethoxyphenyl and 3-methoxy-4-hydroxyphenyl substituents, respectively. Electroactive surfaces result from adsorption of these compounds onto glassy carbon electrodes followed by anodic cycling in acidic media.     

The roles of PGF2α and PGE2 in regression of the corpus luteum after intrauterine infusion of Arcanobacterium pyogenes in cows

To study the effect of bacteria in the uterus on the fate of the corpus luteum (CL), Arcanobacterium pyogenes was inoculated into the uteri of cows on Day 3 (Day 0 = day of spontaneous ovulation). Plasma concentrations of 13,14-dihydro-15-keto-PGF_2α (PGFM), 13,14-dihydro-15-keto-PGE₂ (PGEM) and progesterone (P₄) were determined. In five cows, the developing CL regressed and first-wave dominant follicles, which normally become atretic, ovulated (Group OV) after bacterial inoculation. In another five cows (Group NOV) and five control cows, the developing CL did not regress and first-wave dominant follicles did not ovulate. In Group OV, PGFM concentrations increased by 126.2 pg/mL (from 36.8 ± 7.8 pg/mL on Day 3 to 163 ± 37.2 pg/mL on Day 6), with an increase ratio of 5.8-fold. Conversely, in Group NOV, PGFM had a greater increase of 198.4 pg/mL (from 128.2 ± 27.8 pg/mL on Day 3 to 326.6 ± 115.1 pg/mL on Day 5), but the increase ratio was only 2.3-fold. Although PGEM tended to increase in both groups, raw increases and increase ratios were small. Bacterial inoculation into the uterus stimulated the release of prostaglandins and affected the fate of the CL; in that regard, the CL was affected more by PGF_2α than by PGE₂, and the increase ratio of PGF_2α was more important than the raw increase.     

Syntheses of di-hydrocarbyl derivatives of carbonyl phosphine complexes of iron

Complexes cis,trans-Fe(CO)₂(PMe₃)₂RR′ (R = CH₃, R′ = Ph (2); R = CH₃, R′ = CHCH₂ (3); R = CHCH₂, R′ = Ph (4); R = R′ = CHCH₂ (5); R = R′ = CH₃ (6)) were prepared by reaction of cis,trans-Fe(CO)₂(PMe₃)₂RCl (1) with organolithium reagents LiR′. All complexes were characterized in solution by IR and ¹H, ³¹P and, in a few cases, ¹³C NMR mono- and bi-dimensional spectroscopies. Complexes 5 and 6 were structurally characterized by X-ray diffractometric methods. In solution complexes 2, 3 and 4 undergo slowly coupling of the σ-hydrocarbyl substituents leading to Fe(CO)₃(PMe₃)₂ and other decomposition products. Complex 6 was very stable in solution in the absence of nucleophiles and in the solid state. Complex 5 transformed through intramolecular coupling of the vinyl groups into Fe(CO)(PMe₃)₂(η⁴-butadiene) (7), which was characterized in solution by IR and NMR spectroscopies.