Dimeric and trimeric proanthocyanidins possessing a doubly linked structure from Pavetta owariensis

Pavetannin A-2, a new A-type proanthocyanidin, along with the trimers cinnamtannin B-1, pavetannin B-1, B-2, B-3, B-5 and B-6 have been isolated in their free phenolic form from the stem bark of Pavetta owariensis. Spectral data and partial acid-catalysed degradation established their structures as ent-epicatechin-(4→8,2→O→7)-catechin, epicatechin-(4β→8,2β→O→7)-epicatechin-(4→8)-epicatechin, epicatechin-(4β→8,2β→O→7)-epicatechin-(4β→8)-ent-epicatechin, epicatechin-(4β→8,2β→O→7)-epicatechin-(4β→8)-epicatechin, epicatechin-(4β→6,2β→O→7)-epicatechin-(4β→8)-epicatechin, epicatechin-(4β→6,2β→O→7)-catechin-(4β→8)-epicatechin, epicatechin-(4β→8,2β→O→7)-epicatechin-(4→8)-catechin, respectively.     

Structural characteristics and rheological properties of partially hydrolyzed oat β-glucan: the effects of molecular weight and hydrolysis method

Partial hydrolysates of (1→3)(1→4)-β- -glucan from oats were produced by three hydrolysis methods: acid, cellulase or lichenase. The molecular weights ranged from 31 000 to 237 000 g/mol. Six percent solutions of small molecular weight β-glucans formed elastic gels after 4 days at 4 °C whereas larger molecular weight β-glucans remained viscous liquids after 7 days. The melting temperature of the gels increased as they aged and the peak heat flow temperature, measured by differential scanning calorimetry, was 62±2 °C. Partial hydrolysates produced with cellulase, which was shown to preferentially cleave regions of the molecule with longer contiguous β-(1→4)-linked -glucopyranosyl units, tended to produce more elastic gels with stronger junction zones than partial hydrolysates produced with lichenase which cleaves the β-(1→4) glycosidic 3-o-substituted glucose links. This suggests that β-(1→3)-linked cellotriose sections of the polymer are probably the segments which form the junction zones in the gel network rather than cellulose-like segments.     

Spinach-thylakoid phosphorylation: Studies on the kinetics of changes in photosystem antenna size, spill-over and phosphorylation of light-harvesting chlorophyll a/b protein

The kinetics of LHCP phosphorylation and associated changes in photosystem cross-section and energy ‘spill-over’ from PS II to PS I have been examined in isolated spinach chloroplasts. During an initial phosphorylation period of 3–6 min, in the presence of saturating concentrations of Mg²⁺, the increase in PS I and decrease in PS II cross-section are largely completed, as judged by both measurements of the steady-state redox state of Q and fluorescence yield changes. This corresponds to a period of rapid ³²P incorporation into the low-molecular weight LHCP polypeptide. Subsequent to this initial 3–6-min period there is substantial further phosphorylation of both LHCP polypeptides, which is not accompanied by significant changes in photosystem cross-section, even after the chloroplasts had been unstacked with extensive mixing of PS I and PS II by Mg-removal. It is suggested that there exists a specific ‘mobile’ population of LHCP molecules which is rapidly phosphorylated and which may be enriched in the low-molecular-weight polypeptide. In addition, measurements of the kinetics of the ‘spill-over’ changes upon either Mg²⁺ addition or removal indicate that the continued phosphorylation of LHCP is able to increase the ‘spill-over’ process under favourable ionic conditions.     

Some preliminary results on the action of rhamnogalacturonase on rhamnogalacturonan oligosaccharides from beet pulp

Sugar-beet pulp was saponified and then hydrolysed with 0.1 M HCI at 80°C for 72 h, and a rhamnogalacturonan fraction was isolated by ion-exchange chromatography on AG 1X8 resin. Four individual oligomers, and a mixture of oligomers with higher degrees of polymerization, were obtained by chromatography on BioGel P-4. They all presented the -d-GalAp-(1 [→2)--l-Rhap-(1 → 4)--d-GalAp-(1]_n→2)-l-Rhap structure (with n 2) The five fractions were submitted to hydrolysis with rhamnogalacturonase. The enzyme was active on oligomers with degrees of polymerization f10, and gave as main products -l-Rhap-(1 → 4)--d-GalAp-(1 → 2)--l-Rhap(1 → 4)-d-GalAp and -d-GalAp-(1 → 2)--l- Rhap- (1 → 4) --d- GalAp- (1 → 2) --l-Rhap-(1 → 4)-d-GalAp.     

Induction of lacI mutations in Big Blue Rat-2 cells treated with 1-(2-hydroxyethyl)-1-nitrosourea: a model system for the analysis of mutagenic potential of the hydroxyethyl adducts produced by 1,3-bis (2-chloroethyl)-1-nitrosourea.

We have investigated the genotoxic effects of 1-(2-hydroxyethyl)-1-nitrosourea (HENU). We have chosen this agent because of its demonstrated ability to produce N7-(2-hydroxyethyl) guanine (N7-HOEtG) and O⁶-(2-hydroxyethyl) 2′-deoxyguanosine (O⁶-HOEtdG); two of the DNA alkylation products produced by 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU). For these studies, we have used the Big Blue Rat-2 cell line that contains a lambda/lacI shuttle vector. Treatment of these cells with HENU produced a dose dependent increase in the levels of N7-HOEtG and O⁶-HOEtdG as quantified by HPLC with electrochemical detection. Treatment of Big Blue Rat-2 cells with either 0, 1 or 5 mM HENU resulted in mutation frequencies of 7.2±2.2×10⁻⁵, 45.2±2.9×10⁻⁵ and 120.3±24.4×10⁻⁵, respectively. Comparison of the mutation frequencies demonstrates that 1 and 5 mM HENU treatments have increased the mutation frequency by 6- and 16-fold, respectively. This increase in mutation frequency was statistically significant (P<0.001). Sequence analysis of HENU-induced mutations have revealed primarily G:C→A:T transitions (52%) and a significant number of A:T→T:A transversions (16%). We propose that the observed G:C→A:T transitions are produced by the DNA alkylation product O⁶-HOEtdG. These results suggest that the formation of O⁶-HOEtdG by BCNU treatment contributes to its observed mutagenic properties.     

Determination of the substance P receptor antagonist CP-122, 721 in plasma by narrow-bore high-performance liquid chromatography-ionspray tandem mass spectrometry

A simple, highly sensitive and specific LC-MS-MS assay was developed for the determination of CP-122,721 (I) in rat and human plasma. I and a structural analog, CP-129,943 (II, internal standard), were extracted from plasma with methyl tert.-butyl ether (MTBE). The dried MTBE extracts were reconstituted and analyzed using a narrow-bore (2.1 mm I.D.) YMC basic HPLC column and a mobile phase of acetonitrile-20 mM ammonium acetate, pH 5 (50:50, v/v). Column effluents were monitored by ionspray tandem mass spectrometry. Multiple reaction monitoring (MRM) using the parent to product ion combinations of m/z 381 → 205 and 395 → 219 was used to quantitate I and II, respectively. The assay exhibited a linear dynamic range of 0.2–100 ng/ml. Absolute recoveries from plasma were above 80% for both I and II. The precision and accuracy values for the method were within ±3 and ±9%, respectively. Sample analysis times were less than 5 min from one injection to the next. The assay has proved to be applicable to the pharmacokinetic study of I in rats.     

Are the renal functional changes of human pregnancy caused by prostacyclin?

To investigate the hypothesis of others that the many characteristic physiological alterations of pregnancy are due to prostacyclin, the effects of this vasodilator prostaglandin on renal function and related variables were measured.

Intravenous infusion of prostacyclin (7 ng/kg/min) for 90 minutes in seven healthy male volunteers resulting in a significant fall in diastolic blood pressure and increase in heart rate. No significant change occurred in renal plasma flow or glomerular filtration rate in response to the prostacyclin, but there were significant falls in uric acid clearance (16 ± 1.3 → 10 ± 1.5 ml/min/ 1.73m²) and in free water clearance (4.0 ± 1.1 → −0.5 ± 0.4 ml/min) both of which rose rapidly to pre-infusion levels upon cessation of prostacyclin infusion. Plasma renin and aldosterone levels rose sharply during prostacyclin infusion, falling to pre-infusion levels rapidly afterwards.

Although the haemodynamic alterations were similar, the renal functional effects of prostacyclin infusion were quite different from (and often opposite in direction to) those of normal pregnancy. This acute study therefore does not add support to the hypothesis suggested by others that these alterations in pregnancy are due to prostacyclin, and that deficiency of prostacyclin will lead to the changes of pregnancy-associated hypertension.     

Microbial transformation of baccatin VI and 1beta-hydroxy baccatin I by Aspergillus niger

The biotransformation of baccatin VI (1) and 1β-hydroxybaccatin I (2) with the filamentous fungus Aspergillus niger produced four new taxane diterpenoids taxumairol S₁ (3), taxumairol T₁ (4) and taxumairol S (5), taxumairol T (6), respectively. 1β-Dehydroxybaccatin VI (7) remained unreacted under the same condition.     

Synthesis of the tetrasaccharide repeating-unit of the lipopolysaccharide isolated from pseudomonas maltophilia

The title tetrasacharide having the structure 3-O-Me-β- -Xylp-(1→4)-- -Rhap-(1→4)-- -Rhap-(1→2)- -Rhap was obtained by reaction of the -acetobromo derivative of 4-O-(3-O-methyl-β- -xylopyranosyl)- -rhamnopyranose and benzyl 3,4-di-O-benzyl-2-O-(2,3-O-isopropylidene-- -rhamnopyranosyl)-- -rhamnopyranoside, followed by removal of the protecting groups. The synthesised compounds were characterised on the basis of n.m.r. data.     

Mg2+ ion effect on conformational equilibrium of poly A . 2 poly U and poly A poly U in aqueous solutions

Differential UV spectroscopy and thermal denaturation were used to study the Mg²⁺ ion effect on the conformational equilibrium in poly A · 2 poly U (A2U) and poly A · poly U (AU) solutions at low (0.01 M Na⁺) and high (0.1 M Na⁺) ionic strengths. Four complete phase diagrams were obtained for Mg²⁺–polynucleotide complexes in ranges of temperatures 20–96 °C and concentrations (10⁻⁵–10⁻²) M Mg²⁺. Three of them have a ‘critical’ point at which the type of the conformational transition changes. The value of the ‘critical’ concentration ([Mg_t²⁺]_cr=(4.5±1.0)×10⁻⁵ M) is nearly independent of the initial conformation of polynucleotides (AU, A2U) and of Na⁺ contents in the solution. Such a value is observed for Ni²⁺ ions too. The phase diagram of the (A2U+Mg²⁺) complex with 0.01 M Na⁺ has no ‘critical’ point: temperatures of (3→2) and (2→1) transitions increase in the whole Mg²⁺ range. In (AU+Mg²⁺) phase diagram at 0.01 M Na⁺ the temperature interval in which triple helices are formed and destroyed is several times larger than at 0.1 M Na⁺. Using the ligand theory, a qualitative thermodynamic analysis of the phase diagrams was performed.     

Ricochet inter-ring haptotropic rearrangement of σ-methyl-(η-indenyl) chromium tricarbonyls. Experimental kinetic and theoretical DFT study

σ-Methyl-(η⁵-indenyl) chromium tricarbonyl (III) rearranges quantitatively into η⁶-1-endo-methylindene) chromium tricarbonyl (IV) in C₆D₆ solution at 30–60°C. Methyl group attachment to the positions 2 or 3 of indenyl ligand in (III) has no influence on the activation parameters of this ricochet inter-ring haptotropic rearrangement (ΔG^#=23.6 kcal mol⁻¹; ΔH^#=18.9±0.2 kcal mol⁻¹; ΔS^#=−18.6±0.2 cal K⁻¹ mol⁻¹). (IV) undergoes further irreversible isomerization at 60–120° into (ν⁶-3-methylindene) chromium tricarbonyl (V) with a higher activation barrier (ΔG^#=28.5±0.1 kcal mol⁻¹) via two consecutive [1,5]-sigmatropic hydrogen shifts. The mechanisms of both rearrangements have been studied in detail using density functional theory (DFT) calculations with extended basis sets. Calculations show that the rearrangement (III) → (IV) proceeds in two steps. Methyl group migration from chromium into position 1 of the indenyl ligand is the rate-determining step leading to the formation of the 16-electron intermediate (VII). The calculated activation barrier (E_a=19.6 kcal mol⁻¹) is in good agreement with the experimental one. Further rearrangement (VII) → (V) proceeds via a trimethylenemethane-type transition state (XVIII) with an activation barrier 11.8 kcal mol⁻¹. The coordination of the chromium tricarbonyl group at the six-membered ring has only minor influence on the kinetic parameters of the hydrogen [1,5]-sigmatropic shift in indene.     

Species- and clone-specific responses to environmental stimuli in the cladocerans Bosmina cornuta and B. pellucida - a comparison with Daphnia

We studied the variation of small-scale swimming behaviour in eight Bosmina cornuta and ten B. pellucida clones in response to key environmental factors to test whether swimming behaviour and genotypes are linked in non-Daphnia cladocerans. We quantified (1) the short-term responses to changes in temperature, light intensity and pH, (2) the response to long-term temperature acclimation, and (3) the pH-related survival rates. Vertical swimming activity S was quantified in cuvette experiments as crossings of a line at 2 cm height per individual an hour. S differed significantly among species and conspecific clones. At any temperature, light intensity and pH tested, B. cornuta (clone variation: 40-58 crossings/ind.× h) showed a higher vertical swimming activity than B. pellucida (clone variation: 25-48 crossings/ind.× h). A short-term change of water temperature (range tested: 10-25°C) only affected S of B. cornuta, whereas that of B. pellucida remained unaltered. In contrast, S increased with rising temperature following long-term temperature acclimation (range tested: 10-20°C) in both species. Swimming activity was inversely related to the light intensity (range tested: 60-60,000 lux), but decrease of activity was stronger in B. pellucida (44 → 12 crossings/ind × h) than in B. cornuta (50 → 40 crossings/ind.× h). Short-term changes of pH (range tested: 4-6) did not influence swimming activity in any species, although a prolonged exposure (24 h) to pH 4 was lethal. Thus, Bosmina showed behavioural responses which permit to distinguish between the species and which are related to their seasonal succession and distribution pattern.     

Tachykinins (Substance P, Neurokinin A and Neuropeptide γ) and Neurotensin from the Intestine of the Burmese Python, Python molurus

Conlon, J. M., T. E. Adrian and S. M. Secor. Tachykinins (substance P, neurokinin A and neuropeptide γ,) and neurotensin from the intestine of the burmese python, Python molurus. Peptides 18(10) 1505–1510, 1997.—Peptides with substance P-like immunoreactivity, neurokinin A-like immunoreactivity and neurotensin-like immunoreactivity were isolated in pure form from an extract of the intestine of the Burmese python (Python molurus). The primary structure of python substance P (Arg-Pro-Arg-Pro-Gln-Gln-Phe-Tyr-Gly-Leu-Met-NH₂) shows one amino acid substitution (Phe⁸ → Tyr) compared with chicken/alligator substance P and an additional substitution (Lys³ → Arg) as compared with mammalian substance P. The neurokinin A-like immunoreactivity was separated into two components. Python neuropeptide γ (Asp-Ala-Gly-Tyr-Ser-Pro-Leu-Ser-His-Lys-Arg-His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH₂ shows three substitutions (Gly⁵ → Ser, Gln⁶ → Pro and Ile⁷ → Leu) compared with alligator neuropeptide γ and an additional substitution (His⁴ → Tyr) compared with mammalian neuropeptide γ. Python neurokinin A (His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met.NH₂) is identical to human/chicken/alligator neurokinin A. Python neurotensin (pGlu-Leu-Val-His-Asn-Lys-Ala-Arg-Pro-Tyr-Ile-Leu) is identical to chicken/alligator neurotensin. The data are indicative of differential evolutionary pressure to conserve the amino acid sequences of reptilian gastrointestinal peptides.     

Energy transfer within the isolated light-harvesting B800–850 pigment-protein complex of Rhodobacter sphaeroides

We have used picosecond absorption spectroscopy with low intensity (5 · 10¹¹–5 · 10¹² photons · pulse⁻¹ · cm⁻²) continuously tunable infrared (800–900 nm) pulses to study the energy transfer dynamics in the isolated B800–850 pigment-protein complex of Rhodobacter sphaeroides. Our results suggest the following picture of the energy transfer dynamics: (i) a fast transfer, within approx. 1 ps, from BChl 800 to BChl 850; (ii) transfer among different BChl 800's with a rate which is at the most of the same order of magnitude as that of BChl 800 → BChl 850 transfer; (iii) very fast transfer (k > 1 · 10¹² s⁻¹) between BChl 850 molecules. Assuming Förster type of energy transfer maximum distances of about 22 and 15 Å are obtained for the BChl 800–BChl 850 and BChl 850–BChl 850 separations, respectively.     

Cordiachromes from Auxemma oncocalyx

Further cordiachromes, rel-10,11β-epoxy-11-ethoxy-8-hydroxy-2-methoxy-8aβ-methyl-5,6,7,8,8a,9,10aβ-octahydro-1,4-anthracendione, 6-formyl-2-methoxy-9-methyl-7,8-dihydro-1,4-phenanthrendione, rel-8,11;9,11-diepoxy-1,4-dihydroxy-2-methoxy-8aβ-methyl-5,6,7,8,8a,9,10,10aβ-octahydro-10-anthracenone, rel-9,11-epoxy-1,4,8-trihydroxy-2-methoxy-8aβ-methyl-5,6,7,8,8a,9,10,10aβ-octahydro-10-anthracenone, rel-2″-methoxy-7″-methyl-1″,4″-naphtalendione-(6″→5)-tetrahydropyran-(2-eq→O→2ax)-tetrahydropyran-(5′→6)- 2-methoxy-7-methyl-1,4-naphthalendione, together with the known, allantoin, sitosterol and 3β-O-d-glucopyranosylsitosterol, have been isolated from Auxemma oncocalyx. Their structures were determined from spectral data, including 2D NMR experiments.     

Multi-site phosphorylation in ox-kidney branched-chain 2-oxoacid dehydrogenase complex

Tryptic [³²P]phosphopeptides were prepared from [³²P]phosphorylated ox-kidney branched-chain complex and analysed by high-voltage paper electrophoresis at pH 1.9. In the maximally phosphorylated complex 3 tryptic [³²P]phosphopeptides were identified (TA, TB, TC). R_F-values relative to N⁶-dinitrophenyllysine were (mean ± SEM for 25 obs.): TA, 1.53 ± 0.03; TB, 1.07 ± 0.02; TC, 0.65 ± 0.01. Relative rates of phosphorylation were TA> TB> TC. Phosphorylation of TA reached a maximum when about 66% of the complex was inactivated. Phosphorylation of TB and TC was associated mainly with 66–95% inactivation of the complex.     

A pterocarpan and two isoflavans from alfalfa

(−)6aR,11aR-Dihydro-3-hydroxy-9,10-dimethoxy-6H-benzofuro[3,2c] [1]-benzopyran (10-methoxymedicarpin), (+)-(2,3,4,-trimethoxyphenyl)-2,3-dihydro-7-hydroxy-4H-1-benzopyran (7-hydroxy-2′,3′,4′-trimethoxyisoflavan) and (+)-(2,3,4-trimethoxy-5-hydroxyphenyl)-2,3-dihydro-7-hydroxy-4H-1-benzopyran (7,5′-dihydroxy-2′,3′,4′-trimethoxyisoflavan) were isolated for the first time from dried Medicago sativa hay. Structural assignments were based on ¹H NMR and mass spectra, X-ray crystallography, and optical rotations.     

Six new C21 steroidal glycosides from Asclepias curassavica L

Six new C₂₁ steroidal glycosides, named curassavosides A–F (3–8), were obtained from the aerial parts of Asclepias curassavica (Asclepiadaceae), along with two known oxypregnanes, 12-O-benzoyldeacylmetaplexigenin (1) and 12-O-benzoylsarcostin (2). By spectroscopic methods, the structures of the six new compounds were determined as 12-O-benzoyldeacylmetaplexigenin 3-O-β-d-oleandropyranosyl-(1 → 4)-β-d-digitoxopyranoside (3), 12-O-benzoylsarcostin 3-O-β-d-oleandropyranosyl-(1 → 4)-β-d-digitoxopyranoside (4), sarcostin 3-O-β-d-oleandropyranosyl-(1 → 4)-β-d-canaropyranosyl-(1 → 4)-β-d-oleandropyranosyl-(1 → 4)-β-d-digitoxopyranoside (5), sarcostin 3-O-β-d-oleandropyranosyl-(1 → 4)-β-d-canaropyranosyl-(1 → 4)-β-d-canaropyranosyl-(1 → 4)-β-d-digitoxopyranoside (6), 12-O-benzoyldeacylmetaplexigenin 3-O-β-d-glucopyranosyl-(1 → 4)-β-d-oleandropyranosyl-(1 → 4)-β-d-canaropyranosyl-(1 → 4)-β-d-oleandropyranosyl-(1 → 4)-β-d-digitoxopyranoside (7), and 12-O-benzoylsarcostin 3-O-β-d-glucopyranosyl-(1 → 4)-β-d-oleandropyranosyl-(1 → 4)-β-d-canaropyranosyl-(1 → 4)-β-d-oleandropyranosyl-(1 → 4)-β-d-digitoxopyranoside (8), respectively. All compounds (1–8) were tested for in vitro cytotoxicity; only compound 3 showed weak inhibitory activity against Raji and AGZY cell lines.