Biosynthesis of cardenolides in Digitalis lanata

[8-³H]-Cholesterol was synthesized. A doubly labelled sample of [8-³H, 4-¹⁴C]-cholesterol was administered to Digitalis lanata plants and the cardenolides were isolated. Biosynthesized digitoxigenin and digoxigenin retained all the tritium. Barring the migration of the tritium in biosynthesis the results are interpreted as indicative that neither intermediates with δ7, δ⁷ or δ⁸⁸⁽¹⁴⁾ are participating in the elaboration of cardenolides.     

Cis and Trans isomers of 4-hydroxy-8-sphingenine in plant cerebrosides

Pure cerebroside was isolated from hay and concentrate (extracted oilseed used for cattle feed), and the composition of the long-chain bases determined. The principal base was the cis isomer of 4-hydroxy-8-sphingenine (t18: 1 ^8c) which has not been reported before. The other trihydroxy bases were t18: 1^8t and t18: 0.Allylic and non-allylic dihydroxy bases were found in concentrate cerebroside and identified as d18: 1⁴, d18: 2^{4, x}, d18: 0 and d18: 1^x (x = 8?). Cis and trans isomers of mid-chain double bonds (position x) were tentatively identified, and the dihydroxy bases are probably identical to those recently found in wheat flour cerebroside [2].     

Biosynthesis of mono- and sesqui-terpenes in peppermint from glucose-C and CO2

Labeled glucose and CO₂ are more efficient precursors of monoterpenes in peppermint (Mentha piperita L.) cuttings than is mevalonate, which is the best precursor of sesquiterpenes in this plant. Metabolic turnover of the labeled monoterpenes was observed, in agreement with previous observations. Pulegone derived from ¹⁴CO₂ after 1, 3, 6, 9 and 12 hr of incubation was chemically degraded, and in every case at least 90% of the ¹⁴C-label was found in the seven-carbon fragment containing the isopentenyl pyrophosphate-derived portion of the molecule. The isopropylidene side chain, containing three carbons hypothetically derived from dimethylallyl pyrophosphate, was found to be essentially unlabeled. The results suggest that an endogenous dimethylallyl pyrophosphate pool participates in monoterpene biosynthesis, much as earlier work had suggested that a similar pool participates in sesquiterpene biosynthesis in this plant. These findings are of particular interest because it appears, based on the differential utilization of labeled precursors, that monoterpenes and sesquiterpenes are produced at separate sites in the plant.     

Characterization of aminoalkylphosphonyl cerebrosides in muscle tissue of Turbo cornutus

From muscle tissues of the marine snail (Turbo cornutus) aminoalkylphosphonyl cerebrosides, which had been shown to be present in visceral parts, were isolated.Their structure was determined by degradative methods and by characterization of components by gas chromatography-mass spectrometry.The aminoalkylphosphonyl cerebroside fraction consisted of a major portion of 1-O-[6′-O-(N-methylaminoethylphosphonyl)galactosyl] ceramide and a minor portion of a novel lipid, 1-O-[6′-O-(aminoethylphosphonyl)galactosyl] ceramide.The fatty acids of the fraction were mainly palmitic (53.3%) and 2-hydroxy palmitic acid (14.6%). The long chain bases were mainly dihydroxy C_{22 : 2} (36.6%), C_{18 : 1} (14.6%) and C_{18 : 2} (11.3%), and trihydroxy bases were also found as minor components.     

Biosynthesis of nimbolide from [2-14C,(4R)4-3H1] mevalonic acid lactone in the leaves of Azadirachta indica

Nimbolide was biosynthesized from [2-¹⁴C, (4R)4-³H₁]mevalonic acid lactone in the leaves of Azadirachta indica. The nimbolide had a ³H:¹⁴C ratio of 3:5 which gives support to the suggestion of the involvement of a triterpenoid intermediate with a double bond at the Δ⁸⁽⁹)-position in the biosynthesis of nimbolide.     

Effect of pH on H-2H exchange,H2 production and H2 uptake,catalysed by the membrane-bound hydrogenase of Paracoccus denitrificans

(1) The kinetics of isotope exchange catalysed by the membrane-bound hydrogenase of Paracoccus denitrificans have been studied by measuring H²H, H₂ or ²H₂ produced when the enzyme catalyses the exchange between ²H₂ and H₂O or H₂ and ²H₂O. (2) In the ²H₂-H₂O system the measured rate of H₂ production was always higher than that of H²H. The $\text{H}{}_2\text{H}{}^2\text{H}$ ratio remained constant (about 1.70) in the protein concentration range 0.08–1.32 mg. The very rapid formation of H₂ with respect to H²H is consistent with the hypothesis of a heterolytic cleavage of ²H₂ into a deuteron and an enzyme hydride that can exchange with the solvent. (3) In the H₂-²H₂O system, the exchange rate was much lower than in the ²H₂-H₂O system, indicating a marked isotopic effect of ²H₂O. (4) The H-²H exchange activity, determined from the initial velocity of H²H formation, is optimal at pH 4.5. A second maximum of activity is observed at pH 8.3. The pH value of 4.5 is also the pH optimum for H₂ production while at pH 8.3–8.5 there is a maximum of H₂ oxidation activity. (5) In ordinary H₂O the K_m for hydrogen uptake estimated either from H₂ consumption or from benzyl viologen reduction was 0.06–0.07 μM for both H₂ and ²H₂ indicating a strong affinity of the enzyme for hydrogen at pH 8.3–8.5. Shifting from H₂O to ²H₂O does not affect the K_m of the enzyme for H₂ but lowers the V_max value about 10-fold. The K_m for benzyl viologen and methyl viologen was 0.08 and 2 mM, respectively.     

Free and bound hydroxyl and carboxyl groups in the cutin of Quercus suber leaves

The number of free and bound hydroxyl and carboxyl groups of the cutin of Quercus suber leaves was investigated by the lithium borohydride hydrogenolysis of mesyl-cutin compared with the lithium borohydride hydrogenolysis of untreated cutin. Fifty per cent of the vic-diol groups of the trihydroxy C₁₈ acid component and twenty five per cent of the secondary hydroxyl groups of the dihydroxy C₁₆ acid component are free. The rest of the secondary and all of the primary hydroxyl groups are esterified; all carboxyl groups are esterified.     

Analogs of arachidonic acid methylated at C-7 and C-10 inhibit leukotriene biosynthesis and phospholipase A2 activity

The ability of (all Z)-7,7-dimethyl-5,8,11,14-eico-satetraenoic acid, (all Z)-7,7-dimethyl-5,8,11-eicosatrienoic acid, (Z,Z)-7,7-dimethyl-5,8-eicosadienoic acid, (all Z)-10,10-dimethyl-5,8,11,14-eicosatetraenoic acid, (all Z)-10,10-dimethyl-5,8,11-eicosatrienoic acid, and rac-(Z,Z)-15-hydroxy-7,7-dimethyl-5,8-eicosadienoic acid to inhibit ionophore-induced slow-reacting substance of anaphylaxis (SRS-A) biosynthesis in rat peritoneal cells was studied. It was thought that compounds such as these might inhibit proton abstractions at the 7 or 10 carbon positions on arachidonic acid which are thought to be important in the mechanism of catalysis of Δ⁵-lipoxygenase(Δ⁵-LO). All compounds were found to be potent inhibitors of SRS-A biosynthesis in the in vitro rat peritoneal cell system (IC₅₀ < 10 μM). In fact they were more potent inhibitors in the test system than standard Δ⁵-LO inhibitors such as NDGA and quercetin. To determine if the mechanism of inhibition of the dimethyl arachidonic acid analogs did involve gD⁵-LO inhibition these compounds were evaluated in an assay system utilizing the Δ⁵-LO from rat basophilic leukemia (RBL^?1_cells. It was found, however, that these compounds were much less potent inhibitors of this enzyme (IC₅₀ ～ 100 μM) than standard compounds such as NDGA (IC₅₀ 0.14 μM) and quercetin (IC₅₀, 0.2 μM). The arachidonic acid analogs were subsequently found to be potent inhibitors of phospholipase A₂ (PLA₂) enzymes with IC₅₀'s between 10–20 μM as inhibitors of a snake venom enzyme. In fact these compounds are among the most potent inhibitors of PLA₂ yet studied, having potencies better than standards such as p-bromophenacyl bromide (IC₅₀, 87 μM) and U-10029A (IC₅₀, 36 μM). These results suggest that the methylated arachidonic acid analogs may inhibit SRS-A biosynthesis through inhibiting PLA₂.     

Human mast cells express two leukotriene C4 synthase isoenzymes and the CysLT1 receptor

Cysteinyl-leukotrienes (cys-LTs) are potent smooth muscle contracting agents, especially in the respiratory tract and microcirculation, and play a key role in inflammatory and allergic diseases. The final step in the biosynthesis of LTC₄, the parent compound of cys-LTs, is catalyzed by a specific GSH transferase termed LTC₄ synthase, which is typically expressed in certain bone marrow-derived cells such as eosinophils and mast cells.Here we report that the human mast cell line HMC-1 as well as human mast cells derived from cord blood (CBMC) express a second enzyme capable of synthesizing leukotriene C₄, i.e., microsomal GSH transferase type 2. Furthermore, these cells abundantly express CysLT₁ receptors that are mostly located at the surface of both types of mast cells, as judged by immunohistochemistry. In addition, stimulation of CBMC with LTC₄ and LTD₄ elicits an immediate and dose-dependent (10^?7–10^?11 M) mobilization of intracellular Ca²⁺, which can be blocked with specific CysLT₁ receptor antagonists. Taken together, our data suggest that human mast cells are equipped with two enzymes that can catalyze the committed step in the biosynthesis of cys-LTs. Moreover, the expression of the cognate receptor CysLT₁ suggests that these lipid mediators may be involved in autocrine signaling pathways regulating mast cell functions.     

Simulation of fermentation conditions for vitamin B12 biosynthesis from whey

Vitamin B₁₂ production in fermentation of Propionibacterium shermanii and Propionibacterium arl AKU 1251 in whey permeate medium has been studied. The observed results and simulated expected values obtained by fitting statistical equations to the recorded data showed that 24 h old inoculum, 5 mg iron l^?1 and 4% whey lactose were optimal for vitamin B₁₂ biosynthesis in both strains when fermentation was carried out under anerobic (84 h) and aerobic (84 h) conditions at 30°C. The supplementation of whey medium with 0.5% (NH₄)₂HPO₄ enhanced further the metabolite yield; however, the preference for a mixed carbon source (lactose + d-glucose or lactose + d-fructose) at different levels varied in the strains under study. P. shermanii, under optimal cultural conditions, was found to be a better strain than Propionibacterium arl AKU, 1251 in fermenting whey lactose for product (vitamin B₁₂) formation.     

Pseudojujubogenin,a new sapogenin from Bacopa monniera

Acid hydrolysis of bacoside A, a saponin of Bacopa monniera, afforded ebelin lactone and bacogenin-A₁, while Smith-de Mayo degradation of bacoside A yielded jujubogenin and pseudojujubogenin as sapogenins. The structure of pseudojujubogenin except the absolute configurations at C-20 and C-22 was established by chemical and spectroscopic investigations. Bacoside A was separated by droplet counter-current chromatography into two fractions, A₁ and A₂, which, however, are mixtures, since they both afforded two sapogenins, in different ratios by Smith-de Mayo degradation.     

Bile salt alteration of ion transport across jejunal mucosa

The effect of conjugated dihydroxy and trihydroxy bile salts on electrolyte transport across isolated rabbit jejunal mucosa was studied. Both taurochenodeoxycholic acid and taurocholic acid increased the short-circuit current (I_sc) in bicarbonate-Ringer solution but not in a bicarbonate-free, chloride-free solution. Taurochenodeoxycholic acid was significantly more effective than taurocholic acid in increasing I_sc. The presence of theophylline prevented the taurochenodeoxycholic acid-and taurocholic acid-induced increase in I_sc. Transmural ion fluxes across jejunal mucosa demonstrated that 2 mM taurochenodeoxycholic acid decreased net Na⁺ absorption, increased net Cl⁻ secretion and increased the residual flux (which probably represents HCO₃⁻ secretion). These studies support the hypothesis that cyclic AMP may be a mediator of intestinal electrolyte secretion.     

Somatic variations on a yellow mutant in Nicotiana tabacum L. (a1+/a1a2+/a2) I. Non-reciprocal genetic events occurring in leaf cells

A greenish-yellow mutant was obtained after treatment of seeds of Nicotiana tabacum L. var. Xanthi n.c. with ethyl methanesulfonate (EMS). Two genetically independent mutations (a₁ and a₂) were isolated. The first mutation (a₁) antagonizes the function of its partially dominant a₁⁺ allele. The second mutation (a₂) is amorphous but strongly interacts with a₁.Among the nine possible genotypes at the two loci, five varied in somatic cells. The heterozygous state a₁⁺/a₁ strongly increased the frequency of both spontaneous and induced variations. However, two homozygotes also showed variations.Variants were isolated from induced and spontaneous non-reciprocal and reciprocal variations within paliside tissues by bud induction in vitro. They were genetically tested. In this first paper, only non-reciprocal variations are reported.Green variants from the greenish-yellow (J¹) dihybrid a₁⁺/a₁a₂⁺/a₂ clone had two genotypes: the first was due to true reversions of a₁ to a₁⁺, whereas the second was due to amorphous a₁⁰ mutations from a₁. These a₁⁰ mutations may well be deletions.The lightest yellow variants from J¹ were due to mutations either from a₁⁺ into a₁ or from a₂⁺ into a₂.Deletions at the a₁⁺?a₁ locus led to either yellow variations when a₁⁺ was lost, or to false reversions when the antagonistic allele a₁ was lost.Amorphous alleles at the a₁⁺?a₁ locus were also isolated from tissues other than J⁺. They gave zygotic lethality (s) that probably varied with the size of the deletions. Thus, true reversions and deletions at the a₁⁺?a₁ locus could be distinguished from one another by progeny tests.Other variants showed higher frequencies of spontaneous variations (instability). Somatic changes observed in these unstable systems were due to modifications at the marker loci. The genetic nature of this instability is not yet known.There is strong evidence that the genetic events involved in these non-reciprocal variations were deletions, conversions and point mutations. True reversions from a₁ into a₁⁺ and new mutations from a₁⁺ into a₁ were obtained only from a₁⁺/a₁. It was therefore supposed that the changes observed took place only in heterozygotes, and the conversion hypothesis was made. Attempts are being made to prove that conversions do exist in higher plants, and to find out if this process, as deletions, is induced by radiation.     

NMR study of 17O from H217O in human erythrocytes

Human erythrocytes were incubated in a Ringer's solution enriched with 10–18% H₂¹⁷O. The longitudinal relaxation time (T₁) of the ¹⁷O was determined separately in samples of red cell suspesions, packed cells, and supernatant. The longitudinal relaxation of ¹⁷O in erythrocyte suspensions was non-exponential, reflecting water exchange across the cell membranes as well as relaxation processes inside and outside the cell.The T₁ of intracellular ¹⁷O is 4–5 times shorter than in the supernatant, similar to the enhancement of proton relaxation by hemoglobin in erythrocytes and free solution at the frequency applied (8.13 MHz). This datum is consistent with the thesis that hemoglobin modifies the NMR relaxation behavior of water inside cells and in free solution in the same way.The rate constant

for water exchange was calculated to be 60 and 107 s⁻¹ at 25 and at 37° C, respectively. The apparent activation energy for

over the temperature range 23–37° C was 8.7±1.0 kcal/mole.     

Incorporation of methionine-[methyl-2H3] and mevalonic acid-[2-14C,(4R)-4-3H1] into Phycomyces blakesleeanus sterols

Ergosterol, episterol, 4α-methyl-5α-ergosta-8,24(28)-dien-3β-ol and 24-methylene-24,25-dihydrolanosterol, isolated from Phycomyces blakesleeanus grown in the presence of methionine-[methyl-²H₃], each contained two deuterium atoms; lanosterol, however, was unlabelled. The ¹⁴C:³H atomic ratio of the following sterols isolated from P. blakesleeanus grown in the presence of mevalonic acid-[2-¹⁴C,(4R)-4-³H₁], was: ergosterol, 5:3; episterol, 5:4; ergosta-5,7,24(28)-trien-3β-ol, 5:3; 4α-methyl-5α-ergosta-8,24(28)-dien-3β-ol, 5:4; 24-methylene-24,25-dihydrolanosterol, 6:5; lanosterol, 6:5. The significance of these results in terms of ergosterol biosynthesis is discussed.     

施氮对水稻土N2O释放及反硝化功能基因(narG/nosZ)丰度的影响

以紫潮泥和红黄泥两种不同质地的水稻土壤作为研究对象,通过室内培养试验,分析施用硝态氮肥对N2O释放和反硝化基因(narG/nosZ)丰度的影响,并探讨反硝化基因丰度与N2O释放之间的关系。结果表明,施用硝态氮显著增加两种水稻土的N2O释放量。在72h培养过程中,施氮改变了紫潮泥反硝化基因(narG/nosZ)的丰度,但并未明显影响红黄泥反硝化基因(narG/nosZ)丰度。通过双变量相关分析发现,除了紫潮泥narG基因外,其它的反硝化基因丰度和N2O释放之间并没有显著相关性。     

Effects of pH on the interaction of ligands with the (H+ + K+)-ATPase purified from pig gastric mucosa

The effects of K⁺, Na⁺ and ATP on the gastric (H⁺ + K⁺)-ATPase were investigated at various pH. The enzyme was phosphorylated by ATP with a pseudo-first-order rate constant of 3650 min^?1 at pH 7.4. This rate constant increased to a maximal value of about 7900 min^?1 when pH was decreased to 6.0. Alkalinization decreased the rate constant. At pH 8.0 it was 1290 min^?1. Additions of 5 mM K⁺ or Na⁺, did not change the rate constant at acidic pH, while at neutral or alkaline pH a decrease was observed. Dephosphorylation of phosphoenzyme in lyophilized vesicles was dependent on K⁺, but not on Na⁺. Alkaline pH increased the rate of dephosphorylation. K⁺ stimulated the ATPase and p-nitrophenylphosphatase activities. At high concentrations K⁺ was inhibitory. Below pH 7.0 Na⁺ had little or no effect on the ATPase and p-nitrophenylphosphatase, while at alkaline pH, Na⁺ inhibited both activities. The effect of extravesicular pH on transport of H⁺ was investigated. At pH 6.5 the apparent K_m for ATP was 2.7 μM and increased little when K⁺ was added extravesicularly. At pH 7.5, millimolar concentrations of K⁺ increased the apparent K_m for ATP. Extravesicular K⁺ and Na⁺ inhibited the transport of H⁺. The inhibition was strongest at alkaline pH and only slight at neutral or acidic pH, suggesting a competition between the alkali metal ions and hydrogen ions at a common binding site on the cytoplasmic side of the membrane. Two H⁺-producing reactions as possible candidates as physiological regulators of (H⁺ + K⁺)-ATPase were investigated. Firstly, the hydrolysis of ATP per se, and secondly, the hydration of CO₂ and the subsequent formation of H⁺ and HCO₃^?. The amount of hydrogen ions formed in the ATPase reaction was highest at alkaline pH. The H⁺/ATP ratio was about 1 at pH 8.0. When CO₂ was added to the reaction medium there was no change in the rate of hydrogen ion transport at pH 7.0, but at pH 8.0 the rate increased 4-times upon the addition of 0.4 mM CO₂. The results indicate a possible co-operation in the production of acid between the H⁺ + K⁺-ATPase and a carbonic anhydrase associated with the vesicular membrane.     

Desmosterol als biogenetische vorstufe für convallamarogenin

Labelled 4-¹⁴C-desmosterol, but not cholesterol, is used by plants of Convallaria majalis L. for the biosynthesis of convallamarogenin. It can therefore be concluded that precursors of cholesterol biosynthesis can in some cases be converted directly into steroidal sapogenins, the pathway via cholesterol not always being obligatory.     

Kinetics study of the substitution reaction of fac-[Fe(CN)2(CO)3I] with PPh3

Substitution reaction of fac-[Fe^II(CN)₂(CO)₃I]⁻ with triphenylphosphine (PPh₃) produced mono phosphine substituted complex cis-cis-[Fe^II(CN)₂(CO)₂(PPh₃)I]⁻. Crystal structure of the product showed that carbonyl positioned trans- to iodide was replaced by PPh₃. The substitution reaction was monitored by quantitative infrared spectroscopic method, and the rate law for the substitution reaction was determined to be rate = k[[Fe^II(CN)₂(CO)₂(PPh₃)I]⁻][PPh₃]. Transition state enthalpy and entropy changes were obtained from Eyring equation k = (k_BT/h)exp(−ΔH^≠/RT + ΔS^≠/R) with ΔH^≠ = 119(4) kJ mol⁻¹ and ΔS^≠ = 102(10) J mol⁻¹ K⁻¹. Positive transition state entropy change suggests that the substitution reaction went through a dissociative pathway.