"Universal Warming" protocol for vitrified oocytes to streamline cell exchange for transnational donation programs: a multi-center study.

PURPOSE: To investigate the clinical efficacy of a "Universal Warming" protocol, based on subsequent steps with 1 M and 0.5 M concentration of extracellular cryoprotectant (ECCP), on shipped oocytes. Oocytes are vitrified using different brands of ready-to-use kits which recommend that the use of their own warming kit and combining different vitrification/warming kits may have legal consequences for assisted reproductive (AR) centers, until this practice has been validated with clinical studies. METHODS: Retrospective multi-center transnational observational study. Number of oocytes warmed 1.898. Vitrification performed with vitrification kit (Kitazato, Japan); warming carried out randomly with two different kits: Kitazato warming kit and Vit Kit®-Thaw (FujiFilm Irvine, USA). Warmed oocytes were assigned to 2 groups: KK (Kitazato/Kitazato) 939, and KI (Kitazato/Irvine) 959. Primary endpoint: survival rate. Secondary endpoints: fertilization rate; blastulation rate; implantation rate; live birth rate. RESULTS: Survival was comparable between the groups: 84.6% (795/939) in group KK vs 82.1% (787/959) in group KI. Fertilization rate was lower (P = 0.027) in group KK (75.7%-602/795) than in group KI (80.4%-633/787). Blastulation and implantation and live birth rates were all statistically comparable between the study groups: blastulation rate was 58.5% (352/602) vs 57.8% (366/633); implantation rate was 41.5% (80/193) vs 45.9% (84/183); live birth rate was 52.5% (62/118) in KK and 45.0% (54/120) in KI. CONCLUSION: The use of this "Universal Warming" protocol simplifies vitrified oocyte exchange between AR centers in different countries, and overcomes potential regulatory/commercial/availability differences affecting clinical practice.

Microencapsulation as a tool to counteract the typical low bioavailability of polyphenols in the management of diabetes.

Polyphenols are secondary metabolites widely distributed in many plant foods, such a tea, coffee, chocolate and fruits. The consumption of these compounds is related to the improvement or amelioration of many diseases, including diabetes. Nevertheless, the great barrier to the therapeutic use of polyphenols is the low bioavailability of these compounds once ingested. For that reason, the encapsulation of polyphenols in different matrices may protect them from digestion and improve their release and subsequent absorption to obtain target-specific health effects. Some studies have reported the beneficial effect of encapsulation to increase both bioavailability and bioaccessibility. However, these works have mostly been carried out in vitro and few studies are specifically addressed at improving diabetes. In the current work, an overview of the knowledge related to nanoparticles and their use in the diabetic condition has been reviewed.

Testing the efficacy and efficiency of a single "universal warming protocol" for vitrified human embryos: prospective randomized controlled trial and retrospective longitudinal cohort study.

PURPOSE: To study the efficacy and efficiency of a "universal warming protocol" for vitrified human embryos, based on subsequent steps with 1 and 0.5 M concentration of extracellular cryoprotectant (ECCP). METHOD: Two studies on patients undergoing fertility treatments via ICSI: a prospective randomized controlled trial (RCT) and a retrospective cohort study (CS). SETTING: Private assisted reproductive (AR) center. RCT: duration 01/03/2017-01/10/2017; 315 embryos at blastocyst stage obtained from 169 patients. Each patient's embryos were first randomized for vitrification with two different kits: Vitrification Kit (Kitazato, Japan) and Sage Vitrification Kit (Origio, Denmark). The embryos were randomly warmed with either Kitazato (K) or Sage (S) warming kits, specifically: group A (KK), group B (KS), group C (SK), and group D (SS). PRIMARY OUTCOME MEASURE: survival rate (number of embryos surviving per number of embryos warmed). Secondary: implantation rate (number of embryos implanted per number of embryos transferred). CS: duration 01/01/2013-31/12/2015 embryos from patients' own oocytes; 10/04/2015-31/07/2017 embryos from donors' oocytes. A total of 1055 embryos vitrified at cleavage stage obtained from 631 warming cycles: 847 of these obtained from patients' own oocytes, 208 egg-donation-derived embryos. Each patient's embryos were vitrified and warmed in various combinations of three different vitrification/warming kits: Kitazato (K), Sage (S), or made in-house in our laboratory (H). Vitrification/warming kits from different manufacturers are routinely used in our AR center, and the warming procedures are randomly performed with any available kit on a "first-in-first-out" basis, irrespective of the kit used for vitrification. Group names: KK, KS, SK, SS, SH, HK, HS, HH (embryos from patients' own oocytes); eKK, eKS, eSK, eSS (egg-donation-derived embryos). RESULTS: Cryo-survival rates were comparable in all study groups. RCT. Group A 99.0% (96/97), group B 98.8% (83/84), group C 98.4% (61/62), and group D 98.6% (71/72). CS. Embryos from patients' own oocytes: KK 96.4% (54/56), KS 100.0% (13/13), SK 98.8% (80/81), SS 97.2% (174/179), SH 97.6% (40/41), HK 95.2% (20/21), HS 99.5% (187/188), and HH 97.4% (261/268). Egg-donation-derived embryos: eKK 100.0% (91/91), eKS 98.4% (60/61), eSK 100.0% (26/26), and eSS 96.7 (29/30). Implantation was generally comparable in all study groups-exceptions were in CS: KS vs. SK (P = 0.049), SS (P = 0.012), HS (P = 0.010), HH (P = 0.025); and SH vs. SS (P = 0.042), HS (P = 0.035). CONCLUSION: Worldwide, millions of embryos have been cryopreserved using different vitrification kits; these studies establish that it is possible to combine different kits for vitrification and warming using a universal warming protocol. This can optimize costs, simplify lab routines, and favor embryo exchange between IVF centers. RCT REGISTRATION NUMBER: ISRCTN12342851.

The effect of dietary alterations during rearing on feather condition in broiler breeder females.

In commercial production, broiler breeders are severely feed restricted to maintain healthy BW. This restriction can induce stereotypic behavior, including feather pecking, which has negative welfare implications for both the victim and performer. It has been suggested that the problem may be symptomatic of chronic hunger or the frustration of feeding motivation. In this study, we determined whether feather condition, as an indirect measure of feather pecking, could be improved via dietary manipulation. Six dietary treatments were tested, each with 5 replicate pens of 9 to 12 birds. Control diets (C) were fed on a daily or skip-a-day (SAD) basis. Alternative diets included soybean hulls as a bulking ingredient and calcium propionate (CaP) as an appetite suppressant of either a feed grade (F) or purified (P) quality. Both alternative diets were fed on either a daily or SAD basis. Five or 6 birds were randomly chosen from each pen and feather scored at 10, 14, 20, 26, and 36 wk of age. Six body parts (neck, back, wings, legs, vent area, tail) were given a score from 0 to 5 (0 = no feather damage, and 5 ≥ 50% feather loss with tissue damage). Scores were summed for each bird and averaged for each pen. Data were analyzed with room and feeding frequency as main factors and diet as the subfactor with repeated measures. There was an interaction between diet and time (P < 0.01) with the feather condition of the C birds worsening more quickly in comparison with the F and P birds. There was an interaction between feeding frequency and time (P = 0.015), with SAD-fed birds scoring better than daily-fed birds at 20, 26, and 36 wk. This interaction could indicate that SAD feeding increased satiety after the birds became accustomed to the schedule. Because feather condition was better with the alternative diets, this may indicate a reduction in stereotyped feather pecking with these diets. This suggests that the alternative diets increase satiety compared with the control diets.

The effect of dietary alterations during rearing on growth, productivity, and behavior in broiler breeder females.

Parent stocks of meat birds are severely feed restricted to avoid obesity-related health and fertility problems. This restriction often leads to chronic hunger, accompanied by stereotypic behavior. Research based in the United Kingdom has shown that using diets containing fiber and appetite suppressants may relieve some of the symptoms of hunger. However, few data are available regarding North American-sourced ingredients or nondaily feeding regimens. This study investigated the effects of 2 alternative diets, in combination with 2 feeding frequencies on growth, productivity, and behavior in broiler breeders. Six dietary treatments were tested, each with 5 replicate pens of 12 or 13 birds. Control diets consisted of a commercial crumble, fed on a daily or skip-a-day (SAD) basis. Alternative diets included soybean hulls as a fiber source, and calcium propionate as an appetite suppressant of either a feed-grade or purified quality, fed on either a daily or SAD basis. Birds were weighed weekly and egg production was recorded daily. Video cameras were used to record behavior during and following the morning feeding bout every 2 wk from 11 to 28 wk. Data were analyzed with a mixed model ANOVA, with repeated measures. Diet, feeding frequency, time, or an interaction of the 3 had significant effects on all observed behavior during rearing. These differences appeared to diminish during lay, with most stereotypic behavior no longer present. Very little object pecking and aggression was observed during and immediately following feeding bouts; however, daily-fed control birds still displayed this behavior more often, especially during rearing (P = 0.015). During feeding bouts, SAD birds feather pecked (P = 0.003) and rested more (P = 0.0002) than daily-fed birds. Control birds feather pecked most often (P = 0.033) after feeding bouts. Overall, the feed-grade diet appeared most effective at reducing hunger-related behavior, and the control diet appeared the least effective. There was little conclusive evidence to show that daily feeding was more effective at reducing hunger.

Joint loading decreased by inexpensive and minimalist footwear in elderly women with knee osteoarthritis during stair descent.

OBJECTIVE: Previous studies indicate that flexible footwear, which mimics the biomechanics of walking barefoot, results in decreased knee loads in patients with knee osteoarthritis (OA) during walking. However, the effect of flexible footwear on other activities of daily living, such as descending stairs, remains unclear. Our objective was to evaluate the influence of inexpensive and minimalist footwear (Moleca) on knee adduction moment (KAM) during stair descent of elderly women with and without knee OA. METHODS: Thirty-four elderly women were equally divided into an OA group and a control group (CG). Stair descent was evaluated in barefoot condition, while wearing the Moleca, and while wearing heeled shoes. Kinematics and ground reaction forces were measured to calculate KAM by using inverse dynamics. RESULTS: The OA group experienced a higher KAM during midstance under the barefoot condition (233.3%; P = 0.028), the Moleca (379.2%; P = 0.004), and heeled shoes (217.6%; P = 0.007). The OA group had a similar knee load during early, mid, and late stance with the Moleca compared with the barefoot condition. Heeled shoes increased the knee loads during the early-stance (versus barefoot [16.7%; P < 0.001] and versus the Moleca [15.5%; P < 0.001]), midstance (versus barefoot [8.6%; P = 0.014] and versus the Moleca [9.5%; P = 0.010]), and late-stance phase (versus barefoot [10.6%; P = 0.003] and versus the Moleca [9.2%; P < 0.001]). In the CG, the Moleca produced a knee load similar to the barefoot condition only during the early-stance phase. CONCLUSION: Besides the general foot protection, the inexpensive and minimalist footwear contributes to decreasing knee loads in elderly women with OA during stair descent. The loads are similar to the barefoot condition and effectively decreased when compared with heeled shoes.

Efficiency of aseptic open vitrification and hermetical cryostorage of human oocytes.

The present study reports, as far as is known for the first time, the safety of UV sterilization of liquid nitrogen and hermetical cryostorage of human oocytes by comparing the efficiency of fresh and vitrified sibling oocytes of infertile patients. A prospective randomized study on sibling oocytes of 31 patients was carried out. Metaphase-II oocytes were randomized for intracytoplasmic sperm injection and the supernumerary sibling oocytes were vitrified using a novel Cryotop aseptic procedure (UV liquid nitrogen sterilization and hermetical cryostorage). After unsuccessful attempts with fresh oocytes, vitrified sibling oocytes were injected. Mean outcome measures observed were fertilization, cleavage and top-quality embryo rates. No significant differences were observed between the fresh and vitrified-warmed sibling oocytes: oocyte fertilization was 88.3% versus 84.9%; cleavage 72.6% versus 71.0%; top-quality embryos 33.8% versus 26.3% and mean number of transferred embryos 2.6 ± 0.1 versus 2.5 ± 0.1, respectively. Clinical pregnancy rate per cycle with vitrified-warmed oocytes was 35.5% (implantation rate 17.1%) and seven healthy babies were born. This study demonstrated that UV liquid nitrogen sterilization and hermetical cryostorage does not adversely affect the developmental competence of vitrified oocytes, allowing safe aseptic open vitrification applicable under strict directives on tissue manipulation.

Stereoselective synthesis of trifluoro- and monofluoro-analogues of frontalin and evaluation of their biological activity.

The stereoselective synthesis of both enantiomers of trifluoro frontalin (-)-(1S,5R)- and (+)-(1R,5S)-8, as well as of diastereomeric monofluoro frontalines (-)-(1R,2R,5R)-18 and (-)-(1R,2S,5R)-20, analogues of the bioactive component of the aggregation pheromone of the Scolytidae insect family, has been accomplished starting from (-)-(1R)- and (+)-(1S)-menthyl (S)-toluene-4-sulfinate as a source of chirality and methyl trifluoroacetate or fluoroacetate, respectively, as sources of fluorine. The C-1 stereocenters were installed via stereoselective epoxidation of beta-sulfinyl ketones 2 and 13 with diazomethane. The bicyclic core was obtained by totally stereocontrolled and chemoselective tandem Wacker oxidation/intramolecular ketalization of the intermediate unsatured sulfinyl diols 5, 15, and 19. Axially fluorinated (-)-20 elicited a strong electroantennographic response in laboratory tests on females of Dendroctonus micans, whereas equatorially fluorinated (-)-18 and the trifluoroanalogue (-)-8 showed modest responses. Field trials using (-)-20 were not indicative owing to the locally scarce population of D. micans, but it showed some attractiveness for other Coleoptera families.

Site-directed mutations of human hemoglobin at residue 35beta: a residue at the intersection of the alpha1beta1, alpha1beta2, and alpha1alpha2 interfaces.

Because Tyr35beta is located at the convergence of the alpha1beta1, alpha1beta2, and alpha1alpha2 interfaces in deoxyhemoglobin, it can be argued that mutations at this position may result in large changes in the functional properties of hemoglobin. However, only small mutation-induced changes in functional and structural properties are found for the recombinant hemoglobins betaY35F and betaY35A. Oxygen equilibrium-binding studies in solution, which measure the overall oxygen affinity (the p50) and the overall cooperativity (the Hill coefficient) of a hemoglobin solution, show that removing the phenolic hydroxyl group of Tyr35beta results in small decreases in oxygen affinity and cooperativity. In contrast, removing the entire phenolic ring results in a fourfold increase in oxygen affinity and no significant change in cooperativity. The kinetics of carbon monoxide (CO) combination in solution and the oxygen-binding properties of these variants in deoxy crystals, which measure the oxygen affinity and cooperativity of just the T quaternary structure, show that the ligand affinity of the T quaternary structure decreases in betaY35F and increases in betaY35A. The kinetics of CO rebinding following flash photolysis, which provides a measure of the dissociation of the liganded hemoglobin tetramer, indicates that the stability of the liganded hemoglobin tetramer is not altered in betaY35F or betaY35A. X-ray crystal structures of deoxy betaY35F and betaY35A are highly isomorphous with the structure of wild-type deoxyhemoglobin. The betaY35F mutation repositions the carboxyl group of Asp126alpha1 so that it may form a more favorable interaction with the guanidinium group of Arg141alpha2. The betaY35A mutation results in increased mobility of the Arg141alpha side chain, implying that the interactions between Asp126alpha1 and Arg141alpha2 are weakened. Therefore, the changes in the functional properties of these 35beta mutants appear to correlate with subtle structural differences at the C terminus of the alpha-subunit.

Interface sliding as illustrated by the multiple quaternary structures of liganded hemoglobin.

Initial crystallographic studies suggested that fully liganded mammalian hemoglobin can adopt only a single quaternary structure, the quaternary R structure. However, more recent crystallographic studies revealed the existence of a second quaternary structure for liganded hemoglobin, the quaternary R2 structure. Since these quaternary structures can be crystallized, both must be energetically accessible structures that coexist in solution. Unanswered questions include (i) the relative abundance of the R and R2 structures under various solution conditions and (ii) whether other quaternary structures are energetically accessible for the liganded alpha(2)beta(2) hemoglobin tetramer. Although crystallographic methods cannot directly answer the first question, they represent the most direct and most accurate approach to answering the second question. We now have determined and refined three different crystal structures of bovine carbonmonoxyhemoglobin. These structures provide clear evidence that the dimer-dimer interface of liganded hemoglobin has a wide range of energetically accessible structures that are related to each other by a simple sliding motion. The dimer-dimer interface acts as a "molecular slide bearing" that allows the two alpha beta dimers to slide back and forth without greatly altering the number or the nature of the intersubunit contacts. Since the general stereochemical features of this interface are not unusual, it is likely that interface sliding of the kind displayed by fully liganded hemoglobin plays important structural and functional roles in many other protein assemblies.

Structure and absolute configuration of new acetylenic compounds isolated from cultures of Clitocybe catinus.

Investigations of the extracts of a culture of Clitocybe catinus gave rise to the isolation of new acetylenic diols 1-3. Their structure was determined on the basis of 1H- and 13C-NMR evidence and the absolute configuration elucidated by means of the modified Mosher's method.

The "Non-Oxidative" pummerer reaction: conclusive evidence for S(N)2-type stereoselectivity, mechanistic insight, and synthesis of enantiopure L-alpha-trifluoromethylthreoninate and D-alpha-trifluoromethyl-allo-threoninate(1)

Enantiopure methyl D-alpha-trifluoromethyl-allo-threoninate 18 and L-alpha-trifluoromethylthreoninate 19 were synthesized using (R)-ethyl p-tolylsulfoxide as chiral alpha-hydroxyethyl anion equivalent. The key step was the S(N)2-type replacement of the sulfinyl auxiliary with a hydroxy group, via trifluoroacetic anhydride promoted "non-oxidative" Pummerer reaction (NOPR) of the diastereomeric intermediate beta-sulfinyl amines 14 and 15, obtained by condensation of (R)-ethyl p-tolylsulfoxide 13 with the N-Cbz imine of methyl trifluoropyruvate 12. The conclusive evidence for S(N)2-type stereoselectivity of the NOPR was achieved by X-ray diffraction of both the starting diastereomer 14 and the p-bromobenzoate 25, obtained from the threoninate 19. NMR monitoring of the NOPR performed on 15 allowed the detection of a transient intermediate, which was identified as the four membered cyclic sigma-sulfurane 27. This intermediate spontaneously rearranged (40 min, rt) into the corresponding sulfenamide 17, probably via an intramolecular displacement of the sulfinyl by a trifluoroacetoxy group, with inversion of configuration at the carbon stereocenter. The same process occurred for the diastereomeric beta-sulfinyl amine 14, but the sulfenamide 16 was formed at a very fast rate, thus precluding NMR detection of the corresponding sigma-sulfurane intermediate 26. One-pot treatment of the diastereomeric sulfenamides 16 and 17 with NaBH(4) afforded very good yields of the corresponding threoninates 18 and 19.

Cys-93-betabeta-succinimidophenyl polyethylene glycol 2000 hemoglobin A. Intramolecular cross-bridging of hemoglobin outside the central cavity.

Bis(maleidophenyl)-PEG2000 (Bis-Mal-PEG2000), a new bifunctional protein cross-linker targeted to sulfhydryl groups, introduces intra-tetrameric cross-links into oxy-HbA in nearly quantitative yields. Structural as well as crystallographic analyses of the cross-linked species, Bis-Mal-PEG2000 HbA, identified Cys-93(beta) as the site of intramolecular cross-linking. The cross-bridging had only a limited influence on the O(2) affinity and cooperativity of HbA in 50 mM BisTris acetate, pH 7.4. However, the Bohr effect was reduced by approximately 60%. Bis-Mal-PEG2000 HbA retained sensitivity to the presence of allosteric effectors 2, 3-diphosphoglycerate, IHP, and chloride, albeit to a lesser degree compared with HbA. Crystallographic analysis revealed the overall structure of deoxy-Bis-Mal-PEG2000 HbA to be similar to deoxy-HbA but for the loss of the salt bridge between Asp-94(beta) and His-146(beta). The large influence of the cross-bridging on the alkaline Bohr effect of HbA is consistent with the loss of this salt bridge. Unlike the "central cavity cross-bridges" described previously, the cross-link introduced by Bis-Mal-PEG2000 into HbA is an "outside the central cavity cross-bridge." In view of its oxy-conformational specificity and limited influence on O(2) affinity, this new cross-linking strategy holds promise for the stabilization of new designer low O(2) affinity Hbs generated by recombinant DNA technology for applications as Hb based therapeutics.

Structural and functional properties of human hemoglobins reassembled after synthesis in Escherichia coli.

Human hemoglobin produced in the Escherichia coli coexpression system of Hernan et al. [(1992) Biochemistry 31, 8619-8628] has been transformed into a functionally homogeneous protein whose properties closely approximate those of normal hemoglobin A. Both of the alpha and beta chains of this hemoglobin contain a valine-methionine substitution at position 1 in order to accommodate the difference in specificity of the protein-processing enzymes of procaryotes. Despite extensive purification, functional homogeneity of the E. coli expressed hemoglobin was achieved only by the complete disassembly of the hemoglobin into its component alpha and beta globins and their reassembly in the presence of hemin. The kinetics of CO combination and the thermodynamics of O2 binding and cooperativity of the reassembled alphaV1M-betaV1M hemoglobin closely approximate those of HbA. The alpha globin obtained from the E. coli expressed hemoglobin was also combined with normal human beta chains and hemin to form the alphaV1M variant. The alpha+M variant of HbA, in which the normal N-terminal valine of the alpha chains is preceded by a methionine residue, was prepared by the same procedure. The kinetics of the reactions of CO with the alphaV1M and alpha+M variants are similar to those for HbA. The equilibria of oxygen binding to alphaV1M and HbA are similar whereas alpha+M exhibits a significantly higher oxygen affinity. The three-dimensional structures of alphaV1M and alpha+M offer an explanation for the latter affinity difference. Although the structures of alphaV1M and HbA, which have been determined by X-ray crystallography, are virtually indistinguishable except at the N-terminal residues, that of alpha+M indicates the displacement of a solvent molecule, possibly a chloride ion, from arginine 141alpha. Such an alteration in an anion binding site could result in increased oxygen affinity.

Tsugicoline E, a new polyoxygenated protoilludane sesquiterpene from the fungus laurilia tsugicola1

Tsugicoline E (3) has been isolated from cultures of the Basidiomycetous fungus Laurilia tsugicola and its structure deduced from 1H and 13C NMR and single-crystal X-ray diffraction studies. The suggested absolute configuration is consistent with biogenetic considerations.

Crystal structure of the S-nitroso form of liganded human hemoglobin.

Although numerous reports have documented that the S-nitrosylation of cysteine residues by NO alters the activities of a wide variety of proteins, the direct visualization and the structural consequences of this reversible modification have not yet been reported for any protein. Here we describe the crystal structure of S-nitroso-nitrosylhemoglobin determined at a resolution of 1.8 A. The specific reaction of NO with Cys93beta is confirmed in this structure, and a large S-nitrosylation-induced change in the tertiary structure of the COOH-terminal dipeptides of the beta subunits provides additional insight into the stereochemical mechanism by which blood flow is regulated by the interaction of NO with hemoglobin.

A hydroxytetradecatrienoic acid from Mycosphaerella rubella.

Investigations on the acidic fractions from extracts of a culture of Mycosphaerella rubella led to the isolation of a new unsaturated dihydroxy acid, 6,13-dihydroxytetradeca-2,4,8 trienoic acid, and two of its derivatives, which are probably artifacts. Their structures were determined on the basis of H and C NMR evidence. The absolute configuration of the chiral centres of the new acid was elucidated using the modified Mosher's method.

High-resolution crystal structures of human hemoglobin with mutations at tryptophan 37beta: structural basis for a high-affinity T-state,.

The high-resolution X-ray structures of the deoxy forms of four recombinant hemoglobins in which Trp37(C3)beta is replaced with Tyr (betaW37Y), Ala (betaW37A), Glu (betaW37E), or Gly (betaW37G) have been refined and analyzed with superposition methods that partition mutation-induced perturbations into quaternary structure changes and tertiary structure changes. In addition, a new cross-validation statistic that is sensitive to local changes in structure (a "local Rfree" parameter) was used as an objective measure of the significance of the tertiary structure changes. No significant mutation-induced changes in tertiary structure are detected at the mutation site itself for any of the four mutants studied. Instead, disruption of the intersubunit contacts associated with Trp37(C3)beta results in (1) a change in quaternary structure at the alpha1beta2 interface, (2) alpha subunit tertiary structure changes that are centered at Asp94(G1)alpha-Pro95(G2)alpha, (3) beta subunit tertiary structure changes that are located between residues Asp99(G1)beta and Asn102(G4)beta, (4) increased mobility of the alpha subunit COOH-terminal dipeptide, and (5) shortening of the Fe-Nepsilon2His(F8) bond in the alpha and beta subunits of the betaW37G and betaW37E mutants. In each case, the magnitude of the change in a particular structural parameter increases in the order betaW37Y < betaW37A < betaW37E approximately betaW37G, which corresponds closely to the degree of functional disruption documented in the preceding papers.

Marasmane sesquiterpenes from the basidiomycete Clitocybe hydrogramma.

Investigations of solid cultures of Clitocybe hydrogramma gave rise to the isolation of two new alpha, beta-unsaturated dialdehydes having the marasmane skeleton. Their structures have been determined on the basis of 1H and 13C NMR evidence.

Constituents of Dragon's Blood. 5. Dracoflavans B1, B2, C1, C2, D1, and D2, new A-type deoxyproanthocyanidins.

From Dragon's Blood, a resin produced by plants of the genus Daemonorops (Palmae), six new A-type flavanoid deoxyproanthocyanidins have been isolated. Their structure and stereochemistry, established by chemical degradation and extensive NMR analysis, is consistent with a mechanism of formation common to other constituents of the resin, which involves oxidation of a 6-methylflavan to a quinonemethide, followed by coupling with another flavan moiety.