Isolation and Structures of Minor Metabolites,Cotylenins H and I

Minor fungal metabolites, cotylenins H and I, were isolated from the culture filtrate of an unidentified species of Cladosporium and their structures have been assigned as II and I, respectively, on the basis of the chemical and spectroscopic evidence.     

Some Properties of a Functional Reconstituted Plasmalemma H-ATPase Activated by Fusicoccin

Fusicoccin was shown to stimulate the ATP-driven, intravesicular acidification of liposomes reconstituted with crude fusicoccin receptors and the H⁺-translocating ATPase, both solubilized from maize (Zea mays L.) plasma membrane. The present paper reports optimal conditions for dual reconstitution and fusicoccin activation as well as the biochemical characterization of the effect of fusicoccin on this system. Fusicoccin stimulation of proton pumping was dependent on pH and fusicoccin concentration. Its specificity was demonstrated by the positive effect of two cotylenins that have a high affinity for fusicoccin receptors and by the negative response to 7,9-epideacetylfusicoccin, an inactive fusicoccin derivative. Kinetic measurements at different ATP concentrations showed that fusicoccin increases the V_max of the enzyme. Fusicoccin stimulation of maize H⁺-ATPase was also maintained when receptors from maize were substituted by those from spinach (Spinacia oleracea L.).     

Isolation and Identification of Cotylenins F and G

The structures of cotylenins F and G, isolated from the culture filtrate of a fungus (Cladosporium sp.), have been assigned as IV and V, respectively. Cotylenin G was derived from cotylenin F by treatment with a strong base.     

Cotylenins,Leaf Growth Substances Produced by a Fungus

New biologically active substances named cotylenins A and B were isolated from the culture filtrate of a fungus, strain 501-7w. Isolation and characterization are described in detail. The chemical relationship between the cotylenins has been elucidated.     

Isolation and Identification of a New Glycoside,Phyllodulcin-8-O-β-d-glucose from the Cultured Cells and Fresh Leaves of Amacha (Hydrangea macrophylla Seringe var. Thunbergii Makino)

The structure of cotylenin A, a potent plant-growth stimulant with the most complex molecule in cotylenins from Cladosporium fungus sp. 501-7W, was analyzed again by HMBC experiments and X-ray crystallography of its diacetyl-dihydroderivative. The previously proposed stereostructure of cotylenin A was thus completely confirmed.     

Correlation of cotylenol with the aglycone of fusicoccin

The stereostructure of cotylenol, the aglycone of the cotylenins, has been confirmed by chemical correlation with the aglycone of fusicoccin A.     

Stimulation of Orobanche ramosa seed germination by fusicoccin derivatives: a structure-activity relationship study

A structure-activity relationship study was conducted assaying 25 natural analogues and derivatives of fusicoccin (FC), and cotylenol, the aglycone of cotylenins, for their ability to stimulate the seed germination of the parasitic species Orobanche ramosa. Some of the compounds tested proved to be highly active, being 8,9-isopropylidene of the corresponding FC aglycone and the dideacetyl derivative the most active FC derivatives. In both groups of glucosides and aglycones (including cotylenol), the most important structural feature to impart activity appears to be the presence of the primary hydroxy group at C-19. Furthermore, the functionalities and the conformation of the carbotricyclic ring proved to play a significant role. The dideacetyl derivative of FC, being easily and rapidly obtainable in high yield starting by FC, could be of interest for its practical application as a stimulant of Orobanche ramosa seed germination, inducing the "suicidal germination", an interesting approach for parasitic plant management.     

Intracellular Potassium : A Determinant of the Sodium-Potassium Pump Rate

Normal human red cells which have had their intracellular sodium (Na_c) reduced have a diminished Na-K pump rate, but only if intracellular potassium (K_c) is high. If most of the K_c is replaced by tetramethylammonium or choline, both ouabain-sensitive Na efflux and K influx are significantly increased even with Na_c below normal. Cells with reduced Na_c and high K_c have an unchanged Na efflux if external potassium (K_ext) is removed. In contrast, low-Na, low-K cells have a large ouabain-sensitive Na efflux which shows a normal response to removal of K_ext. Neither low-K nor high-K cells have an altered ouabain-sensitive K efflux. Measurement at constant low Na_c and varying K_c shows the pump Na efflux to be an inverse function of K_c. Thus, in low-Na cells, K_c appears to act as an inhibitor of the pump. Inhibition by high K_c can be seen even when Na_c is normal. The effects attributed to K_c are distinguished experimentally from other variables such as cell volume, adenosine triphosphate concentration, effects of the replacement cations, and the method used to alter intracellular cation concentrations. A role is proposed for K_c, in cooperation with Na_c, in regulating the pump rate of normal human red cells.     

Effectors of metabolic depression in an estivating pulmonate snail (Helix aspersa): whole animal and in vitro tissue studies

 We have examined metabolic depression in the land snail (Helix aspersa) during estivation, and have developed a tissue model of metabolic depression using an in vitro mantle preparation. The metabolic rate of H. aspersa is depressed by 84% in vivo within 4 weeks of onset of estivation, and this metabolic depression is accompanied by a decrease in haemolymph PO₂ and pH, and an increase in haemolymph PCO₂. The in vitro mantle preparation has a stable O₂ consumption and energy charge, and an energy charge similar to that of mantle in vivo. The in vitro mantle is an O₂-conforming tissue, with VO₂ varying curvilinearly with PO₂. Consequently, we have developed a mathematical method of calculating tissue VO₂ at any PO₂. These calculations show that under appropriate incubation conditions of pH and PO₂, the mantle from estivating animals shows a stable in vitro metabolic depression of 48% compared to mantle from control snails. The extrinsic effects of PO₂ and pH account for 70% of the total in vitro metabolic depression of mantle tissue; intrinsic effectors contribute a further 30%. Accepted April 26, 1996     

Cell Synchrony Techniques. II. Analysis of Cell Progression Data

Abstract CHO cells which have been sorted by mitotic detachment, centrifugal elutriation and fluorescence activated cell sorting have been followed for up to 14 hr by flow cytometry to examine their progression characteristics. Mathematical modelling techniques were used to provide quantitative estimates of the cell-cycle parameters. Mitotic detachment gives an 11.2-hr cycle time with mean transit times T_G1, T_s and T_G2M equal to 3.2, 5.6 and 2.4 respectively. Cells prepared by central elutriation in an early G₁ state have a 14-hr cycle time with T_G1, T_s and T_G2M of 5.7, 6.0 and 2.3 hr. Populations prepared by centrifugal elutriation enriched in early S and late S and G₂M have transit times of 2.7, 5.9 and 1.6 hr and 4.9, 6.7 and 2.1 hr with cycle times of 11.2 and 13.2 hr respectively. Cell sorting for a G₁ population gives transit times of 9.8, 8.0 and 3.6 for an overall 21.4-hr cycle time.     

Evidence for a novel circulating alpha 1 protein in tumour-bearing rats. Differentiation from acute phase alpha 1 proteins and from alpha 1 fetoprotein

It has been found in this study that the serum from rats bearing a transplanted dibenzanthracene-induced tumour ($\text{RD}\text{3}$), has a high concentration of alpha₁ proteins compared with normal rat serum. These alpha₁ proteins have been isolated by an immunoabsorption method and have been compared by immunological methods with the acute phase alpha₁ proteins isolated by the same method from the serum of rats presenting an inflammatory reaction. It has been found that the isolated $\text{RD}\text{3}$ alpha₁ proteins were composed of two major proteins: one of these corresponded to an inflammatory protein, the alpha₁-AP-globulin. The other may be a new protein, as it is absent from the serum of rats with an acute phase inflammatory reaction and nor does it correspond to alpha₁ feto-protein, a carcino-embryonic protein presenting the same electrophoretic mobility.     

Diagnostic Confusion Attributable to Spurious Elevation of Both Total Thyroid Hormone and Thyroid Hormone Uptake Measurements in the Setting of Autoantibodies: Case Report and Review of Related Literature

ObjectiveTo review the effect of thyroid autoantibodies on thyroid function assays and to present a case in which thyroid autoantibodies resulted in spurious assay readings for both total thyroid hormone levels and thyroid hormone uptake measurements.MethodsWe present a detailed case, including serial laboratory data, and review the relevant literature.ResultsA 61-year-old man with a history of autoimmune disease presented for evaluation of abnormal results of thyroid function tests. The patient had been treated for hypothyroidism with levothyroxine and was noted to have an elevated total thyroxine (T₄) level in the setting of a low total triiodothyronine (T₃) value and a mildly elevated thyrotropin concentration. He had been referred for evaluation of a presumed deiodinase deficiency that impaired conversion of T₄ to T₃. During treatment with levothyroxine, these test results were confirmed, and the patient was also found to have an elevated T₄ uptake. These findings were initially thought to be due to an excess of transthyretin; however, more extensive testing revealed that the patient had an autoantibody to T₄ that interfered with the assays for both T₄ and T₄ uptake.ConclusionAutoantibodies to both T₃ and T₄ have been described. Such antibodies are not uncommon in patients with thyroid disease. On rare occasions, these antibodies may cause spurious assay readings and obscure the diagnosis. To our knowledge, this is the first report in which both the total T₄ level and the T₄ uptake were elevated because of the presence of autoantibodies. Thyroid hormone autoantibodies must be considered when clinicians encounter patients with unexplained abnormal results of thyroid function tests. (Endocr Pract. 2008; 14:738-742)     

Geochemical constraints on chemolithoautotrophic reactions in hydrothermal systems

Thermodynamic calculations provide the means to quantify the chemical disequilibrium inherent in the mixing of redeuced hydrothermal fluids with seawater. The chemical energy available for metabolic processes in these environments can be evaluated by taking into account the pressure and temperature dependence of the apparent standard Gibbs free energies of reactions in the S-H₂-H₂O system together with geochemical constraints on pH, activities of aqueous sulfur species and fugacities of H₂ and/or O₂. Using present-day mixing of hydrothermal fluids and seawater as a starting point, it is shown that each mole of H₂S entering seawater from hydrothermal fluids represents about 200,000 calories of chemical energy for metabolic systems able to catalyze H₂S oxidation. Extrapolating to the early Earth, which was likely to have had an atmosphere more reduced than at present, shows that this chemical energy may have been a factor of two or so less. Nevertheless, mixing of hydrothermal fluids with seawater would have been an abundant source of chemical energy, and an inevitable consequence of the presence of an ocean on an initially hot Earth. The amount of energy available was more than enough for organic synthesis from CO₂ or CO, and/or polymer formation, indicating that the vicinity of hydrothermal systems at the sea floor was an ideal location for the emergence of the first chemolithoautotrophic metabolic systems.     

Enhanced levels of vitamin B(6) increase aerial organ size and positively affect stress tolerance in Arabidopsis

Vitamin B₆ is an essential nutrient in the human diet derived primarily from plant sources. While it is well established as a cofactor for numerous metabolic enzymes, more recently, vitamin B₆ has been implicated as a potent antioxidant. The de novo vitamin B₆ biosynthesis pathway in plants has recently been unraveled and involves only two proteins, PDX1 and PDX2. To provide more insight into the effect of the compound on plant development and its role as an antioxidant, we have overexpressed the PDX proteins in Arabidopsis, generating lines with considerably higher levels of the vitamin in comparison with other recent attempts to achieve this goal. Interestingly, it was possible to increase the level of only one of the two catalytically active PDX1 proteins at the protein level, providing insight into the mechanism of vitamin B₆ homeostasis in planta. Vitamin B₆ enhanced lines have considerably larger vegetative and floral organs and although delayed in pre‐reproductive development, do not have an altered overall morphology. The vitamin was observed to accumulate in seeds and the enhancement of its levels was correlated with an increase in their size and weight. This phenotype is predominantly a consequence of embryo enlargement as reflected by larger cells. Furthermore, plants that overaccumulate the vitamin have an increased tolerance to oxidative stress providing in vivo evidence for the antioxidant functionality of vitamin B₆. In particular, the plants show an increased resistance to paraquat and photoinhibition, and they attenuate the cell death response observed in the conditional flu mutant.     

2-Oxoamide inhibitors of cytosolic group IVA phospholipase A2 with reduced lipophilicity

Cytosolic GIVA phospholipase A₂ (GIVA cPLA₂) initiates the eicosanoid pathway of inflammation and thus inhibitors of this enzyme constitute novel potential agents for the treatment of inflammatory diseases. Traditionally, GIVA cPLA₂ inhibitors have suffered systemically from high lipophilicity. We have developed a variety of long chain 2-oxoamides as inhibitors of GIVA PLA₂. Among them, AX048 was found to produce a potent analgesic effect. We have now reduced the lipophilicity of AX048 by replacing the long aliphatic chain with a chain containing an ether linked aromatic ring with in vitro inhibitory activities similar to AX048.     

MULTIPLE ORIGINS OF SEX CHROMOSOME FUSIONS CORRELATED WITH CHIASMA LOCALIZATION IN HABRONATTUS JUMPING SPIDERS (ARANEAE: SALTICIDAE)

Entelegyne spiders rarely show fusions yielding neo‐Y chromosomes, which M. J. D. White attributed to a constraint in spiders, namely their proximal chiasma localization acting to upset meiotic segregation in males with fusions. Of the 75 taxa of Habronattus and outgroups studied, 47 have X₁X₂0 sex chromosomes in males, 10 have X₁X₂Y, 15 have X₁X₂X₃Y, 2 have X0, and one has both X₁X₂0 and X₁X₂X₃Y. Chromosome numbers and behavior suggest neo‐Ys formed by an autosome‐X fusion to make X₁X₂Y, with a second fusion to an autosome to make X₁X₂X₃Y. Phylogeny shows at least 8–15 gains (or possibly some losses) of neo‐Y (i.e., X‐autosome fusions), a remarkable number for such a small clade. In contrast to the many X‐autosome fusions, at most one autosome–autosome fusion is indicated. Origins of neo‐Y are correlated significantly with distal localization of chiasmata, supporting White's hypothesis that evolution of neo‐Y systems is facilitated by looser pairing (distal chiasmata) at meiosis. However, an alternative (or contributing) explanation for the correlation is that X‐autosome fusions were selected to permit isolation of male‐favored alleles to the neo‐Y chromosome, aided by distal chiasmata limiting recombination. This intralocus sexual conflict hypothesis could explain both the many X‐autosome fusions, and the stunning complexity of male Habronattus courtship displays.     

Common variant in FUT2 gene is associated with levels of vitamin B12 in Indian population

Vitamin B₁₂ is an essential micronutrient synthesized by microorganisms. Mammals including humans have evolved ways for transport and absorption of this vitamin. Deficiency of vitamin B₁₂ (either due to low intake or polymorphism in genes involved in absorption and intracellular transport of this vitamin) has been associated with various complex diseases. Genome-wide association studies have recently identified several common single nucleotide polymorphisms (SNPs) in fucosyl transferase 2 gene (FUT2) to be associated with levels of vitamin B₁₂—the strongest association was with a non-synonymous SNP rs602662 in this gene. In the present study, we attempted to replicate the association of this SNP (rs602662) in an Indian population since a significant proportion has been reported to have low levels of vitamin B₁₂ in this population. A total of 1146 individuals were genotyped for this SNP using a single base extension method and association with levels of vitamin B₁₂ was assessed in these individuals. Regression analysis was performed to analyze the association considering various confounding factors like for age, sex, diet, hypertension, diabetes mellitus and coronary artery disease status. We found that the SNP rs602662 was significantly associated with the levels of vitamin B₁₂ (p value < 0.0001). We also found that individuals adhering to a vegetarian diet with GG (homozygous major genotype) have significantly lower levels of vitamin B₁₂ in these individuals. Thus, our study reveals that vegetarian diet along with polymorphism in the FUT2 gene may contribute significantly to the high prevalence of vitamin B₁₂ deficiency in India.     

Stoichiometry-driven Protein Folding: A Comment

Abstract

We have found, with the aid of 2-D gel electrophoresis, that double-stranded human telomeric repeat, (T₂AG₃)₁₂·(C₃TA₂)₁₂, being cloned within a plasmid, forms a protonated superhelically-induced structure. Experiments on chemical and enzymatic probing also indicate that the human telomeric repeats adopt an unusual structure. We have proposed an eclectic model for this structure in which four different elements coexist: a non-orthodox intramolecular triplex stabilized by the canonical protonated C · G*C⁺ base-triads and highly enriched by non-canonical base-triads; the intramolecular quadruplex formed by a portion of the G-rich strand; the single-stranded region encompassing a portion of the G-rich strand and, probably, the (C,A)-hairpin formed by a portion of the C-rich strand.     

Crystallization of intact monoclonal antibodies

Attempts were made to crystallize four monoclonal antibodies, one IgG_2ak and three IgG_1k. Using a PEG 3350 screen combined with detergents, and developed from our experiments with an IgG_2ak antibody specific for canine lymphoma cells,^1,2 crystals have now been obtained of two of these four immunoglobulins, an antiphenytoin and an antiphenobarbital antibody. A complex between the antiphenobarbital antibody and its drug antigen crystallized as well. The antibody for phenytoin has, to this point, produced only clustered microcrystals, marginally suitable for X-ray analysis. Single crystals of the IgG_1k antibody against phenobarbital, however, were characterized by X-ray diffraction to be primitive monoclinic, with unit cell dimensions a = 67 Å, b = 193 Å, c = 74 Å, and β = 110°. These crystals have an entire IgG_1k molecule as the asymmetric unit and they diffract to at least 3.2 Å resolution. © 1995 Wiley-Liss, Inc.     

High-dose chromium(III) supplementation has no effects on body mass and composition while altering plasma hormone and triglycerides concentrations

Chromium is generally believed to be an essential element and is often claimed to have value as a weight loss or muscle building agent. Recent studies in humans and rats have failed to demonstrate effects on body composition, although recent studies with pharmacological doses of the cation [Cr(III)₃O(O₂CCH₂CH₃)6(H₂O)₃]⁺ (or Cr3) (≤1 mg Cr/kg body mass) in rats have noted a trend toward body mass loss and fat mass loss. Thus, the effects of large gavage doses of Cr3 (1–10 mg Cr/kg) on body mass, organ mass, food intake, and blood plasma variables (insulin, glucose, leptin, cholesterol, and triglycerides) were examined over a 10-wk period using male Sprague-Dawley rats. No effects on body composition were noted, although Cr3 administration lowered (p<0.05) plasma insulin, leptin, and triglycerides concentrations. As Cr3 is absorbed greater than 10-fold better than commercially available nutritional supplements, the lack of an effect of the Cr(III) compound at these levels of administration clearly indicates that Cr(III) supplements do not have an effect on body composition at any reasonable dosage.