Test of soil extractants for their suitability in predicting Ca/Sr ratios in leaves and stems of sugar maple seedlings

Differential uptake and translocation of Ca and Sr in organisms have been reported, calling into question the use of Sr to track Ca cycling in the environment. We investigated the relationship between Ca/Sr ratios in soil extracts of various strengths (H₂O, NH₄Cl, and NH₄EDTA) and seedlings of sugar maple (Acer saccharum Marsh.) grown from natural regeneration on 37 sites. Our objectives were to determine if Ca/Sr ratios in soil extracts are correlated with those in sugar maple tissues, and what soil extractant best duplicate plant tissue Ca/Sr ratios. Leaves had higher Ca/Sr ratios than stems and the extractants did not produce equal Ca/Sr ratios: H₂O had the lowest Ca/Sr, and NH₄EDTA the highest. The relationships between soil extract Ca/Sr ratios and leaf and stem Ca/Sr ratios were significant and linear, but the slopes differed among extractants. The lowest slope (0.45) was observed for the water extract/leaves and the highest (2.15) for the NH₄EDTA extract/stem with discrimination factors ranging from 0.22 with NH₄EDTA to 1.59 for water. Leaf extracts were more strongly correlated with soil Ca/Sr than stem extracts (R ² of 0.57–0.7 vs. R ² of 0.45–0.6, respectively). These findings support the use of Ca/Sr ratios in plants to track their source of soil Ca, but they highlight the need to calibrate the relationships for the plant tissue and soil extractant used.     

Protective role of Mangifera indica, Cucumis melo and Citrullus vulgaris peel extracts in chemically induced hypothyroidism

An investigation was made to evaluate the pharmacological importance of fruit peel extracts of Mangifera indica (MI), Citrullus vulgaris (CV) and Cucumis melo (CM) with respect to the possible regulation of tissue lipid peroxidation (LPO), thyroid dysfunctions, lipid and glucose metabolism. Pre-standardized doses (200 mg/kg of MI and 100 mg/kg both of CV and CM), based on the maximum inhibition in hepatic LPO, were administered to Wistar albino male rats for 10 consecutive days and the changes in tissue (heart, liver and kidney) LPO and in the concentrations of serum triiodothyronine (T₃), thyroxin (T₄), insulin, glucose, α-amylase and different lipids were examined. Administration of three test peel extracts significantly increased both the thyroid hormones (T₃ and T₄) with a concomitant decrease in tissue LPO, suggesting their thyroid stimulatory and antiperoxidative role. This thyroid stimulatory nature was also exhibited in propylthiouracil (PTU) induced hypothyroid animals. However, only minor influence was observed in serum lipid profile in which CM reduced the concentrations of total cholesterol and low-density lipoprotein-cholesterol (LDL-C), while CV decreased triglycerides and very low-density lipoprotein-cholesterol (VLDL-C). When the combined effects of either two (MI + CV) or three (MI + CV + CM) peel extracts were evaluated in euthyroid animals, serum T₃ concentration was increased in response to MI + CV and MI + CV + CM treatments, while T₄ level was elevated by the combinations of first two peels only. Interestingly, both the categories of combinations increased T₄ levels, but not T₃ in PTU treated hypothyroid animals. Moreover, a parallel increase in hepatic and renal LPO was observed in these animals, suggesting their unsafe nature in combination. In conclusion the three test peel extracts appear to be stimulatory to thyroid functions and inhibitory to tissue LPO but only when treated individually.     

Analysis of 6-Keto PGF1a, 5-hete and LTC4 in rat lung: Comparison of GC/MS,RIA, nad EIA

The recent avaibility of fast and sensitive radioimmunoassay (RIA) and enzyme immunoassay (EIA) procedures to measure icosanoids has led to utilization of these techniques by many investigators. A major concern has been that techniques based on immunoreactivity may lack specificity, in particular if complex biologic fluids or tissue extracts are evaluated. The purpose of this investigation was the comparison of icosanoid measurements obtained either with EIA or RIA with those obtained by gas chromatography/mass spectrometry (GC/MS). Rats were injected with Salmonella enteritidis endotoxin, killed at various times after the injection and the ling extract assayed for 6-keto-PGF_1a, 5-HETE and LTC₄.By EIA lung tissue was found to contain large quantities of 6-keto-PGF_1a after endotoxin stimulation. Comparisons made between EIA and GC/MS analysis showed good correlation between 6-keto-PGF_1a amounts in lung as determined by each technique. It was also determined that little purification of lung extract was needed to obtain reliable quantitation of 6-keto-PGF_1a, probably due to the specificity of the antibody and the large quantity of this prostaglandin produced. Crudely purified (Sep-Park) lung extracts gave 5-HETE levels by RIA which were highly correlated with GC/MS values, but RIA values were 70% higher than those obtained by GC/MS. The presence of other components in lung extract which cross react with this 5-HETE antibody was probably responsible for the higher obtained by RIA. LTC₄ was measured by immunoassay in crude lung extracts, as well as after Sep-Pak purification and HPLC purification. LTC₄ levels were identical in unpurified lung extract and after Sep-Pak purifucation, but decreased substantially after HPLC purification. Thus, by validating the icosanoid immunoassays, we have found that they can give accurate and reproductive results in lung tissue, although LTC₄ and 5-HETE must be purified prior to analysis.     

Survey of pyruvate,phosphate dikinase activity of plants in relation to the C3, C4 and CAM mechanisms of CO2 assimilation

The pyruvate, phosphate dikinase activity (PPD, EC 2.7.9.1) associated with crude extracts of leaf tissue of some C₃ and C₄ plants was determined by phosphoenolpyruvate plus PPi-dependent phosphorylation of AMP. The PPD activity of all C₄ plants examined was > 15 nmol/mg protein/min. Several factors contributed to the underestimation of PPD activity in crude extracts of at least some species. Significant PPD activity (> 0.15 nmol/mg protein/min) was not detected in the majority of C₃ species but several C₃ species and the two CAM species studied exhibited activity in the range 0.4–4 nmol/mg protein/min while the C₃ species Avena sativa showed activity up to 8 nmol/mg protein/min. The oat leaf enzyme was partially purified; it exhibited properties similar to those of partially purified PPD from maize. Leaf extracts of the orchids Cymbidium canaliculatum and C. madidum contained high levels of PPD activity similar to the majority of C₄ plants. PPD activity has also been shown in other previously unstudied species.     

Paper chromatography of auxins and inhibitors in two Nicotiana species and their hybrid

Auxins and auxin inhibitors from tissue extracts of normal Nicotiana plants, Nicotiana glauca, N. langsdorffii and their hybrid (which spontaneously produces tumors) were separated by ascending paper chromatography with n-butanol-distilled water. An Avena curvature test was used for demonstrating growth-promoting and growth-inhibiting substances. IAA could be found in extracts of the parents and the hybrid (R_F 0.75). Hybrid tissue yielded the highest amount (37.1°), N. glauca tissue less (30.8°), and N. langsdorffii tissue the least amount (8.5°) of IAA. A second growth promoter (R_F 0.35) could be separated from the tissue extracts of the parents and the hybrid, but it showed only low activity in the Avena test. Three inhibitors were present in extracts from N. langsdorffii and the hybrid at R_F 0.25, 0.45, and 0.85, whereas N. glauca showed only two of them (R_F 0.25 and 0.85). The inhibitor with an R_F of 0.45 seemed to be identical with the acidic, benzene-insoluble “inhibitor β” of Bennet-Clark and Kefford (1953). The inhibitor (neutral, benzene-soluble) at R_F 0.85 could be found in some tissue extracts of the parents and the hybrid, but showed only little activity in the curvature tests. From neutral and from acidic plant extracts within a pH range of 4.4 to 5.8 a third inhibitor with an R_F of 0.25 could be separated. It seems that the high concentration of natural IAA in the hybrid is regulated by a variety of inhibitors with different specificities in the growth-regulating process. Nicotiana langsdorffii tissue has much less auxin but the same variety of inhibitors as the hybrid, whereas N. glauca tissue contains less auxin than the hybrid and only two of the three inhibitors found in N. langsdorffii and hybrid extracts.     

Determination of DNA Damage in Experimental Liver Intoxication and Role of N-Acetyl Cysteine

The present study aimed at detecting DNA damage and fragmentation as well as histone acetylation depending on oxidative stress caused by CCl₄ intoxication. Also, the protective role of N-acetyl cysteine, a precursor for GSH, in DNA damage is investigated. Sixty rats were used in this study. In order to induce liver toxicity, CCl₄ in was dissolved in olive oil (1/1) and injected intraperitoneally as a single dose (2 ml/kg). N-acetyl cysteine application (intraperitoneal, 50 mg/kg/day) was started 3 days prior to CCl₄ injection and continued during the experimental period. Control groups were given olive oil and N-acetyl cysteine. After 6 and 72 h of CCl₄ injection, blood and liver tissue were taken under ether anesthesia. Nuclear extracts were prepared from liver. Changes in serum AST and ALT activities as well as MDA, TAS, and TOS levels showed that CCl₄ caused lipid peroxidation and liver damage. However, lipid peroxidation and liver damage were reduced in the N-acetyl cysteine group. Increased levels in 8-hydroxy-2-deoxy guanosine and histone acetyltransferase activities, decreased histone deacetylase activities, and DNA breakage detected in nuclear extracts showed that CCl₄ intoxication induces oxidative stress and apoptosis in rat liver. The results of the present study indicate that N-acetyl cysteine has a protective effect on CCl₄-induced DNA damage.     

The structural characterization of proteoglycans of culture aortic smooth muscle cells and arterial wall of the pig

Aortic proteoglycans, from the growth medium of cultured smooth muscle cells and from sequential associative and dissociative extracts of the tissue of origin, the pig aorta, were isolated and purified by precipitation with cetylpiridinium chloride. After isopycnic CsCl gradient centrifugation under associative conditions 94% of the cell-secreted proteoglycans were recuperated in the bottom one fifth (?_av = 1.62 g/ml) fraction. In contrast 80% of the tissue proteoglycans of both extracts, fractionated into two fractions: the bottom one fifth (?_av = 1.60 g/ml) fraction and three fifths (?_av = 1.42 g/ml) fraction. Fractionated tissue proteoglycans were composed predominantly of chondroitin sulfate-dermatan sulfate (83–90%) with lower proportions of heparan sulfate (5–11%) and hyaluronic acid (3–6%) whilst cell-secreted proteoglycans showed a similar glycosaminoglycan composition but with a higher proportion of hyaluronic acid (11–13%). Sepharose 2B and C1-2B chromatography of tissue proteoglycans of high buoyant density showed the presence of only subunit proteoglycans whilst those of intermediate density contained a complex species, partially dissociable in 4 M guanidinium chloride, along with K_av 0.50 subunit species. The latter was also observed for cell-secreted proteoglycans although obtained at high buoyant density. The cell-secreted subunit proteoglycans became separated into two distinct populations when chromatographed on Sepharose 4B and C1-4B, half of which eluted in the column V_o and the rest at a K_av of 0.34.. The majority of subunit macromolecules eluted at the V_o fractions of Sepharose 6B and C1-6B columns. Unlike the major species of cartilage proteoglycans, only approx. 20% of purified arterial proteoglycans formed complexes. This proportion could be increased by only 4–7% by interaction, of a mixture of subunit cell-secreted and tissue-extracted proteoglycans, with hyaluronic acid. These results suggest that proteoglycans secreted by cultured aortic smooth muscle cells and present in the aortic tissue possess certain similar physicochemical properties and are present in the form of complex and several subunit species.     

Antioxidant activities of cultured <Emphasis Type="Italic">Armillariella mellea</Emphasis>

This study aimed to evaluate the antioxidant activities of a cultured medicinal fungus—Armillariella mellea (Vahl. ex Fr.) Karst. (AM). Three antioxidant assay systems, namely cytochrome c, xanthine oxidase inhibition, and FeCl₂-ascorbic acid stimulated lipid peroxidation in rat tissue homogenate tests, were used. Total flavonoid and phenol contents of AM extracts were also analyzed. Results showed that both aqueous (AM-H₂O) and ethanolic (AM-EtOH) extracts of solid state cultured AM showed antioxidant activities in a concentration-dependent manner. At concentrations 1–100 μg/ml, the free radical scavenging activity was 73.7–92.1% for AM-H₂O, and 60.0–90.8% for AM-EtOH. These extracts also showed an inhibitory effect on xanthine oxidase activity, but with a lesser potency (IC₅₀ is 9.17 μg/ml for AM-H₂O and 7.48 μg/ml for AM-EtOH). In general, AM-H₂O showed a stronger antilipid peroxidation activity on different rat’s tissues than AM-EtOH. However, both AM extracts displayed a weak inhibitory effect on lipid peroxidation in plasma. Interestingly, the antilipid peroxidation activity of AM-H₂O (IC₅₀–6.66 μg/ml) in brain homogenate was as good as IC₅₀–5.42 μg/ml. AM-H₂O (80.0 mg/g) possessed a significantly higher concentration of total flavonoids than AM-EtOH (30.0 mg/g), whereas no difference was noted in the total phenol content between these two extracts. These results conclude that AM extracts possess potent free radical scavenging and antilipid peroxidation activities, especially the AM-H₂O in the brain homogenate. Published in Russian in Prikladnaya Biokhimiya i Mikrobiologiya, 2007, Vol. 43, No. 4, pp. 495–500. The text was submitted by the authors in English.     

CHLOROPLAST STRUCTURE AND FUNCTION IN CULTURED TOBACCO TISSUE

A comparative study was made of the ability of cultured pith tissue, leaves of buds induced from callus, and mature leaf tissue of Nicotiana tabacum L. ‘Maryland Mammoth’ to fix carbon, as determined by light-induced C¹⁴O₂ incorporation. Photosynthetic ability was then correlated with the fine structure of chloroplasts from these tissues. The light to dark incorporation ratio for C¹⁴O₂ was at least 3 times as great in the leaf tissue as in growing cultured tissue. The chlorophyll content of the leaf tissue was 10 times as great. The carbon fixation pattern of all the tissues, as determined by radioautographs of chromatogramed extracts, was qualitatively the same. The rate of sucrose synthesis differed greatly, since 20% of the total radioactivity of the extracts from mature leaf tissue appeared in sucrose, while only 1.0% was found in sucrose from callus extracts. The incorporation of C¹⁴O₂ into sugars was inhibited in all the tissue by DCMU (3,4-dichlorophenyl,1, 1-dimethylurea). Cultured tissue past the log phase of growth was intermediate between the younger cultured tissue and the leaf tissue in its chlorophyll content and ability to incorporate C¹⁴O₂ in the light. Proplastids from dark-grown callus possessed stroma lamellae, but prolamellar bodies were not observed. The chloroplasts from growing callus were partially differentiated in comparison with chloroplasts from mature leaf tissue, since each granum had only 4-7 lamellae. Chloroplasts from callus past the log phase of growth were indistinguishable from those in mature leaves. This study establishes that the differentiation of chloroplasts in cultured tissue is a function of the growth rate of the tissue. The growth rate and degree of differentiation of the tissue can be regulated, so a well-defined system is available for the experimental study of chloroplast differentiation.     

Effects of gibberellic acid and uniconazole on the activities of some enzymes of anthocyanin biosynthesis in carrot cell cultures

Gibberellic acid (GA₃) inhibition of anthocyanin accumulation by carrot cell-suspension cultures was reversed by supplying dihydroquercitin or naringenin to the culture and not by supplying 4-coumaric acid or malonic acid. This suggested that gibberellic acid was inhibiting chalcone synthase, chalcone isomerase, or acetyl CoA carboxylase. Acetyl-CoA-carboxylase specific activity was the same in GA₃-treated and untreated cultures and was not detected in cultures treated with uniconazole, an inhibitor of gibberellic acid biosynthesis. Chalcone-isomerase specific activity was lower in GA₃-treated cultures than in untreated cultures and was lower in uniconazole-treated cultures than in the GA₃-treated cultures. The total chalcone synthase activity in extracts from GA₃- and from uniconazole-treated cells was not significantly different from that in extracts of untreated tissue. When these extracts were chromatographed on a Mono Q column, three peaks of chalcone synthase activity were found in extracts of nontreated cells, whereas only two of these peaks were detected in extracts of GA₃-treated cells. The extracts from GA₃-treated cells did not contain the peak of chalcone synthase activity that, in untreated cells, preceded the main peak. The correlation between the absence of this peak and the inhibition of anthocyanin accumulation suggests that this form of chalcone synthase is responsible for anthocyanin synthesis and that GA₃ prevents this form from appearing in the cells.     

Production of a plasmin inhibitory substance by Scopolia japonica suspension cultures

A potent inhibitory agent against human plasmin, fibrinolytic proteinase, has been found in the extracts of callus tissue of Scopolia japonica. Effects of cultural conditions on cell growth and production of the plasmin inhibitory substance by this cell line in suspension cultures were examined in MurashigeSkoog's medium. More than l.5 mg of the inhibitor, as t-amino cyclohexane carboxylic acid, a synthetic plasmin inhibitor, were observed to accumulate per ml of medium containing 0.83 g of NH₄NO₃ and 7.6 g of KNO₃ per liter as well as suitable levels of growth hormones. Addiction of antibiotics and deformers were examined in preliminary tests for large scale cultivation. Semicontinuous culture on a small scale in a glass cylinder, was also tested and growth rate of 1.29 g/liter/day (by dry wt) was obtained. Plasmin inhibitory activities in the extracts of the results intact plant and in cultured cells of S. japonica were compared and the results indicated that cell suspension culture was superior to extraction the natural plant for inhibitor production.     

Biochemical genetics of alcohol dehydrogenase isozymes in the gray short-tailed opossum (Monodelphis domestica)

Polyacrylamide gel-isoelectric focusing (PAGE-IEF) methods were used to examine the multiplicity, tissue distribution, and biochemical genetics of alcohol dehydrogenase (ADH) isozymes among gray short-tailed opossums (Monodelphis domestica). Seven ADH isozymes were resolved and distinguished on the basis of their isoelectric points, tissue distributions, and substrate and inhibitor specificities. ADH1 and ADH2 exhibited Class I properties and were observed in liver (and intestine) extracts. ADH3, ADH4, and ADH5 showed “high-K _m ” (possibly Class IV) properties, with ADH3 and ADH4 exhibiting high activity in cornea, ear, stomach, and esophagus extracts. ADH6 and ADH7 exhibited Class III properties, including activities as formaldehyde dehydrogenases, with each showing different tissue distribution characteristics; ADH6 was widely distributed, and ADH7 was restricted to prostate extracts. An additional form of formaldehyde dehydrogenase (FDH) was observed, which was inactive with hexenol and ethanol as substrates. Isoelectric point variants were observed for ADH3 (three forms) and for ADH4 (two forms), and the inheritance of ADH3 was studied in 15 families ofM. domestica. The data were consistent with codominant inheritance of two alleles (ADH3*A andADH3*B) at a single autosomal locus (designatedADH3) and with a model involving a dimeric ADH isozyme: ADH3 (γ₂ isozyme, forming three dimers designated γ ₂ ¹ , γ¹ γ², and γ ₂ ² in heterozygous individuals).     

Stimulation of ammonium ion excretion in the toad urinary bladder by an extract of human urine

It is well known that ammonium ion excretion is increased during metabolic acidosis in mammals. The purpose of this study was to determine whether we could isolate from human urine during metabolic acidosis a factor that would stimulate NH₄⁺ and/or H⁺ excretion in toad urinary bladder. Extracts of urine from six human subjects collected during NH₄Cl-induced acidosis were prepared. These extracts were tested for their effect on NH₄⁺ excretion in hemibladders mounted between plastic chambers. The extracts significantly increased NH₄⁺ excretion in the toad urinary bladder. We found no effect on H⁺ excretion by these extracts. This ammoniuretic activity was not present in the urine when the same individuals were in metabolic alkalosis. We conclude that during metabolic acidosis a humoral factor is present which stimulates the excretion of NH₄⁺. The factor could act as a permease in the bladder cell or as a stimulator of an NH₄⁺ transport system.     

Vitamin D metabolite-binding proteins in human tissue

Serum and post-microsomal supernatants of human lymphocyte, erythrocyte, skeletal muscle and parathyroid adenoma homogenates were examined for specific binding of 25-hydroxycholecalciferol (25-OHD₃) and 1,25-dihydroxycholecalciferol (1,25-(OH)₂D₃). Muscle, lymphocytes and parathyroid adenomata extracts contained a 6-S 25-OHD₃-binding protein which was not found in erythrocyte extracts, and which was distinct from the smaller serum transport α-globulin. A cathodal, 1,25-(OH)₂D₃-binding protein, which sedimented at 3–4 S was also detected in parathyroid tissue. These observations suggest the possibility of direct physiologic interaction between vitamin D metabolites and nucleated human tissues other than intestine and bone.     

Regulation of Pea Mitochondrial Pyruvate Dehydrogenase Complex : Does Photorespiratory Ammonium Influence Mitochondrial Carbon Metabolism?

Inactivation of the pyruvate dehydrogenase complex catalyzed by pyruvate dehydrogenase kinase was studied using intact mitochondria purified from green leaf tissue of pea (Pisum sativum L.) and dialyzed mitochondrial extracts. Thiamine pyrophosphate was inhibitory in dialyzed extracts but not in intact mitochondria, except in the presence of high concentrations of Na⁺. NH₄⁺, at concentrations as low as 20 micromolar, markedly stimulated inactivation in dialyzed extracts. K⁺ in the range 1 to 10 millimolar also enhanced inactivation. In contrast, Na⁺ was without affect at lower concentrations but was inhibitory at 10 to 100 millimolar levels. The effect of NH₄⁺ is discussed in relation to a possible regulatory interaction between photorespiratory NH₄⁺ production and the entry of carbon into the tricarboxylic acid cycle by way of the pyruvate dehydrogenase complex.     

Metabolomic Profiling Unravels DNA Adducts in Human Breast That Are Formed from Peroxidase Mediated Activation of Estrogens to Quinone Methides

Currently there are three major hypotheses that have been proposed for estrogen induced carcinogenicity, however exact etiology remains unknown. Based on the chemical logic, studies were undertaken to investigate if estrogens could generate quinone methides in an oxidative environment which then could cause DNA damage in humans. In presence of MnO₂ estrogens were oxidized to quinone methides. Surprisingly quinone methides were found to be stable with t_1/2 of 20.8 and 4.5 min respectively. Incubation of estrogens with lactoperoxidase (LPO) and H₂O₂ resulted in formation of respective quinone methides (E₁(E₂)-QM). Subsequent addition of adenine to the assay mixture lead to trapping of E₁(E₂)-QM, resulting in formation of adenine adducts of estrogens, E₁(E₂)-9-N-Ade. Targeted ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) based metabolomic analysis of the breast tissue extracts showed the presence of adenine adducts of estrogens, E₁(E₂)-9-N-Ade, along with other estrogen related metabolites. Identity of E₁(E₂)-N-Ade in LPO assay extracts and breast tissue extracts were confirmed by comparing them to pure synthesized E₁(E₂)-9-N-Ade standards. From these results, it is evident that peroxidase enzymes or peroxidase-like activity in human breast tissue could oxidize estrogens to electrophilic and stable quinone methides in a single step that covalently bind to DNA to form adducts. The error prone repair of the damaged DNA can result in mutation of critical genes and subsequently cancer. This article reports evidence for hitherto unknown estrogen metabolic pathway in human breast, catalyzed by peroxidase, which could initiate cancer.     

Assay of molybdenum cofactor of barley

Conditions for assay of molybdenum cofactor in barley shoot extracts in the presence of molybdate (25 mM N₂MoO₄) and the sulphydryl-group protector, reduced glutathione (5 mM) were optimized. Both total Mo-cofactor (assayed after heat-treatment of cell-free extracts) and ‘free’ Mo-cofactor (assayed in untreated cell-free extracts) were assayed. Compared to control plants grown in the absence of an exogenous nitrogen source total Mo-cofactor levels increased around 70 % when plants were grown for 4 days in the presence of either 15 mM KNO₃ or 15 mM NH₄NO₃. Growth in the presence of 15 mM (NH₄)₂SO₄ did not affect the Mo-cofactor level. Very similar results were seen when plants were transferred to these nitrogen sources for 24 hr after previous growth in the absence of an exogenous nitrogen source. In contrast ‘free’ Mo-cofactor levels of both KNO₃ and NH₄NO₃-treated plants were increased 2-3-fold over untreated controls. Growth in the presence of (NH₄)₂SO₄ did not affect the ‘free’ Mo-cofactor level.     

Bacteriohopanetetrol: abundant lipid in frankia cells and in nitrogen-fixing nodule tissue

An unusual class of lipid with amphiphilic properties has been detected in nodule tissue of Alnus and Ceanothus. High levels of the same lipid (20-50% of total cell lipids) were detected in solvent extracts of Frankia spp. cells. However, the lipid was absent in host roots. The lipid was purified and quantified by high-performance liquid chromatography/flame ionization detector. Phenol-sulfuric acid determinations and proton nuclear magnetic resonance indicated that the purified lipid is not a glycolipid. Mass spectra of the predominant species are consistent with published spectra for bacteriohopanetetrol (C₃₅H₆₂O₄), a pentacyclic triterpenoid, or hopanoid.     

The aggregation states of spinach phosphoribulokinase

Phosphoribulokinase (PRK; EC 2.1.7.19) is active in illuminated chloroplasts and inactive in darkened chloroplasts. This regulatory mechanism is mediated by thioredoxin-dependent reduction of a kinase disulfide in vivo. Extracts of spinach (Spinacia oleracea L.) leaves in the presence of 10 mM dithiothreitol contain a single 80-kDa form of PRK as judged by gel filtration. Gel filtration of thiol-free extracts of light-harvested tissue shows the presence of two inactive forms of PRK, the 80-kDa form and an aggregate (> 550 kDa) form, but treatment of both forms with dithiothreitol restores kinase activity. Gel filtration following extraction of dark-harvested tissue in the absence of dithiotreitol demonstrates the presence of only the heavier form. Inclusion of 400 mM (NH₄)₂SO₄ in the homogenization buffer during extraction of light-harvested tissue suppresses the formation of the high-M _r form of PRK, but does not eliminate the aggregate form observed in extracts of dark-harvested leaves. However, prolonged treatment of extracts from dark-harvested tissue with 400 mM (NH₄)₂SO₄ results in conversion of the high-M _r form of phosphoribulokinase to the low-M _r form. The data are consistent with the heavier form of phosphoribulokinase being the normal in-vivo aggregation state in the dark, while the lighter form is the normal aggregation state in the light.This research was sponsored jointly by the science and education administration of the U.S. Department of Agriculture under Grant No. 88-37130-3722 from the Competitive Research Grants Office and by the Office of Health and Environmental Research, U.S. Department of Energy under Contract DE-AC05-84OR21400 with Martin Marietta Energy Systems Inc., Oak Ridge, Tenn., USA. The author is Postdoctoral Investigator supported by the U.S. Department of Agriculture through Subcontract No. 88-37130-3722 from the Biology Division of Oak Ridge National Laboratory to the University of Tennessee.     

Di(1,N-ethenoadenosine)5′, 5-P,P-tetraphosphate, a fluorescent enzymatically active derivative of Ap4A

Di(1,N⁶-ethenoadenosine) 5′, 5-P¹, P⁴-tetraphosphate, ε-(Ap₄A), a fluorescent analog of Ap₄A has been synthesized by reaction of 2-chloroacetaldehyde with Ap₄A. At neutral pH this Ap₄A analog presents characteristic maxima at 265 and 274 nm, shoulders at ca 260 and 310 nm and moderate fluorescence (λ_exc 307 nm, λ_em 410 nm). Enzymatic hydrolysis of the phosphate backbone produced a slight hyperchromic effect but a notorious increase of the fluorescence emission. Cytosolic extracts from adrenochromaffin tissue as well as cultured chromaffin cells were able to split ε(Ap₄A) and catabolize the resulting ε-nucleotide moieties up to ε-Ado.