Measurement of bacterial sulfate reduction in sediments: Evaluation of a single-step chromium reduction method

A procedure which includes the Total Reduced Inorganic Sulfur (TRIS) in a single distillation step is described for the radiotracer measurement of sulfate reduction in sediments. The TRIS includes both Acid Volatile Sulfide (AVS: H₂S + FeS) and the remaining Chromium Reducible Sulfur (CRS: S⁰, FeS₂). The single-step distillation was simpler and faster than the consecutive distillations of AVS and CRS. It also resulted in higher (4–50%) sulfate reduction rates than those obtained from the sum of³⁵S in AVS and CRS. The difference was largest when the sediment had been dried after AVS but before CRS distillation. Relative to the³⁵S-AVS distillation alone, the³⁵S-TRIS single-step distallation yielded 8–87% higher reduction rates. The separation and recovery of FeS, S⁰ and FeS₂ was studied under three distillation conditions: 1) cold acid, 2) cold acid with Cr²⁺, and 3) hot acid with Cr²⁺. The FeS was recovered by cold acid alone while pyrite was recovered by cold acid with Cr²⁺. A smaller S⁰ fraction, presumably of the finer crystal sizes, was recovered also in the cold acid with Cr²⁺ while most of the S⁰ required hot acid with Cr²⁺ for reduction to H₂S.     

Metabolism of leukotriene B4 by guinea pig eosinophils

The metabolism of leukotriene B₄ (5(S),12(R)-dihydroxy-6-cis-8,10-trans-14-cis-eicosatetraenoic acid) by isolated guinea pig eosinophils was investigated. Incubation of guinea pig eosinophils with [³H]-leukotriene B₄ resulted in the rapid conversion of leukotriene B₄ to several more polar metabolites. Two of these metabolites were identified by ultraviolet spectroscopy and gas chromatography-mass spectrometry as the omega oxidation products 5(S),12(R),20-trihydroxy-6,8,10,14-eicosatetraenoic acid (20-hydroxy-leukotriene B₄) and 5(S),12(R),19-trihydroxy-6,8,10,14-eicosatetraenoic acid (19-hydroxy-leukotriene B₄). Two novel metabolites, 5(S),12(R),18,19-tetrahydroxy-6,8,10,14 eicosatetraenoic acid (18,19-dihydroxy-leukotriene B₄) and 5(S),12(R)-dihydroxy-1,18-dicarboxylic-6,8,10,14,16-octadecapentaenoic acid (Δ16,17–18-carboxy-19,20-dinor-leukotriene B₄) were tentatively identified. The identification of these compounds indicates that guinea pig eosinophils are capable of metabolizing leukotriene B₄ by both omega and beta oxidation. This catabolic activity may play a role in modulating inflammatory reactions by removing the chemoattractant leukotriene B₄ from inflammatory sites.     

Higher Prevalence of Metformin-Induced Vitamin B12 Deficiency in Sulfonylurea Combination Compared with Insulin Combination in Patients with Type 2 Diabetes: A Cross-Sectional Study

Long-term and high-dose treatment with metformin is known to be associated with vitamin B₁₂ deficiency in patients with type 2 diabetes. We investigated whether the prevalence of B₁₂ deficiency was different in patients treated with different combination of hypoglycemic agents with metformin during the same time period. A total of 394 patients with type 2 diabetes treated with metformin and sulfonylurea (S+M group, n = 299) or metformin and insulin (I+M group, n = 95) were consecutively recruited. The vitamin B₁₂ and folate levels were quantified using the chemiluminescent enzyme immunoassay. Vitamin B₁₂ deficiency was defined as vitamin B₁₂≤300 pg/mL without folate deficiency (folate>4 ng/mL). The mean age of and duration of diabetes in the subjects were 59.4±10.5 years and 12.2±6.7 years, respectively. The mean vitamin B₁₂ level of the total population was 638.0±279.6 pg/mL. The mean serum B₁₂ levels were significantly lower in the S+M group compared with the I+M group (600.0±266.5 vs. 757.7±287.6 pg/mL, P<0.001). The prevalence of vitamin B₁₂ deficiency in the metformin-treated patients was significantly higher in the S+M group compared with the I+M group (17.4% vs. 4.2%, P = 0.001). After adjustment for various factors, such as age, sex, diabetic duration, duration or daily dose of metformin, diabetic complications, and presence of anemia, sulfonylurea use was a significant independent risk factor for B₁₂ deficiency (OR = 4.74, 95% CI 1.41–15.99, P = 0.012). In conclusion, our study demonstrated that patients with type 2 diabetes who were treated with metformin combined with sulfonylurea require clinical attention for vitamin B₁₂ deficiency and regular monitoring of their vitamin B₁₂ levels.     

Comparative activity of natural mono-, di- and tri-hydroxy derivatives of arachidonic acid on guinea-pig lungs: Myotropic effect and stimulation of cyclooxygenase activity

The activity of natural 5,6-Dihydroxy-eicosatetraenoic acid (5,6-DiHETE; 2 isomers), 5S,15S-DiHETE, 8S,15S-DiHETE, 5S,12S-DiHETE, Δ⁶-trans-leukotriene B₄, 12-epi-Δ⁶-leukotriene B₄, ω-hydroxy-leukotriene B₄, ω-carboxy-leukotriene B₄, 15S-hydroxy-eicosatetraenoic acid (15S-HETE), 12S-HETE, 5S-HETE and 12S-hydroxy-heptadecatrienoic acid was compared to TLB₄ on the guinea-pig lung parenchymal strip and on the release of prostaglandins and thromboxanes by the perfused guinea-pig lungs. The ω-hydroxy-LTB₄ appeared more potent than LTB₄ both for inducing a contraction and for releasing prostanoids whereas the ω-carboxy-LTB₄ was much less active on the parenchyma and did not release prostanoids at the dose used. All other hydroxy acids tested were either very weakly active or inactive in the two systems used with the exception of the 5,6-DiHETEs which showed significant activity. These di-hydroxy acids induced contractions of the lung parenchymal strip which could be blocked by PFL-55712 but were inactive on the guinea-pig ileum. The 5S-HETE, 12S-HETE and 15S-HETE were also tested for possible myotropic activity on selected smooth muscle preparations. Our results provide further informations on the structural requirements for LTB₄ (and other hydroxy acids) actions on the guinea-pig lungs.     

Dietary consumption of monosodium L‐glutamate induces adaptive response and reduction in the life span of Drosophila melanogaster

Adaptive response is the ability of an organism to better counterattack stress‐induced damage in response to a number of different cytotoxic agents. Monosodium L‐glutamate (MSG), the sodium salt of amino acid glutamate, is commonly used as a food additive. We investigated the effects of MSG on the life span and antioxidant response in Drosophila melanogaster (D. melanogaster). Both genders (1 to 3 days old) of flies were fed with diet containing MSG (0.1, 0.5, and 2.5‐g/kg diet) for 5 days to assess selected antioxidant and oxidative stress markers, while flies for longevity were fed for lifetime. Thereafter, the longevity assay, hydrogen peroxide (H₂O₂), and reactive oxygen and nitrogen species levels were determined. Also, catalase, glutathione S‐transferase and acetylcholinesterase activities, and total thiol content were evaluated in the flies. We found that MSG reduced the life span of the flies by up to 23% after continuous exposure. Also, MSG increased reactive oxygen and nitrogen species and H₂O₂ generations and total thiol content as well as the activities of catalase and glutathione S‐transferase in D. melanogaster (P < .05). In conclusion, consumption of MSG for 5 days by D. melanogaster induced adaptive response, but long‐term exposure reduced life span of flies. This study may therefore have public health significance in humans, and thus, moderate consumption of MSG is advocated by the authors.     

Sulfur cycling in forests

Sulfur is essential for the production of certain amino acids in plants. As amino acid sulfur is the major form of sulfur in trees, there is a strong relationship between organic S and organic N in tree tissue. Sulfur deficiencies occur in parts of southeastern Australia and northwestern North America, remote from pollutant inputs. Since bilogical S requirements of forests are modest (< 5 kg · ha^–1 yr^–1 for net vegetative increment), however, atmospheric S inputs in polluted regions (10–80 kg · ha^–1 yr^–1 ) often exceed not only the forest ecosystem S requirement but also its ability to biologically accumulate S. There is some increase in the SO^2– ₄–S content of forest vegetation in response to elevated atmospheric S inputs, but this capacity is apparently easily saturated. Soil SO^2–2 ₄adsorption is often the dominant feature of S cycling in polluted ecosystems and often accounts for net ecosytem S accumulations.Contribution from a symposium on the role of sulfur in ecosystem processes held August 10, 1983, at the annual meeting of the A.I.B.S., Grand Forks, ND; Myron Mitchell, convenor.     

Inhibition of Vancomycin-Resistant <Emphasis Type="Italic">Enterococcus</Emphasis> by Continuous-Flow Cultures of Human Stool Microflora With and Without Anaerobic Gas Supplementation

A continuous-flow competitive exclusion (CFCE) culture model of human stool microflora was used to examine whether supplemental anaerobic gas is necessary for maintenance of anaerobes and inhibition of vancomycin-resistant Enterococcus (VRE). CFCE cultures of human stool microflora were maintained with supplemental nitrogen, without supplemental nitrogen, or with percolated room air. Cultures with or without supplemental nitrogen maintained >9 log₁₀ CFU mL^–1 of obligate anaerobes and eliminated 10⁶ CFU mL^–1 of VRE. When room air was percolated into the culture, anaerobes were detected at 2 log₁₀ CFU mL^–1, and the same VRE inoculum was not eliminated (P < 0.001). These data demonstrate that human stool CFCE cultures maintain high levels of obligate anaerobes and inhibit VRE without the addition of supplemental anaerobic gas.     

Prostanoid biosynthesis in patients with cystic fibrosis

The urinary excretion rate (ng/h/1.73 m²) of prostanoids was determined with a capillary gas-liquid chromatographic mass spectrometric method in 19 patients with cystic fibrosis (CF) aged 1–29 years. Patients with CF showed an increased excretion of prostaglandin E₂ metabolites (PGE-M) and thromboxane B₂ and its metabolites at all ages. An imbalance in the excretion pattern of thromboxane B₂ metabolites also suggested a relative impairment of β-oxidation. There was no increased excretion of dinor-6-keto-PGF_1α, indicating normal prostacyclin biosynthesis. No correlation was found to genotype, clinical score, lung function or bacterial colonization but a significant negative relation was found between the main prostanoids in the urine and serum phospholipid levels of essential fatty acids. The results show that, contrary to the generally accepted decrease of prostanoid excretion in essential fatty acid deficiency, patients with CF increase their production parallel to the development of the deficiency. Since prostanoid synthesis is rate limited by arachidonic acid release, our data support a previously presented hypothesis about a pathological regulation of the release of arachidonic acid in CF.     

A Novel Hyaluronidase Produced by Bacillus sp. A50

Hyaluronidases are a family of enzymes that degrade hyaluronic acid (hyaluronan, HA) and widely used in many fields. A hyaluronidase producing bacteria strain was screened from the air. 16S ribosomal DNA (16S rDNA) analysis indicated that the strain belonged to the genus Bacillus, and the strain was named as Bacillus sp. A50. This is the first report of a hyaluronidase from Bacillus, which yields unsaturated oligosaccharides as product like other microbial hyaluronate lyases. Under optimized conditions, the yield of hyaluronidase from Bacillus sp. A50 could reach up to 1.5×10⁴ U/mL, suggesting that strain A50 is a good producer of hyaluronidase. The hyaluronidase (HAase-B) was isolated and purified from the bacterial culture, with a specific activity of 1.02×10⁶ U/mg protein and a yield of 25.38%. The optimal temperature and pH of HAase-B were 44°C and pH 6.5, respectively. It was stable at pH 5–6 and at a temperature lower than 45°C. The enzymatic activity could be enhanced by Ca²⁺, Mg²⁺, or Ni²⁺, and inhibited by Zn²⁺, Cu²⁺, EDTA, ethylene glycol tetraacetic acid (EGTA), deferoxamine mesylate salt (DFO), triton X-100, Tween 80, or SDS at different levels. Kinetic measurements of HAase-B towards HA gave a Michaelis constant (K _m) of 0.02 mg/mL, and a maximum velocity (V _max) of 0.27 A ₂₃₂/min. HAase-B also showed activity towards chondroitin sulfate A (CSA) with the kinetic parameters, K _m and V _max, 12.30 mg/mL and 0.20 A ₂₃₂/min respectively. Meanwhile, according to the sequences of genomic DNA and HAase-B’s part peptides, a 3,324-bp gene encoding HAase-B was obtained.     

Organic nitrogen stimulates caulogenesis from hypocotyl callus of Phyllanthus fraternus

Factors involved in promoting caulogenesis from hypocotyl explants of Phyllanthus fraternus were studied. Hypocotyl explants were cultured on B₅ medium supplemented with 2,4-D or NAA in the presence and absence of BAP (at concentrations 0, 10^–7, 10^–6 and 10^–5M). Adventitious shoots differentiated from callus developed from the cut ends of 12.5% of the hypocotyl segments cultured on medium supplemented with 10^–6M BAP in combination with 10^–6M 2,4-D or 10^–6M NAA. Profuse rooting occurred from the hypocotyl explants on medium supplemented with 10^–6M BAP + 10^–6M NAA. Incorporation of casein hydrolysate in B₅ medium along with 10^–6M BAP + 10^–7M 2,4-D enhanced the frequency of cultures with adventitious shoots upto 68.0%. Glutamine, glutamic acid or proline could partially substitute for the effect of casein hydrolysate. Amongst the hypocotyls from 3–14 d old seedlings, the best caulogenesis was obtained with hypocotyls from 7 d old seedlings both in presence or absence of casein hydrolysate. Best rooting of shoots was achieved on half-strength B₅ medium supplemented with 10^–6M IBA. After hardening, plantlets were successfully transferred to the soil.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2, 4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - NAA 1-naphthaleneacetic acid - CH casein hydrolysate - Arg L-arginine - Glu L-glutamic acid - Gln L-glutamine - Leu L-leucine - Lys L-lysine - Pro L-proline     

Dietary vitamin B6 intake modulates colonic inflammation in the IL10−/− model of inflammatory bowel disease

Pyridoxal-5-phosphate, the biologically active form of vitamin B₆, is a cofactor for over 140 biochemical reactions. Although severe vitamin B₆ deficiency is rare, mild inadequacy [plasma pyridoxal 5’-phosphate (PLP) <20 nmol/L] is observed in 19–27% of the US population. Plasma PLP concentrations are inversely related to markers of inflammation such as C-reactive protein. Furthermore, plasma PLP is diminished in those with inflammatory conditions and, in the case of inflammatory bowel disease (IBD), more so in those with active versus quiescent disease. Restricting B₆ intake attenuates IBD pathology in mice; however, the effects of supplementation are unclear. We therefore sought to determine the effects of mild inadequacy and moderate supplementation of B6 on the severity of colonic inflammation. Weanling IL-10^?/? (positive for Helicobacter hepaticus) mice were fed diets containing 0.5 (deficient), 6.0 (replete) or 24 (supplemented) mg/kg pyridoxine HCl for 12 weeks and then assessed for histological and molecular markers of colonic inflammation. Both low and high plasma PLP were associated with a significant suppression of molecular (TNFα, IL-6, IFN-γ, COX-2 and iNOS expression) and histological markers of inflammation in the colon. PLP is required for the breakdown of sphingosine 1-phosphate (S1P), a chemotactic lipid, by S1P lyase. Colonic concentrations of S1P and PLP were significantly and inversely correlated. If confirmed, vitamin B6 supplementation may offer an additional tool for the management of IBD. Although B6 is required in dozens of reactions, its role in the breakdown of S1P may explain the biphasic relationship observed between PLP and inflammation.     

The influence of lipoic acid on adriamycin-induced hyperlipidemic nephrotoxicity in rats

Glutamate, a major excitatory amino acid neurotransmitter is also an endogenous excitotoxin. The present study examined the prolonged and delayed effects of glutamate excitotoxicity on mitochondrial lipid peroxidation and antioxidant parameters in different brain regions, namely, cerebral hemisphere, cerebellum, brain stem and diencephalon. Wistar rats (male) were exposed to monosodium glutamate (MSG) (4 mg × g body wt^–1, i.p.) for 6 consecutive days and sacrificed on 30th and 45th day after last MSG dose. MSG treatment markedly decreased the mitochondrial manganese superoxide-dismutase (Mn-SOD), catalase and reduced glutathione (GSH) content, and increased the lipid peroxidation (LPx), uric acid and glutathione peroxidase (GPx) activity. These results indicate that oxidative stress produced by glutamate in vulnerable brain regions may persist for longer periods and mitochondrial function impairment is an important mechanism of excitatory amino acid mediated neurotoxicity in chronic neurodegeneration.     

Regeneration of plants from callus cultures of Origanum vulgare L.

Investigations were undertaken to achieve rapid multiplication and improvement of Origanum vulgare (a herbaceous, ornamental plant well known for its aromatic and medicinal value) through plant regeneration from callus. The explants (cotyledons, hypocotyl and root segments) excised from 15 d old aseptic seedlings were cultured on Gamborg's B₅ medium supplemented with 2,4-D, NAA and BAP individually and in various combinations (at concentrations of 0,10^–7,10^–6 and 10^–5 M). Best callus induction was noted on medium with 10^–7 M 2,4-D alone. The cotyledonary expiants proved to be the best source for compact and nodulated callus. The subcultured cotyledonary calli showed shoot induction when transferred onto media supplemented with BAP alone orin combination with 10^–7M or 10^–6MNAA. However, 10^–5M NAA completely suppressed the shoot inducing ability of BAP. In general, NAA promoted root induction from all explants used including cotyledonary callus. Best shoot induction was obtained on medium supplemented with 10^–6M BAP+10^–6MNAA. Both IBA and NAA at 10^–6 M proved to be equally effective in induction of roots from the cut ends of 15–20 mm long shoots (excised from callus) in half-strength B₅ liquid medium. Rooted shoots were successfully re-established in soil under controlled conditions.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - IBA indole-3-butyric acid - NAA -naphthaleneacetic acid - BAP 6-benzylaminopurine     

Vitamin B12 (cobalamin) deficiency in elderly patients

VITAMIN B₁₂ OR COBALAMIN DEFICIENCY occurs frequently (> 20%) among elderly people, but it is often unrecognized because the clinical manifestations are subtle; they are also potentially serious, particularly from a neuropsychiatric and hematological perspective. Causes of the deficiency include, most frequently, food-cobalamin malabsorption syndrome (> 60% of all cases), pernicious anemia (15%–20% of all cases), insufficent dietary intake and malabsorption. Food-cobalamin malabsorption, which has only recently been identified as a significant cause of cobalamin deficiency among elderly people, is characterized by the inability to release cobalamin from food or a deficiency of intestinal cobalamin transport proteins or both. We review the epidemiology and causes of cobalamin deficiency in elderly people, with an emphasis on food-cobalamin malabsorption syndrome. We also review diagnostic and management strategies for cobalamin deficiency. Vitamin B₁₂ or cobalamin deficiency occurs frequently among elderly patients,¹ but it is often unrecognized or not investigated because the clinical manifestations are subtle. However, the potential seriousness of the complications (particularly neuropsychiatric and hematological)^1,2,3,4 requires investigation of all patients who present with vitamin or nutritional deficiency. We summarize the current state of knowledge on cobalamin deficiency, with a particular focus on deficiency in elderly people.In gathering information for this article, we systematically searched PubMed for articles published from 1990 to July 2003 (the search strategy is outlined in online Appendix 1 [www.cmaj.ca/cgi/content/full/171/3/251/DC1]). We have also included unpublished data from our working group, the Groupe d''étude des carences en vitamine B12 des Hôpitaux Universitaires de Strasbourg.     

High-performance liquid chromatographic assay of human lymphocyte kynureninase activity levels

Human lymphocyte kynureninase activity was assessed in homogenized cells by determination of 3-hydroxyanthranilic acid production as a function of time after addition of the substrate, 3-hydroxykynurenine. The product, 3-hydroxyanthranilic acid, was determined by isocratic high-performance liquid chromatography and fluorescence detection. Mean (± S.D.) lymphocyte kynureninase activity in a group (n = 12) of vitamin B₆-deficient men was 5.04 ± 0.81 pmol 3-hydroxyanthranilic acid formed per mg protein per min, which was significantly (p=0.005) lower than the 6.69 ± 1.70 pmol 3-hydroxyanthranilic acid formed per mg protein per min in men with a normal vitamin B₆ status. This indicates that lymphocyte kynureninase activity is depressed during a vitamin B₆ deficiency.     

Step-scan FTIR spectroscopy resolves the QA −QB → QAQB − transition in Rb. sphaeroides R26 reaction centres

It is shown that step-scan Fourier transform infrared spectroscopy can be applied to resolve the Q_A ^–Q_B Q_AQ_B ^– transition in Rhodobacter sphaeroides reaction centres with a 5 µs time resolution. In the mid-infrared region (1900 – 1200 cm^–1), transient signals previously assigned to Q_A/B and Q_A/B ^– vibrations, respectively (Brudler et al. 1994; Brudler et al. 1995; Breton and Nabedryk 1996), can be resolved with this new technique. In addition, the three small positive bands in the spectral region of the carboxylic C=O stretching modes of acidic amino acid side chains are also resolved at 1730, 1719 and 1704 cm^–1. A global fit analysis yields two exponentials with half-times of 150 µs and 1.2 ms in agreement with IR spectroscopic studies at single wavenumbers (Hienerwadel et al. 1995), in the UV/VIS and near IR (Tiede et al. 1996, Li et al. 1996). The establishement of the step-scan technique enables a new approach to elucidate the molecular mechanism of this transition.     

Anti-inflammatory drugs, prostanoid and proteoglycan production by cultured bovine articular chondrocytes

The effect of various anti-inflammatory drugs on the production of prostaglandins E₂ and F₂α, 6 keto PGF₁α and thromboxane B₂ by bovine articular chondrocytes was measured by radioimmunoassay. While indomethacin and meclofenamic acid caused a dose-dependent inhibition of all prostanoids measured, the effects of hydrocortisone and colchicine varied with respect to different prostanoids. Hydrocortisone (10⁻⁷M – 10⁻³M) both in the presence and absence of added arachidonic acid, resulted in an inhibition of prostaglandins E₂ and F₂, and to a lesser extent, 6 keto PGF₁α, but T×B₂ production was only slightly inhibited by the drug in the absenced of arachidonic acid and markedly increased in its presence. Colchicine (10⁻⁷M – 10⁻³M) had the opposite effect, causing an inhibition of T×B₂ and stimulating PGE₂ and 6 keto PGF₁α production. These findings suggest that certain anti-inflammatory drugs may, in addition to their action on phospholipase A₂ and cyclo-oxygenase, exert potent effects at the level of the different synthetases. In order to see whether these alterations in relative prostanoid levels affected proteoglycan metabolism, the effect of anti-inflammatory drugs on proteoglycan synthesis by cultured chondrocytes was tested using ³⁵SO₄ labeling methodology. The results showed that the concentrations tested (10⁻⁵M to 10⁻⁷M), indomethacin, dexamethasone, hydrocortisone and colchicine inhibited ³⁵SO₄ incorporation into newly synthesized proteoglycan molecules both in the presence (10⁻⁶M) and absence of exogenous arachidonic acid. In the same concentration range choroquine had no effect.These results do not support the hypothesis of direct prostanoid involvement in the modulation of proteoglycan synthesis in articular cartilage.     

Endothelin receptor in microvessels isolated from human meningiomas: Quantification with radioluminography

Summary 1. We characterized specific¹²⁵I-endothelin-1 (¹²⁵I-ET-1) binding sites in microvessels isolated from human meningiomas, using anin vitro quantitative receptor autoradiographic technique coupled to a radioluminographic imaging plate system.2. This newly developed and highly sensitive method revealed high-affinity ET receptors present in pellet sections of the microvessels from all the meningiomas studied, regardless of histological subtypes (dissociation constant, 1.2 ± 0.3 nM; maximum binding capacity, 185 ± 56 fmol/mg; means ± SE for nine tumors).3. In five cases of meningiomas, ET-3 competed for¹²⁵I-ET-1 binding to microvessels from those tumors with a low affinity [50% inhibiting concentration (IC₅₀) of 1.6 ± 0.4 × 10^–6 M], and a selective ET_B receptor agonist, sarafotoxin S6c, up to 10^–6 M, did not displace ET binding from the sections.4. In the sections of microvessels from four other tumors, biphasic competition curves were obtained in the case of incubation in the presence of increasing concentrations of ET-3, with an IC₅₀ of 1.1 ± 0.2 × 10^–9 M for the high-affinity component and 1.6 ± 0.3 × 10^–6 M for the low-affinity component, respectively. In addition, S6c competed for ET binding to those sections (IC₅₀=2.3 ± 0.2 × 10^–10 M) and 10^–6 M S6c displaced 30% of the control, corresponding to the high-affinity component of competition curves obtained in the presence of ET-3.5. Our results suggest that (a) capillaries in human meningiomas express a large number of high-affinity ET_A (non-ET_B) receptors with a small proportion of ET_B receptors, and (b) ET may have a role in neovascularization, tumor blood flow, and/or function of the blood-tumor barrier in meningioma tissues by interacting with specific receptors present on the surface of the endothelium.     

Amplification of phytochrome induced morphogenesis in plants by the cryptic red light signal (CRS)

The regulation of endogenous levels of ascorbic acid in soybean by far-red absorbing form of phytochrome (Pfr) and by cryptic red light signal (CRS) was studied. Cryptic red light signal is produced by red light pre-irradiation of a photoreceptor other than far-red absorbing form of phytochrome (Pfr) and CRS amplifies the action of phytochrome. The endogenous level of ascorbic acid levels enhanced by phytochrome was amplified by CRS. The lifetime of CRS was from 0 to 2 h and the peak of enhancement of ascorbic acid due to CRS was between 16 to 24 h of dark incubation after the end of the treatment. CRS was found to be ineffective on UV-B enhanced endogenous levels of ascorbic acid.Key words: ascorbic acid, cryptic red light signal, glycine max, phytochrome, ultraviolet-BThe phytochrome mediated morphogenesis involves the conversion of Pr [red absorbing form] to Pfr [far-red absorbing form] and the magnitude of the response is dependent on Pfr/P tot ratio established at the end of the irradiation.¹ In broom Sorghum anthocyanin synthesis induced by red light [R₁] is reversible with far-red light. But a second red pulse [R₂] given after the reversal resulted in increased anthocyanin production compared to the first pulse [R₁]. When the red pulse was repeatedly given after every reversal with far-red, the anthocyanin production increased proportionately to the number of previously given pulses.² Thus red pre-treatment induced a change in the cellular physiological state or change in content of a relevant substance[s] which is designated as Cryptic Red Light Signal [CRS] associated with red signal transduction.² CRS was first characterized in detail in Broom Sorghum as Pfr amplifying signal produced by red pre-irradiation. CRS is inactive in the absence of Pfr but enhances the action of Pfr. CRS escapes reversal when the plants are exposed to far-red and is probably produced by a different species of phytochrome, distinct from the conventional reversible phytochrome.³We have investigated whether CRS influences other phytochrome regulated processes in plants in addition to anthocyanin synthesis. We chose another process, the synthesis of endogenous ascorbic acid, which is also regulated by conventional phytochrome.⁴ In soybean, the endogenous level of ascorbic acid is enhanced by conventional far-red reversible form of phytochrome. In addition, an independent UV-B photoreceptor [non reversible with far-red light] also enhances the endogenous synthesis of ascorbic acid in soybean. By using repeated pulses of red light, we have demonstrated that the Cryptic Red Signal is operative in soybean also and it amplifies the red light induced enhancement in the level of ascorbic acid. That CRS is active only in the presence of Pfr is demonstrated by the fact that pre-irradiation with red light is ineffective in amplifying UV-B induced enhancement of ascorbic acid levels. A similar observation on UV-B induced anthocyanin synthesis has been made in Broom Sorghum.² A separate UV-B photoreceptor independent of phytochrome operates in the plants.⁵ Although CRS is presumably produced by pre-irradiation with red light, it does not enhance UV-B induced anthocyanin synthesis or ascorbic acid synthesis in the absence of formation of Pfr by the second red pulse.The life-time of CRS was determined as 6 h in 20°C and 3 h in 24°C grown seedlings of Broom Sorghum with reference to anthocyanin synthesis.² The life-time of CRS determined in soybean seedlings grown at 25°C was upto 1 h.⁶ Since growing seedlings at a low temperature enhanced the effectiveness of CRS in Broom Sorghum, it was concluded that low temperature may either extend the lifetime of CRS or generate higher amount of CRS.² Although the exact nature of CRS is yet to be analyzed, work in our laboratory has established the universal nature of this signal and evidences have been obtained for CRS effect in promoting red light induced hypocotyls inhibition in Cucumber seedlings and also red light induced synthesis of betacyanins in Amaranthus seedlings (submitted for publication).     

S‐deficiency responsive accumulation of amino acids is mainly due to hydrolysis of the previously synthesized proteins – not to de novo synthesis in Brassica napus

To characterize the mechanisms of amino acid accumulation under sulphur (S)‐deficiency and its physiological significance in Brassica napus, stable isotopes ¹⁵N and ³⁴S were employed. The plants were exposed for 9 days to S‐deficient conditions (0.05 mM vs 1.5 mM sulphate). After 9 days of S‐deficiency, leaf‐osmotic potential and total chlorophyll content significantly decreased. S uptake decreased by 94%, whereas N uptake and biomass were not significantly changed. Using ¹⁵N and ³⁴S labelling, de novo synthesis of amino acids and proteins derived from newly absorbed NO₃^? and SO₄^2? and the content of N and S in the previously synthesized amino acids and proteins were quantified. At the whole plant level, S‐deficiency increased the pool of amino acids but resulted in strong decrease of incorporation of newly absorbed NO₃^? and SO₄^2? into amino acids by 22.2 and 76.6%, respectively, compared to the controls. Total amount of N and S incorporated into proteins also decreased by 28.8 and 62.1%, respectively. The levels of ¹⁴N‐ and ³²S‐proteins (previously synthesized proteins) strongly decreased, mainly in mature leaves. The data thus indicate that amino acid accumulation under short‐term S‐deficiency results from the degradation of previously synthesized proteins rather than from de novo synthesis.