Purification and characterization of the acyl carrier protein of the Streptomyces glaucescens tetracenomycin C polyketide synthase.

The acyl carrier protein (ACP) of the tetracenomycin C polyketide synthase, encoded by the tcmM gene, has been expressed in both Streptomyces glaucescens and Escherichia coli and purified to homogeneity. Expression of the tcmM gene in E. coli results mainly in the TcmM apo-ACP, whereas expression in S. glaucescens yields solely the holo-ACP. The purified holo-TcmM is active in a malonyl coenzyme A:ACP transacylase assay and is labeled by radioactive beta-alanine, confirming that it carries a 4'-phosphopantetheine prosthetic group.     

Osmoregulation in nestling California gulls at Mono Lake,California

• 1.1. Hematocrit (Hct), plasma sodium ([Na]_pl), chloride ([Cl]_pl) and osmotic concentration (Osm_pl); volume and concentration of 1.0MNaCl induced salt gland secretion (SGS); and weights of osmoregulatory organs: kidneys, adrenal glands, and salt glands and nonosmoregulatory organs (liver and heart) were determined in nestling California gulls, Larus californiens (CG), on Krakatoa Islet, Mono Lake, California.
• 2.2. The mean Hct was 40.0% + 1.0%, the mean [Na]_pl and [Cl]_pl were 153.9 ± 0.9 and 110.1 ± 0.5 mM (n = 22); and the mean Osm_pl was 323.6 ± 1.3 mOsm/kg (n = 18).
• 3.3. In CG nestlings with a mean age of 10 days (n = 7), the mean SGS [Na] was 719 ± 19 mM and the birds secreted 81 ± 18% of the injected fluid containing 59 ± 13% of the injected Na. By the mean age of 23 days (n = 7), mean SGS [Na] was slightly higher (790 ± 30 mM than in younger birds (P < 0.05), but the percentage of secreted fluid (54 ± 10%) and Na (42 ± 6%) tended to be less.
• 4.4. In 18 CG nestlings mean organ weights (% body weight) were: kidneys 1.52 ±0.06%; salt glands, 0.13 ±0.01%, and adrenal glands 0.07 ±0.01%.
• 5.5. Nestling CG had significantly greater Hct (P < 0.001), [Na]_pl and [ci]_pl (P < 0.001), Osm_pl, (P < 0.005), and adrenal gland weight (P < 0.01), compared to nestling glaucous-winged Gulls (GWG), L. glaucescens (Hughes, 1984), which nest under cooler, moister conditions. CG kidney weight was smaller (P < 0.001); salt gland weight and salt excretion were the same as GWG.

     

Diet of moreporks (

Population structure, diversity and gene flow in four populations of Aciphylla glaucescens were studied using allozymes. Six of the seven putative loci were polymorphic in at least one population. Within populations the mean percentage of polymorphic loci was 68%. Gene diversity for Aciphylla glaucescens (H-e = 0.258) was greater at the species level compared with other outcrossing, wind pollinated plant species (H-e = 0.162). The mean diversity among populations of A. glaucescens (F-ST = 0.256) was also greater than that reported for most other species with similar breeding systems. Estimated gene flow (N-m) between populations was low, with only 0.72 migrants per generation. The high gene diversity of A. glaucescens may be due to the widespread distribution, obligate outcrossing and high fecundity of this species.     

Overproduction and localization of components of the polyketide synthase of Streptomyces glaucescens involved in the production of the antibiotic tetracenomycin C.

Three proteins, including the beta-keto acyl synthase and the acyl carrier protein, involved in the synthesis of the polyketide antibiotic tetracenomycin C by Streptomyces glaucescens GLA.0 were produced in Escherichia coli by using the T7 RNA polymerase-dependent pT7-7 expression vector. Changing the N-terminal codon usage of two of the genes greatly increased the level of protein produced without affecting mRNA levels, suggesting improvements in translational efficiency. Western immunoblot analysis of cytoplasmic and membrane fractions of S. glaucescens with antibodies raised to synthetic oligopeptides corresponding to the two presumed components of the beta-keto acyl synthase indicated that both proteins were membrane bound; one appears to be proteolytically cleaved before or during association with the membrane. The beta-keto acyl synthase could be detected in stationary-phase cultures but not in rapidly growing cultures, correlating with the time of appearance of tetracenomycin C in the medium.     

Predation of Clitostethus arcuatus (Col.: Coccinellidae) on Trialeurodes vaporariorum (Hem.: Aleyrodidae)

Greenhouse whitefly, Trialeurodes vaporariorum, is a serious pest of glasshouse crops. It shows resistance to different insecticides and growers are interested in finding other useful control methods. This research was carried out to study the predation potential and biology of Clitostethus arcuatus (Rossi) as one of the most important predators of this pest. Adult C. arcuatus were reared on tobacco leaves bearing colonies of greenhouse whitefly eggs under controlled conditions (25±2°C, 65±5% RH and 16 h L:8 h D). Results showed that the average developmental time of the egg, first through fourth instar larva and pupa were 2.82±0.12, 4.47±0.14, 4.54±0.1, 6.3±0.2, 7±0.22 and 3.8±0.13 days, respectively; and longevity of female and male were 66.4±2.6 and 54.9±2.5 days, respectively. The average feeding rates of female, male and larvae (first through fourth) were 61.4±0.7, 27.6±0.9 eggs/day and 12±1.03, 30.3±2.4, 41.3±2, 68.04±2 eggs/day, respectively. The larvae consumed an average of 992.2±36 eggs during the total larval developmental period with a daily mean of 45.8±0.5. A significant difference was shown between the feeding rate of fourth instar larval stages and between sexes. Females, males and one pair of C. arcuatus (♀,♂) consumed an average of 17.2±0.4, 10.6±0.8, 23.1±0.5 nymph/day; 28.5±0.9, 20.3±0.6, 47.2±0.6 pupa/day and 8±0.3, 6.5±0.54, 13.6±0.4 adult/day, respectively. The feeding rate was significantly different among whitefly life stages. Females laid an average of 3±0.23 eggs/day.     

Potency of extract contents from selected tropical chewing sticks against Staphylococcus aureus and Staphylococcus auricularis

Two strains of enteropathogenic Gram-positive bacteria (Staphylococcus auricularis, NIAH 11484 and Staphylococcus aureus, IFO 12732) were tested in vitro for their resistance to extracts from tropical chewing stick species, namely, Garcinia mannii Heck, Masularia accuminata (G. Don) Benth, Zanthoxylum gilletii (DeWild) Waterman, Terminalia glaucescens Plauch ex Benth, Azadiracta indica A. Juss, Anogeissus leiocarpus Guill & Perr and Pseudocedrela kotschyi (Schweinf.). The chewing sticks are normally used for oral hygiene and this is felt to be related to their ability to attack bioagents that could invade the mouth during normal nutrition. Results from our study showed that the antibiotic properties of test chewing stick species vary and are target-microbe-specific. Of the species examined, only T. glaucescens showed appreciable broad antibiotic effect against S. aureus and S. auricularis. Intense antibiotic activity against S. aureus occurred when using a 2.0 g/l extract concentration and a 30-h incubation. T. glaucescens also showed intense activity against S. auricularis at 2.0 g/l concentration, 30-h incubation when all other extracts had lost their potency. A. indica is, however, most effective against S. aureus, showing appreciable antibiotic activity at 0.4 g/l concentration, 30-h incubation. Z. gilletii has no antibiotic activity against any of the test bacteria. The antistaphylococcus compounds in T. glaucescens and A. indica are worthy of isolation and further analyses.     

The tcmVI region of the tetracenomycin C biosynthetic gene cluster of Streptomyces glaucescens encodes the tetracenomycin F1 monooxygenase, tetracenomycin F2 cyclase, and, most likely, a second cyclase.

Certain mutations in the tcmVI region of the Streptomyces glaucescens chromosome affect formation of the D ring of the polyketide antibiotic tetracenomycin C (TCM C). This region lies immediately upstream from the TCM C polyketide synthase genes (tcmKLM), and the nucleotide sequence reveals the presence of three small genes, tcmH, tcmI, and tcmJ. On the basis of the phenotypes of mutants and the effects of these genes, when coupled on a plasmid with the tcmKLMN177 genes (tcmN177 is a 3'-truncated version of tcmN), on the production of TCM intermediates in a TCM- mutant, the tcmH gene encodes the C-5 monooxygenase that converts TCM F1 to TCM D3, the tcmI gene encodes the D-ring cyclase that converts TCM F2 to TCM F1 (mutations in this gene are responsible for the type VI phenotype), and the tcmJ gene most likely encodes the B-ring cyclase that acts in the biosynthesis of TCM F2. Furthermore, it appears that the N-terminal domain of the tcmN gene product (encoded by the tcmN177 gene) acts later in the biosynthesis of TCM F2 than the product of tcmJ, suggesting that the N-terminal domain of the TcmN protein is the C-ring cyclase.     

Biology ofMicroplitis mediator [Hym.: Braconidae] a gregarious endoparasite ofAgrotis segetum [Lep.: Noctuidae]

Microplitis mediator (Haliday) a gregarious endoparasite was recorded for the first time fromAgrotis segetum (Schiff) in Ankara, Turkey. The female parasites found their hosts by responding to the faeces of the caterpillars. An average, females laid 15.5±1.6 eggs in the bodies of their hosts. The newly laid eggs were elongated, oval in shape and 0.23±0.004 mm long and 0.07 mm wide. They hatched in 5, 4 and 3 days at 20±2°C, 25±2°C and 30±2°C respectively when maintained at 60–70% R.H. and 14∶10 light∶dark regime. At the same temperatures, the larval stage lasted for 24.9±0.6, 18.2±0.4 and 17.1±0.5 days respectively. The prepupal stage was completed in 2 days at 25±2°C, whereas the prepupal and pupal (cocoon) stage lasted 10.9±0.2, 7.0±0.1 and 6.2±0.1 days respectively at the temperatures mentioned above. The adults started mating and feeding shortly after emergence. Female parasites started laying after one day, 7–11 hours and 5–7 hours at the temperatures stated above. At these temperatures females lived for 10.8±0.2, 5.4±0.1, 4.6±0.1 days and laid on average 556, 484 and 363 eggs respectively, whereas the males survived 10.5±0.3, 4.7±0.1 and 4.4±0.1 days respectively.      

Potency of extract contents from selected tropical chewing sticks against Staphylococcus aureus and Staphylococcus auricularis

Two strains of enteropathogenic Gram-positive bacteria (Staphylococcus auricularis, NIAH 11484 and Staphylococcus aureus, IFO 12732) were tested in vitro for their resistance to extracts from tropical chewing stick species, namely, Garcinia mannii Heck, Masularia accuminata (G. Don) Benth, Zanthoxylum gilletii (DeWild) Waterman, Terminalia glaucescens Plauch ex Benth, Azadiracta indica A. Juss, Anogeissus leiocarpus Guill & Perr and Pseudocedrela kotschyi (Schweinf.). The chewing sticks are normally used for oral hygiene and this is felt to be related to their ability to attack bioagents that could invade the mouth during normal nutrition. Results from our study showed that the antibiotic properties of test chewing stick species vary and are target-microbe-specific. Of the species examined, only T. glaucescens showed appreciable broad antibiotic effect against S. aureus and S. auricularis. Intense antibiotic activity against S. aureus occurred when using a 2.0 g/l extract concentration and a 30-h incubation. T. glaucescens also showed intense activity against S. auricularis at 2.0 g/l concentration, 30-h incubation when all other extracts had lost their potency. A. indica is, however, most effective against S. aureus, showing appreciable antibiotic activity at 0.4 g/l concentration, 30-h incubation. Z. gilletii has no antibiotic activity against any of the test bacteria. The antistaphylococcus compounds in T. glaucescens and A. indica are worthy of isolation and further analyses.     

苍白秤钩风中脱皮甾酮类化学成分的研究

从中国广西药用植物苍白秤钩风(Diploclisia glaucescens)藤茎的95％乙醇提取物中分离得到5个脱皮甾酮类化合物(1-5)。通过NMR、MS以及化学沟通等方法分别将其结构鉴定为paristerone C-20,22-monoacetonide(1),paristerone(2),eodysterone(3),makisterone C(4)和capisterone(5)。化合物1为首次从自然界分得的天然产物,2,4,5亦为首次从该植物中分得。     

Chonemorphine, stigmasterol, and ecdysterone: steroids isolated through bioassay-directed plant screening programs

In a bioassay-directed screening programme of plants for the identification of active constituents the following steroids were isolated. Chonemorphine was identified as the antiamoebic and antitrichomonad principle of Chonemorpha fragrans. Stigmasterol from Coleus forskohlii and ecdysterone from Diploclisia glaucescens were inactive constituents isolated during the process of purifying the active principles of the plants. D. glaucescens roots are a high-yielding source (0.5% of the dry root weight) of ecdysterone.     

Acid-base, fluid and electrolyte metabolism in the elasmobranch. 3. Oxygen, CO-2, bicarbonate and lactate exchange across the gill

Methods have been developed which permit measurements of cardiac output and sampling of mixed venous and arterial blood in the elasmobrach, Squalus acanthias. These methods have been utilized to characterize some of the parameters involved in gill gas exchange. A total of 17 animals have been studied with the following results. Cardiac output: 1.49±0.57 1/kg/hr.; V?_o2: 38.3±22.1 ml/kg/hr.; V?_co2: 32.1±19.0 ml/kg/hr.; RER: 0.92±0.17; PaO₂: 104±27 mmHg; Pv?_o2: 18±8.8 mmHg; PaCO₂: 2.9±0.75 mmHg; Pv?_co2: 4.6±1.1 mmHg; CaO₂: 3.8±0.75 vol. %; Cv?_o2: 1.26±0.59 vol. %; pHa: 7.52±0.10 units; Pv?: 7.42±0.11 units; [HCO₃?]_a: 3.92± 1.14 meq/1; [HCO₃?]v?: 4.80±1.15 meq/1; [dissolved CO₂]_a: 0.17± 0.04 mM/1; [dissolved CO₂]v?: 0.24±0.04 mM/1; [lactate?]_a: 13.47±7.17 meq/1; [Lactate?]v?: 13.01±8.18 meq/1. Some of the mechanisms involved in gill gas exchange have been characterized.     

The production and secretion of cortisol by baboon neonates.

The metabolic clearance rate (MCR), transfer constants (p), production (PR) and secretion (SR) rates of cortisol (F) andrortisone (E) were determined by the continuous infusion of {1,2³H}F and {4-¹⁴C}E into 5 neonates delivered prior to the parturition by cesarean section (164–179 days; term = 184 days) and into 5 newborns delivered spontaneously per vagina at term (166 – 187 days). In spontaneously delivered animals, MCR-E (X ± SE, 34.3 ± 7.0 1/day/kg was greater (P < 0.001) than MCR-F (14.9 ± 1.5 1/day/kg), pF to E (59.7 ± 8.9%) exceeded (P < 0.001) pE to F (17.8 ± 3.0%) and the percentage of F bound to serum proteins other than albumin (57.5 ± 6.2) was greater (P < 0.001) than that of E (27.0 ± 10.3) Although the serum E level (25.6 ± 3.6 μg/100 ml) was similar to that of F (33.5 ± 8.0 μg/100 ml), the PR-E (6.4 ± 1.3 μ/min/kg) was greater (P < 0.001) than PR-F (3.3 ± 0.5 μ/min/kg). Approximately eighty-five percent of the E and 65% of the F produced orginated by secretion.In animals delivered by cesarean section, the serum F concentration (32.4 ± 6.7 μ/100ml), pE to F (13.4 ± 2.8%) pF to E (80.0 ± 12.2%) PR-E (4.5 ± 0.2 μ/min/kg) and SR-E (3.9 ± 0.3 μ/min/kg) were not different from values for spontaneously delivered animals. Serum E levels (35.9 ± 1.6 μ/100 ml) were higher but MCR-F (6.7 ± 0.6 1/day/kg) and MCR-E (18.2 ± 0.41/day/kg) lower in neonates delivered by cesarean section. Serum Cortisol binding capacity (μg F bound/100 ml) was greater (P < 0.025) in neonates delivered by cesarean section (23.6 ± 2.6) than in spontaneously delivered animals (14.4 ± 2.0). As a result of these changes in F and E dynamics, PR-F (1.4 ± 0.3 μ/min/kg) and SR-F (0.9 ± 0.2 μ/min/kg) in neonates delivered by cesarean section were lower (P< 0.01) than corresponding values in spontaneously delivered newborns.It is concluded that the greater F secretion in animals delivered spontaneously than those delivered by cesarean section probably results from increased fetal adrenal 3β-hydroxysteroid dehydrogenase-isomerase activity, which as previously reported, occurs in late gestation in this species.     

Microsporidium goeldichironomi n. sp. and Microsporidium chironomi n. sp. (Microsporida: Apansporoblastina): Two new microsporidia from Florida chironomids

Two new species of Microsporida belonging to the genus Microsporidium are described. Microsporidium goeldichironomi n. sp. parasitizes the fat body of Goeldichironomus holoprasinus and Microsporidium chironomi n. sp. infects Chironomus attenuatus. Both microsporidia form uninucleate spores from rosette-shaped sporonts. M. goeldichironomi sporonts form 4, 6, 8, 10, 12, 16, and possibly more spores. Two shapes of spores are produced, oval, or slightly pyriform spores measuring 3.70 ± 0.09 × 2.49 ± 0.13 μm and pyriform spores measuring 3.74 ± 0.44 × 2.04 ± 0.17 μm. Electron micrographs show that both types of spores are uninucleate, have 8 to 11 polar filament coils and a lamellate polaroplast showing several distinct regions. M. chironomi spores are pyriform and are often joined at the posterior end in groups of two or four. They measure 4.12 ± 0.37 × 2.45 ± 0.26 μm. The spores are uninucleate, have six to seven polar filament coils and a lamellate polaroplast showing two distinct regions. Neither species can be transmitted per os and thus are assumed to be transovarially transmitted. No pansporoblastic membrane is present in either species.     

Survival ofVarroa jacobsoni Oud. (Acari: Varroidae) away from its living hostApis mellifera L.

Mite survival was measured on eight different substrates: cloth, wax comb, dead drones, dead workers, metal, wood, pollen and control. Trials were conducted at both 26°C and 13°C. Substrate type, temperature and their interactions significantly affected the survival ofV. jacobsoni. Longest survival (71±1.1 h) was recorded on dead worker bees (Apis mellifera L.) held at 26°C. Shortest survivals were observed on cloth (21±1.3 h), no substrate (21±1.3 h), metal (20±1.1 h) and pollen (18±1.3 h), all maintained at 13°C.     

Secretion of tyrosinase in Streptomyces glaucescens

In Streptomyces glaucescens, the intracellular and the extracellular enzyme forms of tyrosinase were found to be indentical in molecular weight (29 000), in copper content (0.21%), in the 19 amino acids at the amino-terminal end and in the ratio of cresolase to catecholase activity (0,005). The tyrosinase secretion process exhibited a constant rate of 0.15 units h-1 (mg protein)-1. Under highly induced conditions intracellular tyrosinase was accumulated. Mutations responsible for the non-melanogenic, tyrosinase-positive non-secretor mutant type are located chromosomally on the upper right arc of the S. glaucescens map near the ade-1 marker.     

Studies on camel semen. II. Biochemical characteristics

The mean concentrations of fructose, citric acid, total proteins and lipids in the seminal plasma of camel were 23.5±2.5, 9.8±2.9, 775±15 and 87±32 mg/ml respectively. The corpus prostatae seemed to be the principal source of fructose and citric acid, smaller amounts are contributed by the bulbourethral glands, ampulla ductus deferentis and pars disseminata of the prostate. Electrophoretic fractionation of seminal plasma proteins by capillary isotachophoresis revealed eight protein bands similar to those of blood serum. Electrofocusing in polyacrylamide gels on the other hand showed the presence of eighteen fractions with minor variations from fractions obtained from blood serum.     

Residual effects of four pesticides on the predatory bug,Orius albidipennis Reut. (Hem.: Anthocoridae)

Residual effects of abamectin (0.02%), propargite (0.1%), dichlorvos (DDVP) (0.15%) and pymetrozine (0.15%) were determined on Orius albidipennis at laboratory conditions. Planted cucumbers were sprayed with the highest recommended concentrations of all insecticides and experimental treatments were monitored during 20 days based on the period of pesticides residues. On the first day after spraying, the greatest of mortality belonged to dichlorvos and pymetrozine (97 ± 1.22, 57 ± 3.1, respectively, p < 0.01), while abamectin and propargite had the highest mortality after four (44 ± 1.87 and 24 ± 2.91, respectively, p < 0.01) and eight (51 ± 2.91 and 17 ± 2.23, respectively, p < 0.01) days after spraying. At day 16th, post treatment, abamectin had the highest residual mortalities on O. albidipennis (15 ± 1.58, p < 0.01) and finally mortality results at the 20th day showed there is no significant difference between insecticides (p < 0.01).