Affinity purification of plasmid DNA directly from crude bacterial cell lysates

We have shown previously that a sequence-specific DNA-binding protein based on the Lac repressor protein can isolate pre-purified DNA efficiently from simple buffer solution but our attempts to purify plasmids directly from crude starting materials were disappointing with unpractically low DNA yields. We have optimized tbe procedure and present a simple affinity methodology whereby plasmid DNA is purified directly by mixing two crude cell lysates, one cell lysate containing the plasmid and the other the protein affinity ligand, without the need for treatment by RNaseA. After IMAC chromatography, high purity supercoiled DNA is recovered in good yields of 100-150 μg plasmid per 200 mL shake flask culture. Moreover, the resulting DNA is free from linear or open-circular plasmid DNA, genomic DNA, RNA, and protein, to the limits of our detection. Furthermore, we show that lyophilized affinity ligand can be stored at room temperature and re-hydrated for use when required.

A review of hydrothermal liquefaction bio-crude properties and prospects for upgrading to transportation fuels

Hydrothermal liquefaction (HTL) presents a viable route for converting a vast range of materials into liquid fuel, without the need for pre-drying. Currently, HTL studies produce bio-crude with properties that fall short of diesel or biodiesel standards. Upgrading bio-crude improves the physical and chemical properties to produce a fuel corresponding to diesel or biodiesel. Properties such as viscosity, density, heating value, oxygen, nitrogen and sulphur content, and chemical composition can be modified towards meeting fuel standards using strategies such as solvent extraction, distillation, hydrodeoxygenation and catalytic cracking. This article presents a review of the upgrading technologies available, and how they might be used to make HTL bio-crude into a transportation fuel that meets current fuel property standards.

IgE-Associated IGHV Genes from Venom and Peanut Allergic Individuals Lack Mutational Evidence of Antigen Selection

Antigen selection of B cells within the germinal center reaction generally leads to the accumulation of replacement mutations in the complementarity-determining regions (CDRs) of immunoglobulin genes. Studies of mutations in IgE-associated VDJ gene sequences have cast doubt on the role of antigen selection in the evolution of the human IgE response, and it may be that selection for high affinity antibodies is a feature of some but not all allergic diseases. The severity of IgE-mediated anaphylaxis is such that it could result from higher affinity IgE antibodies. We therefore investigated IGHV mutations in IgE-associated sequences derived from ten individuals with a history of anaphylactic reactions to bee or wasp venom or peanut allergens. IgG sequences, which more certainly experience antigen selection, served as a control dataset. A total of 6025 unique IgE and 5396 unique IgG sequences were generated using high throughput 454 pyrosequencing. The proportion of replacement mutations seen in the CDRs of the IgG dataset was significantly higher than that of the IgE dataset, and the IgE sequences showed little evidence of antigen selection. To exclude the possibility that 454 errors had compromised analysis, rigorous filtering of the datasets led to datasets of 90 core IgE sequences and 411 IgG sequences. These sequences were present as both forward and reverse reads, and so were most unlikely to include sequencing errors. The filtered datasets confirmed that antigen selection plays a greater role in the evolution of IgG sequences than of IgE sequences derived from the study participants.

Crude oil and agricultural futures: An analysis of correlation dynamics

Correlations between oil and agricultural commodities have varied over previous decades, impacted by renewable fuels policy and turbulent economic conditions. We estimate smooth transition conditional correlation models for 12 agricultural commodities and WTI crude oil. While a structural change in correlations occurred concurrently with the introduction of biofuel policy, oil and food price levels are also key influences. High correlation between biofuel feedstocks and oil is more likely to occur when food and oil price levels are high. Correlation with oil returns is strong for biofuel feedstocks, unlike with other agricultural futures, suggesting limited contagion from energy to food markets.

Analysis of Crude Protein and Allergen Abundance in Peanuts (Arachis hypogaea cv. Walter) from Three Growing Regions in Australia

The effects of plant growth conditions on concentrations of proteins, including allergens, in peanut (Arachis hypogaea L.) kernels are largely unknown. Peanuts (cv. Walter) were grown at five sites (Taabinga, Redvale, Childers, Bundaberg, and Kairi) covering three commercial growing regions in Queensland, Australia. Differences in temperature, rainfall, and solar radiation during the growing season were evaluated. Kernel yield varied from 2.3 t/ha (Kairi) to 3.9 t/ha (Childers), probably due to differences in solar radiation. Crude protein appeared to vary only between Kairi and Childers, whereas Ara h 1 and 2 concentrations were similar in all locations. 2D-DIGE revealed significant differences in spot volumes for only two minor protein spots from peanuts grown in the five locations. Western blotting using peanut-allergic serum revealed no qualitative differences in recognition of antigens. It was concluded that peanuts grown in different growing regions in Queensland, Australia, had similar protein compositions and therefore were unlikely to show differences in allergenicity.

Primary structure of a cytotoxin-like basic protein from Naja naja naja (Indian Cobra) venom

The complete amino acid sequence of a cytotoxin-like basic protein (CLBP) from the venom of Naja naja naja (Indian Cobra) was determined by manual degradation using a 4-dimethylaminoazobenzene-4'-isothiocyanate double-coupling method. Peptide fragments obtained by chemical cleavage with cyanogen bromide and enzymic cleavages with trypsin and Staphylococcus aureus proteases for sequence analysis were purified by reversed-phase chromatography. The total number of amino acid residues was 61, with leucine as the C-terminal residue. (C) Munksgaard 1995.

Characterization of Major Zinc Containing Myonecrotic and Procoagulant Metalloprotease `Malabarin' from Non Lethal Trimeresurus malabaricus Snake Venom with Thrombin Like Activity: Its Neutralization by Chelating Agents

A major myonecrotic zinc containing metalloprotease `malabarin' with thrombin like activity was purified by the combination of gel permeation and anion exchange chromatography from T. malabaricus snake venom. MALDI-TOF analysis of malabarin indicated a molecular mass of 45.76 kDa and its N-terminal sequence was found to be Ile-Ile-Leu-Pro(Leu)-Ile-Gly-Val-Ile-Leu(Glu)-Thr-Thr. Atomic absorption spectral analysis of malabarin raveled the association of zinc metal ion. Malabarin is not lethal when injected i.p. or i.m. but causes extensive hemorrhage and degradation of muscle tissue within 24 hours. Sections of muscle tissue under light microscope revealed hemorrhage and congestion of blood vessel during initial stage followed by extensive muscle fiber necrosis with elevated levels of serum creatine kinase and lactate dehydrogenase activity. Malabarin also exhibited strong procoagulant action and its procoagulant action is due to thrombin like activity; it hydrolyzes fibrinogen to form fibrin clot. The enzyme preferentially hydrolyzes A alpha followed by B beta subunits of fibrinogen from the N-terminal region and the released products were identified as fibrinopeptide A and fibrinopeptide B by MALDI. The myonecrotic, fibrinogenolytic and subsequent procoagulant activities of malabarin was neutralized by specific metalloprotease inhibitors such as EDTA, EGTA and 1, 10-phenanthroline but not by PMSF a specific serine protease inhibitor. Since there is no antivenom available to neutralize local toxicity caused by T. malabaricus snakebite, EDTA chelation therapy may have more clinical relevance over conventional treatment.

Conformational Diversity in Contryphans from Conus Venom: cis-trans Isomerisation and Aromatic/Proline Interactions in the 23-Membered Ring of a 7-Residue Peptide Disulfide Loop

Conformational diversity or shapeshifting in cyclic peptide natural products can, in principle, confer a single molecular entity with the property of binding to multiple receptors. Conformational equilibria have been probed in the contryphans, which are peptides derived from Conus venom possessing a 23-membered cyclic disulfide moiety. The natural sequences derived from Conus inscriptus, GCV(D)LYPWC* (In936) and Conus loroisii, GCP(D)WDPWC* (Lo959) differ in the number of proline residues within the macrocyclic ring. Structural characterisation of distinct conformational states arising from cis-trans equilibria about Xxx-Pro bonds is reported. Isomerisation about the C2-P3 bond is observed in the case of Lo959 and about the Y5-P6 bond in In936. Evidence is presented for as many as four distinct species in the case of the synthetic analogue V3P In936. The Tyr-Pro-Trp segment in In936 is characterised by distinct sidechain orientations as a consequence of aromatic/proline interactions as evidenced by specific sidechain-sidechain nuclear Overhauser effects and ring current shifted proton chemical shifts. Molecular dynamics simulations suggest that Tyr5 and Trp7 sidechain conformations are correlated and depend on the geometry of the Xxx-Pro bond. Thermodynamic parameters are derived for the cis trans equilibrium for In936. Studies on synthetic analogues provide insights into the role of sequence effects in modulating isomerisation about Xxx-Pro bonds.

Effect of crude oil and some petroleum products on Clarias Gariepinus fingerlings (Catfish: Claridae)

Ninety (90) hatchery bred fingerlings of Clarias gariepinus (mean weight: 0.96 ± 0.1g) were randomly placed in 15 plastic baths (25 litres each) at the Research laboratory and were exposed to different concentrations of oil products to determine their effects on the fish, to facilitate inferential deductions that will enhance effective aquatic environmental management. Three (3) replicate basins of 5 experimental treatments (crude oil, petrol oil, kerosene oil, engine oil and control) were used at a concentration of 1.25ml. L-1. The control experiment was devoid of oil treatment. Six (6) fingerlings were placed in each replicate basin, flooded with 20 litres of clean tap water and fed with nutrafin cichilid food, 2 times daily at 3% body weight. The results showed that the feeding behaviour and swimming performances of fish were reduced after 24 hours of the addition of the various oil pollutants. Mortality of fingerlings in the oiled basins increased as the hours of exposure increased (i.e. 24, 48, 72 and 96 hours). Recovery was not immediate in the treated basin while surviving fingerlings in the control basins grew up to post-fingerlings after 90 days (3 months). There were significant differences (P<0.01 and P<0.05) in the effect of crude oil and the petroleum products on the mortality rate of C. gariepinus when exposed to oil pollutants at 1.25ml. L-1 concentration

Aspects of mineral composition of male and female Heterobranchus bidorsalis adults exposed to different concentrations of Bonny-light crude oil

Studies were carried out to assess some macro and trace elements of mineral composition of the male and female Heterobranchus bidorsalis adults exposed to graded concentrations (1.00-8.00m/L-1) of Bonny-light crude oil (BLCO). The experiment was monitored for 4 days (toxicity) and 42days (recovery) periods. Significant decreases (P < 0.05)in the sodium (Na), potassium (K), magnesium (Mg), calcium (Ca), phosphorus (P), zinc (Zn), iron (Fe), vanadium (Va), lead (Pb) and manganese (Mn) contents of the male H. bidorsalis corresponded with the increasing concentrations of BLCO. In contrast, the female fishes recorded significant increases (P < 0.05) in the values of the above elements in their tissues as the concentrations of BLCO increased. Furthermore, the values of Na, K, Mg, Ca, P, Zn, Fe, Va, Pb and Mn recorded in the male fishes where generally lower than those of their female counterparts and the control fish. Increased values of these elements were also recorded during the recovery periods (days 14, 28 and 42) of this study in the magnitudes of 15% at day 14, 20% at day 28 and 20% at day 42. This implied that the removal of crude oil stress during this period improved the quantity of these minerals deposited in the fish tissues. The highest percent proportion of Zn and the lowest proportion of Pb recorded in both male and female H. bidorsalis adults agreed with the report of other workers for other fish species. KEYWORDS: Heterobranchus bidorsalis, Mineral composition, Bonny-light crude oil, Toxicity, Recovery.

Mass breeding of silver carp in a cement cistern with crude H.C.G.

The paper presents a resume on experiments carried on mass breeding of the silver carp Hypophthalmichthys molitrix in a cement cistern through administration of crude human chorionic gonadotropin. The experiments proves that human chorionic gonadotropin (H.C.G.) can be used for mass breeding of the fish in simple enclosures, with facilities of some input and output of water and a dose of 5 mg/kg and 18 mg/kg of body weight, for males and females respectively are required for the purpose. Observations were made by using pituitary glands in similar way. For successful breeding of about 80 kg female together, the expenditure towards cost of pituitary gland and H.C.G. are respectively 275 and 117 Rs.

Effects of natural gas pipeline condensate and crude oil spills, and comments on remediation, with emphasis on south Louisiana salt marshes: a review

This report reviews some of the natural ecological processes at work within a salt marsh as they relate to a spill of natural gas condensate - a mixture of aliphatic hydrocarbons, n-hexane, benzene, toluene, and xylene. It also reviews the environmental impacts of some of the components of natural gas condensate as well as related compounds (crude oil, higher molecular weight hydrocarbons, polycyclic aromatic hydrocarons - PAHs, linear alkyl-benzenes - LABs, etc.) on salt marsh ecosystems in southern Louisiana and elsewhere in the world. The behavior and persistence of these compounds once they have entered the environment is also considered.