Evaluation of a method to measure conjugal transfer of recombinant DNA in soil slurries

Release of recombinant microbes into the environment necessitates an evaluation of their ability to transfer genetic material. The present report evaluates a method to detect conjugal DNA plasmid transfer in soil slurries under various environmental conditions. DonorPseudomonas cepacia containing pR388::Tn1721 andP. cepacia recipient cultures were coincubated in soil slurries containing autoclaved or natural soil and treated with one or more of 14 experimental conditions. Conjugal mating frequency (transconjugants per initial donor) ranged from 4.8×10^–1 to 1.9×10^–7. Highest numbers of transconjugants, 1.5×10⁷ colony forming units/ml soil slurry, were observed following incubation at 35°C with an enriched nutrient supplement added to the soil. Low numbers of transconjugants, 10³ colony forming units/ml soil slurry, were observed when mating pairs were subjected to low nutrient or pH stress even though initial donor and recipient populations were maintained at high levels. This test system provides a simple way to estimate effects of changing environmental factors on plasmid transfer rates and on the survival of recombinant microorganisms. By use of soil collected from sites proposed to receive genetically engineered microorganisms, preliminary risk assessments can be obtained regarding the potential for gene transfer and microorganism survival with this soil slurry test system.     

Comparability of two tests of olfactory functioning

In the first of two comparisons, 50 patients who came to theConnecticut Chemosensory Clinical Research Center (CCCRC) withvarious chemosensory complaints were evaluated by a dual threshold/odoridentification test administered in the CCCRC and by the self-administeredUniversity of Pennsylvania Smell Identification Test (UPSIT).Agreement between tests proved high, with a correlation coefficientof 0.92. The UPSIT correlated better with the odor identificationcomponent than with the threshold component of the CCCRC test.This disparity stemmed largely from limitations in the reliabilityof the threshold component. An increase in the stringency ofthe criterion for determination of threshold increased the reliabilityof this component and, in a second comparison with a total of58 patients and controls, the correlation between the UPSITand CCCRC test increased. With the more stringent criterion,the correlation equalled 0.96. ²Present address: International Flavors and Fragrances, Researchand Development, Union Beach, NJ 07735, USA     

Different pituitary beta-endorphin and adrenal cortisol response to ethanol in individuals with high and low risk for future development of alcoholism

The purpose of the present studies was to investigate the activity of the adrenal gland and the pituitary beta-endorphin system in individuals from families with a 3 generation history of alcoholism, High Risk group, or from families without history of alcoholism, Low Risk group. All subjects had a medical examination, a drinking behavior personal interview and the Michigan Alcoholism Screening Test. Individuals with medical problems or excessive drinking were not included in the study. On the day of testing, a blood sample was taken at 9:00 a.m., then the subject drank a placebo drink or an ethanol solution (0.5 g ethanol/kg B.Wt.). Additional blood samples were taken at 15, 45 and 120 minutes post-drink. Results indicated that individuals of the High Risk group had lower basal levels of beta-endorphin like immunoreactivity (beta-EPLIR) than individuals of the Low Risk group. The dose of 0.5 g ethanol/kg B.Wt. induced an increase in the plasma content of beta-EPLIR of the High Risk group, but not of the Low Risk group. In the Low Risk group ethanol did not induce an increase above the 9:00 a.m. levels, however, it attenuated the beta-endorphin decrease overtime, observed following the placebo drink. Analysis of beta-endorphin-like peptides in the plasma of the High Risk group, with Sephadex G-75 chromatography indicated that the major component of the plasma beta-EPLIR was beta-lipotropin. Plasma cortisol levels, following ethanol intake, presented a small increase in the High Risk group but not in the Low Risk group. Both groups presented similar blood alcohol levels. The basal levels of immunoreactive cortisol and beta-endorphin in the plasma of individuals who were alcoholics, but had been abstinent for at least six months prior to testing were similar to the levels of the High Risk group. Thus there are differences both in the basal levels and in the response of the cortisol and the pituitary beta-endorphin system to an acute ethanol challenge between the two groups.     

The use of amphipathic maleimides to study membrane-associated proteins

A series of amphiphilic polymethylenecarboxymaleimides has been synthesized for use as sulfhydryl reagents applicable to membrane proteins. Physical properties of the compounds which are relevant to their proposed mode of action have been determined. By comparing rates of reaction in aqueous and aprotic solvents, the compounds have been shown to react exclusively with the thiolate ion. The effects of the reagents on three membrane-associated proteins are reported, and in two cases a comparative study has been made of the effects on the proteins in the absence of membranes. A mechanism is proposed whereby the reagents are anchored at the lipid/water interface by the negatively charged carboxyl group, thus siting the reactive maleimide in a plane whose depth is defined by the length of the reagent. Supporting evidence for this model is provided by the inability of the reagents to traverse membranes, and variation of their inhibitory potency with chain length when the proteins are embedded in the membrane, but not when extracted into solution. As examples of general use of the reagents to probe sulfhydryl groups in membrane proteins, the reagents have been used to (a) determine the depths in the membrane at which two populations of sulfhydryl groups occur in the mitochondrial phosphate transporter; (b) locate a single sulfhydryl associated with the active site ofD--hydroxybutyrate dehydrogenase in the inner mitochondrial membrane; (c) examine sulfhydryl groups in theD-3-glyceraldehyde phosphate dehydrogenase associated with the human red blood cell membrane.     

Lipid composition and food quality of some freshwater phytoplankton for cladoceran zooplankters

The nutritional value of several planktonic algae was testedby means of feeding trials with three cladoceran zooplankters.The algae were monocultures and included two blue-greens, fourgreens and four flagellates with a size range of 5–48µm. The specific growth rates of the zooplankters werechosen as the measure of the nutritional value of the algae.The three cladocerans showed large differences in growth ratein the different algae, but the two cryptomonads were withoutdoubt best suited as food for all. The fatty acid compositionfor the cryptomonads were different from the other algae. Theycontained high percentages of the polyunsaturated fatty acids20:5æ3 (EPA) and 22:6æ3 (DHA), which also are commonin fish. It is suggested that the lipid composition is a probablefactor determining the nutritional quality of the algae.     

Isolation of a crustaceann-acetyl-d-glucosamine-1-phosphate transferase and its activation by phospholipids

Summary Then-acetyl-d-glucosamine-1-phosphate: dolichol phosphate transferase fromArtemia has been partially purified and characterized. The enzyme is solubilized from crude microsomes using Triton X-100, and after detergent removal appears to be associated with phospholipids. Using dolichol phosphate and UDP-n-acetyl-d-glucosamine as substrates, the enzyme catalyzes the formation of dolichol-pyrophosphate-n-acetyl-d-glucosamine. the product identity has been verified by TLC and paper chromatography following mild acid hydrolysis. Under the incubation conditions used only one product is made, i.e., Dol-p-p-GlcNAc. The formation of product is linear with increasing amounts of added protein and with time of incubation. The enzyme requires magnesium ions for activity. Activity of the enzyme is stimulated 6-fold by exogenous dolichol phosphate and is also stimulated by added phospholipids, with optimal activity being obtained in the presence of mixtures of phosphatidylcholine and phosphatidylglycerol. Enzymatic activity is not increased upon addition of GDP-mannose or dolichol phosphate mannose. The enzyme is rapidly inactivated by exposure to several detergents, including Triton X-100 and deoxycholate. The activity is inhibited by tunicamycin and by the purified B₂ homologue of this antibiotic. Other antibiotic inhibitors such as diumycin and polyoxin D have little effect on the enzyme. Both the microsomal and solubilized enzyme preparations are inactivated by 70% upon treatment with phospholipase A₂; activity may be restored by addition of phospholipids. Following hydrophobic interaction chromatography on Phenyl Sepharose, gel filtration chromatography on Sepharose CL-4B indicated that the enzyme, purified 81-fold, contained phophatidylcholine and phosphatidyl-ethanolamine.Abbreviations SDS sodium dodecyl sulfate - PMSF phenyl methanesulfonylfluoride - HEPES 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid - GlcNAc N-acetyl-d-glucosamine - Dol-PP-GlcNAc dolichol pyrophosphate N-acetyl-d-glucosamine - Dol-P-man dolichol-phosphate-mannose - Dol-PP- (GlcNAc)₂ dolichol-pyrophosphate-di-N- acetylchitobiose - DMSO dimethylsulfoxide - C:M (2:1) chloroform:methanol (2:1) - C:M:W (10:10:3) chloroform:methanol:water (10:10:3) - GlcNAc-1-P N-acetyl-d-glucosamine-1-phosphate - Dol-P dolichol phosphate - EGTA ethylene glycol bis (b-aminoethyl ether)-NNNN tetraacetic acid