An evaluation of several adjuvant emulsion regimens for the production of polyclonal antisera in rabbits.

Emulsion adjuvants have been used for production of polyclonal antisera in rabbits (Oryctolagus cunniculi) for decades. Complete Freund's adjuvant has a reputation as a very effective immunoenhancer, but adverse physiological effects, including fever, inflammation and sterile abscess formation, have prompted a search for alternatives to complete Freund's. In this study, we quantitatively compared five adjuvant regimens: (a) a primary inoculation with complete Freund's followed by three boosts with incomplete Freund's; (b) four serial inoculations of incomplete Freund's adjuvant augmented with 6-bromoguanisine; (c) four serial inoculations with RIBI's MPL + TDM + CWS adjuvant emulsion; (d) four serial inoculations with Montanide ISA 50 emulsion; and (e) four serial inoculations with Montanide ISA 70 emulsion. We chose a small (12 amino acid) chain polypeptide coupled to bovine serum albumin as our test antigen. When compared, no system could be seen to be significantly better than a regimen of a primary immunization with complete Freund's adjuvant followed by serial reimmunization with incomplete Freund's adjuvant. The commercially available RIBI adjuvant produced significantly lower antibody levels, while other systems produced essentially equivalent levels. With all five adjuvants, antibody quantities plateaued after the second injection and further immunization did not increase titers significantly. Boost injections did yield greater intradermal tissue reaction than primary inoculations, and intramuscular inoculum volumes of 0.4 cc caused chronic lesions still detectable by the gross necropsy 2 weeks after the final injection.     

NMR studies of the backbone protons and secondary structure of pentapeptide and heptapeptide substrates bound to bovine heart protein kinase

The conformations of enzyme-bound pentapeptide (Arg-Arg-Ala-Ser-Leu) and heptapeptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) substrates of protein kinase have been studied by NMR in quaternary complexes of the type (Formula: see text). Paramagnetic effects of Mn2+ bound at the inhibitory site of the catalytic subunit on the longitudinal relaxation rates of backbone Ca protons, as well as on side-chain protons of the bound pentapeptide and heptapeptide substrates, have been used to determine Mn2+ to proton distances which range from 8.2 to 12.4 A. A combination of the paramagnetic probe-T1 method with the Redfield 2-1-4-1-2 pulse sequence for suppression of the water signal has been used to measure distances from Mn2+ to all of the backbone amide (NH) protons of the bound pentapeptide and heptapeptide substrates, which range from 6.8 to 11.1 A. Paramagnetic effects on the transverse relaxation rates yield rate constants for peptide exchange, indicating that the complexes studied by NMR dissociate rapidly enough to participate in catalysis. Model-building studies based on the Mn2+-proton distances, as well as on previously determined distances from Cr3+-AMPPCP to side-chain protons [Granot, J., Mildvan, A.S., Bramson, H. N., & Kaiser, E. T. (1981) Biochemistry 20, 602], rule out alpha-helical, beta-sheet, beta-bulge, and all possible beta-turn conformations within the bound pentapeptide and heptapeptide substrates. The distances are fit only by extended coil conformations for the bound peptide substrates with a minor difference between the pentapeptides and heptapeptides in the phi torsional angle at Arg3C alpha and in psi at Arg2C alpha. An extended coil conformation, which minimizes the number of interactions within the substrate, would facilitate enzyme-substrate interaction and could thereby contribute to the specificity of protein kinase.     

Leaf disc transformation of cultivated tomato (L. esculentum) using Agrobacterium tumefaciens

The leaf disc transformation/regeneration system was modified for tomato (L. esculentum). Both leaf explants and cotyledon/hypocotyl sections can be used to regenerate transformed plants. We have obtained over 300 transgenic plants from eight tomato cultivars. We have evidence for both single and multi-copy insertions of the T-DNA, and have demonstrated inheritance of the T-DNA insert in the expected Mendelian ratios. Several heterologous promoters function in tomato. A reduced efficiency of transformation was observed with binary T-DNA vectors as compared to co-integrate T-DNA vectors. The ease of the leaf disc method makes tomato a premier experimental organism for plant biotechnology.Abbreviations BA benzyl adenine - IAA indole acetic acid - LB Luria Broth     

Electron paramagnetic resonance studies of heme c and its nitrosyl derivative in Vibrio (Achromobacter) fischeri nitrite reductase

Interactions of Vibrio (formerly Achromobacter) fischeri nitrite reductase were studied by electron paramagnetic resonance spectroscopy. The spectrum of the oxidized enzyme showed a number of features which were attributed to two low-spin ferric hemes. These comprised an unusual derivative peak at g = 3.7 and a spectrum at g = 2.88, 2.26, and 1.51. Neither heme was reactive in the oxidized state with the substrate nitrite and with cyanide and azide. When frozen under turnover conditions (i.e., reduction in the presence of excess nitrite), the enzyme showed the spectrum of a nitrosyl heme derivative. The g = 2.88, 2.26, and 1.51 signals reappeared partially on reoxidation by nitrite, indicating that the nitrosyl species which remained arose from the g = 3.7 heme. The nitrosyl derivative showed a 14N nuclear hyperfine splitting, Az = 1.65 mT. The nitrosyl derivative was produced by treatment of the oxidized nitrite reductase with nitric oxide or hydroxylamine. Exchange of nitric oxide between the nitrosyl derivative and NO gas in solution was observed by using the [15N]nitrosyl compound. A possible reaction cycle for the enzyme is discussed, which involves reduction of the enzyme followed by binding of nitrite to one heme and formation of the nitrosyl intermediate.     

Flight muscle hypertrophy and ecophysiological variation of Yellow wagtail Motacilla flava races at Lake Chad

During the few days before emigration to the Palaearctic, the weight of Yellow wagtails wintering in Africa doubles. Most of the increase is due to the deposition of fat, as is well known, but a part of it is shown to result from hypertrophy of the pectoral muscles. The same applies to other Palaearctic birds, four warblers and a hirundine, that winter in Africa.
Eleven subspecies of M. flava occur at Lake Chad in spring. The four black-headed ones have more restricted habitat requirements than some of the others; however, no differences in diet have been found. There is no evidence of hyperphagia and little of diet change when fattening. Broadly, the races that have farthest to migrate leave earliest, and their sexual recrudescence and fattening are early.
Yellow wagtails wintering at or passing through Lake Chad breed mainly in eastern Europe (from the Adriatic to the Gulf of Finland, between about 12° and 30°E). They probably migrate on a great circle route, and during the flight lose one gram of weight, mainly fat, per 200 km.     

Application of Urine Analysis to Diagnosis and Treatment of Heroin Addiction

Experience with urine analysis for morphine using thin-layer chromatography in 310 cases of real or possible heroin abuse showed that it was valuable not only in detecting improper drug use but also in monitoring treatment. The results of this test can be available routinely in 24, and exceptionally in five hours. A negative result implies that the subject has taken less than 10 mg. of heroin in the past 24 hours.     

THE EVOLUTION AND SYSTEMATICS OF BEE-EATERS (MEROPIDAE)

Behavioural and ecological characters are used in addition to structural ones in considering the systematics of the Meropidae. The two species of Nyctyornis are the most primitive extant forms. Meropogon is retained as a monotypic genus for forsteni, and all other bee-eaters are placed in the single genus Merops, being ecologically and morphologically rather uniform except in details of wing and tail structure, which should be considered only of specific importance. M. breweri and M. oreobates are thought to be secondarily rather than primarily forest species. With the submergence of Aerops and Melittophagus, and the monotypic genera Bombylonax and Dicrocercus, which are considered to be closely related to the pusillus species-group, Merops is enlarged to 21 species which are uniform except in tail-shape, wing formula and throat feather structure. These characters are of specific importance only, and have a mosaic distribution within the genus. Their use in the definition of the formerly recognized genera results in an artificial classification. The proposed delimitation of species-groups within Merops differs somewhat from previous arrangements, and affinities argued in the text are summarized in Fig. 15, which shows superspecies and species-groups. The species recognized are formally tabulated below. The Meropidae probably originated in southeast Asian forest and spread through former forest to Africa. Only in Africa was the open-country environment invaded, and speciation in the savanna was in two main directions, producing small sedentary and large migratory species. Representatives of both types returned to Asia in open country. From the present distribution of species it is inferred that speciation has proceeded under the main influences of (1) isolation of a population and its habitat in Pleistocene Africa and (2) isolation of migrants away from their breeding range. The distributions of species with wide or rather limited ranges are discussed in terms of physiological adaptation and ecological competition. Opinion has not been expressed on the validity of subspecies, which have been discussed only in delimiting controversial species. In the following summary, the only subspecies named are those affected by changes from Peters' scheme (cf. Table 1). Superspecies are bracketed. Nyctyornis amicta N. athertoni Meropogon forsteni Merops guloris M. mülleri M. bulocki M. bullockoides M. pusillus M. variegatus (?loringi, oariegatus, lafresnayii, bangweoloensis) M. oreobates M. hirundineus M. breweri M. revoilii M. albicollis M. orientalis M. boehmi M. viridis M. superciliosus (persicus, chrysocercus, superciliosus) M. philippinus (philippinus, salvadorii) M. ornatus M. apiaster M. leschenaulti M. malimbicus M. nubicus (nubicus, nubicoides)