Experimental dissociation of food intake and plasma beta-endorphin following 2-deoxy-D-glucose in rats

The present studies were undertaken to further assess the role of plasma beta-endorphin (beta-EP) in the hyperphagia induced by the glucose antimetabolite, 2-deoxy-D-glucose (2-DG). Plasma concentrations of immunoreactive beta-EP (ir-beta-EP) were measured at the end of the first hour of feeding in all animals treated with 400 mg/kg 2-DG. Previous studies had shown a consistent, positive association between 2-DG hyperphagia and plasma ir-beta-EP concentrations, but the present data revealed dissociations between hyperphagia and plasma ir-beta-EP. Dexamethasone administration blocked the 2-DG-induced rise in plasma ir-beta-EP, but had no effect on the 2-DG hyperphagia measured at 1 hour. Forced drinking of a 2% NaCl solution decreased 2-DG hyperphagia, but not the 2-DG induced rise in plasma ir-beta-EP. Thus, elevations in plasma ir-beta-EP are not necessary for the full expression of 2-DG-induced hyperphagia in dexamethasone-treated rats. Furthermore, decreased feeding responses to 2-DG could coexist with increased levels of plasma ir-beta-EP in NaCl-treated normal rats. Elevations in plasma ir-beta-EP do not appear to be the critical opiate link in 2-DG induced hyperphagia.     

The effects of mispair and nonpair correction in hybrid DNA on base ratios (G + C content) and total amounts of DNA

Base ratios and total DNA amounts can vary substantially between and within higher taxa and genera, and even within species. Gene conversion is one of several mechanisms that could cause such changes. For base substitutions, disparity in conversion direction is accompanied by an equivalent disparity in base ratio at the heterozygous site. Disparity in the direction of gene conversion at meiosis is common and can be extreme. For transitions (which give purine [R]/pyrimidine [Y] mispairs) and for transversions giving unlike R/R and Y/Y mispairs in hybrid DNA, this disparity could give slow but systematic changes in G + C percentage. For transversions giving like R/R and Y/Y mispairs, it could change AT/TA and CG/GC ratios. From the extent of correction direction disparity, one can deduce properties of repair enzymes, such as the ability (1) to excise preferentially the purine from one mispair and the pyrimidine from the other for two different R/Y mispairs from a single heterozygous site and (2) to excise one base preferentially from unlike R/R or Y/Y mispairs. Frame-shifts usually show strong disparity in conversion direction, with preferential cutting of the nonlooped or the looped-out strand of the nonpair in heterozygous h-DNA. The opposite directions of disparity for frame-shifts and their intragenic suppressors as Ascobolus suggest that repair enzymes have a strong, systematic bias as to which strand is cut. The conversion spectra of mutations induced with different mutagens suggest that the nonlooped strand is preferentially cut, so that base additions generally convert to mutant and deletions generally convert to wild-type forms. Especially in nonfunctional or noncoding DNA, this could cause a general increase in DNA amounts. Conversion disparity, selection, mutation, and other processes interact, affecting rates of change in base ratios and total DNA.      

Monoclonal antibodies to plant plasma-membrane antigens

Murine monoclonal antibodies to membrane antigens were generated by immunization with a crude cellular membrane preparation from suspension-cultured cells of Nicotiana glutinosa L. From a panel of thirteen monoclonal antibodies, seven were found to be directed against antigens present on the plasma-membrane by immunofluorescence visualization of antibody binding to the surface of isolated protoplasts. The corresponding set of plasma-membrane antigen(s) were present in root, shoot and leaf tissue and some but not all of these antigens were of wide species distribution, being found in Nicotiana tabacum L., N. plumbaginifolia L., Glycine max L., Phaseolus vulgaris L. and Triticum aestivum L. Topologically specific labeling of intact protoplasts with a monoclonal antibody reactive with an epitope present on the plasma-membrane specifically labeled a membrane fraction which equilibrated at a density of 1.14 kg/l following centrifugation in a sucrose gradient. In addition to use as biochemical markers for fractionation and molecular characterization of plasma-membranes, these monoclonal antibodies provide the basis for new selection tools in plant cell and gene manipulations.     

Bradyrhizobium japonicum mutants defective in root-nodule bacteroid development and nitrogen fixation

The genome of the slow-growing Bradyrhizobium japonicum (strain 110) was mutagenized with transposon Tn5. A total of 1623 kanamycin/streptomycin resistant derivatives were screened in soybean infection tests for nodulation (Nod) and symbiotic nitrogen fixation (Fix). In this report we describe 14 strains possessing a stable, reproducible Nod⁺Fix^- phenotype. These strains were also grown under microaerobic culture conditions to test them for free-living nitrogen fixation activity (Nif). In addition to strains having reduced Fix and Nif activities, there were also strains that had reduced symbiotic Fix activity but were Nif⁺ ex planta.Analysis of the genomic structure revealed that the majority of the strains had a single Tn5 insertion without any further apparent physical alteration. A few strains had additional insertions (by Tn5 or IS50), or a deletion, or had cointegrated part of the vector used for Tn5 mutagenesis. One of the insertions was found in a known nif gene (nifD) whereas all other mutations seem to affect different, hitherto unknown genes or operons.Several mutant strains had an altered nodulation phenotype, inducing numerous, small, widely distributed nodules. Light and electron microscopy revealed that most of these mutants were defective in different stages of bacteroid development and/or bacteroid persistence. The protein patterns of the mutants were inspected by two-dimensional gel electrophoresis after labelling microaerobic cultures with l-(³⁵S)methionine. Of particular interest were mutants lacking a group of proteins the synthesis of which was known to be under oxygen control. Such strains can be regarded as potential regulatory mutants.     

Properties of narrow-range ampholytes isolated from wide-range ampholyte preparations

A simple procedure for obtaining useful narrow-pH-range ampholytes from inexpensive laboratory-synthesized ampholytes by preparative isoelectric focusing in Pevikon is described. The narrow range ampholytes prepared in this way are comparable to commercial ampholyte preparation as judged by conductivity, buffer capacity, pH gradient formation, and resolving power. These inexpensive narrow-range ampholytes are particularly well suited to preparative isoelectric focusing applications requiring large quantities of ampholytes.     

Effect of mutations and deletions in a bicistronic mRNA on the synthesis of influenza B virus NB and NA glycoproteins.

The mRNA derived from influenza B virus RNA segment 6 is functionally bicistronic and encodes the NB and NA glycoproteins in different, overlapping reading frames. NB protein synthesis is initiated at the 5'-proximal AUG codon, and 4 nucleotides downstream there is a second AUG codon which is used to initiate NA protein synthesis. The nucleotide sequence context of the first AUG codon conforms closely with the established 5'-CC(A/G)CCAUGG-3' consensus sequence (M. Kozak, Nucleic Acids Res. 15:8125-8148, 1987), which should favor initiation of NB protein synthesis at this site, yet NB and NA are found to accumulate in approximately equal amounts in infected cells. To determine the features important for allowing initiation at the second 5'-proximal AUG codon, we made changes in the 5'-terminal region of the mRNA, including deletions, insertions, and site-specific mutations. The recombinant DNA molecules were expressed in eucaryotic cells, and the accumulation of NB and NA was quantitated. The data indicate that changes in the immediate sequence around the first AUG codon do not make a large difference in the amounts of NB and NA that accumulate, but that when the first AUG codon is displaced from its normal position it is now quite efficient at preventing downstream initiation events. In addition, the data indicate that an element of the B/NB/NA mRNA 5' untranslated leader region acts in cis to enhance the expression of NB and NA.     

Functional dissection of a bean chalcone synthase gene promoter in transgenic tobacco plants reveals sequence motifs essential for floral expression

Expression of chalcone synthase (CHS), the first enzyme in the flavonoid branch of the phenylpropanoid biosynthetic pathway in plants, is induced by developmental cues and environmental stimuli. We used plant transformation technology to delineate the functional structure of the French bean CHS15 gene promoter during plant development. In the absence of an efficient transformation procedure for bean, Nicotiana tabacum was used as the model plant. CHS15 promoter activity, evaluated by measurements of -d-glucuronidase (GUS) activity, revealed a tissue-specific pattern of expression similar to that reported for CHS genes in bean. GUS activity was observed in flowers and root tips. Floral expression was confined to the pigmented part of petals and was induced in a transient fashion. Fine mapping of promoter cis-elements was accomplished using a set of promoter mutants generated by unidirectional deletions or by site-directed mutagenesis. Maximal floral and root-specific expression was found to require sequence elements located on both sides of the TATA-box. Two adjacent sequence motifs, the G-box (CACGTG) and H-box (CCTACC(N)₇CT) located near the TATA-box, were both essential for floral expression, and were also found to be important for root-specific expression. The CHS15 promoter is regulated by a complex interplay between different cis-elements and their cognate factors. The conservation of both the G-box and H-box in different CHS promoters emphasizes their importance as regulatory motifs.