Identification of mitochondrial proteins on two-dimensional electrophoresis gels of extracts of adult Drosophila melanogaster

Several hundred proteins have been resolved on two-dimensional gels of extracts of [³⁵S]methionine-labeled adult Drosophila melanogaster. 27 of these polypeptides disappear from the gel pattern after feeding the K⁺ ionophore nonactin. These proteins have been identified as mitochondrial, since the two-dimensional gel pattern of extracts of isolated mitochondria correlates well with the pattern of the proteins missing from that of nonactin-treated flies. Nine new proteins also appear on the two-dimensional gels of the extracts from the nonactin-treated flies. Apparently, these nine proteins are precursors of the mature mitochondrial forms. These particular data support the concept that processing of many of the cytoplasmically synthesized mitochondrial proteins requires a specific membrane potential, and that some of these proteins are modified intramitochondrially. However, using [³⁵S]methionine incorporation techniques, not all labeled polypeptides disappear from mitochondria during such treatment. Feeding similarly radiolabeled flies with chloramphenicol, an inhibitor of mitochondrial protein synthesis, results in the disappearance of only one protein from the gel pattern with the concurrent appearance of a ‘new’ high-molecular-weight polypeptide. Collectively, these data show that a specific group of [³⁵S]methionine-labeled mitochondrial proteins can be identified by selective inhibition of mitochondrial function in whole cell protein maps of adult D. melanogaster.     

The impact of ticks on pheasant territoriality

Pheasants are competent reservoir hosts for the Lyme disease spirochaete, Borrelia burgdorferi s.l., and carry large, but highly over-dispersed, infestations of the vector ticks, Ixodes ricinus . The effects of experimental reduction of tick infestation levels on the survival and territorial behaviour of male pheasants were studied. Over three years in two woodlands in southern England, birds were marked individually and half were fitted with a slow-release acaricide, which substantially reduced their tick burdens from March to August. Acaricide treatment affected reproductive success but had no discernible impact on the survival rates of male pheasants. The degree of wattle inflation by males, an indicator of territorial status and a correlate of harem acquisition, was significantly greater among treated males. In each year, a significantly higher proportion of treated (overall 44%) than control (22%) males acquired harems. Males that acquired females ranged over small areas on field edges. By contrast, those with no females ranged more widely in woods and the adjoining fields, increasing their exposure to questing ticks. The relative contribution of such roving males to tick-borne pathogen transmission may thus increase.     

Construction of an ordered overlapping library of bacteriophage P1 DNA in phage vector lambda D69

A library of bacteriophage P1 DNA was constructed in the phage vector lambda D69. The DNA of some 150 randomly chosen lambda-P1 hybrid phages containing P1 DNA fragments 5-10 kb in size was analyzed by restriction endonuclease digestion using enzymes EcoRI, BglII, and BamHI that cleave P1 DNA at known positions on the physical map of P1. Approximately one third of the phages contained P1 DNA inserts having two or more restriction sites for any one of these enzymes, thus allowing the location of the insert to be determined with respect to the physical map. Genetic tests allowed detection of lambda-P1 hybrid phages possessing inserts with functional P1 ban and CmR genes. A subset of 18 phages was analyzed in more detail; their P1 DNA inserts comprise an ordered collection of overlapping P1 DNA fragments that cover almost 98% of the P1 genome.     

Can a simple function for the dielectric response model electrostatic effects in globular proteins?

The relationship between the effective dielectric constant that models the electrostatic effect from a charged side chain in a protein was evaluated both experimentally and theoretically. Experimental values were obtained from the shifts in pKa that resulted from point mutations of side chains in subtilisin. Theoretical values were obtained from an iterative solution to Poisson's equation that considers the dielectric response of the protein and the solvent together with charge positions. There is no simple relationship between the effective dielectric constant and the distance from the charge responsible for the interactions. For some charge positions a linear but not a direct proportional relationship of the effective dielectric with distance of separation was observed. Thus, simple models such as a linear distance-dependence for the dielectric response are not suitable to evaluate electrostatic effects in proteins.     

Effects of nucleotides and divalent cations on phospholipase activity in Saccharomyces cerevisiae

Divalent cations activate the lysophospholipase and transacylase reactions catalyzed by the same enzymes in the yeast Saccharomyces cerevisiae. The activation was observed at neutral pH, but not at the pH optimum of lysophospholipase/transacylase, near 3.5. Adenine nucleotides, especially AMP and ADP, are strong inhibitors of the same group of enzymes. Half maximal inhibition by AMP was found at a concentration of about 20 M. The inhibition by nucleotides in low concentrations is enhanced by divalent cations.     

Aestival dormancy in the cabbage moth Mamestra brassicae L. (Lepidoptera: Noctuidae)

Summary In a geographically wide distribution the life cycles of different populations of the cabbage moth Mamestra brossicae are adapted to a remarkable diversity of climatic conditions. This is undoubtedly a proof of its success in adaptation. Some populations living in regions characterized by a drought period interrupting the growth season are capable of distinguishing between one critical day length signalling the onset of the drought period and another signalling the end of the growth season. This study, therefore, is primarily concerned with the geographical patterns in the variability of the adaptional responses of populations exposed to environmental conditions requiring different strategies and tactics in, synchronizing individual, life cycles. It is also a contribution to our understanding of evolutionary mechanisms maintaining median responses to photoperiodically inductive day lengths in geographically different populations. The populations investigated originated from regions differing in predictability of the incidence, onset and duration of a drought period: Freiburg (48.0°N, Southern Germany), Avignon (44.0°N, Southern France), and Argelès (42.5°N, Southern France). Geographical variation with respect to both onset and duration of a drought period consequently results in clinal variation of the variability of innate day length thresholds triggering aestival dormancy and of innate duration of aestivation. In this paper we considered the influence of geographically changing temperatures on aestival dormancy induction. Even in southern populations of M. brassicae a temperature dependent switch off-mechanism exists which prevents aestival dormancy under certain environmental conditions. The effective temperatures vary geographically, too. What the geographical patterns in adaptive responses really are, is discussed.This research was supported by the Deutsche Forschungsgemeinschaft (Sa 259/3-1)     

Nerve growth factor (NGF) regulates adult rat cultured dorsal root ganglion neuron responses to the excitotoxin capsaicin

An overlap between subpopulations of nerve growth factor (NGF)-responsive and capsaicin-sensitive dorsal root ganglion (DRG) sensory neurons has been suggested from a number of in vivo studies. To examine this apparent link in more detail, we compared the effects of capsaicin on adult rat DRG neurons cultured in the presence or absence of NGF. Capsaicin sensitivity was assessed histochemically by a cobalt staining method, by measuring capsaicin-induced 45Ca2+ uptake, and by electrophysiological recording of capsaicin-evoked membrane currents. When cultured with NGF, approximately 50% of these adult DRG neurons were capsaicin-sensitive, whereas adult sympathetic neurons or ganglionic nonneuronal cells were insensitive. DRG cultures grown in the absence of NGF, however, were essentially unresponsive to capsaicin. Capsaicin sensitivity could be regained fully within 4-6 days of replacement of NGF. These results indicate that, at least in vitro, NGF can modify the capsaicin sensitivity of adult DRG neurons.     

Stachyose synthesis in mature leaves of Cucumis melo. Purification and characterization of stachyose synthase (EC 2.4.1.67)

Galactinol: raffinose-6-galactosyltransferase (EC 2.4.1.67), a stachyose synthase, was extracted from mature leaves of Cucumis melo cv. Ranjadew and was purified to homogeneity by (NH₄)₂SO₄ precipitation, ion-exchange chromatography, gel-filtration and non-denaturing polyacrylamide gel electrophoresis. A specific activity of 516 kat · mg^-1 and a 160-fold purification was achieved. The pH optimum of the enzyme reaction was found to be 6.8 in sodium-phosphate buffer, and the temperature optimum 32° C. The purified enzyme was very sensitive towards SH-poisons but its reaction was hardly affected by changes in the ion composition of the assay medium. The two-substrate enzyme was specific for galactinol and raffmose; uridine-diphosphate galactose and p-nitrophenyl--d-galactoside as well as melibiose were not accepted by the purified enzyme. Stachyose synthesis was competitively inhibited by concentrations >4 mM raffinose as well as 2.5 mM galactinol. The K _m values determined under non-saturating conditions were 3.3 mM for raffinose and 7.7 mM for galactinol. Myoinositol was a strong competitive inhibitor with a K _i of 1.8mM. Galactinol was hydrolyzed in the absence of raffinose with a K _m of 0.8 mM. The pure enzyme is a protein with a molecular weight of at least 95 kDa and an isoelectric point of 5.1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of two subunits of 45 and 50 kDa. Polyclonal antibodies from rabbit were obtained which were specific for the native enzyme but cross-reacted with other proteins separated under denaturing conditions.Abbreviations DEAE diethylaminoethyl - DTT dithiothreitol - FPLC fast protein liquid chromatography - HPLC high-performance liquid chromatography - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate This work was supported by Deutsche Forschungsgemeinschaft. The gift of galactinol by Dr. T. Schweizer (Nestlé, Switzerland) is gratefully acknowledged.     

Biosynthesis of gallotannins

Cell-free extracts from leaves of Rhus typhina L. (sumach) were found to transfer the 1-O-galloyl moiety of l,6-di-O-galloyl-β-d-glucose to the 2-position of the same compound, yielding 1,2,6-tri-O-galloyl-β-d-glucose and leaving 6-O-galloylglucose as the deacylated by-product. The enzyme catalyzing this ‘disproportionation’ was purified almost 1700-fold. It had a molecular weight of approx. 56 000, a K _m value of 11.5 mM, was stable between pH 4.5 and 6.5, and most active at pH 5.9 and 40° C. The systematic name “1,6-di-O-galloyl-glucose: 1,6-di-O-galloylglucose 2-O-galloyltransferase” (EC 2.3.1.) was proposed for this new enzyme whose detection provided evidence that, in addition to β-glucogallin (1-O-galloyl-β-d-glucose), higher substituted glucose esters also have the potential to serve as acyl donors in the biosynthesis of gallotannins.     

No evidence for linkage of autosomal dominant proximal spinal muscular atrophies to chromosome 5q markers

Summary Two recent articles have reported the linkage of a gene for recessive spinal muscular atrophy (SMA) on the chromosome region 5q11.2–13.3. Our data show no linkage of the dominantly inherited forms of SMA to this chromosome region.