Horizontal transmission, vertical inactivation, and stochastic loss of mariner-like transposable elements

Horizontal transmission has been well documented as a major mechanism for the dissemination of mariner-like elements (MLEs) among species. Less well understood are mechanisms that limit vertical transmission of MLEs resulting in the "spotty" or discontinuous distribution observed in closely related species. In this article we present evidence that the genome of the common ancestor of the melanogaster species subgroup of Drosophila contained an MLE related to the mellifera (honey bee) subfamily. Horizontal transmission, approximately 3-10 MYA, is strongly suggested by the observation that the sequence of the MLE in Drosophila erecta is 97% identical in nucleotide sequence with that of an MLE in the cat flea, Ctenocephalides felis. The D. erecta MLE has a spotty distribution among species in the melanogaster subgroup. The element has a high copy number in D. erecta and D. orena, a moderate copy number in D. teissieri and D. yakuba, and was apparently lost ("stochastic loss") in the lineage leading to D. melanogaster, D. simulans, D. mauritiana, and D. sechellia. In D. erecta, most copies are concentrated in the heterochromatin. Two copies from D. erecta, denoted De12 and De19, were cloned and sequenced, and they appear to be nonfunctional ("vertical inactivation"). It therefore appears that the predominant mode of MLE evolution is vertical inactivation and stochastic loss balanced against occasional reinvasion of lineages by horizontal transmission.      

Stress and ACTH increase circulating concentrations of 3 alpha-androstanediol in female rats.

The present experiments were carried out to determine what physiological conditions are responsible for the acute increases in serum levels of 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-androstanediol, 3 alpha-Adiol) which are seen in the intact estrous female rat within 15-30 min after mating. Blood samples were obtained from proestrus rats immediately before and 20 min after injection of exogenous hormones or initiation of stress procedures, and plasma concentrations of 3 alpha-Adiol and/or progesterone (P) were measured in these samples by RIA. Intravenous injections of ovine LH (5, 15, or 45 micrograms) or saline resulted in equivalent significant increases in plasma 3 alpha-Adiol 20 min after injection. In contrast, dose-dependent increases in 3 alpha-Adiol were seen after intravenous injection of 0 (acidic saline vehicle), 2, 4, or 8 ng ACTH1-24 to dexamethasone-pretreated rats. The highest ACTH1-24 dose also resulted in a significant increase in plasma P concentration. In a third experiment, a significant increase in 3 alpha-Adiol concentration above baseline was seen at 20 min after onset of restraint stress; in this case plasma P concentrations did not increase significantly. Finally, blood samples were obtained after onset of ether/jugular venipuncture stress two days after ovariectomy (ovx), adrenalectomy (adx), or ovx+adx on diestrus. The plasma 3 alpha-Adiol response to stress was normal in intact sham-operated and non-operated groups of controls, but was significantly diminished in the ovx and the adx groups to 28.4% of that shown by the intact animals. Circulating 3 alpha-Adiol concentrations were undetectable in 22/26 samples obtained in the ovx+adx group. These data demonstrate that plasma concentrations of 3 alpha-Adiol increase in response to stress or ACTH but not LH, and that both the ovary and the adrenal contribute to this increase.     

A neutron Laue diffraction study of endothiapepsin: implications for the aspartic proteinase mechanism.

Current proposals for the catalytic mechanism of aspartic proteinases are largely based on X-ray structures of bound oligopeptide inhibitors possessing nonhydrolyzable analogues of the scissile peptide bond. However, the positions of protons on the catalytic aspartates and the ligand in these complexes have not been determined with certainty. Thus, our objective was to locate crucial protons at the active site of an inhibitor complex since this will have major implications for a detailed understanding of the mechanism of action. We have demonstrated that high-resolution neutron diffraction data can be collected from crystals of the fungal aspartic proteinase endothiapepsin bound to a transition state analogue (H261). The neutron structure of the complex has been refined at a resolution of 2.1 A to an R-factor of 23.5% and an R(free) of 27.4%. This work represents the largest protein structure studied to date by neutron crystallography at high resolution. The neutron data demonstrate that 49% of the main chain nitrogens have exchanged their hydrogen atoms with D2O in the mother liquor. The majority of residues resisting exchange are buried within core beta-sheet regions of the molecule. The neutron maps confirm that the protein has a number of buried ionized carboxylate groups which are likely to give the molecule a net negative charge even at very low pH, thereby accounting for its low pI. The functional groups at the catalytic center have clearly undergone H-D exchange despite being buried by the inhibitor occupying the active site cleft. Most importantly, the data provide convincing evidence that Asp 215 is protonated and that Asp 32 is the negatively charged residue in the transition state complex. This has an important bearing on mechanistic proposals for this class of proteinase.     

Fungal virulence studies come of age

Sophisticated molecular biological research has revealed many virulence attributes in at least four pathogenic fungi, but the future study of fungal virulence requires investigators to distinguish between molecules that directly interact with the host, molecules that regulate these, and molecules that are always required for fungal growth and survival, independent of the host.     

The X-ray structure of yeast 5-aminolaevulinic acid dehydratase complexed with two diacid inhibitors

The structures of 5-aminolaevulinic acid dehydratase complexed with two irreversible inhibitors (4-oxosebacic acid and 4,7-dioxosebacic acid) have been solved at high resolution. Both inhibitors bind by forming a Schiff base link with Lys 263 at the active site. Previous inhibitor binding studies have defined the interactions made by only one of the two substrate moieties (P-side substrate) which bind to the enzyme during catalysis. The structures reported here provide an improved definition of the interactions made by both of the substrate molecules (A- and P-side substrates). The most intriguing result is the novel finding that 4,7-dioxosebacic acid forms a second Schiff base with the enzyme involving Lys 210. It has been known for many years that P-side substrate forms a Schiff base (with Lys 263) but until now there has been no evidence that binding of A-side substrate involves formation of a Schiff base with the enzyme. A catalytic mechanism involving substrate linked to the enzyme through Schiff bases at both the A- and P-sites is proposed.     

The effect of estrogen on muscle damage biomarkers following prolonged aerobic exercise in eumenorrheic women

This study assessed the influence of estrogen (E₂) on muscle damage biomarkers [skeletal muscle - creatine kinase (CK); cardiac muscle - CK-MB] responses to prolonged aerobic exercise. Eumenorrheic women (n=10) who were physically active completed two 60-minute treadmill running sessions at ∼60-65% maximal intensity during low E₂ (midfollicular menstrual phase) and high E₂ (midluteal menstrual phase) hormonal conditions. Blood samples were collected prior to exercise (following supine rest), immediately post-, 30 min post-, and 24 hours post-exercise to determine changes in muscle biomarkers. Resting blood samples confirmed appropriate E₂ hormonal levels Total CK concentrations increased following exercise and at 24 hours post-exercise were higher in the midfollicular low E₂ phase (p<0.001). However, CK-MB concentrations were unaffected by E₂ level or exercise (p=0.442) resulting in the ratio of CK-MB to total CK being consistently low in subject responses (i.e., indicative of skeletal muscle damage). Elevated E₂ levels reduce the CK responses of skeletal muscle, but had no effect on CK-MB responses following prolonged aerobic exercise. These findings support earlier work showing elevated E₂ is protective of skeletal muscle from exercise-induced damage associated with prolonged aerobic exercise.     

Iron deficiency in lentil: Yield loss and geographic distribution in a germplasm collection

Iron deficiency symptoms are observed on some genotypes of lentil (Lens culinaris Medikus) grown in calcareous soil. A germplasm collection of 3512 accessions originating from 18 countries was characterized for iron deficiency in a Calcic Rhodoxeralf soil at ICARDA, Tel Hadya, Syria in the 1979/80 season. At 105 days after sowing, 592 accessions, representing 16.9% of the collection, showed chlorosis symptoms characteristic of iron (Fe) deficiency. The Fe deficiency was verified by foliar application of Fe-chelate. Germplasm from different countries showed differences in iron deficiency, with those accessions exhibiting symptoms of iron deficiency mostly originating from relatively warm climates such as India (37.5% accessions showing Fe deficiency) and Ethiopia (30%). Populations from those Mediterranean countries where lentil originated (Syria and Turkey) exhibited Fe-deficiency symptoms only at very low frequencies. Fe-deficiency induced chlorosis was positively correlated with cold susceptibility. Fe chlorosis was transient, the deficiency symptoms largely disappearing during reproductive growth at a time, coinciding with increases in soil temperature and daylength-conditions favorable for plant growth. In Indian germplasm, mild deficiency symptoms did not lead to reduced seed yield, but there was a major yield reduction of 47% in those accessions with the most severe symptoms. Straw yields was reduced commensurately with the severity of symptoms. ei]Section editor: B G Rolfe     

X-ray, neutron and NMR studies of the catalytic mechanism of aspartic proteinases

Current proposals for the catalytic mechanism of aspartic proteinases are largely based on X-ray structures of bound oligopeptide inhibitors possessing non-hydrolysable analogues of the scissile peptide bond. Until recent years, the positions of protons on the catalytic aspartates and the ligand in these complexes had not been determined with certainty due to the inadequate resolution of these analyses. There has been much interest in locating the catalytic protons at the active site of aspartic proteinases since this has major implications for detailed understanding of the mechanism of action and the design of improved transition state mimics for therapeutic applications. In this review we discuss the results of studies which have shed light on the locations of protons at the catalytic centre. The first direct determination of the proton positions stemmed from neutron diffraction data collected from crystals of the fungal aspartic proteinase endothiapepsin bound to a transition state analogue (H261). The neutron structure of the complex at a resolution of 2.1 A provided evidence that Asp 215 is protonated and that Asp 32 is the negatively charged residue in the transition state complex. Atomic resolution X-ray studies of inhibitor complexes have corroborated this finding. A similar study of the native enzyme established that it, unexpectedly, has a dipeptide bound at the catalytic site which is consistent with classical reports of inhibition by short peptides and the ability of pepsins to catalyse transpeptidation reactions. Studies by NMR have confirmed the findings of low-barrier and single-well hydrogen bonds in the complexes with transition state analogues.