MAGGY,a retrotransposon in the genome of the rice blast fungusMagnaporthe grisea

Full-length copies of a previously described repetitive DNA sequence (CH2-8) were isolated from the genome of theMagnaporthe grisea strain 2539. One copy of the complete element was sequenced and found to resemble agypsy-like LTR retrotransposon. We named this element MAGGY (MAGnaporthe GYpsy-like element). MAGGY contains two internal ORFs putatively encoding Gag, Pol and Env-like proteins which are similar to peptides encoded by retroelements identified in other filamentous fungi. MAGGY was found to be widely distributed amongM. grisea isolates from geographically dispersed locations and different hosts. It was present in high copy number in the genomes of all nine rice-pathogenic isolates examined. By contrast,M. grisea strains isolated from other Gramineae were found to possess varying copy numbers of MAGGY and in some cases the element was completely absent. The wide distribution of MAGGY suggests that this element invaded the genome ofM. grisea prior to the evolution of rice-specific form(s). It may since have been horizontally transmitted to other sub-specific groups. One copy of MAGGY, corresponding to the element we sequenced, was located at identical locations in the genomes of geographically dispersed strains, suggesting that this copy of the element is a relatively ancient insertion.     

Transformation frequencies are enhanced and vector DNA is targeted during retransformation of Leptosphaeria maculans, a fungal plant pathogen.

Summary Leptosphaeria maculans, a fungal pathogen of Brassica spp., was successfully transformed with the vector pAN8-1, encoding phleomycin resistance. Protoplasts of a vigorous Phleo^r transformant were then retransformed using the partially homologous vector, pAN7-1 which encodes hygromycin B resistance. Retransformation of this strain to hygromycin resistance occurred at frequencies that were consistently twofold higher than with the original recipient strain. Linearised pAN7-1 DNA transformed phleomycin-resistant protoplasts at higher frequencies still. All the transformants that were tested retained a phleomycin-resistant phenotype (20/20). Molecular analysis of five transformants generated with circular pAN7-1 DNA indicated that in four cases the pAN7-1 vector had integrated into pAN8-1 sequences. These results suggest that transformation frequencies in L. maculans are limited by the ability of vector DNA to integrate into the genome. Hence, construction of strains with target sites for integration may prove to be a generally useful method for improving transformation frequencies of poorly characterised filamentous fungi, particularly when using heterologous vectors. This would greatly facilitate the identification of genes by transfer of gene libraries and the standardisation of chromosomal location effects in studies of expression of nested promoter deletions.     

Cell sodium-induced recruitment of Na(+)-K(+)-ATPase pumps in rabbit cortical collecting tubules is aldosterone-dependent

The influence of intracellular sodium concentration ( [Na+]i) on the number of Na(+)-K(+)-ATPase pumps was examined in cortical collecting tubules (CCD) of kidneys from rabbits in different aldosterone conditions. Specific [3H]ouabain binding was measured in isolated CCD with various [Na+]i. Experiments were performed on adrenalectomized rabbits receiving only a substitutive dose of dexamethasone and on adrenalectomized rabbits replete with aldosterone. In aldosterone-replete rabbits, the number of binding sites increased linearly with [Na+]i, from 16 fmol/nl tubular volume at 15 mM Na+i to 39 fmol/nl tubular volume at 140 mM Na+i. Neither actinomycin D (5 microM) nor cycloheximide (10 microM) prevented this [Na+]i-dependent increase. In adrenalectomized rabbits, the number of ouabain-binding sites was reduced and did not increase with [Na+]i. These results are in favor of the presence of a "latent" pool of pumps in CCD, rapidly recruited under [Na+]i influence. Aldosterone appears to be required for the constitution and/or activation of this pool.     

Effect of cell sodium on Na+/K(+)-ATPase-dependent sodium efflux in cortical collecting tubule of rabbits under different aldosterone status

Aldosterone increased the tubular volume in cortical collecting tubules (CCD) of rabbit kidney. It modulated the rate of cell sodium accumulation, under condition of ATPase inhibition (4 degrees C, in the absence of K+). In contrast, the relationship between Na+/K(+)-ATPase-dependent Na+ extrusion rate and intracellular Na+ concentration (Nai+) was similar in control, adrenalectomized, and aldosterone-treated adrenalectomized animals: Na+ extrusion rate increased with Nai+, up to 70 mM Nai+, and then plateaued. This indicates that aldosterone does not modify the characteristics of Nai(+)-dependent Na+ extrusion rate by the Na+/K(+)-ATPase pump in CCD.     

An astigmatid defence volatile against a phytoseiid mite

Numerous semiochemicals have been isolated from several species of astigmatid mites with various identified or unidentified functions. Alarm pheromonal activity is widespread with neryl formate and neral, being the most common compounds eliciting alarm response in conspecifics. The cosmopolitan astigmatid mite Suidasia medanensis (= S. pontifica) Oudemans (Acari: Suidasidae) has been reported to use neral as an alarm pheromone, but neral can also act as an allomone towards predators of oribatid mites. Suidasia medanensis can be utilised as a factitious prey for mass‐rearing of the phytoseiid predatory mite Amblyseius (= Typhlodromips) swirskii (Athias‐Henriot) (Acari: Phytoseiidae), which is used for biological control of insect and mite pests in protected crops. This study investigated the potential defence properties of the S. medanensis volatiles against A. swirskii, comparing the repellency to pollen‐reared (naïve) vs. S. medanensis‐reared (experienced) predators using a synthetic blend of the isomers neral and geranial (1:1) as a model compound. In a repellency bioassay, the synthetic blend elicited a significant repellence to A. swirskii with no difference between naïve and experienced predators. During capture success studies, S. medanensis under repeated attack could release sufficient quantities of the defence volatile to deter 1–5 attacks from A. swirskii, whereas hexane‐treated S. medanensis artificially depleted of volatiles were significantly more vulnerable to an attack. This is the first report of an astigmatid defence volatile with repellent activity to a phytoseiid mite and the starting point to understanding semiochemical interactions in any current or novel factitious predator‐prey mass‐rearing system.     

Functional role and immunocytochemical localization of the gamma a and gamma b forms of the Na,K-ATPase gamma subunit

The gamma subunit of the Na,K-ATPase is a member of the FXYD family of type 2 transmembrane proteins that probably function as regulators of ion transport. Rat gamma is present primarily in the kidney as two main splice variants, gamma(a) and gamma(b), which differ only at their extracellular N termini (TELSANH and MDRWYL, respectively; Kuster, B., Shainskaya, A., Pu, H. X., Goldshleger, R., Blostein, R., Mann, M., and Karlish, S. J. D. (2000) J. Biol. Chem. 275, 18441-18446). Expression in cultured cells indicates that both variants affect catalytic properties, without a detectable difference between gamma(a) and gamma(b). At least two singular effects are seen, irrespective of whether the variants are expressed in HeLa or rat alpha1-transfected HeLa cells, i.e. (i) an increase in apparent affinity for ATP, probably secondary to a left shift in E(1) <--> E(2) conformational equilibrium and (ii) an increase in K(+) antagonism of cytoplasmic Na(+) activation. Antibodies against the C terminus common to both variants (anti-gamma) abrogate the first effect but not the second. In contrast, gamma(a) and gamma(b) show differences in their localization along the kidney tubule. Using anti-gamma (C-terminal) and antibodies to the rat alpha subunit as well as antibodies to identify cell types, double immunofluorescence showed gamma in the basolateral membrane of several tubular segments. Highest expression is in the medullary portion of the thick ascending limb (TAL), which contains both gamma(a) and gamma(b). In fact, TAL is the only positive tubular segment in the medulla. In the cortex, most tubules express gamma but at lower levels. Antibodies specific for gamma(a) and gamma(b) showed differences in their cortical location; gamma(a) is specific for cells in the macula densa and principal cells of the cortical collecting duct but not cortical TAL. In contrast, gamma(b) but not gamma(a) is present in the cortical TAL only. Thus, the importance of gamma(a) and gamma(b) may be related to their partially overlapping but distinct expression patterns and tissue-specific functions of the pump that these serve.     

Oleate and linoleate enhance the growth-promoting effects of insulin-like growth factor-I through a phospholipase D-dependent pathway in arterial smooth muscle cells

Diabetes causes accelerated atherosclerosis and subsequent cardiovascular disease through mechanisms that are poorly understood. We have previously shown, using a porcine model of diabetes-accelerated atherosclerosis, that diabetes leads to an increased accumulation and proliferation of arterial smooth muscle cells in atherosclerotic lesions and that this is associated with elevated levels of plasma triglycerides. We therefore used the same model to investigate the mechanism whereby diabetes may stimulate smooth muscle cell proliferation. We show that lesions from diabetic pigs fed a cholesterol-rich diet contain abundant insulin-like growth factor-I (IGF-I), in contrast to lesions from non-diabetic pigs. Furthermore, two fatty acids common in triglycerides, oleate and linoleate, enhance the growth-promoting effects of IGF-I in smooth muscle cells isolated from these animals. These fatty acids accumulate predominantly in the membrane phospholipid pool; oleate accumulates preferentially in phosphatidylcholine and phosphatidylethanolamine, whereas linoleate is found mainly in phosphatidylethanolamine. The growth-promoting effects of oleate and linoleate depend on phospholipid hydrolysis by phospholipase D and subsequent generation of diacylglycerol. Thus, concurrent increases in levels of IGF-I and triglyceride-derived oleate and linoleate in lesions may contribute to accumulation and proliferation of smooth muscle cells and lesion progression in diabetes-accelerated atherosclerosis.