HvHMA2, a P(1B)-ATPase from Barley, Is Highly Conserved among Cereals and Functions in Zn and Cd Transport

Manipulation of crops to improve their nutritional value (biofortification) and optimisation of plants for removal of toxic metals from contaminated soils (phytoremediation) are major goals. Identification of membrane transporters with roles in zinc and cadmium transport would be useful for both aspects. The P(1B)-ATPases play important roles in heavy metal allocation and detoxification in Arabidopsis and it is now important to elucidate their roles in monocots. We identified nine P(1B)-ATPases in barley and this study focuses on the functional characterization of HvHMA2, providing evidence for its role in heavy metal transport. HvHMA2 was cloned using information from EST analysis and 5' RACE. It possesses the conserved aspartate that is phosphorylated during the reaction cycle of P-type pumps and has motifs and key residues characteristic of P(1B)-ATPases, falling into the P(1B-2) subclass. Homologous sequences occur in three major sub-families of the Poaceae (Gramineae). Heterologous expression in Saccharomyces cerevisiae demonstrates that HvHMA2 functions as a Zn and Cd pump. Mutagenesis studies show that proposed cation coordination sites of the P(1B-2) pumps are crucial for the metal responses conferred by HvHMA2 in yeast. HvHMA2 expression suppresses the Zn-deficient phenotype of the Arabidopsis hma2hma4 mutant indicating that HvHMA2 functions as a Zn pump in planta and could play a role in root to shoot Zn transport. When expressed in Arabidopsis, HvHMA2 localises predominantly to the plasma membrane.     

The cortisol response to hypobaric hypoxia at rest and post-exercise

High altitude exposure normally leads to a marked natriuresis and diuresis. Acute mountain sickness is often associated with fluid retention, to which an elevated cortisol may contribute. Most investigators report a rise in resting cortisol with ascent, but little data exist regarding the cortisol response to a day trekking. We therefore measured salivary cortisol during ascent to > 5000 m in a cohort of between 42-45 subjects following a 6-h trek (samples taken between 15:30-16:30 h) and between 15-20 subjects at rest (morning samples taken between 08:00-09:00 h). Morning resting cortisol [nmol/l, mean±sd, (range)] was 5.5±2.9 (2.13-13.61) at 1300 m; 4.7±6.8 (1.4-27.02) at 3400 m, and significantly (p=0.002) rose between 4270 m [3.5±2.1 (1.4-8.34)] and 5150 m [14.5±30.3 (1.9-123.1)]. Post-exercise cortisol [nmol/l, mean±sd, (range)] dropped between 3400 m [7±6 (1.5-33.3)] and 4270 m [4.2±4.8 (1.4-29.5)] (p=0.001) followed by a significant rise in post-exercise cortisol between 4270 m [4.2±4.8 (1.4-29.5)] and 5 150 m [9.2±10.2 (1.4-61.3)] (p<0.001). There were no significant associations between severity of acute mountain sickness and cortisol levels. There was a significant though weak correlation between cortisol post-exercise at 5150 m and oxygen saturation at 5150 m (rho= - 0.451, p=0.004). In conclusion, this is the largest cohort to have their resting and post-exercise cortisol levels ascertained at high altitude. We confirm the previous findings of an elevated resting morning cortisol at > 5000 m, but present the novel finding that the cortisol response to a day trekking at HA appears suppressed at 4270 m.     

Detection and quantification of Lyme spirochetes using sensitive and specific molecular beacon probes

Background  

Lyme disease, caused by Borrelia burgdorferi, affects a large number of people in both the USA and Europe. The mouse is a natural host for this spirochete and is widely used as a model system to study Lyme pathogenesis mechanisms. Since disease manifestations often depend upon the spirochete burden in a particular tissue, it is critical to accurately measure the bacterial number in infected tissues. The current methods either lack sensitivity and specificity (SYBR Green), or require independent analysis of samples in parallel to quantitate host and bacterial DNA (TaqMan). We have developed a novel molecular beacon-based convenient multiplex real-time quantitative PCR assay to identify and detect small numbers of B. burgdorferi in infected mouse tissues.     

Structure, function, and motility of vacuoles in filamentous fungi

Current information on the structure and function of motile tubular vacuoles in Pisolithus tinctorius and other fungi is reviewed. The use of fluorochromes to label the vacuole lumen is evaluated and observations on the structure and motility of vacuoles in P. tinctorius are differentiated from possible artifacts. The styryl dyes FM4-64 and MDY-64, used in yeast to demonstrate endocytosis, show little or no labeling of internal membranes in undamaged P. tinctorius cells. This agrees with our data showing that other probes for endocytosis such as Lucifer yellow CH are not taken up by hyphal tip cells. Overall, the observations do not support endocytosis in hyphal tips. It has been suggested that tubular vacuole systems carry out longitudinal transport, and evidence in favor of this hypothesis is evaluated. New data are presented to show that many of the large vacuoles in subapical cells are attached to the plasma membrane and are relatively immobile, while video sequences show movement of fluorochrome in pulses along a series of several large vacuoles, all interconnected via tubules. Tubular vacuoles from thick sections of hyphae processed under anhydrous conditions are shown by X-ray microanalysis to contain relatively high levels of P and K, as seen previously in the larger vacuoles. These results provide further evidence for a role of the tubular vacuoles in longitudinal transport of P. Copyright 1998 Academic Press.     

Effect of amantadine on L-2-14C-dopa metabolism in parkinsonism

A metabolic study of the effects of amantadine hydrochloride on the fate of orally administered L-2-¹⁴C-dopa is described in three subjects with Parkinson's disease. Serum and urine distribution of radioactivity were determined in catecholamine, dopa, methoxydopa, and phenol carboxylic acid fractions prior to and after the administration of amantadine 100 mg daily for 4 weeks. Amantadine moderately decreased serum and urinary radioactivity during the first 2 hrs. Further, amantadine administration markedly decreased the urinary, but not serum, catecholamine fraction. Concomitantly, a two fold rise in urinary phenol carboxylic acids fraction from base line values was noted. It is concluded that amantadine may decrease extracerebral metabolism of levodopa and reduce the extent of its metabolism to its catecholamine metabolites.