PCR with degenerate primers amplifies a subgenomic DNA fragment from the endoglucanase gene(s) of Torula thermophila, a thermophilic fungus

The aim of this study was to enable the polymerase chain reaction (PCR) amplification of DNA fragments within endoglucanase gene(s) of Torula thermophila, by using degenerate primers so that the amplified fragment(s) could be used as homologous probe(s) for cloning of full-length endoglucanase gene(s). The design of the degenerate PCR primers was mainly based on the endoglucanase sequences of other fungi. The endoglucanase gene sequence of Humicola insolens was the only sequence from a thermophilic fungus publicly available in the literature. Therefore, the endoglucanase sequences of the two Trichoderma species, Trichoderma reesei and Trichoderma longibrachiatum, were used to generalize the primers. PCR amplification of T. thermophila genomic DNA with these primers resilied in a specific amplification. The specificity of the amplified fragment was shown by Southern hybridization analysis using egl3 gene of T. reesei as probe. This result suggested that the degenerate primers used in this study may be of value for studies aimed at cloning of endoglucanase genes from a range of related fungi.     

Genetic diversity for grain nutrients in wild emmer wheat: potential for wheat improvement

Background and Aims

Micronutrient malnutrition, particularly zinc and iron deficiency, afflicts over three billion people worldwide due to low dietary intake. In the current study, wild emmer wheat (Triticum turgidum ssp. dicoccoides), the progenitor of domesticated wheat, was tested for (1) genetic diversity in grain nutrient concentrations, (2) associations among grain nutrients and their relationships with plant productivity, and (3) the association of grain nutrients with the eco-geographical origin of wild emmer accessions.

Methods

A total of 154 genotypes, including wild emmer accessions from across the Near Eastern Fertile Crescent and diverse wheat cultivars, were characterized in this 2-year field study for grain protein, micronutrient (zinc, iron, copper and manganese) and macronutrient (calcium, magnesium, potassium, phosphorus and sulphur) concentrations.

Key Results

Wide genetic diversity was found among the wild emmer accessions for all grain nutrients. The concentrations of grain zinc, iron and protein in wild accessions were about two-fold greater than in the domesticated genotypes. Concentrations of these compounds were positively correlated with one another, with no clear association with plant productivity, suggesting that all three nutrients can be improved concurrently with no yield penalty. A subset of 12 populations revealed significant genetic variation between and within populations for all minerals. Association between soil characteristics at the site of collection and grain nutrient concentrations showed negative associations between soil clay content and grain protein and between soil-extractable zinc and grain zinc, the latter suggesting that the greatest potential for grain nutrient minerals lies in populations from micronutrient-deficient soils.

Conclusions

Wild emmer wheat germplasm offers unique opportunities to exploit favourable alleles for grain nutrient properties that were excluded from the domesticated wheat gene pool.     

Exogenous salicylate application affects the lead and copper accumulation characteristics of Lemna gibba L

Previous studies have shown that salicylates can change the ion permeability of root cells. Therefore the possible effects of exogenous salicylate application on lead (Pb) and copper (Cu) accumulation and its protective role against DNA damage due to metal exposure in Lemna gibba were studied. L. gibba was exposed to 5, 10, and 25 microM Pb and Cu for six days in the presence and absence of sodium salicylate (SA) (0.1, 0.5, and 1 mM). At all concentrations tested, SA application decreased Pb accumulation. On the other hand, application of 0.5 mM SA increased Cu accumulation. SA did not reduce DNA damage resulting from Pb and Cu toxicity. In summary, SA may be useful for reducing Pb accumulation, and application of SA at 0.5 mM may be useful for the phytoextraction of Cu.     

Amiodarone induces a caffeine-inhibited,MID1-depedent rise in free cytoplasmic calcium in Saccharomyces cerevisiae

Calcium signalling is involved in myriad cellular processes such as mating morphogenesis. Mating in yeast induces changes in cell morphology with a concomitant increase in calcium uptake that is dependent on the MID1 and CCH1 genes. Mid1p and Cch1p are believed to function in a capacitive calcium entry (CCE)-like process. Amiodarone alters mammalian calcium channel activity but, despite its clinical importance, its molecular mechanisms are not clearly defined. We have shown previously that amiodarone has fungicidal activity against a broad array of fungi. We show here that amiodarone causes a dramatic increase in cytoplasmic calcium ([Ca2+]cyt) in Saccharomyces cerevisiae. The majority of this increase is dependent on extracellular Ca2+ nonetheless, a significant increase in [Ca2+]cyt is still induced by amiodarone when no uptake of extracellular Ca2+ can occur. The influx of extracellular Ca2+ may be a direct effect of amiodarone on a membrane transporter or may be by a CCE mechanism. Uptake of the extracellular Ca2+ is inhibited by caffeine and reduced in strains deleted for the mid1 gene, but not in cells deleted for cch1. Our data are the first demonstrating control of yeast calcium channels by amiodarone and caffeine.     

Effects of zinc deficiency and supplementation on malondialdehyde and glutathione levels in blood and tissues of rats performing swimming exercise

The aim of the study was to investigate the effects of zinc deficiency and supplementation on lipid peroxidation and glutathione levels in blood and in some tissues of rats performing swimming exercise. Forty adult male Sprague-Dawley rats were divided into four groups: group 1, zinc-deficient consisted of swimming rats; group 2 consisted of zinc-supplemented swimming rats; groups 3 and 4 were the swimming and nonswimming controls, respectively. The levels of malondialdehyde and glutathione were measured after 4 wk of zinc-deficient or zinc-supplemented diet and 30 min of swimming exercise daily. The erythrocyte glutathione levels of groups 2 and 4 were significantly higher than those of groups 1 and 3 (p<0.01). The plasma malondialdehyde level of group 1 was significantly higher than all other groups. The glutathione levels in liver, kidney, striated muscle, and testes of group 2 were higher than in the other groups (p<0.01) and higher in kidney and striated muscle of group 3 than in groups 1 and 4 (p<0.01). The tissue malondialdehyde levels of striated muscle, liver, kidney, and testes of group 1 were significantly higher than for all other groups (p<0.01). Our findings suggest that both swimming exercise and zinc deficiency result in an increase of lipid peroxidation in tissues and that zinc supplementation prevents these alterations by the activation of the antioxidant system.     

Comparison of five methods for determination of total plasma protein concentration

Quantitation of exact total protein content is often a key step and is common to many applications in general biochemistry research and routine clinical laboratory practice. Before embarking on any type of protein analysis, particularly comparative techniques, it is important to accurately quantitate the amount of protein in the sample. In order to assess the quality of total protein estimation results, five methods were tested and were applied to the same pooled plasma sample. For this aim, Bradford (Coomassie Brilliant Blue), Lowry (Folin-Ciocalteau), Biüret, Pesce and Strande (Ponceau-S/TCA), and modified method of Schaffner-Weismann (Amido Black 10B) were used. The last two methods employ simultaneous precipitation of proteins with the acid containing dye solutions followed by dissolution of precipitate in a NaOH solution. It is shown that each assay has advantages and disadvantages relative to sensitivity, ease of performance, acceptance in literature, accuracy and reproducibility/coefficient of variation. All of the methods tested show a CV % < 6. Besides pooled plasma, a known concentration of human serum albumin was also analyzed and discussed by means of standardization of plasma total protein content.     

May primary empty sella turcica be a cause of isolated ACTH deficiency? A case report and the review of related literature

Isolated ACTH deficiency is an uncommon cause of secondary adrenocortical insufficiency and accompaniment with primary empty sella has been reported in several cases. We present a case of isolated ACTH deficiency associated with empty sella. A sixty-two year old woman was admitted to our endocrine clinic with complaints of weakness, fatigue, weight loss, nausea, vomiting, and lack of appetite for about one month. Physical examination indicated orthostatic hypotension and epigastric tenderness. Laboratory investigations revealed hypoglycemia, hyponatremia and anemia, in addition low plasma cortisole and ACTH levels. Serum cortisole responses to short and prolonged ACTH stimulation were tested and partial and accurate responses were obtained, respectively. Plasma ACTH and serum cortisole levels failed to respond after intravenous injection of human corticotropin releasing hormone. Other hypophysial hormone levels were within the normal reference ranges. Although cranial and abdominal computerized tomography images were evaluated as normal, cranial magnetic resonance imaging of the pituitary gland revealed 'primary empty sella turcica'. Replacement therapy with methylprednisolon resulted in the improvement of hypoglycemia, hyponatremia and clinical symptoms. Based on these results, the patient was diagnosed as isolated ACTH deficiency and was scheduled for follow up by our outpatient clinic. Our report is consistent with other reports pointing out that primary empty sella may be responsible for pathogenesis of isolated ACTH deficiency.     

Salicylic acid induced changes on some physiological parameters symptomatic for oxidative stress and mineral nutrition in maize (Zea mays L.) grown under salinity

It has been proposed that salicylic acid (SA) acts as an endogenous signal molecule responsible for inducing abiotic stress tolerance in plants. The effect of varying salicylic acid (SA) supply (0, 0.1, 0.5 and 1.0mM) on growth, mineral uptake, membrane permeability, lipid peroxidation, H(2)O(2) concentration, UV-absorbing substances, chlorophyll and carotenoid concentrations of NaCl (40 mM) stressed maize (Zea mays L.) was investigated. Exogenously applied SA increased plant growth significantly both in saline and non-saline conditions. As a consequence of salinity stress, lipid peroxidation, measured in terms of malondialdehyde (MDA) content and membrane permeability was decreased by SA. UV-absorbing substances (UVAS) and H(2)O(2) concentration were increased by increasing levels of SA. SA also strongly inhibited Na(+) and Cl(-) accumulation, but stimulated N, Mg, Fe, Mn and Cu concentrations of salt stressed maize plants. These results suggest that SA could be used as a potential growth regulator to improve plant salinity stress resistance.