Characterization of tryptophan high affinity transport system in pinealocytes of the rat. Day-night modulation

Summary.  Tryptophan is required in the pineal gland for the formation of serotonin, precursor of melatonin biosynthesis. The level of this amino acid in the serum and in the pineal gland of the rat undergoes a circadian rhythm, and reduced plasma tryptophan concentration decreases secretion of melatonin in humans. Tryptophan is transported into the cells by the long chain neutral amine acid system T and by the aromatic amino acid system T. The high affinity component of [³H]tryptophan uptake was studied in pinealocytes of the rat. Inhibition was observed in the presence of phenylalanine or tyrosine, but not in the presence of neutral amino acids, alanine, glycine, serine, lysine or by 2-aminobicyclo[2,2,1]-heptane-2-carboxylic acid, a substrate specific for system L. The transport of tryptophan was temperature-dependent and trans-stimulated by phenylalanine and tyrosine, but was energy-, sodium-, chloride-, and pH-independent. In addition, the sulphydryl agent N-ethylmaleimide did not modify the high affinity transport of tryptophan in pinealocytes. The kinetic parameters were not significantly different at 12:00 as compared to 24:00 h. The treatment with the inhibitor of tryptophan hydroxylase, p-chlorophenylalanine, produced an increase in the maximal velocity of the uptake and a reduction in the affinity at 12:00, but not at 24:00 h, probably indicating that during the day, the formation of serotonin in the pineal gland is favoured by elevating the uptake of tryptophan, whereas at 24:00 h other mechanisms, such as induction of enzymes are taking place. High affinity tryptophan uptake in the rat pineal gland occurs through system T and is upregulated during the day when the availability of serotonin is reduced. Received March 15, 2001 Accepted July 8, 2002 Published online January 20, 2003 Acknowledgements This work was supported by the Grant S1-3490 from Consejo Nacional de Investigaciones Cientificas y Tecnológicas (CONICIT), Venezuela. We appreciate the secretarial assistance of Mrs. Isabel Otaegui. Carmen I. Gutiérrez is a PhD Student from Ciencias Fisiológicas, Facultad de Medicina, Universidad Central de Venezueta (UCV), Caracas, and supported by Universidad Francisco de Miranda, Coro, Falcón, Venezuela. Joseph Glykys is a Medical Student from Universidad de Carabobo, Valencia, Venezuela, and an Assistant Student of Centro de Estudios Avanzados, IVIC. Authors' address: Dr. Lucimey Lima, Laboratorio de Neuroquímica, Centro de Biofísica y Bioquímica, Instituto Venezolano de Investigaciones Científicas, Apdo. 21827, Caracas 1020-A, Venezuela, Fax: 58-212-504-1295, E-mail: llima@cbb.ivic.ve     

Two African swine fever virus proteins derived from a common precursor exhibit different nucleocytoplasmic transport activities

African swine fever virus (ASFV), a large icosahedral deoxyvirus, is the causative agent of an economically relevant hemorrhagic disease that affects domestic pigs. The major purpose of the present study was to investigate the nuclear transport activities of the ASFV p37 and p14 proteins, which result from the proteolytic processing of a common precursor. Experiments were performed by using yeast-based nucleocytoplasmic transport assays and by analysis of the subcellular localization of different green fluorescent and Myc fusion proteins in mammalian cells. The results obtained both in yeast and mammalian cells clearly demonstrated that ASFV p14 protein is imported into the nucleus but not exported to the cytoplasm. The ability of p37 protein to be exported from the nucleus to the cytoplasm of both yeast and mammalian cells was also demonstrated, and the results clearly indicate that p37 nuclear export is dependent on the interaction of the protein with the CRM-1 receptor. In addition, p37 was shown to exhibit nuclear import activity in mammalian cells. The p37 protein nuclear import and export abilities described here constitute the first report of a nucleocytoplasmic shuttling protein encoded by the ASFV genome. Overall, the overlapping results obtained for green fluorescent protein fusions and Myc-tagged proteins undoubtedly demonstrate that ASFV p37 and p14 proteins exhibit nucleocytoplasmic transport activities. These findings are significant for understanding the role these proteins play in the replication cycle of ASFV.     

Cultural transmission of ethnobotanical knowledge in a rural community of northwestern Patagonia, Argentina

In the present study we analyzed medicinal and edible plant utilization in Cuyin Manzano, a small rural population located near the Andean forests of Argentina. We also studied where and when plant knowledge was learned, who the principal transmitters were, and how people were taught. The participants were interviewed individually and at random, by means of a semi-structured questionnaire. Interviews were carried out in 16 families in order to examine the present use of wild plants. The inhabitants of Cuyin Manzano cited 87 plants: 63 medicinal and 24 edible species. They mentioned on average 31 ± 10 species per person. Similar patterns of plant use were found in young and old people alike, irrespective of gender. Learning about useful plants took place at an early age as a result of family tradition. This local knowledge is acquired and taught “by doing,” and is mostly transmitted vertically through family dissemination. Wild plant learning implies the acquisition of plants' physical and functional features as well as their environmental characteristics.     

High-resolution mapping reveals linkage between genes in common bean cultivar Ouro Negro conferring resistance to the rust,anthracnose, and angular leaf spot diseases

Key message

Co-segregation analysis and high-throughput genotyping using SNP, SSR, and KASP markers demonstrated genetic linkage between Ur-14 and Co-3 ⁴ /Phg-3 loci conferring resistance to the rust, anthracnose and angular leaf spot diseases of common bean.

Abstract

Rust, anthracnose, and angular leaf spot are major diseases of common bean in the Americas and Africa. The cultivar Ouro Negro has the Ur-14 gene that confers broad spectrum resistance to rust and the gene cluster Co-3 ⁴ /Phg-3 containing two tightly linked genes conferring resistance to anthracnose and angular leaf spot, respectively. We used co-segregation analysis and high-throughput genotyping of 179 F_2:3 families from the Rudá (susceptible) × Ouro Negro (resistant) cross-phenotyped separately with races of the rust and anthracnose pathogens. The results confirmed that Ur-14 and Co-3 ⁴ /Phg-3 cluster in Ouro Negro conferred resistance to rust and anthracnose, respectively, and that Ur-14 and the Co-3 ⁴ /Phg-3 cluster were closely linked. Genotyping the F_2:3 families, first with 5398 SNPs on the Illumina BeadChip BARCBEAN6K_3 and with 15 SSR, and eight KASP markers, specifically designed for the candidate region containing Ur-14 and Co-3 ⁴ /Phg-3, permitted the creation of a high-resolution genetic linkage map which revealed that Ur-14 was positioned at 2.2 cM from Co-3 ⁴ /Phg-3 on the short arm of chromosome Pv04 of the common bean genome. Five flanking SSR markers were tightly linked at 0.1 and 0.2 cM from Ur-14, and two flanking KASP markers were tightly linked at 0.1 and 0.3 cM from Co-3 ⁴ /Phg-3. Many other SSR, SNP, and KASP markers were also linked to these genes. These markers will be useful for the development of common bean cultivars combining the important Ur-14 and Co-3 ⁴ /Phg-3 genes conferring resistance to three of the most destructive diseases of common bean.

     

Evolved distal tail carbohydrate binding modules of Lactobacillus phage J‐1: a novel type of anti‐receptor widespread among lactic acid bacteria phages

Bacteriophage replication requires specific host‐recognition. Some siphophages harbour a large complex, the baseplate, at the tip of their non‐contractile tail. This baseplate holds receptor binding proteins (RBPs) that can recognize the host cell‐wall polysaccharide (CWPS) and specifically attach the phage to its host. While most phages possess a dedicated RBP, the phage J‐1 that infects Lactobacillus casei seemed to lack one. It has been shown that the phage J‐1 distal tail protein (Dit) plays a role in host recognition and that its sequence comprises two inserted modules compared with ‘classical’ Dits. The first insertion is similar to carbohydrate‐binding modules (CBMs), whereas the second insertion remains undocumented. Here, we determined the structure of the second insertion and found it also similar to several CBMs. Expressed insertion CBM2, but not CBM1, binds to L. casei cells and neutralize phage attachment to the bacterial cell wall and the isolated and purified CWPS of L. casei BL23 prevents CBM2 attachment to the host. Electron microscopy single particle reconstruction of the J‐1 virion baseplate revealed that CBM2 is projected at the periphery of Dit to optimally bind the CWPS receptor. Taken together, these results identify J‐1 evolved Dit as the phage RBP.     

Purification, characterization and antifungal properties of a lipid-transfer protein from sunflower (Helianthus annuus) seeds

An antifungal protein from Helianthus annuus L. seeds (Ha-AP10) has been purified to homogeneity and characterized. Ha-AP10 purification was performed by gel filtration, cation exchange chromatography and reverse phase HPLC. Its molecular mass was estimated to be 10 kDa and western blot analyses suggest that it has an extracellular location. The N-terminal sequence of Ha-AP10 showed strong homology to some plant lipid-transfer proteins (LTPs). Antifungal tests have demonstrated that Ha-AP10 exerts a fungistatic effect. It completely inhibits the germination of spores of the fungal pathogen Fusarium solani f. sp. eumartii at a concentration of 40 μg ml⁻¹ and produces a 50% growth inhibition at 6.5 μg ml⁻¹ (0.65 μ M ). These data place Ha-AP10 among the most potent antifungal LTPs described so far.     

The predatory behavior of wintering <Emphasis Type="Italic">Accipiter</Emphasis> hawks: temporal patterns in activity of predators and prey

Studies focused on how prey trade-off predation and starvation risk are prevalent in behavioral ecology. However, our current understanding of these trade-offs is limited in one key respect: we know little about the behavior of predators. In this study, we provide some of the first detailed information on temporal patterns in the daily hunting behavior of bird-eating Accipiter hawks and relate that to their prey. During the winters of 1999–2004, twenty-one sharp-shinned hawks (A. striatus) and ten Cooper’s hawks (A. cooperii) were intensively radio tracked in rural and urban habitats in western Indiana, USA. Cooper’s hawks left roost before sunrise and usually returned to roost around sunset, while sharp-shinned hawks left roost at sunrise or later and returned to roost well before sunset. An overall measure of Cooper’s-hawk-induced risk (a composite variable of attack rate and activity patterns) generally reflected the timing of prey activity, with peaks occurring around sunrise and sunset. In contrast, risk induced by the smaller sharp-shinned hawk did not strongly reflect the activity of their prey. Specifically, an early morning peak in prey activity did not correspond to a period with intense hawk activity. The lack of early morning hunting by sharp-shinned hawks may reflect the high risk of owl-induced predation experienced by these hawks. The net effect of this intraguild predation may be to “free” small birds from much hawk-induced predation risk prior to sunrise. This realization presents an alternative to energetics as an explanation for the early morning peak in small bird activity during the winter.     

Candidiasis: Predisposing Factors,Prevention, Diagnosis and Alternative Treatment

Candidiasis is the most common opportunistic yeast infection. Candida species and other microorganisms are involved in this complicated fungal infection, but Candida albicans continues to be the most prevalent. In the past two decades, it has been observed an abnormal overgrowth in the gastrointestinal, urinary and respiratory tracts, not only in immunocompromised patients, but also related to nosocomial infections and even in healthy individuals. There is a widely variety of causal factors that contribute to yeast infection which means that candidiasis is a good example of a multifactorial syndrome. Due to rapid increase in the incidence in these infections, this is the subject of numerous studies. Recently, the focus of attention is the treatment and, above all, the prevention of those complications. The diagnosis of candidiasis could become quite complicated. Prevention is the most effective “treatment,” much more than eradication of the yeast with antifungal agents. There are several aspects to consider in the daily routine that can provide a strength protection. However, a therapeutic approach is necessary when the infection is established, and therefore, other alternatives should be explored. This review provides an overview on predisposition factors, prevention and diagnosis of candidiasis, highlighting alternative approaches for candidiasis treatment.     

Effect of vasoactive intestinal peptide, peptide histidine isoleucine and growth hormone-releasing factor-40 on bombesin-like immunoreactivity, somatostatin and gastrin release from the perfused rat stomach

Bombesin-like immunoreactivity (BLI) has been demonstrated in neurons of the gastrointestinal tract and gastric BLI secretion can be demonstrated in response to the classical neurotransmitter acetylcholine. Since structurally related peptides VIP, PHI and GRF have to be considered as peptidergic neurotransmitters it was of interest to determine their effect on gastric BLI secretion. Additionally, somatostatin (SLI) and gastrin secretion was examined. The isolated stomach of overnight fasted rats was perfused with Krebs-Ringer buffer via the celiac artery and the effluent was collected via the portal vein. The gastric lumen was perfused with isotonic saline at pH7 or pH2. All four peptides were tested at a dose of 10(-11) M and 10(-8) M at both pH levels and in addition the effect of VIP and PHI was examined at 10(-14) M and 10(-12) M during luminal pH2. At luminal pH7 VIP and PHI stimulated SLI release at 10(-8) M but had no effect on BLI or gastrin secretion. rGRF and hpGRF were both ineffective on SLI and gastrin release while rGRF inhibited and hpGRF stimulated BLI secretion. This effect was not dose related. At luminal pH2 all four peptides stimulated BLI secretion. Stimulation by PHI was already observed at a dose of 10(-14) M while VIP elicited a stimulatory effect at 10(-12) M. PHI at the two lowest concentrations of 10(-14) and 10(-12) M elicited a stimulation of SLI and gastrin release while the same doses of VIP and the higher doses of all four peptides had no effect on SLI and gastrin secretion at an acidic intraluminal pH.(ABSTRACT TRUNCATED AT 250 WORDS)