Functional and structural characterization of the first prokaryotic member of the L-amino acid transporter (LAT) family: a model for APC transporters

We have identified YkbA from Bacillus subtilis as a novel member of the L-amino acid transporter (LAT) family of amino acid transporters. The protein is approximately 30% identical in amino acid sequence to the light subunits of human heteromeric amino acid transporters. Purified His-tagged YkbA from Escherichia coli membranes reconstituted in proteoliposomes exhibited sodium-independent, obligatory exchange activity for L-serine and L-threonine and also for aromatic amino acids, albeit with less activity. Thus, we propose that YkbA be renamed SteT (Ser/Thr exchanger transporter). Kinetic analysis supports a sequential mechanism of exchange for SteT. Freeze-fracture analysis of purified, functionally active SteT in proteoliposomes, together with blue native polyacrylamide gel electrophoresis and transmission electron microscopy of detergent-solubilized purified SteT, suggest that the transporter exists in a monomeric form. Freeze-fracture analysis showed spherical particles with a diameter of 7.4 nm. Transmission electron microscopy revealed elliptical particles (diameters 6 x 7 nm) with a distinct central depression. To our knowledge, this is the first functional characterization of a prokaryotic member of the LAT family and the first structural data on an APC (amino acids, polyamines, and choline for organocations) transporter. SteT represents an excellent model to study the molecular architecture of the light subunits of heteromeric amino acid transporters and other APC transporters.     

The molecular bases of cystinuria and lysinuric protein intolerance

Cystinuria and lysinuric protein intolerance are inherited aminoacidurias caused by defective amino-acid transport activities linked to a family of heteromeric amino-acid transporters (HATs). HATs comprise two subunits: co-expression of subunits 4F2hc and y(+)LAT-1 induces the efflux of dibasic amino acids from cells, whereas co-expression of subunits rBAT and b(o,+)AT induces the renal reabsorption and intestinal absorption of cystine and dibasic amino acids at the brush border of epithelial cells. Recently, the role of b(o,+)AT (SLC7A9) in cystinuria (non Type I) and the role of y(+)LAT-1 (SLC7A7) in lysinuric protein intolerance have been demonstrated.     

Natal dispersal in great bustards: the effect of sex, local population size and spatial isolation

1. We investigated the causes of natal dispersal in four Spanish areas where 35 breeding groups of the polygynous great bustard Otis tarda were monitored intensively. A total of 392 juveniles were radio-tracked between 1991 and 2006 by ground and via aeroplane to avoid potential biases derived from the non-detection of long-distance dispersers. 2. We explored 10 explanatory variables that were related to individual phenotypic features, habitat and conspecific traits in terms of group size and breeding performance, and spatial distribution of available breeding groups. Probability of group change and natal dispersal distances were investigated separately through multifactorial analyses. 3. Natal dispersal occurred in 47.8% of the birds and median natal dispersal distance of dispersers was 18.1 km (range 4.97-178.42 km). Sex largely determined the dispersal probability, with 75.6% of males being dispersers and 80.0% of females being philopatric, in contrast to the general pattern of female-biased dispersal found in most avian species. 4. Both the frequency of natal dispersal and dispersal distances were affected by the spatial distribution of breeding groups. More isolated groups showed a higher proportion of philopatric individuals, the effect being more evident in males than in females. This implies a reduction in gene flow in fragmented populations, as most genetic exchange is achieved through male dispersal. Additionally, dispersers hatched in more isolated groups tended to exhibit longer dispersal distances, which increases the associated energetic costs and mortality risks. 5. The dispersal decision was influenced by the number of conspecifics in the natal group. The individual probability of natal dispersal was related inversely to the size of the natal group, which supports the balanced dispersal model and the conspecific attraction hypothesis. 6. Overall, our results provide a good example of phenotypic plasticity and reinforce the current view that dispersal is an evolutionary complex trait conditioned by the interaction of individual, social and environmental causes that vary between individuals and populations.     

Effect of exogenous melatonin on the ovary, the embryo and the establishment of pregnancy in sheep

Administration of melatonin to advance the breeding season in sheep has been widely used, since this hormone conveys the photoperiodic signal to the reproductive neuroendocrine axis. An increased lambing percentage has been reported following such treatment during anoestrus, which could be mediated through a higher rate of embryonic survival, either by an improvement in luteal function or by a reduction in the antiluteolytic mechanisms. The aim of this article is to review the body of knowledge on the effect of melatonin on the ovine ovary, the embryo and the establishment of pregnancy. Some studies using synchronized ewes have found that melatonin treatments during anoestrus do not improve ovulation rate by modifying the timing of follicle emergence, but increasing the number of ovulatory follicles by decreasing the atresia of medium and large follicles. On the other hand, the addition of melatonin to the in vitro maturation medium does not improve oocyte maturation rate in oocytes from sheep ovaries recovered either in anoestrus or in the breeding season. However, a luteotrophic effect of melatonin at either short or medium term has been reported. We have recently observed that melatonin implants tend to improve the survival of embryos collected from ewes after superovulation in anoestrus. More specifically, melatonin induced a significant reduction of the number and rate of non-viable (degenerate and retarded) embryos. Preliminary data from our laboratory suggest that the uterine sensitivity to progesterone - in terms of progesterone receptor expression - of superovulated ewes could be reduced by melatonin treatment. It can be concluded that the success of exogenous melatonin as a means to improve lamb production of sheep is due, at least in part, to an improvement of luteal support and embryonic survival.     

Inhibitors such as staurosporine,H-7 or polymyxin B cannot be used in skeletal muscle to prove the role of protein kinase C on insulin action

The precise role of protein kinase C in insulin action in skeletal muscle is not well defined. Based on the fact that inhibitors of protein kinase C block some insulin effects, it has been concluded that some of the biological actions of insulin are mediated via protein kinase C. In this study, we present evidence that inhibitors of protein kinase C such as staurosporine, H-7 or polymyxin B cannot be used to ascertain the role of protein kinase C in skeletal muscle. This is based on the following experimental evidences: a) staurosporine, H-7 and polymyxin B markedly block in muscle the effect of insulin on System A transport activity; however, this effect of insulin is not mimicked in muscle by TPA-induced stimulation of protein kinase C, b) H-7 and polymyxin B block insulin action on System A transport activity in an additive manner to the inhibitory effect of phorbol esters, c) staurosporine, H-7 and polymyxin B block the effect of insulin on lactate production, a process that is activated by insulin and TPA in an additive fashion, and d) staurosporine completely blocks the tyrosine kinase activity of insulin receptors partially purified from rat skeletal muscle.Abbreviations MeAIB a-(methyl)aminoisobutyric acid - TPA 12-O-tetradecanoylphorbol-13-acetate - H-7 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine     

Poly(A)+ RNA from rabbit intestinal mucosa induces b0,+ and y+ amino acid transport activities in Xenopus laevis oocytes.

Injection of poly(A)+ RNA (mRNA) isolated from rabbit intestinal mucosa into Xenopus laevis oocytes results in an increase in sodium-independent uptake of L-[3H]leucine, L-[35S]cystine, and L-[3H]arginine. This uptake activity is related to an mRNA species corresponding to the recently isolated rabbit kidney cortex cDNA clone rBAT (related to b0,+ amino acid transporter; Bertran, J., Werner, A., Stange, G., Markovich, D., Moore, M. L., Biber, J., Testar, X., Zorzano, A., Palacin, M., and Murer, H. (1992) Proc. Natl. Acad. Sci. U.S.A. 281, 717-723) and to a protein involved in amino acid transport via system y+. This conclusion is based on the following observations: 1) mRNA isolated from mucosa of duodenum, jejunum, and ileum, but not from colon, induces sodium-independent uptake of L-leucine, L-cystine, and L-arginine. 2) In Northern blot analysis, mRNA isolated from mucosa of duodenum, jejunum, and ileum, but not from colon, hybridizes to an rBAT cDNA probe, with signals of 2.2-2.3 kilobases and 3.7-3.9 kilobases. 3) mRNA isolated from mucosa of jejunum induces sodium-independent uptake of L-leucine and L-cysteine which shows an inhibition pattern corresponding to system b0,+; the inhibition pattern of mRNA-induced uptake of L-arginine is compatible with the contribution of system b0,+ and y+. 4) Hybrid depletion with an rBAT antisense oligonucleotide greatly prevents the mRNA-dependent induction of uptake of L-cystine (greater than 90%) and of L-leucine (approximately 75%); it reduces to about 50% the induction of L-arginine uptake. 5) After separation of mRNA on a sucrose density gradient, the fractions resulting in expression of b0,+ transport activity were also those hybridizing with rBAT cDNA; induction of transport activity from these fractions was also sensitive to hybrid depletion. 6) The mRNA-induced component of L-arginine uptake which is resistant to rBAT hybrid depletion is inhibited by L-homoserine, only in the presence of sodium; thus, it is related to a system y(+)-like activity.     

Studies on the function of TM20, a transmembrane protein present in cereal embryos

The possible function of the maize transmembrane protein TM20 in hormone transport has been investigated using immunological methods and by microinjection of TM20 cRNA in Xenopus oocytes. The existence of a similar gene in rice indicates that the overall structure of the deduced protein is conserved between these two cereals. An antibody raised against a conserved motif, in a loop between two transmembrane domains, locates the protein (TM20) in differentiating provascular cells in maize embryo. The protein has a polarized distribution within the cell in the most differentiated stages of development. Xenopus laevis oocytes microinjected with TM20 appear to modify transport activities across the plasma membrane. These results are discussed in relation to other transport proteins that influence plant development.     

Alarming decline and range reduction of the highly threatened Great Bustard Otis tarda in Morocco

A Great Bustard Otis tarda survey carried out in spring 2015 in Morocco confirmed the decline of this highly endangered population. Bustards were only seen at two of the seven leks occupied ten years ago. The total number of birds counted was 40-44, which represents a 40% decline over the last decade. The sex-ratio was still strongly female-biased (1 male: 3 females), but less than in previous surveys, which suggests that trophy hunting has not been the major mortality cause in recent times. The productivity was 0.29-0.33 juveniles per female, the highest ever recorded in this population, suggesting that breeding success doesn’t represent the main problem for the survival of this population. Based on the recent development of the power line network at some areas, the main threat today is probably collision with power lines. Reducing this mortality cause should be considered a high conservation priority.