Seasonal effects on the endocrine pattern of semi-captive female Asian elephants (Elephas maximus): timing of the anovulatory luteinizing hormone surge determines the length of the estrous cycle.

Better breeding strategies for captive Asian elephants in range countries are needed to increase populations; this requires a thorough understanding of their reproductive physiology and factors affecting ovarian activity. Weekly blood samples were collected for 3.9 years from 22 semi-captive female Asian elephants in Thai elephant camps to characterize LH and progestin patterns throughout the estrous cycle. The duration of the estrous cycle was 14.6+/-0.2 weeks (mean+/-S.E.M.; n=71), with follicular and luteal phases of 6.1+/-0.2 and 8.5+/-0.2 weeks, respectively. Season had no significant effect on the overall length of the estrous cycle. However, follicular and luteal phase lengths varied among seasons and were negatively correlated (r=-0.658; P<0.01). During the follicular phase, the interval between the decrease in progestin concentrations to baseline and the anovulatory LH (anLH) surge varied in duration (average 25.9+/-2.0 days, range 7-41, n=23), and was longer in the rainy season (33.4+/-1.8 days, n=10) than in both the winter (22.2+/-4.5 days, n=5; P<0.05) and summer (18.9+/-2.6 days, n=8; P<0.05). By contrast, the interval between the anLH and ovulatory LH (ovLH) surge was more consistent (19.0+/-0.1 days, range 18-20, n=14). Thus, seasonal variation in estrous cycle characteristics were mediated by endocrine events during the early follicular phase, specifically related to timing of the anLH surge. Overall reproductive hormone patterns in Thai camp elephants were not markedly different from those in western zoos. However, this study was the first to more closely examine how timing of the LH surges impacted estrous cycle length in Asian elephants. These findings, and the ability to monitor reproductive hormones in range countries (and potentially in the field), should improve breeding management of captive and semi-wild elephants.

A phosphate transporter expressed in arbuscule-containing cells in potato.

Arbuscular mycorrhizas are the most common non-pathogenic symbioses in the roots of plants. It is generally assumed that this symbiosis facilitated the colonization of land by plants. In arbuscular mycorrhizas, fungal hyphae often extend between the root cells and tuft-like branched structures (arbuscules) form within the cell lumina that act as the functional interface for nutrient exchange. In the mutualistic arbuscular-mycorrhizal symbiosis the host plant derives mainly phosphorus from the fungus, which in turn benefits from plant-based glucose. The molecular basis of the establishment and functioning of the arbuscular-mycorrhizal symbiosis is largely not understood. Here we identify the phosphate transporter gene StPT3 in potato (Solanum tuberosum). Functionality of the encoded protein was confirmed by yeast complementation. RNA localization and reporter gene expression indicated expression of StPT3 in root sectors where mycorrhizal structures are formed. A sequence motif in the StPT3 promoter is similar to transposon-like elements, suggesting that the mutualistic symbiosis evolved by genetic rearrangements in the StPT3 promoter.

Pht2;1 encodes a low-affinity phosphate transporter from Arabidopsis.

An Arabidopsis genomic sequence was recently shown to share similarity with bacterial and eukaryotic phosphate (Pi) transporters. We have cloned the corresponding cDNA, which we named Pht2;1, and subsequently performed gene expression studies and functional analysis of the protein product. The cDNA encodes a 61-kD protein with a putative topology of 12 transmembrane (TM) domains interrupted by a large hydrophilic loop between TM8 and TM9. Two boxes of eight and nine amino acids, located in the N- and C-terminal domains, respectively, are highly conserved among species across all kingdoms (eubacteria, archea, fungi, plants, and animals). The Pht2;1 gene is predominantly expressed in green tissue, the amount of transcript staying constant in leaves irrespective of the Pi status of the shoot; in roots, however, there is a marginal increase in mRNA amounts in response to Pi deprivation. Although the protein is highly similar to eukaryotic sodium-dependent Pi transporters, functional analysis of the Pht2;1 protein in mutant yeast cells indicates that it is a proton/Pi symporter dependent on the electrochemical gradient across the plasma membrane. Its fairly high apparent K(m) for Pi (0.4 mM) and high mRNA content in the shoot, especially in leaves, suggest a role for shoot organs in Pi loading. Pht2;1 thus differs from members of the recently described plant Pi transporter family in primary structure, affinity for Pi, and presumed function.

Functional analysis and cell-specific expression of a phosphate transporter from tomato.

For a better understanding of the molecular and biochemical processes involved in orthophosphate (Pi) uptake at the root/soil interface, we cloned a Pi-transporter c DNA (LePT1) from a root air-specific cDNA library of tomato (Lycopersicon esculentum Mill.). The corresponding protein belongs to the growing family of ion transporters with twelve putative transmembrane domains. It is highly homologous to recently isolated Pi transporters from higher plants, yeast and fungi. When expressed in a Pi-uptake-deficient yeast mutant, the L. esculentum phosphate transporter 1 (LePT1) protein exhibits an apparent Km of 31 MicroM. The transporter is still active at submicromolar Pi concentrations and mediates highest Pi uptake at pH 5. The activity of LePT1 is dependent on the electrochemical membrane potential mediated by the yeast P-type H + - ATPase. Transcript levels of LePT1 in tomato seedlings are detectable in all vegetative organs under Pi-sufficient conditions, with highest concentrations in root hairs. In situ hybridization studies demonstrate cell-specific expression of LePT1 in the tomato root. The LePT1 mRNA is detectable in peripheral cell layers such as rhizodermal and root cap cells. Under Pi-deprivation condition, mRNA levels are also detectable in young stelar tissue. This work presents molecular and biochemical evidence for distinct root cells playing an important role in Pi acquisition at the root/soil interface.

Tetramerization of the AKT1 plant potassium channel involves its C-terminal cytoplasmic domain.

All plant channels identified so far show high conservation throughout the polypeptide sequence except in the ankyrin domain which is present only in those closely related to AKT1. In this study, the architecture of the AKT1 protein has been investigated. AKT1 polypeptides expressed in the baculovirus/Sf9 cells system were found to assemble into tetramers as observed with animal Shaker-like potassium channel subunits. The AKT1 C-terminal intracytoplasmic region (downstream from the transmembrane domain) alone formed tetrameric structures when expressed in Sf9 cells, revealing a tetramerization process different from that of Shaker channels. Tests of subfragments from this sequence in the two-hybrid system detected two kinds of interaction. The first, involving two identical segments (amino acids 371-516), would form a contact between subunits, probably via their putative cyclic nucleotide-binding domains. The second interaction was found between the last 81 amino acids of the protein and a region lying between the channel hydrophobic core and the putative cyclic nucleotide-binding domain. As the interacting regions are highly conserved in all known plant potassium channels, the structural organization of AKT1 is likely to extend to these channels. The significance of this model with respect to animal cyclic nucleotide-gated channels is also discussed.

Cystic fibrosis transmembrane conductance regulator mutations that disrupt nucleotide binding.

Increasing evidence suggests heterogeneity in the molecular pathogenesis of cystic fibrosis (CF). Mutations such as deletion of phenylalanine at position 508 (delta F508) within the cystic fibrosis transmembrane conductance regulator (CFTR), for example, appear to cause disease by abrogating normal biosynthetic processing, a mechanism which results in retention and degradation of the mutant protein within the endoplasmic reticulum. Other mutations, such as the relatively common glycine-->aspartic acid replacement at CFTR position 551 (G551D) appear to be normally processed, and therefore must cause disease through some other mechanism. Because delta F508 and G551D both occur within a predicted nucleotide binding domain (NBD) of the CFTR, we tested the influence of these mutations on nucleotide binding by the protein. We found that G551D and the corresponding mutation in the CFTR second nucleotide binding domain, G1349D, led to decreased nucleotide binding by CFTR NBDs, while the delta F508 mutation did not alter nucleotide binding. These results implicate defective ATP binding as contributing to the pathogenic mechanism of a relatively common mutation leading to CF, and suggest that structural integrity of a highly conserved region present in over 30 prokaryotic and eukaryotic nucleotide binding domains may be critical for normal nucleotide binding.

Affinity purification of insoluble recombinant fusion proteins containing glutathione-S-transferase.

Prokaryotic expression of polypeptides as fusion proteins with glutathione-S-transferase has recently been reported as a one-step means of purifying recombinant protein. The usefulness of the glutathione-S-transferase/glutathione-agarose system, however, is significantly limited by the frequent synthesis of recombinant proteins in insoluble form by Escherichia coli. We have found that for 5 separate fusion proteins containing glutathione-S-transferase and different domains of the large cystic fibrosis transmembrane conductance regulator, all were packaged in insoluble form by E. coli. Insolubility of these products made them inaccessible to one-step purification utilizing this scheme requires proper folding of recombinant glutathione-S-transferase to allow recognition on glutathione affinity agarose, we investigated the suitability of several alternative approaches for converting insoluble recombinant fusion proteins to a soluble form amenable to glutathione-agarose affinity purification. Low-temperature induction of fusion protein synthesis, but not incubation with anion-exchange resins, led to improved one-step purification of glutathione-S-transferase fusion proteins from E. coli cell lysate using mild, nondenaturing conditions. Solubilization in 8 mol/L urea, but not with other chaotropic agents or detergents, also allowed preparative yields of affinity-purified fusion protein. These techniques increase the usefulness of this recombinant protein purification scheme, and should be broadly applicable to diverse polypeptides synthesized as fusions with glutathione-S-transferase.