Role Of Active Transport Research Articles

Neighbourhood walkability and body mass index in children: Evidence from the Millennium Cohort Study in Wales

BackgroundOverweight and obesity in children continues to increase. Yet, the role of active transport, namely walking, in mitigating these trends remains unclear. This study examined the cross-sectional association between walkability and children's Body Mass Index (BMI) and how this varies by socio-economic and lifestyle characteristics. MethodsWe analysed BMI for 14-year-old children living in Wales from the UK Millennium Cohort Study. Children were categorised as healthy weight, overweight or obese using the British 1990 cut-off points. Walkability was assessed using the Wales Active Living Environments (Wal-ALE) database, categorised as 1 (least walkable environments) to 5 (most walkable environments). We applied multinomial logistic regression analysis and adjusted for ethnicity, having a limiting longstanding illness, parental BMI, socio-economic circumstances, and lifestyle characteristics. ResultsChildren were more likely to be obese if they lived in areas classed as more walkable in unadjusted analysis [Relative Risk Ratio (RRR) = 1.72 (95%CI = 1.15–2.58)] and following adjustment for ethnicity, limiting longstanding illness and parental BMI [RRR = 1.83 (95%CI = 1.12–3.00)]). Significant associations remained even after further adjustment for lifestyle characteristics and socio-economic circumstances [RRR = 1.76 (95%CI = 1.05–2.96)]. Further, children were more likely to be obese if their parents were overweight or obese and if they were living in poverty. Children were less likely to be obese if they spent 3 or more days per week in moderate-to-vigorous physical activity (MVPA) and if they ate breakfast every day vs. some days or never. ConclusionFindings demonstrate that walkable environments are not associated with lower rates of obesity in children, indicating that the relationship between the built environment and child health is complex and requires further study.

What is significant for engagement in cycling and walking in South Korea? Applying value-belief-norm theory

The value-belief-norm (VBN) theory has been widely used to describe pro-sustainability behavior. Despite being used to examine issues of sustainable and public transport behavior, research using the VBN has largely overlooked the pro-environmental behavior of cyclists and walkers, which is extremely significant given the role of active transport in environmental and human health. Therefore, this study creates and verifies a conceptually comprehensive active transport (cycling/walking) research model employing VBN theory, with a moderating role of cycling/walking and comparing cyclists and walkers, and utilizing PLS-SEM, multi-group analysis (MGA), necessary condition analysis (NCA), and fuzzy set Qualitative Comparative Analysis (fsQCA). Results revealed that value has a great effect on belief in walking/biking. However, belief has a stronger impact on personal norm than social norm, and also personal norm shows twice the impact on behavioral intention than social norm. Behavior to engage in cycling and walking is greatly influenced by behavioral intention. Walkers have a stronger relationship between belief and personal norm than cyclists. Cyclists have higher prediction power with social norm, while walkers have a higher prediction power with belief, personal norm, behavioral intention, and behavior. Importantly for sustainable transport planning and interventions, the cycling and walking groups distinctively different from each other in terms of solutions for outcome high level of active transport behavior.

Role of active transport of potassium to leaves in the mechanisms of tolerance to salinity in common bean (Phaseolus vulgaris L.)

Biochemical markers are of great utility in screening for salt tolerance of crops. In common beans (Phaseolus vulgaris), lower levels of proline under stress have been associated with a better stress resistance of cultivars. In the present study, the responses to salinity have been analysed in six cultivars of common beans: four local landraces from Spain and two experimental lines from Cuba. Proline was used for ranking the relative tolerance of the cultivars, confirming a previous study which reported as more stress-tolerant two of the Spanish landraces. Total soluble sugars concentrations varied with treatments and between genotypes, but it was difficult to assess their role in stress tolerance of the analysed plants. Sodium concentration in leaves was the lowest in one of the two salt-resistant cultivars, and potassium did not vary or even increased under salt stress in all of them, except for the most susceptible one, where a drop of this cation was registered under 150 mM NaCl. Changes in malondialdehyde (MDA) contents did not indicate salt-induced membrane peroxidation resulting from secondary oxidative stress; consequently, accumulation of total phenolic compounds and flavonoids, as an antioxidant defence mechanism, was not detected. These results highlight the reliability of using proline as a biochemical marker of salt stress in common beans and the importance of the mechanism related to potassium transport to leaves in conferring stress tolerance to some common bean cultivars.

Workshop 4 report: Developing inter-modal transport systems

Workshop 4 explored the development of inter-modal transport systems in developed and developing economies worldwide, focusing on their characteristics, approach, design, performance, relative merits, challenges faced and their evaluation. The workshop examined the social, political, institutional, regulatory, and operational challenges in providing inter-modal transport. The workshop was based on thematic areas covering the key questions of: What is meant by inter-modal transport systems? What are the challenges to creating an inter-modal transport system? What is the role of active transport? How do we address the challenges and what are the recommendations for discussion at Thredbo 15?

A Putative Bacterial ABC Transporter Circumvents the Essentiality of Signal Peptidase.

ABSTRACTThe type I signal peptidase of Staphylococcus aureus, SpsB, is an attractive antibacterial target because it is essential for viability and extracellularly accessible. We synthesized compound 103, a novel arylomycin-derived inhibitor of SpsB with significant potency against various clinical S. aureus strains (MIC of ~1 µg/ml). The predominant clinical strain USA300 developed spontaneous resistance to compound 103 with high frequency, resulting from single point mutations inside or immediately upstream of cro/cI, a homolog of the lambda phage transcriptional repressor cro. These cro/cI mutations led to marked (>50-fold) overexpression of three genes encoding a putative ABC transporter. Overexpression of this ABC transporter was both necessary and sufficient for resistance and, notably, circumvented the essentiality of SpsB during in vitro culture. Mutation of its predicted ATPase gene abolished resistance, suggesting a possible role for active transport; in these bacteria, resistance to compound 103 occurred with low frequency and through mutations in spsB. Bacteria overexpressing the ABC transporter and lacking SpsB were capable of secreting a subset of proteins that are normally cleaved by SpsB and instead were cleaved at a site distinct from the canonical signal peptide. These bacteria secreted reduced levels of virulence-associated proteins and were unable to establish infection in mice. This study reveals the mechanism of resistance to a novel arylomycin derivative and demonstrates that the nominal essentiality of the S. aureus signal peptidase can be circumvented by the upregulation of a putative ABC transporter in vitro but not in vivo.

Is unphosphorylated Rex, as multifunctional protein of HTLV-1, a fully intrinsically disordered protein? An in silico study

Intracellularlocation of a viral unspliced mRNA in host cell is a crucial factor for normal life of the virus. Rex is a neucleo-cytoplasmic shuffling protein of Human T-cell Leukemia Virus-1(HTLV-1)which has important role in active transport of cargo-containing RNA from nucleus to cytoplasm. Therefore, it plays a crucial role in the disease development by the virus. In spite of its importance, the 3d-structurephosphorylated and unphosphorylated of this protein has not been determined. In this study, first we predicted whether Rex protein is an ordered or disordered protein. In second step protein 3Dstructure of Rex was obtained. The content of disorder-promoting amino acids, flexibility, hydrophobicity, short linear motifs (SLiMs) and protein binding regions and probability of Rex crystallization were calculated by various In Silico methods. The3D models of Rex protein were obtained by various In Silico methods, such as homology modeling, threading and ab initio, including; I-TASSER, LOMETS, SPARSKS, ROBBETA and QUARK servers. By comparing and analyzing Qmean, z-scores and energy levels of selected models, the best structures with highest favored region in Ramachandran plot (higher than 90%) was refined with MODREFINER software. In silico analysis of Rex physicochemical properties and also predicted SLiMs and binding regions sites confirms that unphosphorylated Rex protein in HTLV-1 as Rev protin in HIV is wholly disordered protein belongs to the class of intrinsically disordered proteins with extended disorder (native coils, native pre-molten globules).

Abstract 782: Transmembrane 2-3 loop domain of the proton coupled folate transporter (PCFT) forms a novel “reentrant loop” structure and is functionally important

Abstract The proton coupled folate transporter (PCFT) is a proton-folate symporter with a twelve transmembrane domain (TMD) structure. Human PCFT (hPCFT) is highly expressed in many solid tumors and has an acidic pH optimum, suggesting its high activity in the acidic tumor microenvironment. Our recent studies have focused on exploiting PCFT for targeting solid tumors with novel cytotoxic antifolates. Predicted topology models suggest that the loop domain between TMDs 2 and 3 resides in the cytosol. However, mutations in TMD2-3 loop residues Asp109 or Arg113 result in loss of PCFT activity, suggesting an important role in transport activity. Additionally, TMD2 may itself form a part of the substrate binding domain, suggesting functional importance for TMD2 residues. Using site-directed mutagenesis, we replaced each of the seven cysteine (cys) residues of hPCFT with serine, creating functional “cys-less” PCFT for use in substituted cys accessibility methods (SCAM). We then used site-directed mutagenesis to create 34 single cys mutants spanning TMD2 and the TMD 2-3 loop region (positions 85-118) in a cys-less PCFT background. These mutants were transfected into R1-11 HeLa cells, which express no endogenous folate transporters. We measured [3H]methotrexate uptake at pH 5.5 as a readout of PCFT activity and confirmed PCFT protein expression by western blotting. All 34 mutants were detected on westerns and 29 mutants were active, while W107C, D109C, G112C, R113C, and R114C were inactive. Using functionally active mutants, we performed pull-down assays with 2-aminoethyl methanethiosulfonate-biotin (MTSEA-biotin) and steptavidin beads, in order to determine the aqueous accessibility of each mutant. We found that mutants G93C, F94W, G105C, A106C, S110C, and L116C reacted with MTSEA-biotin, suggesting that these residues are aqueous-accessible. For G93C, F94C, and L116C, MTSEA-biotin labeling was inhibited by pemetrexed pretreatment, and MTSEA-biotin treatment inhibited transport activity of G93C and F94C. Our results suggest that the TMD 2-3 loop domain is aqueous accessible and must form a novel functionally important “reentrant loop” structure. Additionally, residues located in TMD2 (Gly93 and Phe94) appear to play a key role in substrate binding to PCFT. Better understanding of functional and structural determinants of PCFT membrane transport will be critical to the development of novel PCFT-selective antifolate drugs for targeting solid tumors. Citation Format: Michael R. Wilson, Zhanjun Hou, Larry H. Matherly. Transmembrane 2-3 loop domain of the proton coupled folate transporter (PCFT) forms a novel “reentrant loop” structure and is functionally important. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 782. doi:10.1158/1538-7445.AM2014-782

Carbon partitioning to berries in water stressed grapevines: The role of active transport in leaves and fruits

Although imposed water stress is a common agricultural practice worldwide, the physiological and molecular responses of grapevine leaves and fruits, and their interactions, in relation to carbon partitioning remain unknown. We have assessed, in field grown grapevines, the effect of four deficit irrigation regimes, from veraison through to the end of the season, on daily and seasonal non-structural carbon stocks and assimilation in leaves and sugar content in berries, along with the transcript profile for sugar transport proteins in leaves and berries. Average midday xylem water potentials along the season ranged from mild to severe water stress, i.e., −0.7MPa to −1.05MPa, respectively. In all the treatments, berries reached equal sugar concentration 20–35 days after veraison because of a proportional effect on berry volume and sugar content per berry. In berries, mild water stress accelerated the sugar accumulation increasing the abundance of VvSUC27, VvHT3 and VvHT5, only strictly around veraison. Transcripts abundance in berries did not match sugar uptake rate since, VvSUC11, VvSUC12, VvHT5, as well as the cell wall invertase VvCWI, kept rising after berries were filled. In leaves, when berries reached maximal sugar content, export was transiently reduced resulting in starch accumulation. Water stress increased the gene expression for sucrose transporters known to code for mesophyll cell proteins in leaves, without affecting the transcript abundance for the phloem loading protein. The latter suggests that mild water stress triggers active sugar transport in the source tissues as a means for supporting the sugar accumulation in berries under depressed carbon assimilation by leaves.

A novel sodium–glucose co-transporter 2 gene (SGLT2) mutation contributes to the abnormal expression of SGLT2 in renal tissues in familial renal glucosuria

Familial renal glucosuria (FRG) is characterized by persistent glucosuria in the presence of normal serum glucose concentrations and the absence of other impairments of tubular function [1]. Mutations in the sodium–glucose cotransporter 2 gene (SGLT2, OMIM: 182381) have recently been confirmed as responsible for the vast majority of FRG cases [2–4]. Because not all individuals with SGLT2 heterozygous mutations show increased glucose excretion, the inheritance pattern of FRG has been explained as a codominant trait with variable penetrance [2, 5]. However, no direct evidence for SGLT2 heterozygous mutations in renal tissues has yet been obtained. In the current study, a 50-year-old male patient was referred to the renal division of our hospital because of repeated glucosuria. A review of his systems and a physical examination were completely normal. The result of a quantitative urine glucose test was 4.8 g/24 h. Urine and blood biochemistry were otherwise unremarkable. Routine analysis of renal specimens using indirect immunofluorescence, and light and electron microscopy revealed no apparent glomerular or tubulointerstitial lesions. A novel SGLT2 missense mutation was identified in the patient (229G [ C: G77R), and the amino acid residue affected (77G) was found to be highly conserved among human SGLT subtypes and across SGLT2 homologs in multiple species. This mutation was not detected in 200 chromosomes derived from 100 healthy, unrelated individuals, indicating that it is not a common polymorphism. SGLT2 expression in renal biopsy specimens was detected by immunofluorescence staining and observed using a confocal microscope (Olympus viewer 1000, Tokyo, Japan). The expression of SGLT2 in the apical side of the proximal convoluted tubule was discontinuously decreased compared with both normal renal tissue from a tumor patient following partial nephrectomy and disease controls (biopsy tissue from patients with minimal change disease or diabetic nephropathy) (Fig. 1). Because our patient showed clinical manifestations (urine glucose excretion, 4.8 g/day), this implies that the mutation causes clinically relevant SGLT2 dysfunction. We therefore inferred that the mutation affects transport activity by altering the processing of the protein and impairing its insertion into the plasma membrane. Thus, an abnormal distribution of mutant SGLT2 might play a key role in transport activity. Together with previous results, this suggests that the inheritance of renal glucosuria is best described as co-dominant, and its variable penetrance may relate to the compensatory capacity of wild-type SGLT2. To the best of our knowledge, altered SGLT2 expression caused by a heterozygous mutation in SGLT2 has not been previously reported. Our study therefore provides valuable clues regarding the effect of SGLT2 genotype–phenotype changes in FRG families. L. Yu and P. Hou are co-first authors and have contributed equally to the work.

In Silico Prediction of Inhibition of Promiscuous Breast Cancer Resistance Protein (BCRP/ABCG2)

BackgroundBreast cancer resistant protein has an essential role in active transport of endogenous substances and xenobiotics across extracellular and intracellular membranes along with P-glycoprotein. It also plays a major role in multiple drug resistance and permeation of blood-brain barrier. Therefore, it is of great importance to derive theoretical models to predict the inhibition of both transporters in the process of drug discovery and development. Hitherto, very limited BCRP inhibition predictive models have been proposed as compared with its P-gp counterpart.Methodology/Principal FindingsAn in silico BCRP inhibition model was developed in this study using the pharmacophore ensemble/support vector machine scheme to take into account the promiscuous nature of BCRP. The predictions by the PhE/SVM model were found to be in good agreement with the observed values for those molecules in the training set (n = 22, r 2 = 0.82, = 0.73, RMSE = 0.40, s = 0.24), test set (n = 97, q 2 = 0.75–0.89, RMSE = 0.31, s = 0.21), and outlier set (n = 16, q 2 = 0.72–0.91, RMSE = 0.29, s = 0.17). When subjected to a variety of statistical validations, the developed PhE/SVM model consistently met the most stringent criteria. A mock test by HIV protease inhibitors also asserted its predictivity.Conclusions/SignificanceIt was found that this accurate, fast, and robust PhE/SVM model can be employed to predict the BCRP inhibition of structurally diverse molecules that otherwise cannot be carried out by any other methods in a high-throughput fashion to design therapeutic agents with insignificant drug toxicity and unfavorable drug–drug interactions mediated by BCRP to enhance clinical efficacy and/or circumvent drug resistance.

Allosteric Regulation of Transport Activity by Heterotrimerization of Arabidopsis Ammonium Transporter Complexes in Vivo

Ammonium acquisition by plant roots is mediated by AMMONIUM TRANSPORTERs (AMTs), ubiquitous membrane proteins with essential roles in nitrogen nutrition in all organisms. In microbial and plant cells, ammonium transport activity is controlled by ammonium-triggered feedback inhibition to prevent cellular ammonium toxicity. Data from heterologous expression in yeast indicate that oligomerization of plant AMTs is critical for allosteric regulation of transport activity, in which the conserved cytosolic C terminus functions as a trans-activator. Employing the coexpressed transporters AMT1;1 and AMT1;3 from Arabidopsis thaliana as a model, we show here that these two isoforms form functional homo- and heterotrimers in yeast and plant roots and that AMT1;3 carrying a phosphomimic residue in its C terminus regulates both homo- and heterotrimers in a dominant-negative fashion in vivo. (15)NH4(+) influx studies further indicate that allosteric inhibition represses ammonium transport activity in roots of transgenic Arabidopsis expressing a phosphomimic mutant together with functional AMT1;3 or AMT1;1. Our study demonstrates in planta a regulatory role in transport activity of heterooligomerization of transporter isoforms, which may enhance their versatility for signal exchange in response to environmental triggers.

Deposition, Diffusion and Transport Mechanism of Dry Powder Microparticulate Salbutamol, at the Respiratory Epithelia

The deposition, dissolution and transport of salbutamol base (SB) and salbutamol sulfate (SS) inhalation powders were investigated using the Calu-3 air interface cell culture model and Franz diffusion cell. Drug uptake by cells was studied with respect to deposited dose, drug solubility and hydrophobicity. Furthermore, the role of active transport via organic cationic transporters (OCTs) was studied. SB and SS were processed to have similar diameters (3.09 ± 0.06 μm and 3.07 ± 0.03 μm, respectively) and were crystalline in nature. Analysis of drug wetting, dissolution and diffusion using a conventional in vitro Franz cell (incorporating a cell culture support Transwell polyester membrane) showed diffusion of SB to be slower than that of SS (98.57 ± 4.23 μg after 4 h for SB compared to 98.57 ± 4.01 μg after 15 min for SS). Such observations suggest dissolution to be the rate-limiting step. In comparison, the percentage transfer rate using the air interface Calu-3 cell model suggested SB transport to be significantly faster than SS transport (92.02 ± 4.47 μg of SB compared to 63.76 ± 8.84 μg of SS transported over 4 h), indicating that passive diffusion through the cell plays a role in transport. Furthermore, analysis of SB and SS transport, over a range of deposited doses, suggested the transport rate in the Franz diffusion cell to be limited by wetting of the particle and dissolution into the medium. However, for the cell monolayer, the cell membrane properties regulate the diffusion and transport rate. Analysis of the drug transport in the presence of triethylamine (TEA), a known inhibitor of OCTs, resulted in a significant decrease in drug transport, suggesting an active transport mechanism. The presence of OCTs in this cell line was further validated by Western blot analysis. Finally, the transport of SS from a commercial product (Ventolin Rotacaps) was studied and showed good agreement with the model SS system studied here.

Structure of a fucose transporter in an outward-open conformation

The major facilitator superfamily (MFS) transporters are an ancient and widespread family of secondary active transporters. In Escherichia coli, the uptake of l-fucose, a source of carbon for microorganisms, is mediated by an MFS proton symporter, FucP. Despite intensive study of the MFS transporters, atomic structure information is only available on three proteins and the outward-open conformation has yet to be captured. Here we report the crystal structure of FucP at 3.1 Å resolution, which shows that it contains an outward-open, amphipathic cavity. The similarly folded amino and carboxyl domains of FucP have contrasting surface features along the transport path, with negative electrostatic potential on the N domain and hydrophobic surface on the C domain. FucP only contains two acidic residues along the transport path, Asp 46 and Glu 135, which can undergo cycles of protonation and deprotonation. Their essential role in active transport is supported by both in vivo and in vitro experiments. Structure-based biochemical analyses provide insights into energy coupling, substrate recognition and the transport mechanism of FucP.

Dynamics of nonmembranous cell components: Role of active transport along microtubules

Here we discuss some common mechanisms of microtubule-dependent active transport of nonmembranous components in animal cells. We summarize data about mRNA, cytoskeletal elements, structural proteins, and signaling complexes transport. We also characterize the series of molecular interactions that connect nonmembranous cargoes and microtubules and describe the regulatory pathways for these interactions.

A comparison of uptake of metformin and phenformin mediated by hOCT1 in human hepatocytes

Metformin, a biguanide that has been used to treat type 2 diabetes mellitus, is reportedly transported into human hepatocytes by human organic cation transporter 1 (hOCT1). The objective of this study was to investigate differences in the hepatic uptake of metformin and phenformin, a biguanide derivative similar to metformin. Special focus was on the role of active transport into cells. Experiments were therefore performed using human cryopreserved hepatocytes and hOCT1 expressing oocytes. Both biguanides proved to be good substrates for hOCT1. However, phenformin exhibited a much higher affinity and transport activity, with a marked difference in uptake kinetics compared with metformin. Both biguanides were transported actively by hOCT1, with the active transport components much greater than passive transport components in both cases, suggesting that functional changes in hOCT1 might affect the transport of both compounds to the same degree. This study for the first time produced detailed comparative findings for uptake profiles of metformin and phenformin in human hepatocytes and hOCT1 expressing oocytes. It is considered that hOCT1 may not be the only key factor that determines the frequency of metformin and phenformin toxicity, considering the major contribution of this transporter to the total hepatic uptake and comparable width of their therapeutic concentrations.

Multi-drug resistance 1 polymorphism is associated with reduced risk of gastric cancer in the Japanese population

Host genetic factors play a key role in gastric carcinogenesis, but the mechanism has not been clarified. The multi-drug resistance 1 (MDR1) gene mediates the expression of P-glycoprotein, which has a role in active transport of various substrates, including xenobiotics, and thus has a protective function in various tissues and organs like gastrointestinal epithelial cells. C3435T polymorphism in exon 26 of the MDR1 gene influences P-glycoprotein expression and activity in the gastrointestinal tract. We investigated the influences of MDR1 gene polymorphism on the risk of gastric cancer. The study was performed on 157 patients with gastric cancer (GC) and 104 patients without GC as the control group. C3435T polymorphism of MDR1 was investigated by PCR-RFLP in all of the patients. The MDR1 3435 TT genotype showed a significantly higher frequency in controls than in GC patients (OR = 0.43; 95% CI = 0.23-0.79). There were no significant differences of the CT and CC genotype frequencies between GC patients and controls. We also found that the 3435TT genotype of MDR1 was associated with a lower risk of non-cardiac cancer (OR = 0.42; 95% CI = 0.23-0.79), middle-third cancer (OR = 0.36; 95% CI = 0.17-0.77), advanced cancer (OR = 0.31; 95% CI = 0.13-0.73), venous invasion (OR = 0.30; 95% CI = 0.10-0.91), and lymph node metastasis (OR = 0.28; 95% CI = 0.13-0.65). Our data suggest that 3435T/T polymorphism of MDR1 is associated with a reduced risk of gastric cancer in the Japanese population.

Evidence for Coupled Biogenesis of Yeast Gap1 Permease and Sphingolipids: Essential Role in Transport Activity and Normal Control by Ubiquitination

Current models for plasma membrane organization integrate the emerging concepts that membrane proteins tightly associate with surrounding lipids and that biogenesis of surface proteins and lipids may be coupled. We show here that the yeast general amino acid permease Gap1 synthesized in the absence of sphingolipid (SL) biosynthesis is delivered to the cell surface but undergoes rapid and unregulated down-regulation. Furthermore, the permease produced under these conditions but blocked at the cell surface is inactive, soluble in detergent, and more sensitive to proteases. We also show that SL biogenesis is crucial during Gap1 production and secretion but that it is dispensable once Gap1 has reached the plasma membrane. Moreover, the defects displayed by cell surface Gap1 neosynthesized in the absence of SL biosynthesis are not compensated by subsequent restoration of SL production. Finally, we show that down-regulation of Gap1 caused by lack of SL biogenesis involves the ubiquitination of the protein on lysines normally not accessible to ubiquitination and close to the membrane. We propose that coupled biogenesis of Gap1 and SLs would create an SL microenvironment essential to the normal conformation, function, and control of ubiquitination of the permease.

Effect of block deletions in the C-terminus on the functional expression of human anion exchanger 1 (AE1)

The human anion exchanger 1 (AE1) is the most abundant integral membrane protein in red cells and is responsible for the exchange of Cl(-) for HCO(3)(-). However, the detailed role played by the AE1 C-terminal region in the anion translocation and membrane trafficking process remains unclear. In this paper, we created four mutants in the human AE1 C-terminus by deletion of the residues Ala(891)-Phe(895), Asp(896)-Glu(899), Asp(902)-Glu(906) and Val(907)-Val(911), to investigate the role of these sequences in functional expression of AE1. WT AE1 and its deletion mutant constructs were expressed in HEK 293 cells. Western blotting showed that deletions of Ala(891)-Phe(895), Asp(896)-Glu(899), and Val(907)-Val(911) induced high expression of AE1, whereas loss of Asp(902)-Glu(906) results in stable low expression. Pulse chase assays of WT AE1 and its mutants showed that the stability of protein is unaffected by the levels of expression of the AE1 and its mutants. Ala(891)-Phe(895), Asp(902)-Glu(906) and Val(907)-Val(911) mutants exhibited lower levels of trafficking to the plasma membrane compared with WT AE1, while the Asp(896)-Glu(899) mutant was more highly expressed at the plasma membrane. The decreased ability of the mutants to mediate Cl(-)/HCO(3)(-) exchange in transfected cells revealed that the deletion sequences have an important role in transport activity. These results demonstrate that the studied residues in the AE1 C-terminus differently affect the expression, membrane trafficking and functional folding of AE1.

Transport kinetics of zinc, copper, selenium, and iron in perfused human placental lobule in vitro.

Transport characteristics of essential trace elements as zinc, copper, selenium and iron have been studied in maternal-fetal direction in normal pregnancies, using in vitro perfusion of human placental lobules. Solutions of trace elements corresponding to twice the physiological concentrations were injected (100 microl bolus) into the maternal arterial perfusate. Serial perfusate samples were collected every 30 sec from venous outflows for a study period of 5 min. Concentrations of these trace elements and their transport kinetics were determined. Transport fractions (TF) of zinc, copper, selenium and iron averaged 0.21, 0.49, 0.55 and 0.10% of maternal load respectively. Other parameters such as area under the curve, clearance, elimination constant, absorption and elimination rates showed some significant differences between the various elements. Copper and selenium appear to be transported passively in maternal-fetal direction, while for iron and zinc, role of active transport for transfer across the human placental membrane cannot be discounted. We speculate that alterations in copper: zinc TR50 (transport rate for 50% efflux) and TF ratios could serve as useful indicators for assessing placental transport status of these essential elements in complicated pregnancy states.

Sequential control signals determine arm and trunk contributions to hand transport during reaching in humans.

When reaching towards objects placed outside the arm workspace, the trunk assumes an active role in transport of the hand by contributing to the extent of movement while simultaneously maintaining the direction of reach. We investigated the spatial-temporal aspects of the integration of the trunk motion into reaching. Specifically, we tested the hypothesis that the efficiency ('gain') of the arm-trunk co-ordination determining the contribution of the trunk to the extent of hand movement may vary substantially with the phase of reaching. Sitting subjects made fast pointing movements towards ipsi- and a contralateral targets placed beyond the reach of the right arm so that a forward trunk motion was required to assist in transporting the hand to the target. Sight of the arm and target was blocked before the movement onset. In randomly selected trials, the trunk motion was unexpectedly prevented by an electromagnet. Subjects were instructed to make stereotypical movements whether or not the trunk was arrested. In non-perturbed trials, most subjects began to move the hand and trunk simultaneously. In trunk-blocked trials, it was impossible for the hand to cover the whole pointing distance but the hand trajectory and velocity profile initially matched those from the trials in which the trunk motion was free, approximately until the hand reached its peak velocity. The arm inter-joint co-ordination substantially changed in response to the trunk arrest at a minimal latency of 40 ms after the perturbation onset. The results suggest that when the trunk was free, the influence of the trunk motion on the hand trajectory and velocity profile was initially neutralized by appropriate changes in the arm joint angles. Only after the hand had reached its peak velocity did the trunk contribute to the extent of pointing. Previous studies suggested that the central commands underlying the transport component of arm movements are completed when the hand reaches peak velocity. These studies, together with the present finding that the trunk only begins to contribute to the hand displacement at peak hand velocity, imply that the central commands that determine the contributions of the arm and the trunk to the transport of the hand are generated sequentially, even though the arm and trunk move in parallel.