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Histidine is an important carbon and nitrogen source of γ‐proteobacteria and can affect bacteria–host interactions. The mechanisms of histidine uptake are only partly understood. Here, we analyze functional properties of the putative histidine transporter HutT of the soil bacterium Pseudomonas putida. The hutT gene is part of the histidine utilization operon, and the gene product belongs to the amino acid‐polyamine‐organocation (APC) family of secondary transporters. Deletion of hutT severely impairs growth of P. putida on histidine, suggesting that the encoded transporter is the major histidine uptake system of P. putida. Transport experiments with cells and purified and reconstituted protein indicate that HutT functions as a high‐affinity histidine : proton symporter with high specificity for the amino acid. Substitution analyses identified amino acids crucial for HutT function. [ABSTRACT FROM AUTHOR]

Membrane transporters allow the selective transport of otherwise poorly permeable solutes across the cell membrane and thus, play a key role in maintaining cellular homeostasis in all kingdoms of life. Importantly, these proteins also serve as important drug targets. Over the last decades, major progress in structural biology methods has elucidated important structure-function relationships in membrane transporters. However, structures obtained using methods such as X-ray crystallography and high-resolution cryogenic electron microscopy merely provide static snapshots of an intrinsically dynamic, multi-step transport process. Therefore, there is a growing need for developing new experimental approaches capable of exploiting the data obtained from the high-resolution snapshots in order to investigate the dynamic features of membrane proteins. Here, we present the basic principles of hydrogen-deuterium exchange mass-spectrometry (HDX-MS) and recent advancements in its use to study membrane transporters. In HDX-MS experiments, minute amounts of a protein sample can be used to investigate its structural dynamics under native conditions, without the need for chemical labelling and with practically no limit on the protein size. Thus, HDX-MS is instrumental for resolving the structure-dynamic landscapes of membrane proteins in their apo (ligand-free) and ligand-bound forms, shedding light on the molecular mechanism underlying the transport process and drug binding.

Membrane transporters allow the selective transport of otherwise poorly permeable solutes across the cell membrane and thus, play a key role in maintaining cellular homeostasis in all kingdoms of life. Importantly, these proteins also serve as important drug targets. Over the last decades, major progress in structural biology methods has elucidated important structure-function relationships in membrane transporters. However, structures obtained using methods such as X-ray crystallography and high-resolution cryogenic electron microscopy merely provide static snapshots of an intrinsically dynamic, multi-step transport process. Therefore, there is a growing need for developing new experimental approaches capable of exploiting the data obtained from the high-resolution snapshots in order to investigate the dynamic features of membrane proteins. Here, we present the basic principles of hydrogen-deuterium exchange mass-spectrometry (HDX-MS) and recent advancements in its use to study membrane transporters. In HDX-MS experiments, minute amounts of a protein sample can be used to investigate its structural dynamics under native conditions, without the need for chemical labelling and with practically no limit on the protein size. Thus, HDX-MS is instrumental for resolving the structure-dynamic landscapes of membrane proteins in their apo (ligand-free) and ligand-bound forms, shedding light on the molecular mechanism underlying the transport process and drug binding. [ABSTRACT FROM AUTHOR]

There is significant interest in the secondary transport of the critically ill and injured patients. Inter-hospital transfer to a major emergency centers should be initiated soon after arriving at the primary hospital in order to reduce delaying definitive care. Major trauma requiring an inter-hospital transfer can be recognized by: certain vital sign markers; the presence of a specific physiological or anatomical injury; deterioration associated with a high-risk mechanism of injury; or being a high-risk patient. The aim of guidelines and protocols is to guide and support early management and transfer for specific injuries including spinal trauma, burns, traumatic brain injury, obstetric and pediatric trauma. Secondary transport should only occur if it can improve the patient's care and outcome. It should be undertaken in a manner that does not jeopardize the level and quality of care. Early identification of critical illness or major trauma criteria requiring inter-hospital transfer should be achieved using the defined triage guidelines. Patients with the presence of a high-risk mechanism of injury or a comorbid are at risk of major trauma complications. Physiological stability during the transfer requires careful pretransfer assessment and optimization of the patient. Good communication between referring and receiving medical staff is very important and have been shown to be effective. Emergency medicine has a key role in the organization of secondary transportation. [ABSTRACT FROM AUTHOR]

Abstract MdfA is an interesting member of a large group of secondary multidrug (Mdr) transporters. Through genetic, biochemical and biophysical studies of MdfA, many challenging aspects of the multidrug transport phenomenon have been addressed. This includes its ability to interact with chemically unrelated drugs and how it utilizes energy to drive efflux of compounds that are not only structurally, but also electrically, different. Admittedly, however, despite all efforts and a recent pioneering structural contribution, several important mechanistic issues of the promiscuous capabilities of MdfA still seek better molecular and dynamic understanding. [ABSTRACT FROM AUTHOR]

Aim: The chance of a good result of mechanical thrombectomy (MT) for a large cerebral artery occlusion is directly proportional to the speed of its execution. The aim was to fi nd diff erences in outcome in patients transported primarily and secondarily to the Comprehensive Stroke Center (CSC) of the University Hospital Hradec Králové. Patients and Methods: Out of 258 consecutive patients, 171 were transported to the CSC for MT primarily and 87 secondarily, from the primary stroke center. Clinical data, including intervals from the onset of cerebral ischemia and arrival to the center and then to treatment, were compared with clinical trials and the STRATIS registry and in both groups. The indicator of treatment eff ectiveness was the achievement of successful recanalization (TICI ≥ 2b) and self-suffi ciency in 3 months (mRS ≤ 2). Results: Despite the fact that in secondarily transported patients MT was started and completed signifi cantly later (271.7 ± 96.8 min vs. 175.7 ± 63.9 min, and 321.3 ± 107.6 min vs. 226.4 ± 72.3 min, resp.; both p < 0.0001), these patients had a statistically insignifi cant tendency towards a higher proportion of self-suffi ciency (63.2 vs. 46.8%) and lower mortality (14.9 vs. 21.1%). They were statistically signifi cantly younger (68.8 ± 13.2 vs. 73.4 ± 12.8 years; p = 0.007), with a lower proportion of patients > 80 years (17.0 vs. 33.3%; p = 0.043) and a higher proportion of tandem pathologies (24.1 vs. 11.7%; p = 0.036). Conclusion: Our results suggest that selection associated with secondary transport excludes a group of more risky patients from endovascular treatment. [ABSTRACT FROM AUTHOR]

Secondary multidrug (Mdr) transporters utilize ion concentration gradients to actively remove antibiotics and other toxic compounds from cells. The model Mdr transporter MdfA from Escherichia coli exchanges dissimilar drugs for protons. The transporter should open at the cytoplasmic side to enable access of drugs into the Mdr recognition pocket. Here we show that the cytoplasmic rim around the Mdr recognition pocket represents a previously overlooked important regulatory determinant in MdfA. We demonstrate that increasing the positive charge of the electrically asymmetric rim dramatically inhibits MdfA activity and sometimes even leads to influx of planar, positively charged compounds, resulting in drug sensitivity. Our results suggest that unlike the mutants with the electrically modified rim, the membrane-embedded wild-type MdfA exhibits a significant probability of an inward-closed conformation, which is further increased by drug binding. Since MdfA binds drugs from its inward-facing environment, these results are intriguing and raise the possibility that the transporter has a sensitive, drug-induced conformational switch, which favors an inward-closed state. [ABSTRACT FROM AUTHOR]

Emergency medical services (EMS) typically transports patients to the nearest emergency department (ED). After initial presentation, children who require specialized care must undergo secondary transport, exposing them to additional risks and delaying definitive treatment. EMS direct transport protocols exist for major trauma and certain adult medical conditions, however the same cannot be said for pediatric medical conditions or injuries that do not meet trauma center criteria ('minor trauma'). To explore the utility of such future protocols, we sought to first describe the pediatric secondary transport population and examine prehospital risk factors for secondary transport. Methods: Pediatric secondary transport patients aged 0-18 years were identified. Patients meeting state EMS trauma protocol criteria or who were clinically unstable were excluded. Data were abstracted by chart review of EMS, community hospital ED, and specialty hospital records. Patients were compared to control patients with similar conditions who did not require secondary transport. Results: This study identified 211 medical or minor trauma pediatric secondary transport patients between 2013 and 2014. The three most prevalent conditions were seizure (n = 52), isolated orthopedic injury (n = 49), and asthma/respiratory distress (n = 27). Increased odds of secondary transport for seizure patients were associated with administration of supplemental oxygen, glucose measurement, and online medical direction; for isolated orthopedic injuries, online medical direction; and for asthma/respiratory distress, administration of supplemental oxygen, and online medical direction. Decreased odds of secondary transport for seizure patients were associated with a higher GCS; for isolated orthopedic injuries, increased age and oxygen saturation; and for asthma/respiratory distress, administration of albuterol only. Conclusions: Children with seizures, isolated orthopedic injuries, and asthma/respiratory distress comprised the majority of the medical or minor trauma pediatric secondary transport population. Each of those conditions had specific risk factors for secondary transport. This study's results provide information to guide future prospective studies and the development of direct transport protocols for those populations. [ABSTRACT FROM AUTHOR]

Bor1p is a secondary transporter in yeast that is responsible for boron transport. Bor1p belongs to the SLC4 family which controls bicarbonate exchange and pH regulation in animals as well as borate uptake in plants. The SLC4 family is more distantly related to members of the Amino acid-Polyamine-organoCation (APC) superfamily, which includes well studied transporters such as LeuT, Mhp1, AdiC, vSGLT, UraA, SLC26Dg. Their mechanism generally involves relative movements of two domains: a core domain that binds substrate and a gate domain that in many cases mediates dimerization. To shed light on conformational changes governing transport by the SLC4 family, we grew helical membrane crystals of Bor1p from Saccharomyces mikatae and determined a structure at ∼6 Å resolution using cryo-electron microscopy. To evaluate the conformation of Bor1p in these crystals, a homology model was built based on the related anion exchanger from red blood cells (AE1). This homology model was fitted to the cryo-EM density map using the Molecular Dynamics (MD) Flexible Fitting method and then relaxed by all-atom MD simulation in explicit solvent and membrane. Mapping of water accessibility indicates that the resulting structure represents an inward-facing conformation. Comparisons of the resulting Bor1p model with the X-ray structure of AE1 in an outward-facing conformation, together with MD simulations of inward-facing and outward-facing Bor1p models, suggest rigid body movements of the core domain relative to the gate domain. These movements are consistent with the rocking-bundle transport mechanism described for other members of the APC superfamily. [ABSTRACT FROM AUTHOR]

Multidrug transporters are membrane proteins that catalyze efflux of antibiotics and other toxic compounds from cells, thereby conferring drug resistance on various organisms. Unlike most solute transporters that transport a single type of compound or similar analogues, multidrug transporters are extremely promiscuous. They transport a broad spectrum of dissimilar drugs and represent a serious obstacle to antimicrobial or anticancer chemotherapy. Many challenging aspects of multidrug transporters, which are unique, have been studied in detail, including their ability to interact with chemically unrelated drugs, and how they utilize energy to drive efflux of compounds that are not only structurally but electrically different. A new and surprising dimension of the promiscuous nature of multidrug transporters has been described recently: they can move long molecules through the membrane in a processive manner.

The common human pathogen Salmonella enterica takes up citrate as a nutrient via the sodium symporter SeCitS. Uniquely, our 2.5 Å x-ray structure of the SeCitS dimer shows three different conformations of the active protomer. One protomer is in the outside-facing state. Two are in different inside-facing states. All three states resolve the substrates in their respective binding environments. Together with comprehensive functional studies on reconstituted proteoliposomes, the structures explain the transport mechanism in detail. Our results indicate a six-step process, with a rigid-body 31° rotation of a helix bundle that translocates the bound substrates by 16 Å across the membrane. Similar transport mechanisms may apply to a wide variety of related and unrelated secondary transporters, including important drug targets.

Secondary active transporters are critical for neurotransmitter clearance and recycling during synaptic transmission and uptake of nutrients. These proteins mediate the movement of solutes against their concentration gradients, by using the energy released in the movement of ions down pre-existing concentration gradients. To achieve this, transporters conform to the so-called alternating-access hypothesis, whereby the protein adopts at least two conformations in which the substrate binding sites are exposed to either the outside or inside of the membrane, but not both simultaneously. Structures of a bacterial homolog of neuronal glutamate transporters, GltPh, in several different conformational states have revealed that the protein structure is asymmetric in the outward- and inward-open states, and that the conformational change connecting them involves a elevator-like movement of a substrate binding domain across the membrane. The structural asymmetry is created by inverted-topology repeats, i.e., structural repeats with similar overall folds whose transmembrane topologies are related to each other by two-fold pseudo-symmetry around an axis parallel to the membrane plane. Inverted repeats have been found in around three-quarters of secondary transporter folds. Moreover, the (a)symmetry of these systems has been successfully used as a bioinformatic tool, called repeat-swap modeling to predict structural models of a transporter in one conformation using the known structure of the transporter in the complementary conformation as a template. Here, we describe an updated repeat-swap homology modeling protocol, and calibrate the accuracy of the method using GltPh, for which both inward- and outward-facing conformations are known. We then apply this repeat-swap homology modeling procedure to a concentrative nucleoside transporter, VcCNT, which has a three-dimensional arrangement related to that of GltPh. The repeat-swapped model of VcCNT predicts that nucleoside transport also occurs via an elevator-like mechanism.

Secondary active transporters are critical for neurotransmitter clearance and recycling during synaptic transmission and uptake of nutrients. These proteins mediate the movement of solutes against their concentration gradients, by using the energy released in the movement of ions down pre-existing concentration gradients. To achieve this, transporters conform to the so-called alternating-access hypothesis, whereby the protein adopts at least two conformations in which the substrate binding sites are exposed to one or other side of the membrane, but not both simultaneously. Structures of a bacterial homolog of neuronal glutamate transporters, GltPh, in several different conformational states have revealed that the protein structure is asymmetric in the outward- and inward-open states, and that the conformational change connecting them involves a elevator-like movement of a substrate binding domain across the membrane. The structural asymmetry is created by inverted-topology repeats, i.e., structural repeats with similar overall folds whose transmembrane topologies are related to each other by two-fold pseudo-symmetry around an axis parallel to the membrane plane. Inverted repeats have been found in around three-quarters of secondary transporter folds. Moreover, the (a)symmetry of these systems has been successfully used as a bioinformatic tool, called "repeat-swap modeling" to predict structural models of a transporter in one conformation using the known structure of the transporter in the complementary conformation as a template. Here, we describe an updated repeatswap homology modeling protocol, and calibrate the accuracy of the method using GltPh, for which both inward- and outward-facing conformations are known. We then apply this repeat-swap homology modeling procedure to a concentrative nucleoside transporter, VcCNT, which has a three-dimensional arrangement related to that of GltPh. The repeat-swapped model of VcCNT predicts that nucleoside transport also occurs via an elevator-like mechanism. [ABSTRACT FROM AUTHOR]

The objective of this study was to develop a predictive model for travel speed of softwood sawlog timber transport (STT) vehicles over a range of forest and provincial roads of varying condition for the South African forestry industry. Data was accumulated from both the Eastern Cape/KwaZulu-Natal and Mpumalanga forest regions of South Africa. Vehicle location and payload data were collected remotely using a combination of GPS tracking and remotely sensed data. Road condition, including road width, was assessed for each identified road segment in-field according to a visual condition indicator (VCI) index. Two STT contractors were selected from each forest region transporting pine sawlogs directly from compartment roadside landings to either a processing plant or log storage area. Five STT vehicle types, representative of transport operations in the two forest regions, were assessed. Principal component analysis was conducted to determine the degree of communality between the respective predictor variables combining road width and VCI as one factor, and truck maximum power and the percentage of (legal) maximum payload as a second factor. Comparisons of the correlations between average speed and the respective predictor variables showed that road width and percentage of maximum (legal) payload had the highest correlations. Multiple linear regression of these two factor variables were used in the model showing both variables as significant (p< 0.05) with an adjustedr2value of 0.52. [ABSTRACT FROM PUBLISHER]

Bilayer lipids contribute to the stability of membrane transporters and are crucially involved in their proper functioning. However, the molecular knowledge of how surrounding lipids affect membrane transport is surprisingly limited and despite its general importance is rarely considered in the molecular description of a transport mechanism. One reason is that only few atomic resolution structures of channels or transporters reveal a functional interaction with lipids, which are difficult to detect in X-ray structures per se. Overcoming these difficulties, we report here on a new structure of the osmotic stress-regulated betaine transporter BetP in complex with anionic lipids. This lipid-associated BetP structure is important in the molecular understanding of osmoregulation due to the strong dependence of activity regulation in BetP on the presence of negatively charged lipids. We detected eight resolved palmitoyl-oleoyl phosphatidyl glycerol (PG) lipids mimicking parts of the membrane leaflets and interacting with key residues in transport and regulation. The lipid-protein interactions observed here in structural detail in BetP provide molecular insights into the role of lipids in osmoregulated secondary transport. [ABSTRACT FROM AUTHOR]

Multidrug transporters are integral membrane proteins that use cellular energy to actively extrude antibiotics and other toxic compounds from cells. The multidrug/proton antiporter MdfA from Escherichia coli exchanges monovalent cationic substrates for protons with a stoichiometry of 1, meaning that it translocates only one proton per antiport cycle. This may explain why transport of divalent cationic drugs by MdfA is energetically unfavorable. Remarkably, however, we show that MdfA can be easily converted into a divalent cationic drug/≥2 proton-antiporter, either by random mutagenesis or by rational design. The results suggest that exchange of divalent cationic drugs with two (or more) protons requires an additional acidic residue in the multidrug recognition pocket of MdfA. This outcome further illustrates the exceptional promiscuous capabilities of multidrug transporters. [ABSTRACT FROM AUTHOR]

Abstract: Na+/solute symporters are essential membrane integrated proteins that couple the flow of Na+ ions driven by electrochemical Na+ gradients to the transport of solutes across biological membranes. Here, we used a combination of molecular modeling techniques and evolutionary conservation analysis to construct and validate a first model of the Na+/proline symporter PutP of Escherichia coli based on the crystal structure of the bacterial Na+/galactose symporter vSGLT. Ligand docking experiments were employed to gain information about residues involved in proline binding. The proposed model is consistent with the available experimental data and was further validated by amino acid substitutions and kinetic and protein chemical analyses. Combination of the results of molecular modeling and functional studies predicts the location and organization of the Na+ and proline binding sites. Remarkably, as proposed computationally and discovered here experimentally, residues Y140, W244, and Y248 of transmembrane segments 4 and 7 are found to be particularly important for PutP function and suggested to participate in proline binding and/or gating. [Copyright &y& Elsevier]

Primary and secondary transport of citrate has been extensively studied in pathogenic and non-pathogenic bacteria. Primary transporters of citrate complexed with metal ions, particularly Fe, have also garnered attention, with the fec system of E. coli being a classic example. In contrast, little is known about secondary transporters of metal-citrate complexes. Recently, a family of proteins responsible for secondary metal-citrate transport in bacteria was discovered and designated as the CitMHS transporter family. Several members have been functionally characterized to date and serve as the foundation for understanding this family. Three subfamilies have been categorized, depending on the main metal ion transported. These subfamilies are the Mg2+-citrate transporter, the Ca2+-citrate transporter, and the Fe3+-citrate transporter. Each subfamily is believed to be substrate-selective due to the metal-citrate complexes being abundantly present in their environment and/or the ability of the complex to be metabolized by the organism. The implication of this family in the pathogenic access to Fe, information about transcriptional control, putative structure, predicted family members, members characterized to date and potential use in bioremediation are discussed. [ABSTRACT FROM AUTHOR]

Abstract: Multidrug transporters are membrane proteins that expel a wide spectrum of cytotoxic compounds from the cell. Through this function, they render cells resistant to multiple drugs. These transporters are found in many different families of transport proteins, of which the largest is the major facilitator superfamily. Multidrug transporters from this family are highly represented in bacteria and studies of them have provided important insight into the mechanism underlying multidrug transport. This review summarizes the work carried out on these interesting proteins and underscores the differences and similarities to other transport systems. [Copyright &y& Elsevier]

Many opportunistic bacteria that infect the upper respiratory tract decorate their cell surface with phosphorylcholine to support colonisation and outgrowth. These surface modifications require the active import of choline from the host environment, a process thought to be mediated by a family of dedicated integral membrane proteins that act as choline permeases. Here, we present the expression and purification of the archetype of these choline transporters, LicB from Haemophilus influenzae. We show that LicB can be recombinantly produced in Escherichia coli and purified to homogeneity in a stable, folded state using the detergent n -dodecyl-β- d -maltopyranoside. Equilibrium binding studies with the fluorescent ligand dansylcholine suggest that LicB is selective towards choline, with reduced affinity for acetylcholine and no apparent activity towards other small molecules including glycine, carnitine and betaine. We also identify a conserved sequence motif within the LicB family and show that mutations within this motif compromise protein structure and function. Our results are consistent with previous observations that LicB is a specific high-affinity choline transporter, and provide an experimental platform for further studies of this permease family. • The LicB family of integral membrane proteins contribute to bacterial colonisation of human airways • LicB from H. influenzae is expressed in recombinant E. coli membranes • Recombinant LicB is successfully solubilised and purified in the surfactant DDM • Ligand binding assays support the assignment of LicB as a selective, high-affinity choline permease [ABSTRACT FROM AUTHOR]

Abstract: Selected residues of transmembrane domain (TM) IX were previously shown to play key roles in ligand binding and transport in members of the Na+/solute symporter family. Using the Na+/proline transporter PutP as a model, a complete Cys scanning mutagenesis of TM IX (positions 324 to 351) was performed here to further investigate the functional significance of the domain. G328, S332, Q345, and L346 were newly identified as important for Na+-coupled proline uptake. Placement of Cys at one of these positions altered K m(pro) (S332C and L346C, 3- and 21-fold decreased, respectively; Q345C, 38-fold increased), K 0.5(Na+) (S332C, 13-fold decreased; Q345C, 19-fold increased), and/or V max [G328C, S332C, Q345C, and L346C, 3-, 22-, 2-, and 8-fold decreased compared to PutP(wild type), respectively]. Membrane-permeant N-ethylmaleimide inhibited proline uptake into cells containing PutP with Cys at distinct positions in the middle (T341C) and cytoplasmic half of TM IX (C344, L347C, V348C, and S351C) and had little or no effect on all other single Cys PutP variants. The inhibition pattern was in agreement with the pattern of labeling with fluorescein-5-maleimide. In addition, Cys placed into the cytoplasmic half of TM IX (C344, L347C, V348C, and S351C) was protected from fluorescein-5-maleimide labeling by proline while Na+ alone had no effect. Membrane-impermeant methanethiosulfonate ethyltrimethylammonium modified Cys in the middle (A337C and T341C) and periplasmic half (L331C) but not in the cytoplasmic half of TM IX in intact cells. Furthermore, Cys at the latter positions was partially protected by Na+ but not by proline. Based on these results, a model is discussed according to which residues of TM IX participate in the formation of ligand-sensitive, hydrophilic cavities in the protein that may reconstitute part of the Na+ and/or proline translocation pathway of PutP. [Copyright &y& Elsevier]

Aims: To determine the decision time (DT) in the Emergency Department (ED) to establish the factors that prolong DT. Methods: This was a prospective study performed between November 17 and December 18, 2005, involving patients who had been admitted to the ED and whose transfer to other hospitals was decided. We used "The Patient Transfer Questionnaire", which included the date of transfer, demographics of the patient, and the physician's decision of the time of transfer. The statistical variables were assessed using SPSS. Results: A total of 507 patients were included, of whom 53.4% were male. The most frequent diagnoses in transferred infants, children, adolescents, and adults were infection, convulsion or epilepsy, acute abdomen, and coronary syndrome, respectively. There was a significant correlation between age groups and the main diagnosis (trauma vs. no trauma). The mean DT was 37.7 ± 1.6 min. The longest DT was due to a lack of critical care beds; the shortest DT was determined for infants. The differences in DT between age groups, reasons for transport, and the patients' in-hospital consultation before the decision to transport were statistically significant. Age groups were found to be the most significant indicators in the DT. Conclusion: The organization of the services in the hospital directly affects the secondary DT. [ABSTRACT FROM AUTHOR]

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The bacteria facilitating enhanced biological phosphorus removal gain a selective advantage from intracellularly stored polymer-driven substrate uptake under anaerobic conditions during sequential anaerobic : aerobic cycling. Mechanisms for these unusual membrane transport processes were proposed and experimentally validated using selective inhibitors and highly-enriched cultures of a polyphosphate-accumulating organism, Accumulibacter, and a glycogen-accumulating organism, Competibacter. Acetate uptake by both Accumulibacter and Competibacter was driven by a proton motive force (PMF). Stored polymers were used to generate the PMF – Accumulibacter used phosphate efflux through the Pit transporter, while Competibacter generated a PMF by proton efflux through the ATPase and fumarate reductase in the reductive TCA cycle. [ABSTRACT FROM AUTHOR]

Abstract: Transport of patients may be divided into primary and secondary. Primary transport is the movement of patients from the scene of an accident or the onset of illness to hospital, where their presenting problems can be assessed, stabilized and treated. Secondary transport is the movement of the patient after initial assessment and may be to specialized diagnostic or therapeutic facilities within the same hospital (intra-hospital) or another hospital (inter-hospital). In the UK, the secondary transfer of critically ill patients started under the auspices of the Clinical Shock Study Group in Glasgow. This specialized transfer team has been responsible for most of the inter-hospital transfers in the West of Scotland for almost 35 years. Early publications from this Group outlined the physiological changes that could be expected during the transfer of critically ill patients and these principles form the basis of the clinical management of such patients. In the UK, just over 10,000 critically ill patients require secondary transfer each year. Despite the logistic advantages and improved patient outcome when transfers are undertaken by specialist teams, there are few such teams and most transfers are undertaken by the staff of the referring hospital, who do not regularly perform such tasks. In the UK, where intensive care facilities are relatively sparse, the concept of ICUs working together as a critical care network was introduced to maximize the use of resources following a government review in 2000. The National Intensive Care Bed Register and biannual KH03a census of critical care beds emphasizes the political interest in this area. [Copyright &y& Elsevier]

Ser57 in the Na+/proline permease of Escherichia coli has been replaced with alanine, cysteine, glycine. or threonine, and properties of the corresponding putP mutants have been analyzed. Although Ser57 is not essential for activity, the amino acid side chain at this position is critical for proline uptake. Thus, alanine, cysterine. glycine. or threonine in place of Ser57 reduces the initial rate of proline transport under standard conditions to less than 10% of the wild-type value. In addition, substitution of Ser57 in the Na+ /proline permease reduces the sensitivity of E. coli cells to the toxic proline analogs L-azetidine- 2-carboxylate and 3,4-dehydro-D,L-proline. Replacement of Ser57 with alanine or cysteine results in apparent affinities for proline that are reduced by more than two orders of magnitude. and permeases with threonine and glycme in place of Ser57 yield apparent affinities reduced by a factor of 60 and 1 8. respectively, relative to wild-type, in contrast. all of the Ser57 replacements analysed cause only small changes in Vmax values. All permease molecules containing Ser57 substitutions are inserted into the membrane in amounts comparable to the wild-type protein as shown by immunoblot analysis. These results indicate that alterations of proline transport and sensitivity to toxic proline analogs have to he attributed primarily to defects in substrate binding. It is suggested that the serine residue at position 57 of the permease is located within the substrate-binding domain of the protein. [ABSTRACT FROM AUTHOR]

When Corynebacterium glutamicum is grown with a sufficient nitrogen supply, urea crosses the cytoplasmic membrane by passive diffusion. A permeability coefficient for urea diffusion of 9 × 10–7 cm s–1 was determined. Under conditions of nitrogen starvation, an energy-dependent urea uptake system was synthesized. Carrier-mediated urea transport was catalyzed by a secondary transport system linked with proton motive force. With a K m for urea of 9 μM, the affinity of this uptake system was much higher than the affinity of urease towards its substrate ( K m approximately 55 mM urea). The maximum uptake velocity depended on the expression level and was relatively low [2–3.5 nmol min–1 (mg dry wt.)–1]. [ABSTRACT FROM AUTHOR]

Secondary metabolic-energy-generating systems generate a proton motive force (pmf) or a sodium ion motive force (smf) by a process that involves the action of secondary transporters. The (electro)chemical gradient of the solute(s) is converted into the electrochemical gradient of protons or sodium ions. The most straightforward systems are the excretion systems by which a metabolic end product is excreted out of the cell in symport with protons or sodium ions (energy recycling). Similarly, solutes that were accumulated and stored in the cell under conditions of abundant energy supply may be excreted again in symport with protons when conditions become worse (energy storage). In fermentative bacteria, a proton motive force is generated by fermentation of weak acids, such as malate and citrate. The two components of the pmf, the membrane potential and the pH gradient, are generated in separate steps. The weak acid is taken up by a secondary transporter either in exchange with a fermentation product (precursor/product exchange) or by a uniporter mechanism. In both cases, net negative charge is translocated into the cell, thereby generating a membrane potential. Decarboxylation reactions in the metabolic breakdown of the weak acid consume cytoplasmic protons, thereby generating a pH gradient across the membrane. In this review, several examples of these different types of secondary metabolic energy generation will be discussed. [ABSTRACT FROM AUTHOR]

Nitrosomonas europaea is capable of incorporating exogenously supplied amino acids. Studies in whole cells revealed that at least eight amino acids are actively accumulated, probably by the action of three different transport systems, each with high affinity (μ molar range) for several amino acids. Evidence for the action of secondary mechanisms of transport was obtained from efflux, counterflow and exchange experiments. More detailed information was obtained from studies in liposomes in which solubilized integral membrane proteins of N. europaea were incorporated. Uptake of l-alanine in these liposomes could be driven by artificially imposed pH gradients and electrical potentials, but not by chemical sodium-ion gradients. These observations indicate that l-alanine is transported by a H/alanine symport system. The ecological significance of secondary amino acid transport systems in autotrophic ammonium-oxidizing bacteria is discussed. [ABSTRACT FROM AUTHOR]

Acetate is effectively taken up by whole cells of Corynebacterium glutamicum via a specific carrier with a pH optimum of 8. The K of acetate uptake was 50 μM and the V 25-35 nmol/mg dw min. The activation energy was determined to be 70 kJ/mol. Acetate uptake was competitively inhibited by propionate with a K of about 30 μM and blocked by addition of sulfhydryl reagents. The transport activity was clearly dependent on the membrane potential, but independent of the presence of Na-ions. It is concluded that uptake of acetate proceeds by a secondary, proton coupled mechanism. [ABSTRACT FROM AUTHOR]

Lactic acid bacteria play an essential role in many food fermentation processes. They are anaerobic organisms which obtain their metabolic energy by substrate phosphorylation. In addition three secondary energy transducing processes can contribute to the generation of a proton motive force: proton/substrate symport as in lactic acid excretion, electrogenic precursor/product exchange as in malolactic and citrolactic fermentation and histidine/histamine exchange, and electrogenic uniport as in malate and citrate uptake in Leuconostoc oenos. In several of these processes additional H+ consumption occurs during metabolism leading to the generation of a pH gradient, internally alkaline. Lactic acid bacteria have also developed multidrug resistance systems. In Lactococcus lactis three toxin excretion systems have been characterized: cationic toxins can be excreted by a toxin/proton antiport system and by an ABC-transporter. This cationic ABC-transporter has surprisingly high structural an d functional analogy with the human MDR1-(P-glycoprotein). For anions an ATP-driven ABC-like excretion systems exist. [ABSTRACT FROM AUTHOR]

The solute/sodium symporter family (SSS family; TC 2.A.21; SLC5) consists of integral membrane proteins that use an existing sodium gradient to drive the uphill transport of various solutes, such as sugars, amino acids, vitamins, or ions across the membrane. This large family has representatives in all three kingdoms of life. The human sodium/iodide symporter (NIS) and the sodium/glucose transporter (SGLT1) are involved in diseases such as iodide transport defect or glucose-galactose malabsorption. Moreover, the bacterial sodium/proline symporter PutP and the sodium/sialic acid symporter SiaT play important roles in bacteria–host interactions. This review focuses on the physiological significance and structural and functional features of prokaryotic members of the SSS family. Special emphasis will be given to the roles and properties of proteins containing an SSS family domain fused to domains typically found in bacterial sensor kinases. [ABSTRACT FROM AUTHOR]

Objective: To describe characteristics of encounters in U.S. emergency departments (EDs) brought by interfacility transport by emergency medical services (EMS) from other EDs or urgent care settings.Methods: We performed a cross-sectional study of the National Hospital Ambulatory Medical Care Survey, a multistage probability survey of nonfederal of visits to U.S. EDS. We evaluated patients who were brought to the ED as an interfacility transport by EMS from another ED or urgent care setting between 2014 and 2017. We report demographics, clinical characteristics and treatment factors of ED encounters brought interfacility transport and assessed factors associated with discharge from the receiving ED.Results: Of 562.9 million ED encounters during the assessed period, 4.5 million were brought by interfacility transport by EMS (1.1 million per year). This represented 0.8% (95% CI 0.6-1.0%) of all ED encounters and 5.3% (95% CI 4.4-6.3%) of ED encounters transported by EMS. Most encounters brought by interfacility transport were adults (85%) who were publicly insured (62%). 39% had at least one abnormal vital sign. Most encounters received diagnostic testing (84%) and were seen within 30 min of presentation (61%). 54% were admitted, and 36% were discharged from the ED. Encounters without chronic complex conditions and with normal triage vital signs were associated with ED discharge (p < 0.01).Discussion: Interfacility transports between EDs transported by EMS account for <1% of ED encounters in the U.S. Nearly 40% of such encounters are ultimately discharged. Further research is needed to identify a low-risk cohort among patients in need of secondary transport. [ABSTRACT FROM AUTHOR]