Drug-abbreviated infections of <Emphasis Type="Italic">Trichostrongylus colubriformis</Emphasis> and development of immunity in jirds (<Emphasis Type="Italic">Meriones unguiculatus</Emphasis>)

The objective of this study was to examine the development and the duration of immunity achieved with drug-abbreviated infections of Trichostrongylus colubriformis in jirds (Meriones unguiculatus). Jirds were primarily infected either by trickle infection with 6 × 100 infective larvae (L₃) of T. colubriformis at 3-day intervals or by a single infection with 600 L₃. On day 35 post-infection, one batch of jirds from each group was autopsied; the others were treated with oxfendazole at a dose of 5 mg/kg and were challenged with 1,000 L₃ on either day 7 or day 42 post-treatment. All jirds were autopsied at 17 days post-challenge. Trickle infection resulted in lower levels of egg production during the primary infection period. The systemic IgM and IgG antibody response was significantly stronger in trickle- and single-infected groups as compared with the negative control group (P < 0.01–P < 0.05). Significantly higher levels of intestinal IgA were demonstrated in trickle- and single-infected groups than in the negative control group (P < 0.01). Numbers of mucosal mast cells increased following infection, but this was not dependent on the type of immunisation. After challenge the extent of worm reduction was greater in trickle-infected than in single-infected subgroups. The IgM and IgG response was significantly stronger in challenged subgroups as compared with negative control subgroups (P < 0.01). However, the IgG response was weaker in control challenged subgroups than in challenged subgroups (P < 0.01). There was a negative correlation between the IgG response and the worm burden after the second challenge (r=−0.73). The acquired immunity to T. colubriformis infection in jirds developed within 5 weeks of primary infection. The level of immunity was higher after trickle infection than after single infection. Furthermore, the immunity persisted for at least 6 weeks after oxfendazole treatment in the absence of a worm burden and larval intake, which is very similar to the situation in domestic ruminant hosts. Received: 25 October1999 / Accepted: 23 December 1999     

Difficult laryngoscopy and tracheal intubation due to calcified stylohyoid ligaments

Two cases of difficult laryngoscopy and tracheal intubation caused by calcified stylohyoid ligaments are presented. Neither patient exhibited a skin crease over the hyoid bone. It is suggested that inability to lift up the epiglottis from the posterior pharyngeal wall be taken as a more useful sign of this condition than the presence of the skin crease.     

Quantitative relationships of Candida albicans infections and dressing patterns in Nigerian women.

Candida albicans colony counts were far higher in patients with vaginitis wearing tight fitting clothing than in patients wearing loose fitting clothing. In Ile-Ife, Nigeria, tight fitting dresses, woolen and corduroy jeans, coupled with nylon underwear, appear to create an environment favorable to Candida albicans colonization.     

Protective dietary factors in experimental ulceration��study of some Nigerian cereals and tubers

Rats fed on a supplement of millet, Guinea-corn, rice or maize given in addition to laboratory stock diet showed a high degree of protection against experimental ulceration following indomethacin administration. A higher degree of protection was shown when the rats were fed with the mixture of the four cereals and laboratory stock diet. The tubers did not offer protection, while a mixture of beans and millet, Guinea-corn, unpolished rice, and maize offered no protection. The significance of such findings with regard to the geographical distribution of duodenal ulcer in Nigeria is discussed.     

Multiconformation continuum electrostatics analysis of the NhaA Na+/H+ antiporter of Escherichia coli with functional implications

Sodium proton antiporters are essential enzymes that catalyze the exchange of sodium ions for protons across biological membranes. Protonations and deprotonations of individual amino acid residues and of clusters formed by these residues play an important role in activating these enzymes and in the mechanism of transport. We have used multiconformation continuum electrostatics method to investigate the protonation states of residues in the sodium proton exchanger NhaA from Escherichia coli, the structure of which has been determined recently by x-ray crystallography. Our calculations identify four clusters of electrostatically tightly interacting residues as well as long-range interactions between residues required for activation. The importance of many of these residues has been demonstrated by the characterization of site-directed mutants. A number of residues with extreme pKa values, including several of the "pH sensor," can only undergo protonation/deprotonation reactions subsequent to conformational changes. The results of the calculations provide valuable information on the activation of the antiporter and the role of individual amino acid residues, and provide a solid framework for further experiments.     

An angular motion of a conserved four-helix bundle facilitates alternating access transport in the TtNapA and EcNhaA transporters

There is ongoing debate regarding the mechanism through which cation/proton antiporters (CPAs), like Thermus thermophilus NapA (TtNapA) and Escherichia coli NapA (EcNhaA), alternate between their outward- and inward-facing conformations in the membrane. CPAs comprise two domains, and it is unclear whether the transition is driven by their rocking-bundle or elevator motion with respect to each other. Here we address this question using metadynamics simulations of TtNapA, where we bias conformational sampling along two axes characterizing the two proposed mechanisms: angular and translational motions, respectively. By applying the bias potential for the two axes simultaneously, as well as to the angular, but not the translational, axis alone, we manage to reproduce each of the two known states of TtNapA when starting from the opposite state, in support of the rocking-bundle mechanism as the driver of conformational change. Next, starting from the inward-facing conformation of EcNhaA, we sample what could be its long-sought-after outward-facing conformation and verify it using cross-linking experiments.

A secondary active transporter harnesses the electrochemical gradient created by the movement of an ion across a biological membrane to facilitate the transport of another ion or molecule against its electrochemical gradient. Structural data obtained throughout the past decade have revealed that these transporters share some common structural features and can be classified under three main structural folds: the MFS fold (major facilitator superfamily), the LeuT fold (leucine transporters), and the NhaA fold (Na⁺/H⁺ antiporters) (1, 2).When folded, these transporters are organized in two functional domains: 1) a mobile core domain that harbors an ion-binding site and 2) a dimerization/oligomerization domain (1). To carry out their function, they alternate between at least two main states—inward-facing and outward-facing (IF and OF) —such that their binding sites are accessible from different sides of the membrane in each state in what is known as an alternating access model (3). Extensive research has been devoted to elucidating the mechanisms underlying the alternating access model in secondary active transporters. In particular, studies of the LeuT fold have suggested that a four-helix bundle in the core domain undergoes significant conformational changes during transport (4). Notably, this four-helix bundle is structurally conserved across many transporters, suggesting that it may be implicated in the conformational changes underlying their function as well.Herein, using simulations, we contribute new insights with regard to the alternating access mechanism of the cation/proton antiporter (CPA) superfamily of secondary active transporters. CPAs exchange monovalent cations with protons, and their malfunction is associated with a growing number of human pathologies (5) affecting millions worldwide. However, there is still an ongoing debate regarding the structural mechanism underlying the alternating access model in CPAs (6, 7). One possibility is a rocking-bundle mechanism, which involves a tilting movement of the core domain relative to a stationary dimerization domain on the plane of the membrane; this mechanism was originally proposed on the basis of the structure of the bacterial LeuT transporter (4) (Fig. 1). An alternative possibility is the elevator mechanism, first described in a bacterial homolog of the glutamate transporter Glt_Ph. Glt_Ph is a member of the proton/sodium glutamate symport protein fold (1) that shares some similar structural features with the MFS, LeuT, and NhaA folds. This mechanism involves a prominent vertical movement of the transporter’s core domain along the membrane’s normal, which is accompanied by a tilting movement (8) (Fig. 1).Open in a separate windowFig. 1.Rocking bundle vs. elevator mechanism. Schematic representation of TtNapA oriented in the membrane such that the periplasm is at the top and the cytoplasm is at the bottom. Transmembrane helices are shown as cylinders, where the core domain is designated in orange and the dimerization domain in green. The core domain’s direction of movement relative to the dimerization domain that correlates with each of the two mechanisms underlying the alternating access model for cation/proton transport is marked with black arrows.Thus far, three-dimensional structures of four CPAs have been determined experimentally (6, 9–11). Most notably, the structure of the CPA member NapA from Thermus thermophilus (TtNapA) was solved in both IF and OF states (6). Consistent with observations obtained for LeuT (4), these structures revealed that the main rearrangements between the alternating states of TtNapA occur in the conserved four-helix bundle in the core domain (6). This bundle includes the two discontinuous transmembrane helices (TMs) 4 and 11 (2), as well as TM-5, which includes the ion-binding site, and TM-12; the numbering of the helices follows the prevalent helix nomenclature of Escherichia coli NhaA (EcNhaA), which will be used throughout the text (12). A comparison of the two oppositely facing structures of TtNapA reveals an angular motion of one domain relative to the other that is also accompanied by a 6-Å vertical displacement of the antiporter’s core domain relative to the membrane and 7 Å relative to the dimer domain (6). This is consistent with the elevator mechanism proposed for Glt_Ph that involves the two types of motions. It was thus suggested that the structural data favor the elevator mechanism in CPAs.While this interpretation is straightforward, the extreme conformations represented by the crystal structures do not provide any direct information about the actual conformational changes that take place during the transport processes. Accordingly, on its own, the structural information obtained for TtNapA is insufficient for determining whether the angular motion, vertical translation, or both underlie the alternating access model of cation/proton antiport in CPAs. Okazaki et al. (13) used molecular-dynamics (MD) simulations to predict the transition path between the IF and OF conformations of an archaeal member of the CPA superfamily. Their work demonstrated how insightful simulations can be in CPAs, but it did not address the question of which mode of motion is essential for the IF–OF transition.Here, we used metadynamics, an enhanced sampling MD protocol, to study the conformational dynamics of two CPA members, TtNapA and EcNhaA, embedded in a lipid bilayer. We focused on the structurally conserved four-helix bundle in an attempt to predict which mode of motion is most likely to drive the IF–OF transition in CPAs. Specifically, we applied external potential to bias the conformational sampling along the two axes of motion in TtNapA that underlie the rocking-bundle and elevator mechanisms. The first axis corresponds to an angular motion of the conserved four-helix bundle in the antiporter’s core domain relative to a separate four-helix bundle in the dimerization domain, and the second axis corresponds to a vertical translation of the four-helix bundle along the membrane normal. By applying the bias potential for both axes simultaneously, as well as for the angular motion axis alone, we successfully reproduced each of the two known states of TtNapA when starting from the opposite state, with accuracy of between 0.80 and 1.51 Å. Importantly, although both types of motions are observed, our results suggest that conformational changes depend mainly on the angular motion rather than on the translation. This dominance of the angular motions in determining the IF-to-OF transition is more in line with the rocking-bundle mechanism of ion/proton antiport. Furthermore, by biasing the conformational sampling of the IF conformation of EcNhaA along the angular motion axis, we sampled a conformation that seems to fit the long-sought-after OF conformation of this protein, and we verified it experimentally. Accordingly, in addition to providing a better understanding of the transport mechanism of CPAs, our findings point to the potential of computational methods to predict, for example, additional conformations for CPA members with only one known experimental structure, as well as to obtain more metastable states.     

Post dural puncture headache in obstetric patients: experience from a West African teaching hospital

BACKGROUND: This prospective, non-randomised study examined the frequency and severity of post dural puncture headache in 96 Ghanaian women who consented to spinal anaesthesia for caesarean section at the Korle Bu Teaching Hospital, Accra, Ghana. METHOD: Spinal anaesthesia was performed using 22-gauge (n = 22), 25-gauge (n = 46) or 26-gauge (n = 38) Quincke needles. Patients were followed up to determine the incidence and severity of post spinal headache. RESULT: The overall incidence of post dural puncture headache was 8.3%, but was significantly higher (33%) in patients in whom 22-gauge Quincke needles were used than in the other two groups (4% and 5% respectively: P = 0.003). Most patients rated their headache as mild to moderate on a 10-cm visual analogue scale. CONCLUSIONS: In view of the high incidence of headache and the need for treatment associated with the use of the 22-gauge Quincke needle, we recommend that this should not be used in the obstetric population. We are also aware that the incidence of post dural puncture headache could be further reduced by the use of small calibre pencil-point needles but these are currently very expensive and many obstetric units in developing countries may not be able to afford them.