Anion modulation of the slowly activating vacuolar channel

A series of n-alkanols and phenyl-substituted n-alkanols (Φ-alkanols) of increasing chain length and phenol were characterized for their ability to block action potentials (APs) in frog sciatic nerves. APs were recorded using the single sucrose-gap method. The degree of AP attenuation when the nerve was exposed to different concentrations of an alcohol was used to construct dose-response curves. The reciprocals of the half-blocking doses (ED₅₀s) were used to obtain a measure of the potency of the alcohols. For n-alkanols and Φ-alkanols, increasing the chain length by the addition of a methylene group increased the potency on average by 3.1 for both groups of alkanols. The addition of a phenyl group caused a potency increase that ranged between the values of 77 and 122. The ED₅₀ for both groups of alkanols could not be solely predicted by the log octanol-water partition coefficient (K _OW ). Using linear solvation energy relations (LSER), the log ED₅₀ could be described as a linear combination of the intrinsic (van der Waals) molar volume (V _I ), polarity (P), and hydrogen bond acceptor basicity (β) and donor acidity (α). Size alone could not predict the ED₅₀ for both n-alkanols and Φ-alkanols. The results are consistent with the hypothesis that alkanols bind to and interact with Na channels to cause AP block. Phenyl group addition to an alkanol markedly increases the molecule's potency. Received: 11 August 2000/Revised: 21 December 2000     

Bilayer reconstitution of voltage-dependent ion channels using a microfabricated silicon chip

Painted bilayers containing reconstituted ion channels serve as a well defined model system for electrophysiological investigations of channel structure and function. Horizontally oriented bilayers with easy solution access to both sides were obtained by painting a phospholipid:decane mixture across a cylindrical pore etched into a 200-microm thick silicon wafer. Silanization of the SiO(2) layer produced a hydrophobic surface that promoted the adhesion of the lipid mixture. Standard lithographic techniques and anisotropic deep-reactive ion etching were used to create pores with diameters from 50 to 200 microm. The cylindrical structure of the pore in the partition and the surface treatment resulted in stable bilayers. These were used to reconstitute Maxi K channels in the 100- and 200-microm diameter pores. The electrophysiological characteristics of bilayers suspended in microchips were comparable with that of other bilayer preparations. The horizontal orientation and good voltage clamping properties make the microchip bilayer method an excellent system to study the electrical properties of reconstituted membrane proteins simultaneously with optical probes.     

A new approach to improve acoustic trapping effectiveness for Aedes aegypti (Diptera: Culicidae)

Monitoring mosquito populations is essential to designing and implementing control strategies. Recent strategies based on releasing biologically modified mosquitoes have increased the need to effectively monitor mosquito abundance. Unfortunately, existing surveillance traps are of limited value due to their high cost and low capture rates. Here, we report the results of experiments designed to evaluate the effectiveness of an acoustic trap prototype. Stimuli synthesized from recordings of Ae. aegypti wingbeat signals and pure tones were evaluated as attractants to males in indoor and semi‐field conditions. Overall, the acoustic trap´s efficacy differed significantly between indoor and semi‐field conditions. After two hours of indoor recapture, ～69% of males were collected from acoustic traps broadcasting pure tones while ～78% of males were collected using synthesized wingbeat signals. Under semi‐field conditions, however, acoustic traps collected less than ～1.7% of the males released. Increasing the intensity of the signals up to 90 dB (SPL re. 20 uPa at 1 m from the trap) did not improve the capture rate under semi‐field conditions. Overall, our results indicate that acoustic signals synthesized from recordings of wingbeats can be used to enhance capture of male Ae. aegypti.     

Chemokine/cytokine production by mononuclear cells from human lymphoid tissues and their modulation by Mycobacterium tuberculosis antigens

The majority of knowledge about the role of cytokines and chemokines in controlling Mycobacterium tuberculosis infection mainly derives from animal models. In humans, this knowledge is still mainly limited to the blood compartment or accessible lymphoid organs, such as tonsils. Here, we studied cytokine and chemokine production and their modulation by M. tuberculosis antigens in mononuclear cells from human blood, spleen and hilar lung lymph nodes. Results show that the kinetics and magnitude of cytokine and chemokine production varied according to the tissue of cell origin. Mycobacterium tuberculosis antigens enhanced cytokine and chemokine production in blood, but the enhancement was restricted in spleen and hilar lung lymph node cells. We show, for the first time in humans, differences in cytokine and chemokine microenvironments according to lymphoid tissues, and suggest that these differences may affect the way cells respond to M. tuberculosis infection.     

The eleventh reported case of Mulvihill-Smith syndrome in the literature

Background

The Mulvihill-Smith Syndrome was first recognized in 1975. After the recognition of the Mulvihill-Smith Syndrome, ten cases have been described.

Case presentation

This article describes the eleventh case of this syndrome in a male patient, 24 years-old with short stature and microcephaly with mild cognitive impairment, deafness and allergic conjunctivitis. The patient was hospitalized several times for repeated infections, and the presence of multiple melanocytic nevi on his skin was noticed.

Conclusions

Based on the entire set of signs and symptoms presented in our study, it was diagnosed the patient with Mulvihill-Smith Syndrome.     

Generation of “LYmph Node Derived Antibody Libraries” (LYNDAL) for selecting fully human antibody fragments with therapeutic potential

The development of efficient strategies for generating fully human monoclonal antibodies with unique functional properties that are exploitable for tailored therapeutic interventions remains a major challenge in the antibody technology field. Here, we present a methodology for recovering such antibodies from antigen-encountered human B cell repertoires. As the source for variable antibody genes, we cloned immunoglobulin G (IgG)-derived B cell repertoires from lymph nodes of 20 individuals undergoing surgery for head and neck cancer. Sequence analysis of unselected “LYmph Node Derived Antibody Libraries” (LYNDAL) revealed a naturally occurring distribution pattern of rearranged antibody sequences, representing all known variable gene families and most functional germline sequences. To demonstrate the feasibility for selecting antibodies with therapeutic potential from these repertoires, seven LYNDAL from donors with high serum titers against herpes simplex virus (HSV) were panned on recombinant glycoprotein B of HSV-1. Screening for specific binders delivered 34 single-chain variable fragments (scFvs) with unique sequences. Sequence analysis revealed extensive somatic hypermutation of enriched clones as a result of affinity maturation. Binding of scFvs to common glycoprotein B variants from HSV-1 and HSV-2 strains was highly specific, and the majority of analyzed antibody fragments bound to the target antigen with nanomolar affinity. From eight scFvs with HSV-neutralizing capacity in vitro, the most potent antibody neutralized 50% HSV-2 at 4.5 nM as a dimeric (scFv)₂. We anticipate our approach to be useful for recovering fully human antibodies with therapeutic potential.     

Pore Mutations in Ammonium Transporter AMT1 with Increased Electrogenic Ammonium Transport Activity

AMT/Mep ammonium transporters mediate high affinity ammonium/ammonia uptake in bacteria, fungi, and plants. The Arabidopsis AMT1 proteins mediate uptake of the ionic form of ammonium. AMT transport activity is controlled allosterically via a highly conserved cytosolic C terminus that interacts with neighboring subunits in a trimer. The C terminus is thus capable of modulating the conductivity of the pore. To gain insight into the underlying mechanism, pore mutants suppressing the inhibitory effect of mutations in the C-terminal trans-activation domain were characterized. AMT1;1 carrying the mutation Q57H in transmembrane helix I (TMH I) showed increased ammonium uptake but reduced capacity to take up methylammonium. To explore whether the transport mechanism was altered, the AMT1;1-Q57H mutant was expressed in Xenopus oocytes and analyzed electrophysiologically. AMT1;1-Q57H was characterized by increased ammonium-induced and reduced methylammonium-induced currents. AMT1;1-Q57H possesses a 100× lower affinity for ammonium (K_m) and a 10-fold higher V_max as compared with the wild type form. To test whether the trans-regulatory mechanism is conserved in archaeal homologs, AfAmt-2 from Archaeoglobus fulgidus was expressed in yeast. The transport function of AfAmt-2 also depends on trans-activation by the C terminus, and mutations in pore-residues corresponding to Q57H of AMT1;1 suppress nonfunctional AfAmt-2 mutants lacking the activating C terminus. Altogether, our data suggest that bacterial and plant AMTs use a conserved allosteric mechanism to control ammonium flux, potentially using a gating mechanism that limits flux to protect against ammonium toxicity.All organisms depend on an adequate supply of nutrients, especially nitrogen. For microorganisms and plants, which are able to assimilate ammonium, NH₄⁺ represents the sole bioavailable nitrogen form. (Nitrate use requires enzymatic conversion to ammonia.) Plants preferentially take up ammonium; however, overaccumulation of NH₄⁺ is toxic to microorganisms and plants (1, 2.) Levels above 50 μm become toxic for the central nervous system of most mammals (3, 4). A precise homeostasis of the cellular levels of ammonium is therefore critical.Plant ammonium uptake is mediated by low affinity/high capacity and high affinity/low capacity transporters (5). Nonselective cation channels (2), potassium channels (6), and members of the aquaporin family appear to be able to mediate NH₃/NH₄⁺ low affinity uptake (7–9). High affinity uptake by transporters of the AMT/Mep superfamily is essential at supply levels in the micromolar to low millimolar range (10–12). AMT/Mep ammonium transporter genes were originally identified in yeast and plants by complementation of a yeast mutant deficient in ammonium uptake (13, 14). In contrast to potassium channels, which do not effectively differentiate between potassium and ammonium, AMTs are highly selective for ammonium and its methylated form, methylammonium (MeA).⁶ Plant AMT1 ammonium transporters were shown to be electrogenic when expressed in Xenopus oocytes, suggesting transport of charged NH₄⁺ or co-transport of NH₃ with a proton (15). Quantitation of charge movement and tracer uptake demonstrated that AMT1 transports exclusively the ionic form, i.e. each transported ¹⁴C-MeA molecule corresponded to the transfer of a single positive elementary charge across the membrane (16). The high affinity and low capacity of AMT1, which is too slow to be classified as a channel, suggests that it rather functions as a transporter, with significant conformational changes limiting its turnover numbers. Interestingly, it has been suggested that the bacterial homologs use a different mechanism, in that they mediate transport of uncharged NH₃ (17), although this hypothesis has been disputed (18, 19).Biochemical as well as structural analyses of bacterial and archaeal AMTs revealed a highly stable and conserved trimeric complex (15). Each monomer is composed of 11 transmembrane helices (TMHs) that form a noncontinuous channel through which the substrate can pass. Highly conserved residues are observed in positions that are likely crucial for function: a tryptophan located in a central extracellular surface cleft is thought to be part of a selectivity filter, discriminating K⁺ ions and water molecules from NH₄⁺ via a cation-π interaction and H-bonds via neighboring residues. Below this cleft, a pair of phenylalanines is assumed to function as a gate that blocks the entrance of the channel, which, after that point, appears open to the cytoplasmic side. Two histidines on helices V and VI are in H-bonding distance and line the central part of the channel pathway.Similar to the bacterial Na⁺/leucine and the Na⁺/arabinose transporters (20, 21), AMT monomers are built from an ancient duplication of a subunit of five TMHs, organized as a pseudo-2-fold axis in the membrane plane; in the case of the AMT/Meps, an additional 11th segment M11 (5 + 5 + 1), a 50-Å α-helix, belts the surface of the monomer at an angle of ∼50° relative to the normal vector of the membrane plane and connects to the cytosolic C terminus (17, 23, 24). Recent findings demonstrate that AMTs can exist in active and inactive states, probably controlled by phosphorylation of residues in the conserved C terminus (25).⁷ In the Arabidopsis thaliana AMT1, an allosteric trans-activation is mediated through the interaction of the C termini with cytosolic loops of the neighboring subunits in a trimer (25). This finding is consistent with a novel regulatory mechanism that can provide for rapid shut-off of transport. This feedback loop may potentially be important for protection against ammonium toxicity by limiting peak output, namely ammonium uptake capacity at high external supply. Analysis of >900 AMT homologs shows that the C terminus is highly conserved from cyanobacteria to fungi and plants, indicating that the regulatory mechanism may be conserved (25).A suppressor screen using inactive mutants carrying a mutation in the cytosolic C terminus of AMT1;1 identified mutants that had lost their strict dependence on allosteric trans-activation (25). Here, we show that, when expressed in yeast, some of these mutants show increased ammonium transport capacity. Electrophysiological analysis of one of the pore mutants, AMT1;1-Q57H, demonstrates that transport is still electrogenic and that the increased ammonium sensitivity is due to a conversion from a saturable high affinity kinetic profile to low affinity and high capacity uptake kinetics. Mutation of the corresponding glutamine residue (Q53H) also suppresses an inactive mutant of the archaeal Archaeoglobus fulgidus AfAmt-2, demonstrating the conservation of these mechanisms from archaea to higher plants.     

Patterns of MADS-box gene expression mark flower-type development in Gerbera hybrida (Asteraceae)

Background  

The inflorescence of the cut-flower crop Gerbera hybrida (Asteraceae) consists of two principal flower types, ray and disc, which form a tightly packed head, or capitulum. Despite great interest in plant morphological evolution and the tractability of the gerbera system, very little is known regarding genetic mechanisms involved in flower type specification. Here, we provide comparative staging of ray and disc flower development and microarray screening for differentially expressed genes, accomplished via microdissection of hundreds of coordinately developing flower primordia.