How can the aromatic side-chains modulate the conductance of the gramicidin channel? A new approach using non-coded amino acids.

In order to elucidate the role of the aromatic side-chains in the mechanism of transduction of monovalent cations through the channel of linear gramicidin, two series of analogues containing non-coded aromatic amino acids were synthesized. In the first series, the four tryptophans were replaced by either four L-3-(8-quinolyl)alanyl or four L-3-(4-quinolyl)alanyl residues and single channel conductance measurements showed that these substitutions led to a strong lowering of the channel conductance, which is attributed to a modification of the orientation of the aromatic side-chains due to an increase of their hydrophobicity. In the second series, the analogues contained both tryptophyl and naphthylalanyl residues in various amounts and positions. The single channel conductance data indicated that the conductance was mainly governed by the number of polar residues (Trp) and not by their positions. The conformational consequences of these results are discussed together with their influence on the energy profile of the gramicidin channel.

A common V delta 2-D delta 2-D delta 3 T cell receptor gene rearrangement in precursor B acute lymphoblastic leukaemia.

Despite their apparent commitment to the B lymphocytic lineage, human precursor B cell acute lymphoblastic leukaemias (ALL) frequently rearrange their T cell antigen receptor (TCR) alpha, beta and gamma chain genes. Since these three genes are active sites of rearrangement in precursor B cell neoplasms, it seemed that the recently discovered fourth TCR gene, delta, might be similarly rearranged. To investigate this possibility, a series of precursor B cell leukaemias was analysed for rearrangements at the delta chain gene locus, using probes of the variable, joining, and constant regions of the delta chain gene. The majority of precursor B cell ALLs in this series (25/32, 78%) showed rearrangement or deletion of one or more TCR delta genes. This contrasts sharply with a series of 16 mature B cell neoplasms (chronic lymphocytic leukaemia) in which no TCR delta gene rearrangements were detected. An unusual TCR delta rearrangement, rarely observed in normal or neoplastic T cells, was seen in the majority (14/18) of precursor B cell ALLs with TCR delta rearrangements. In contrast to the utilization ov V delta 1 in T cell ALL, detailed restriction mapping of precursor B ALL revealed an incomplete rearrangement without involvement of J delta segments. Direct genomic sequencing was performed on one example and demonstrated a nonproductive V delta 2-D delta 2-D delta 3 recombination in this precursor B ALL. We conclude that the TCR delta chain gene is an active locus in precursor B cell neoplasia, involves an unusual type of rearrangement and provides a clonal tumour marker for diagnosis of precursor B ALL.

Effects of challenge with trichostrongylid nematode larvae on immunologically resistant grazing yearling sheep in South Australia.

Paddocks near Gawler, South Australia, were grazed by newly weaned sheep which received either no anthelmintic treatment, regular 3-weekly treatment or a single treatment in February. A decline in the faecal egg count of untreated sheep in autumn associated with the ingestion of infective larvae indicated that the sheep had developed a resistance to reinfection. At the end of the experiment, 10 sheep were experimentally infected with Trichostrongylus vitrinus larvae to demonstrate this resistance. Untreated sheep grew less wool and had lower body weights when compared with treated animals and were extremely 'daggy'. A single treatment in February did not prevent production losses or scouring.

The effect of point mutations on energy profiles in a model of the nicotinic acetylcholine receptor (AChR) channel.

Energy profiles are calculated, using energy optimization computations, for a sodium cation in the AChR channel and four of its mutants, alpha E241D, beta E247Q, delta E255Q and alpha E241Q, using the model developed previously. The relative energy location of the calculated profiles confirms and specifies the role of each of the Glu residues found in the anionic ring at the bottom of the MII helices. The structural analysis of the results allows the understanding of the differences observed in the conductances for the wild-type and mutant alpha E241D, or for the mutants beta E247Q and delta E255Q in spite of the identity of the global charge of both channels in each couple. The striking correlation observed between the average relative energy location of the profiles and the conductance data appears to provide confirmation of the essential structural features adopted in the model, in particular the inclusion of the Glu(Gln in gamma)-Lys residues in the alpha-helical stretch of the MII helices and the overall location of the internal residues.

A modified technique for the estimation of the number of infective nematode larvae present on pasture, and its application in the field under South Australian conditions.

A modified technique for the recovery of larvae from pasture is described involving two centrifugations of pasture washings in a solution of potassium iodide. On average, the technique recovered 96% of larvae and was simple to perform. At three sites in South Australia (rainfall 550-330 mm year-1), the numbers of larvae present on pasture each month using this technique was compared with results obtained using one or four tracer sheep. The general pattern of larval availability was similar using the two methods, but discrepancies were noted. In some instances, particularly when numbers of larvae were low, pasture sampling underestimated the numbers of larvae available to sheep; in other situations, when the numbers of larvae were high, tracer sheep probably underestimated the number being ingested. Increasing the numbers of tracer sheep from one to four did not appreciably increase the correspondence between the two methods.

The intrinsic molecular potential of glyceryl monooleate layers and its effect on the conformation and orientation of an inserted molecule: example of gramicidin A.

It is shown by explicit calculation that the distribution of the atomic charges in the constituent molecules of a lipid monolayer or bilayer of glyceryl monooleate creates an intrinsic potential difference between the head region and the hydrocarbon region which tends to repel positive charges towards the exterior and attract negative charges to the interior. The analogies and differences between a bilayer and a monolayer are analyzed. The possible consequences of the intrinsic potential gradient in a lipid layer on the preferred orientation and conformation of a polar neutral molecule are illustrated on the case of a gramicidin A monomer.

Gamma/delta lineage relationship within a consecutive series of human precursor T-cell neoplasms.

We analyzed the gene rearrangements associated with the newly described delta T-cell receptor (TCR) gene from a series of 19 consecutive precursor T-cell (lymphoblastic) neoplasms that represent discrete stages surrounding the TCR gene rearrangement process. Significantly, the delta TCR gene showed rearrangement in most (13 of 19) of these T cells, and in addition it was rearranged in two cells displaying no rearrangement for any other TCR gene. Our survey showed three types of delta gene rearrangements associated with cell-surface TCR expression that presumably represent usage of three V delta genes. This analysis demonstrates (1) a major subclass of human precursor T-cell neoplasms belonging to the gamma/delta T-cell receptor-rearranging subtype; (2) a narrow repertoire of human V delta gene usage; and (3) the utility of delta gene rearrangements as a diagnostic clonal marker in precursor T lymphoblastic neoplasms.

A possible model for the inner wall of the acetylcholine receptor channel.

A structural model of the inner wall of the acetylcholine receptor (AChR) channel is developed using assumptions derived from the results of the recent labelling experiments of the MII helices by noncompetitive blockers. The assumption of steric blocking of the channel by chlorpromazine (CPZ) in the neighbourhood of the labelled serines imposes the MII helices to be in contact at this level and allows the calculation of their minimal interaxial distance. The assumption that CPZ diffuses to this position through the upper crowded part of the channel imposes that the helices are more distant in this region and permits the determination of a tilt of about 7 degrees with respect to the central axis. Electrostatic potentials are used to demonstrate the effect of the charged residues at the exit of the pore. A discussion is given on the possible aptitude of MI to satisfy the contacts necessary with the MII/s at the different heights of the model.

Effects of temperature and relative humidity on development and survival of the free-living stages of Trichostrongylus colubriformis, T. rugatus and T. vitrinus.

Temperatures of 4 and 10 degrees C reduced the rate of hatching of eggs and the development of the pre-infective stages of T. colubriformis and T. rugatus, but not T. vitrinus. Eggs of T. rugatus hatched at 4 degrees C, but did not develop to the infective stage. At 20 and 30 degrees C, no differences were detected between species in egg hatching or the development of infective larvae. Third-stage larvae of T. rugatus survived for significantly longer periods of time at 33 and 56% relative humidities at a temperature of 20 degrees C than did either T. colubriformis or T. vitrinus. At 30 degrees C, differences were less marked, but T. rugatus and T. colubriformis survived longer than T. vitrinus. The results are discussed in relationship to the known distribution of these species in eastern Australia.

Energy profiles in the acetylcholine receptor (AChR) channel. The MII-helix model and the role of the remaining helices.

It is demonstrated by theoretical computations that no favorable energy profile for cation transfer can be obtained in a model of the AChR channel constructed with the sole five MII helices of the inner wall. A favorable profile is obtained upon including the effect of the remaining helices of the five subunits. The decisive role, for the exit of the ion, of the charged residues situated at the N-terminal of the MII segments, established before, is underlined further. The role of the other elements of the channel wall (peptide carbonyl oxygens, hydrocarbon residues and polar side chains) is analyzed.

The effects on merino lambs of chronic infection with Trichostrongylus rugatus.

Four groups of six 5-month-old merino lambs were exposed to infection with 12,000 larvae of Trichostrongylus rugatus per week and one group was killed at each of 3, 6, 9 and 12 weeks. Numbers of adult worms increased in an approximately linear manner throughout the experiment; the percentage of larvae developing to adults remained between 35 and 25% throughout the course of the experiment. Sub-total villus atrophy was evident in the anterior small intestine of each group of infected sheep and significant reductions occurred in the plasma concentrations of albumin and phosphate. Serum globulin and calcium concentrations rose in infected animals. No clinical signs were observed in infected sheep, no alteration in bone histology was detected and no decrease in growth rate occurred.

Comparison of the effects of infection with Trichostrongylus colubriformis, T. vitrinus and T. rugatus in merino lambs.

Groups of six worm-free merino lambs were infected with 100,000 larvae of T. colubriformis, T. vitrinus, T. rugatus or a mixture of the three species and were slaughtered 21 days later. Nematodes were distributed predominantly in the first 3 m of the small intestine where they caused subtotal villus atrophy. T. vitrinus caused severe erosion of the epithelium; erosion was less severe with T. colubriformis infections and mild with T. rugatus infections. Plasma concentrations of albumin declined during the experiment and were most marked in sheep infected with T. vitrinus. Plasma phosphate concentrations declined during infection with all three species, while plasma concentrations of globulin and calcium declined only in sheep infected with T. vitrinus. Food intake was reduced in all groups, but was most marked with sheep infected with T. vitrinus. T. vitrinus appeared to be more pathogenic than the other species.

Conformation and pairing properties of the N-terminal fragments of trichorzianine and alamethicin: a theoretical study.

Energy optimizations are carried out on the N-terminal fragment of trichorzianine in comparison to that of alamethicin. The results indicate that the helical character of the (Ac...Pro13) sequence of trichorzianine (TA IIIc) is essentially alpha with a bend in the helix axis in the end proline region, a structure comparable to the optimal alpha-helical structure of the corresponding segment (Ac...Pro14) of alamethicin AI. However, two weak n----n + 3 interactions coexist in trichorzianine with the alpha-helical n----n + 4 hydrogen bonds. The possible role of the glutamine side-chains in pairing such segments together is considered.

Energy profile of Cs+ in gramicidin A in the presence of water. Problem of the ion selectivity of the channel.

The effect of water present at the mouth and inside the channel of Gramicidin A on the energy profile calculated for a caesium ion is determined. The total optimal interaction energy computed for the system GA-Cs+-(22 waters) leads to an energy profile characterized by a deep minimum at 11 A followed by an entrance energy barrier of 7 Kcal/mol expanding until 9 A from the center. After this point, a second minimum less deep than the previous one is observed, itself followed by a central barrier. The shape of the profile at the entrance is governed by the balance between the progressive desolvation process of the ion and the increase of favorable hydrogen bond interactions implying both the water molecules and GA. The comparison of this energy profile with that obtained in vacuo shows that the presence of water molecules does not modify the pathway of the ion which, owing to its size, is constrained essentially to remain on the channel axis. The comparison Na+ versus Cs+ indicates that although the phenomena involved are globally the same, differences between the two profiles appear due firstly to the difference in the affinity of the two ions for water and secondly to their respective size. This last difference implies that the number of water molecules present in the interior of the channel during the cation progression is reduced roughly by one in the case of caesium. The desolvation barrier computed for Cs+ is half the corresponding value for Na+, a result in agreement with the observed selectivity.

Theoretical study of the packing of alpha-helices into possible transmembrane bundles. Sequences including alanines, leucines and serines.

Energy optimizations are carried out on packages of two and five alpha-helices containing leucines on their faces of contact and made otherwise of alanines. The effect of these bulky side-chains on the optimal arrangements is analysed and compared to the results previously obtained for pure poly(L-alanine) packages; the essential pairing properties are conserved (near antiparallelism, preponderous role of the non-bonded interactions, possibility of existence of parallel pairs); five alpha-helices made of 8 alanines and 6 leucines (three on each interface) can pack in different stable P5L bundles including various holes, according to the tilt and relative sliding of the helices. Substitution of serines to the alanines lying on the inner wall affects very little the interhelix packing. The seryl side chains adapt their conformation at best to their surroundings. The P5L packages can be used to represent individual subunits arranged in 'superbundles' around a central pit in a channel-forming protein.

Experimental and theoretical study of gramicidin P, an analog of gramicidin A with a methylamine C-terminal.

Gramicidin P (a gramicidin A in which the ethanolamine C-terminus is replaced by methylamine) was synthesized and shown to have the same single-channel conductance behavior as gramicidin A. The results are discussed in connection with the energy profile computed in the presence of water in comparison with the corresponding profile for gramicidin A.