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Tools for genome-wide rapid identification of peptide-major histocompatibility complex targets of T-cell receptors (TCR) are not yet universally available. We present a new antigen screening method, the T-synapse (Tsyn) reporter system, which includes antigen-presenting cells (APC) with a Fas-inducible NF-κB reporter and T cells with a nuclear factor of activated T cells (NFAT) reporter. To functionally screen for target antigens from a cDNA library, productively interacting T cell-APC aggregates were detected by dual-reporter activity and enriched by flow sorting followed by antigen identification quantified by deep sequencing (Tsyn-seq). When applied to a previously characterized TCR specific for the E7 antigen derived from human papillomavirus type 16 (HPV16), Tsyn-seq successfully enriched the correct cognate antigen from a cDNA library derived from an HPV16-positive cervical cancer cell line. Tsyn-seq provides a method for rapidly identifying antigens recognized by TCRs of interest from a tumor cDNA library. See related Spotlight by Makani and Joglekar, p. 515., (©2024 American Association for Cancer Research.)

This communication reports the effect of alphaxalone on motor somatosensory evoked potential (SEPs) in a rhesus macaque. The animal was deeply anaesthetised with an infusion of ketamine, medetomidine, midazolam and alfentanil. The median nerve was stimulated, and SEPs were recorded from the motor cortex. The successive administration of three doses of alphaxalone (0.5, 1 and 2 mg/kg) induced an increase of the latency time and a decrease of the amplitude of the SEPs. However, the structure of the waveforms was conserved, and hence alphaxalone might represent a suitable general anaesthetic option in neuroscience research as well as veterinary or human medicine.

Anatomical studies report a large proportion of fine myelinated fibers in the primate pyramidal tract (PT), while very few PT neurons (PTNs) with slow conduction velocities (CV) (<~10 m/s) are reported electrophysiologically. This discrepancy might reflect recording bias toward fast PTNs or prevention of antidromic invasion by recurrent inhibition (RI) of slow PTNs from faster axons. We investigated these factors in recordings made with a polyprobe (32 closely-spaced contacts) from motor cortex of anesthetized rats (n = 2) and macaques (n = 3), concentrating our search on PTNs with long antidromic latencies (ADLs). We identified 21 rat PTNs with ADLs >2.6 ms and estimated CV 3-8 m/s, and 67 macaque PTNs (>3.9 ms, CV 6-12 m/s). Spikes of most slow PTNs were small and present on only some recording contacts, while spikes from simultaneously recorded fast-conducting PTNs were large and appeared on all contacts. Antidromic thresholds were similar for fast and slow PTNS, while spike duration was considerably longer in slow PTNs. Most slow PTNs showed no signs of failure to respond antidromically. A number of tests, including intracortical microinjection of bicuculline (GABAA antagonist), failed to provide any evidence that RI prevented antidromic invasion of slow PTNs. Our results suggest that recording bias is the main reason why previous studies were dominated by fast PTNs., (© The Author(s) 2019. Published by Oxford University Press.)

The Stomacher is very widely used in food and medical research for extracting tissues. To determine whether nematode larvae were disrupted by the Stomacher, L3 larvae of Haemonchus contortus were homogenised for up to 40 min at full power but no larval disruption occurred. Therefore, tissue from the mucosa and submucosa of the caecum of horses collected from a licenced abattoir was treated to determine whether inhibited cyathostomin larvae could be extracted. The optimum time on full power for a 10-g sample was 20 min, and in three out of five caecal samples from different horses, significantly more larvae were recovered than with 6 h pepsin HCl digestion. It is concluded that the Stomacher provides a simple fast method of extracting inhibited nematode larvae from gastrointestinal tissues in the horse that could replace digestion with pepsin HCl.

The ability to lactate is a process restricted to mammals and is necessary for the survival of nonhuman mammals. Female mice carrying a null mutation in the winged helix transcription factor Foxb1 (previously Mf3/Fkh5/TWH) have lactation defects on inbred genetic backgrounds. To determine the cellular basis of the Foxb1 lactation defect we have inserted a tau-lacZ lineage marker into the locus to follow the fate of Foxb1 expressing cells. This approach has revealed that Foxb1 is expressed in epithelial cells of developing and adult mammary glands as well as previously described regions of the central nervous system. Mammary glands from C57BL/6 Foxb1-/- mice have incomplete lobuloalveolar development. In addition, the tau-lacZ lineage label was used to determine that the mammillothalamic tract was lost in all Foxb1-/- mice. Finally, morphological defects in the inferior colliculi of the midbrain in Foxb1-/- mice correlate with the inability to lactate, suggesting that the midbrain defect, but not the loss of the mammillothalamic tract, may be responsible for the lactation defect., (Copyright 2001 Wiley-Liss, Inc.)

A bifunctional enzyme, 2-hydroxyhepta-2,4,-diene-l,7-dioate isomerase/5-oxopent-3-ene-1,2,5-tricarboxylate decarboxylase from the homoprotocatechaute (HPC) degradative pathway of Escherichia coli has been crystallized, using polyethylene glycol as a precipitant. The enzyme, of molecular weight 44 514 Da forms crystals belonging to the orthorhombic space group P2(1)2(1)2(1) with cell dimensions a = 106, b = 127 and c = 139 A. The crystals diffract to at least 2.2 A resolution using synchrotron radiation. A complete native data set has been collected to 3.3 A resolution. The Matthews number calculated for a single molecule in the asymmetric unit is outside the normally acceptable limits and the aggregation state of the molecules in the crystal was investigated using self-rotation function studies, the results show features which are consistent with a tetramer in the asymmetric unit, giving a V(m) value of 2.7 A(3) Da(-1).

gammabeta-crystallin is a structural protein of the eye lens with a role in the maintenance of an even distribution of protein and water over distances around the wavelength of light, preserving lens transparency. The structure of the 174-residue bovine protein has already been determined at room temperature to 1.47 A resolution. By flash freezing the protein crystals, data have now been collected to a nominal resolution limit of 1.2 A as radiation damage was essentially eliminated. The protein-water model has been refined against this data using the program RESTRAIN converging to an R factor of 18.5% with all data. Atomic positions are clearly indicated in the electron-density maps. Discrete bimodal disorder has been visualized for a few side chains. Out of a total of 498 water molecules present in the crystal asymmetric unit, 394 have been modelled and refined at unit occupancy. The solvent structure is extremely well ordered with an average B value of 23.4 A(2). Partially occupied sites have been identified where disorder in the protein induces concomitant disorder in the local solvent structure. The solvent structure covers 97% of the solvent-exposed surface of the protein in the crystal. 126 water molecules are distributed in second and higher hydration shells. There are networks of hydrogen-bonded solvent extending up to 64 molecules in a network, comprising trimers and tetramers as well as five- and six-membered water-ring structures. The hydration of the protein surface is dominated by arginine and aspartate side chains. Extensive cages of highly ordered solvent molecules are also observed around exposed non-polar groups.

OppA is a 58.8 kDa bacterial transport protein involved in the transport of peptides across the cytoplasmic membrane of Gram-negative bacteria. It binds peptides from two to five residues in length but with little sequence specificity. OppA from Salmonella typhimurium has been cloned and expressed in E. coli and the protein cocrystallized with uranyl acetate, producing two distinct crystal forms with different uranium sites. Multiple-wavelength data collected about the uranium L(III) edge have been collected at the Daresbury Synchrotron Radiation Source (SRS) to a nominal resolution limit of 2.3 A. Maximum-likelihood phasing methods have been used in phase determination from the multiple-wavelength data giving a readily interpretable electron-density map, without any density modification. The electron-density map, calculated at 2.3 A resolution shows OppA to be a bilobal, principally beta-stranded, three-domain protein. The tri-lysine ligand molecule can be clearly seen in the peptide-binding site between the two lobes.

Crystals of C-reactive protein from rat have been grown both with and without calcium. Two major components of the protein have been resolved on the basis of their calcium-dependent fine specificity for monophosphate esters. Crystals grown without calcium are tetragonal, space group P42(1)2 with unit cell parameters a = b = 163.81(9)A and c = 125.21(6)A, and diffract X-rays to 3.0 A resolution. The rotation function specifies a molecular 5-fold symmetry axis at 24 degrees away from c in the (110) plane.

Pancreatic polypeptide has been extracted and sequenced from a wide range of species. The 36-residue polypeptides have some hormonal characteristics, and show a high degree of sequence homology. Two recently isolated polypeptides, from porcine gut and brain, also show a high degree of sequence homology with the pancreatic polypeptides. It was proposed that these polypeptides were members of a related family. The X-ray determined structure of one member of the family, turkey pancreatic polypeptide, is known to high resolution, but there is no structural information for the others. Studies designed to give an insight into the tertiary structure of these related molecules have been carried out, including model building using interactive computer graphics, circular dichroic spectroscopy and secondary structure prediction using a variety of algorithms. The results indicate that a compact globular conformation, similar to that observed in turkey pancreatic polypeptide may be adopted by all molecules and that this may be more highly conserved than the individual amino acid sequences.

The sequences of urokinase (UK) and tissue-type plasminogen activator (TPA) were aligned with those of chymotrypsin, trypsin, and elastase according to their 'structurally conserved regions'. In spite of its trypsin-like specificity UK was model-built on the basis of the chymotrypsin structure because of a corresponding disulfide pattern. The extra disulfide bond falls to cysteines 50 and 111d. Insertions can easily be accommodated at the surface. As they occur similarly in both, UK and TPA, a role in plasminogen recognition may be possible. Of the functional positions known to be involved in substrate or inhibitor binding, Asp 97, Lys 143 and Arg 217 (Leu in TPA) may contribute to plasminogen activating specificity. PTI binding may in part be impaired by structural differences at the edge of the binding pocket.

aPP is a 36-amino acid polypeptide which forms a stable globular structure stabilised by hydrophobic interactions between a polyproline-like helix and an alpha-helix. Crystals contain dimers and are crosslinked by coordination through zinc ions leading to a well-ordered lattice which diffracts X-rays to a resolution of 0.98A. This gives a 5:1 ratio of observations-to-parameters even when anisotropic thermal ellipsoids defined by six parameters for each non-hydrogen atom were refined using least-squares techniques. Rigid body refinement of groups within the polypeptide was also undertaken. The relationship of the principal axes of individual thermal ellipsoids and the librations of rigid side groups to features of secondary, tertiary, and quaternary structures of aPP and its interactions with water molecules are described.

Serum amyloid A protein (SAA), an apolipoprotein of high density lipoprotein (HDL), is an acute phase protein thought to be the precursor of amyloid fibrils in reactive systemic (AA) amyloidosis. A prediction of the secondary structure of the human serum amyloid protein SAA1(alpha) is presented. The prediction was based upon one-dimensional Fourier analysis of the amino acid sequence together with sequence matching to known structural motifs. The results were compared with those from prediction algorithms based upon statistical techniques. Our findings are consistent with available experimental data. They include the putative identification of the amino-terminal 11 residues as the functionally important lipid-binding site of SAA and of a likely, neutral, calcium-binding sequence: Gly48-Pro49-Gly50-Gly51. Sequence comparisons between SAA and protein tyrosine kinases, phospholipases A2 and delta-crystallin, all of which bind both calcium and phospholipid, revealed significant homologies that support our proposals concerning structure-function relationships in SAA.