Fine specificity of HLA-B27 cellular allorecognition. HLA-B27f is a functional variant distinguishable by cytolytic T cell clones

Three HLA-B27 allospecific cytolytic T lymphocyte (CTL) clones were isolated by limiting dilution of HLA-B27-negative responder cells stimulated with HLA-B27.1-positive lymphoblastoid cells. These clones displayed three distinct reaction patterns when tested for their lytic ability against target cells expressing various structurally defined HLA-B27 subtypes. One of the clones was specific for HLA-B27.1; a second CTL clone reacted only with B27.1 and, less efficiently, with B27.2; the third clone recognized both B27.1 and B27f targets but not cells expressing any other B27 subtype. These results indicate that HLA-B27f is a functional variant amenable to differential recognition by alloreactive CTL. A correlation of the structure of the HLA-B27 subtypes with the reactivity of these clones revealed that multiple B27-specific alloreactive CTL are activated against epitopes of the HLA-B27.1 molecule sharing common structural features. This illustrates the complexity and fine specificity of the allogeneic CTL response against class I HLA antigens and suggests that their immunodominant regions are those which are capable of eliciting a diverse polyclonal response against each of these regions, rather than inducing the selective expansion of a single T cell clone.     

Calcium antagonists and heat-induced hepatic injury

Several laboratories have demonstrated the value of the isolated perfused rat liver as a suitable model for heat-induced hepatic injury in vivo. Membrane changes caused by perfusion of rat livers at 42 degrees C for 90 min were similar to those induced by toxic chemicals or hypoxia. In an evaluation of several categories of drugs reported to reduce cell injury, calcium antagonists (nifedipine, dantrolene, and verapamil), were evaluated for their therapeutic potential for heat injury. Isolated rat livers were perfused at 42 degrees C for 90 min with and without calcium antagonists. Livers were also perfused at 37 degrees C. Potassium and transaminase leakage, bile production and ultrastructure were used to evaluate their responses. Neither of the three calcium antagonists significantly improved any of the functional parameters measured. However, dantrolene produced dilated or vesicular rough endoplasmic reticulum in the heated livers. These changes suggest selective intracellular action on endoplasmic reticulum of heated livers. Ring-shaped mitochondria and vesicular endoplasmic reticulum were observed in the heated, verapamil-treated livers, but these could not be quantitatively distinguished from controls. Nifedipine did not appear to alter intracellular membranes, but did increase bile production.     

Characterization of messenger RNA populations of Crithidia fasciculata

Cells of Crithidia fasciculata taken from exponentially growing cultures display a relatively high content of poly(A) RNA (roughly 45% of the total) in a nonpolysomal compartment. The proportion of this fraction is significantly higher than that of the free ribosomal units. This RNA is translationally competent and is indistinguishable from polysomal mRNA by various criteria. The significance of these findings is discussed with respect to the protein synthesis capability of the cell and the metabolic relationships between the cytoplasmic compartments of mRNA.     

Application of a statistical design to the optimization of culture medium for recombinant interferon-gamma production by Chinese hamster ovary cells

Summary The importance of serum-free medium components on the growth of Chinese hamster ovary (CHO) cells and production of recombinant human interferon(IFN)-gamma was investigated. The complexity of the medium led to the adoption of a statistical optimization approach based on a Plackett-Burman design. From this analysis a set of nutritional components was identified as important for cell growth and recombinant protein production. Glycine was identified as an important determinant of specific growth rate, whereas for cell production bovine serum albumin (BSA), phenylalanine and tyrosine were also identified as important. BSA, sodium pyruvate, glutamate, methionine, proline, histidine, hydroxyproline, tyrosine and phenylalanine were shown to be important for IFN-gamma production. Other medium components, such as insulin, arginine, aspartate and serine produced an inhibitory effect on both cell growth and IFN-gamma production. The effect of the stimulatory nutrients as a whole group was tested by increasing their concentration in the medium. A significant improvement in specific cell growth rate, cell production and IFN-gamma production (up to 45%) was achieved on both shake-flask and fermentor cultures. An increase in the medium concentration of the negative variables had only a small inhibitory effect (approximately 10%) on the same parameters. Analysis of the effects of the group of stimulatory amino acids and BSA on CHO cell growth showed that the effect of the former was independent of BSA.Correspondence to: A. T. Bull     

Weed Mapping in Early-Season Maize Fields Using Object-Based Analysis of Unmanned Aerial Vehicle (UAV) Images

The use of remote imagery captured by unmanned aerial vehicles (UAV) has tremendous potential for designing detailed site-specific weed control treatments in early post-emergence, which have not possible previously with conventional airborne or satellite images. A robust and entirely automatic object-based image analysis (OBIA) procedure was developed on a series of UAV images using a six-band multispectral camera (visible and near-infrared range) with the ultimate objective of generating a weed map in an experimental maize field in Spain. The OBIA procedure combines several contextual, hierarchical and object-based features and consists of three consecutive phases: 1) classification of crop rows by application of a dynamic and auto-adaptive classification approach, 2) discrimination of crops and weeds on the basis of their relative positions with reference to the crop rows, and 3) generation of a weed infestation map in a grid structure. The estimation of weed coverage from the image analysis yielded satisfactory results. The relationship of estimated versus observed weed densities had a coefficient of determination of r²=0.89 and a root mean square error of 0.02. A map of three categories of weed coverage was produced with 86% of overall accuracy. In the experimental field, the area free of weeds was 23%, and the area with low weed coverage (<5% weeds) was 47%, which indicated a high potential for reducing herbicide application or other weed operations. The OBIA procedure computes multiple data and statistics derived from the classification outputs, which permits calculation of herbicide requirements and estimation of the overall cost of weed management operations in advance.     

Population genetics in the american tropics. VIII. The courtship isolation within Drosophila paulistorum incipient “species”

The so called races of Drosophila paulistorum from South America are so deeply isolated ethologically that although still partially bridged by gene flow through hybridization, they should be considered as 6 separate sibling species just as the willistoni group to which D. paulistorum itself belongs. The present article emphasizes the way in which isolation is implemented.This paper is dedicated to him on his 72nd birthday for his many contributions to our field.     

Early and Middle Pleistocene hominins from Atapuerca (Spain) show differences in dental developmental patterns

The Bayesian statistical approach considers teeth as forming a developmental module, as opposed to a tooth-by-tooth analysis. This approach has been employed to analyze Upper Pleistocene hominins, including Neandertals and some anatomically modern humans, but never earlier populations. Here, we show its application on five hominins from the TD6.2 level of the Gran Dolina site (Homo antecessor, Early Pleistocene) and the Sima de los Huesos site (Middle Pleistocene) of the Sierra de Atapuerca (Burgos, northern Spain). Our results show an advanced development of the third molars in both populations with respect to modern Homo sapiens. In addition, the Sima de los Huesos hominins differ from H. sapiens and H. antecessor in the relatively advanced development of their second molar. The relative mineralization of I1/M1 in H. antecessor appears to be similar to that of modern humans, as opposed to that of Neandertals, which appear to be unique. These observations, combined with reduced enamel formation times and the advanced development of the third molars, appear to indicate a shorter ontogenetic period in the hominins from Gran Dolina and Sima de los Huesos in comparison to modern human average.     

Structural basis for the modulation of plant cytosolic triosephosphate isomerase activity by mimicry of redox‐based modifications

Reactive oxidative species (ROS) and S‐glutathionylation modulate the activity of plant cytosolic triosephosphate isomerases (cTPI). Arabidopsis thaliana cTPI (AtcTPI) is subject of redox regulation at two reactive cysteines that function as thiol switches. Here we investigate the role of these residues, AtcTPI‐Cys13 and At‐Cys218, by substituting them with aspartic acid that mimics the irreversible oxidation of cysteine to sulfinic acid and with amino acids that mimic thiol conjugation. Crystallographic studies show that mimicking AtcTPI‐Cys13 oxidation promotes the formation of inactive monomers by reposition residue Phe75 of the neighboring subunit, into a conformation that destabilizes the dimer interface. Mutations in residue AtcTPI‐Cys218 to Asp, Lys, or Tyr generate TPI variants with a decreased enzymatic activity by creating structural modifications in two loops (loop 7 and loop 6) whose integrity is necessary to assemble the active site. In contrast with mutations in residue AtcTPI‐Cys13, mutations in AtcTPI‐Cys218 do not alter the dimeric nature of AtcTPI. Therefore, modifications of residues AtcTPI‐Cys13 and AtcTPI‐Cys218 modulate AtcTPI activity by inducing the formation of inactive monomers and by altering the active site of the dimeric enzyme, respectively. The identity of residue AtcTPI‐Cys218 is conserved in the majority of plant cytosolic TPIs, this conservation and its solvent‐exposed localization make it the most probable target for TPI regulation upon oxidative damage by reactive oxygen species. Our data reveal the structural mechanisms by which S‐glutathionylation protects AtcTPI from irreversible chemical modifications and re‐routes carbon metabolism to the pentose phosphate pathway to decrease oxidative stress.