Skin graft rejection elicited by beta 2-microglobulin as a minor transplantation antigen involves multiple effector pathways: role of Fas-Fas ligand interactions and Th2-dependent graft eosinophil infiltrates

Beta(2)-microglobulin (beta(2)m)-derived peptides are minor transplantation Ags in mice as beta(2)m-positive skin grafts (beta(2)m(+/+)) are rejected by genetically beta(2)m-deficient recipient mice (beta(2)m(-/-)). We studied the effector pathways responsible for the rejection induced by beta(2)-microglobulin-derived minor transplantation Ags. The rejection of beta(2)m(+/+) skin grafts by naive beta(2)m(-/-) mice was dependent on both CD4 and CD8 T cells as shown by administration of depleting mAbs. Experiments performed with beta(2)m(-/-)CD8(-/-) double knockout mice grafted with a beta(2)m(+/+) MHC class I-deficient skin showed that sensitized CD4 T cells directed at beta(2)m peptides-MHC class II complexes are sufficient to trigger rapid rejection. Rejection of beta(2)m(+/+) grafts was associated with the production of IL-5 in vitro, the expression of IL-4 and IL-5 mRNAs in the grafted tissue, and the presence within rejected grafts of a considerable eosinophil infiltrate. Blocking IL-4 and IL-5 in vivo and depleting eosinophils with an anti-CCR3 mAb prevented graft eosinophil infiltration and prolonged beta(2)m(+/+) skin graft survival. Lymphocytes from rejecting beta(2)m(-/-) mice also displayed an increased production of IFN-gamma after culture with beta(2)m(+/+) minor alloantigens. In vivo neutralization of IFN-gamma inhibited skin graft rejection. Finally, beta(2)m(+/+) skin grafts harvested from B6(lpr/lpr) donor mice, which lack a functional Fas molecule, survived longer than wild-type beta(2)m(+/+) skin grafts, showing that Fas-Fas ligand interactions are involved in the rejection process. We conclude that IL-4- and IL-5-dependent eosinophilic rejection, IFN-gamma-dependent mechanisms, and Fas-Fas ligand interactions are effector pathways in the acute rejection of minor transplantation Ags.     

Mad2 and BubR1 function in a single checkpoint pathway that responds to a loss of tension

The spindle checkpoint monitors microtubule attachment and tension at kinetochores to ensure proper chromosome segregation. Previously, PtK1 cells in hypothermic conditions (23 degrees C) were shown to have a pronounced mitotic delay, despite having normal numbers of kinetochore microtubules. At 23 degrees C, we found that PtK1 cells remained in metaphase for an average of 101 min, compared with 21 min for cells at 37 degrees C. The metaphase delay at 23 degrees C was abrogated by injection of Mad2 inhibitors, showing that Mad2 and the spindle checkpoint were responsible for the prolonged metaphase. Live cell imaging showed that kinetochore Mad2 became undetectable soon after chromosome congression. Measurements of the stretch between sister kinetochores at metaphase found a 24% decrease in tension at 23 degrees C, and metaphase kinetochores at 23 degrees C exhibited higher levels of 3F3/2, Bub1, and BubR1 compared with 37 degrees C. Microinjection of anti-BubR1 antibody abolished the metaphase delay at 23 degrees C, indicating that the higher kinetochore levels of BubR1 may contribute to the delay. Disrupting both Mad2 and BubR1 function induced anaphase with the same timing as single inhibitions, suggesting that these checkpoint genes function in the same pathway. We conclude that reduced tension at kinetochores with a full complement of kinetochore microtubules induces a checkpoint dependent metaphase delay associated with elevated amounts of kinetochore 3F3/2, Bub1, and BubR1 labeling.     

EB1 targets to kinetochores with attached,polymerizing microtubules

Microtubule polymerization dynamics at kinetochores is coupled to chromosome movements, but its regulation there is poorly understood. The plus end tracking protein EB1 is required both for regulating microtubule dynamics and for maintaining a euploid genome. To address the role of EB1 in aneuploidy, we visualized its targeting in mitotic PtK1 cells. Fluorescent EB1, which localized to polymerizing ends of astral and spindle microtubules, was used to track their polymerization. EB1 also associated with a subset of attached kinetochores in late prometaphase and metaphase, and rarely in anaphase. Localization occurred in a narrow crescent, concave toward the centromere, consistent with targeting to the microtubule plus end-kinetochore interface. EB1 did not localize to kinetochores lacking attached kinetochore microtubules in prophase or early prometaphase, or upon nocodazole treatment. By time lapse, EB1 specifically targeted to kinetochores moving antipoleward, coupled to microtubule plus end polymerization, and not during plus end depolymerization. It localized independently of spindle bipolarity, the spindle checkpoint, and dynein/dynactin function. EB1 is the first protein whose targeting reflects kinetochore directionality, unlike other plus end tracking proteins that show enhanced kinetochore binding in the absence of microtubules. Our results suggest EB1 may modulate kinetochore microtubule polymerization and/or attachment.     

Role of chemical and visual cues in food recognition by leatherback posthatchlings (Dermochelys coriacea L)

We raised leatherback posthatchlings in the laboratory for up to 7 weeks to study the role of visual and chemical cues in food recognition and food-seeking behavior. Turtles were reared on a formulated (artificial gelatinous) diet and had no contact with test materials until experiments began. Subjects were presented with visual cues (a plastic jellyfish; white plastic shapes [circle, square, diamond] similar in surface area to the plastic model), chemical cues (homogenates of lion's mane jellyfish, Cyanea capillata; moon jellyfish, Aurelia aurita; and a ctenophore, Ocyropsis sp., introduced through a water filter outflow), and visual and chemical cues presented simultaneously. Visual stimuli evoked an increase in swimming activity, biting, diving, and orientation toward the object. Chemical cues elicited an increase in biting, and orientation into water currents (rheotaxis). When chemical and visual stimuli were combined, turtles ignored currents and oriented toward the visual stimuli. We conclude that both cues are used to search for, and locate, food but that visual cues may be of primary importance. We hypothesize that under natural conditions turtles locate food visually, then, as a consequence of feeding, associate chemical with visual cues. Chemical cues then may function alone as a feeding attractant.     

Development and progression of malignancy in human colon tissues are correlated with expression of specific Ca(2+)-binding S100 proteins

The expression levels of seven different S100 proteins (S100A1, S100A2, S100A3, S100A4, S100A5, S100A6, and S100B) were characterized by immunohistochemistry in the epithelial versus connective tissues of a series of 35 colon specimens, including 6 normal samples, 5 adenomas with low-grade dysplasia, 5 adenomas with high-grade dysplasia, and 19 cancers. The results showed that S100A2, S100A3, and S100B proteins could not (or only marginally) be detected in colon tissues. On the other hand, the expression of S100A6 increased in epithelial tissues directly proportional to the increase of malignancy. The percentage of epithelial (or connective tissue) cells expressing S100A4 significantly decreased as the malignancy grade increased. The expression level of S100A1 proteins was somewhat higher in the connective tissues of normal cases and adenomas with low-grade dysplasia than in adenomas with high-grade dysplasia and cancers. This pattern of expression was not observed in epithelial tissues. While the node-positive cancers did not express S100A1, about half of the node-negative specimens did. The expression levels of S100A5 were similar in different epithelial tissues. However, in the connective tissues the expression levels decreased inversely proportional to the increase in pathological grading of the specimens. Therefore, the present study implicates several S100 proteins as useful tools for histochemical typing of colon cancer malignancy development.     

Characterization of ligands for galectins, natural galactoside-binding immunoglobulin G subfractions and sarcolectin and also of the expression of calcyclin in thyroid lesions

The purpose of this study was to characterize ligands for galectins, natural galactoside-binding immunoglobulin G subfractions and sarcolectin and also the expression of calcyclin in various benign and malignant thyroid lesions. The extent of the binding of eight glycochemical probes was quantitatively assessed using computer-assisted microscopy on 76 thyroid lesions including 10 not-otherwise-specified multinodular goiters (S_MNG), 11 multinodular goiters with adenomatous hyperplasia (AH_MNG), 8 normomacrovesicular (NM_ADE) and 12 microvesicular (MIC_ADE) adenomas, and 9 papillary (P_CAR), 10 follicular variants of papillary (FvarP_CAR), 7 follicular (F_CAR) and 9 anaplastic (A_CAR) carcinomas. The 8 histochemical probes included 5 animal lectins (including galectins and sarcolectin), 1 polyclonal antibody (raised against calcyclin) and 2 immunoglobulin G subfractions from human serum with selectivity to alpha- and beta-galactosyl residues. The results show that multinodular goiters with adenomatous hyperplasia exhibited histochemical characteristics intermediate to those of normal multinodular goiters and microvesicular adenomas. Normomacrovesicular adenomas behaved very distinctly from microvesicular ones. Microvesicular adenomas were more closely related to differentiated thyroid carcinomas than any other type of benign thyroid lesions of epithelial origin. Papillary and follicular carcinomas seemed to represent the two extremes of the same biological entity with the follicular variant of the papillary carcinoma serving as a biological link between these two extremes. Anaplastic carcinomas behaved in a significantly different manner when compared to the differentiated forms of thyroid carcinomas. The results suggest that the patterns of expression of the glycoconjugates investigated in the present study may constitute useful tools for characterizing lesions in the human thyroid.     

Phylogenetic analysis of the vertebrate glycoprotein hormone family including new sequences of sturgeon (Acipenser baeri) beta subunits of the two gonadotropins and the thyroid-stimulating hormone

The beta subunits of the two gonadotropins (GTH1 and GTH2) and of the thyroid-stimulating hormone (TSH) of a chondrostean fish, Acipenser baeri, were cloned. These new sequences and selected representative members of beta subunits of vertebrate glycoprotein hormones, including tetrapod follicle-stimulating hormones (FSH) and luteinizing hormones (LH), allowed us to infer the phylogenetic relationships within this family. Both distance matrix and maximum parsimony methods were used on both nucleotide and amino acid sequences, with bootstrapping evaluation over 1000 replicates. The four trees obtained had highly similar topologies. In each case, three monophylogenetic lineages, TSH, GTH1-FSH, and GTH2-LH were clearly identified. The three monophylogenetic lineages were supported by 21-23 specific characters at the amino acid level, out of a total of 121 characters. The resolved topologies within each monophyletic hormone cluster were congruent with the known phylogenetic relationships between the related species. The inferred parental relationships within gonadotropins are in agreement with data concerning their biological functions. The present study demonstrates that GTH1 and GTH2 are the actinopterygian homologues of tetrapod FSH and LH, respectively.     

Influence of impeller type on power input in fermentation vessels

Prior investigations comparing radial flow Rushton impellers with axial flow hydrofoil impellers (Maxflo T and A315) were extended at the pilot scale. Six types of impellers (disk-style Rushton, Prochem Maxflo T hydrofoils of three diameters pumping downwards and A315 hydrofoils pumping upwards and downwards) were compared for qualitative differences in power number behavior with Reynolds' number, single versus double impeller power draw, gassed power reduction with aeration number and gas hold-up. Power measurements were obtained using watt transducers which, although limited in accuracy and prone to interferences, were able to provide useful qualitative monitoring results. Measurements were conducted using three model liquid systems: water, glycerol and Melojel (soluble starch). Apparent viscosities for actual Streptomyces cultivations were estimated using measured gassed power values and the experimental relationships obtained for gassed/ungassed power to aeration number and power number to Reynolds' number for the glycerol model system. Results confirmed the lower power number and lower shear environment for hydrofoil impellers, yet suggested useful trends for various process parameters and process fluids.     

Influence of impeller type on mass transfer in fermentation vessels

Radial flow Rushton impellers were compared qualitatively with axial flow hydrofoil impellers (Maxflo T and A315) at the pilot scale. Six types of impellers were compared for qualitative differences in mass transfer. Measurements were conducted using three model systems: water, glycerol and Melojel (soluble starch). Power measurements were obtained using watt transducers, which although limited in accuracy and prone to interferences, were able to provide useful qualitative monitoring results. While there was little effect of impeller type on mass transfer as measured by the rapid pressure increase technique, significant qualitative differences were observed using the rapid temperature increase technique specifically for the Melojel and glycerol model systems. The Miller correlation, relating gassed-to-ungassed power, was used effectively to qualitatively evaluate the power drop upon gassing for both the model systems and a Streptomyces fermentation for the various impeller types. A high oxygen demand Streptomcyes fermentation then was conducted in fermenters possessing each type of impeller. Performance was not adequate with the A315 impellers pumping upwards and the small diameter Maxflo T impellers. Peak titers and profiles of the estimated apparent broth viscosity varied depending upon the impeller type. Mass transfer rates generally declined with higher viscosities when other fermentation operating conditions where held constant. Overall, values for OUR, k _L a, P _g /V _L and other calculated mass transfer and power input quantities for the A315 pumping upwards and undersized Maxflo T (D _T /D _I ?=?2.3) impellers were at the lower end of the range obtained for the larger Maxflo T (D _T /D _I ?=?1.8–2.0) and A315 impellers pumping downwards. Rushton impellers generally behaved qualitatively similar to hydrofoil impellers based on these calculated quantities.     

Influence of D-galactosamine on the kinetics of metabolic processes for two intermediate metabolites, 9-hydroxybenzo(a)pyrene and 3-hydroxybenzo(a)pyrene, in 3T3 and RTG2 cells

PAH metabolism is known to proceed in two successive steps, the first step resulting in the production of activated metabolites which are subsequently transformed by the different pathways involved in the second step. Microspectrofluorometry enables the study of the kinetics of these steps in living intact cells into which no imbalance has been artificially introduced. We used this technique to check the influence of pre-incubation with D-galactosamine on the kinetics of the detoxification step. 9- and 3-hydroxybenzo(a)pyrene (OH-B(a)P) were selected as fluorescent substrates because they are potential substrates for the different pathways of the second step. The physiological cell status was controlled at the level of the intrinsic cellular fluorescence. Pre-incubation with D-galactosamine results in a strong decrease of the experimental rate constants characteristic of the metabolism of 9- and 3-OH-B(a)P in both RTG2 and 3T3 cells. Moreover, such pre-incubation leads to a strong decrease of the transitory intracellular accumulation of 3-O-glucuronide when 3-OH-B(a)P is used as substrate for 3T3 cells. Nevertheless, it cannot be said that both phenols cannot be used as substrates by MFOs and STase, at least in rigorous experimental conditions.