Selective breeding of miniature swine leads to an increased rate of acceptance of MHC-identical, but not of class I-disparate, renal allografts.

Previous work from this laboratory demonstrated that tolerance to MHC-identical or class I-disparate renal allografts develops in approximately one third of miniature swine without exogenous immunosuppression. A back-cross study indicated that rejection of MHC-identical transplants due to minor Ag was controlled by one or possibly two non-MHC-linked, autosomal dominant Ir genes. According to this hypothesis, and assuming complete penetrance, graft acceptors would be homozygous recessive at the relevant Ir loci, as would their offspring. Alternatively, if the gene(s) were incompletely penetrant, then two acceptors could give rise to a rejector. However, a high rate of MHC-identical graft acceptance would still be expected in the offspring of acceptors even if the Ir gene(s) were incompletely penetrant. To test this hypothesis and to obtain a higher frequency of acceptor animals for studies of tolerance, a program of selective breeding of renal allograft acceptors was begun. In the present paper, we assess the effect of selective breeding on renal graft acceptance. The analysis indicates a marked increase in the rate of MHC-identical graft acceptance, from 27.3% (n = 24) for the earliest of the four chronologic subgroups assessed to 64.5% (n = 33) for the most recent subgroup (p less than 0.0001). Calculations of kinship revealed that the increased acceptance of MHC-identical grafts was not the result of differences between acceptors and rejectors in donor/recipient consanguinity. Class I-disparate grafts (n = 128) were similarly stratified chronologically and compared. Unlike MHC-identical grafts, the rate of acceptance of class I-disparate grafts has not changed over time. We conclude that rejector/acceptor status with respect to class I MHC incompatibility is determined by genetic factors in addition to those that control responses to minor antigen incompatibilities only.     

Nitrogen deposition enhances moss growth,but leads to an overall decline in habitat condition of mountain moss‐sedge heath

Ecosystems are subject to multiple, natural and anthropogenic environmental influences, including nitrogen (N) deposition, land use and climate. Assessment of the relative importance of these influences on biodiversity and ecosystem functioning is crucial for guiding policy and management decisions to mitigate global change; yet, few studies consider multiple drivers. In the UK, ongoing loss of the internationally important arctic/alpine moss‐sedge community, Racomitrium heath, has been linked to elevated N deposition, high grazing pressures and their combination; however, the relative importance of these drivers remains unclear. We used environmental gradients across the habitat's European distribution (UK, Faroes, Norway and Iceland) to investigate the relative impact of N deposition and grazing pressure, as well as climate, on the condition of the dominant moss species, Racomitrium lanuginosum. Key variables including tissue chemistry, growth and cover were measured at 36 sites, and multiple linear regressions were used to examine the relative importance of the drivers across sites. Our results clearly show that regional variation in the condition of R. lanuginosum across Europe is primarily associated with the impacts of N deposition, with climate (air temperature) and grazing pressure playing secondary roles. In contrast to previous experimental studies, we found moss growth to be stimulated by elevated N deposition; this apparent discrepancy may result from the use of artificially high N concentrations in many experiments. Despite increased growth rates, we found that moss mat depth and cover declined in response to N deposition. Our results suggest that this is due to increased decomposition of material in the moss mat, which ultimately leads to loss of moss cover and habitat degradation. This study clearly demonstrates both the key role of N deposition in degradation of Racomitrium heath and the importance of observational studies along natural gradients for testing predictions from experimental studies in the real world.     

Characterization of an endooligopeptidase A-like protein in PC12 cells: Activity modulation by cAMP but not by basic fibroblast growth factor

Endooligopeptidase A is a putative neuropeptide-metabolizing enzyme. It converts small enkephalin-containing peptides into the corresponding enkephalins and inactivates biopeptides such as bradykinin and neurotensin in vitro. We investigated the presence of endooligopeptidase A in PC12 cells. This cell line was derived from a rat pheochromocytoma tumor and resembles fetal chromaffin cell. Depending on the supplements added to the cell culture, this cell line can be differentiated into mature chromaffin cell or sympathetic neuron-like cell. Endooligopeptidase A activity was measured in soluble cellular extracts using a specific fluorogenic substrate QF-ERP7. The PC12 endooligopeptidase A-like activity shared similar but not identical biochemical properties with rabbit brain endooligopeptidase A. Similarly to rabbit brain endooligopeptidase A, the PC12 endooligopeptidase A-like activity was enhanced by DTT, totally inhibited by DTNB and 1-10 Phenanthroline, partially inhibited by cFP-AAF-pAb, and not affected by PMSF. Furthermore, the PC12 endooligopeptidase A-like activity displayed identical elution profile as rabbit brain endooligopeptidase A in gel filtration and anion-exchange chromatography. In addition, an antiserum raised against rabbit brain endooligopeptidase A cross-reacted with a 71 kDa component from PC12 cell extracts in Western blotting and was also able to partially neutralize the PC12 endooligopeptidase A-like activity. Treatment of PC12 cells with basic fibroblast growth factor (bFGF), a neurotrophic factor for this cell line, did not modify the specific activity of this enzyme. However, cAMP analogs decreased the specific activity of the enzyme. These results indicate the presence of an endooligopeptidase A-like activity in PC12 cells which is modulated by cAMP but not by bFGF.     

In vivo inhibition of NF-kappa B in T-lineage cells leads to a dramatic decrease in cell proliferation and cytokine production and to increased cell apoptosis in response to mitogenic stimuli, but not to abnormal thymopoiesis.

To understand the role of NF-kappa B complexes in T cell development and activation, we have generated transgenic mice in which RelA and c-Rel complexes were selectively inhibited in the T-lineage cells by specific expression of a trans-dominant form of I kappa B alpha. Transgene expression did not affect the thymic development, but led to lowered numbers of splenic T cells and to a dramatic decrease in the ex vivo proliferative response of splenic T lymphocytes. Analysis of IL-2 and IL-2R alpha expression demonstrated that the perturbation of the proliferation response was not attributable to an abnormal expression of these genes. In contrast, expression of IL-4, IL-10, and IFN-gamma was strongly inhibited in the transgenic T cells. The proliferative deficiency of the transgenic T cells was associated with an increased apoptosis. These results point out the involvement of NF-kappa B/Rel family proteins in growth signaling pathways by either regulating proteins involved in the IL-2 signaling or by functionally interfering with the cell cycle progression.     

Induction of SM-20 in PC12 cells leads to increased cytochrome c levels,accumulation of cytochrome c in the cytosol,and caspase-dependent cell death

Sympathetic neurons deprived of nerve growth factor (NGF) release cytochrome c into the cytosol and undergo caspase-dependent cell death through a process that requires de novo gene expression. Expression of the SM-20 gene increases after NGF withdrawal, and ectopic SM-20 expression induces cell death in NGF-maintained neurons. To further evaluate the mechanism by which SM-20 promotes cell death, we developed a PC12-derived cell line in which SM-20 expression can be induced by addition of doxycycline to the culture medium. Induction of SM-20 in either undifferentiated or NGF-differentiated cells resulted in cell death. Cell death was accompanied by an increase in caspase activity and was inhibited by the caspase inhibitor zVAD-FMK. Analysis of cytochrome c in cytosolic and mitochondria-enriched subcellular fractions revealed that induction of SM-20 led to the accumulation of cytochrome c in the cytosol. Surprisingly, SM-20 expression also resulted in a selective increase in the total amount of cytochrome c protein. Thus, induction of SM-20 expression appears to affect both the amount and subcellular localization of cytochrome c in PC12 cells. These results suggest that SM-20 promotes caspase-dependent cell death through a mechanism involving cytochrome c.     

Overexpression of HuD, but not of its truncated form HuD I+II, promotes GAP-43 gene expression and neurite outgrowth in PC12 cells in the absence of nerve growth factor

We have previously shown that the RNA-binding protein HuD binds to a regulatory element in the growth-associated protein (GAP)-43 mRNA and that this interaction involves its first two RNA recognition motifs (RRMs). In this study, we investigated the functional significance of this interaction by overexpression of human HuD protein (pcHuD) or its truncated form lacking the third RRM (pcHuD I+II) in PC12 cells. Morphological analysis revealed that pcHuD cells extended short neurites containing GAP-43-positive growth cones in the absence of nerve growth factor (NGF). These processes also contained tubulin and F-actin filaments but were not stained with antibodies against neurofilament M protein. In correlation with this phenotype, pcHuD cells contained higher levels of GAP-43 without changes in levels of other NGF-induced proteins, such as SNAP-25 and tau. In mRNA decay studies, HuD stabilized the GAP-43 mRNA, whereas HuD I+II did not have any effect either on GAP-43 mRNA stability or on the levels of GAP-43 protein. Likewise, pcHuD I+II cells showed no spontaneous neurite outgrowth and deficient outgrowth in response to NGF. Our results indicate that HuD is sufficient to increase GAP-43 gene expression and neurite outgrowth in the absence of NGF and that the third RRM in the protein is critical for this function.     

Transforming growth factor-alpha expression is enhanced in human mammary epithelial cells transformed by an activated c-Ha-ras protooncogene but not by the c-neu protooncogene, and overexpression of the transforming growth factor-alpha complementary DNA leads to transformation

MCF-10A cells are a spontaneously immortalized normal human mammary epithelial cell line. MCF-10A cells were transfected with two expression vector plasmids containing either a human point-mutated c-Ha-ras protooncogene or the rat c-neu protooncogene. c-Ha-ras-transfected MCF-10A cells grow as colonies in soft agar, exhibit a 3- to 4-fold increase in their growth rate in serum-free medium, and show a reduced mitogenic response to exogenous epidermal growth factor (EGF) or transforming growth factor-alpha (TGF alpha) as compared to MCF-10A cells. c-Ha-ras-transfected MCF-10A cells express a 4- to 8-fold increase in TGF alpha mRNA levels and secrete 4- to 6-fold more TGF alpha protein as compared to MCF-10A cells. Addition of either an anti-TGF alpha neutralizing monoclonal antibody or an anti-EGF receptor blocking monoclonal antibody to the Ha-ras-transformed MCF-10A cells produces a 50 to 80% inhibition of colony formation of these cells in soft agar. c-neu-transfected MCF-10A cells grown in soft agar and exhibit an increase in their growth rate in serum-free medium at a level comparable to that observed in Ha-ras-transformed MCF-10A cells. Addition of an anti-c-erbB-2 monoclonal antibody inhibits the anchorage-independent growth of these cells in soft agar. However, c-neu-transformed MCF-10A cells show no increase in TGF alpha secretion and no change in their responsiveness to exogenous EGF or TGF alpha. A recombinant retroviral vector containing the human TGF alpha gene was also introduced into MCF-10A cells. TGF alpha-infected MCF-10A cells secrete 15- to 20-fold more TGF alpha protein than MCF-10A cells, form colonies in soft agar, exhibit an enhanced growth rate in serum-free medium, and show a decreased mitogenic response to exogenous EGF or TGF alpha at a level equivalent to Ha-ras-transformed MCF-10A cells. Growth of TGF alpha-infected MCF-10A cells in soft agar is completely inhibited by anti-TGF alpha neutralizing or anti-EGF receptor blocking monoclonal antibodies. These results suggest that TGF alpha is an intermediary in the transformation of human mammary epithelial cells by an activated c-Ha-ras gene, but not by the c-neu gene, and demonstrate that overexpression of this growth factor is able to transform immortalized human mammary epithelial cells which also express a sufficient complement of functional EGF receptors.     

Postembryonic development of the sea spider Ammothella biunguiculata (Pycnogonida,Ammotheidae) endoparasitic to an actinian Entacmaea quadricolor (Anthozoa,Stichodactylidae) in Izu Peninsula,Japan

Postembryonic developmental stages of an endoparasitic pycnogonid, Ammothella biunguiculata in Izu Peninsula, Japan are described. Eleven stages were identified beginning with a protonymphon larva attached to the male oviger. We found endoparasitic individuals in the host actinian from the second to tenth instar, and forms in the ninth stage to adult were found free-living. This indicates a transition from being endoparasitic to free-living during the ninth to tenth instar stages. The first instar protonymphon attached to the adult male oviger has a gland duct on the anterior margin of each chelifore scape which completely disappears with the second instar. The disappearance of the chelifore gland duct coincides with the beginning of an endoparasitic stage in the development of this species. Although the larval morphology and the postembryonic development of pycnogonids have been summarized by several authors, the present study concludes that much remains to be learnt.     

Appearance of ossification centers of the lower arm,wrist, lower leg,and ankle in immature orangutans and chimpanzees with an assessment of the relationship of ossification to dental development

This study examines the appearance of the secondary ossification centers in the lower arms, wrists, lower legs, and ankles of a cross-sectional sample of 20 infant orangutans and chimpanzees (15 of known age). The number of tarsal and carpal centers is analyzed relative to the degree of M₁ development and the weight of individual animals. Variation in the appearance of these ossification centers is discussed relative to these variables and others. In addition, a sequence of appearance is established for the carpal and tarsal ossification centers in the orangutan and data is presented on the status of these centers in a fetal and newborn gorilla. Study results indicate that 1) there is variation in the number of secondary epiphyses present in animals of similar ages; 2) tarsal ossification is completed prior to carpal ossification in the orangutan; 3) there are indications of a relationship between weight and the number of ossification centers present in animals of similar age; and 4) there appears to be no evidence of specific relationships between carpal and tarsal development and M₁ development. © 1996 Wiley-Liss, Inc.     

Heterologous expression of a<Emphasis Type="Italic"> keto</Emphasis>hexokinase in potato plants leads to inhibited rates of photosynthesis,severe growth retardation and abnormal leaf development

In the present paper we investigated the effect of heterologous expression of a rat liver ketohexokinase in potato (Solanum tuberosum L.) plants with the aim of investigating the role of fructose 1-phosphate in plant metabolism. Plants were generated that contained appreciable activity of ketohexokinase but did not accumulate fructose 1-phosphate. They were, however, characterised by a severe growth retardation and abnormal leaf development. Studies of ¹⁴CO₂ assimilation and metabolism, and of the levels of photosynthetic pigments, revealed that these lines exhibited restricted photosynthesis. Despite this fact, the levels of starch and soluble sugars remained relatively constant. Analysis of intermediates of starch and sucrose biosynthesis revealed large increases in the triose phosphate and fructose 1,6-bisphosphate pools but relatively unaltered levels of inorganic phosphate and 3-phosphoglycerate, and these lines were also characterised by an accumulation of glyceraldehyde. The transformants neither displayed consistent changes in the activities of Calvin cycle enzymes nor in enzymes of sucrose synthesis but displayed a metabolic profile partially reminiscent of that brought about by end-product limitation, but most likely caused by an inhibition of photosynthesis brought about by the accumulation of glyceraldehyde. Analysis of the metabolite contents in lamina and vein fractions of the leaf, and of the enzymes of carbohydrate oxidation indicate that the phloem-enriched veins of ketohexokinase-expressing leaves tend toward hypoxia and indicate a problem of phloem transport.Abbreviations CaMV Cauliflower mosaic virus - DHAP Dihydroxyacetone phosphate - F1P Fructose 1-phosphate - FBP Fructose 1,6-bisphosphate - KHK Ketohexokinase - NADP-GAPdH NADP-dependent glyceraldehyde-3-phosphate dehydrogenase - PFP Pyrophosphate: fructose 6-phosphate 1-phosphotransferase - 3PGA 3-Phosphoglycerate - PEP Phosphoenolpyruvate - Rubisco Ribulose 1,5-bisphosphate carboxylase/oxygenase - SPS Sucrose phosphate synthase - SuSy Sucrose synthase     

Grafting of <Emphasis Type="Italic">Cucumis sativus</Emphasis> onto <Emphasis Type="Italic">Cucurbita ficifolia</Emphasis> leads to improved plant growth,increased light utilization and reduced accumulation of reactive oxygen species in chilled plants

The effects of chilling at 14 and 7°C on plant growth, CO₂ assimilation, light allocation, photosynthetic electron flux and antioxidant metabolism were examined in cucumber (Cucumis sativus L. cv. Jinyan No. 4, CS) plants with figleaf gourd (Cucurbita ficifolia Bouché, CF) and cucumber as rootstocks, respectively. Growth inhibition by chilling at 7°C was characterized by irreversible inhibition of CO₂ assimilation in grafted plants with cucumber as rootstock and scion (CS/CS) but this effect was significantly alleviated by grafting onto CF roots (CS/CF). Chilled CS/CF plants exhibited a higher photosynthetic activity and lower proportion of energy dissipation than chilled CS/CS plants. Chilling resulted in a greater decrease in the electron flux in photosystem (PS) II (J _PSII) than the rate of energy dissipation either via light-dependent (J _NPQ) or via constitutive thermal dissipation and fluorescence (J _f,D) in CS/CS plants. In parallel with the reduction in J _PSII, electron flux to oxygenation (J _o) and carboxylation by Rubisco (J _c) all decreased significantly whilst alternative electron flux in PS II (J _a) increased, especially in CS/CS plants. Moreover, CS/CF plants exhibited higher activity of antioxidant enzymes, lower antioxidant content and less membrane peroxidation relative to CS/CS plants after chilling.