E infα supk transgene in B10 mice suppresses the development of myasthenia gravis

Mice bearing the H-2 ^bhaplotype are susceptible to the development of experimental autoimmune myasthenia gravis (EAMG), induced by acetylcholine receptor (AChR) autoimmunity. One of the genes influencing EAMG susceptibility has been mapped to the A ^blocus of the major histocompatibility complex, and the A chain has been implicated in the pathogenesis. Mice of the H-2 ^bhaplotype, including C57BL/10 (B10), have a genomic deletion of the E gene and therefore fail to express the E molecule on their cell surface. To test the hypothesis that failure to express the cell surface E molecule in B10 mice contributes to EAMG pathogenesis, E _inf ^supk transgenic B10 mice expressing the T molecule were examined. Expression of the E molecule in E _inf ^supk transgenic B10 mice partially prevented the development of EAMG.     

Synthesis, surface deposition, and secretion of immunoglobulins by Abelson virus-transformed lymphosarcoma cell lines.

Three Abelson virus-transformed lymphoma cell lines were established in tissue culture and the immunoglobulin biosynthesis by these cell lines was studied. Two of the cell lines (ABLS-1 and ABLS-5) were found to synthesize monomeric IgM molecules which were deposited in the cell membrane, probably to serve as an antigen receptor. The third cell line (ABLS-8) was found to synthesize membrane-associated IgM as well as cellular IgG molecules. In addition, these cell lines were found to synthesize a protein of 35,000 molecular weight which is also membrane-associated and which has the capability to bind the immunoglobulin (MAID). It is speculated that this protein might play a role in adapting the receptor immunoglobulin molecule to the hydrophobic environment of the cell membrane. The kinetics of amino acid incorporation into immunoglobulins by these cell lines show that they produce immunoglobulins at a rate which is two orders of magnitude smaller than plasmacytoma cells (MOPC 104E). These results suggest that Abelson virus transforms thymus-independent lymphocytes in various stages of maturation and these lymphocytes might be of B cell origin. The T lymphoma (P1798) used as a control cell line was found occasionally to produce minute amounts of immunoglobulin.     

Activation characteristics of transient receptor potential ankyrin 1 and its role in nociception

Transient receptor potential (TRP) ankyrin 1 (TRPA1) is a Ca(2+)-permeant, nonselective cationic channel. It is predominantly expressed in the C afferent sensory nerve fibers of trigeminal and dorsal root ganglion neurons and is highly coexpressed with the nociceptive ion channel transient receptor potential vanilloid 1 (TRPV1). Several physical and chemical stimuli have been shown to activate the channel. In this study, we have used electrophysiological techniques and behavioral models to characterize the properties of TRPA1. Whole cell TRPA1 currents induced by brief application of lower concentrations of N-methyl maleimide (NMM) or allyl isothiocyanate (AITC) can be reversed readily by washout, whereas continuous application of higher concentrations of NMM or AITC completely desensitized the currents. The deactivation and desensitization kinetics differed between NMM and AITC. TRPA1 current amplitude increased with repeated application of lower concentrations of AITC, whereas saturating concentrations of AITC induced tachyphylaxis, which was more pronounced in the presence of extracellular Ca(2+). The outward rectification exhibited by native TRPA1-mediated whole cell and single-channel currents was minimal as compared with other TRP channels. TRPA1 currents were negatively modulated by protons and polyamines, both of which activate the heat-sensitive channel, TRPV1. Interestingly, neither protein kinase C nor protein kinase A activation sensitized AITC-induced currents, but each profoundly sensitized capsaicin-induced currents. Current-clamp experiments revealed that AITC produced a slow and sustained depolarization as compared with capsaicin. TRPA1 is also expressed at the central terminals of nociceptors at the caudal spinal trigeminal nucleus. Activation of TRPA1 in this area increases the frequency and amplitude of miniature excitatory or inhibitory postsynaptic currents. In behavioral studies, intraplantar and intrathecal administration of AITC induced more pronounced and prolonged changes in nociceptive behavior than those induced by capsaicin. In conclusion, the characteristics of TRPA1 we have delineated suggest that it might play a unique role in nociception.     

Diabetic Peripheral Neuropathy: Role of Reactive Oxygen and Nitrogen Species

The prevalence of diabetes has reached epidemic proportions. There are two forms of diabetes: type 1 diabetes mellitus is due to auto-immune-mediated destruction of pancreatic β-cells resulting in absolute insulin deficiency and type 2 diabetes mellitus is due to reduced insulin secretion and or insulin resistance. Both forms of diabetes are characterized by chronic hyperglycemia, leading to the development of diabetic peripheral neuropathy (DPN) and microvascular pathology. DPN is characterized by enhanced or reduced thermal, chemical, and mechanical pain sensitivities. In the long-term, DPN results in peripheral nerve damage and accounts for a substantial number of non-traumatic lower-limb amputations. This review will address the mechanisms, especially the role of reactive oxygen and nitrogen species in the development and progression of DPN.     

Relative Biological Effectiveness of HZE Particles for Chromosomal Exchanges and Other Surrogate Cancer Risk Endpoints

The biological effects of high charge and energy (HZE) particle exposures are of interest in space radiation protection of astronauts and cosmonauts, and estimating secondary cancer risks for patients undergoing Hadron therapy for primary cancers. The large number of particles types and energies that makeup primary or secondary radiation in HZE particle exposures precludes tumor induction studies in animal models for all but a few particle types and energies, thus leading to the use of surrogate endpoints to investigate the details of the radiation quality dependence of relative biological effectiveness (RBE) factors. In this report we make detailed RBE predictions of the charge number and energy dependence of RBE’s using a parametric track structure model to represent experimental results for the low dose response for chromosomal exchanges in normal human lymphocyte and fibroblast cells with comparison to published data for neoplastic transformation and gene mutation. RBE’s are evaluated against acute doses of γ-rays for doses near 1 Gy. Models that assume linear or non-targeted effects at low dose are considered. Modest values of RBE (<10) are found for simple exchanges using a linear dose response model, however in the non-targeted effects model for fibroblast cells large RBE values (>10) are predicted at low doses <0.1 Gy. The radiation quality dependence of RBE’s against the effects of acute doses γ-rays found for neoplastic transformation and gene mutation studies are similar to those found for simple exchanges if a linear response is assumed at low HZE particle doses. Comparisons of the resulting model parameters to those used in the NASA radiation quality factor function are discussed.     

B7-1 costimulatory molecule is critical for the development of experimental autoimmune myasthenia gravis

Following immunization with acetylcholine receptor (AChR), MHC class II-restricted, AChR-specific CD4 cell activation is critical for the development of experimental autoimmune myasthenia gravis (EAMG) in C57BL/6 mice. To study the contributions of B7-1 and B7-2 costimulatory molecules in EAMG, B7-1, B7-2, and B7-1/B7-2 gene knockout (KO) mice were immunized with Torpedo AChR in CFA. Compared with wild-type C57BL6 mice, B7-1 and B7-1/2 KO mice were resistant to EAMG development. B7-1 KO mice had reduced anti-AChR Ab compared with C57BL/6 mice. However, neither B7-1 nor B7-2 gene disruption impaired AChR-induced or dominant alpha(146-162) peptide-induced in vitro lymphoproliferative responses. Blocking of the B7-1 or B7-2 molecule by specific mAbs in vivo led to a reduction in the AChR-specific lymphocyte response, and the reduction was more pronounced in mice treated with anti-B7-2 Ab. The findings implicate B7-1 molecules as having a critical role in the induction of EAMG, and the resistance of B7-1 KO mice is associated with suppressed humoral, rather than suppressed AChR-specific, T cell responses. The data also point to B7-2 molecules as being the dominant costimulatory molecules required for AChR-induced lymphocyte proliferation.     

Genetic evidence for involvement of classical complement pathway in induction of experimental autoimmune myasthenia gravis

Abs to acetylcholine receptor (AChR) and complement are the major constituents of pathogenic events causing neuromuscular junction destruction in both myasthenia gravis (MG) and experimental autoimmune MG (EAMG). To analyze the differential roles of the classical vs alternative complement pathways in EAMG induction, we immunized C3(-/-), C4(-/-), C3(+/-), and C4(+/-) mice and their control littermates (C3(+/+) and C4(+/+) mice) with AChR in CFA. C3(-/-) and C4(-/-) mice were resistant to disease, whereas mice heterozygous for C3 or C4 displayed intermediate susceptibility. Although C3(-/-) and C4(-/-) mice had anti-AChR Abs in their sera, anti-AChR IgG production by C3(-/-) mice was significantly suppressed. Both C3(-/-) and C4(-/-) mice had reduced levels of B cells and increased expression of apoptotis inducers (Fas ligand, CD69) and apoptotic cells in lymph nodes. Immunofluorescence studies showed that the neuromuscular junction of C3(-/-) and C4(-/-) mice lacked C3 or membrane attack complex deposits, despite having IgG deposits, thus providing in vivo evidence for the incapacity of anti-AChR IgGs to induce full-blown EAMG without the aid of complements. The data provide the first direct genetic evidence for the classical complement pathway in the induction of EAMG induced by AChR immunization. Accordingly, severe MG and other Ab- and complement-mediated diseases could be effectively treated by inhibiting C4, thus leaving the alternative complement pathway intact.     

Industrial microbiology of solar salt production

Solar salterns can be modeled as giant outdoor chemostats, much like a series of dams on a slow-moving river. Microorganisms and their products play an essential, but sometimes uncharacterized, role in salt production in these ponds, from seawater salinity up through NaCl saturation. They may physically affect the evaporation process and their by-products may chemically modify or bind with dissolved ions. Many solar salt facilities engage microbiologists to establish monitoring programs for analyses of nutrients, standing crop and associated biological variables in the ponds. Other solar salt companies engage microbiologists only when there are “crises” in the ponds that interfere with salt production. Journal of Industrial Microbiology & Biotechnology (2002) 28, 42–47 DOI: 10.1038/sj/jim/7000173 Received 20 May 2001/ Accepted in revised form 13 June 2001     

An unusual halotolerant alpha-type carbonic anhydrase from the alga Dunaliella salina functionally expressed in Escherichia coli

A 60-kDa, salt-inducible, internally duplicated alpha-type carbonic anhydrase (Dca) is associated with the plasma membrane of the extremely salt-tolerant, unicellular, green alga Dunaliella salina. Unlike other carbonic anhydrases, Dca remains active over a very broad range of salinities (0-4M NaCl), thus representing a novel type of extremely halotolerant enzyme. To elucidate the structural principles of halotolerance, structure-function investigations of Dca have been initiated. Such studies require considerable amounts of the enzyme, and hence, large-scale algal cultivation. Furthermore, the purified enzyme is often contaminated with other, co-purifying algal carbonic anhydrases. Expression in heterologous systems offers a means to produce, and subsequently purify, sufficiently large amounts of Dca required for activity and structural studies. Attempts to over-express Dca in the Escherichia coli BL21(DE3)pLysS strain, after optimizing various expression parameters, produced soluble, but weakly active protein, composed of fully reduced and variably -S-S- cross-linked chains (each of the Dca repeats contains a pair of cysteine residues, presumably forming a disulfide bond). However, when the E. coli Origami B(DE3)pLysS strain was used as a host, a functionally active enzyme with proper disulfide bonds was formed in good yield. Affinity-purified recombinant Dca resembled the native enzyme from D. salina in activity and salt tolerance. Hence, this expression system offers a means of pursuing detailed studies of this extraordinary protein using biochemical, biophysical, and crystallographic approaches.     

Rapid and unexpected effects of piscivore introduction on trophic position and diet of perch (Perca flavescens) in lakes recovering from acidification and metal contamination

1. Yellow perch (Perca flavescens) are often the only surviving fish species in acidified lakes. We studied four lakes along a gradient of recovery from acidification and that had different food web complexities. All had abundant yellow perch, two had low piscivore abundance, one had a well‐established piscivore population and one was manipulated by introducing piscivorous smallmouth bass (Micropterus dolomieu). We hypothesised that there would be strong effects on perch abundance, behaviour and diet induced by the presence of piscivores. 2. In the manipulated lake, the bass reduced yellow perch abundance by 75% over a 2‐year period. Concomitantly, perch use of the pelagic habitat fell from 48 to 40%. 3. In contrast to findings from less disturbed systems, yellow perch in the littoral zone of the manipulated lake did not strongly shift from zooplankton to benthic food sources after the arrival of piscivores. Diet analysis using stable carbon isotopes revealed a strong continued reliance on zooplankton in all lakes, independent of the degree of piscivory. The failure to switch to benthos in the refuge area of the littoral zone is most likely related to the depauperate benthos communities in these formerly acidified lakes. 4. Yellow perch in lakes recovering from acidification face a considerable ecological challenge as the necessary switch to benthic diet is hindered by a low abundance of benthos. The arrival of piscivores in these recovering lakes imposes further restrictions on perch access to food items. We infer that future recovery of perch populations (and higher trophic levels) will have to be preceded by the re‐establishment of diverse benthic macroinvertebrate communities in these lakes.