The effect of weak 50 Hz magnetic fields on the number of free oxygen radicals in rat lymphocytes in vitro

The aim of the work was verification of the hypothesis that weak power frequency (50 Hz) magnetic fields (MF) affected the number of free oxygen radicals in living biological cells and that these changes could be qualitatively explained by the radical pair mechanism. The experiments were performed on rat lymphocytes. One-hour exposure to 50 Hz MF at 20, 40, or 200 microT flux densities was performed inside a pair of Helmholtz coils with axis along or crosswise to the Earth's static MF. Iron ions (FeCl2) were used as a stimulator of the oxidation processes. Oxygen radicals were measured by fluorimetry using a DCF-DA fluorescent probe. Only in the lymphocytes exposed at 40 microT MF directed along the Earth's static MF there was a decrease of fluorescence in relation to non-exposed samples. Our observation seems to confirm the hypothesis that low level power frequency MF affects oxidative processes which occur in living biological cells and that this effect can be explained by the radical pair mechanism.     

Nitric oxide regulates retinal vascular tone in humans

The purpose of the present study was to investigate the contribution of basal nitric oxide (NO) on retinal vascular tone in humans. In addition, we set out to elucidate the role of NO in flicker-induced retinal vasodilation in humans. Twelve healthy young subjects were studied in a three-way crossover design. Subjects received an intravenous infusion of either placebo or NG-monomethyl-L-arginine (L-NMMA; 3 or 6 mg/kg over 5 min), an inhibitor of NO synthase. Thereafter, diffuse luminance flicker was consecutively performed for 16, 32, and 64 s at a frequency of 8 Hz. The effect of L-NMMA on retinal arterial and venous diameter was assessed under resting conditions and during the hyperemic flicker response. Retinal vessel diameter was measured with a Zeiss retinal vessel analyzer. L-NMMA significantly reduced arterial diameter (3 mg/kg: -2%; 6 mg/kg: -4%, P < 0.001) and venous diameter (3 mg/kg: -5%; 6 mg/kg: -8%, P < 0.001). After placebo infusion, flicker induced a significant increase in retinal vessel diameter (P < 0.001). At a flicker duration of 64 s, arterial diameter increased by 4% and venous diameter increased by 3%. L-NMMA did not abolish these hyperemic responses but blunted venous vasodilation (P = 0.017) and arterial vasodilation (P = 0.02) in response to flicker stimulation. Our data indicate that NO contributes to basal retinal vascular tone in humans. In addition, NO appears to play a role in flicker-induced vasodilation of the human retinal vasculature.     

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.     

Fluorescence resonance energy transfer analysis of cytochromes P450 2C2 and 2E1 molecular interactions in living cells

The molecular organization of microsomal cytochromes P450 (P450s) and formation of complexes with P450 reductase have been studied previously with isolated proteins and in reconstituted systems. Although these studies demonstrated that some P450s oligomerize in vitro, neither oligomerization nor interactions of P450 with P450 reductase have been studied in living cells. Here we have used fluorescence resonance energy transfer (FRET) to study P450 oligomerization and binding to P450 reductase in live transfected cells. Cytochrome P450 2C2, but not P450 2E1, forms homo-oligomeric structures, and this self-association is mediated by the signal-anchor sequence. Because P450 2C2, in contrast to P450 2E1, is directly retained in the endoplasmic reticulum (ER), these results could suggest that oligomerization may prevent transport from the ER. However, P450 2C1 signal-anchor sequence chimera defective in ER retention also formed oligomers, and chimera containing the cytoplasmic domain of P450 2C2, which is directly retained in the ER, did not exhibit self-oligomerization, which indicates that oligomerization is not correlated with direct retention. By using FRET, we have also detected binding of P450 2C2 and P450 2E1 to P450 reductase. In contrast to self-oligomerization, the catalytic domain can mediate an interaction of P450 2C2 with P450 reductase. These results suggest that microsomal P450s may differ in their quaternary structure but that these differences do not detectably affect interaction with the reductase or transport from the ER.     

A characterization of the egg capsules of Anacroneuria starki and A. talamanca (Plecoptera: Perlidae), with a suggestion about the distribution of stoneflies in the Tropics

In this study, the structure of egg capsules of two species of Neotropical Perlidae: Anacroneuria starki Fenoglio and Morisi (2001 a) and A. talamanca Stark (1998), were examined. Eggs were studied using a scanning electron microscope. A morphological characterization of these eggshells is presented: layers, attachment structures and other anatomical features are described. The egg capsules of the two species differ in some aspects, but both are characterized by thin-layered egg coverings (vitelline envelope, chorion and extrachorion). On the basis of these observations, the importance of eggshell structure for the biogeographical distribution of Plecoptera in the tropics is discussed. A key role for egg capsules in the adaptation process of Plecoptera to aquatic environments of the Neotropics is hypothesized.     

Immunological characterization of the Campylobacter jejuni 72Dz/92 cjaD gene product and its fusion with B subunit of E. coli LT toxin

Campylobacter jejuni 72Dz/92 cjaD gene, orthologue of C. jejuni NCTC 11168 cj0113, C. jejuni M275 omp18 and C. jejuni ATCC 29428 omp18, has been cloned, sequenced and analysed from the viewpoint of its immunological attributes. Neither the 5' nor 3' fragment of the cjaD encodes protein capable of reacting with anti-Campylobacter antibodies. Several fusions of the cjaD with eltB, which encodes B subunit of the E. coli LT toxin, have been constructed. The hybrid proteins, which differ in respect to their cellular localization, retain the ability to react with GM1 and are recognized by the antibodies specific for both moieties of the proteins. The fusion protein equipped with signal sequence, reveals a stronger affinity to GM1 than its equivalent which is unable to cross the inner membrane. Two recombinant plasmids (pUWM405 expressing both LTB and CjaD proteins and pUWM299 containing cjaD gene fused into 3' end of Escherichia coli eltB gene lacking signal sequence) were introduced into avirulent Salmonella enterica serovar Typhimurium strain where they are stably maintained.     

Vaccination of macaques with long-standing SIVmac251 infection lowers the viral set point after cessation of antiretroviral therapy

A cohort of rhesus macaques with long-standing SIVmac251 infection (> or =5 mo) was treated with continuous antiretroviral therapy (ART). A group of eight macaques was vaccinated with or without simultaneous administration of low dose IL-2 with the highly attenuated poxvirus vector (NYVAC) vaccine candidate expressing the SIVmac structural gag-pol-env (gpe) genes and a novel chimeric fusion protein derived from the rev-tat-nef (rtn) regulatory genes. Control groups consisted of mock-vaccinated macaques or animals treated only with IL-2. Vaccination significantly expanded both virus-specific CD4(+) and CD8(+) T cell responses, and IL-2 further increased the vaccine-induced response to an immunodominant Gag epitope. Following antiretroviral treatment interruption, the viral set point was significantly lower in vaccinated than in control macaques for at least 4 consecutive mo, and viral containment was inversely correlated with vaccine-induced, virus-specific CD4(+) and CD8(+) T cell responses. These data provide the proof of concept that therapeutic vaccination before cessation of ART may be a feasible approach in the clinical management of HIV-1 infection.