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Soamsan, a traditional Korean medicine, enhances antigen-specific immune responses in low-responder mice via the combined activity of glycoproteins and endotoxins.

Soamsan is a traditional anti-cancer treatment in oriental medicine. It is thought that this material modulates immune responses. To determine whether Soamsan treatment has any effect on the induction of antigen-specific immune responses, C57BL/6 mice, which are low-responders to hen egg-white lysozyme (HEL), were injected with HEL, and their specific immune responses were measured while they were fed Soamsan. Oral administration of Soamsan enhanced the anti-HEL antibody response as well as the T-cell proliferative response to the antigen. Analyses of the HEL-specific antibody isotypes showed that Soamsan treatment resulted in increased levels of HEL-specific antibodies, irrespective of isotype. In particular, however, HEL-specific antibodies of the IgG2b, IgG3, and IgM isotypes, which are associated with direct stimulation of B cells, were significantly increased by the Soamsan treatment. Stimulation of C57BL/6 splenocytes in vitro showed that the presence of Soamsan significantly augmented the proliferative activity induced by both B and T cell mitogens. This augmentation was associated with glycoprotein(s) with a molecular weight mass of about 100 kDa, as well as with endotoxin-like compounds. These results suggest that Soamsan modulates and enhances antigen-specific immune responses.

Adjuvants, Immunologic↗

Hydrophobic probe binding of beta-lactoglobulin in the native and molten globule state induced by high pressure as affected by pH, KIO(3) and N-ethylmaleimide.

High hydrostatic pressure (HHP) at 500 MPa and 50 degrees C induces beta-LG into the molten globule state. Retinol, cis-parinaric acid (CPA), and 1-anilino-naphthalene-8-sulfonate (ANS) fluorescence from pH 2.5 to 10.5 in the presence of the native and molten globule states of beta-LG indicate that retinol binds to beta-LG in the calyx, CPA at the surface hydrophobic site, and ANS in multiple hydrophobic sites. HHP treatment results in a decrease of beta-LG affinity for retinol and CPA, suggesting conformational changes in the calyx and surface hydrophobic site of beta-LG during HHP treatment. beta-LG treated by HHP in the presence of N-ethylmaleimide (NEM) retains retinol affinity, suggesting that NEM protects the calyx conformation of beta-LG during HHP treatment. HHP treatment of beta-LG in the presence of KIO(3) exhibits a great decrease of CPA affinity compared to HHP-treated beta-LG in the absence of KIO(3), suggesting the formation of non-native disulfide bonding at the CPA binding site.

Anilino Naphthalenesulfonates↗

Improved procedure for the oxidative cleavage of olefins by OsO4-NaIO4.

[reaction: see text] Oxidative cleavage of olefins by OsO(4)-NaIO(4) sometimes suffers from low yields due to the formation of side products. It is found that the addition of 2,6-lutidine can suppress the side reactions and dramatically improve the yield of this classic reaction.

Aldehydes↗

Low intracellular pH and chemical agents slow inactivation gating in sodium channels of muscle.

Excitation of nerve or muscle requires an orderly opening and closing of molecular pores, the ionic channels, in the plasma membrane. During the action potential, Na channels are opened (activated) by the advancing wave of depolarisation, contributing a pulse of inward sodium current, and then are closed again (inactivated) by the continued depolarisation. As one approach both to obtaining molecular information on the Na channel and towards further defining the recently discovered kinetic interactions of the inactivation and activation gating steps, we have surveyed here the effects of chemical agents reported to slow or prevent Na channel inactivation. We find that many of the agents studied by others on invertebrate giant axons or vertebrate nerve act on our frog skeletal muscle preparation. In addition, we have discovered that simply lowering the intracellular pH nearly eliminates inactivation. The activation mechanism seems to resist modification.

Animals↗

Phosphatidylinositol kinase. A component of the chromaffin-granule membrane.

Phosphorylation of bovine chromaffin granules by ATP leads to the formation of diphosphoinositide in the granule membrane. Both phosphatidylinositol kinase and its substrate are components of this membrane, and triphosphoinositide is not formed under the conditions of the assay. The reaction is Mg(2+)-dependent and is stimulated by Mn(2+) and F(-) ions. The initial reaction is rapid, with a broad pH profile and a ;transition' temperature for its activation energy at 27 degrees C. The apparent K(m) for ATP is 5mum. ATP, N-ethylmaleimide, Cu(2+) ions and NaIO(4) are inhibitory. The phospholipids of chromaffin-granule membranes have been analysed: 6.8% of the lipid P is found in phosphatidylinositol, and only 2-3% in phosphatidylserine. Comparison of the rate of phosphorylation of intact and lysed granules suggests that the sites for phosphorylation are on the outer (cytoplasmic) surface of the granules, and diphosphoinositide may therefore make an important contribution to the charge of the chromaffin granule in vivo.

Adenosine Triphosphate↗

Uptake and release of ferritin iron. Surface effects and exchange within the crystalline core.

The uptake and subsequent release of iron by apoferritin and ferritin was studied by using labelled iron ((59)Fe). The experimental results are consistent with predictions arising from a model system developed in the interpretation of previous experiments. In this model, uptake and release of ferritin iron is controlled by the available surface area of the small crystalline particles of hydrous ferric oxide found within the ferritin molecule. Evidence is also presented for the exchange of Fe(3+) ions among the various cation sites within these crystallites.

Acetates↗

Characterization of plasma-membrane glycoproteins from functional domains of the rat hepatocyte.

Plasma-membrane glycoproteins from the three different functional domains of the rat hepatocyte were radioactively labelled by oxidation with NaIO4, followed by reduction with NaB3H4. Analysis of the radioactively labelled glycoproteins by polyacrylamide-gel electrophoresis revealed the presence of at least 12 major sialoglycoproteins in each different region of the hepatocyte surface. The Mr-110 000 component was homogeneously distributed over the plasma membrane, whereas the Mr-90 000 polypeptide was only located at the sinusoidal face. These radiolabelled glycoproteins were solubilized in 1% Triton X-100, and the soluble fraction was subjected to affinity chromatography on Sepharose-conjugated wheat-germ agglutinin (WGA). The labelled glycoproteins were poorly bound to WGA. Membrane glycoproteins were also labelled by the galactose oxidase/NaB3H4 method. The results show that the polypeptides with apparent Mr 170 000 from the sinusoidal, 230 000 from the canalicular and 170 000 from the lateral membranes were specifically labelled. When the membranes were treated with neuraminidase and galactose oxidase/NaB3H4, the electrophoretic patterns showed changes in the apparent Mr values of the glycoproteins, owing to loss of sialic acid, and a clear increase in labelling in the sinusoidal and canalicular membranes compared with the lateral membranes. When these labelled membranes were solubilized in 1% Triton X-100 and subjected to affinity chromatography on Sepharose-conjugated Ricinus communis agglutinin and/or Lens culinaris agglutinin, the results showed that the former columns efficiently bound the radiolabelled glycoproteins, whereas the latter columns bound poorly. The results show that there is a differential distribution of glycoproteins along the hepatocyte's surface.

Animals↗

Recruitment of the gamma-tubulin ring complex to Drosophila salt-stripped centrosome scaffolds.

Extracting isolated Drosophila centrosomes with 2 M KI generates salt-resistant scaffolds that lack the centrosomal proteins CP190, CP60, centrosomin, and gamma-tubulin. To clarify the role of these proteins in microtubule nucleation by centrosomes and to identify additional centrosome components required for nucleation, we have developed an in vitro complementation assay for centrosome function. Centrosome aster formation is reconstituted when these inactive, salt-stripped centrosome scaffolds are supplemented with a soluble fraction of a Drosophila embryo extract. The CP60 and CP190 can be removed from this extract without effect, whereas removing the gamma-tubulin destroys the complementing activity. Consistent with these results, we find no evidence that these three proteins form a complex together. Instead, gamma-tubulin is found in two distinct protein complexes of 240,000 and approximately 3,000,000 D. The larger complex, which is analogous to the Xenopus gamma-tubulin ring complex (gammaTuRC) (Zheng, Y., M.L. Wong, B. Alberts, and T. Mitchison. 1995. Nature. 378:578-583), is necessary but not sufficient for complementation. An additional factor found in the extract is required. These results provide the first evidence that the gammaTuRC is required for microtubule nucleation at the centrosome.

Animals↗

Ultraviolet irradiation of canine dendritic cells prevents mitogen-induced cluster formation and lymphocyte proliferation.

The objectives of this study were to characterize canine peripheral blood dendritic cells (DC), to compare their functional role in lectin (Con-A) induced and oxidative (NaIO4-induced) mitogenesis, and to investigate the effect of ultraviolet (UV) irradiation on DC function. We used, sequentially, Ficoll-Hypaque fractionation, discontinuous percoll gradients, rosette, sedimentation with human red blood cells, adherence to plastic and cytofluorometric sorting after labeling with an anti-Ia monoclonal antibody (7.2) to obtain cells of low buoyant density that were rosette-negative, plastic-non-adherent, Ia-positive, nonspecific-esterase-negative, and nonphagocytic. By transmission and scanning electron microscopy these cells had large irregular nuclei, numerous spherical mitochondria, elongated cytoplasmatic processes, and lacked phagosomes. These features clearly separate these cells from canine monocytes and characterize them as DC. These cells were capable of reconstituting the proliferative responses of accessory-cell-depleted autologous lymphocytes to both Con-A and NaIO4. The responses to both mitogens showed a similar time course: an initial cluster formation of lymphocytes with DC was followed by lymphocyte blastogenesis (48-72 hr) and proliferation (72-96 hr). UV irradiation of DC before culture completely abrogated accessory activity for the response to both Con-A and NaIO4, and neither cluster formation nor blast transformation or proliferation occurred. These data provide evidence that a UV-sensitive step of direct interaction of lymphocytes and DC is involved in the mechanism of T cell activation and proliferation.

Animals↗

Iodide.

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Disaster Planning↗

Pharmacoproteomics in the development of personalised medicine in Age-related Macular Degeneration (PHARPRO-AMD) study protocol.

INTRODUCTION: Age-related macular degeneration (AMD) is the leading cause of irreversible vision loss among people over 55 years of age globally, being neovascular AMD (nAMD) its most aggressive form. Its treatment consists of the use of drugs that block vascular endothelial growth factor (anti-VEGF). Proteomics may allow the identification of differentially expressed proteins between responders and non-responders to each anti-VEGF drug. Thus, the objective of Pharmacoproteomics in the development of personalised medicine in Age-related Macular Degeneration (PHARPRO-AMD) is to find new proteomic biomarkers, predictive of response to antiangiogenic treatment in patients with nAMD. METHODS AND ANALYSIS: PHARPRO-AMD is a nationwide, multicentre, prospective, observational study. Treatment-naïve patients with nAMD starting anti-VEGF therapy will be enrolled and followed up for 2 years. During this period, clinical variables will be gathered to classify treatment response. In addition, blood, tear and vitreous and aqueous humour samples will be collected and will undergo a ZenoSWATH proteomic analysis. Relevant biomarkers identified and response classification will be used to perform a multivariate logistic regression and construct receiver operating characteristic curves. RESULTS: The study is expected to identify a panel of proteomic biomarkers predictive of anti-VEGF treatment response. Integrating data from invasive and non-invasive biological samples may enhance clinical applicability. Once validated, these biomarkers could support the design of future clinical trials on biomarker-guided therapies, helping to optimise treatment regimens and improve visual outcomes. CONCLUSIONS: The PHARPRO-AMD study aims to provide proof-of-concept for biomarker-guided anti-VEGF therapy in nAMD, potentially improving vision outcomes. A notable limitation is the exclusion of patients with visual acuity above 73 Early Treatment of Diabetic Retinopathy Study letters, a criterion chosen to reduce potential ceiling effects and improve response assessment accuracy. ETHICS AND DISSEMINATION: Approved by the Galician Network of Ethics Committees, with nationwide validity. Anonymised data will be deposited in open-access repositories and published in peer-reviewed journals. TRIAL REGISTRATION NUMBER: Spanish Clinical Studies Registry (REec) (0033-2024-OBS).

Humans↗