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The adenine nucleotide translocator protein (ANT) is the first well-characterized mitochondrial polypeptide to be identified as an antigen for antimitochondrial autoantibodies (AMA) in PBC sera. Because of the potential use for a highly purified antigen as a tool in studying the actiology of PBC, we have undertaken an assessment of the quantitative importance of ANT as a PBC-specific mitochondrial antigen. Immunoblotting and ELISA techniques were used. Both methods reveal PBC antibodies against isolated rat liver ANT. However, competitive ELISA experiments using purified rat liver ANT as the competing antigen show that anti-ANT antibodies in PBC scrum comprise only a fraction of the total AMA. Furthermore, both ELISA and immunoblotting experiments show that rat liver ANT is not a specific-antigen for PBC autoantibodies. Sera from patients with SLE, chronic active hepatitis, and sera from normal, control patients, have nearly the same, or higher. ANT antibody titres. Thus, ANT is not a good candidate as an antigen for the diagnosis of PBC. [ABSTRACT FROM AUTHOR]

Anti-mitochondrial autoantibodics (AMA) from patients with primary biliary cirrhosis (PBC) were analysed for fine specificity by immunoblotting and enzyme-linked immunosorbent assay (ELISA). Inhibition ELISA showed that complex l (NADH-ubiquinone reductase) from beef heart mitochondria completely inhibited the binding of AMA to mitochondrial inner membranes (SMP), indicating that the major mitochondrial antigens are located in complex l. Immunoblot analysis of beef heart SMP, complex 1 and the iron sulphur (IP) subtraction of complex l revealed several antigens, one of which (75 kDa) reacted with all PBC sera but not with the additional autoimmune sera tested. Resolution of SMP or complex 1 by two-dimensional electrophoresis yielded in both preparations a polypeptide of 75 kDa with an isoelectric point of 6.4, which reacted with PBC serum and with rabbit antisera raised against the 75,000 subunit of complex 1. In immunoblot experiments, the antigenicity of the 75,000 polypeptide in SMP, complex I, and the IP subtraction is increased by prior reduction of the sample with mercaptoethanol. This suggests a similarity to the PBC-specific 'M-2' antigen, which is also sensitive to sulphur reagents. The data indicate that the 75 kDa polypeptide of complex I is a major mitochondrial antigen binding AMA in PBC sera, and allows us to identify the location and probable function of the PBC antigen. [ABSTRACT FROM AUTHOR]

A monoclonal antibody specific for the major primary biliary cirrhosis (PBC)-associated mitochondrial antigen (subunit I of NADH-ubiquinone reductase) was produced and used to study the binding sites recognized by anti-mitochondrial autoantibodies (AMA) in PBC sera. Immunization of mice with purified beef heart mitochondrial inner membranes resulted in one monoclonal of mice with purified beef heart mitochondrial inner membranes resulted in one monoclonal antibody which reacted with mitochondrial proteins. This antibody (PBC-MoAb), which was of the IgG2b subclass with kappa light chains, exhibited a pattern of immunofluores-cence reactivity with rat kidney, human thyroid, and cultured human epithelial cells (Hep-2) similar to that obtained with sera from PBC patients. Similar binding patterns between PBC-MoAb and AMA were also found in western blot analysis using mitochondria as antigen. Both types of antibodies revealed a major antigen of 75kDa, a minor antigen of 60kDa, and a third antigen (70 kDa), which was detected only in samples that had not been boiled prior to electrophoresis. Furthermore, optimal binding of the PBC-MoAb and AMA to the 75 and 70 kDa antigens required reduction of the antigen with mercaptoethanol prior to electrophoresis. Competition ELISA experiments were conducted to compare the epitopes recognized by PBC-MoAb and AMA. Of 28 PBC sera tested. 27 inhibited the binding of PBC-MoAb to mitochondrial inner membranes by almost 100% and one serum inhibited binding by 50%, indicating that most PBC sera contain autoantibodies reactive with the same or a closely related antibody binding site as the PBC-MoAb, PBC-MoAb inhibited AMA binding to the inner membrane by more than 80% in 10 sera, 60-80% in 11 sera, and 40-59% in seven sera, with an average inhibition of 71%. Our observations strongly indicate that anti-mitochondrial autoantibody binding sites are restricted to a highly immunogenic epitope on the major PBC-specific antigen (NADH-ubiquinone reductase subunit I), and that the anti-mitochondrial monoclonal anti-body obtained has a specificity identical with the human PBC-specific M2 type anti-mitochondrial autoantibody. [ABSTRACT FROM AUTHOR]

Primary biliary cirrhosis (PBC)-specific antigens were purified from beef heart mitochondria by immunoaffinity chromatography. Three major polypeptides (75, 60, and 40 kDa) were detected in the purified antigen fraction both by Coomassie blue staining and by western blot analysis. The 75 kDa antigen was identified as a subunit of Complex I (NADM-ubiquinone reductase) by the following criteria: (1) antibodies against the purified 75 kDa subunit of beef heart Complex I react with the immunoaffinity-purified 75 kDa antigen, (2) the 75 kDa subunit present in isolated Complex I, like that in the immunoaffinity-purified antigen, reacts with PBC sera only after reduction with mercaptoethanol, and (3) the 75 kDa antigen is enriched in isolated Complex I. A relationship between the 75 kDa and the 60 and 40 kDa antigens is suggested, since optimal binding of anti-mitochondrial autoantibodies (AMA) to the latter antigens also requires prior reduction with mercaptoethanol. A fourth major antigen (70 kDa) was also detected by western blot analysis, but only in samples that had not been boiled prior to electrophoresis. This antigen, which is also present in isolated Complex I, resembles the 75,60, and 40 kDa antigens in its response in mercaptoethanol and its reaction with antibodies against the 75 kDa subunit Complex I. A scheme is presented which relates all of the PBC antigens to the parent 75 kDa subunit of Complex I, probably as proteolytic products of the latter. [ABSTRACT FROM AUTHOR]

Plant-fungal interactions are ubiquitous across ecosystems and contribute significantly to plant ecology and evolution. All orchids form obligate symbiotic relationships with specific fungi for germination and early growth, and the distribution of terrestrial orchid species has been linked to occurrence and abundance of specific orchid mycorrhizal fungi (OMF) in the soil. The availability of OMF can therefore be a habitat requirement that is relevant to consider when establishing management and conservation strategies for threatened orchid species, but knowledge on the spatial distribution of OMF in soil is limited. We here studied the mycorrhizal associations of three terrestrial orchid species ( Anacamptis pyramidalis , Orchis purpurea and Platanthera chlorantha ) found in a local orchid diversity hotspot in eastern Denmark, and investigated the abundance of the identified mycorrhizal fungi in the surrounding soil. We applied ITS metabarcoding to samples of orchid roots, rhizosphere soil and bulk soil collected at three localities, supplemented with standard barcoding of root samples with OMF specific primers, and detected 22 Operational Taxonomic Units (OTUs) putatively identified as OMF. The three orchid species displayed different patterns of OMF associations, supporting the theory that association with specific fungi constitutes part of an orchid's ecological niche allowing co-occurrence of many species in orchid-rich habitats. The identified mycorrhizal partners in the basidiomycete families Tulasnellaceae and Ceratobasidiaceae (Cantharallales) were detected in low abundance in rhizosphere soil, and appeared almost absent from bulk soil at the localities. This finding highlights our limited knowledge of the ecology and trophic mode of OMF outside orchid tissues, as well as challenges in the detection of specific OMF with standard methods. Potential implications for management and conservation strategies are discussed., Competing Interests: The authors declare no competing interests., (© 2024 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

In the billion-dollar global illegal wildlife trade, rosewoods have been the world's most trafficked wild product since 2005. Dalbergia cochinchinensis and Dalbergia oliveri are the most sought-after rosewoods in the Greater Mekong Subregion. They are exposed to significant genetic risks and the lack of knowledge on their adaptability limits the effectiveness of conservation efforts. Here, we present genome assemblies and range-wide genomic scans of adaptive variation, together with predictions of genomic offset to climate change. Adaptive genomic variation was differentially associated with temperature and precipitation-related variables between the species, although their natural ranges overlap. The findings are consistent with differences in pioneering ability and in drought tolerance. We predict their genomic offsets will increase over time and with increasing carbon emission pathway but at a faster pace in D. cochinchinensis than in D. oliveri . These results and the distinct gene-environment association in the eastern coastal edge of Vietnam suggest species-specific conservation actions: germplasm representation across the range in D. cochinchinensis and focused on hotspots of genomic offset in D. oliveri . We translated our genomic models into a seed source matching application, seedeR , to rapidly inform restoration efforts. Our ecological genomic research uncovering contrasting selection forces acting in sympatric rosewoods is of relevance to conserving tropical trees globally and combating risks from climate change.

Tree diversity in Asia's tropical and subtropical forests is central to nature-based solutions. Species vulnerability to multiple threats, which affect provision of ecosystem services, is poorly understood. We conducted a region-wide, spatially explicit assessment of the vulnerability of 63 socioeconomically important tree species to overexploitation, fire, overgrazing, habitat conversion, and climate change. Trees were selected for assessment from national priority lists, and selections were validated by an expert network representing 20 countries. We used Maxent suitability modeling to predict species distribution ranges, freely accessible spatial data sets to map threat exposures, and functional traits to estimate threat sensitivities. Species-specific vulnerability maps were created as the product of exposure maps and sensitivity estimates. Based on vulnerability to current threats and climate change, we identified priority areas for conservation and restoration. Overall, 74% of the most important areas for conservation of these trees fell outside protected areas, and all species were severely threatened across an average of 47% of their native ranges. The most imminent threats were overexploitation and habitat conversion; populations were severely threatened by these factors in an average of 24% and 16% of their ranges, respectively. Our model predicted limited overall climate change impacts, although some study species were likely to lose over 15% of their habitat by 2050 due to climate change. We pinpointed specific natural areas in Borneo rain forests as hotspots for in situ conservation of forest genetic resources, more than 82% of which fell outside designated protected areas. We also identified degraded areas in Western Ghats, Indochina dry forests, and Sumatran rain forests as hotspots for restoration, where planting or assisted natural regeneration will help conserve these species, and croplands in southern India and Thailand as potentially important agroforestry options. Our results highlight the need for regionally coordinated action for effective conservation and restoration., (© 2021 The Authors. Conservation Biology published by Wiley Periodicals LLC on behalf of Society for Conservation Biology.)

Indochina is a biodiversity hot spot and harbors a high number of endemic species, most of which are poorly studied. This study explores the genetic structure and reproductive system of the threatened endemic timber species Dalbergia cochinchinensis and Dalbergia oliveri using microsatellite data from populations across Indochina and relates it to landscape characteristics and life-history traits. We found that the major water bodies in the region, Mekong and Tonle Sap, represented barriers to gene flow and that higher levels of genetic diversity were found in populations in the center of the distribution area, particularly in Cambodia. We suggest that this pattern is ancient, reflecting the demographic history of the species and possible location of refugia during earlier time periods with limited forest cover, which was supported by signs of old genetic bottlenecks. The D. oliveri populations had generally high levels of genetic diversity (mean H e  = 0.73), but also strong genetic differentiation among populations (global G ST  = 0.13), while D. cochinchinensis had a moderate level of genetic diversity (mean H e  = 0.55), and an even stronger level of differentiation (global G ST  = 0.25). These differences in genetic structure can be accounted for by a higher level of gene flow in D. oliveri due to a higher dispersal capacity, but also by the broader distribution area for D. oliveri , and the pioneer characteristics of D. cochinchinensis . This study represents the first detailed analysis of landscape genetics for tree species in Indochina, and the found patterns might be common for other species with similar ecology.

The genus Dalbergia contains many valuable timber species threatened by illegal logging and deforestation, but knowledge on distributions and threats is often limited and accurate species identification difficult. The aim of this study was to apply DNA barcoding methods to support conservation efforts of Dalbergia species in Indochina. We used the recommended rbcL, matK and ITS barcoding markers on 95 samples covering 31 species of Dalbergia, and tested their discrimination ability with both traditional distance-based as well as different model-based machine learning methods. We specifically tested whether the markers could be used to solve taxonomic confusion concerning the timber species Dalbergia oliveri, and to identify the CITES-listed Dalbergia cochinchinensis. We also applied the barcoding markers to 14 samples of unknown identity. In general, we found that the barcoding markers discriminated among Dalbergia species with high accuracy. We found that ITS yielded the single highest discrimination rate (100%), but due to difficulties in obtaining high-quality sequences from degraded material, the better overall choice for Dalbergia seems to be the standard rbcL+matK barcode, as this yielded discrimination rates close to 90% and amplified well. The distance-based method TaxonDNA showed the highest identification rates overall, although a more complete specimen sampling is needed to conclude on the best analytic method. We found strong support for a monophyletic Dalbergia oliveri and encourage that this name is used consistently in Indochina. The CITES-listed Dalbergia cochinchinensis was successfully identified, and a species-specific assay can be developed from the data generated in this study for the identification of illegally traded timber. We suggest that the use of DNA barcoding is integrated into the work flow during floristic studies and at national herbaria in the region, as this could significantly increase the number of identified specimens and improve knowledge about species distributions.

The prognosis for patients suffering from cardiovascular and many other diseases can be substantially improved if diagnosed at an early stage. High performance diagnostic testing using disposable microfluidic chips can provide a platform for realizing this vision. Amic AB (Uppsala, Sweden) has developed a new microfluidic test chip for sandwich immunoassays fabricated by injection molding of the cycloolefin-copolymer Zeonor. A highly ordered array of micropillars within the fluidic chip distributes the sample solution by capillary action. Since wetting of the pillar array surface is the only driving force for liquid distribution precise control of the surface chemistry is crucial. In this work we demonstrate a novel protocol for surface hydrophilization and antibody immobilization on cycloolefin-copolymer test chips, based on direct silanisation of the thermoplastic substrate. Dextran is subsequently covalently coupled to amino groups, thus providing a coating with a low contact angle suitable for antibody immobilization. The contact angle of dextran coated chips is stable for at least two months, which enables production of large batches that can be stored for extended periods of time. We demonstrate the utility of the presented platform and surface chemistry in a C-reactive protein assay with a detection limit of 2.6 ng ml(-1), a dynamic range of 10(2) and a coefficient of variance of 15%.

Background: A simple point-of-care method for measuring leukocyte counts in a doctor's office or emergency room could be of great importance. We developed a protocol for measuring cell count by disrupting the cell membrane and analyzing specific proteins within the cells and used it to analyze proteins from eosinophils and neutrophils., Methods: Lateral immunochromatographic (ICR) assays have been developed for eosinophil protein X (EPX) and human neutrophil lipocalin (HNL) as measures of the concentration of eosinophils and neutrophils. The correlation between the lateral ICR assays and cell counting of eosinophils and neutrophils was performed manually and with an automated cell counter. RIA assays measuring the same analytes were also compared with the results from cell counting and lateral ICR assays., Results: The optimized assays showed analytical detection limits below the clinical ranges of 3.36 microg/L and 2.05 microg/L for EPX and HNL, respectively. The recovery was 114.8%-122.8% for EPX and 94.5%-96.9% for HNL. The imprecision was 3%-17% CV for EPX over the whole range and 5%-16% CV for HNL. The correlation coefficients between manually counted cells and lateral ICR assays were 0.9 and 0.83 for EPX and HNL, respectively., Conclusion: The numbers of eosinophils and neutrophils in small amounts of blood can be estimated in the point-of-care setting by means of fast lateral ICR assays of EPX and HNL.

A new variant of human growth hormone was recently found [Pavlu, B. & Gellerfors, P. (1993) Bioseparation 3, 257-265]. We report here the identification and the structural determination of this variant. The variant, which is formed during the expression of human growth hormone in Escherichia coli, was found to be more hydrophobic than rhGH as judged by its prolonged elution time by hydrophobic interaction chromatography. The rhGH hydrophobic variant (rhGH-HV) was isolated and subjected to trypsin digestion and RP-HPLC analysis, resulting in an altered retention time of one single tryptic peptide as compared to the corresponding fragment of rhGH. This tryptic peptide constitutes the C-terminus (aa 179-191) of hGH and contains one of the two disulfide bridges in hGH, viz. Cys182-Cys189. Amino acid sequences and composition analyses of the tryptic peptide from rhGH-HV (Tv18-19) and the corresponding tryptic peptide from rhGH (T18+19) were identical. Electrospray mass spectrometry (ES MS) of Tv18+19 isolated from rhGH-HV revealed a monoisotopic mass increase of 32.7, as compared to T18+19 from rhGH. A synthetic Tv18+19 peptide having a trisulfide bridge between Cys182 and Cys189 showed identical fragment in ES/MS compared to Tv18+19 isolated from rhGH-HV, i.e. m/z 617.7 and 682.9. These fragments are formed through a unique cleavage in the trisulfide (Cys182-SSS-Cys189) bridge not found in the corresponding T18+19 disulfide peptide. Furthermore, the synthetic Tv18+19 co-eluted in RP-HPLC with Tv18+19 isolated from rhGH-HV. Two-dimensional NMR spectroscopy of the synthetic T18+19 and Tv18+19 peptides were performed. Using these data all protons were assigned. The major chemical shift changes (delta delta > 0.05 ppm) observed were for the beta-protons of Cys182 and Cys189 in Tv18+19 as compared to T18+19. CD spectroscopy data were also in agreement with the above results. Based on these physico-chemical data rhGH-HV has been structurally defined as a trisulfide variant of rhGH. The receptor binding properties of rhGH-HV was studied by a biosensor device, BIAcore. The binding capacity of rhGH-HV was similar to rhGH with a binding stoichiometry to the rhGHBP of 1:1.6 and 1:1.5, respectively, indicating that the trisulfide modification did not affect its receptor binding properties.

We used two-color and three-color flow cytometric analysis to study phenotypical activation and functional subsets of T and natural killer cells in the blood and liver tissue of patients with primary biliary cirrhosis, other chronic liver diseases and the blood of healthy subjects. The changes in blood lymphocyte phenotype in patients with primary biliary cirrhosis and other chronic liver diseases were similar and comprised elevated relative or absolute numbers of activated human leukocyte antigen-DR + T subset (CD4+ and CD8+) cells and DR+ natural killer-like (CD16+) cells. B cell (CD19+) numbers were normal. In primary biliary cirrhosis a selective reduction in T cells of suppressor-inducer (CD45RA + CD4 + ) type was registered. The human leukocyte antigen-DR expression among CD4+ T cell subsets was investigated further in primary biliary cirrhosis and healthy controls using triple antibody flow cytometric analysis. Phenotypical cell activation was confined to helper T cells of the primed, memory (CD45RO + CD4+) type. The decrease in suppressor-inducer T cells in primary biliary cirrhosis was paralleled by a reciprocal increase in primed memory T cells. Several significant differences were observed when blood and liver-infiltrating cells from primary biliary cirrhosis patients were compared. In the liver tissue, the CD4/CD8 ratio was decreased, the relative activation of T-subset cells and NK cells was further increased, the suppressor-inducer T subset was further depressed and the primed memory T subset was increased. The cytotoxic T-cell subset (CD11b-) dominated within the CD8+ population. In liver tissue from other chronic liver disease subjects, a lower CD4/CD8 ratio was found compared with primary biliary cirrhosis.(ABSTRACT TRUNCATED AT 250 WORDS)

The nature of mitochondrial PBC-related antigens has been investigated with radioimmunoassay (RIA) and immunoblotting methods. The major antigen(s) was located by RIA in beef heart mitochondria, submitochondrial particles, chloroform-extracted F1-ATPase and Complex III. Cross-competition RIA experiments showed that the same antigen is present in all the above samples but at different concentrations. The antigen is not present in purified F1-ATPase, cytochrome oxidase, the oligomycin sensitivity conferring protein (OSCP), Factor6, or the Transhydrogenase. Immunoblot analysis of the above mitochondrial proteins revealed two PBC-related antigens (apparent molecular weights of 70 KD and 60 KD) whose distribution in the various proteins and protein complexes correlated well with the antigens determined by RIA. Immunoblot analysis of mitochondrial antigens was carried out using sera from normal subjects and from patients with PBC and with different autoimmune diseases (AID). Only PBC sera reacted with the 70 KD and 60 KD antigens. The PBC antigen detected by RIA in submitochondrial particles and the chloroform-F1-ATPase could be blocked by Mersalyl, suggesting its relationship to the mitochondrial 'M2' antigen. Furthermore, the antigenicity of the 70 KD peptide was shown by immunoblotting to be dependent upon mercaptoethanol. Thus, not only is the antigenicity of the 70 KD component dependent on a sulphur group, but the sulphur must be in the reduced form.

1. The synthesis of cytochrome oxidase was studied in isolated rat hepatocytes labeled in vitro. Labeled whole cells, isolated mitochondria, microsomes and the post microsomal supernatant were treated with antisera to rat liver holo-cytochrome oxidase, and the subunits were adsorbed onto Sepharose-protein A. 2. Seven peptides, corresponding to subunits of rat liver cytochrome oxidase, were immunoabsorbed from mitochondria isolated from cells labeled in the absence of inhibitors. Two peptides, corresponding to subunits I (45 500 daltons) and II (26 000 daltons), were labeled in mitochondria isolated from cycloheximide-treated cells. Labeling of these peptides was inhibited by chloramphenicol. Peptides I and II correspond to the two most heavily labeled mitochondrial translation products found in submitochondrial particles. Possible explanations for the lack of labeling of a third mitochondrially translated subunit are discussed. Labeling of the five smallest peptides was inhibited by cyclohexamide but not by chloramphenicol. 3. Peptide I appears in the holoenzyme later than the other six peptides after a pulse-chase. It is not labeled in the immunoabsorbed cytochrome oxidase after a 30 min pulse with [35S]-methionine, but appears after a 3 h chase with unlabeled methionine. Labeling of the other subunits showed no further increase after the chase.

Antibodies against isolated beef-heart ubiquinol--cytochrome c reductase (complex III) have been characterized. Antibodies to complex III react strongly with isolated beef heart complex III and intact beef heart mitochondria, as shown by immunodiffusion and rocket electrophoresis experiments. The complex III content of intact mitochondria can be quantitated with rocket electrophoresis using isolated complex III as a standard. Antibodies to complex III also react with beef liver mitochondria and with both heart and liver mitochondria from rats. The latter are very weak antigens compared to beef heart material. Antibodies to complex III do not react with respiratory chain complexes I and IV, or F1-ATPase from beef heart mitochondria, but gives a slight, but variable, reaction with complex II and the membrane fraction isolated from complex V (oligomycin-sensitive ATPase). Antigenic sites are located on at least five of the seven peptides of complex III. These peptides are presumably lacking in respiratory chain complexes which do not react with antibodies to complex III, and are assumed to be uniquely located in complex III. Antiserum against complex III inhibitis duroquinol--cytochrome c reductase activity in isolated complex III and in complex III incorporated into phospholipid vesicles. Oxidation of NADH and succinate is not affected in submitochondrial particles treated with 6-times more antibody than required for complete inhibition of enzyme activity in free complex III or in complex III-phospholipid vesicles.

Thyrostatic drug treatment of Graves' disease suppresses excessive thyroid hormone synthesis and causes a parallel decrease in serum thyroid autoantibody levels. The mechanism of this immunosuppression is unknown. We studied methimazole-induced immunoregulatory effects prospectively in 14 patients with Graves' disease treated for up to six months. The numbers of circulating activated, HLA-DR-positive T helper/inducer cells decreased gradually, from 8.3+1.7 percent (+SD) to 1.0+1.7 percent (P less than 0.001). HLA-DR-positive T suppressor/cytotoxic cells increased transiently at one month, from 2.0+1.9 percent to 12.6+6.4 percent (P less than 0.001), and returned to 2.9+3.7 percent at six months. Methimazole did not alter the HLA-DR expression of T cells in vitro. In two patients, the helper activity of T cells in inducing autoantibody secretion in vitro was substantially reduced after one month of methimazole treatment. Before treatment, large proportions of thyroid-infiltrating T-cell subsets expressed the activation markers HLA-DR, interferon-gamma, and interleukin-2 receptors, which were partially lost during therapy. Methimazole treatment was accompanied by a gradual reduction in circulating levels of thyrotropin-receptor, microsomal, and thyroglobulin autoantibodies. These results are compatible with the view that methimazole-induced immunoregulation in Graves' disease is mediated by a direct inhibitory effect on thyrocytes. This inhibition is in turn accompanied by marked changes in the proportions of activated T helper-like and T suppressor-like cells. This altered T-cell activation profile reflects, at least in part, the functional suppression of autoantibody production observed in methimazole-treated patients with Graves' disease.

Cerebrospinal fluid (CSF) markers of brain damage are potentially capable of providing quantitative information about the extent of certain neurological injury. The presence of such markers in CSF after brain damage is transient and it is essential to understand their kinetics if they are to be used in clinical practice. In the present study, the CSF concentrations of two neurospecific proteins. S-100 protein and neuron-specific enolase (NSE), were determined in rats before and repeatedly after one of two types of experimental brain damage: traumatic cortical injury and focal cerebral ischemia induced by middle cerebral artery (MCA) occlusion. The two types of experimental brain damage resulted in significant differences in the kinetics of S-100 and NSE concentrations in CSF. Cortical contusion was followed by a rapid increase in both S-100 and NSE and a peak occurred in both after about 7 1/2 hours, at which time the values declined toward normal. A second, smaller peak was seen after about 1 1/2 days. The increase and decrease in S-100 and NSE levels in CSF was slower after MCA occlusion; a peak was seen after 2 to 4 days. Furthermore, S-100 was generally higher than NSE after trauma, whereas after MCA occlusion the NSE concentration was slightly higher than the S-100 value. These results support the use of CSF markers for estimation of the extent of brain damage in experimental models and forms a basis for the understanding of their kinetics, which is important for their use in clinical practice.

We have utilized a one step ion-exchange (FPLC Mono Q) purification procedure for the isolation of human thyroid peroxidase (TPO). The purified TPO had the properties of a major microsomal antigen and inhibited the binding of human microsomal autoantibodies to thyroid microsomal membranes. The isolated TPO was free from thyroglobulin and showed, compared with crude microsomal proteins, a reduced background binding with control sera in enzyme-linked immunoassay (ELISA). SDS-gel electrophoresis of the isolated TPO detected one major band with an apparent molecular weight of 105 kD. The antigenicity of the protein was demonstrated by immunoblotting using sera from patients with autoimmune thyroiditis. These results demonstrate that FPLC Mono Q chromatography offers a rapid, quantitative and precise method for large scale purification of TPO with retained enzymatic and antigenic activity for use in ELISA and for further studies on the structure and function of this protein.