Diagnose of the ingestion of Asclepias mellodora St. Hil. by sheep through microhistological analysis of their digestive contents

Asclepias mellodora St. Hil. is a native acute toxic species frequent in the grasslands of the Buenos Aires province, Argentina, whose toxicity had not been assessed until now. This study evaluates the minimal lethal dose of this species for sheep, and the possibility of microscopically recognizing its fragments in gastrointestinal contents as a complementary diagnostic tool in necropsies. Three Frisona sheep (average LW=55±4.5 kg) were dosed via an esophageal tube with each one of the following doses of asclepias: 8.0, 5.0, 2.0 and 0.8 g DM.kg LW-1. Sheep poisoned with the three higher doses died between 10 and 85 h after intoxication, but those receiving the lower dose did not. During necropsies we: 1) determined the dry weight of the contents of rumen+reticulum, omasum+abomasum, and large intestine, 2) estimated the percentages of asclepias fragments by microanalysis correcting for digestion effects on fragment recognition, and 3) calculated the total mass of asclepias in the digestive tract of each animal. For the three higher doses, the mass of asclepias identified in the total ingesta was 12.3±3.4% of the amount supplied, possibly because of the strong diarrhea its ingestion produced. The percentages of asclepias in rumen+reticulum did not differ from the average quantified for the entire tract. The results of this study indicate that the minimal lethal doses of asclepias for sheep is between 2.0 and 0.8g DM·kg LW-1, and that the microhistological analysis of the rumen+reticulum, the easiest region to sample, can be used to confirm the ingestion of this toxic species, although the estimated percentage will be not a good estimator of the ingested percentage.

Feasibility of ASH DEC‐ process in treating sewage sludge and manure ash in Finland

In Finland the thermal treatment of sewage sludge has been moderate in 21th century. The reason has been the high moisture content of sludge. During 2005-2008, 97-99% of sewage sludge was utilized in landscaping and agriculture. However agricultural use has been during 2005-2007 less than 3 %. The aim of national waste management plan is that by 2016 100% of sludge is used either as soil amendment or energy. The most popular utilization method for manure is spreading it on arable land. The dry manures such as poultry manure and horse manure could also be used in incineration. The ashes could be used as fertilizers and while it is not suitable as a starter fertilizer, it is suitable in maintaining P levels in the soil. One of the main drivers for more efficient nutrient management is the eutrophication in lakes and the Baltic See. ASH DEC process can be used in concentrating phosphorus rich ashes while separating the heavy metals that could be included. ASH DEC process uses thermochemical treatment to produce renewable phosphate for fertilizer production. The process includes mixing of ashes and chlorine donors and subsequent treatment in rotary kiln for 20 min in temperature of 900 – 1 050 oC. The heavy metals evaporate and P-rich product is obtained. The toxic substances are retained in air pollution control system in form of mixed metal hydroxides. The aim of conducting this study is to estimate the potential of ASH DEC process in treating phosphorus rich ashes in Finland. The masses considered in are sewage sludge, dry manure from horses, and poultry and liquid pig manure. To date the usual treatment method for sewage sludge in Finland is composting or anaerobic digestion. Part of the amount of produced sewage sludge (800 kt/a fresh mass and 160 kt/a TS) could also be incinerated and the residual ashes used in ASH DEC process. Incinerating only manure can be economically difficult to manage because the incineration of manure is in Finland considered as waste incineration. Getting a permit for waste incineration is difficult and also small scale waste incineration is too expensive. The manure could act as an additional feedstock in counties with high density of animal husbandry where the land area might not be enough for spreading of manure. Now when the manure acts as a supplementary feedstock beside sludge, the ash can’t be used directly as fertilizer. Then it could be used in ASH DEC process. The perquisite is that the manure producers could pay for the incineration, which might prove problematic.

Waste-to-Energy Scenarios in the China Context

Waste incineration plants are increasingly established in China. A low heating value and high moisture content, due to a large proportion of biowaste in the municipal solid waste (MSW), can be regarded as typical characteristics of Chinese MSW. Two incineration technologies have been mainly established in China: stoker grate and circular fluidized bed (CFB). Both of them are designed to incinerate mixed MSW. However, there have been difficulties to reach the sufficient temperature in the combustion process due to the low heating value of the MSW. That is contributed to the usage of an auxiliary fossil fuel, which is often used during the whole incineration process. The objective of this study was to design alternative Waste-to-energy (WTE) scenarios for existing WTE plants with the aim to improve the material and energy efficiency as well as the feasibility of the plants. Moreover, the aim of this thesis was to find the key factors that affect to the feasibility of the scenarios. Five different WTE plants were selected as study targets. The necessary data for calculation was gained from literature as well as received from the operators of the target WTE plants. The created scenarios were based on mechanical-biological treatment (MBT) technologies, in which the produced solid recovered fuel (SRF) was fed as an auxiliary fuel into a WTE plant replacing the fossil fuel. The mechanically separated biowaste was treated either in an anaerobic digestion (AD) plant, a biodrying plant, a thermal drying plant, or a combined AD plant + thermal drying plant. An interactive excel spreadsheet based computation tool was designed to estimate the viability of the scenarios in different WTE cases. The key figures of the improved material and energy efficiency, such as additional electricity generated and avoided waste for landfill, were got as results. Furthermore, economic indicators such as annual profits (or costs), payback period, and internal rate of return (IRR) were gained as results. The results show that the AD scenario was the most profitable in most of the cases. The current heating value of MSW and the tipping fee for the received MSW appeared as the most important factor in terms of feasibility.

Potential utilization ways of recovered chemical products from digestate

The study evaluates the potential application of chemical substances, obtained from biogas plants` by-products. Through the anaerobic digestion process with biogas the large amount of digestate is produced. This digestate mainly consists on the organic matter with the high concentration of nutrients such as nitrogen and phosphorus. During ammonia stripping and phosphorus precipitation the products- ammonia water, ammonium sulfate, ammonium nitrate, ferrous phosphate, aluminum phosphate, calcium phosphate and struvite can be recovered. These chemicals have potential application in different industrial sectors. According to Finnish market and chemicals properties, the most perspective industrial applications were determined. Based on the data, obtained through the literature review and market study, the ammonia water was recognized as a most perspective recovered substances. According to interview provided among Finnish companies, ammonia water is used for flue gas treatment in SNCR technology. This application has a large scale in the framework of Finnish industrial sectors. As well nitrogen with phosphorous can be used as a source of nutrients in the biological wastewater treatment plants of paper mills.

Vitamin D receptor alleles and bone mineral density in a normal premenopausal Brazilian female population

Studies on the association between vitamin D receptor (VDR) polymorphism and bone mineral density (BMD) in different populations have produced conflicting results probably due to ethnic differences in the populations studied. The Brazilian population is characterized by a very broad genetic background and a high degree of miscegenation. Of an initial group of 164, we studied 127 women from the city of São Paulo, aged 20 to 47 years (median, 31 years), with normal menses, a normal diet and no history of diseases or use of any medication that could alter BMD. VDR genotype was assessed by PCR amplification followed by BsmI digestion of DNA isolated from peripheral leukocytes. BMD was measured using dual energy X-ray absorptiometry (Lunar DPX) at the lumbar site (L2-L4) and femoral neck. Most of the women (77.6%) were considered to be of predominantly European ancestry (20.6% of them reported also native American ancestry), 12.8% were of African-Brazilian ancestry and 9.6% of Asian ancestry, 41.0% (52) were classified as bb, 48.8% (62) as Bb and 10.2% (13) as BB. The BB, Bb and bb groups did not differ in age, height, weight, body mass index or age at menarche. Lumbar spine BMD was significantly higher in the bb group (1.22 ± 0.16 g/cm²) than in the BB group (1.08 ± 0.14; P<0.05), and the Bb group presented an intermediate value (1.17 ± 0.15). Femoral neck BMD was higher in the bb group (0.99 ± 0.11 g/cm²) compared to Bb (0.93 ± 0.12) and BB (0.90 ± 0.09) (P<0.05). These data indicate that there is a significant correlation between the VDR BsmI genotype and BMD in healthy Brazilian premenopausal females.

Cloning, expression and purification of recombinant streptokinase: partial characterization of the protein expressed in Escherichia coli

We cloned the streptokinase (STK) gene of Streptococcus equisimilis in an expression vector of Escherichia coli to overexpress the profibrinolytic protein under the control of a tac promoter. Almost all the recombinant STK was exported to the periplasmic space and recovered after gentle lysozyme digestion of induced cells. The periplasmic fraction was chromatographed on DEAE Sepharose followed by chromatography on phenyl-agarose. Active proteins eluted between 4.5 and 0% ammonium sulfate, when a linear gradient was applied. Three major STK derivatives of 47.5 kDa, 45 kDa and 32 kDa were detected by Western blot analysis with a polyclonal antibody. The 32-kDa protein formed a complex with human plasminogen but did not exhibit Glu-plasminogen activator activity, as revealed by a zymographic assay, whereas the 45-kDa protein showed a Km = 0.70 µM and kcat = 0.82 s-1, when assayed with a chromogen-coupled substrate. These results suggest that these proteins are putative fragments of STK, possibly derived from partial degradation during the export pathway or the purification steps. The 47.5-kDa band corresponded to the native STK, as revealed by peptide sequencing

Aging and immunoglobulin isotype patterns in oral tolerance

In the present review we address oral tolerance as an important biological phenomenon and discuss how it is affected by aging. Other factors such as frequency of feeding and previous digestion of the antigen also seem to influence the establishment of oral tolerance. We also analyze immunoglobulin isotypes of specific antibodies formed by tolerant and immunized animals of different ages submitted to different conditions of oral antigen administration. Isotypic patterns were studied as a parameter for assessing the pathways of B and T cell interactions leading to antibody production

Oral tolerance induction with altered forms of ovalbumin

As a T cell-dependent phenomenon, oral tolerance is not expected to depend necessarily on native configuration of antigens. We investigated the induction of oral tolerance with modified ovalbumin (Ova). Oral administration of heat-denatured (HD-Ova) and cyanogen bromide-degraded ovalbumin was less effective than native Ova in inducing oral tolerance in B6D2F1 mice. HD-Ova was effective in suppressing delayed-type hypersensitivity (DTH) reactions but did not suppress specific antibody formation. Injection of Ova directly into the stomach, but not into the ileum or cecum, suppressed subsequent immunization to DTH reactions. Gavage with protease inhibitors (aprotinin or ovomucoid) before gavage with Ova was ineffective in blocking tolerance induction. Treatment with hydroxyurea to destroy cycling cells 24 h before gavage with Ova blocked oral tolerance induction and also the possibility to passively transfer tolerance to naive recipients with the serum of mice gavaged with Ova 1 h before. The implications of these findings about oral tolerance induction are discussed

Biogas production in regional integrated biodegradable waste treatment – Possibilities for improving energy performance and reducing GHG emissions

The greatest threat that the biodegradable waste causes on the environment is the methane produced in landfills by the decomposition of this waste. The Landfill Directive (1999/31/EC) aims to reduce the landfilling of biodegradable waste. In Finland, 31% of biodegradable municipal waste ended up into landfills in 2012. The pressure of reducing disposing into landfills is greatly increased by the forthcoming landfill ban on biodegradable waste in Finland. There is a need to discuss the need for increasing the utilization of biodegradable waste in regional renewable energy production to utilize the waste in a way that allows the best possibilities to reduce GHG emissions. The objectives of the thesis are: (1) to find important factors affecting renewable energy recovery possibilities from biodegradable waste, (2) to determine the main factors affecting the GHG balance of biogas production system and how to improve it and (3) to find ways to define energy performance of biogas production systems and what affects it. According to the thesis, the most important factors affecting the regional renewable energy possibilities from biodegradable waste are: the amount of available feedstock, properties of feedstock, selected utilization technologies, demand of energy and material products and the economic situation of utilizing the feedstocks. The biogas production by anaerobic digestion was seen as the main technology for utilizing biodegradable waste in agriculturally dense areas. The main reason for this is that manure was seen as the main feedstock, and it can be best utilized with anaerobic digestion, which can produce renewable energy while maintaining the spreading of nutrients on arable land. Biogas plants should be located close to the heat demand that would be enough to receive the produced heat also in the summer months and located close to the agricultural area where the digestate could be utilized. Another option for biogas use is to upgrade it to biomethane, which would require a location close to the natural gas grid. The most attractive masses for biogas production are municipal and industrial biodegradable waste because of gate fees the plant receives from them can provide over 80% of the income. On the other hand, directing gate fee masses for small-scale biogas plants could make dispersed biogas production more economical. In addition, the combustion of dry agricultural waste such as straw would provide a greater energy amount than utilizing them by anaerobic digestion. The complete energy performance assessment of biogas production system requires the use of more than one system boundary. These can then be used in calculating output–input ratios of biogas production, biogas plant, biogas utilization and biogas production system, which can be used to analyze different parts of the biogas production chain. At the moment, it is difficult to compare different biogas plants since there is a wide variation of definitions for energy performance of biogas production. A more consistent way of analyzing energy performance would allow comparing biogas plants with each other and other recovery systems and finding possible locations for further improvement. Both from the GHG emission balance and energy performance point of view, the energy consumption at the biogas plant was the most significant factor. Renewable energy use to fulfil the parasitic energy demand at the plant would be the most efficient way to reduce the GHG emissions at the plant. The GHG emission reductions could be increased by upgrading biogas to biomethane and displacing natural gas or petrol use in cars when compared to biogas CHP production. The emission reductions from displacing mineral fertilizers with digestate were seen less significant, and the greater N2O emissions from spreading digestate might surpass the emission reductions from displacing mineral fertilizers.

Fractures of the proximal femur: correlation with vitamin D receptor gene polymorphism

Fractures are the feared consequences of osteoporosis and fractures of the proximal femur (FPF) are those that involve the highest morbidity and mortality. Thus far, evaluation of bone mineral density (BMD) is the best way to determine the risk of fracture. Genetic inheritance, in turn, is one of the major determinants of BMD. A correlation between different genotypes of the vitamin D receptor (VDR) and BMD has been recently reported. On this basis, we decided to determine the importance of the determination of VDR genotype in the presence of an osteoporotic FPF in a Brazilian population. We studied three groups: group I consisted of 73 elderly subjects older than 65 years (78.5 ± 7.2 years) hospitalized for nonpathological FPF; group II consisted of 50 individuals older than 65 years (72.9 ± 5.2 years) without FPF and group III consisted of 98 young normal Brazilian individuals aged 32.6 ± 6.6 years (mean ± SD). Analysis of VDR gene polymorphism by restriction fragment length polymorphism (RFLP) was performed by PCR amplification followed by BsmI digestion of DNA isolated from peripheral leukocytes. The genotype distribution in group I was 20.5% BB, 42.5% Bb and 37% bb and did not differ significantly from the values obtained for group II (16% BB, 36% Bb and 48% bb) or for group III (10.2% BB, 47.6% Bb and 41.8% bb). No differences in genotype distribution were observed between sexes or between the young and elderly groups. We conclude that determination of VDR polymorphism is of no practical use for the prediction of FPF. Other nongenetic factors probably start to affect bone mass, the risk to fall and consequently the occurrence of osteoporotic fractures with advancing age.

Methylmercury intoxication and histochemical demonstration of NADPH-diaphorase activity in the striate cortex of adult cats

The effects of methylmercury (MeHg) on histochemical demonstration of the NADPH-diaphorase (NADPH-d) activity in the striate cortex were studied in 4 adult cats. Two animals were used as control. The contaminated animals received 50 ml milk containing 0.42 µg MeHg and 100 g fish containing 0.03 µg MeHg daily for 2 months. The level of MeHg in area 17 of intoxicated animals was 3.2 µg/g wet weight brain tissue. Two cats were perfused 24 h after the last dose (group 1) and the other animals were perfused 6 months later (group 2). After microtomy, sections were processed for NADPHd histochemistry procedures using the malic enzyme method. Dendritic branch counts were performed from camera lucida drawings for control and intoxicated animals (N = 80). Average, standard deviation and Student t-test were calculated for each data group. The concentrations of mercury (Hg) in milk, fish and brain tissue were measured by acid digestion of samples, followed by reduction of total Hg in the digested sample to metallic Hg using stannous chloride followed by atomic fluorescence analysis. Only group 2 revealed a reduction of the neuropil enzyme activity and morphometric analysis showed a reduction in dendritic field area and in the number of distal dendrite branches of the NADPHd neurons in the white matter (P<0.05). These results suggest that NADPHd neurons in the white matter are more vulnerable to the long-term effects of MeHg than NADPHd neurons in the gray matter.

HFE gene mutations in coronary atherothrombotic disease

Although iron can catalyze the production of free radicals involved in LDL lipid peroxidation, the contribution of iron overload to atherosclerosis remains controversial. The description of two mutations in the HFE gene (Cys282Tyr and His63Asp) related to hereditary hemochromatosis provides an opportunity to address the question of the association between iron overload and atherosclerosis. We investigated the prevalence of HFE mutations in 160 survivors of myocardial infarction with angiographically demonstrated severe coronary atherosclerotic disease, and in 160 age-, gender- and race-matched healthy control subjects. PCR amplification of genomic DNA followed by RsaI and BclI restriction enzyme digestion was used to determine the genotypes. The frequency of the mutant Cys282Tyr allele was identical among patients and controls (0.022; carrier frequency, 4.4%), whereas the mutant His63Asp allele had a frequency of 0.143 (carrier frequency, 27.5%) in controls and of 0.134 (carrier frequency, 24.5%) in patients. Compound heterozygotes were found in 2 of 160 (1.2%) controls and in 1 of 160 (0.6%) patients. The finding of a similar prevalence of Cys282Tyr and His63Asp mutations in the HFE gene among controls and patients with coronary atherothrombotic disease, indirectly questions the possibility of an association between hereditary hemochromatosis and atherosclerosis.

Production, isolation and characterization of bioactive peptides with antihypertensive properties from rapeseed and potato protein

Consumers’ increasing awareness of healthiness and sustainability of food presents a great challenge to food industry to develop healthier, biologically active and sustainable food products. Bioactive peptides derived from food proteins are known to possess various biological activities. Among the activities, the most widely studied are antioxidant activities and angiotensin I converting enzyme (ACE) inhibitory activity related to blood pressure regulation and antihypertensive effects. Meanwhile, vast amounts of byproducts with high protein content are produced in food industry, for example potato and rapeseed industries. The utilization of these by-products could be enhanced by using them as a raw material for bioactive peptides. The objective of the present study was to investigate the production of bioactive peptides with ACE inhibitory and antioxidant properties from rapeseed and potato proteins. Enzymatic hydrolysis and fermentation were utilized for peptide production, ultrafiltration and solid-phase extraction were used to concentrate the active peptides, the peptides were fractionated with liquid chromatographic processes, and the peptides with the highest ACE inhibitory capacities were putified and analyzed with Maldi-Tof/Tof to identify the active peptide sequences. The bioavailability of the ACE inhibitory peptides was elucidated with an in vitro digestion model and the antihypertensive effects in vivo of rapeseed peptide concentrates were investigated with a preventive premise in 2K1C rats. The results showed that rapeseed and potato proteins are rich sources of ACE inhibitory and antioxidant peptides. Enzymatic hydrolysis released the peptides effectively whereas fermentation produced lower activities.The native enzymes of potato were also able to release ACE inhibitory peptides from potato proteins without the addition of exogenous enzymes. The rapeseed peptide concentrate was capable of preventing the development of hypertension in vivo in 2K1C rats, but the quality of rapeseed meal used as raw material was found to affect considerably the antihypertensive effects and the composition of the peptide fraction.

Stability of Natural Colorants of Plant Origin

In recent years, there have been studies that show a correlation between the hyperactivity of children and use of artificial food additives, including colorants. This has, in part, led to preference of natural products over products with artificial additives. Consumers have also become more aware of health issues. Natural food colorants have many bioactive functions, mainly vitamin A activity of carotenoids and antioxidativity, and therefore they could be more easily accepted by the consumers. However, natural colorant compounds are usually unstable, which restricts their usage. Microencapsulation could be one way to enhance the stability of natural colorant compounds and thus enable better usage for them as food colorants. Microencapsulation is a term used for processes in which the active material is totally enveloped in a coating or capsule, and thus it is separated and protected from the surrounding environment. In addition to protection by the capsule, microencapsulation can also be used to modify solubility and other properties of the encapsulated material, for example, to incorporate fat-soluble compounds into aqueous matrices. The aim of this thesis work was to study the stability of two natural pigments, lutein (carotenoid) and betanin (betalain), and to determine possible ways to enhance their stability with different microencapsulation techniques. Another aim was the extraction of pigments without the use of organic solvents and the development of previously used extraction methods. Stability of pigments in microencapsulated pigment preparations and model foods containing these were studied by measuring the pigment content after storage in different conditions. Preliminary studies on the bioavailability of microencapsulated pigments and sensory evaluation for consumer acceptance of model foods containing microencapsulated pigments were also carried out. Enzyme-assisted oil extraction was used to extract lutein from marigold (Tagetes erecta) flower without organic solvents, and the yield was comparable to solvent extraction of lutein from the same flowers. The effects of temperature, extraction time, and beet:water ratio on extraction efficiency of betanin from red beet (Beta vulgaris) were studied and the optimal conditions for maximum yield and maximum betanin concentration were determined. In both cases, extraction at 40 °C was better than extraction at 80 °C and the extraction for five minutes was as efficient as 15 or 30 minutes. For maximum betanin yield, the beet:water ratio of 1:2 was better, with possibly repeated extraction, but for maximum betanin concentration, a ratio of 1:1 was better. Lutein was incorporated into oil-in-water (o/w) emulsions with a polar oil fraction from oat (Avena sativa) as an emulsifier and mixtures of guar gum and xanthan gum or locust bean gum and xanthan gum as stabilizers to retard creaming. The stability of lutein in these emulsions was quite good, with 77 to 91 percent of lutein being left after storage in the dark at 20 to 22°C for 10 weeks whereas in spray dried emulsions the retention of lutein was 67 to 75 percent. The retention of lutein in oil was also good at 85 percent. Betanin was incorporated into the inner w1 water phase of a water1-in-oil-inwater2 (w1/o/w2) double emulsion with primary w1/o emulsion droplet size of 0.34 μm and secondary w1/o/w2 emulsion droplet size of 5.5 μm and encapsulation efficiency of betanin of 89 percent. In vitro intestinal lipid digestion was performed on the double emulsion, and during the first two hours, coalescence of the inner water phase droplets was observed, and the sizes of the double emulsion droplets increased quickly because of aggregation. This period also corresponded to gradual release of betanin, with a final release of 35 percent. The double emulsion structure was retained throughout the three-hour experiment. Betanin was also spray dried and incorporated into model juices with different pH and dry matter content. Model juices were stored in the dark at -20, 4, 20–24 or 60 °C (accelerated test) for several months. Betanin degraded quite rapidly in all of the samples and higher temperature and a lower pH accelerated degradation. Stability of betanin was much better in the spray dried powder, with practically no degradation during six months of storage in the dark at 20 to 24 °C and good stability also for six months in the dark at 60 °C with 60 percent retention. Consumer acceptance of model juices colored with spray dried betanin was compared with similar model juices colored with anthocyanins or beet extract. Consumers preferred beet extract and anthocyanin colored model juices over juices colored with spray dried betanin. However, spray dried betanin did not impart any off-odors or off-flavors into the model juices contrary to the beet extract. In conclusion, this thesis describes novel solvent-free extraction and encapsulation processes for lutein and betanin from plant sources. Lutein showed good stability in oil and in o/w emulsions, but slightly inferior in spray dried emulsions. In vitro intestinal lipid digestion showed a good stability of w1/o/w2 double emulsion and quite high retention of betanin during digestion. Consumer acceptance of model juices colored with spray dried betanin was not as good as model juices colored with anthocyanins, but addition of betanin to real berry juice could produce better results with mixture of added betanin and natural berry anthocyanins could produce a more acceptable color. Overall, further studies are needed to obtain natural colorants with good stability for the use in food products.

Aggrecan structure in amphibian cartilage

The structure of the large proteoglycan present in the bullfrog epiphyseal cartilage was studied by immunochemical and biochemical methods. The isolated monomer showed a polydisperse behavior on Sepharose CL2B, with a peak at Kav = 0.14. Chondroitin sulfate chains were identified by HPLC analysis of the products formed by chondroitinase digestion and mercuric acetate treatment. These chains have approximately 38 disaccharides, a Di45:Di68 ratio of 1.6 and GalNAc4S + GalNAc4,6S are the main non-reducing terminals. Keratan sulfate was identified by the use of two monoclonal antibodies in Western blots after chondroitinase ABC treatment. A keratan sulfate-rich region (~110 kDa) was isolated by sequential treatment with chondroitinase ABC and proteases. We also employed antibodies in Western blotting experiments and showed that the full length deglycosylated core protein is about 300 kDa after SDS-PAGE. Domain-specific antibodies revealed the presence of immunoreactive sites corresponding to G1/G2 and G3 globular domains and the characterization of this large proteoglycan as aggrecan. The results indicate the high conservation of the aggrecan domain structure in this lower vertebrate.