A truncating mutation in the laminin-332α chain highlights the role of the LG45 proteolytic domain in regulating keratinocyte adhesion and migration.

BACKGROUND: Altered function of laminin-332 (α3ß3γ2) consequent to mutations in the LAMA3, LAMB3 and LAMC2 genes causes junctional epidermolysis bullosa non-Herlitz (JEB-nH). JEB-nH patients suffer from skin blistering and have an increased risk of developing aggressive skin carcinomas in adulthood. Laminin-332 is proteolytically processed and its extracellular mature form lacks the α3 chain C-terminal globules 4 and 5 (LG45). The LG45 tandem has cell adhesion and protumorigenic properties. However, mutations that affect this domain are very rare and their functional effects in patients have not been explored to date. OBJECTIVE: To characterize molecularly an adult patient with JEB-nH and altered laminin-332 expression presenting multiple skin carcinomas, and to analyse LG45-mediated biological functions using keratinocytes from the patient. METHODS: A mutational search in laminin-332 genes was performed by hetero-duplex analysis. LAMA3 mRNA and laminin-332 protein levels in patient keratinocytes were investigated by real-time reverse transcriptase polymerase chain reaction and radioimmunoprecipitation assay, respectively. Keratinocyte migration was examined by scratch and Boyden chamber assays. RESULTS: We identified a homozygous LAMA3 mutation, p.Leu1648TrpfsX32, which truncates the last 45 amino acids of the carboxyl terminal LG5 subdomain. Gene expression studies revealed that the mutant transcripts were stable and even increased, precursor laminin-332 molecules were retained intracellularly and the amount of mature extracellular heterotrimers was reduced to about 50%. Finally, the patient's keratinocytes migrated faster than normal keratinocytes. CONCLUSIONS: Structural disruption of LG5 highlights the critical functions of the LG45 proteolytic region in precursor laminin-332 secretion and keratinocyte adhesion and migration. Perturbation of LG45 function might explain the non-aggressive behaviour of carcinomas in this patient.

Induction of senescence pathways in Kindler syndrome primary keratinocytes.

BACKGROUND: Individuals with Kindler syndrome (KS) have loss-of-function mutations in the FERMT1 gene that encodes the focal adhesion component kindlin-1. The major clinical manifestation of KS is epidermal atrophy (premature skin ageing). This phenotypic feature is thought to be related to the decreased proliferation rate of KS keratinocytes; nevertheless, molecular mediators of such abnormal behaviour have not been fully elucidated. OBJECTIVES: To investigate how kindlin-1 deficiency affects the proliferative potential of primary human keratinocytes. METHODS: We serially cultivated nine primary KS keratinocyte strains until senescence and determined their lifespan and colony-forming efficiency (CFE) at each serial passage. The expression of molecular markers of stemness and cellular senescence were investigated by immunoblotting using cell extracts of primary keratinocyte cultures from patients with KS and healthy donors. In another set of experiments, kindlin-1 downregulation in normal keratinocytes was obtained by small interfering RNA (siRNA) technology. RESULTS: We found that KS keratinocytes exhibited a precocious senescence and strongly reduced clonogenic potential. Moreover, KS cultures showed a strikingly increased percentage of aborted colonies (paraclones) already at early passages indicating an early depletion of stem cells. Immunoblotting analysis of KS keratinocyte extracts showed reduced levels of the stemness markers p63 and Bmi-1, upregulation of p16 and scant amounts of hypophosphorylated Rb protein, which indicated cell cycle-arrested status. Treatment of normal human primary keratinocytes with siRNA targeting kindlin-1 proved that its deficiency was directly responsible for p63, Bmi-1 and pRb downregulation and p16 induction. CONCLUSIONS: Our data directly implicate kindlin-1 in preventing premature senescence of keratinocytes.

Metal accumulation and binding protein induction in Mytilus galloprovincialis, Scapharca inaequivalvis, and Tapes philippinarum from the Lagoon of Venice.

Heavy metal pollution is known to be widespread in the sediments of the Lagoon of Venice. Therefore, physiological parameters influenced by this form of contamination were examined. The bivalve molluscs blue mussel (Mytilus galloprovincialis), ark clam (Scapharca inaequivalvis), and Japanese littleneck (Tapes philippinarum) were sampled in two sites (Marghera, Chioggia) every 3 months for 1 year. The digestive gland and gills of each bivalve were analyzed. The concentrations of essential and nonessential metals (including chromium, manganese, iron, cobalt, nickel, copper, zinc, and cadmium) were determined. Because glutathione and metallothioneins (MTs) are involved in metal homeostasis and detoxification, their concentrations were evaluated in relation to metal concentrations. Results show that (1) all three studied species accumulate metals to a considerable extent, with some species-specific differences between the digestive gland and gills; (2) gills are a good tissue to evaluate pollution by examining the MT content. In particular, the correlation between Zn and MT levels in the gills indicates that M. galloprovincialis and S. inaequivalvis are sentinel organisms and can be used specifically for Zn pollution; (3) T. philippinarum accumulates Cu in the digestive gland more readily than the other two bivalves and therefore has the highest MT.

Identification, cloning and characterisation of a novel copper-metallothionein in tetrahymena pigmentosa. Sequencing of cDNA and expression.

The protist Tetrahymena pigmentosa accumulates large amounts of metal ions, particularly cadmium and copper. This capability is linked to the induction of metallothioneins (MTs), cysteine-rich metal-binding proteins found in protists, plants and animals. The present study focuses on a novel inducible MT-isoform isolated from Tetrahymena after exposure to a non-toxic dose of copper. The cDNA sequence was determined utilising the partial peptide sequence of purified protein. The Cu-MT cDNA encodes 96 amino acids containing 28 cysteine residues (29%) arranged in motifs characteristic of the metal-binding regions of vertebrate and invertebrate MTs. Both the amino acid and nucleotide sequences differ, not only from other animal MTs, but also from the previously characterised Tetrahymena Cd-MT. Both MTs contain the structural pattern GTXXXCKCXXCKC, which may be proposed as a conservative sequence of Tetrahymena MTs. Cu-dependent regulation of MT expression was also investigated by measuring MT-mRNA and MT levels. MT synthesis occurs very quickly and MT contents increase with Cu accumulation. The induction of Cu-MT mRNA is very rapid, with no observable lag period, and is characterised by transient fluctuation, similar to that described for Cd-MT mRNA. The data reported here indicate that, also in the unicellular organism Tetrahymena, two very different MT isoforms, which perform different biological functions, are expressed according to the inducing metal, Cu or Cd.

Oxidative burst and metallothionein as a scavenger in macrophages.

UNLABELLED: The role of metallothionein (MT) in the scavenging of superoxide radicals (*O2-) generated by macrophages has been examined. The present work has focused on the effects of added cadmium, a known inducer of MT biosynthesis, on determined amounts of superoxide radicals produced by in vitro cultured rat peritoneal macrophages on their stimulation with phorbol-12-myristate-13-acetate (PMA). The levels of superoxide radicals (*O2-) have been found to decrease when cadmium was added to cells exposed to PMA. However, substantially lower levels of MT have been determined in this case compared to cells untreated with PMA. This effect could be reversed by incubation of the PMA and cadmium-treated cells with a reducing agent, 2-mercaptoethanol (2-ME). Results suggest that *O2- caused thiolate oxidation and subsequent metal loss, thus reducing the cellular MT content as quantified by the silver saturation METHOD: This conclusion is supported by cell-free experiments in which the oxidation of rabbit MT-I by a xanthine/xanthine-oxidase system could be reversed by its subsequent reduction with 2-ME. The data presented provide direct evidence of the involvement of MT in scavenging superoxide radicals in living cells.

Ion exchange chromatographic determination of olpadronate, phosphate, phosphite, chloride and methanesulfonic acid.

A method based on single column ion chromatography with UV detection was developed for purity testing and assay of monosodium olpadronate. The analyte aqueous solution is precipitated with methanol to enhance the impurities/olpadronate molar ratio, thus improving purity determination at trace levels. The resulting solution is injected into a standard chromatographic system with UV detector in indirect mode with a Waters IC Pak HR column using diluted nitric acid as the mobile phase. The method was fully validated according to ICH guidelines for the determination of phosphite, phosphate, chloride and methanesulfonic acid in olpadronate being suitable for purity testing and assay.

Mechanisms of paraneoplastic syndromes in mice bearing a spontaneous lung adenocarcinoma.

Paraneoplastic syndromes are rarely described in animal models. It may be useful to have a suitable experimental model to study the mechanisms by which they are produced. In this study, we describe a murine lung adenocarcinoma, P07, which presents hypercalcemia, leukocytosis and cachexia. We determined the presence of PTHrP in plasma as well as GM-CSF produced by P07 cells. TNF-alpha, which is responsible for cachexia, could neither be detected in serum nor in P07 cell supernatants. We conclude that this model, which shows paraneoplastic syndromes similar to those of lung tumor patients, should be useful to study the pathways and significance of these signs.

Cadmium metallothionein gene of Tetrahymena pyriformis.

A genomic sequence from Tetrahymena pyriformis, encoding a cadmium-induced metallothionein has been cloned. The gene encodes a transcript of 487 bases, with an intronless coding region of 324 nt, using TGA as the stop codon, TAA coding for glutamine, and the translational initiation sequence AAAATGG. Two regions of internal similarity in the coding sequence support the hypothesis that the Tetrahymena protein arose by gene duplication. The sequences of untranslated regions show some similarities with nematode MT-1 and MT-2 transcripts. Sequence of 525 bases upstream of the transcription start contains a TATA box, a CAAT box reverse complement, and many short stretches partially matching the AP-1 and ACE-1 binding sites, but no characteristic sequences found in other metallothionein promoters.

Tetrahymena pyriformis: a tool for toxicological studies. A review.

Among protozoa, Tetrahymena pyriformis is the most commonly ciliated model used for laboratory research. After a brief description of the morphology and biology of Tetrahymena pyriformis, this article focuses on the most important and recent investigations performed with this species in toxicology and ecotoxicology. The methodological features of its culture, and main tests, based on cell growth rate, biochemical markers, behavioral changes and motility, are discussed. Examples of xenobiotics (organic and inorganic substances, pharmaceutical drugs, water pollutants) tested with Tetrahymena pyriformis are also given.

[Morphological studies of hydroxyapatite crystals exposed to disodium pamidronate]. / Estudios morfológicos de los cristales de hidroxiapatita tratados con pamidronato disódico.

"In vitro" effects of disodium pamidronate on hydroxyapatite crystals morphology, and some "in vivo" data from bone powder of tibia and vertebrae from treated young and mature rabbits are here reported. Hydroxyapatite, synthesized following Rigoli et al method, and bone powder from rabbits were studied with X-ray, infrared and raman emission techniques for crystallographic analysis. Adsorption studies were also performed with a balanced solution of hydroxyapatite exposed to different times, 48, 120 and 168 hours and concentrations 1 x 10(-5) M, 3 x 10(-5) M, 8 x 10(-5) M y 1 x 10(-4) M of pamidronate. Infrared and raman spectrometry were not conclusive due to technical bias, but X-ray difractograms showed pure hydroxyapatite crystals in an hexagonal system. At constant time, pamidronate concentrations were varied, showing after 48 hours of exposition, a slight growth in the 002 plane, an aleatoric behavior in 213 and a marked increase in 004. After 120 hours, 002 plane is steady with a net growth in 004 and 213. After 168 hours, the 3 mentioned planes grow in proportion to pamidronate concentrations, tending to enlarge the crystal shape. Plane 13 markedly grow with pamidronate 8 x 10(-5) M a 1 x 10(-4) M, which are biologically high concentrations. Potentiometric assessments, in the 1 x 10(-5) to 1 x 10(-4) M range of concentrations show that bisphosphonate was completely adsorbed to the crystals. Additional "in vivo" observations showed changes in bone powder crystals isolated from pamidronate treated young animals, involving a growing of planes 002 and 211, in samples from both epiphysis and diaphysis, regarding untreated samples. Changes were more evident at epiphysis. In mature rabbits, it was shown a decrease in basal plane 002 and growing at 210, 211 and 310 with a trend to enlarge the crystal shape in diaphysis and to shorten it in vertebrate spongiosa. The "in vivo" doses are equivalent to those used by Ferretti et al. in intact rats with pamidronate low dose groups, showing an improvement of bone material properties and stiffness. Thus, it may rather be lower than the "in vitro" used concentrations. In concordance with above experimental conditions it can be concluded that bisphosphonates exert morphological changes in hydroxyapatite crystals, in a dose dependent manner, at least when high concentrations are used. In addition, it is postulated that changes observed on "in vivo" samples may be the result with other adaptative factors as for example the local mechanical usage. The latter data were limited, and should be studied with more details if an extrapolation to the bisphosphonate treated osteoporotic women is intended. Finally, it is suggested that any agent that changes BMU activity (all known anti-osteoporotic drugs) may potentially modify the quality of hydroxyapatite crystals, affecting in turn the bone resistance to fracture, independently from the quantity of bone mass gained. Thus, to help predicting the consequences on skeletal fragility, there is a need to know the direct or indirect effect of drugs on bone crystals.

[Experimental effects of olpadronate and pamidronate on bone mass]. / Efectos experimentales de olpadronato y pamidronato sobre la masa ósea.

Ovariectomy and immobilization in rats have demonstrated to be useful models for osteopenia and they are considered to mimic some aspects of human osteoporosis associated with a deficit of ovarian hormones and the absence of mechanical function (disuse of the bone). Pamidronate (APD) and Olpadronate (OLPA), a new dimethylated aminobisphosphonate, on a continuous oral scheme (APD: 8 and OLPA: 0.8 mg/kg/day) or on an intermittent parenteral scheme (APD: 1.25 and OLPA: 0.075 mg/kg every 15 days) did effectively prevent the trabecular bone loss caused by immobilization (unilateral sciaticectomy), by lack of ovarian stimuli (bilateral ovariectomy) or by both approaches. There were no signs of deterioration in the cortical bone mass. In a model of preestablished osteopenia, caused by estrogen deprivation, OLPA stopped the progression of the bone mass loss (0.5 mg/kg/i.v. every 15 days) and restored (0.30-0.60 mg/kg/i.v. every 15 days) the bone mineral density which had been affected (trabecular and cortical). The different activity of OLPA and APD on trabecular and cortical regions of long bones seems to accompany their different responses because of negative stimulus: better responses were more evident in the trabecular bone which proved to be more labile. In these "in vivo" models of OLPA's efficacy was similar to APD's but it was roughly 5-10 times more potent. OLPA has a high safety margin. Therefore, it could advantageously be used in those bone diseases which benefit with the use of bisphosphonates.

[Clinical application of bisphosphonate's pharmacokinetic principles]. / Aplicación clínica de los principios farmacocinéticos de los bisfosfonatos.

Bisphosphonates are a group of osteotropic substances able to modulate bone metabolism in different ways. They all display similar pharmacokinetic characteristics when administered in proportional dosages and assessed by similar methods. With the exception of olpadronate which is soluble in water, bisphosphonates have poor solubility, and may easily precipitate in the digestive media. In spite of their low digestive absorption (Bioavailability: 0.3-5%), they are effectively administered by oral route. Once in plasma they distribute rapidly, being uptaked by mineralized tissues, plasma proteins or eliminated by renal filtration in few minutes. The fraction retained in bones may be stored for long periods (from months up to 10 years depending on the compound) in an apparent inactive compartment. The risks of newly released molecules may be related to the potency of the drug. Within the clinical range of doses, bisphosphonates are not retained in soft tissues. This may explain the lack of extraskeletal collateral effects. Plasma blood levels are scarcely related to the clinical activity. Therefore, dosage may be guided better by biochemical markers of the bone disease than by the standard kinetic variables. On the other hand, dose is independent from age and/or body weight. Only renal impairment may induce additional dose adjustments.

[Preclinical toxicology of bisphosphonates]. / Toxicología preclínica de bisfosfonatos.

Bisphosphonates regulate bone turnover by inhibiting osteoclastic bone resorption. Due to their pharmacodynamic and pharmacokinetic characteristics, bisphosphonates have a special pharmacotoxicological profile related to their high degree of specificity: low or non-existent distribution in soft tissues and strong affinity for calcified tissues. Some general conclusions may be drawn from the pre-clinical toxicological studies, whose main aim is to identify the toxicity target organ/s and estimate the safety margins of a "prospective therapeutic agent" in laboratory animals. They are based on our own results and on data from the available literature as regards various bisphosphonates: Alendronate, Clodronate, Etidronate, Olpadronate and Pamidronate. Generally, very high doses of bisphosphonates are required to produce in different levels and incidence various extra-skeletical toxic side effects: local reaction, hypocalcemia (and its consequences on the cardiovascular system and the possibility of tetany), affection of the dental structures and renal dysfunction. Most of side effects may be related to the low solubility in biological fluids, the formation of calcium complexes, the potent inhibitory effect of endogenous or induced bone resorption as well as to its main excretion pathway. Some other side effects (on the eye, lungs and liver), may be related to repeated excessive high doses. A safety margin of 200 to 300 : 1 between the "toxic" and "pharmacological" doses may be estimated if the total quantity of Olpadronate given to various animal species in toxicological studies and in pharmacodynamic experimental models (osteopenias due to estrogen deprivation or immobilization and retinoid-induced hypercalcemia) is considered. If the toxic doses in animals are related to the highest doses suggested for human beings, then the ratio increases from 300 to 1000 : 1 depending on the pathology and the route of administration. As regards their effect on the bone, experimental data with the new bisphosphonates suggest a significant dissociation between pharmacologically active doses and those ones producing defective mineralization. The excessive inhibition of bone remodelling, due to the use of high doses in normal animals, is the natural consequence of the pharmacological effect of this family of compounds. A bisphosphonate's toxic potential effect on bone should not be evaluated in normal animals but in particular situations with a high bone turnover. Furthermore, the doses should be adjusted in order to regulate the magnitude of bone remodelling inhibition so as to take it to a normal level without totally suppressing it. Potency, safety margins, doses and proper administration schemes, should be considered as key elements for the optimum use of the therapeutic potentiality of these compounds.

[Tolerability of oral bisphosponates in patients with osteoporosis and other osteopathies]. / Tolerabilidad digestiva de los bisfosfonatos orales en pacientes con osteoporosis y otras osteopatías medicas.

Oral nitrogen containing bisphosphonates (NCB) are effective drugs to inhibit bone metabolism turnover in osteoporosis and other bone diseases. Notwithstanding, some digestive disturbances create concern on the long term acceptance of the oral route. Side effects are mainly caused by low absorption and poor solubility in digestive content. Therefore the compound may precipitate and irritate the mucosas. Furthermore, the administered amount of a particular molecule, its intrinsic potency to irritate digestive walls and the degree of exposition to such sensitive tissue are other facts that combined, may determine the clinical tolerability. Thus, a single factor cannot predict the clinical tolerability. Pamidronate, alendronate and olpadronate are the main NCB under clinical usage. Alendronate is 10 times more potent than pamidronate but possesses a similar slight solubility (2.4 vs 3.0% W/V respectively). It also seems to be more (3 times fold) ulcerogenic in experimental assays. The current available pamidronate formulation protects from esophagus and gastric exposition. Up to now and until randomized clinical trials be performed the selection of the most tolerated aminobisphosphonate in clinical practice will depend on the interplay of many factors (table 1 shows a hypothetical view). Moreover, different patients may react dissimilarly depending on their sensitivity to a particular factor. Olpadronate is free-soluble (24% W/V), almost equipotent with alendronate (figure 1) and seems to lack relevant irritation potential, but clinical data is on its early phases and is still not available. Micronization of the bisphosphonate preparation may be of help to improve tolerability as shown with newer pamidronate oral formulations. The current clinical published data shows more or less the same safety profile for pamidronate (only when enteric coated capsules are used) as alendronate, with more than 90% of patients complying with long term treatments. Anyway the trials are not entirely comparable as said before. Some other pamidronate formulations proved to be intolerable and have not been accepted. Identifying the many factors of oral NCB's digestive tolerability may help with their clinical management. And in those countries where the two compounds are available they may alternatively be used in the sensitive patients. Finally, extra-digestive side effects, not commented in this article, should also be weighted when selecting a bisphosphonate.

Evidence of a cadmium-thionein and the glycine cleavage system in Oxytricha granulifera.

This work presents the further purification of a Cd-linking protein in Oxytricha granulifera by reverse-phase chromatography. This protein contains 25% cysteine and no aromatic amino acid. It may be considered as a chelatin with some similarity to metallothioneins. During the purification of another Cd-linking compound, we were able to demonstrate that the H protein precursor of glycine cleavage is present in Oxytricha. This is the first finding of the presence of this system in Protozoa.

Purification and primary structure of metallothioneins induced by cadmium in the protists Tetrahymena pigmentosa and Tetrahymena pyriformis.

Tetrahymena pyriformis and Tetrahymena pigmentosa grown in the presence of a non-toxic dose of cadmium, accumulate the metal in the cytosol. Purification by gel-permeation, ion-exchange and reverse-phase high-performance liquid chromatography showed that the metal is bound principally to newly formed proteins with ultraviolet spectra and cysteine contents similar to those of Cd(2+)-metallothioneins from multicellular organisms. The isolated proteins revealed that the two species of ciliates each express two Cd(2+)-isothioneins. The primary structures determined by both Edman degradation and mass spectrometry revealed that the equivalent proteins from T. pyriformis and T. pigmentosa have identical sequences and that the two isoforms in each species differ only by the presence or absence of a lysine residue at the N-terminus. The development of automated mass spectrometric sequence analysis algorithms combined with an accurate determination of the molecular mass allowed the rapid confirmation of the sequences. The Tetrahymena metallothionein sequences are unusually long (105 and 104 amino acids) and show a unique internal homology which suggests that the proteins arose by gene duplication. The chains contain 31 cysteine residues, 15 of which are arranged in motifs characteristic of the mammalian metallothioneins; the remaining residues show several unique repeating motifs, which could have interesting consequences for the tertiary structure of the metal-binding sites. Amino acid sequences of Tetrahymena metallothioneins have some similarity with other eukaryotic metallothioneins. A comparison on the basis of optimised FASTA scores, shows a closer relationship with horse metallothionein-1B.

Cadmium-thionein in Tetrahymena thermophila and Tetrahymena pyriformis.

The treatment of Tetrahymena thermophila with cadmium causes a reduction in growth rate according to dose; almost all the metal is accumulated in the cytosol where the Zn content is also increased threefold. Bio-Gel and Water 160 (HPLC) column chromatography show that Cd and Zn are bound to a protein with an ultraviolet (UV) spectrum that appears to be similar to that of Cd-metallothioneins isolated by higher organisms, but its molecular weight is greater: about 28 000 D, comparable to that of metallothionein isolated from Tetrahymena pyriformis. Further purification of these proteins by ion exchange chromatography revealed the presence of two peaks, considered as two isoforms of the metallothioneins present in both T. thermophila and T. pyriformis (MT 1 and MT 2). Their amino acid analyses confirmed that they are different isometallothioneins, MT 1 and MT 2, with about 30% cysteine, and aspartic acid, glycine and lysine as major amino acids. From our analyses we may conclude that Tetrahymena pyriformis MTs are similar to those present in invertebrates and vertebrates, while Tetrahymena thermophila MTs are peculiar in that they have cyclic amino acid histidine in both MT 1 and MT 2; furthermore, aromatic amino acid phenylalanine is also present in MT 2.

Biochemical and ultrastructural data on Tetrahymena pyriformis treated with copper and cadmium.

The addition of copper (10 micrograms ml-1) or cadmium (5 micrograms ml-1) to the medium is well tolerated by Tetrahymena pyriformis GL. Both metals are accumulated by cells, cadmium to a greater extent than copper. The growth rate is not affected and from the micrographs it is evident that the ultrastructure is not altered by the treatments. After 3 days of culture the macronucleus contains dense masses of chromatin and numerous nucleolar fusion bodies. Granules, cytolysomes and many food vacuoles are present in both control and treated cells. Cadmium induces the formation of a chelating protein; the amino acid analyses and the ultraviolet spectrum indicate that it is similar to the metallothionein isolated by higher organisms. The molecular weight of native protein is about 27,000. After treatment by sulphitolysis or oxidation we obtained a peak of molecular weight at about 6,000. The treatment with copper does not appear to induce metallothioneins or other chelatins. The high tolerance of Tetrahymena towards cadmium is believed to be due to the formation of a Cd-Zn metallothionein. The different chelating proteins induced by copper and cadmium in other groups of Protozoa and the different detoxification mechanisms present in these organisms are discussed.

Chelatins in Euglena gracilis and Ochromonas danica.

Amino acid analysis was performed on low molecular weight copper binding proteins purified from two species of Protozoa after exposure to a high level of this metal. The compound from Ochromonas is similar to Cu-chelatins. The two peptides from Euglena have a different molecular weight and a very dissimilar amino acid composition. Peptide No. 1 has a peculiar composition with a high content of aspartic acid and arginine. Some speculations are made about its detoxification role in comparison with other compounds found in blue-green algae.